CELLS, ISLETS AND ORGANOIDS THAT EVADE IMMUNE DETECTION AND AUTOIMMUNITY, METHODS OF PRODUCTION AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. Provisional Application No. 62/795,284, filed on January 22, 2019, and U.S. Provisional Application No. 62/745,086, filed on October 12, 2018, the entire contents of each of which are incorporated by reference herein in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This invention was made with government support under Grant Nos. DK057978, DK090962, HL088093, HL105278 and ES010337 awarded by the National Institutes of Health, and Grant No. P30 014195 awarded by the National Institutes of Health and the National Cancer Institute. The government has certain rights in the invention.

BACKGROUND

For the treatment of insulin dependent diabetes, such as type 1 diabetes and late-stage type 2 diabetes, the shortage of human islets limits the number of patients who can benefit from this therapy. Despite progress in the field of in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into b-like cells, the b-like cells generated in this manner typically exhibit impairments in glucose-stimulated insulin secretion (GSIS) and

mitochondrial metabolic function, as well as detection and destruction by a recipient’s immune system following administration. Thus, further improvements to the maturation process are required to fully capture pancreatic islet physiology and the generation of functional and lasting organoids.

Needed in the art are methods for generating functional human organs that survive transplant for the treatment of diseases, as well as new platforms for drug-screening and disease modeling to provide new treatment strategies and therapeutics for patients with organ failure.

SUMMARY OF THE DESCRIBED EMBODIMENTS

Provided are compositions and methods for generating an immunoprotected cell, islet, organoid, or islet-like organoid, including, but not limited to, a human pancreatic islet organoid or a pancreatic organoid, in particular, a human islet-like organoid (abbreviated as “HILO” herein), that survives and evades detection by the immune system (autoimmunity) following administration to or transplant or implant in a subject. In an embodiment, the cell, islet, organoid, islet-like organoid (and cells therein) expresses interferon gamma (IFNy)- receptors. In an embodiment, the cell, islet, organoid, or islet-like organoid (and cells therein) is human.

In an aspect, a method of increasing survival or reducing cell death of a transplanted donor cell is provided in which the method comprises contacting the donor cell with multiple intermittent exposures to interferon gamma (IFNy) over a given time period, e.g., a time period of at least 24 hours, thereby increasing survival of the transplanted donor cell. In an embodiment, the the transplanted donor cell is an organoid cell, an islet cell, an islet-like organoid cell, or a b-like islet cell. In an embodiment, the transplanted donor cell is syngeneic to the subject who receives the transplant. In an embodiment, the transplanted donor cell is autologous to the subject who receives the transplant. In an embodiment, the transplanted donor cell is allogeneic or xenogeneic to the subject who receives the transplant. In an embodiment, the transplanted donor cell is an interferon gamma (IFNy) receptor expressing cell. In an embodiment, the transplanted donor cell is a human cell.

In another aspect, a method of generating an immunoprotected cell, islet, or organoid that survives detection by immune system cells, e.g., T cells or B cell, is provided in which the method comprises subjecting an interferon gamma (IFNy) receptor-expressing cell, islet, or organoid, or cells thereof, to multiple intermittent exposure to IFNy over a given time period, e.g., a time period of at least 24 hours, thereby inducing expression of an immune checkpoint protein by the cell, islet, or organoid and allowing said cell, islet, or organoid to survive immune detection or autoimmunity.

In an aspect, the human islet-like organoid (HILO) and the cells comprising the HILO, namely, beta (P)-like cells, express or are induced to express following exposure to IFNy one or more molecules involved in modulating the immune response or autoimmunity, such as an immune checkpoint protein, to overcome immune rejection or autoimmunity of “non-self’ cells or HILOs introduced into, e.g., transplanted or implanted, into a subject. In an embodiment, the immune checkpoint protein is PD-L1. In an embodiment, the subject into whom HILOs are introduced, transplanted, or implanted has diabetes. In an

embodiment, the subject into whom HILOs are introduced, transplanted, or implanted has type 1, type 2 diabetes, or late stage type 2 diabetes. In an embodiment, the subject into whom HILOs are introduced, transplanted, or implanted has type 1 diabetes. In an embodiment, the subject into whom HILOs are introduced, transplanted, or implanted is a human subject or patient. In an embodiment, the one or more immune checkpoint protein is recombinantly expressed in the introduced, transplanted, or implanted cells or HILOs. The terms“transplant” and“implant” may be used interchangeably herein to refer to cells, islets, or organoids (and cells therein) that are introduced or transferred into a subject by procedures practiced in the medical arts to effect or provide a function therein, especially a therapeutic function to treat a disease, disorder or pathology.

In one aspect, a method of generating a pancreatic islet organoid is provided in which induced pluripotent stem cell (iPSC)-derived beta (p)-like cells are cultured in a 3- dimensional matrix containing gellan gum, thereby generating a pancreatic islet organoid in which the organoid cells express one or more checkpoint proteins. Also provided is a cell culture including an iPSC-derived beta-like cell, which expresses one or more immune checkpoint proteins, in a three-dimensional matrix containing gellan gum. In an

embodiment, the one or more immune checkpoint proteins is PD-L1.

In an aspect, a cell culture including a human iPSC-derived beta-like cell, a human adipose-derived stem cell (hADSC), and a human umbilical vein endothelial cell (HUVEC) in a three-dimensional matrix containing gellan gum is provided, in which the cells of the culture express one or more immune checkpoint proteins.

In various embodiments of any aspect delineated herein, the cell culture includes an adipose-derived stem cell and/or an endothelial cell.

In an aspect, a pancreatic islet-like organoid containing an iPSC-derived beta-like cell which expresses one or more immune checkpoint proteins is provided, wherein the organoid is vascularized and exhibits glucose-stimulated insulin secretion (GSIS) and wherein the cells of the organoid and the organoid express one or more immune checkpoint proteins. In an embodiment, the pancreatic islet-like organoid is a human pancreatic islet-like organoid. In an embodiment the one or more immune checkpoint proteins is PD-L1.

In an aspect, a pancreatic islet organoid containing an iPSC-derived beta (B)-like cell, an iPSC-derived alpha (a) cell, an iPSC-derived delta (d) cell, an iPSC-derived duct cell, an adipose-derived stem cell (hADSC), and an endothelial cell wherein the iPSC cell expresses one or more immune checkpoint proteins, the organoid is vascularized and exhibits glucose- stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, and glucagon secretion is provided.

In a related aspect, a non-human organism transplanted or implanted with the organoid of any aspect delineated herein is provided.

In an aspect, a method of treating a pancreatic disease in a subject is provided, in which a pancreatic islet organoid, or HILO, is introduced or transplanted or implanted into the subject, wherein the pancreatic islet organoid, or HILO, contains iPSC-derived beta-like cells, which express one or more immune checkpoint proteins to evade immune detection; wherein the pancreatic islet organoid, or HILO, is vascularized and exhibits glucose- stimulated insulin secretion (GSIS). In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the subject is human and the pancreatic islet organoid, or HILO, is generated from human tissue or cells.

In an aspect, a method of treating type 1 diabetes in a subject is provided, in which a pancreatic islet organoid, or HILO, is introduced, transplanted, or implanted into the subject, wherein the pancreatic islet organoid, or HILO, contains iPSC-derived beta-like cells, which express one or more immune checkpoint proteins to evade immune detection; wherein the pancreatic islet organoid, or HILO, is vascularized and exhibits glucose-stimulated insulin secretion (GSIS). In an embodiment, the pancreatic islet organoid, or HILO, expresses a checkpoint protein to evade immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the subject is human and the pancreatic islet organoid, or HILO, is generated from human tissue or cells.

In an aspect, a pancreatic islet organoid or HILO is provided, in which the pancreatic islet organoid or HILO is generated by culturing an induced pluripotent stem cell (iPSC)- derived beta-like cell in a 3-dimensional matrix containing gellan gum. In an embodiment, the pancreatic islet organoid, or HILO, expresses one or more immune checkpoint proteins to evade immune detection. In an embodiment, the subject is human and the pancreatic islet organoid, or HILO, is generated from human tissue or cells. In an embodiment, the one or more immune checkpoint proteins is PD-L1.

Provided in another aspect is a pancreatic organoid or HILO generated by culturing an induced pluripotent stem cell (iPSC)-derived beta-like cell and an iPSC-derived exocrine component cell in a 3-dimensional matrix containing gellan gum. In an embodiment, the pancreatic islet organoid, or HILO, expresses one or more immune checkpoint proteins to evade immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the subject is human and the pancreatic islet organoid, or HILO, is generated from human tissue or cells.

Provided in another aspect is a pancreatic organoid or HILO generated by culturing an induced pluripotent stem cell (iPSC)-derived beta-like cell and an iPSC-derived exocrine component cell in a culture medium, such as a 3 -dimensional matrix containing gellan gum and an agent that stimulates expression and production of a checkpoint protein in the cells of the pancreatic organoid (b-cells) or HILO. Without wishing to be bound by theory, the PD- Ll is produced in the b-cells or HILO through the mechanism of transcriptional memory. In an embodiment, the culture medium or matrix comprises interferon gamma (IFNy). In an embodiment, the pancreatic islet organoid, or HILO, expresses one or more immune checkpoint proteins to evade immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the subject is human and the pancreatic islet organoid, or HILO, is generated from human tissue or cells.

In another aspect, the invention provides a liver organoid generated by culturing an induced pluripotent stem cell (iPSC)-derived hepatocyte in a 3-dimensional matrix containing gellan gum; wherein the iPSC-derived hepatocyte expresses one or more immune checkpoint proteins such that the liver organoid evades immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1.

In another aspect, the invention provides a heart organoid generated by culturing an induced pluripotent stem cell (iPSC)-derived cardiomyocyte in a 3-dimensional matrix containing gellan gum wherein the iPSC-derived cardiomyocyte expresses one or more immune checkpoint proteins such that the heart organoid evades immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1.

In another aspect, the invention provides an intestinal organoid generated by culturing an induced pluripotent stem cell (iPSC)-derived intestinal cell in a 3-dimensional matrix containing gellan gum, wherein the iPSC-derived intestinal cell expresses one or more immune checkpoint proteins such that the intestinal organoid evades immune detection. In an embodiment, the one or more immune checkpoint proteins is PD-L1.

In various embodiments of any aspect delineated herein, the method involves culturing the iPSC-derived beta-like cell, which expresses one or more immune checkpoint proteins, with an adipose-derived stem cell and/or an endothelial cell. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In various embodiments of any aspect delineated herein, the method involves culturing the iPSC-derived beta-like cell, which expresses one or more immune checkpoint proteins, with an iPSC-derived alpha-like cell, an iPSC-derived delta-like cell, and/or an iPSC-derived duct-like cell.

In various embodiments of any aspect delineated herein, the pancreatic islet organoid contains an iPSC-derived alpha-like cell, an iPSC-derived delta-like cell, and/or an iPSC- derived duct-like cell. In various embodiments of any aspect delineated herein, the pancreatic islet organoid includes an adipose-derived stem cell and/or an endothelial cell. In various embodiments of any aspect delineated herein, the pancreatic islet organoid exhibits KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, c-peptide expression, and/or glucagon secretion. In various embodiments of any aspect delineated herein, the pancreatic islet organoid expresses one or more of the beta cell transcription factors Pdxl, MafA, Pax4, Pax6, NeuroDl, Nkx6-l, Gata6, and Foxa2. In certain embodiments, the pancreatic islet organoid contains an iPSC-derived beta-like cell, which expresses one or more immune checkpoint proteins, an iPSC-derived alpha cell, an iPSC-derived delta cell, an iPSC-derived duct cell, an adipose-derived stem cell (hADSC), and an endothelial cell, where the organoid is vascularized and exhibits glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, and glucagon secretion. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In various embodiments of any aspect delineated herein, the pancreatic islet organoid is surrounded by an iPSC-derived exocrine component.

In various embodiments, the iPSC-derived exocrine component expresses one or more of the markers PDX1, Nkx6-l, and Ptfl.

In various embodiments of any aspect delineated herein, the liver organoid expresses one or more of the markers AFP, ALB, and Cyp3a7. In various embodiments of any aspect delineated herein, the liver organoid exhibits insulin signaling, insulin resistance by palmitic acids, and lipid accumulation.

In various embodiments of any aspect delineated herein, the heart organoid expresses one or more of the markers hMlc2a, hNkx2-5, alphaMHC and KCNQ1. In various embodiments of any aspect delineated herein, the heart organoid exhibits cardiac beating.

In various embodiments of any aspect delineated herein, the intestinal organoid expresses one or more of the markers CDX2, Muc2, and Lgr5. In various embodiments of any aspect delineated herein, the intestinal organoid exhibits budding in response to R- spondin.

In various embodiments of any aspect delineated herein, the iPSC-derived beta-like cell, iPSC-derived alpha-like cell, iPSC-derived delta-like cell, and/or iPSC-derived duct-like cell is human. In various embodiments of any aspect delineated herein, the iPSC-derived beta-like cell, iPSC-derived exocrine component cell, iPSC-derived hepatocyte, iPSC-derived cardiomyocyte, or iPSC-derived intestinal cell is human. In various embodiments, the adipose-derived stem cell is a human adipose-derived stem cell (hADSC). In various embodiments of any aspect delineated herein, the endothelial cell is a human umbilical vein endothelial cell (HUVEC). In various embodiments, the organoids are generated from human cells.

In various embodiments of any aspect delineated herein, the pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid, or intestinal organoid, contains an adipose-derived stem cell and/or an endothelial cell. In various embodiments of any aspect delineated herein, the pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid, or intestinal organoid is vascularized.

In another aspect, the invention provides a method of generating a pancreatic islet organoid of HILO, the method comprising culturing an induced pluripotent stem cell (iPSC)- derived beta-like cell, which expresses one or more immune checkpoint proteins, in a medium comprising Wnt4 or Wnt5a protein. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the induced pluripotent stem cell (iPSC)- derived beta-like cell is cultured in a 3 -dimensional matrix. In an embodiment of the foregoing aspect, the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. In a particular embodiment, the medium comprises recombinant human Wnt4 protein. In another particular embodiment, the medium comprises recombinant human Wnt5a protein.

In a particular embodiment, a Wnt4- or Wnt5-induced human islet organoid or HILO is a mature islet or a mature HILO.

In another aspect the invention provides a cell culture comprising a human iPSC- derived beta-like cell, which expresses one or more immune checkpoint proteins, and Wnt4 or Wnt5a protein. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the human iPSC-derived beta-like cell is in a three-dimensional matrix comprising gellan gum. In an embodiment, the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. In a particular embodiment, the medium comprises recombinant human Wnt4 protein. In another particular embodiment, the medium comprises recombinant human Wnt5a protein. In a particular embodiment, a Wnt4- or Wnt5-induced human islet organoid or HILO is a mature islet or a mature HILO.

In another aspect, the invention provides a pancreatic islet organoid comprising an iPSC-derived beta-like cell, which expresses one or more immune checkpoint proteins, cultured in medium comprising Wnt4 or Wnt5a protein, wherein the organoid is vascularized and exhibits glucose-stimulated insulin secretion (GSIS). In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the organoid further exhibits KC1- stimulated insulin secretion or glucose stimulated insulin secretion. In an embodiment, the pancreatic islet organoid expresses Fltp and Esrrg genes. In an embodiment, the Wnt4 or Wnt5a protein is a recombinant human Wnt4 or Wnt5a protein. In a particular embodiment, the medium comprises recombinant human Wnt4 protein. In another particular embodiment, the medium comprises recombinant human Wnt5a protein. In a particular embodiment, a Wnt4- or Wnt5-induced human islet organoid or HILO is a mature islet or a mature HILO.

In another aspect, the invention provides a non-human organism transplanted or implanted with the organoid defined in the above described aspects.

In another aspect, the invention provides a method of enhancing self organization of adipose-derived stem cells (ADSCs) for generating an induced pluripotent stem cell (iPSC)- derived organoid, which evades immune surveillance and rejection, the method comprising culturing the ADSCs in a 3-dimensional (3-D) culture matrix medium comprising a Wnt5a protein. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment of the method, the ADSCs are cultured in a 3-D culture matrix comprising gellan gum. In an embodiment, the ADSCs are cultured in the 3-D culture matrix medium comprising a Wnt5 protein and an iPSC-derived cell selected from an iPSC-derived beta-like cell, an iPSC-derived exocrine component cell, an iPSC-derived hepatocyte, an iPSC-derived cardiomyocyte, or an iPSC-derived intestinal cell which expresses one or more immune checkpoint inhibitor proteins. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment of the method, the iPSC-derived organoid is selected from a pancreatic islet organoid, pancreatic organoid, a liver organoid, a heart organoid, or an intestinal organoid. In an embodiment of the method, the induced pluripotent stem cell (iPSC)-derived organoid is a human induced pluripotent stem cell (hiPSC)-derived organoid. In an embodiment of the method, the Wnt5a protein is a recombinant human Wnt5a protein. In an embodiment of the method, the pancreatic islet organoid, pancreatic organoid, liver organoid, heart organoid, or intestinal organoid is derived from an iPSC-derived cell selected from an iPSC-derived beta-like cell, an iPSC-derived exocrine component cell, an iPSC- derived hepatocyte, an iPSC-derived cardiomyocyte, or an iPSC-derived intestinal cell, respectively. In an embodiment, of any of the above, the iPSC-derived cell is human.

In another aspect, the invention provides a method of enhancing self organization of adipose-derived stem cells (ADSCs) for generating a pancreatic islet or pancreatic organoid that evades immune rejection or autoimmunity, comprising culturing ADSCs, which express one or more immune checkpoint proteins, in medium comprising Wnt5a protein. In an embodiment, the one or more immune checkpoint proteins is PD-L1. In an embodiment, the ADSCs are cultured in a 3 -dimensional matrix comprising gellan gum. In another embodiment, the Wnt5a protein a recombinant human Wnt5a protein.

In another aspect, the invention provides a pancreatic islet organoid, pancreatic organoid, a liver organoid, a heart organoid, or intestinal organoid produced by any of the above-delineated methods and embodiments thereof.

In various aspects of any of the foregoing embodiments, the immune checkpoint protein, or the one or more immune checkpoint proteins, or a fragment or portion of the immune checkpoint protein that binds to cognate ligand, is recombinantly expressed in or molecularly introduced into the cells of an organoid, (e.g., b-like cells that constitute HILOs) which express the one or more checkpoint proteins as membrane surface proteins that bind to a cognate ligand on an immune cell, e.g., a T cell, that is involved in autoimmunity, or that reacts against a foreign or‘non-self cell, so as to suppress or block the T cell response (an allogeneic immune response or autoimmune response) and thus evade immune system surveillance and rejection in a recipient.

In embodiments, the cells of an organoid, (e.g., b-like cells that constitute HILOs) express one or more checkpoint proteins or molecules that bind to cognate ligands on the surface of an immune cell to suppress allogeneic immune activity or autoimmunity against the cells and the organoid. In a particular embodiment, the cells of an organoid, (e.g., a b-like cell) and the organoid (e.g., HILO) express the immune checkpoint protein PD-L1, programmed cell-death ligand 1, which binds to PD-l, programmed cell-death protein 1, which is expressed, for example, on T cells. PD-L2, programmed cell-death ligand 2, also binds to PD-l, but with a different Kd. In other embodiments, the cells of an organoid, (e.g., a b-like cell) and the organoid (e.g., HILO) are molecularly engineered to express a molecule that binds a checkpoint protein expressed on the surface of an immune cell, such as a T cell (e.g., an effector T cell), wherein the checkpoint protein expressed on the surface of an immune cell is CTLA-4 (cytotoxic T-lymphocyte protein 4, also called CD152); LAG-3, lymphocyte activation gene 3 protein; KIR, killer cell immunoglobulin-like receptor; IDOl, indoleamine 2,3-dioxygenase 1; 4-1BB, a tumor necrosis factor receptor superfamily member 9, (also known as CD137); GITR,“glucocorticoid-induced TNFR family related gene; TIM- 3,“T-cell immunoglobulin domain and mucin domain;” 0X40, tumor necrosis factor receptor superfamily member 4, (also known as CD 134); A2AR, adenosine A2A receptor; B7-H3 (also called CD276); B7-H4 (also called VTCN1); B7-1/B7-2; BTLA (also called CD272); VISTA,“V-domain Ig suppressor of T cell activation;” or a combination of any of the foregoing.

In an aspect of any of the foregoing embodiments, the immune checkpoint protein comprises all, or a portion, e.g., the extracellular domain, of the checkpoint protein (also called a“checkpoint molecule” herein). In a particular embodiment, the immune checkpoint protein is PD-L1 or a binding portion thereof. In an embodiment, the checkpoint protein is the extracellular domain of the PD-L1 protein.

Another aspect provides a human induced pluripotent stem cell (hiPSC), human beta (P)-cell, or human islet-like organoid (HILO) generated therefrom, molecularly engineered to express one or more immune checkpoint proteins that bind to a cognate ligand on an immune cell, such as a T cell. In an embodiment, the one or more immune checkpoint proteins expressed by a hiPSC, human beta (p)-cell, or human islet-like organoid (HILO) binds to an immune cell-expressed cognate ligand selected from programmed cell-death protein 1 (PD- 1); cytotoxic T-lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG- 3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3 -di oxygenase 1 (IDOl); tumor necrosis factor receptor superfamily member 9 (4-1BB); glucocorticoid-induced TNFR family related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4, (0X40); adenosine A2A receptor (A2AR); B7-H3; B7-H4; B7-1/B7-2; BTLA; V-domain Ig suppressor of T cell activation (VISTA); or a combination of any of the foregoing. In a particular embodiment, the hiPSC, human beta (P)-cell, or HILO expresses the immune checkpoint protein, programmed cell- death protein-ligand 1 (PD-L1), which binds to PD-l .

In another aspect, a method of generating cells, islets, organoids that survive and have reduced cell death following transplantation, implantation, or transfer is provided in which the method comprises: (a) contacting interferon gamma (IFNy)-receptor expressing cells, islets, or organoids with interferon gamma (FFNy) for at least 0.5 hour or at least one hour at a predetermined time point; and (b) repeating step (a) at least about two times during a time period of about or equal to 72-hours; wherein the cells, islets, or organoids are maintained in the absence of IFNy between times of contact with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in the cells, islets, or organoids. In an embodiment of the method, the cells, islets, organoids or cells are contacted with IFNy for a time period selected from about or equal to at least 0.5 hour, at least 1 hour, at least 2 hours, or more than 2 hours in step (a). In another embodiment of the method, the cells, islets, or organoids are contacted with IFNy for a time period selected from about or equal to 0.5 hour, or about or equal to 1 hour, or about or equal to 2 hours or about or equal to 12 hours in step (a). In another embodiment of the method, step (a) is repeated at least three times for at least about 0.5 hour each time, or for at least about 1 hour each time, or for at least about 2 hours each time in the about or equal to 72-hour time period of step (b). In another embodiment of the method, the cells, islets, or organoids are washed to remove the presence of IFNy between step (a) and step (b). In another embodiment of the method, IFNy is used in an amount of 1- 25 ng/ml. In another embodiment of the method, IFNy is used in an amount of 10 ng/ml. In another embodiment of the method, PD-L1 expression in the cells, islets, or organoids is maintained following step (b) for greater than about or equal to 7 days. In an embodiment, sustained expression of PD-L1 comprises about or equal to 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer, of PD-Ll expression in a cell.

In another aspect, a method of generating islets, or organoids and the cells thereof that survive and have reduced cell death following transplant, implant or transfer is provided, in which the method comprises: (a) contacting interferon gamma (IFNy)-receptor expressing islets or organoids and the cells thereof with interferon gamma (IFNy) in an amount of about 1 ng/ml to 25 ng/ml for greater than 1 hour at a first time point during a given time period, e.g., a time period of about or equal to 24-hours; and (b) contacting the islets or organoids and the cells thereof with IFNy in an amount of about 1 ng/ml to 25 ng/ml for greater about 0.5-1 hour or longer at two or more additional time points during a following time period, e.g., a 48-hour time period, following step (a); wherein said islets or organoids are washed and rested in medium in the absence of IFNy between being contacted with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in said islets or organoids. In an embodiment of the method, the islets or organoids are contacted with IFNy in an amount of 10 ng/ml for at least 2 hours in step (a) and step (b). In another embodiment of the method, the islets or organoids are contacted with IFNy for at least about 2 hours at 3 timepoints during the 72-hour timeperiod.

In an embodiment of any of the above-denoted methods, the cells, islets, or organoids are human cells, islets, or organoids. In another embodiment of the above methods, the organoids are HILOs or human HILOs. In another embodiment of the above methods, the islets are human cadaveric islets which are protected from destruction or clearance by the immune system.

In another aspect, a method of generating human cells, islets, or human islet like organoids (HILOs) that evade immune detection or autoimmunity is provided in which the method involves (a) contacting the human cells, islets or HILOs with interferon gamma (IFNy) for greater than one hour at predetermined time point; repeating step (a) at least two times during a given time period, e.g., a 72-hour time period; wherein the human cells, islets, or HILOs are maintained in the absence of IFNy between times of contact with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in the human islets or HILOs. In an embodiment of the method, the human cells, islets, or HILOs are contacted with IFNy for 2 hours or more in step (a). In another embodiment of the method, the human cells, islets, or HILOs are contacted with IFNy for 2 hours or 12 hours in step (a). In another embodiment of the method, step (a) is repeated three times for at least 2 hours each time in the given time period, i.e., a 72-hour time period. In another embodiment of the method, the human cells, islets, or HILOs are washed to remove IFNy between step (a) and step (b). In another embodiment of the method, IFNy is used in an amount of 1-25 ng/ml. In another

embodiment of the method, IFNy is used in an amount of 10 ng/ml. In another embodiment of the method, PD-L1 expression in the islets or HILOs is maintained or sustained following step (b) for greater than 7 days. In another aspect, a method of generating human cells, islets or human islet like organoids (HILOs) that evade immune detection or autoimmunity is provided in which the method involves (a) contacting the human cells, islets or HILOs with interferon gamma (IFNy) in an amount of about 1 ng/ml to 25 ng/ml for greater than 1 hour at a first time point during a given time period, e.g., a 24-hour time period; and (b) contacting the human cells, islets or HILOs with IFNy in an amount of about 1 ng/ml to 25 ng/ml for greater than 1 hour at at least two additional time points during a next given time period, e.g., a 48-hour time period, following step (a); wherein the human cells, islets, or HILOs are washed and rested in medium in the absence of IFNy between being contacted with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in the human islets or HILOs. In an embodiment of the method, the human islets or HILOs are contacted with interferon gamma (IFNy) in an amount of 10 ng/ml for at least 2 hours in step (a) and step (b). In another embodiment of the method, the human islets or HILOs are contacted with interferon gamma (IFNy) for at least 2 hours at 3 different intervals (time points) during a given time period, such as a 72-hour time period. In an embodiment of the method of the foregoing aspects, the human islets or HILOs are mature human islets or HILOs. In an embodiment, sustained expression of PD-L1 comprises about or equal to 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer, ofPD-Ll expression in a cell. In embodiments of the method, the cells comprise cardiac cells, colon cells, kidney cells, bladder cells, liver cells (hepatocytes), esophageal cells, gastrointestinal cells, gastric (stomach) cells, lung cells, ovarian cells, cervical cells, uterine cells, testicular cells, pancreatic cells, pancreatic b cells, retinal cells, corneal cells, brain cells, muscle cells, hematopoietic cells, immune cells (B cells, T cells), chimeric antigen receptor-T cells (CAR- T cells), bone marrow cells, mononuclear cells, neurons, neuronal cells, insulin-producing pancreatic b cells derived from human skin cells, umbilical cord blood (UCB) cells, adipose derived mesenchymal stromal (stem) cells, cardiac stem cells, colon stem cells, kidney stem cells, liver (hepatocyte) stem cells, gastrointestinal stem cells, gastric stem cells, lung stem cells, pancreatic stem cells, pancreatic b stem cells, muscle stem cells, hematopoietic stem cells, immune cell (T cell or B cell) stem cells, bone marrow stem cells, CD133+ stem cells, CD34+ hematopoietic cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, umbilical cord mesenchymal stem cells, retinal stem cells, neuronal stem cells, ectoderm- derived neuronal cells, immortalized dopaminergic neuronal precursor cells and organoids generated from or containing said cells. In an embodiment of the method, the organoids comprise cardiac organoids, intestinal/gastrointestinal organoids, colonic organoids, hepatic organoids, kidney organoids, bladder organoids, ovarian organoids, cervical organoids, neural organoids, or pulmonary (lung) organoids.

In an embodiment of the methods of any of the above-delineated aspects, the interferon gamma (IFNy)-receptor expressing cells, islets, or organoids are contacted with IFNy in culture medium or a physiologically acceptable solution, or in a three-dimensional matrix. In an embodiment, the the interferon gamma (IFNy)-receptor expressing cells, islets, or organoids are contacted with IFNy in a three-dimensional (3D) matrix, e.g., gellan gum, as described herein.

In another aspect, a method of generating an islet-like organoid that evades immune detection or autoimmunity is provided, in which the method comprises culturing endocrine progenitor cells in a three-dimensional matrix comprising Wnt4 or Wnt5a protein for a time sufficient to generate a multicellular islet-like organoid comprising two or more cell types selected from beta (b) cells, alpha (a) cells, delta (d) cells, epsilon (e) cells and duct-like cells; wherein the islet-like organoid secretes insulin in response to glucose; and subjecting the islet-like organoid to multiple intermittent exposure to interferon gamma (IFNy) over a given time period, e.g., a time period of at least 24 hours; thereby inducing sustained expression of an immune checkpoint protein by the islet-like organoid and allowing the islet-like organoid to evade immune detection or autoimmunity. In an embodiment of the method, the islet-like organoid is exposed to IFNy at least two times over at least a two-day time period. In another embodiment of the method, the islet-like organoid is exposed to IFNy at least three times over a three-day time period. In another embodiment of the method, the islet-like organoid is exposed to IFNy for greater than one hour at least two times over a two-day time period. In another embodiment of the method, the islet-like organoid is exposed to IFNy for greater than one hour at least three times over a three-day time period. In another embodiment of the method, the islet-like organoid is exposed to IFNy for two hours at least two times over a two-day time period. In another embodiment of the method, the islet-like organoid is exposed to IFNy for two hours at least three times over a three-day time period. In embodiments of the method, the the islet-like organoid is intermittently exposed to IFNy over a time period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer. In another aspect, a method of generating an islet-like organoid that evades immune detection or autoimmunity is provided, in which the method comprises culturing endocrine progenitor cells which recombinantly express an immune checkpoint protein in a three- dimensional matrix comprising Wnt4 or Wnt5a protein for a time sufficient to generate a multicellular islet-like organoid comprising two or more cell types selected from beta (b) cells, alpha (a) cells, delta (d) cells, epsilon (e) cells and duct-like cells; wherein the islet-like organoid secretes insulin in response to glucose and wherein the islet-like organoid evades immune detection and autoimmunity. In an embodiment, recombinant expression of the immune checkpoint protein results from transduction of islet-like organoid cells with a vector containing a polynucleotide encoding the immune checkpoint protein.

In an embodiment of the methods of the foregoing aspects, the three-dimensional matrix comprises a human Wnt4 protein, a recombinant human Wnt4 protein, a human Wnt5 protein, or a recombinant human Wnt5a protein. In a particular embodiment, the three- dimensional matrix comprises a recombinant human Wnt4 protein.

In an embodiment of the foregoing methods of generating an islet-like organoid that evades immune detection or autoimmunity, the three-dimensional matrix comprises gellan gum. In an embodiment, the three-dimensional matrix comprises recombinant human Wnt4 protein. In embodiments of the foregoing methods, the immune checkpoint protein binds to an immune cell-expressed cognate ligand selected from programmed cell-death protein 1 (PD-l); cytotoxic T-lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG-3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3-dioxygenase 1 (IDOl); tumor necrosis factor receptor superfamily member 9 (4-1BB); glucocorticoid- induced TNFR family related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4, (0X40); adenosine A2A receptor (A2AR); B7-H3; B7-H4; B7-1/B7-2; BTLA; V-domain Ig suppressor of T cell activation (VISTA); or a combination of any of the foregoing. In a particular embodiment, the immune checkpoint protein is programmed death ligand- 1 (PD-L1).

In an embodiment of the methods of the foregoing aspects, the endocrine progenitor cells are selected from induced pluripotent stem cells (iPSCs), embryonic pluripotent stem cells (ePSCs), and/or pancreatic progenitor cells.

In an embodiment of the methods of the foregoing aspects, the the endocrine progenitor cells express at least one of neurogenin 3, neurodl, Nkx2.2 and Pax4 biomarkers. In an embodiment of the methods of the foregoing aspects, the islet-like organoid is a human islet-like organoid (HILO). In a particular embodiment, the islet-like organoid is vascularized. In a particular embodiment, the islet-like organoid further comprises an adipose-derived stem cell and/or an endothelial cell. In an embodiment, the adipose-derived stem cell is a human adipose-derived stem cell (hADSC) and/or the endothelial cell is a human umbilical vein endothelial cell (HUVEC).

In an embodiment of the methods of the foregoing aspects, the islet-like organoid further exhibits at least one of KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, glucagon secretion.

In an embodiment of the methods of the foregoing aspects, the islet-like organoid expresses a beta cell lineage marker selected from the group consisting of NKX2-2 ,

NEUROD1, RFX6 , GCK, INS , NKX6-1, UCN3 , MAFB and SYT4 and an ARX alpha cell lineage marker.

In an embodiment of the methods of the foregoing aspects, the islet-like organoid exhibits increased expression of Estrogen Related Receptor gamma (ERRy).

In another embodiment of the methods of the foregoing aspects, the islet-like organoid exhibits increased oxidative metabolism characterized by increased oxygen consumption rate (OCR) and decreased cellular acidification rate (ECAR).

In an embodiment of the methods of the foregoing aspects, the islet-like organoid is a pancreatic islet organoid, a pancreatic organoid, a liver organoid, a heart organoid, or intestinal organoid. In a particular embodiment of the methods, the islet-like organoid is a human pancreatic islet organoid.

In another aspect, a method of generating a human islet like organoid (HILO) that evades immune detection or autoimmunity is provided, in which the method comprises (a) culturing endocrine progenitor cells in culture medium or a three-dimensional matrix comprising Wnt4 or Wnt5a protein for a time sufficient to generate a multicellular human islet-like organoid comprising two or more cell types selected from beta (b) cells, alpha (a) cells, delta (d) cells, epsilon (e) cells and duct-like cells; wherein the human islet-like organoid secretes insulin in response to glucose; (b) contacting the HILO of step (a) with interferon gamma (IFNy) two or three times for greater than one hour each time over a total time period of at least 48-72 hours; wherein the human islets or HILOs are maintained in the absence of IFNy between times of contact with IFNy; and wherein steps (a) and (b) induce sustained expression of immune checkpoint protein programmed death ligand-l (PD-L1) in the HILO. In an embodiment of the method, the HILO is contacted with IFNy for 2 hours in step (b). In another embodiment of the method, the HILO is contacted with IFNy two times for two hours each time, over at least 48 hours. In another embodiment of the method, the HILO is contacted with IFNy three times for two hours each time, over at least 72 hours. In another embodiment of the method, the endocrine progenitor cells are selected from induced pluripotent stem cells (iPSCs), embryonic pluripotent stem cells (ePSCs), and/or pancreatic progenitor cells. In another embodiment of the method, the endocrine progenitor cells express at least one of neurogenin 3, neurodl, Nkx2.2 and Pax4 biomarkers. In another embodiment of the method, the HILO is vascularized and exhibits increased oxidative metabolism characterized by increased oxygen consumption rate (OCR) and decreased cellular acidification rate (ECAR).

In an embodiment of the methods of the foregoing aspects, IFNy is used in an amount of 1-25 ng/ml. In an embodiment of the methods of the foregoing aspects, IFNy is used in an amount of 10 ng/ml. In an embodiment of the methods of the foregoing aspects, PD-L1 expression in the islet-like organoid or HILO is maintained for greater than 7 days.

In an aspect, a human islet-like organoid or pancreatic islet organoid having sustained expression of an immune checkpoint protein is produced by the method as described in the above-delineated aspects. In an embodiment, the human islet-like organoid or pancreatic islet organoid exhibits sustained expression of the immune checkpoint protein PD-L1.

In another aspect is provided a human islet-like organoid (HILO) derived from endocrine progenitor cells cultured in culture medium or a three-dimensional matrix comprising Wnt4 or Wnt5 protein and comprising multi-lineage cells comprising at least two of beta (b) cells, alpha (a) cells, delta (d) cells, epsilon (e) cells and duct-like cells, wherein the HILO is vascularized, exhibits glucose-stimulated insulin secretion (GSIS) and exhibits sustained expression of an immune checkpoint protein. In an embodiment, the human islet- like organoid (HILO) is a pancreatic islet-like organoid or a pancreatic organoid. In an embodiment, the human islet-like organoid (HILO) further exhibits KCl-stimulated insulin secretion or glucose stimulated insulin secretion. In another embodiment, the three- dimensional matrix for culturing the human islet-like organoid (HILO) comprises gellan gum. In another embodiment, the three-dimensional matrix for culturing the human islet-like organoid (HILO) comprises recombinant human Wnt4 protein. In an embodiment, the human islet-like organoid (HILO) is derived from endocrine progenitor cells which are selected from induced pluripotent stem cells (iPSCs), embryonic pluripotent stem cells (ePSCs), and/or pancreatic progenitor cells. In an embodiment, the endocrine progenitor cells express at least one of neurogenin 3, neurodl, Nkx2.2 and Pax4 biomarkers. In an embodiment, the human islet-like organoid (HILO) expresses FLTP and ESRR gamma genes. In an embodiment, the human islet-like organoid (HILO) further comprises an adipose- derived stem cell and/or an endothelial cell. In a particular embodiment, the adipose-derived stem cell is a human adipose-derived stem cell (hADSC) and/or the endothelial cell is a human umbilical vein endothelial cell (HUVEC). In another embodiment, the human islet- like organoid (HILO) further exhibits KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, or glucagon secretion. In another embodiment, the human islet-like organoid (HILO) expresses a beta cell lineage marker selected from the group consisting OΪNKC2-2, NEUROD1, RFX6 , GCK, INS, NKX6-1 , UCN3, MAFB and SYT4 and an ARX alpha cell lineage marker. In another embodiment, the human islet-like organoid (HILO) is a pancreatic HILO that expresses a beta cell transcription factor selected from the group consisting of Pdxl, MafA, Pax4, Pax6, NeuroDl, Nkx6-l, Gata6, and Foxa2. In embodiments, the human islet-like organoid (HILO) exhibit sustained expression of an immune checkpoint protein which binds to an immune cell-expressed cognate ligand selected from programmed cell-death protein 1 (PD-l); cytotoxic T-lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG-3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3 -dioxygenase 1 (IDOl); tumor necrosis factor receptor superfamily member 9 (4-1BB); glucocorticoid-induced TNFR family related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4, (0X40); adenosine A2A receptor (A2AR); B7-H3; B7-H4; B7-1/B7- 2; BTLA; V-domain Ig suppressor of T cell activation (VISTA); or a combination of any of the foregoing. In an embodiment, the human islet-like organoid (HILO) of any one of claims 40-54, wherein the immune checkpoint protein is programmed death ligand-l (PD-L1).

In another aspect is provided a non-human organism transplanted or implanted with the human islet-like organoid, pancreatic islet organoid, or HILO as described in the foregoing aspects delineated above. In an embodiment, the non-human organism is a mammal. In an embodiment, the non-human organism is a mouse. In another aspect, a method of treating a pancreatic disease in a subject is provided, in which the method comprises transplanting or implanting an islet-like organoid or a pancreatic islet organoid into the subject, wherein the islet-like organoid or a pancreatic islet organoid comprises endocrine progenitor cell-derived, multi-lineage cells including beta, alpha, delta, epsilon cells, duct-like cells, or a combination thereof, is vascularized, exhibits glucose- stimulated insulin secretion (GSIS) and exhibits sustained expression of an immune checkpoint protein to evade immune detection or autoimmunity.

In another aspect, a method of treating type 1 diabetes in a subject is provided, in which the method comprises transplanting or implanting an islet-like organoid or a pancreatic islet organoid into the subject, wherein the islet-like organoid or a pancreatic islet organoid comprises endocrine progenitor cell-derived multi-lineage cells including beta, alpha, delta, epsilon cells, duct-like cells, or a combination thereof, is vascularized, exhibits glucose- stimulated insulin secretion (GSIS) and exhibits sustained expression of an immune checkpoint protein to evade immune detection or autoimmunity.

In an embodiment of the methods delineated in the above-described aspects, the islet- like organoid or pancreatic islet organoid further exhibits KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, or glucagon secretion. In an embodiment of the methods delineated in the above-described aspects, the islet-like organoid or pancreatic islet organoid expresses a beta cell lineage marker selected from the group consisting of NKX2-2, NEUROD1 , RFX6 , GCK, INS , NKX6-1, UCN3 , MAFB and SYT4 and an ARX alpha cell lineage marker. In an embodiment of the methods delineated in the above- described aspects, the endocrine progenitor cells are selected from induced pluripotent stem cells (iPSCs), embryonic pluripotent stem cells (ePSCs), and/or pancreatic progenitor cells.

In an embodiment, the endocrine progenitor cells express at least one of neurogenin 3, neurodl, Nkx2.2 and Pax4 biomarkers. In an embodiment of the methods delineated in the above-described aspects, the islet-like organoid or pancreatic islet organoid expresses a beta cell transcription factor selected from the group consisting of Pdxl, MafA, Pax4, Pax6, NeuroDl, Nkx6-l, Gata6, and Foxa2. In an embodiment of the treatment methods as described in the above-delineated aspects, the immune checkpoint protein binds to an immune cell-expressed cognate ligand selected from programmed cell-death protein 1 (PD- 1); cytotoxic T-lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG- 3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3 -di oxygenase 1 (IDOl); tumor necrosis factor receptor superfamily member 9 (4-1BB); glucocorticoid-induced TNFR family related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4, (0X40); adenosine A2A receptor (A2AR); B7-H3; B7-H4; B7-1/B7-2; BTLA; V-domain Ig suppressor of T cell activation (VISTA); or a combination of any of the foregoing. In a particular embodiment, the immune checkpoint protein is programmed death ligand-l (PD-L1). In an embodiment of the treatment methods as described in the above-delineated aspects, the islet-like organoid or pancreatic islet organoid is produced by a method described in the aspects hereinabove. In an embodiment of the treatment methods as described in the above-delineated aspects, the islet- like organoid or pancreatic islet organoid is the organoid as described in the above-delineated aspects. In an embodiment of the treatment methods as described in the above-delineated aspects, an immunosuppressive agent is administered to the subject. In an embodiment of the treatment methods as described in the above-delineated aspects, the subject is human. In an embodiment of the treatment methods as described in the above-delineated aspects, the pancreatic disease is type 1 diabetes or type 2 diabetes.

In another aspect, a method of cell transplantation is provided, in which the method comprises administering to a subject in need thereof an immunoprotected cell, human islet- like organoid or pancreatic islet organoid as described in the above-delineated aspects. In an embodiment, the immunoprotected cell, human islet-like organoid or pancreatic islet organoid is syngeneic, autologous, allogeneic or xenogeneic to the subject receiving the transplant.

In another aspect, a kit containing an immunoprotected cell, human islet-like organoid or pancreatic islet organoid as described in the above-delineated aspects, or a

pharmaceutically acceptable composition comprising the immunoprotected cell, human islet- like organoid or pancreatic islet organoid is provided. In an embodiment, the kit contains an immunoprotected cell, human islet-like organoid or pancreatic islet organoid that is syngeneic, autologous, allogeneic, or xenogeneic.

Other features and advantages will be apparent from the detailed description of the embodiments and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention pertains. The following references provide one of skill in the pertinent art with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By“AFP polypeptide” or“alpha-fetoprotein” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI

Accession No. NP_00l 125.1 and having a biological activity of an AFP polypeptide.

Exemplary biological activities of an AFP polypeptide include binding to copper, nickel, fatty acids, and bilirubin. The amino acid sequence provided at NCBI Accession No.

NP 001125.1 is shown below:

1 MKWVESIFLI FLLNFTESRT LHRNEYGIAS ILDSYQCTAE ISLADLATIF FAQFVQEATY

61 KEVSKMVKDA LTAIEKPTGD EQSSGCLENQ LPAFLEELCH EKEILEKYGH SDCCSQSEEG

121 RHNCFLAHKK PTPASIPLFQ VPEPVTSCEA YEEDRETFMN KFIYEIARRH PFLYAPTILL

181 WAARYDKIIP SCCKAENAVE CFQTKAATVT KELRESSLLN QHACAVMKNF GTRTFQAITV

241 TKLSQKFTKV NFTEIQKLVL DVAHVHEHCC RGDVLDCLQD GEKIMSYICS QQDTLSNKIT

301 ECCKLTTLER GQCIIHAEND EKPEGLSPNL NRFLGDRDFN QFSSGEKNIF LASFVHEYSR

361 RHPQLAVSVI LRVAKGYQEL LEKCFQTENP LECQDKGEEE LQKYIQESQA LAKRSCGLFQ

421 KLGEYYLQNA FLVAYTKKAP QLTSSELMAI TRKMAATAAT CCQLSEDKLL ACGEGAADII

481 IGHLCIRHEM TPWPGVGQC CTSSYANRRP CFSSLWDET YVPPAFSDDK FIFHKDLCQA

541 QGVALQTMKQ EFLINLVKQK PQITEEQLEA VIADFSGLLE KCCQGQEQEV CFAEEGQKLI

601 SKTRAALGV

By“AFP polynucleotide” is meant a polynucleotide encoding a AFP polypeptide or fragment thereof. An exemplary AFP polynucleotide sequence is provided at NCBI Ref: NM_00l 134.2. The sequence provided at NCBI Ref: NM_00l 134.2 is reproduced below:

1 atattgtgct tccaccactg ccaataacaa aataactagc aaccatgaag tgggtggaat

61 caattttttt aattttccta ctaaatttta ctgaatccag aacactgcat agaaatgaat

121 atggaatagc ttccatattg gattcttacc aatgtactgc agagataagt ttagctgacc

181 tggctaccat attttttgcc cagtttgttc aagaagccac ttacaaggaa gtaagcaaaa

241 tggtgaaaga tgcattgact gcaattgaga aacccactgg agatgaacag tcttcagggt

301 gtttagaaaa ccagctacct gcctttctgg aagaactttg ccatgagaaa gaaattttgg

361 agaagtacgg acattcagac tgctgcagcc aaagtgaaga gggaagacat aactgttttc

421 ttgcacacaa aaagcccact ccagcatcga tcccactttt ccaagttcca gaacctgtca

481 caagctgtga agcatatgaa gaagacaggg agacattcat gaacaaattc atttatgaga

541 tagcaagaag gcatcccttc ctgtatgcac ctacaattct tctttgggct gctcgctatg

601 acaaaataat tccatcttgc tgcaaagctg aaaatgcagt tgaatgcttc caaacaaagg

661 cagcaacagt tacaaaagaa ttaagagaaa gcagcttgtt aaatcaacat gcatgtgcag 721 taatgaaaaa ttttgggacc cgaactttcc aagccataac tgttactaaa ctgagtcaga

781 agtttaccaa agttaatttt actgaaatcc agaaactagt cctggatgtg gcccatgtac

841 atgagcactg ttgcagagga gatgtgctgg attgtctgca ggatggggaa aaaatcatgt

901 cctacatatg ttctcaacaa gacactctgt caaacaaaat aacagaatgc tgcaaactga

961 ccacgctgga acgtggtcaa tgtataattc atgcagaaaa tgatgaaaaa cctgaaggtc

1021 tatctccaaa tctaaacagg tttttaggag atagagattt taaccaattt tcttcagggg

1081 aaaaaaatat cttcttggca agttttgttc atgaatattc aagaagacat cctcagcttg

1141 ctgtctcagt aattctaaga gttgctaaag gataccagga gttattggag aagtgtttcc

1201 agactgaaaa ccctcttgaa tgccaagata aaggagaaga agaattacag aaatacatcc

1261 aggagagcca agcattggca aagcgaagct gcggcctctt ccagaaacta ggagaatatt

1321 acttacaaaa tgcgtttctc gttgcttaca caaagaaagc cccccagctg acctcgtcgg

1381 agctgatggc catcaccaga aaaatggcag ccacagcagc cacttgttgc caactcagtg

1441 aggacaaact attggcctgt ggcgagggag cggctgacat tattatcgga cacttatgta

1501 tcagacatga aatgactcca gtaaaccctg gtgttggcca gtgctgcact tcttcatatg

1561 ccaacaggag gccatgcttc agcagcttgg tggtggatga aacatatgtc cctcctgcat

1621 tctctgatga caagttcatt ttccataagg atctgtgcca agctcagggt gtagcgctgc

1681 aaacgatgaa gcaagagttt ctcattaacc ttgtgaagca aaagccacaa ataacagagg

1741 aacaacttga ggctgtcatt gcagatttct caggcctgtt ggagaaatgc tgccaaggcc

1801 aggaacagga agtctgcttt gctgaagagg gacaaaaact gatttcaaaa actcgtgctg

1861 ctttgggagt ttaaattact tcaggggaag agaagacaaa acgagtcttt cattcggtgt

1921 gaacttttct ctttaatttt aactgattta acactttttg tgaattaatg aaatgataaa

1981 gacttttatg tgagatttcc ttatcacaga aataaaatat ctccaaatgt ttccttttca

2041 aaaaaaaaaa aaaaaaa

By“ALB polypeptide” or“albumin” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No.

NP 000468.1 and having a biological activity of ALB polypeptide. Exemplary biological activities of ALB polypeptide include binding to fatty acids, calcium ions, sodium ions, potassium ions, hormones, and bilirubin; stabilization of extracellular fluid volume; and, transport of plasma zinc. The amino acid sequence provided at NCBI Accession No.

NP 000468.1 is shown below:

1 MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF

61 EDHVKLWEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP

121 ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF

181 FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV

241 ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK

301 ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR

361 RHPDYSWLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE

421 QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSW

481 LNQLCVLHEK TPVSDRVTKC CTESLWRRP CFSALEVDET YVPKEFNAET FTFHADICTL

541 SEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV

601 AASQAALGL By“ALB polynucleotide” is meant a polynucleotide encoding a ALB polypeptide or fragment thereof. An exemplary AFP polynucleotide sequence is provided at NCBI Ref: NM_000477.5. The sequence provided at NCBI Ref: NM_000477.5 is reproduced below:

1 agtatattag tgctaatttc cctccgtttg tcctagcttt tctcttctgt caaccccaca

61 cgcctttggc acaatgaagt gggtaacctt tatttccctt ctttttctct ttagctcggc

121 ttattccagg ggtgtgtttc gtcgagatgc acacaagagt gaggttgctc atcggtttaa

181 agatttggga gaagaaaatt tcaaagcctt ggtgttgatt gcctttgctc agtatcttca

241 gcagtgtcca tttgaagatc atgtaaaatt agtgaatgaa gtaactgaat ttgcaaaaac

301 atgtgttgct gatgagtcag ctgaaaattg tgacaaatca cttcataccc tttttggaga

361 caaattatgc acagttgcaa ctcttcgtga aacctatggt gaaatggctg actgctgtgc

421 aaaacaagaa cctgagagaa atgaatgctt cttgcaacac aaagatgaca acccaaacct

481 cccccgattg gtgagaccag aggttgatgt gatgtgcact gcttttcatg acaatgaaga

541 gacatttttg aaaaaatact tatatgaaat tgccagaaga catccttact tttatgcccc

601 ggaactcctt ttctttgcta aaaggtataa agctgctttt acagaatgtt gccaagctgc

661 tgataaagct gcctgcctgt tgccaaagct cgatgaactt cgggatgaag ggaaggcttc

721 gtctgccaaa cagagactca agtgtgccag tctccaaaaa tttggagaaa gagctttcaa

781 agcatgggca gtagctcgcc tgagccagag atttcccaaa gctgagtttg cagaagtttc

841 caagttagtg acagatctta ccaaagtcca cacggaatgc tgccatggag atctgcttga

901 atgtgctgat gacagggcgg accttgccaa gtatatctgt gaaaatcaag attcgatctc

961 cagtaaactg aaggaatgct gtgaaaaacc tctgttggaa aaatcccact gcattgccga

1021 agtggaaaat gatgagatgc ctgctgactt gccttcatta gctgctgatt ttgttgaaag

1081 taaggatgtt tgcaaaaact atgctgaggc aaaggatgtc ttcctgggca tgtttttgta

1141 tgaatatgca agaaggcatc ctgattactc tgtcgtgctg ctgctgagac ttgccaagac

1201 atatgaaacc actctagaga agtgctgtgc cgctgcagat cctcatgaat gctatgccaa

1261 agtgttcgat gaatttaaac ctcttgtgga agagcctcag aatttaatca aacaaaattg

1321 tgagcttttt gagcagcttg gagagtacaa attccagaat gcgctattag ttcgttacac

1381 caagaaagta ccccaagtgt caactccaac tcttgtagag gtctcaagaa acctaggaaa

1441 agtgggcagc aaatgttgta aacatcctga agcaaaaaga atgccctgtg cagaagacta

1501 tctatccgtg gtcctgaacc agttatgtgt gttgcatgag aaaacgccag taagtgacag

1561 agtcaccaaa tgctgcacag aatccttggt gaacaggcga ccatgctttt cagctctgga

1621 agtcgatgaa acatacgttc ccaaagagtt taatgctgaa acattcacct tccatgcaga

1681 tatatgcaca ctttctgaga aggagagaca aatcaagaaa caaactgcac ttgttgagct

1741 cgtgaaacac aagcccaagg caacaaaaga gcaactgaaa gctgttatgg atgatttcgc

1801 agcttttgta gagaagtgct gcaaggctga cgataaggag acctgctttg ccgaggaggg

1861 taaaaaactt gttgctgcaa gtcaagctgc cttaggctta taacatcaca tttaaaagca

1921 tctcagccta ccatgagaat aagagaaaga aaatgaagat caaaagctta ttcatctgtt

1981 tttctttttc gttggtgtaa agccaacacc ctgtctaaaa aacataaatt tctttaatca

2041 ttttgcctct tttctctgtg cttcaattaa taaaaaatgg aaagaatcta atagagtggt

2101 acagcactgt tatttttcaa agatgtgttg ctatcctgaa aattctgtag gttctgtgga

2161 agttccagtg ttctctctta ttccacttcg gtagaggatt tctagtttct tgtgggctaa

2221 ttaaataaat cattaatact cttctaaaaa aaaaaaaaaa aaaa

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. By“altered” is meant an increase or decrease. An increase is any positive change, e.g., by at least about 5%, 10%, or 20%; by at least about 25%, 50%, 75%, or even by 100%, 200%, 300% or more. A decrease is a negative change, e.g., a decrease by at least about 5%, 10%, or 20%; by at least about 25%, 50%, 75%; or even an increase by 100%, 200%, 300% or more.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By“CDX2 polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_00l256.3 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_00l256.3 is shown below:

1 MYVSYLLDKD VSMYPSSVRH SGGLNLAPQN FVSPPQYPDY GGYHVAAAAA AAANLDSAQS 61 PGPSWPAAYG APLREDWNGY APGGAAAAAN AVAHGLNGGS PAAAMGYSSP ADYHPHHHPH 121 HHPHHPAAAP SCASGLLQTL NPGPPGPAAT AAAEQLSPGG QRRNLCEWMR KPAQQSLGSQ 181 VKTRTKDKYR WYTDHQRLE LEKEFHYSRY ITIRRKAELA ATLGLSERQV KIWFQNRRAK 241 ERKINKKKLQ QQQQQQPPQP PPPPPQPPQP QPGPLRSVPE PLSPVSSLQA SVSGSVPGVL 301 GPTGGVLNPT VTQ

By“CDX2 polynucleotide” is meant a polynucleotide encoding a CDX2 polypeptide or fragment thereof. An exemplary CDX2 polynucleotide sequence is provided at NCBI Ref: NM_00l265.4. The sequence provided at NCBI Ref: NM_00l265.4 is reproduced below:

1 ctccaaccat tggtgtctgt gtcattacta atagagtctt gtaaacactc gttaatcacg

61 gaaggccgcc ggcctggggc tccgcacgcc agcctgtggc gggtcttccc cgcctctgca

121 gcctagtggg aaggaggtgg gaggaaagaa ggaagaaagg gagggaggga ggaggcaggc

181 cagagggagg gaccgcctcg gaggcagaag agccgcgagg agccagcgga gcaccgcggg

241 ctggggcgca gccacccgcc gctcctcgag tcccctcgcc cctttccctt cgtgcccccc

301 ggcagcctcc agcgtcggtc cccaggcagc atggtgaggt ctgctcccgg accctcgcca

361 ccatgtacgt gagctacctc ctggacaagg acgtgagcat gtaccctagc tccgtgcgcc

421 actctggcgg cctcaacctg gcgccgcaga acttcgtcag ccccccgcag tacccggact

481 acggcggtta ccacgtggcg gccgcagctg cagcggcagc gaacttggac agcgcgcagt

541 ccccggggcc atcctggccg gcagcgtatg gcgccccact ccgggaggac tggaatggct

601 acgcgcccgg aggcgccgcg gccgccgcca acgccgtggc tcacggcctc aacggtggct 661 ccccggccgc agccatgggc tacagcagcc ccgcagacta ccatccgcac caccacccgc

721 atcaccaccc gcaccacccg gccgccgcgc cttcctgcgc ttctgggctg ctgcaaacgc

781 tcaaccccgg ccctcctggg cccgccgcca ccgctgccgc cgagcagctg tctcccggcg

841 gccagcggcg gaacctgtgc gagtggatgc ggaagccggc gcagcagtcc ctcggcagcc

901 aagtgaaaac caggacgaaa gacaaatatc gagtggtgta cacggaccac cagcggctgg

961 agctggagaa ggagtttcac tacagtcgct acatcaccat ccggaggaaa gccgagctag

1021 ccgccacgct ggggctctct gagaggcagg ttaaaatctg gtttcagaac cgcagagcaa

1081 aggagaggaa aatcaacaag aagaagttgc agcagcaaca gcagcagcag ccaccacagc

1141 cgcctccgcc gccaccacag cctccccagc ctcagccagg tcctctgaga agtgtcccag

1201 agcccttgag tccggtgtct tccctgcaag cctcagtgtc tggctctgtc cctggggttc

1261 tggggccaac tgggggggtg ctaaacccca ccgtcaccca gtgacccacc gggttctgca

1321 gcggcagagc aattccaggc tgagccatga ggagcgtgga ctctgctaga ctcctcagga

1381 gagacccctc ccctcccacc cacagccata gacctacaga cctggctctc agaggaaaaa

1441 tgggagccag gagtaagaca agtgggattt ggggcctcaa gaaatatact ctcccagatt

1501 tttacttttt cccatctggc tttttctgcc actgaggaga cagaaagcct ccgctgggct

1561 tcattccgga ctggcagaag cattgcctgg actgaccaca ccaaccaggc cttcatcctc

1621 ctccccagct cttctcttcc tagatctgca ggctgcacct ctggctagag ccgaggggag

1681 agagggactc aagggaaagg caagcttgag gccaagatgg ctgctgcctg ctcatggccc

1741 tcggaggtcc agctgggcct cctgcctccg ggcaggcaag gtttacactg cggaagccaa

1801 aggcagctaa gatagaaagc tggactgacc aaagactgca gaacccccag gtggcctgcg

1861 tcttttttct cttcccttcc cagaccagga aaggcttggc tggtgtatgc acagggtgtg

1921 gtatgagggg gtggttattg gactccaggc ctgaccaggg ggcccgaaca gggacttgtt

1981 tagagagcct gtcaccagag cttctctggg ctgaatgtat gtcagtgcta taaatgccag

2041 agccaacctg gacttcctgt cattttcaca atcttggggc tgatgaagaa gggggtgggg

2101 ggagtttgtg ttgttgttgc tgctgtttgg gttgttggtc tgtgtaacat ccaagccaga

2161 gtttttaaag ccttctggat ccatgggggg agaagtgata tggtgaaggg aagtggggag

2221 tatttgaaca cagttgaatt ttttctaaaa agaaaaagag ataaatgagc tttccagatt

2281 tcagattctg tatttatctt cagattttgt ctgcaactat tttttatttt ttaaagaaat

2341 gaaatatctt caaaaaaaaa aaaaaaaaaa

By“CYP3 A7 polypeptide” or“cytochrome P450” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_000756.3 and having monooxygenase activity. The amino acid sequence provided at NCBI Accession No. NP_000756.3 is shown below:

1 MDLIPNLAVE TWLLLAVSLI LLYLYGTRTH GLFKKLGIPG PTPLPFLGNA LSFRKGYWTF 61 DMECYKKYRK VWGIYDCQQP MLAITDPDMI KTVLVKECYS VFTNRRPFGP VGFMKNAISI 121 AEDEEWKRIR SLLSPTFTSG KLKEMVPIIA QYGDVLVRNL RREAETGKPV TLKHVFGAYS 181 MDVITSTSFG VSIDSLNNPQ DPFVENTKKL LRFNPLDPFV LSIKVFPFLT PILEALNITV 241 FPRKVISFLT KSVKQIKEGR LKETQKHRVD FLQLMIDSQN SKDSETHKAL SDLELMAQSI 301 IFIFAGYETT SSVLSFIIYE LATHPDVQQK VQKEIDTVLP NKAPPTYDTV LQLEYLDMW 361 NETLRLFPVA MRLERVCKKD VEINGMFIPK GVWMIPSYV LHHDPKYWTE PEKFLPERFS 421 KKNKDNIDPY IYTPFGSGPR NCIGMRFALV NMKLALVRVL QNFSFKPCKE TQIPLKLRFG 481 GLLLTEKPIV LKAESRDETV SGA

By“CYP3A7 polynucleotide” is meant a polynucleotide encoding a CYP3 A7 polypeptide or fragment thereof. An exemplary AFP polynucleotide sequence is provided at NCBI Ref: NM_000765.4. The sequence provided at NCBI Ref: NM_000765.4 is reproduced

1 aatcactgct gtgcagggca ggaaagctcc acacacacag cccagcaaac agcagcacgc

61 tgctgaaaaa aagactcaga ggagagagat aaggaaggaa agtagtgatg gatctcatcc

121 caaacttggc cgtggaaacc tggcttctcc tggctgtcag cctgatactc ctctatctat

181 atggaacccg tacacatgga ctttttaaga agcttggaat tccagggccc acacctctgc

241 cttttttggg aaatgctttg tccttccgta agggctattg gacgtttgac atggaatgtt

301 ataaaaagta tagaaaagtc tggggtattt atgactgtca acagcctatg ctggctatca

361 cagatcccga catgatcaaa acagtgctag tgaaagaatg ttattctgtc ttcacaaacc

421 ggaggccttt cgggccagtg ggatttatga aaaatgccat ctctatagct gaggatgaag

481 aatggaagag aatacgatca ttgctgtctc caacattcac cagcggaaaa ctcaaggaga

541 tggtccctat cattgcccag tatggagatg tgttggtgag aaatctgagg cgggaagcag

601 agacaggcaa gcctgtcacc ttgaaacacg tctttggggc ctacagcatg gatgtgatca

661 ctagcacatc atttggagtg agcatcgact ctctcaacaa tccacaagac ccctttgtgg

721 aaaacaccaa gaagctttta agatttaatc cattagatcc attcgttctc tcaataaaag

781 tctttccatt ccttacccca attcttgaag cattaaatat cactgtgttt ccaagaaaag

841 ttataagttt tctaacaaaa tctgtaaaac agataaaaga aggtcgcctc aaagagacac

901 aaaagcaccg agtggatttc cttcagctga tgattgactc tcagaattca aaagactctg

961 agacccacaa agctctgtct gatctggagc tcatggccca atcaattatc tttatttttg

1021 ctggctatga aaccacgagc agtgttctct ccttcattat atatgaactg gccactcacc

1081 ctgatgtcca gcagaaagtg cagaaggaaa ttgatacagt tttacccaat aaggcaccac

1141 ccacctatga tactgtgcta cagttggagt atcttgacat ggtggtgaat gaaacactca

1201 gattattccc agttgctatg agacttgaga gggtctgcaa aaaagatgtt gaaatcaatg

1261 ggatgtttat tcccaaaggg gtggtggtga tgattccaag ctatgttctt catcatgacc

1321 caaagtactg gacagagcct gagaagttcc tccctgaaag gttcagtaaa aagaacaagg

1381 acaacataga tccttacata tacacaccct ttggaagtgg acccagaaac tgcattggca

1441 tgaggtttgc tctcgtgaac atgaaacttg ctctagtcag agtccttcag aacttctcct

1501 tcaaaccttg taaagaaaca cagatccccc tgaaattacg ctttggagga cttcttctaa

1561 cagaaaaacc cattgttcta aaggctgagt caagggatga gaccgtaagt ggagcctgat

1621 ttccctaagg acttctggtt tgctctttaa gaaagctgtg ccccagaaca ccagagacct

1681 caaattactt tacaaataga accctgaaat gaagacgggc ttcatccaat gtgctgcata

1741 aataatcagg gattctgtac gtgcattgtg ctctctcatg gtctgtatag agtgttatac

1801 ttggtaatat agaggagatg accaaatcag tgctggggaa gtagatttgg cttctctgct

1861 tctcatagga ctatctccac cacccccagt tagcaccatt aactcctcct gagctctgat

1921 aacataatta acatttctca ataatttcaa ccacaatcat taataaaaat aggaattatt

1981 ttgatggctc taacagtgac atttatatca tgtgttatat ctgtagtatt ctatagtaag

2041 ctttatatta agcaaatcaa taaaaacctc tttacaaaag taaaaaaaaa aaaaaaaaa

“Autologous” refers to biological material, e.g., autologous cells, tissues, islets, organoids, or islet-like organoids, that are obtained or derived from the same individual, subject, or patient. By way of example, autologous transplants (e.g., donor cells, tissues, organs, islets, organoids, or islet-like organoids) involve one individual, subject, or patient as both donor and recipient. “Syngeneic” refers to cells, tissues, organs, islets, organoids, islet-like organoids, or organisms (or other biological material) that are genetically similar or identical, (and of the same species) and thus, are immunologically compatible.

Syngeneic donor biological material is typically so closely related that transplantation does not provoke an immune response in the recipient. ‘‘Allogeneic” refers to biological material, e.g., donor allogeneic cells, tissues, organs, islets, organoids, or islet-like

organoids, that is genetically dissimilar to the recipient. Allogeneic biological material is typically obtained or derived from individuals of the same species. In addition, allogeneic biological material may be from an unrelated donor or from a donor matched as to MHC or HLA histocompatibility antigen type(s) with that of the recipient. “Xenogeneic” refers to biological material (e.g., cells, tissues, organs, islets, organoids, or islet-like organoids) that are derived or obtained from individuals of a different species. By way of example, autologous, syngeneic, allogeneic, or xenogeneic cells, tissues, organs, islets, organoids, or islet-like organoids may be used for transplant or implant, particularly, those generated by the methods involving IFNy treatment (e.g., MPS IFNy treatment) as described herein to yield long-term, immune evasive, transplanted or implanted biological material. In an embodiment, such biological material is obtained or generated from a living donor

(individual, subject, or organism). In an embodiment, such biological material is obtained or generated from a nonliving donor, e.g., cadaveric human islets or donor-matched cadaveric human islets.

As used herein, the term "carrier" refers to a physiologically acceptable diluent, excipient, buffer, or vehicle with which a composition (e.g., a physiologically acceptable or pharmaceutical composition), e.g., comprising a cell, islet, islet-like organoid, or organoid, may be administered to a subject or in which it may be stored. Pharmaceutical and pharmaceutically acceptable carriers include sterile liquids, such as medium, saline, buffers, and the like. In embodiments, the physiologically acceptable carriers are used in

pharmaceutical compositions that are administered to or transplanted into a subject, including, but not limited to, a human subject or patient. In some embodiments, water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers (and pharmaceutical compositions) are known and used by practitioners in the art and are described in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.

Gennaro, Lippincott Williams & Wilkins, 2000, and later editions thereof.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

As used herein, the term "immune response" refers to a subject’s immune system response or reaction to one or more antigens, (e.g., an immunogenic protein or peptide), and/or the epitopes of the antigens, recognized by the immune system as foreign, allogeneic, or heterologous. Immune responses include both cell-mediated immune responses (i.e., responses mediated by effector T cells, such as antigen-specific or non-specific T-cells, such as CD8+ T-cells, Thl cells, Th2 cells, and Thl7 cells) as well as humoral immune responses (i.e., responses characterized by B-cell activation and the production of antigen-specific antibodies). The term "immune response" encompasses both the innate immune responses to an antigen or immunogen, as well as memory responses that are a result of acquired immunity and can involve either B cells or T cells, or both.

By“immune checkpoint protein” or“immune checkpoint molecule,” or simply, “checkpoint protein or molecule” is meant a protein or molecule that can either induce or hinder activation of T cells, or a particular process in a cellular or immune system pathway, e.g., to prevent errors or an abnormal or pathological activity or condition. In an immune response, the crucial interaction between antigen presenting cells (APCs) and T-cells is tightly regulated by a‘three signal model’: (1) display of a surface complex consisting of an antigen bound on a major histocompatibility complex (MHC) protein class I or II (MHC I or II) molecule to a T-cell receptor (TCR) on a T-cell (CD8+ or CD4+); (2) costimulation by immune checkpoint proteins and (3) cytokines. Immune checkpoint proteins comprise costimulatory and inhibitory proteins that can either induce or inhibit activation of T-cells. Naive T-cells that only receive signal 1 without costimulatory signal 2 become anergic or die through apoptosis. The engagement of costimulatory ligand/receptor pairs triggers an accumulation of receptors and protein complexes at the center of the immunological synapse, which then amplifies and enhances the duration of TCR signaling (Wulfing, C. and Davis, M.M., 1998, Science , 282:2266-2269). The cytokine environment, signal 3, then induces naive CD4+ T-cells to differentiate into various T-cell subsets, such as T helper (Th)l cells, Th2 cells, Thl7 cells and regulatory T-cells (Tregs), each of which produce and release a distinct set of cytokines upon activation. (Foks, A.C. and Kuiper, T, 2017, Br. ./. Pharmacol ., 174:3940-3955). The immune system provides a large variety of stimulatory and inhibitory immune checkpoint proteins (signal 2), and each pathway has its own unique effect on the fate of individual immune cells. Signaling through stimulatory immune checkpoint proteins can promote cell survival, cell cycle progression and differentiation to effector and memory cells, while inhibitory immune checkpoint protein signaling can terminate these processes directly or indirectly by the induction of Tregs. Costimulation can be provided in cis, i.e., both signals 1 and 2 are provided by the same APC, or in trans, i.e., signal 2 is provided by a different or‘bystander’ APC than signal 1 (Roska, A.K. and Lipsky, P.E., 1985, J. Immunol., 135:2953-2961; Liu, Y. and Janeway, C.A., Jr., 1992, Proc. Natl. Acad. Sci. USA, 89:3845- 3849; Ding, L. and Shevach, E.M., 1994, Eur. J. Immunol., 24:859-866).

Checkpoint proteins are regulators of the immune system and frequently are bound by or interact with ligands (cognate ligands), which may cause a given effect, e.g., cell stimulation, anergy, or apoptosis. In an embodiment, the immune checkpoint protein is one which binds a cognate ligand (e.g., a receptor ligand) on an immune cell surface, e.g., a T cell surface receptor. In a specific embodiment, the immune checkpoint protein is PD-L1 or a binding portion thereof, where the cognate ligand of PD-L1 is PD-l expressed on the surface of T cells. In an embodiment, the checkpoint protein is the extracellular domain of the checkpoint protein.

The term“cognate ligand” refers to the specific binding partner, binding member, or ligand with which an immune checkpoint protein specifically interacts or with which it specifically binds. For example, a specific ligand to which a receptor protein binds or with which it interacts is a“cognate ligand” for that receptor protein. Similarly, the receptor protein is a cognate ligand for a specific ligand molecule or protein.

By“constitutive expression” is meant expression of a gene that is transcribed continually compared to a facultative gene which is only transcribed as needed. Genes that are constitutive] y expressed are transcribed in an ongoing manner, with control limited to that which is directly associated with the metabolic state of a cell, tissue, or organism. The level of expression of a constitutively expressed gene may be modified, e.g., via post- transcriptional or post-translational modification. In an embodiment, the gene is PD-L1 that encodes the PD-Ll polypeptide.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected. By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

“Differentiation” refers to the developmental process of lineage commitment.

Differentiation can be assayed by measuring an increase in one or more cell specific markers relative to their expression in a corresponding undifferentiated control cell. A“lineage” refers to a pathway of cellular development, in which precursor or“progenitor” cells undergo progressive physiological changes to become a specified cell type having a characteristic function. In some embodiments, the cell type is a beta cell. In some embodiments, the cell type is an alpha cell, delta cell, or duct cell. In some other embodiments, the cell type is a hepatocyte. In still other embodiments, the cell type is a cardiomyocyte. In some embodiments, the cell type is an intestinal cell. Differentiation occurs in stages, whereby cells gradually become more specified until they reach full maturity, which is also referred to as“terminal differentiation.” A“terminally differentiated cell” is a cell that has committed to a specific lineage, and has reached the end stage of differentiation (i.e., a cell that has fully matured). In some embodiments, an induced pluripotent stem cell (iPSC) is differentiated into a beta-like cell, an alpha-like cell, a delta-like cell, or a duct-like cell. In some other embodiments, an induced pluripotent stem cell (iPSC) is differentiated into a hepatocyte, cardiomyocyte, or intestinal cell.

A“de-differentiated cell” is a cell in which the process of differentiation has been, at least to some degree, reversed. De-differentiation can be assayed, for example, by identifying a reduction in the expression of one or more cell specific markers relative to their expression in a corresponding control cell. Alternatively, de-differentiation can be assayed by measuring an increase in one or more markers typically expressed in an embryonic stem cell, a pluripotent or multi-potent cell type, or expressed at an earlier stage of development.

In some embodiments, the de-differentiated cell is an induced pluripotent stem cell (iPSC).

In certain embodiments, the de-differentiated cell is a human induced pluripotent stem cell (iPSC). By“disease” is meant any condition or disorder that adversely affects, damages or interferes with the normal function of a cell, tissue, or organ, or a part of the body, such as autoimmunity or autoimmune disease. Examples of diseases include type 1 diabetes, type 2 diabetes, and pancreatic cancer. An autoimmune disease is one in which the body produces immune cells (e.g., effector T cells or NK cells) and/or antibodies produced by B cells that immunologically react against (attack) its own tissues or organs (or tissue or organ transplants or implants), leading to the deterioration, and, in some cases, to the destruction of the tissue or organ (or tissue or organ transplant or implant).

By "effective amount" is meant the amount of a therapeutic agent or organoid required to ameliorate the symptoms of a disease in a subject relative to an untreated subject. The effective amount of a therapeutic used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. In some embodiments, the therapeutic organoid is a pancreatic islet organoid. In some other embodiments, an effective amount of a pancreatic islet organoid is administered to a subject having type 1 or type 2 diabetes.

By“ESRRG polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No.

NP 001230448.1 and having nuclear hormone receptor activity. The amino acid sequence provided at NCBI Accession No. NP_001230448.1 is shown below:

1 MSNKDRHIDS SCSSFIKTEP SSPASLTDSV NHHSPGGSSD ASGSYSSTMN GHQNGLDSPP

61 LYPSAPILGG SGPVRKLYDD CSSTIVEDPQ TKCEYMLNSM PKRLCLVCGD IASGYHYGVA

121 SCEACKAFFK RTIQGNIEYS CPATNECEIT KRRRKSCQAC RFMKCLKVGM LKEGVRLDRV

181 RGGRQKYKRR IDAENSPYLN PQLVQPAKKP YNKIVSHLLV AEPEKIYAMP DPTVPDSDIK

241 ALTTLCDLAD RELWIIGWA KHIPGFSTLS LADQMSLLQS AWMEILILGV VYRSLSFEDE

301 LVYADDYIMD EDQSKLAGLL DLNNAILQLV KKYKSMKLEK EEFVTLKAIA LANSDSMHIE

361 DVEAVQKLQD VLHEALQDYE AGQHMEDPRR AGKMLMTLPL LRQTSTKAVQ HFYNIKLEGK

421 VPMHKLFLEM LEAKV

By“ESRRG polynucleotide” is meant a polynucleotide encoding a ESRRG polypeptide or fragment thereof. An exemplary ESRRG polynucleotide sequence is provided at NCBI Ref: NM 001243519.1. The sequence provided at NCBI Ref: NM_001243519.1 is reproduced below:

1 aagctccaat cggggcttta agtccttgat taggagagtg tgagagcttt ggtcccaact

61 ggctgtgcct ataggcttgt cactaggaga acatttgtgt taattgcact gtgctctgtc 121 aaggaaactt tgatttatag ctggggtgca caaataatgg ttgccggtcg cacatggatt

181 cggtagaact ttgccttcct gaatcttttt ccctgcacta cgaggaagag tagacttgaa

241 tgagacctgc ctcatcagtc atgggatcat agtgtcacag atggaaaagc aactatcagc

301 tgaattgtac tgaactacac acttggctaa ttcatcttat tgctctacac atctaaagga

361 aggctcattc tgttcttgga gtctagacag catcaggagt tgggctcagt gaacaaaact

421 ttaatgtcta gagcatttat gagggtttta atgattggaa aatctatcct gagaatgtgg

481 tcaccatatg tgacagcctt gctttctatc ttgtcttcag tttctggggc ttctctgcag

541 aatgtcaaac aaagatcgac acattgattc cagctgttcg tccttcatca agacggaacc

601 ttccagccca gcctccctga cggacagcgt caaccaccac agccctggtg gctcttcaga

661 cgccagtggg agctacagtt caaccatgaa tggccatcag aacggacttg actcgccacc

721 tctctaccct tctgctccta tcctgggagg tagtgggcct gtcaggaaac tgtatgatga

781 ctgctccagc accattgttg aagatcccca gaccaagtgt gaatacatgc tcaactcgat

841 gcccaagaga ctgtgtttag tgtgtggtga catcgcttct gggtaccact atggggtagc

901 atcatgtgaa gcctgcaagg cattcttcaa gaggacaatt caaggcaata tagaatacag

961 ctgccctgcc acgaatgaat gtgaaatcac aaagcgcaga cgtaaatcct gccaggcttg

1021 ccgcttcatg aagtgtttaa aagtgggcat gctgaaagaa ggggtgcgtc ttgacagagt

1081 acgtggaggt cggcagaagt acaagcgcag gatagatgcg gagaacagcc catacctgaa

1141 ccctcagctg gttcagccag ccaaaaagcc atataacaag attgtctcac atttgttggt

1201 ggctgaaccg gagaagatct atgccatgcc tgaccctact gtccccgaca gtgacatcaa

1261 agccctcact acactgtgtg acttggccga ccgagagttg gtggttatca ttggatgggc

1321 gaagcatatt ccaggcttct ccacgctgtc cctggcggac cagatgagcc ttctgcagag

1381 tgcttggatg gaaattttga tccttggtgt cgtataccgg tctctttcgt ttgaggatga

1441 acttgtctat gcagacgatt atataatgga cgaagaccag tccaaattag caggccttct

1501 tgatctaaat aatgctatcc tgcagctggt aaagaaatac aagagcatga agctggaaaa

1561 agaagaattt gtcaccctca aagctatagc tcttgctaat tcagactcca tgcacataga

1621 agatgttgaa gccgttcaga agcttcagga tgtcttacat gaagcgctgc aggattatga

1681 agctggccag cacatggaag accctcgtcg agctggcaag atgctgatga cactgccact

1741 cctgaggcag acctctacca aggccgtgca gcatttctac aacatcaaac tagaaggcaa

1801 agtcccaatg cacaaacttt ttttggaaat gttggaggcc aaggtctgac taaaagctcc

1861 ctgggccttc ccatccttca tgttgaaaaa gggaaaataa acccaagagt gatgtcgaag

1921 aaacttagag tttagttaac aacatcaaaa atcaacagac tgcactgata atttagcagc

1981 aagactatga agcagctttc agattcctcc ataggttcct gatgagtttc tttctacttt

2041 ctccatcatc ttctttcctc tttcttccca catttctctt tctctttatt ttttctcctt

2101 ttcttctttc acctccctta tttctttgct tctttcattc ctagttccca ttctccttta

2161 ttttcttccc gtctgcctgc cttctttctt ttctttacct actctcattc ctctcttttc

2221 tcatccttcc ccttttttct aaatttgaaa tagctttagt ttaaaaaaaa atcctccctt

2281 ccccctttcc tttccctttc tttccttttt ccctttcctt ttccctttcc tttcctttcc

2341 tcttgacctt ctttccatct ttctttttct tccttctgct gctgaacttt taaaagaggt

2401 ctctaactga agagagatgg aagccagccc tgccaaagga tggagatcca taatatggat

2461 gccagtgaac ttattgtgaa ccatactgtc cccaatgact aaggaatcaa agagagagaa

2521 ccaacgttcc taaaagtaca gtgcaacata tacaaattga ctgagtgcag tattagattt

2581 catgggagca gcctctaatt agacaactta agcaacgttg catcggctgc ttcttatcat

2641 tgcttttcca tctagatcag ttacagccat ttgattcctt aattgttttt tcaagtcttc

2701 caggtatttg ttagtttagc tactatgtaa ctttttcagg gaatagttta agctttattc

2761 attcatgcaa tactaaagag aaataagaat actgcaattt tgtgctggct ttgaacaatt

2821 acgaacaata atgaaggaca aatgaatcct gaaggaagat ttttaaaaat gttttgtttc

2881 ttcttacaaa tggagatttt tttgtaccag ctttaccact tttcagccat ttattaatat

2941 gggaatttaa cttactcaag caatagttga agggaaggtg catattatca cggatgcaat

3001 ttatgttgtg tgccagtctg gtcccaaaca tcaatttctt aacatgagct ccagtttacc

3061 taaatgttca ctgacacaaa ggatgagatt acacctacag tgactctgag tagtcacata

3121 tataagcact gcacatgaga tatagatccg tagaattgtc aggagtgcac ctctctactt 3181 gggaggtaca attgccatat gatttctagc tgccatggtg gttaggaatg tgatactgcc

3241 tgtttgcaaa gtcacagacc ttgcctcaga aggagctgtg agccagtatt catttaagag

3301 gcaataaggc aaatgccaga attaaaaaaa aaaatcatca aagacagaaa atgcctgacc

3361 aaattctaaa acctaatcca tataagttta ttcatttagg aatgttcgtt taaattaatc

3421 tgcagttttt accaagagct aagccaatat atgtgctttt caaccagtat tgtcacagca

3481 tgaaagtcaa gtcaggttcc agactgttaa gaggtgtaat ctaatgaaga aatcaattag

3541 atgccccgaa atctacagtc gctgaataac caataaacag taacctccat caaatgctat

3601 accaatggac cagtgttagt agctgctccc tgtattatgt gaacagtctt attctatgta

3661 cacagatgta attaaaattg taatcctaac aaacaaaaga aatgtagttc agcttttcaa

3721 tgtttcatgt ttgctgtgct tttctgaatt ttatgttgca ttcaaagact gttgtcttgt

3781 tcttgtggtg tttggattct tgtggtgtgt gcttttagac acagggtaga attagagaca

3841 atattggatg tacaattcct caggagacta cagtagtata ttctattcct taccagtaat

3901 aaggttcttc ctaataataa ttaagagatt gaaactccaa acaagtattc attatgaaca

3961 gatacacatc aaaatcataa taatattttc aaaacaagga ataatttctc taatggttta

4021 ttatagaata ccaatgtata gcttagaaat aaaactttga atatttcaag aatatagata

4081 agtctaattt ttaaatgctg tatatatggc tttcactcaa tcatctctca gatgttgtta

4141 ttaactcgct ctgtgttgtt gcaaaacttt ttggtgcaga ttcgtttcca aaactattgc

4201 tactttgtgt gctttaaaca aaataccttg ggttgatgaa acatcaaccc agtgctagga

4261 atactgtgta tctatcatta gctatatggg actatattgt agattgtggt ttctcagtag

4321 agaagtgact gtagtgtgat tctagataaa tcatcattag caattcattc agatggtcaa

4381 taacttgaaa tttatagctg tgataggagt tcagaaattg gcacatccct ttaaaaataa

4441 caacagaaaa tacaactcct gggaaaaaag gtgctgattc tataagatta tttatatatg

4501 taagtgttta aaaagattat tttccagaaa gtttgtgcag ggtttaagtt gctactattc

4561 aactacacta tatataaata aaatatatac aatatataca ttgttttcac tgtatcacat

4621 taaagtactt gggcttcaga agtaagagcc aaccaactga aaacctgaga tggagatatg

4681 ttcaaagaat gagatacaat tttttagttt tcagtttaag taactctcag cattacaaaa

4741 gagtaagtat ctcacaaata ggaaataaaa ctaaaacgtg gatttaaaaa gaactgcacg

4801 ggctttaggg taaatgctca tcttaaacct cactagaggg aagtcttctc aagtttcaag

4861 caagaccatt tacttaatgt gaagttttgg aaagttataa aggtgtatgt tttagccata

4921 tgattttaat tttaattttg cttcttttag gttcgttctt atttaaagca atatgattgt

4981 gtgactcctt gtagttacac ttgtgtttca atcagatcag attgttgtat ttattccact

5041 attttgcatt taaatgataa cataaaagat ataaaaaatt taaaactgct atttttctta

5101 tagaagagaa aatgggtgtt ggtgattgta ttttaattat ttaagcgtct ctgtttacct

5161 gcctaggaaa acattttatg gcagtcttat gtgcaaagat cgtaaaagga caaaaaattt

5221 aaactgctta taataatcca ggagttgcat tatagccagt agtaaaaata ataataataa

5281 taataaaacc atgtctatag ctgtagatgg gcttcacatc tgtaaagcaa tcaattgtat

5341 atttttgtga tgtgtaccat actgtgtgct ccagcaaatg tccatttgtg taaatgtatt

5401 tattttatat tgtatatatt gttaaatgca aaaaggagat atgattctgt aactccaatc

5461 agttcagatg tgtaactcaa attattatgc ctttcaggat gatggtagag caatattaaa

5521 caagcttcca cttttgactg ctaaaaaaaa aaaaaaaaa

As used herein,“endocrine” refers to secretion of an agent (e.g., a hormone) into a bloodstream.“Exocrine” refers to secretion of an agent into an epithelial surface by way of a duct.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By“FOXA2 polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No. NP 068556.2 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_068556.2 is shown below:

1 MHSASSMLGA VKMEGHEPSD WSSYYAEPEG YSSVSNMNAG LGMNGMNTYM SMSAAAMGSG 61 SGNMSAGSMN MSSYVGAGMS PSLAGMSPGA GAMAGMGGSA GAAGVAGMGP HLSPSLSPLG 121 GQAAGAMGGL APYANMNSMS PMYGQAGLSR ARDPKTYRRS YTHAKPPYSY ISLITMAIQQ 181 SPNKMLTLSE IYQWIMDLFP FYRQNQQRWQ NSIRHSLSFN DCFLKVPRSP DKPGKGSFWT 241 LHPDSGNMFE NGCYLRRQKR FKCEKQLALK EAAGAAGSGK KAAAGAQASQ AQLGEAAGPA 301 SETPAGTESP HSSASPCQEH KRGGLGELKG TPAAALSPPE PAPSPGQQQQ AAAHLLGPPH 361 HPGLPPEAHL KPEHHYAFNH PFSINNLMSS EQQHHHSHHH HQPHKMDLKA YEQVMHYPGY 421 GSPMPGSLAM GPVTNKTGLD ASPLAADTSY YQGVYSRPIM NSS

By“FOXA2 polynucleotide” is meant a polynucleotide encoding a FOXA2 polypeptide or fragment thereof. An exemplary FOXA2 polynucleotide sequence is provided at NCBI Ref: NM_02l784.4. The sequence provided at NCBI Ref: NM_02l784.4 is reproduced

1 cccgcccact tccaactacc gcctccggcc tgcccaggga gagagaggga gtggagccca

61 gggagaggga gcgcgagaga gggagggagg aggggaeggt gctttggctg actttttttt

121 aaaagagggt gggggtgggg ggtgattgct ggtcgtttgt tgtggctgtt aaattttaaa

181 ctgccatgca ctcggcttcc agtatgctgg gagcggtgaa gatggaaggg cacgagccgt

241 ccgactggag cagctactat gcagagcccg agggctactc ctccgtgagc aacatgaacg

301 ccggcctggg gatgaacggc atgaacacgt acatgagcat gtcggcggcc gccatgggca

361 gcggctcggg caacatgagc gcgggctcca tgaacatgtc gtcgtacgtg ggcgctggca

421 tgagcccgtc cctggcgggg atgtcccccg gcgcgggcgc catggcgggc atgggcggct

481 cggccggggc ggccggcgtg gcgggcatgg ggccgcactt gagtcccagc ctgagcccgc

541 tcggggggca ggcggccggg gccatgggcg gcctggcccc ctacgccaac atgaactcca

601 tgagccccat gtacgggcag gcgggcctga gccgcgcccg cgaccccaag acctacaggc

661 gcagctacac gcacgcaaag ccgccctact cgtacatctc gctcatcacc atggccatcc

721 agcagagccc caacaagatg ctgacgctga gcgagatcta ccagtggatc atggacctct

781 tccccttcta ccggcagaac cagcagcgct ggcagaactc catccgccac tcgctctcct

841 tcaacgactg tttcctgaag gtgccccgct cgcccgacaa gcccggcaag ggctccttct

901 ggaccctgca ccctgactcg ggcaacatgt tcgagaacgg ctgctacctg cgccgccaga

961 agcgcttcaa gtgcgagaag cagctggcgc tgaaggaggc cgcaggcgcc gccggcagcg

1021 gcaagaaggc ggccgccgga gcccaggcct cacaggctca actcggggag gccgccgggc

1081 cggcctccga gactccggcg ggcaccgagt cgcctcactc gagcgcctcc ccgtgccagg

1141 agcacaagcg agggggeetg ggagagctga aqqqqacqcc ggctgcggcg ctgagccccc

1201 cagagccggc gccctctccc gggcagcagc agcaggccgc ggcccacctg ctgggcccgc

1261 cccaccaccc gggcctgccg cctgaggccc acctgaagcc ggaacaccac tacgccttca

1321 accacccgtt ctccatcaac aacctcatgt cctcggagca gcagcaccac cacagccacc

1381 accaccacca accccacaaa atggacctca aggcctacga acaggtgatg cactaccccg

1441 gctacggttc ccccatgcct ggcagcttgg ccatgggccc ggtcacgaac aaaacgggcc

1501 tggacgcctc gcccctggcc gcagatacct cctactacca gggggtgtac tcccggccca 1561 ttatgaactc ctcttaagaa gacgacggct tcaggcccgg ctaactctgg caccccggat

1621 cgaggacaag tgagagagca agtgggggtc gagactttgg ggagacggtg ttgcagagac

1681 gcaagggaga agaaatccat aacaccccca ccccaacacc cccaagacag cagtcttctt

1741 cacccgctgc agccgttccg tcccaaacag agggccacac agatacccca cgttctatat

1801 aaggaggaaa acgggaaaga atataaagtt aaaaaaaagc ctccggtttc cactactgtg

1861 tagactcctg cttcttcaag cacctgcaga ttctgatttt tttgttgttg ttgttctcct

1921 ccattgctgt tgttgcaggg aagtcttact taaaaaaaaa aaaaaatttt gtgagtgact

1981 cggtgtaaaa ccatgtagtt ttaacagaac cagagggttg tactattgtt taaaaacagg

2041 aaaaaaaata atgtaagggt ctgttgtaaa tgaccaagaa aaagaaaaaa aaagcattcc

2101 caatcttgac acggtgaaat ccaggtctcg ggtccgatta atttatggtt tctgcgtgct

2161 ttatttatgg cttataaatg tgtattctgg ctgcaagggc cagagttcca caaatctata

2221 ttaaagtgtt atacccggtt ttatcccttg aatcttttct tccagatttt tcttttcttt

2281 acttggctta caaaatatac aggcttggaa attatttcaa gaaggaggga gggataccct

2341 gtctggttgc aggttgtatt ttattttggc ccagggagtg ttgctgtttt cccaacattt

2401 tattaataaa attttcagac ataaaaaa

By“GATA6 polypeptide” is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_005248.2 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_005248.2 is shown below:

1 MALTDGGWCL PKRFGAAGAD ASDSRAFPAR EPSTPPSPIS SSSSSCSRGG ERGPGGASNC

61 GTPQLDTEAA AGPPARSLLL SSYASHPFGA PHGPSAPGVA GPGGNLSSWE DLLLFTDLDQ

121 AATASKLLWS SRGAKLSPFA PEQPEEMYQT LAALSSQGPA AYDGAPGGFV HSAAAAAAAA

181 AAASSPVYVP TTRVGSMLPG LPYHLQGSGS GPANHAGGAG AHPGWPQASA DSPPYGSGGG

241 AAGGGAAGPG GAGSAAAHVS ARFPYSPSPP MA GAAREPG GYAAAGSGGA GGVSGGGSSL

301 AAMGGREPQY SSLSAARPLN GTYHHHHHHH HHHPSPYSPY VGAPLTPAWP AGPFETPVLH

361 SLQSRAGAPL PVPRGPSADL LEDLSESREC WCGSIQTPL WRRDGTGHYL CNACGLYSKM

421 NGLSRPLIKP QKRVPSSRRL GLSCANCHTT TTTLWRRNAE GEPVCNACGL YMKLHGVPRP

481 LAMKKEGIQT RKRKPKNINK SKTCSGNSNN SIPMTPTSTS SNSDDCSKNT SPTTQPTASG

541 AGAPVMTGAG ESTNPENSEL KYSGQDGLYI GVSLASPAEV TSSVRPDSWC ALALA

By“GATA6 polynucleotide” is meant a polynucleotide encoding a GATA6 polypeptide or fragment thereof. An exemplary KCNK3 polynucleotide sequence is provided at NCBI Ref: NM_005257.5. The sequence provided at NCBI Ref: NM_005257.5 is reproduced below:

1 agttccgacc cacagcctgg cacccttcgg cgagcgctgt ttgtttaggg ctcggtgagt

61 ccaatcagga gcccaggctg cagttttccg gcagagcagt aagaggcgcc tcctctctcc

121 tttttattca ccagcagcgc ggcgcagacc ccggactcgc gctcgcccgc tggcgccctc

181 ggcttctctc cgcgcctggg agcaccctcc gccgcggccg ttctccatgc gcagcgcccg

241 cccgaggagc tagacgtcag cttggagcgg cgccggaccg tggatggcct tgactgacgg

301 cggctggtgc ttgccgaagc gcttcggggc cgcgggtgcg gacgccagcg actccagagc

361 ctttccagcg cgggagccct ccacgccgcc ttcccccatc tcttcctcgt cctcctcctg

421 ctcccggggc ggagagcggg gccccggcgg cgccagcaac tgcgggacgc ctcagctcga

481 cacggaggcg gcggccggac ccccggcccg ctcgctgctg ctcagttcct acgcttcgca

541 tcccttcggg gctccccacg gaccttcggc gcctggggtc gcgggccccg ggggcaacct 601 gtcgagctgg gaggacttgc tgctgttcac tgacctcgac caagccgcga ccgccagcaa

661 gctgctgtgg tccagccgcg gcgccaagct gagccccttc gcacccgagc agccggagga

721 gatgtaccag accctcgccg ctctctccag ccagggtccg gccgcctacg acggcgcgcc

781 cggcggcttc gtgcactctg cggccgcggc ggcagcagcc gcggcggcgg ccagctcccc

841 ggtctacgtg cccaccaccc gcgtgggttc catgctgccc ggcctaccgt accacctgca

901 ggggtcgggc agtgggccag ccaaccacgc gggcggcgcg ggcgcgcacc ccggctggcc

961 tcaggcctcg gccgacagcc ctccatacgg cagcggaggc ggcgcggctg gcggcggggc

1021 cgcggggcct ggcggcgctg gctcagccgc ggcgcacgtc tcggcgcgct tcccctactc

1081 tcccagcccg cccatggcca acggcgccgc gcgggagccg ggaggctacg cggcggcggg

1141 cagtgggggc gcgggaggcg tgagcggcgg cggcagtagc ctggcggcca tgggcggccg

1201 cgagccccag tacagctcgc tgtcggccgc gcggccgctg aacgggacgt accaccacca

1261 ccaccaccac caccaccacc atccgagccc ctactcgccc tacgtggggg cgccactgac

1321 gcctgcctgg cccgccggac ccttcgagac cccggtgctg cacagcctgc agagccgcgc

1381 cggagccccg ctcccggtgc cccggggtcc cagtgcagac ctgctggagg acctgtccga

1441 gagccgcgag tgcgtgaact gcggctccat ccagacgccg ctgtggcggc gggacggcac

1501 cggccactac ctgtgcaacg cctgcgggct ctacagcaag atgaacggcc tcagccggcc

1561 cctcatcaag ccgcagaagc gcgtgccttc atcacggcgg cttggattgt cctgtgccaa

1621 ctgtcacacc acaactacca ccttatggcg cagaaacgcc gagggtgaac ccgtgtgcaa

1681 tgcttgtgga ctctacatga aactccatgg ggtgcccaga ccacttgcta tgaaaaaaga

1741 gggaattcaa accaggaaac gaaaacctaa gaacataaat aaatcaaaga cttgctctgg

1801 taatagcaat aattccattc ccatgactcc aacttccacc tcttctaact cagatgattg

1861 cagcaaaaat acttccccca caacacaacc tacagcctca ggggcgggtg ccccggtgat

1921 gactggtgcg ggagagagca ccaatcccga gaacagcgag ctcaagtatt cgggtcaaga

1981 tgggctctac ataggcgtca gtctcgcctc gccggccgaa gtcacgtcct ccgtgcgacc

2041 ggattcctgg tgcgccctgg ccctggcctg agcccacgcc gccaggaggc agggagggct

2101 ccgccgcggg cctcactcca ctcgtgtctg cttttgtgca gcggtccaga cagtggcgac

2161 tgcgctgaca gaacgtgatt ctcgtgcctt tattttgaaa gagatgtttt tcccaagagg

2221 cttgctgaaa gagtgagaga agatggaagg gaagggccag tgcaactggg cgcttgggcc

2281 actccagcca gcccgcctcc ggggcggacc ctgctccact tccagaagcc aggactagga

2341 cctgggcctt gcctgctatg gaatattgag agagattttt taaaaaagat tttgcatttt

2401 gtccaaaatc atgtgcttct tctgatcaat tttggttgtt ccagaatttc ttcatacctt

2461 ttccacatcc agatttcatg tgcgttcatg gagaagatca cttgaggcca tttggtacac

2521 atctctggag gctgagtcgg ttcatgaggt ctcttatcaa aaatattact cagtttgcaa

2581 gactgcattg taactttaac atacactgtg actgacgttt ctcaaagttc atattgtgtg

2641 gctgatctga agtcagtcgg aatttgtaaa cagggtagca aacaagatat ttttcttcca

2701 tgtatacaat aattttttta aaaagtgcaa tttgcgttgc agcaatcagt gttaaatcat

2761 ttgcataaga tttaacagca ttttttataa tgaatgtaaa cattttaact taatggtact

2821 taaaataatt taaaagaaaa atgttaactt agacattctt atgcttcttt tacaactaca

2881 tcccatttta tatttccaat tgttaaagaa aaatatttca agaacaaatc ttctctcagg

2941 aaaattgcct ttctctattt gttaagaatt tttatacaag aacaccaata tacccccttt

3001 attttactgt ggaatatgtg ctggaaaaat tgcaacaaca ctttactacc taacggatag

3061 catttgtaaa tactctaggt atctgtaaac actctgatga agtctgtata gtgtgactaa

3121 cccacaggca ggttggttta cattaatttt tttttttgaa tgggatgtcc tatggaaacc

3181 tatttcacca gagttttaaa aataaaaagg gtattgtttt gtcttctgta cagtgagttc

3241 cttccctttt caaagctttc tttttatgct gtatgtgact atagatattc atataaaaca

3301 agtgcacgtg aagtttgcaa aatgctttaa ggccttcctt tcaaagcata gtccttttgg

3361 agccgttttg taccttttat accttggctt atttgaagtt gacacatggg gttagttact

3421 actctccatg tgcattgggg acagttttta taagtgggaa ggactcagta ttattatatt

3481 tgagatgata agcattttgt ttgggaacaa tgcttaaaaa tattccagaa agttcagatt

3541 ttttttcttt gtgaatgaaa tatattctgg cccacgaaca gggcgatttc ctttcagttt

3601 tttccttttg caacgtgcct tgaagtctca aagctcacct gaggttgcag acgttacccc 3661 caacagaaga taggtagaaa tgattccagt ggcctctttg tattttcttc attgttgagt 3721 agatttcagg aaatcaggag gtgtttcaca atacagaatg atggccttta actgtgaaaa 3781 aaaaa

By“gellan gum” is meant a polysaccharide having a straight chain with a repeating unit that has any one of the following molecular structures:

Gellan gum - high acyl form

In the foregoing structures,“Ac” refers to an acetate group and“Gly” refers to a glycerate group and“M+” is a monovalent cation. In some embodiments, the gellan gum is KELCOGEL® gellan gum.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By“immunosuppressive agent” or“immunosuppressant” is meant an agent that inhibits or prevents an immune reaction, such as rejection, of a transplanted or implanted organ, islet, or organoid in a subject. Examples of immunosuppressants include, but are not limited to, basilizimab, antithymocyte globulin, alemtuzumab, prednisone, azathioprine, mycophenolate, cyclosporine, sirolimus, methotrexate, interferon, and tacrolimus.

By“induced pluripotent stem cell” or“iPSC” is meant a differentiated somatic cell that acquires pluripotency by the exogenous expression of one or more transcription factors in the cell. An“iPSC-derived cell” is a cell derived from an induced pluripotent stem cell.

An“iPSC-derived beta-like cell,”“iPSC-derived alpha-like cell,”“iPSC-derived delta-like cell,” or“iPSC-derived duct-like cell” is a cell derived from an induced pluripotent stem cell and has characteristics of a beta cell, alpha cell, delta cell, or duct cell, respectively.

“Interferon gamma (IFNY) receptor-expressing” cells (e.g., donor cells), islets, organoids (and the cells therein) refer to cells, islets, organoids (and the cells therein) that express IFNy receptor on their surface in an amount or level sufficient to respond to IFNy following contact or exposure to IFNy, e.g., MPS IFNy exposure according to the methods described herein, and, in turn, to express or upregulate expression of a checkpoint protein encoding gene or a checkpoint protein, e.g. PD-L1 (PD-L1 marker protein). In an embodiment, PD-L1 protein is expressed on the surface of the cells (cell membrane expression). In an embodiment, the expression or upregulation of the checkpoint protein, e.g., PD-L1 is sustained, e.g. for greater than or equal to 1, 2, 3, 4, 5, 6, or 7 days or longer.

In an embodiment, the expression or upregulation of the checkpoint protein, e.g., PD-L1 is sustained, e.g. for greater than or equal to 7 days or longer (e.g., more than 1, 2, 3, 4, 5, 6 weeks, or longer). The expression of PD-L1 or the level of expression of PD-L1 in or on cells, for example, may be detected or determined by any assay that is routinely known or used by those skilled in the art to detect or determine levels of proteins or polynucleotides, e.g., without limitation, enzymatic, fluorescent, chemiluminescent or

electrochemiluminescent immunoassay, flow cytometry, spectrometry (mass spectrometry); PCR, or RNA or DNA detection methods.

Intermittent exposure as used herein refers to repeated exposure, e.g., short repeated exposure, of cells, islets, organoids (islet-like organoids, e.g., human islet-like organoids, and the cells therein), especially of interferon-gamma (IFNy) receptor-expressing cells, islets, organoids (islet-like organoids and the cells therein), to multiple pulses, e.g., short repeated pulses, called multiple pulse stimulation (MPS), of IFNy, as used in the described protocols to generate immunoprotected cells, islets, or organoids that survive and have reduced cell death, e.g., evade immune detection, following transplantation, implantation, or transfer, as described herein. The duration of each of the repeated pulses of IFNy exposure is typically a short time period, such as minutes or a few hours, rather than a prolonged period of time. By way of example, the exposure to IFNy may comprise a time period of 0.5 hour, 1 hour, 2 hours, or 3 hours, and the like, multiple times over a given or overall time period, e.g., hours (e.g., 2, 4, 6, 12, 24, 36, 48, 72, 144, or more hours, or intervals therebetween), days (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days), or weeks (1, 2, 3, 4, 5, 6, 7, 8, or more weeks), as described herein.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. The preparation can be at least 75%, at least 90%, and at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By“KCNK3 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_002237.1 and having potassium channel activity. The amino acid sequence provided at NCBI Accession No. NP_002237.l is shown below:

1 MKRQNVRTLA LIVCTFTYLL VGAAVFDALE SEPELIERQR LELRQQELRA RYNLSQGGYE 61 ELERWLRLK PHKAGVQWRF AGSFYFAITV ITTIGYGHAA PSTDGGKVFC MFYALLGIPL 121 TLVMFQSLGE RINTLVRYLL HRAKKGLGMR RADVSMANMV LIGFFSCIST LCIGAAAFSH 181 YEHWTFFQAY YYCFITLTTI GFGDYVALQK DQALQTQPQY VAFSFVYILT GLTVIGAFLN 241 LWLRFMTMN AEDEKRDAEH RALLTRNGQA GGGGGGGSAH TTDTASSTAA AGGGGFRNVY 301 AEVLHFQSMC SCLWYKSREK LQYSIPMIIP RDLSTSDTCV EQSHSSPGGG GRYSDTPSRR 361 CLCSGAPRSA ISSVSTGLHS LSTFRGLMKR RSSV

By“KCNK3 polynucleotide” is meant a polynucleotide encoding a KCNK3 polypeptide or fragment thereof. An exemplary KCNK3 polynucleotide sequence is provided at NCBI Ref: NM_002246.2. The sequence provided at NCBI Ref: NM_002246.2 is reproduced

1 ggcggcggcg gcggcggcgg ccccgggcgc tgagcgggtg cccggcgcgg agagcggcga

61 gcgcagccat gccccaggcc gcctccgggg cagcagcagc ggcggccggg gccgaggcgc

121 gggccggggg cgccgggggg ccggcggcgg cccgggcggg acgatgaagc ggcagaacgt

181 gcgcacgctg gcgctcatcg tgtgcacctt cacctacctg ctggtgggcg ccgcggtctt

241 cgacgcgctg gagtcggagc ccgagctgat cgagcggcag cggctggagc tgcggcagca

301 ggagctgcgg gcgcgctaca acctcagcca gggcggctac gaggagctgg agcgcgtcgt

361 gctgcgcctc aagccgcaca aggccggcgt gcagtggcgc ttcgccggct ccttctactt

421 cgccatcacc gtcatcacca ccatcggcta cgggcacgcg gcacccagca cggatggcgg

481 caaggtgttc tgcatgttct acgcgctgct gggcatcccg ctcacgctcg tcatgttcca

541 gagcctgggc gagcgcatca acaccttggt gaggtacctg ctgcaccgcg ccaagaaggg

601 gctgggcatg cggcgcgccg acgtgtccat ggccaacatg gtgctcatcg gcttcttctc

661 gtgcatcagc acgctgtgca tcggcgccgc cgccttctcc cactacgagc actggacctt

721 cttccaggcc tactactact gcttcatcac cctcaccacc atcggcttcg gcgactacgt

781 ggcgctgcag aaggaccagg ccctgcagac gcagccgcag tacgtggcct tcagcttcgt

841 ctacatcctt acgggcctca cggtcatcgg cgccttcctc aacctcgtgg tgctgcgctt

901 catgaccatg aacgccgagg acgagaagcg cgacgccgag caccgcgcgc tgctcacgcg

961 caacgggcag gcgggcggcg gcggaggggg tggcagcgcg cacactacgg acaccgcctc

1021 atccacggcg gcagcgggcg gcggcggctt ccgcaacgtc tacgcggagg tgctgcactt

1081 ccagtccatg tgctcgtgcc tgtggtacaa gagccgcgag aagctgcagt actccatccc

1141 catgatcatc ccgcgggacc tctccacgtc cgacacgtgc gtggagcaga gccactcgtc

1201 gccgggaggg ggcggccgct acagcgacac gccctcgcga cgctgcctgt gcagcggggc

1261 gccacgctcc gccatcagct cggtgtccac gggtctgcac agcctgtcca ccttccgcgg 1321 cctcatgaag cgcaggagct ccgtgtgact gccccgaggg gcctggagca cctgggggcg

1381 cgggcggggg acccctgctg ggaggccagg agactgcccc tgctgccttc tgcccagtgg

1441 gaccccgcac aacatccctc accactctcc cccagcaccc ccatctccga ctgtgcctgc

1501 ttgcaccagc cggcaggagg ccgggctctg aggacccctg gggcccccat cggagccctg

1561 caaattccga gaaatgtgaa acttggtggg gtcagggagg aaaggcagaa gctgggagcc

1621 tcccttccct ttgaaaatct aagaagctcc cagtcctcag agaccctgct ggtacccaga

1681 cccccacctt cggaggggac ttcatgttcc gtgtacgttt gcatctctat ttatacctct

1741 gtcctgctag gtctcccacc ttcccttggt tccaaaagcc agggtgtcta tgtccaagtc

1801 acccctactc agccccactc cccttcctca tccccagctg tgtctcccaa cctcccttcg

1861 tgttgttttg catggctttg cagttatgga gaaagtggaa acccagcagt ccctaaagct

1921 ggtccccaga aagcaggaca gaaagaagga gggacaggca ggcagcagga ggggcgagct

1981 gggaggcagg aggcagcggc ctgtcagtct gcagaatggt cgcactggag gttcaagcta

2041 actggcctcc agccacattc tcatagcagg taggacttca gccttccaga cactgccctt

2101 agaatctgga acagaagact tcagactcac cataattgct gataattacc cactcttaaa

2161 tttgtcgagt gatttttagc ctctgaaaac tctatgctgg ccactgattc ctttgagtct

2221 cacaaaaccc tacttaggtc atcagggcag gagttctcac tcccatttta cagatgagaa

2281 tactgaggcc tggacaggtg aagtgaccag agagcaaaag gcaaaggggt gggggetggg

2341 tgcagtggct cacacctgta ttcccaacac ttttggaggc tgaggttgga ggattgcttg

2401 agcccaggaa tttgagacca gcctaggtga catagtgaga ccccatctct acaaaaaata

2461 aaaaattaac caggtgtggt ggcacgtgcc tgggagtccc agcgacttgg gaggctgagg

2521 tgggaggatt gtttgagcct gggaggtcga ggctgtagtg agccctgatt gcaccactgt

2581 actccagcct gggtgacagg gcaagaccct gtctcaaaaa aaaaaaaaaa aatggcaaag

2641 ggagacaaga gcccagcctg cttgttgcta gccaaagtgt tctttccttc cagcttggcc

2701 tgctcttaaa agcaaagctc ctgcagtgta catcctggca ttgtgtggct acctgggttt

2761 taaaccagaa tcagaagtcc cggatcagag ggcactgctg aggttcagcc tcttctcttc

2821 ttggccagga ggcagcagct ctgaatgggc ccctgaggct gcacaggggc ctttgtcact

2881 ggggcgcatg cttacaaaca gtgcagttct tgggaccgag gtaagcaggg ctgggtctca

2941 tggcagaaag gccaggatct ggggctctag gaatttggga attgggcaga gtggccaaga

3001 aagctggcag gcatatccta tgggacatca cacctggcac cattgtcatt gttggtgcct

3061 gtgtcccaag tagctagtga taagctgagg ctgcagcaag aaacaccctt cccaggtggg

3121 ggagtttgga ccagaggtgc cctctgccca ccacacctgc aacccagaag cccagatgga

3181 acgcagctga cgaaggtgat gcttgaggct cacttttggg gccccacagc tggagccggt

3241 ataatgactg ggacaacatc aaggggtgga tgaggggcct ctcctcccgc aacactgcct

3301 tcccatgctg ttcccctgcc agctccttaa cactgccgac caaggccagc cctggcattc

3361 agggaaattg gagggcagca cccgtagggt ggccagcctc aggccccacc ccagctgtgt

3421 cctctagtct ctggggaccc ctggggggaa gaagtctacc ctgcttgtga gtcccgtctc

3481 agtgtggagg aactggctgc acgtgggacc tgaaggtgcc ctctgtgttt atgttggggg

3541 tgggggggca gtgctggctg cctctgtcct gtgtgtgacc ctgccctcga agggtcctgt

3601 cctgtcagtc ccgagggagc cacaaccaaa gctgcggaga gaaggtgggg aagggtgcag

3661 aatggccgtg gggcacagcg tggcagactg ttcagtctct gctgggtctt tcctagggac

3721 ctggaaggcc agtgttgctt ccccctcact ccctttcact gcaggcagcc tctctgcttc

3781 cccaatgcct tatgcctggg cacactgcca cagaatatgc aatatgtgtg ggtgaccatg

3841 ccctcacgac cacaccccca ccccgggcag cccccggact ccaaaggtcg tggctgccac

3901 agcctccctc agctcttcct gcctatctgt cttcacactg agaatggcgc ccaataaatg

3961 ctatccacgg agaccagg

By“KCNQ1 polypeptide” is meant a protein or fragment thereof having at least%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity the sequence provided at NCBI Accession No. NP_000209.2 (isoform 1) or NP 861463.1 (isoform 2) and having potassium channel activity. The amino acid sequence provided at NCBI Accession No. NP_000209.2 is shown below:

1 MAAASSPPRA ERKRWGWGRL PGARRGSAGL AKKCPFSLEL AEGGPAGGAL YAPIAPGAPG

61 PAPPASPAAP AAPPVASDLG PRPPVSLDPR VSIYSTRRPV LARTHVQGRV YNFLERPTGW

121 KCFVYHFAVF LIVLVCLIFS VLSTIEQYAA LATGTLFWME IVLWFFGTE YWRLWSAGC

181 RSKYVGLWGR LRFARKPISI IDLIVWASM WLCVGSKGQ VFATSAIRGI RFLQILRMLH

241 VDRQGGTWRL LGSWFIHRQ ELITTLYIGF LGLIFSSYFV YLAEKDAWE SGRVEFGSYA

301 DALWWGWTV TTIGYGDKVP QTWVGKTIAS CFSVFAISFF ALPAGILGSG FALKVQQKQR

361 QKHFNRQIPA AASLIQTAWR CYAAENPDSS TWKIYIRKAP RSHTLLSPSP KPKKSVWKK

421 KKFKLDKDNG VTPGEKMLTV PHITCDPPEE RRLDHFSVDG YDSSVRKSPT LLEVSMPHFM

481 RTNSFAEDLD LEGETLLTPI THISQLREHH RATIKVIRRM QYFVAKKKFQ QARKPYDVRD

541 VIEQYSQGHL NLMVRIKELQ RRLDQSIGKP SLFISVSEKS KDRGSNTIGA RLNRVEDKVT

601 QLDQRLALIT DMLHQLLSLH GGSTPGSGGP PREGGAHITQ PCGSGGSVDP ELFLPSNTLP

661 TYEQLTVPRR GPDEGS

By“KCNQ1 polynucleotide” is meant a polynucleotide encoding a KCNQ1 polypeptide or fragment thereof. An exemplary KCNQ1 polynucleotide sequence is provided at NCBI Ref: NM_0002l8.2. The sequence provided at NCBI Ref: NM_0002l8 .2 is reproduced below:

1 gcggcggggc tggcagcagt ggctgcccgc actgcgcccg ggcgctcgcc ttcgctgcag 61 ctcccggtgc cgccgctcgg gccggccccc cggcaggccc tcctcgttat ggccgcggcc 121 tcctccccgc ccagggccga gaggaagcgc tggggttggg gccgcctgcc aggcgcccgg 181 cggggcagcg cgggcctggc caagaagtgc cccttctcgc tggagctggc ggagggcggc 241 ccggcgggcg gcgcgctcta cgcgcccatc gcgcccggcg ccccaggtcc cgcgccccct 301 gcgtccccgg ccgcgcccgc cgcgccccca gttgcctccg accttggccc gcggccgccg 361 gtgagcctag acccgcgcgt ctccatctac agcacgcgcc gcccggtgtt ggcgcgcacc 421 cacgtccagg gccgcgtcta caacttcctc gagcgtccca ccggctggaa atgcttcgtt 481 taccacttcg ccgtcttcct catcgtcctg gtctgcctca tcttcagcgt gctgtccacc 541 atcgagcagt atgccgccct ggccacgggg actctcttct ggatggagat cgtgctggtg 601 gtgttcttcg ggacggagta cgtggtccgc ctctggtccg ccggctgccg cagcaagtac 661 gtgggcctct gggggegget gcgctttgcc cggaagccca tttccatcat cgacctcatc 721 gtggtcgtgg cctccatggt ggtcctctgc gtgggctcca aggggeaggt gtttgccacg 781 tcggccatca ggggcatccg cttcctgcag atcctgagga tgctacacgt cgaccgccag 841 ggaggcacct ggaggctcct gggctccgtg gtcttcatcc accgccagga gctgataacc 901 accctgtaca tcggcttcct gggcctcatc ttctcctcgt actttgtgta cctggctgag 961 aaggacgcgg tgaacgagtc aggccgcgtg gagttcggca gctacgcaga tgcgctgtgg 1021 tggggggtgg tcacagtcac caccatcggc tatggggaca aggtgcccca gacgtgggtc 1081 gggaagacca tcgcctcctg cttctctgtc tttgccatct ccttctttgc gctcccagcg 1141 gggattcttg gctcggggtt tgccctgaag gtgcagcaga agcagaggca gaagcacttc 1201 aaccggcaga tcccggcggc agcctcactc attcagaccg catggaggtg ctatgctgcc 1261 gagaaccccg actcctccac ctggaagatc tacatccgga aggccccccg gagccacact 1321 ctgctgtcac ccagccccaa acccaagaag tctgtggtgg taaagaaaaa aaagttcaag 1381 ctggacaaag acaatggggt gactcctgga gagaagatgc tcacagtccc ccatatcacg 1441 tgcgaccccc cagaagagcg gcggctggac cacttctctg tcgacggcta tgacagttct 1501 gtaaggaaga gcccaacact gctggaagtg agcatgcccc atttcatgag aaccaacagc 1561 ttcgccgagg acctggacct ggaaggggag actctgctga cacccatcac ccacatctca 1621 cagctgcggg aacaccatcg ggccaccatt aaggtcattc gacgcatgca gtactttgtg 1681 gccaagaaga aattccagca agcgcggaag ccttacgatg tgcgggacgt cattgagcag

1741 tactcgcagg gccacctcaa cctcatggtg cgcatcaagg agctgcagag gaggctggac

1801 cagtccattg ggaagccctc actgttcatc tccgtctcag aaaagagcaa ggatcgcggc

1861 agcaacacga tcggcgcccg cctgaaccga gtagaagaca aggtgacgca gctggaccag

1921 aggctggcac tcatcaccga catgcttcac cagctgctct ccttgcacgg tggcagcacc

1981 cccggcagcg gcggcccccc cagagagggc ggggcccaca tcacccagcc ctgcggcagt

2041 ggcggctccg tcgaccctga gctcttcctg cccagcaaca ccctgcccac ctacgagcag

2101 ctgaccgtgc ccaggagggg ccccgatgag gggtcctgag gaggggatgg ggctggggga

2161 tgggcctgag tgagagggga ggccaagagt ggccccacct ggccctctct gaaggaggcc

2221 acctcctaaa aggcccagag agaagagccc cactctcaga ggccccaata ccccatggac

2281 catgctgtct ggcacagcct gcacttgggg gctcagcaag gccacctctt cctggccggt

2341 gtgggggccc cgtctcaggt ctgagttgtt accccaagcg ccctggcccc cacatggtga

2401 tgttgacatc actggcatgg tggttgggac ccagtggcag ggcacagggc ctggcccatg

2461 tatggccagg aagtagcaca ggctgagtgc aggcccaccc tgcttggccc agggggettc

2521 ctgaggggag acagagcaac ccctggaccc cagcctcaaa tccaggaccc tgccaggcac

2581 aggcagggca ggaccagccc acgctgacta cagggccgcc ggcaataaaa gcccaggagc

2641 ccatttggag ggcctgggcc tggctccctc actctcagga aatgctgacc catgggcagg

2701 agactgtgga gactgctcct gagcccccag cttccagcag gagggacagt ctcaccattt

2761 ccccagggca cgtggttgag tggggggaac gcccacttcc ctgggttaga ctgccagctc

2821 ttcctagctg gagaggagcc ctgcctctcc gcccctgagc ccactgtgcg tggggctccc

2881 gcctccaacc cctcgcccag tcccagcagc cagccaaaca cacagaaggg gactgccacc

2941 tccccttgcc agctgctgag ccgcagagaa gtgacggttc ctacacagga caggggttcc

3001 ttctgggcat tacatcgcat agaaatcaat aatttgtggt gatttggatc tgtgttttaa

3061 tgagtttcac agtgtgattt tgattattaa ttgtgcaagc ttttcctaat aaacgtggag

3121 aatcacaggc tgggctgggc actgctctca ccttggttcc tggggcatcc atggggtctc

3181 tcacagacag gacccctgca gttcccctgg aagcagtgcc caggtggctg tggaatagga

3241 acgctaaaaa aaaaaaaaaa aa

By“LGR5 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_003658. l (isoform 1),

NR_001264155.1 (isoform 2), or NR_001264156.1 (isoform 3) and having transmembrane signaling receptor activity or G-protein coupled receptor activity. The amino acid sequence provided at NCBI Accession No. NP_003658.1 is shown below:

1 MDTSRLGVLL SLPVLLQLAT GGSSPRSGVL LRGCPTHCHC EPDGRMLLRV DCSDLGLSEL 61 PSNLSVFTSY LDLSMNNISQ LLPNPLPSLR FLEELRLAGN ALTYIPKGAF TGLYSLKVLM 121 LQNNQLRHVP TEALQNLRSL QSLRLDANHI SYVPPSCFSG LHSLRHLWLD DNALTEIPVQ 181 AFRSLSALQA MTLALNKIHH IPDYAFGNLS SLWLHLHNN RIHSLGKKCF DGLHSLETLD 241 LNYNNLDEFP TAIRTLSNLK ELGFHSNNIR SIPEKAFVGN PSLITIHFYD NPIQFVGRSA 301 FQHLPELRTL TLNGASQITE FPDLTGTANL ESLTLTGAQI SSLPQTVCNQ LPNLQVLDLS 361 YNLLEDLPSF SVCQKLQKID LRHNEIYEIK VDTFQQLLSL RSLNLAWNKI AIIHPNAFST 421 LPSLIKLDLS SNLLSSFPIT GLHGLTHLKL TGNHALQSLI SSENFPELKV IEMPYAYQCC 481 AFGVCENAYK ISNQWNKGDN SSMDDLHKKD AGMFQAQDER DLEDFLLDFE EDLKALHSVQ 541 CSPSPGPFKP CEHLLDGWLI RIGVWTIAVL ALTCNALVTS TVFRSPLYIS PIKLLIGVIA 601 AWMLTGVSS AVLAGVDAFT FGSFARHGAW WENGVGCHVI GFLSIFASES SVFLLTLAAL 661 ERGFSVKYSA KFETKAPFSS LKVIILLCAL LALTMAAVPL LGGSKYGASP LCLPLPFGEP 721 STMGYMVALI LLNSLCFLMM TIAYTKLYCN LDKGDLENIW DCSMVKHIAL LLFTNCILNC 781 PVAFLSFSSL INLTFISPEV IKFILLVWP LPACLNPLLY ILFNPHFKED LVSLRKQTYV 841 WTRSKHPSLM SINSDDVEKQ SCDSTQALVT FTSSSITYDL PPSSVPSPAY PVTESCHLSS 901 VAFVPCL

By“LGR5 polynucleotide” is meant a polynucleotide encoding a LGR5 polypeptide or fragment thereof. An exemplary LGR5 polynucleotide sequence is provided at NCBI Ref: NM_003667.3. The sequence provided at NCBI Ref: NM_003667.3 is reproduced below:

1 aaaaaacgag cgtgcaagca gagatgctgc tccacaccgc tcaggccgcg agcagcagca

61 aggcgcaccg ccactgtcgc cgctgcagcc agggctgctc cgaaggccgg cgtggcggca

121 accggcacct ctgtccccgc cgcgcttctc ctcgccgccc acgccgtggg gtcaggaacg

181 cggcgtctgg cgctgcagac gcccgctgag ttgcagaagc ccacggagcg gcgcccggcg

241 cgccacggcc cgtagcagtc cggtgctgct ctccgcccgc gtccggctcg tggcccccta

301 cttcgggcac catggacacc tcccggctcg gtgtgctcct gtccttgcct gtgctgctgc

361 agctggcgac cgggggcagc tctcccaggt ctggtgtgtt gctgaggggc tgccccacac

421 actgtcattg cgagcccgac ggcaggatgt tgctcagggt ggactgctcc gacctggggc

481 tctcggagct gccttccaac ctcagcgtct tcacctccta cctagacctc agtatgaaca

541 acatcagtca gctgctcccg aatcccctgc ccagtctccg cttcctggag gagttacgtc

601 ttgcgggaaa cgctctgaca tacattccca agggagcatt cactggcctt tacagtctta

661 aagttcttat gctgcagaat aatcagctaa gacacgtacc cacagaagct ctgcagaatt

721 tgcgaagcct tcaatccctg cgtctggatg ctaaccacat cagctatgtg cccccaagct

781 gtttcagtgg cctgcattcc ctgaggcacc tgtggctgga tgacaatgcg ttaacagaaa

841 tccccgtcca ggcttttaga agtttatcgg cattgcaagc catgaccttg gccctgaaca

901 aaatacacca cataccagac tatgcctttg gaaacctctc cagcttggta gttctacatc

961 tccataacaa tagaatccac tccctgggaa agaaatgctt tgatgggctc cacagcctag

1021 agactttaga tttaaattac aataaccttg atgaattccc cactgcaatt aggacactct

1081 ccaaccttaa agaactagga tttcatagca acaatatcag gtcgatacct gagaaagcat

1141 ttgtaggcaa cccttctctt attacaatac atttctatga caatcccatc cagtttgttg

1201 ggagatctgc ttttcaacat ttacctgaac taagaacact gactctgaat ggtgcctcac

1261 aaataactga atttcctgat ttaactggaa ctgcaaacct ggagagtctg actttaactg

1321 gagcacagat ctcatctctt cctcaaaccg tctgcaatca gttacctaat ctccaagtgc

1381 tagatctgtc ttacaaccta ttagaagatt tacccagttt ttcagtctgc caaaagcttc

1441 agaaaattga cctaagacat aatgaaatct acgaaattaa agttgacact ttccagcagt

1501 tgcttagcct ccgatcgctg aatttggctt ggaacaaaat tgctattatt caccccaatg

1561 cattttccac tttgccatcc ctaataaagc tggacctatc gtccaacctc ctgtcgtctt

1621 ttcctataac tgggttacat ggtttaactc acttaaaatt aacaggaaat catgccttac

1681 agagcttgat atcatctgaa aactttccag aactcaaggt tatagaaatg ccttatgctt

1741 accagtgctg tgcatttgga gtgtgtgaga atgcctataa gatttctaat caatggaata

1801 aaggtgacaa cagcagtatg gacgaccttc ataagaaaga tgctggaatg tttcaggctc

1861 aagatgaacg tgaccttgaa gatttcctgc ttgactttga ggaagacctg aaagcccttc

1921 attcagtgca gtgttcacct tccccaggcc ccttcaaacc ctgtgaacac ctgcttgatg

1981 gctggctgat cagaattgga gtgtggacca tagcagttct ggcacttact tgtaatgctt

2041 tggtgacttc aacagttttc agatcccctc tgtacatttc ccccattaaa ctgttaattg

2101 gggtcatcgc agcagtgaac atgctcacgg gagtctccag tgccgtgctg gctggtgtgg

2161 atgcgttcac ttttggcagc tttgcacgac atggtgcctg gtgggagaat ggggttggtt

2221 gccatgtcat tggttttttg tccatttttg cttcagaatc atctgttttc ctgcttactc

2281 tggcagccct ggagcgtggg ttctctgtga aatattctgc aaaatttgaa acgaaagctc

2341 cattttctag cctgaaagta atcattttgc tctgtgccct gctggccttg accatggccg

2401 cagttcccct gctgggtggc agcaagtatg gcgcctcccc tctctgcctg cctttgcctt

2461 ttggggagcc cagcaccatg ggctacatgg tcgctctcat cttgctcaat tccctttgct 2521 tcctcatgat gaccattgcc tacaccaagc tctactgcaa tttggacaag ggagacctgg

2581 agaatatttg ggactgctct atggtaaaac acattgccct gttgctcttc accaactgca

2641 tcctaaactg ccctgtggct ttcttgtcct tctcctcttt aataaacctt acatttatca

2701 gtcctgaagt aattaagttt atccttctgg tggtagtccc acttcctgca tgtctcaatc

2761 cccttctcta catcttgttc aatcctcact ttaaggagga tctggtgagc ctgagaaagc

2821 aaacctacgt ctggacaaga tcaaaacacc caagcttgat gtcaattaac tctgatgatg

2881 tcgaaaaaca gtcctgtgac tcaactcaag ccttggtaac ctttaccagc tccagcatca

2941 cttatgacct gcctcccagt tccgtgccat caccagctta tccagtgact gagagctgcc

3001 atctttcctc tgtggcattt gtcccatgtc tctaattaat atgtgaagga aaatgttttc

3061 aaaggttgag aacctgaaaa tgtgagattg agtatatcag agcagtaatt aataagaaga

3121 gctgaggtga aactcggttt aaaaaccaaa aaagaatctc tcagttagta agaaaaggct

3181 gaaaacctct tgatacttga gagtgaatat aagtctaaat gctgctttgt ataatttgtt

3241 cagctaaggg atagatcgat cacactattt aagtgagccc agatcaaaaa agcagattga

3301 aattttcttt agaaaagatt ctccatgatt tgaattgcat tctctttaaa ctcaccaatg

3361 taatcatttt gggaggaggg agaacccact tgctttccaa atgggtttat ttaaacccac

3421 aaactcaaga ggttgttggg ggaattagga aaataagggt tttcaatgac ctacattgct

3481 aggtagaggc tgtgatccat gggatttcat tctaatgacc atgtgaagat gtttgagtcc

3541 tcctttgcct ttcctcagaa agaatccttc taaggcacaa atcccttaga tggataatgt

3601 aaggtattgt taactcactc atattgagat catttttaga gataccaggt tttatgtatc

3661 agcactagat ggttccaccc tcatgggata aaactgctta caagtatttt gaaagaaaaa

3721 ctgaccaaaa ttcttaaatt gttactaagg caatcatgca caggtgacgt atgtcttatc

3781 tgatttgttt ttaactcctt ggtgcccaaa gctcagaagg gaattccact gccagcaatg

3841 aacatacctg gaaaagaaag taagcaatct gggatttttt ttctgggtta gtaaagaatt

3901 tttgcaataa gttttatcag ttgattcaaa ctgatgtgca tcttaatgat caaatgtgca

3961 cattacataa attaagtcca ctgatacaac ttcttacaca tgtatctcta gtagctctgg

4021 caaacccaat atctgacacc actttggact caagagactc agtaacgtat tatcctgttt

4081 atttagcttg gttttagctg tgttctctct ggataaccca cttgatgtta ggaacattac

4141 ttctctgctt attccatatt aatactgtgt taggtatttt aagaagcaag ttattaaata

4201 agaaaagtca aagtattaat tcttaccttc tattatccta tattagcttc aatacatcca

4261 aaccaaatgg ctgttaggta gatttatttt tatataagca tgtttatttt gatcagatgt

4321 tttaacttgg atttgaaaaa atacatttat gagatgtttt ataagatgtg taaatataga

4381 actgtattta ttactatagt aaaggttcag taacattaag gaccatgata atgataataa

4441 accttgtaca gtggcatatt ctttgattta tattgtgttt ctctgcccat tttctttaaa

4501 ttcattaact gtatatatgt aaatatatag tacttgtaaa tagattccaa atttgctttt

4561 ctattgggta aaaaataaat ttgtaataaa atgtgtgact atgaaacaaa aaaaaaaaaa

4621 aaaaa

By“LDHA polypeptide” or“lactate dehydrogenase A polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_005557.l (isoform 1), NP_00l 128711.1 (isoform 2), NP_00l 158886.1 (isoform 3), NP_00l 158887.1 (isoform 4), or P_00H58888.l (isoform 5) and having

dehydrogenase activity. The amino acid sequence provided at NCBI Accession No.

NP_005557.l is shown below:

1 MATLKDQLIY NLLKEEQTPQ NKITWGVGA VGMACAISIL MKDLADELAL VDVIEDKLKG 61 EMMDLQHGSL FLRTPKIVSG KDYNVTANSK LVIITAGARQ QEGESRLNLV QRNWIFKFI 121 IPNWKYSPN CKLLIVSNPV DILTYVAWKI SGFPKNRVIG SGCNLDSARF RYLMGERLGV 181 HPLSCHGWVL GEHGDSSVPV WSGMNVAGVS LKTLHPDLGT DKDKEQWKEV HKQWESAYE 241 VIKLKGYTSW AIGLSVADLA ESIMKNLRRV HPVSTMIKGL YGIKDDVFLS VPCILGQNGI 301 SDLVKVTLTS EEEARLKKSA DTLWGIQKEL QF

By“LDHA polynucleotide” or“lactate dehydrogenase A polynucleotide” is meant a polynucleotide encoding a LDHA polypeptide or fragment thereof. An exemplary LDHA polynucleotide sequence is provided at NCBI Ref: NM_005566.3. The sequence provided at

NCBI Ref: NM_005566.3 is reproduced below:

1 gtctgccggt cggttgtctg gctgcgcgcg ccacccgggc ctctccagtg ccccgcctgg 61 ctcggcatcc acccccagcc cgactcacac gtgggttccc gcacgtccgc cggccccccc 121 cgctgacgtc agcatagctg ttccacttaa ggcccctccc gcgcccagct cagagtgctg 181 cagccgctgc cgccgattcc ggatctcatt gccacgcgcc cccgacgacc gcccgacgtg 241 cattcccgat tccttttggt tccaagtcca atatggcaac tctaaaggat cagctgattt 301 ataatcttct aaaggaagaa cagacccccc agaataagat tacagttgtt ggggttggtg 361 ctgttggcat ggcctgtgcc atcagtatct taatgaagga cttggcagat gaacttgctc 421 ttgttgatgt catcgaagac aaattgaagg gagagatgat ggatctccaa catggcagcc 481 ttttccttag aacaccaaag attgtctctg gcaaagacta taatgtaact gcaaactcca 541 agctggtcat tatcacggct ggggcacgtc agcaagaggg agaaagccgt cttaatttgg 601 tccagcgtaa cgtgaacatc tttaaattca tcattcctaa tgttgtaaaa tacagcccga 661 actgcaagtt gcttattgtt tcaaatccag tggatatctt gacctacgtg gcttggaaga 721 taagtggttt tcccaaaaac cgtgttattg gaagcggttg caatctggat tcagcccgat 781 tccgttacct aatgggggaa aggctgggag ttcacccatt aagctgtcat gggtgggtcc 841 ttggggaaca tggagattcc agtgtgcctg tatggagtgg aatgaatgtt gctggtgtct 901 ctctgaagac tctgcaccca gatttaggga ctgataaaga taaggaacag tggaaagagg 961 ttcacaagca ggtggttgag agtgcttatg aggtgatcaa actcaaaggc tacacatcct 1021 gggctattgg actctctgta gcagatttgg cagagagtat aatgaagaat cttaggcggg 1081 tgcacccagt ttccaccatg attaagggtc tttacggaat aaaggatgat gtcttcctta 1141 gtgttccttg cattttggga cagaatggaa tctcagacct tgtgaaggtg actctgactt 1201 ctgaggaaga ggcccgtttg aagaagagtg cagatacact ttgggggatc caaaaggagc 1261 tgcaatttta aagtcttctg atgtcatatc atttcactgt ctaggctaca acaggattct 1321 aggtggaggt tgtgcatgtt gtccttttta tctgatctgt gattaaagca gtaatatttt 1381 aagatggact gggaaaaaca tcaactcctg aagttagaaa taagaatggt ttgtaaaatc 1441 cacagctata tcctgatgct ggatggtatt aatcttgtgt agtcttcaac tggttagtgt 1501 gaaatagttc tgccacctct gacgcaccac tgccaatgct gtacgtactg catttgcccc 1561 ttgagccagg tggatgttta ccgtgtgtta tataacttcc tggctccttc actgaacatg 1621 cctagtccaa cattttttcc cagtgagtca catcctggga tccagtgtat aaatccaata 1681 tcatgtcttg tgcataattc ttccaaagga tcttattttg tgaactatat cagtagtgta 1741 cattaccata taatgtaaaa agatctacat acaaacaatg caaccaacta tccaagtgtt 1801 ataccaacta aaacccccaa taaaccttga acagtgacta ctttggttaa ttcattatat 1861 taagatataa agtcataaag ctgctagtta ttatattaat ttggaaatat taggctattc 1921 ttgggcaacc ctgcaacgat tttttctaac agggatatta ttgactaata gcagaggatg 1981 taatagtcaa ctgagttgta ttggtaccac ttccattgta agtcccaaag tattatatat 2041 ttgataataa tgctaatcat aattggaaag taacattcta tatgtaaatg taaaatttat 2101 ttgccaactg aatataggca atgatagtgt gtcactatag ggaacacaga tttttgagat 2161 cttgtcctct ggaagctggt aacaattaaa aacaatctta aggcagggaa aaaaaaaaaa 2221 aaaaaa By“MAFA polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP 963883.2 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_963883.2 is shown below:

1 MAAELAMGAE LPSSPLAIEY WDFDLMKFE VKKEPPEAER FCHRLPPGSL SSTPLSTPCS 61 SVPSSPSFCA PSPGTGGGGG AGGGGGSSQA GGAPGPPSGG PGAVGGTSGK PALEDLYWMS 121 GYQHHLNPEA LNLTPEDAVE ALIGSGHHGA HHGAHHPAAA AAYEAFRGPG FAGGGGADDM 181 GAGHHHGAHH AAHHHHAAHH HHHHHHHHGG AGHGGGAGHH VRLEERFSDD QLVSMSVREL 241 NRQLRGFSKE EVIRLKQKRR TLKNRGYAQS CRFKRVQQRH ILESEKCQLQ SQVEQLKLEV 301 GRLAKERDLY KEKYEKLAGR GGPGSAGGAG FPREPSPPQA GPGGAKGTAD FFL

By“MAFA polynucleotide” is meant a polynucleotide encoding a MAFA polypeptide or fragment thereof. An exemplary MAFA polynucleotide sequence is provided at NCBI

Ref: NM 201589.3. The sequence provided at NCBI Ref: NM_20l589.3 is reproduced below:

1 gcgcggccgg gcgcgggccc cgggcgatgg ccgcggagct ggcgatgggc gccgagctgc

61 ccagcagccc gctggccatc gagtacgtca acgacttcga cctgatgaag ttcgaggtga

121 agaaggagcc tcccgaggcc gagcgcttct gccaccgcct gccgccaggc tcgctgtcct

181 cgacgccgct cagcacgccc tgctcctccg tgccctcctc gcccagcttc tgcgcgccca

241 gcccgggcac cggcggcggc ggcggcgcgg ggggcggcgg cggctcgtct caggccgggg

301 gcgcccccgg gccgccgagc ggggg ^cccc g gcgccgtcgg gggcacctcg gggaagccgg

361 cgctggagga tctgtactgg atgagcggct accagcatca cctcaacccc gaggcgctca

421 acctgacgcc cgaggacgcg gtggaggcgc tcatcggcag cggccaccac ggcgcgcacc

481 acggcgcgca ccacccggcg gccgccgcag cctacgaggc tttccgcggc ccgggcttcg

541 cgggcggcgg cggagcggac gacatgggcg ccggccacca ccacggcgcg caccacgccg

601 cccaccatca ccacgccgcc caccaccacc accaccacca ccaccaccat ggcggcgcgg

661 gacacggcgg tggcgcgggc caccacgtgc gcctggagga gcgcttctcc gacgaccagc

721 tggtgtccat gtcggtgcgc gagctgaacc ggcagctccg cggcttcagc aaggaggagg

781 tcatccggct caagcagaag cggcgcacgc tcaagaaccg cggctacgcg cagtcctgcc

841 gcttcaagcg ggtgcagcag cggcacattc tggagagcga gaagtgccaa ctccagagcc

901 aggtggagca gctgaagctg gaggtggggc gcctggccaa agagcgggac ctgtacaagg

961 agaaatacga gaagctggcg ggccggggcg gccccgggag cgcgggcggg gccggtttcc

1021 cgcgggagcc ttcgccgccg caggccggtc ccggcggggc caagggcacg gccgacttct

1081 tcctgtaggc gccggacccc gagcccgcgc cgccgtcgcc ggggacaagt tcgcgcaggc

1141 ctctcggggc ctcggctcgg actccgcggt acaggacgtg gacaccaggc ccggcccggc

1201 cgtgctggcc ccggtgccaa gtctgcgggc gcggggctgg aggccccttc gctcccggtc

1261 cccgttcgcg cgcgtcggcc cgggtcgccg tcctgaggtt gagcggagaa cggtgatttc

1321 taaggaaact tgagccaggt ctaacttctt tccaagcgtc cgcttgtaca tacgttgaac

1381 gtggttctcc gttcccacct tcgccctgcc agcctagagg gaccgcgctg ccgtcccttc

1441 ccgggtggcc cctgcctgcc cccgccctcc ttcgttctct tctcagcctc cctttccttg

1501 ccttttttaa cttcccctcc ccgttttaaa atcggtctta ttttcgaagt atttataatt

1561 attatgcttg gtgattagaa aagaaaacct tggaggaagc cccttctttc cccagccggg

1621 gtccgccctc agtcgcgagt cacagcatga gtcgctcgcc aggaggggcc cggcccctgc

1681 ctgccccctc cccgcttgcc cccgaccctg ctaccggcgt tccttggagg tcgaagccag

1741 ggacgtcacc cgtgctgtgt ccaggcctgc tgtcctacta tgctcaaccg ggggtggggg 1801 gaggggggtg agtcctgtgc tcagtcgggt gggggetggc ccggatcccg agctgctgtc

1861 tctctatgca ccagaacata tctgtaactc ctggggaaat acatcttgtt ttaaccttca

1921 agagaagtga aagaaaaaag taatgcacag tatttctagc agaaaatttt tttttttaag

1981 aggaggcttg ggccagagcc ttctggcatg gggcgggtgg agaaagtgtt tttattttaa

2041 tttaaattgt gtttcgtttt gtttgtggaa tctttcttta atgettegte gctctttgga

2101 ctagccggga gagagggcga ggaggcgggt gctccaggcc ctgtaggctg ggccaggcgc

2161 ctgggggatc tgcccgtttt cggaggccct caggggccat cagtgggatt ccagccgctc

2221 cacacccctc ccctgagcac tcggagtgga aggcgcgccg actcgttgaa agttttgttg

2281 tgtagttggt tttcgttgag ttcttttttc atttgctacg aaactgagaa aaagaaaaaa

2341 atacacaaaa taaatctgtt cagatccaag tea

As used herein, a“marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder or that is associated with a particular cell type. In some embodiments, a marker for a beta cell is Pdxl, MafA, Pax4, Pax6, NeuroDl, Nkx6-l, Gata6, or Foxa2. In some embodiments, a marker for a hepatocyte is AFP, ALB, or Cyp3a7. In some other embodiments, a marker for a cardiomyocyte is hMlc2a, hNkx2-5, alphaMHC or KCNQ1. In still other embodiments, a marker for a small intestine cell is CDX2, Muc2, or Lgr5.

By“alphaMHC polypeptide” or“myosin heavy chain (MHC) alpha polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_002462.2 and having actin binding activity. The amino acid sequence provided at NCBI Accession No. NP_002462.2 is shown below:

1 MTDAQMADFG AAAQYLRKSE KERLEAQTRP FDIRTECFVP DDKEEFVKAK ILSREGGKVI

61 AETENGKTVT VKEDQVLQQN PPKFDKIEDM AMLTFLHEPA VLFNLKERYA AWMIYTYSGL

121 FCVTWPYKW LPVYNAEWA AYRGKKRSEA PPHIFSISDN AYQYMLTDRE NQSILITGES

181 GAGKTWTKR VIQYFASIAA IGDRGKKDNA NANKGTLEDQ IIQANPALEA FGNAKTVRND

241 NSSRFGKFIR IHFGATGKLA SADIETYLLE KSRVIFQLKA ERNYHIFYQI LSNKKPELLD

301 MLLVTNNPYD YAFVSQGEVS VASIDDSEEL MATDSAFDVL GFTSEEKAGV YKLTGAIMHY

361 GNMKFKQKQR EEQAEPDGTE DADKSAYLMG LNSADLLKGL CHPRVKVGNE YVTKGQSVQQ

421 VYYSIGALAK AVYEKMFNWM VTRINATLET KQPRQYFIGV LDIAGFEIFD FNSFEQLCIN

481 FTNEKLQQFF NHHMFVLEQE EYKKEGIEWT FIDFGMDLQA CIDLIEKPMG IMSILEEECM

541 FPKATDMTFK AKLYDNHLGK SNNFQKPRNI KGKQEAHFSL IHYAGTVDYN ILGWLEKNKD

601 PLNETWALY QKSSLKLMAT LFSSYATADT GDSGKSKGGK KKGSSFQTVS ALHRENLNKL

661 MTNLRTTHPH FVRCIIPNER KAPGVMDNPL VMHQLRCNGV LEGIRICRKG FPNRILYGDF

721 RQRYRILNPV AIPEGQFIDS RKGTEKLLSS LDIDHNQYKF GHTKVFFKAG LLGLLEEMRD

781 ERLSRIITRM QAQARGQLMR IEFKKIVERR DALLVIQWNI RAFMGVKNWP WMKLYFKIKP

841 LLKSAETEKE MATMKEEFGR IKETLEKSEA RRKELEEKMV SLLQEKNDLQ LQVQAEQDNL

901 NDAEERCDQL IKNKIQLEAK VKEMNERLED EEEMNAELTA KKRKLEDECS ELKKDIDDLE

961 LTLAKVEKEK HATENKVKNL TEEMAGLDEI IAKLTKEKKA LQEAHQQALD DLQVEEDKW

1021 SLSKSKVKLE QQVDDLEGSL EQEKKVRMDL ERAKRKLEGD LKLTQESIMD LENDKLQLEE

1081 KLKKKEFDIN QQNSKIEDEQ VLALQLQKKL KENQARIEEL EEELEAERTA RAKVEKLRSD

1141 LSRELEEISE RLEEAGGATS VQIEMNKKRE AEFQKMRRDL EEATLQHEAT AAALRKKHAD 1201 SVAELGEQID NLQRVKQKLE KEKSEFKLEL DDVTSNMEQI IKAKANLEKV SRTLEDQANE

1261 YRVKLEEAQR SLNDFTTQRA KLQTENGELA RQLEEKEALI SQLTRGKLSY TQQMEDLKRQ

1321 LEEEGKAKNA LAHALQSARH DCDLLREQYE EETEAKAELQ RVLSKANSEV AQWRTKYETD

1381 AIQRTEELEE AKKKLAQRLQ DAEEAVEAW AKCSSLEKTK HRLQNEIEDL MVDVERSNAA

1441 AAALDKKQRN FDKILAEWKQ KYEESQSELE SSQKEARSLS TELFKLKNAY EESLEHLETF

1501 KRENKNLQEE ISDLTEQLGE GGKNVHELEK VRKQLEVEKL ELQSALEEAE ASLEHEEGKI

1561 LRAQLEFNQI KAEIERKLAE KDEEMEQAKR NHQRWDSLQ TSLDAETRSR NEVLRVKKKM

1621 EGDLNEMEIQ LSHANRMAAE AQKQVKSLQS LLKDTQIQLD DAVRANDDLK ENIAIVERRN

1681 NLLQAELEEL RAWEQTERS RKLAEQELIE TSERVQLLHS QNTSLINQKK KMESDLTQLQ

1741 SEVEEAVQEC RNAEEKAKKA ITDAAMMAEE LKKEQDTSAH LERMKKNMEQ TIKDLQHRLD

1801 EAEQIALKGG KKQLQKLEAR VRELEGELEA EQKRNAESVK GMRKSERRIK ELTYQTEEDK

1861 KNLLRLQDLV DKLQLKVKAY KRQAEEAEEQ ANTNLSKFRK VQHELDEAEE RADIAESQW

1921 KLRAKSRDIG AKQKMHDEE

By“alphaMHC polynucleotide” is meant a polynucleotide encoding a alphaMHC polypeptide or fragment thereof. An exemplary alphaMHC polynucleotide sequence is provided at NCBI Ref: NM_00247l.3. The sequence provided at NCBI Ref: NM_00247l.3 is reproduced below:

1 agatagagag actcctgcgg cccagattct tcaggattct ccgtgaaggg ataaccaggg

61 gaagcaccaa gatgaccgat gcccagatgg ctgactttgg ggcagcggcc cagtacctcc

121 gcaagtcaga gaaggagcgt ctagaggccc agacccggcc ctttgacatt cgcactgagt

181 gcttcgtgcc cgatgacaag gaagagtttg tcaaagccaa gattttgtcc cgggagggag

241 gcaaggtcat tgctgaaacc gagaatggga agacggtgac tgtgaaggag gaccaggtgt

301 tgcagcagaa cccacccaag ttcgacaaga ttgaggacat ggccatgctg accttcctgc

361 acgagcccgc ggtgcttttc aacctcaagg agcgctacgc ggcctggatg atatatacct

421 actcgggcct cttctgtgtc actgtcaacc cctacaagtg gctgccggtg tacaatgccg

481 aggtggtggc cgcctaccgg ggcaagaaga ggagtgaggc cccgccccac atcttctcca

541 tctccgacaa cgcctatcag tacatgctga cagatcggga gaaccagtcc atcctcatca

601 cgggagaatc eggggegggg aagactgtga acaccaagcg tgtcatccag tactttgcca

661 gcattgcagc cataggtgac cgtggcaaga aggacaatgc caatgcgaac aagggcaccc

721 tggaggacca gatcatccag gccaaccccg ctctggaggc cttcggcaat gccaagactg

781 tccggaacga caactcctcc cgctttggga aattcattag gatccacttt ggggccactg

841 gaaagctggc ttctgcagac atagagacct acctgctgga gaagtcccgg gtgatcttcc

901 agctgaaagc tgagagaaac taccacatct tctaccagat tctgtccaac aagaagccgg

961 agttgctgga catgctgctg gtcaccaaca atccctacga ctacgccttc gtgtctcagg

1021 gagaggtgtc cgtggcctcc attgatgact ccgaggagct catggccacc gatagtgcct

1081 ttgacgtgct gggcttcact tcagaggaga aagctggcgt ctacaagctg acgggagcca

1141 tcatgcacta cgggaacatg aagttcaagc agaagcagcg ggaggagcag gcggagccag

1201 acggcaccga agatgctgac aagtcggcct acctcatggg gctgaactca gctgacctgc

1261 tcaaggggct gtgccaccct cgggtgaaag tgggcaacga gtatgtcacc aaggggcaga

1321 gcgtgcagca ggtgtactac tccatcgggg ctctggccaa ggcagtgtat gagaagatgt

1381 tcaactggat ggtgacgcgc atcaacgcca ccctggagac caagcagcca cgccagtact

1441 tcataggagt cctggacatc gctggcttcg agatcttcga cttcaacagc tttgagcagc

1501 tctgcatcaa cttcaccaac gagaagctgc agcagttctt caaccaccac atgttcgtgc

1561 tggagcagga ggagtacaag aaggagggca ttgagtggac attcattgac tttggcatgg

1621 acctgcaggc ctgcattgac ctcatcgaga agcccatggg catcatgtcc atcctggagg

1681 aggagtgcat gttccccaag gccactgaca tgaccttcaa ggccaagctg tacgacaacc

1741 acctgggcaa gtccaacaat ttccagaagc cacgcaacat caaggggaag caggaagccc

1801 acttctccct gatccactac gccggcactg tggactacaa catcctgggc tggctggaaa 1861 aaaacaagga tcctctcaac gagactgttg tggccctgta ccagaagtcc tccctcaagc

1921 tcatggccac tctcttctcc tcctacgcaa ctgccgatac tggggacagt ggtaaaagca

1981 aaggaggcaa gaaaaagggc tcatccttcc agacggtgtc ggctctccac cgggaaaatc

2041 tcaacaagct aatgaccaac ctgaggacca cccatcctca ctttgtgcgt tgcatcatcc

2101 ccaatgagcg gaaggctcca ggggtgatgg acaaccccct ggtcatgcac cagctgcgct

2161 gcaatggcgt gctggagggc atccgcatct gcaggaaggg cttccccaac cgcatcctct

2221 acggggactt ccggcagagg tatcgcatcc tgaacccagt ggccatccct gagggacagt

2281 tcattgatag caggaagggg acagagaagc tgctcagctc tctggacatt gatcacaacc

2341 agtacaagtt tggccacacc aaggtgttct tcaaggcagg gctgcttggg ctgctggagg

2401 agatgcggga tgagaggctg agccgcatca tcacgcgcat gcaggcccaa gcccggggcc

2461 agctcatgcg cattgagttc aagaagatag tggaacgcag ggatgccctg ctggtaatcc

2521 agtggaacat tcgggccttc atgggggtca agaattggcc ctggatgaag ctctacttca

2581 agatcaagcc gctgctgaag agcgcagaga cggagaagga gatggccacc atgaaggaag

2641 agttcgggcg catcaaagag acgctggaga agtccgaggc tcgccgcaag gagctggagg

2701 agaagatggt gtccctgctg caggagaaga atgacctgca gctccaagtg caggcggaac

2761 aagacaacct caatgatgct gaggagcgct gcgaccagct gatcaaaaac aagattcagc

2821 tggaggccaa agtaaaggag atgaatgaga ggctggagga tgaggaggag atgaacgcgg

2881 agctcactgc caagaagcgc aagctggaag acgagtgctc agagctcaag aaggacattg

2941 atgacctgga gctgacactg gccaaggtgg agaaggagaa gcatgcaaca gagaacaagg

3001 tgaagaacct aacagaggag atggctgggc tggatgaaat catcgctaag ctgaccaagg

3061 agaagaaagc tctacaagag gcccatcagc aggccctgga tgaccttcag gttgaggaag

3121 acaaggtcaa cagcctgtcc aagtctaagg tcaagctgga gcagcaggtg gatgatctgg

3181 agggatccct agagcaagag aagaaggtgc gcatggacct ggagcgagca aagcggaaac

3241 tggagggcga cctgaagctg acccaggaga gcatcatgga cctggaaaat gataaactgc

3301 agctggaaga aaagcttaag aagaaggagt ttgacattaa tcagcagaac agtaagattg

3361 aggatgagca ggtgctggcc cttcaactac agaagaaact gaaggaaaac caggcacgca

3421 tcgaggagct ggaggaggag ctggaggccg agcgcaccgc cagggctaag gtggagaagc

3481 tgcgctcaga cctgtctcgg gagctggagg agatcagcga gcggctggaa gaggccggcg

3541 gggccacgtc cgtgcagatc gagatgaaca agaagcgcga ggccgagttc cagaagatgc

3601 ggcgggacct ggaggaggcc acgctgcagc acgaggccac tgccgcggcc ctgcgcaaga

3661 agcacgccga cagcgtggcc gagctgggcg agcagatcga caacctgcag cgggtgaagc

3721 agaagctgga gaaggagaag agcgagttca agctggagct ggatgacgtc acctccaaca

3781 tggagcagat catcaaggcc aaggcaaacc tggagaaagt gtctcggacg ctggaggacc

3841 aggccaatga gtaccgcgtg aagctagaag aggcccaacg ctccctcaat gatttcacca

3901 cccagcgagc caagctgcag accgagaatg gagagttggc ccggcagcta gaggaaaagg

3961 aggcgctaat ctcgcagctg acccggggga agctctctta tacccagcaa atggaggacc

4021 tcaaaaggca gctggaggag gagggcaagg cgaagaacgc cctggcccat gcactgcagt

4081 cggcccggca tgactgcgac ctgctgcggg agcagtacga ggaggagaca gaggccaagg

4141 ccgagctgca gcgcgtcctg tccaaggcca actcggaggt ggcccagtgg aggaccaagt

4201 atgagacgga cgccattcag cggactgagg agctcgaaga ggccaaaaag aagctggccc

4261 agcggctgca ggatgccgag gaggccgtgg aggctgttaa tgccaagtgc tcctcactgg

4321 agaagaccaa gcaccggcta cagaatgaga tagaggactt gatggtggac gtagagcgct

4381 ccaatgctgc tgctgcagcc ctggacaaga agcagagaaa ctttgacaag atcctggccg

4441 agtggaagca gaagtatgag gagtcgcagt ctgagctgga gtcctcacag aaggaggctc

4501 gctccctcag cacagagctc ttcaagctca agaacgccta cgaggagtcc ctggagcacc

4561 tagagacctt caagcgggag aacaagaacc ttcaggagga aatctcggac cttactgagc

4621 agctaggaga aggaggaaag aatgtgcatg agctggagaa ggtccgcaaa cagctggagg

4681 tggagaagct ggagctgcag tcagccctgg aggaggcaga ggcctccctg gagcacgagg

4741 agggcaagat cctccgggcc cagctagagt tcaaccagat caaggcagag atcgagcgga

4801 agctggcaga gaaggacgag gagatggaac aggccaagcg caaccaccag cgggtggtgg

4861 actcgctgca gacctccctg gatgcagaga cacgcagccg caacgaggtc ctgagggtga 4921 agaagaagat ggaaggagac ctcaatgaga tggagatcca gctcagccac gccaaccgca

4981 tggctgccga ggcccagaag caagtcaaga gcctccagag cttgctgaag gacacccaga

5041 tccagctgga cgatgcggtc cgtgccaacg acgacctgaa ggagaacatc gccatcgtgg

5101 agcggcgcaa caacctgctg caggctgagc tggaggagct gcgtgccgtg gtggagcaga

5161 cagagcggtc ccggaagctg gcggagcagg agctgattga gaccagcgag cgggtgcagc

5221 tgctgcattc ccagaacacc agcctcatca accagaagaa gaagatggag tcggatctga

5281 cccagctcca gtcggaagtg gaggaggcag tgcaggagtg cagaaacgcc gaggagaagg

5341 ccaagaaggc catcacggat gccgccatga tggcagagga gctgaagaag gagcaggaca

5401 ccagcgccca cctggagcgc atgaagaaga acatggagca gaccattaag gacctgcagc

5461 accggctgga cgaggccgag cagatcgccc tcaagggagg caagaagcag ctgcagaagc

5521 tggaagcgcg ggtgcgggag ctggagggtg agctggaggc cgagcagaag cgcaacgcag

5581 agtcggtgaa gggcatgagg aagagcgagc ggcgcatcaa ggagctcacc taccagacag

5641 aggaagacaa aaagaacctg ctgcggctac aggacctggt ggacaagctg caactgaagg

5701 tcaaggccta caagcgccag gccgaggagg cggaggagca agccaacacc aacctgtcca

5761 agttccgcaa ggtgcagcat gagctggatg aggcagagga gcgggcggac atcgctgagt

5821 cccaggtcaa caagcttcga gccaagagcc gtgacattgg tgccaagcaa aaaatgcacg

5881 atgaggagtg acactgcctc gggaacctca ctcttgccaa cctgtaataa atatgagtgc

5941 c

By“MLC2A polypeptide” or“human MLSC2A (hMLC2A) polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No.

NP 067046.1 and having calcium binding activity. The amino acid sequence provided at NCBI Accession No. NP_067046.l is shown below:

1 MASRKAGTRG KVAATKQAQR GSSNVFSMFE QAQIQEFKEA FSCIDQNRDG IICKADLRET 61 YSQLGKVSVP EEELDAMLQE GKGPINFTVF LTLFGEKLNG TDPEEAILSA FRMFDPSGKG 121 VWKDEFKQL LLTQADKFSP AEVEQMFALT PMDLAGNIDY KSLCYIITHG DEKEE

By“MLC2A polynucleotide” is meant a polynucleotide encoding a MLC2A polypeptide or fragment thereof. An exemplary MLC2A polynucleotide sequence is provided at NCBI Ref: NM_02l223.2. The sequence provided at NCBI Ref: NM_02l223.2 is reproduced below:

1 tctgcagaga gaatggccag caggaaggcg gggacccggg gcaaggtggc agccaccaag 61 caggcccaac gtggttcttc caacgtcttt tccatgtttg aacaagccca gatacaggag 121 ttcaaagaag ccttcagctg tatcgaccag aatcgtgatg gcatcatctg caaggcagac 181 ctgagggaga cctactccca gctggggaag gtgagtgtcc cagaggagga gctggacgcc 241 atgctgcaag agggcaaggg ccccatcaac ttcaccgtct tcctcacgct ctttggggag 301 aagctcaatg ggacagaccc cgaggaagcc atcctgagtg ccttccgcat gtttgacccc 361 agcggcaaag gggtggtgaa caaggatgag ttcaagcagc ttctcctgac ccaggcagac 421 aagttctctc cagctgaggt ggagcagatg ttcgccctga cacccatgga cctggcgggg 481 aacatcgact acaagtcact gtgctacatc atcacccatg gagacgagaa agaggaatga 541 ggggcagggc caggcccacg gggggg ^cacc tcaataaact ctgttgcaaa attggaaaaa 601 aaaaaaaaaa aaaaaaaaa By“MUC2 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_002448.3 and having and having a biological activity of a MUC2 polypeptide. Exemplary biological activities of a MUC2 polypeptide include polymerization into a gel and coating of epithelia of the intestines and other mucus membrane-containing organs. The amino acid sequence provided at NCBI

Accession No. NP_0024 8.3 is shown below:

1 MGLPLARLAA VCLALSLAGG SELQTEGRTR NHGHNVCSTW GNFHYKTFDG DVFRFPGPCD 61 YNFASDCRGS YKEFAVHLKR GPGQAEAPAG VESILLTIKD DTIYLTRHLA VLNGAWSTP 121 HYSPGLLIEK SDAYTKVYSR AGLTLMWNRE DALMLELDTK FRNHTCGLCG DYNGLQSYSE 181 FLSDGVLFSP LEFGNMQKIN QPDWCEDPE EEVAPASCSE HRAECERLLT AEAFADCQDL 241 VPLEPYLRAC QQDRCRCPGG DTCVCSTVAE FSRQCSHAGG RPGNWRTATL CPKTCPGNLV 301 YLESGSPCMD TCSHLEVSSL CEEHRMDGCF CPEGTVYDDI GDSGCVPVSQ CHCRLHGHLY 361 TPGQEITNDC EQCVCNAGRW VCKDLPCPGT CALEGGSHIT TFDGKTYTFH GDCYYVLAKG 421 DHNDSYALLG ELAPCGSTDK QTCLKTWLL ADKKKNVWF KSDGSVLLNE LQVNLPHVTA 481 SFSVFRPSSY HIMVSMAIGV RLQVQLAPVM QLFVTLDQAS QGQVQGLCGN FNGLEGDDFK 541 TASGLVEATG AGFANTWKAQ STCHDKLDWL DDPCSLNIES ANYAEHWCSL LKKTETPFGR 601 CHSAVDPAEY YKRCKYDTCN CQNNEDCLCA ALSSYARACT AKGVMLWGWR EHVCNKDVGS 661 CPNSQVFLYN LTTCQQTCRS LSEADSHCLE GFAPVDGCGC PDHTFLDEKG RCVPLAKCSC 721 YHRGLYLEAG DVWRQEERC VCRDGRLHCR QIRLIGQSCT APKIHMDCSN LTALATSKPR 781 ALSCQTLAAG YYHTECVSGC VCPDGLMDDG RGGCWEKEC PCVHNNDLYS SGAKIKVDCN 841 TCTCKRGRWV CTQAVCHGTC SIYGSGHYIT FDGKYYDFDG HCSYVAVQDY CGQNSSLGSF 901 SIITENVPCG TTGVTCSKAI KIFMGRTELK LEDKHRWIQ RDEGHHVAYT TREVGQYLW 961 ESSTGIIVIW DKRTTVFIKL APSYKGTVCG LCGNFDHRSN NDFTTRDHMV VSSELDFGNS 1021 WKEAPTCPDV STNPEPCSLN PHRRSWAEKQ CSILKSSVFS ICHSKVDPKP FYEACVHDSC 1081 SCDTGGDCEC FCSAVASYAQ ECTKEGACVF WRTPDLCPIF CDYYNPPHEC EWHYEPCGNR 1141 SFETCRTING IHSNISVSYL EGCYPRCPKD RPIYEEDLKK CVTADKCGCY VEDTHYPPGA 1201 SVPTEETCKS CVCTNSSQW CRPEEGKILN QTQDGAFCYW EICGPNGTVE KHFNICSITT 1261 RPSTLTTFTT ITLPTTPTTF TTTTTTTTPT SSTVLSTTPK LCCLWSDWIN EDHPSSGSDD 1321 GDRETFDGVC GAPEDIECRS VKDPHLSLEQ LGQKVQCDVS VGFICKNEDQ FGNGPFGLCY 1381 DYKIRWCCW PMDKCITTPS PPTTTPSPPP TSTTTLPPTT TPSPPTTTTT TPPPTTTPSP 1441 PITTTTTPPP TTTPSPPIST TTTPPPTTTP SPPTTTPSPP TTTPSPPTTT TTTPPPTTTP 1501 SPPTTTPITP PASTTTLPPT TTPSPPTTTT TTPPPTTTPS PPTTTPITPP TSTTTLPPTT 1561 TPSPPPTTTT TPPPTTTPSP PTTTTPSPPT ITTTTPPPTT TPSPPTTTTT TPPPTTTPSP 1621 PTTTPITPPT STTTLPPTTT PSPPPTTTTT PPPTTTPSPP TTTTPSPPIT TTTTPPPTTT 1681 PSSPITTTPS PPTTTMTTPS PTTTPSSPIT TTTTPSSTTT PSPPPTTMTT PSPTTTPSPP 1741 TTTMTTLPPT TTSSPLTTTP LPPSITPPTF SPFSTTTPTT PCVPLCNWTG WLDSGKPNFH 1801 KPGGDTELIG DVCGPGWAAN ISCRATMYPD VPIGQLGQTV VCDVSVGLIC KNEDQKPGGV 1861 IPMAFCLNYE INVQCCECVT QPTTMTTTTT ENPTPPTTTP ITTTTTVTPT PTPTGTQTPT 1921 TTPITTTTTV TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP 1981 TPTGTQTPTT TPITTTTTVT PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT 2041 TTTTVTPTPT PTGTQTPTTT PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT 2101 QTPTTTPITT TTTVTPTPTP TGTQTPTTTP _ITTTTTVT PT PTPTGTQTPT TTPITTTTTV 2161 TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT 2221 TPITTTTTVT PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT 2281 PTGTQTPTTT PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT 2341 TTTVTPTPTP TGTQTPTTTP ITTTTTVTPT PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ 2401 TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT TPITTTTTVT

2461 PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT

2521 PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT TTTVTPTPTP

2581 TGTQTPTTTP ITTTTTVTPT PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ TPTTTPITTT

2641 TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT TPITTTTTVT PTPTPTGTQT

2701 PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT PITTTTTVTP

2761 TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT TTTVTPTPTP TGTQTPTTTP

2821 _ITTTTTVT PT PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT

2881 GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT TPITTTTTVT PTPTPTGTQT PTTTPITTTT

2941 TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT PITTTTTVTP TPTPTGTQTP

3001 TTTPITTTTT VTPTPTPTGT QTPTTTPITT TTTVTPTPTP TGTQTPTTTP ITTTTTVTPT

3061 PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT GTQTPTTTPI

3121 TTTTTVTPTP TPTGTQTPTT TPITTTTTVT PTPTPTGTQT PTTTPITTTT TVTPTPTPTG

3181 TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT PITTTTTVTP TPTPTGTQTP TTTPITTTTT

3241 VTPTPTPTGT QTPTTTPITT TTTVTPTPTP TGTQTPTTTP ITTTTTVTPT PTPTGTQTPT

3301 TTPITTTTTV TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP

3361 TPTGTQTPTT TPITTTTTVT PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT

3421 TTTTVTPTPT PTGTQTPTTT PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT

3481 QTPTTTPITT TTTVTPTPTP TGTQTPTTTP _ITTTTTVT PT PTPTGTQTPT TTPITTTTTV

3541 TPTPTPTGTQ TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT

3601 TPITTTTTVT PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT

3661 PTGTQTPTTT PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT

3721 TTTVTPTPTP TGTQTPTTTP ITTTTTVTPT PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ

3781 TPTTTPITTT TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT TPITTTTTVT

3841 PTPTPTGTQT PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT

3901 PITTTTTVTP TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT TTTVTPTPTP

3961 TGTQTPTTTP ITTTTTVTPT PTPTGTQTPT TTPITTTTTV TPTPTPTGTQ TPTTTPITTT

4021 TTVTPTPTPT GTQTPTTTPI TTTTTVTPTP TPTGTQTPTT TPITTTTTVT PTPTPTGTQT

4081 PTTTPITTTT TVTPTPTPTG TQTPTTTPIT TTTTVTPTPT PTGTQTPTTT PITTTTTVTP

4141 TPTPTGTQTP TTTPITTTTT VTPTPTPTGT QTPTTTPITT TTTVTPTPTP TGTQTGPPTH

4201 TSTAPIAELT TSNPPPESST PQTSRSTSSP LTESTTLLST LPPAIEMTST APPSTPTAPT

4261 TTSGGHTLSP PPSTTTSPPG TPTRGTTTGS SSAPTPSTVQ TTTTSAWTPT PTPLSTPSII

4321 RTTGLRPYPS SVLICCVLND TYYAPGEEVY NGTYGDTCYF VNCSLSCTLE FYNWSCPSTP

4381 SPTPTPSKST PTPSKPSSTP SKPTPGTKPP ECPDFDPPRQ ENETWWLCDC FMATCKYNNT

4441 VEIVKVECEP PPMPTCSNGL QPVRVEDPDG CCWHWECDCY CTGWGDPHYV TFDGLYYSYQ

4501 GNCTYVLVEE ISPSVDNFGV YIDNYHCDPN DKVSCPRTLI VRHETQEVLI KTVHMMPMQV

4561 QVQVNRQAVA LPYKKYGLEV YQSGINYWD IPELGVLVSY NGLSFSVRLP YHRFGNNTKG

4621 QCGTCTNTTS DDCILPSGEI VSNCEAAADQ WLVNDPSKPH CPHSSSTTKR PAVTVPGGGK

4681 TTPHKDCTPS PLCQLIKDSL FAQCHALVPP QHYYDACVFD SCFMPGSSLE CASLQAYAAL

4741 CAQQNICLDW RNHTHGACLV ECPSHREYQA CGPAEEPTCK SSSSQQNNTV LVEGCFCPEG

4801 TMNYAPGFDV CVKTCGCVGP DNVPREFGEH FEFDCKNCVC LEGGSGIICQ PKRCSQKPVT

4861 HCVEDGTYLA TEVNPADTCC NITVCKCNTS LCKEKPSVCP LGFEVKSKMV PGRCCPFYWC

4921 ESKGVCVHGN AEYQPGSPVY SSKCQDCVCT DKVDNNTLLN VIACTHVPCN TSCSPGFELM

4981 EAPGECCKKC EQTHCIIKRP DNQHVILKPG DFKSDPKNNC TFFSCVKIHN QLISSVSNIT

5041 CPNFDASICI PGSITFMPNG CCKTCTPRNE TRVPCSTVPV TTEVSYAGCT KTVLMNHCSG

5101 SCGTFVMYSA KAQALDHSCS CCKEEKTSQR EWLSCPNGG SLTHTYTHIE SCQCQDTVCG

5161 LPTGTSRRAR RSPRHLGSG

By“MUC2 polynucleotide” is meant a polynucleotide encoding a MUC2 polypeptide or fragment thereof. An exemplary MUC2 polynucleotide sequence is provided at NCBI Ref: NM_002457.3. The sequence provided at NCBI Ref: NM_002457.3 is reproduced below:

1 caacccacac cgcccctgcc agccaccatg gggctgccac tagcccgcct ggcggctgtg 61 tgcctggccc tgtctttggc agggggetcg gagctccaga cagagggcag aacccgaaac 121 cacggccaca acgtctgcag cacctggggc aacttccact acaagacctt cgacggggac 181 gtcttccgct tccccggccc ctgcgactac aacttcgcct ccgactgccg aggctcctac 241 aaggaatttg ctgtgcacct gaagcggggt ccgggccagg ctgaggcccc cgccggggtg 301 gagtccatcc tgctgaccat caaggatgac accatctacc tcacccgcca cctggctgtg 361 cttaacgggg ccgtggtcag caccccgcac tacagccccg ggctgctcat tgagaagagc 421 gatgcctaca ccaaagtcta ctcccgcgcc ggcctcaccc tcatgtggaa ccgggaggat 481 gcactcatgc tggagctgga cactaagttc cggaaccaca cctgtggcct ctgcggggac 541 tacaacggcc tgcagagcta ttcagaattc ctctctgacg gcgtgctctt cagtcccctg 601 gagtttggga acatgcagaa gatcaaccag cccgatgtgg tgtgtgagga tcccgaggag 661 gaggtggccc ccgcatcctg ctccgagcac cgcgccgagt gtgagaggct gctgaccgcc 721 gaggccttcg cggactgtca ggacctggtg ccgctggagc cgtatctgcg cgcctgccag 781 caggaccgct gccggtgccc gggcggtgac acctgcgtct gcagcaccgt ggccgagttc 841 tcccgccagt gctcccacgc cggcggccgg cccgggaact ggaggaccgc cacgctctgc 901 cccaagacct gccccgggaa cctggtgtac ctggagagcg gctcgccctg catggacacc 961 tgctcacacc tggaggtgag cagcctgtgc gaggagcacc gcatggacgg ctgtttctgc 1021 ccagaaggca ccgtatatga cgacatcggg gacagtggct gcgttcctgt gagccagtgc 1081 cactgcaggc tgcacggaca cctgtacaca ccgggccagg agatcaccaa tgactgcgag 1141 cagtgtgtct gtaacgctgg ccgctgggtg tgcaaagacc tgccctgccc cggcacctgt 1201 gccctggaag gcggctccca catcaccacc ttcgatggga agacgtacac cttccacggg 1261 gactgctact atgtcctggc caagggtgac cacaacgatt cctacgctct cctgggcgag 1321 ctggccccct gtggctccac agacaagcag acctgcctga agacggtggt gctgctggct 1381 gacaagaaga agaatgtggt ggtcttcaag tccgatggca gtgtactgct caacgagctg 1441 caggtgaacc tgccccacgt gaccgcgagc ttctctgtct tccgcccgtc ttcctaccac 1501 atcatggtga gcatggccat tggcgtccgg ctgcaggtgc agctggcccc agtcatgcaa 1561 ctctttgtga cactggacca ggcctcccag gggcaggtgc agggcctctg cgggaacttc 1621 aacggcctgg aaggtgacga cttcaagacg gccagcgggc tggtggaggc cacgggggcc 1681 ggctttgcca acacctggaa ggcacagtca acctgccatg acaagctgga ctggttggac 1741 gatccctgct ccctgaacat cgagagcgcc aactacgccg agcactggtg ctccctcctg 1801 aagaagacag agaccccctt tggcaggtgc cactcggctg tggaccctgc tgagtattac 1861 aagaggtgca aatatgacac gtgtaactgt cagaacaatg aggactgcct gtgcgccgcc 1921 ctgtcctcct acgcgcgcgc ctgcaccgcc aagggcgtca tgctgtgggg ctggcgggag 1981 catgtctgca acaaggatgt gggctcctgc cccaactcgc aggtcttcct gtacaacctg 2041 accacctgcc agcagacctg ccgctccctc tccgaggccg acagccactg tctcgagggc 2101 tttgcgcctg tggacggctg cggctgccct gaccacacct tcctggacga gaagggccgc 2161 tgcgtacccc tggccaagtg ctcctgttac caccgcggtc tctacctgga ggcgggggac 2221 gtggtcgtca ggcaggaaga acgatgtgtg tgccgggatg ggcggctgca ctgtaggcag 2281 atccggctga tcggccagag ctgcacggcc ccaaagatcc acatggactg cagcaacctg 2341 actgcactgg ccacctcgaa gccccgagcc ctcagctgcc agacgctggc cgccggctat 2401 taccacacag agtgtgtcag tggctgtgtg tgccccgacg ggctgatgga tgacggccgg 2461 ggtggctgcg tggtggagaa ggaatgccct tgcgtccata acaacgacct gtattcttcc 2521 ggcgccaaga tcaaggtgga ctgcaatacc tgcacctgca agagaggacg ctgggtgtgc 2581 acccaggctg tgtgccatgg cacctgctcc atttacggga gtggccacta catcaccttt 2641 gacgggaagt actacgactt tgacggacac tgctcctacg tggctgttca ggactactgc 2701 ggccagaact cctcactggg ctcattcagc atcatcaccg agaacgtccc ctgtggcact 2761 acgggcgtca cctgctccaa ggccatcaag atcttcatgg ggaggacgga gctgaagttg 2821 gaagacaagc accgtgtggt gatccagcgt gatgagggtc accacgtggc ctacaccacg 2881 cgggaggtgg gccagtacct ggtggtggag tccagcacgg gcatcatcgt catctgggac

2941 aagaggacca ccgtgttcat caagctggct ccctcctaca agggcaccgt gtgtggcctg

3001 tgtgggaact ttgaccaccg ctccaacaac gacttcacca cgcgggacca catggtggtg

3061 agcagcgagc tggacttcgg gaacagctgg aaggaggccc ccacctgccc agatgtgagc

3121 accaaccccg agccctgcag cctgaacccg caccgccgct cctgggccga gaagcagtgc

3181 agcatcctca aaagcagcgt gttcagcatc tgccacagca aggtggaccc caagcccttc

3241 tacgaggcct gtgtgcacga ctcgtgctcc tgtgacacgg gtggggactg tgagtgcttc

3301 tgctctgccg tggcctccta cgcccaggag tgtaccaaag agggggcctg cgtgttctgg

3361 aggacgccgg acctgtgccc catattctgc gactactaca accctccgca tgagtgtgag

3421 tggcactatg agccatgtgg gaaccggagc ttcgagacct gcaggaccat caatggcatc

3481 cactccaaca tctccgtgtc ctacctggag ggctgctacc cccggtgccc caaggacagg

3541 cccatctatg aggaggatct gaagaagtgt gtcactgcag acaagtgtgg ctgctatgtc

3601 gaggacaccc actacccacc tggagcatcg gttcccaccg aggagacctg caagtcctgc

3661 gtgtgtacca actcctccca agtcgtctgc aggccggagg aaggaaagat tcttaaccag

3721 acccaggatg gcgccttctg ctactgggag atctgtggcc ccaacgggac ggtggagaag

3781 cacttcaaca tctgttccat tacgacacgc ccgtccaccc tgaccacctt caccaccatc

3841 accctcccca ccacccccac caccttcacc actaccacca ccaccaccac cccgacctcc

3901 agcacagttt tatcaacaac tccgaagctg tgctgcctct ggtctgactg gatcaatgag

3961 gaccacccca gcagtggcag cgacgacggt gaccgagaaa catttgatgg ggtctgcggg

4021 gcccctgagg acatcgagtg caggtcggtc aaggatcccc acctcagctt ggagcagcta

4081 ggccagaagg tgcagtgtga tgtctctgtt gggttcattt gcaagaatga agaccagttt

4141 ggaaatggac catttggact gtgttacgac tacaagatac gtgtcaattg ttgctggccc

4201 atggataagt gtatcaccac tcccagccct ccaactacca ctcccagccc tccaccaacc

4261 agcacgacca cccttccacc aaccaccacc cccagccctc caaccaccac cacaaccacc

4321 cctccaccaa ccaccacccc cagccctcca ataaccacca cgaccacccc tccaccaacc

4381 accactccca gccctccaat aagcaccaca accacccctc caccaaccac cactcccagc

4441 cctccaacca ccactcccag ccctccaacc accactccca gccctccaac aaccaccaca

4501 accacccctc caccaaccac cactcccagc cctccaacga ctacgcccat cactccacca

4561 gccagcacta ccacccttcc accaaccacc actcccagcc ctccaacaac caccacaacc

4621 acccctccac caaccaccac tcccagtcct ccaacgacta cgcccatcac tccaccaacc

4681 agcactacta cccttccacc aaccaccact cccagccctc caccaaccac cacaaccacc

4741 cctccaccaa ccaccactcc cagccctcca acaaccacca ctcccagtcc tccaacaatc

4801 accacaacca cccctccacc aaccaccact cccagccctc caacaacgac cacaaccacc

4861 cctccaccaa ccaccactcc cagccctcca acgactacac ccatcactcc accaaccagc

4921 actaccaccc ttccaccaac caccactccc agccctccac caaccaccac aaccacccct

4981 ccaccaacca ccactcccag ccctccaaca accaccactc ccagccctcc aataaccacc

5041 acaaccaccc ctccaccaac caccactccc agctctccaa taaccaccac tcccagccct

5101 ccaacaacca ccatgaccac cccttcacca accaccaccc ccagctctcc aataaccacc

5161 acaaccaccc cttcctcaac taccactccc agccctccac caaccaccat gaccacccct

5221 tcaccaacca ccactcccag ccctccaaca accaccatga ccacccttcc accaaccacc

5281 acttccagcc ctctaacaac tactcctcta cctccatcaa taactcctcc tacattttca

5341 ccattctcaa cgacaacccc tactacccca tgcgtgcctc tctgcaattg gactggctgg

5401 ctggattctg gaaaacccaa ctttcacaaa ccaggtggag acacagaatt gattggagac

5461 gtctgtggac caggctgggc agctaacatc tcttgcagag ccaccatgta tcctgatgtt

5521 cccattggac agcttggaca aacagtggtg tgtgatgtct ctgtggggct gatatgcaaa

5581 aatgaagacc aaaagccagg tggggtcatc cctatggcct tctgcctcaa ctacgagatc

5641 aacgttcagt gctgtgagtg tgtcacccaa cccaccacca tgacaaccac caccacagag

5701 aacccaactc cgccaaccac gacacccatc accaccacca ctacggtgac cccaacccca

5761 acacccaccg gcacacagac cccaaccacg acacccatca ccaccaccac tacggtgacc

5821 ccaaccccaa cacccaccgg cacacagacc ccaaccacga cacccatcac caccaccact

5881 acggtgaccc caaccccaac acccaccggc acacagaccc caaccacgac acccatcacc 5941 accaccacta cggtgacccc aaccccaaca cccaccggca cacagacccc aaccacgaca

6001 cccatcacca ccaccactac ggtgacccca accccaacac ccaccggcac acagacccca

6061 accacgacac ccatcaccac caccactacg gtgaccccaa ccccaacacc caccggcaca

6121 cagaccccaa ccacgacacc catcaccacc accactacgg tgaccccaac cccaacaccc

6181 accggcacac agaccccaac cacgacaccc atcaccacca ccactacggt gaccccaacc

6241 ccaacaccca ccggcacaca gaccccaacc acgacaccca tcaccaccac cactacggtg

6301 accccaaccc caacacccac cggcacacag accccaacca cgacacccat caccaccacc

6361 actacggtga ccccaacccc aacacccacc ggcacacaga ccccaaccac gacacccatc

6421 accaccacca ctacggtgac cccaacccca acacccaccg gcacacagac cccaaccacg

6481 acacccatca ccaccaccac tacggtgacc ccaaccccaa cacccaccgg cacacagacc

6541 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc

6601 acacagaccc caaccacgac acccatcacc accaccacta cggtgacccc aaccccaaca

6661 cccaccggca cacagacccc aaccacgaca cccatcacca ccaccactac ggtgacccca

6721 accccaacac ccaccggcac acagacccca accacgacac ccatcaccac caccactacg

6781 gtgaccccaa ccccaacacc caccggcaca cagaccccaa ccacgacacc catcaccacc

6841 accactacgg tgaccccaac cccaacaccc accggcacac agaccccaac cacgacaccc

6901 atcaccacca ccactacggt gaccccaacc ccaacaccca ccggcacaca gaccccaacc

6961 acgacaccca tcaccaccac cactacggtg accccaaccc caacacccac cggcacacag

7021 accccaacca cgacacccat caccaccacc actacggtga ccccaacccc aacacccacc

7081 ggcacacaga ccccaaccac gacacccatc accaccacca ctacggtgac cccaacccca

7141 acacccaccg gcacacagac cccaaccacg acacccatca ccaccaccac tacggtgacc

7201 ccaaccccaa cacccaccgg cacacagacc ccaaccacga cacccatcac caccaccact

7261 acggtgaccc caaccccaac acccaccggc acacagaccc caaccacgac acccatcacc

7321 accaccacta cggtgacccc aaccccaaca cccaccggca cacagacccc aaccacgaca

7381 cccatcacca ccaccactac ggtgacccca accccaacac ccaccggcac acagacccca

7441 accacgacac ccatcaccac caccactacg gtgaccccaa ccccaacacc caccggcaca

7501 cagaccccaa ccacgacacc catcaccacc accactacgg tgaccccaac cccaacaccc

7561 accggcacac agaccccaac cacgacaccc atcaccacca ccactacggt gaccccaacc

7621 ccaacaccca ccggcacaca gaccccaacc acgacaccca tcaccaccac cactacggtg

7681 accccaaccc caacacccac cggcacacag accccaacca cgacacccat caccaccacc

7741 actacggtga ccccaacccc aacacccacc ggcacacaga ccccaaccac gacacccatc

7801 accaccacca ctacggtgac cccaacccca acacccaccg gcacacagac cccaaccacg

7861 acacccatca ccaccaccac tacggtgacc ccaaccccaa cacccaccgg cacacagacc

7921 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc

7981 acacagaccc caaccacgac acccatcacc accaccacta cggtgacccc aaccccaaca

8041 cccaccggca cacagacccc aaccacgaca cccatcacca ccaccactac ggtgacccca

8101 accccaacac ccaccggcac acagacccca accacgacac ccatcaccac caccactacg

8161 gtgaccccaa ccccaacacc caccggcaca cagaccccaa ccacgacacc catcaccacc

8221 accactacgg tgaccccaac cccaacaccc accggcacac agaccccaac cacgacaccc

8281 atcaccacca ccactacggt gaccccaacc ccaacaccca ccggcacaca gaccccaacc

8341 acgacaccca tcaccaccac cactacggtg accccaaccc caacacccac cggcacacag

8401 accccaacca cgacacccat caccaccacc actacggtga ccccaacccc aacacccacc

8461 ggcacacaga ccccaaccac gacacccatc accaccacca ctacggtgac cccaacccca

8521 acacccaccg gcacacagac cccaaccacg acacccatca ccaccaccac tacggtgacc

8581 ccaaccccaa cacccaccgg cacacagacc ccaaccacga cacccatcac caccaccact

8641 acggtgaccc caaccccaac acccaccggc acacagaccc caaccacgac acccatcacc

8701 accaccacta cggtgacccc aaccccaaca cccaccggca cacagacccc aaccacgaca

8761 cccatcacca ccaccactac ggtgacccca accccaacac ccaccggcac acagacccca

8821 accacgacac ccatcaccac caccactacg gtgaccccaa ccccaacacc caccggcaca

8881 cagaccccaa ccacgacacc catcaccacc accactacgg tgaccccaac cccaacaccc

8941 accggcacac agaccccaac cacgacaccc atcaccacca ccactacggt gaccccaacc 9001 ccaacaccca ccggcacaca gaccccaacc acgacaccca tcaccaccac cactacggtg

9061 accccaaccc caacacccac cggcacacag accccaacca cgacacccat caccaccacc

9121 actacggtga ccccaacccc aacacccacc ggcacacaga ccccaaccac gacacccatc

9181 accaccacca ctacggtgac cccaacccca acacccaccg gcacacagac cccaaccacg

9241 acacccatca ccaccaccac tacggtgacc ccaaccccaa cacccaccgg cacacagacc

9301 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc

9361 acacagaccc caaccacgac acccatcacc accaccacta cggtgacccc aaccccaaca

9421 cccaccggca cacagacccc aaccacgaca cccatcacca ccaccactac ggtgacccca

9481 accccaacac ccaccggcac acagacccca accacgacac ccatcaccac caccactacg

9541 gtgaccccaa ccccaacacc caccggcaca cagaccccaa ccacgacacc catcaccacc

9601 accactacgg tgaccccaac cccaacaccc accggcacac agaccccaac cacgacaccc

9661 atcaccacca ccactacggt gaccccaacc ccaacaccca ccggcacaca gaccccaacc

9721 acgacaccca tcaccaccac cactacggtg accccaaccc caacacccac cggcacacag

9781 accccaacca cgacacccat caccaccacc actacggtga ccccaacccc aacacccacc

9841 ggcacacaga ccccaaccac gacacccatc accaccacca ctacggtgac cccaacccca

9901 acacccaccg gcacacagac cccaaccacg acacccatca ccaccaccac tacggtgacc

9961 ccaaccccaa cacccaccgg cacacagacc ccaaccacga cacccatcac caccaccact

10021 acggtgaccc caaccccaac acccaccggc acacagaccc caaccacgac acccatcacc

10081 accaccacta cggtgacccc aaccccaaca cccaccggca cacagacccc aaccacgaca

10141 cccatcacca ccaccactac ggtgacccca accccaacac ccaccggcac acagacccca

10201 accacgacac ccatcaccac caccactacg gtgaccccaa ccccaacacc caccggcaca

10261 cagaccccaa ccacgacacc catcaccacc accactacgg tgaccccaac cccaacaccc

10321 accggcacac agaccccaac cacgacaccc atcaccacca ccactacggt gaccccaacc

10381 ccaacaccca ccggcacaca gaccccaacc acgacaccca tcaccaccac cactacggtg

10441 accccaaccc caacacccac cggcacacag accccaacca cgacacccat caccaccacc

10501 actacggtga ccccaacccc aacacccacc ggcacacaga ccccaaccac gacacccatc

10561 accaccacca ctacggtgac cccaacccca acacccaccg gcacacagac cccaaccacg

10621 acacccatca ccaccaccac tacggtgacc ccaaccccaa cacccaccgg cacacagacc

10681 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc

10741 acacagaccc caaccacgac acccatcacc accaccacta cggtgacccc aaccccaaca

10801 cccaccggca cacagacccc aaccacgaca cccatcacca ccaccactac ggtgacccca

10861 accccaacac ccaccggcac acagacccca accacgacac ccatcaccac caccactacg

10921 gtgaccccaa ccccaacacc caccggcaca cagaccccaa ccacgacacc catcaccacc

10981 accactacgg tgaccccaac cccaacaccc accggcacac agaccccaac cacgacaccc

11041 atcaccacca ccactacggt gaccccaacc ccaacaccca ccggcacaca gaccccaacc

11101 acgacaccca tcaccaccac cactacggtg accccaaccc caacacccac cggcacacag

11161 accccaacca cgacacccat caccaccacc actacggtga ccccaacccc aacacccacc

11221 ggcacacaga ccccaaccac gacacccatc accaccacca ctacggtgac cccaacccca

11281 acacccaccg gcacacagac cccaaccacg acacccatca ccaccaccac tacggtgacc

11341 ccaaccccaa cacccaccgg cacacagacc ccaaccacga cacccatcac caccaccact

11401 acggtgaccc caaccccaac acccaccggc acacagaccc caaccacgac acccatcacc

11461 accaccacta cggtgacccc aaccccaaca cccaccggca cacagacccc aaccacgaca

11521 cccatcacca ccaccactac ggtgacccca accccaacac ccaccggcac acagacccca

11581 accacgacac ccatcaccac caccactacg gtgaccccaa ccccaacacc caccggcaca

11641 cagaccccaa ccacgacacc catcaccacc accactacgg tgaccccaac cccaacaccc

11701 accggcacac agaccccaac cacgacaccc atcaccacca ccactacggt gaccccaacc

11761 ccaacaccca ccggcacaca gaccccaacc acgacaccca tcaccaccac cactacggtg

11821 accccaaccc caacacccac cggcacacag accccaacca cgacacccat caccaccacc

11881 actacggtga ccccaacccc aacacccacc ggcacacaga ccccaaccac gacacccatc

11941 accaccacca ctacggtgac cccaacccca acacccaccg gcacacagac cccaaccacg

12001 acacccatca ccaccaccac tacggtgacc ccaaccccaa cacccaccgg cacacagacc 12061 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc

12121 acacagaccc caaccacgac acccatcacc accaccacta cggtgacccc aaccccaaca

12181 cccaccggca cacagacccc aaccacgaca cccatcacca ccaccactac ggtgacccca

12241 accccaacac ccaccggcac acagacccca accacgacac ccatcaccac caccactacg

12301 gtgaccccaa ccccaacacc caccggcaca cagaccccaa ccacgacacc catcaccacc

12361 accactacgg tgaccccaac cccaacaccc accggcacac agaccccaac cacgacaccc

12421 atcaccacca ccactacggt gaccccaacc ccaacaccca ccggcacaca gaccccaacc

12481 acgacaccca tcaccaccac cactacggtg accccaaccc caacacccac cggcacacag

12541 accccaacca cgacacccat caccaccacc actacggtga ccccaacccc aacacccacc

12601 ggcacacaga ccgggccccc cacccacaca agcacagcac cgattgctga gttgaccaca

12661 tccaatcctc cgcctgagtc ctcaacccct cagacctctc ggtccacctc ttcccctctc

12721 acggagtcaa ccacccttct gagtacccta ccacctgcca ttgagatgac cagcacggcc

12781 ccaccctcca cacccacggc acccacgacc acgagcggag gccacacact gtctccaccg

12841 cccagcacca ccacgtcccc tccaggcacc cccactcgcg gtaccacgac tgggtcatct

12901 tcagccccca cccccagcac tgtgcagacg accaccacca gtgcctggac ccccacgccg

12961 accccactct ccacacccag catcatcagg accacaggcc tgaggcccta cccttcctct

13021 gtgcttatct gctgtgtcct gaacgacacc tactacgcac caggtgagga ggtgtacaac

13081 ggcacatacg gagacacctg ttatttcgtc aactgctcac tgagctgtac gttggagttc

13141 tataactggt cctgcccatc cacgccctcc ccaacaccca cgccctccaa gtcgacgccc

13201 acgccttcca agccatcgtc cacgccctcc aagccgacgc ccggcaccaa gccccccgag

13261 tgcccagact ttgatcctcc cagacaggag aacgagactt ggtggctgtg cgactgcttc

13321 atggccacgt gcaagtacaa caacacggtg gagatcgtga aggtggagtg tgagccgccg

13381 cccatgccca cctgctccaa cggcctccaa cccgtgcgcg tcgaggaccc cgacggctgc

13441 tgctggcact gggagtgcga ctgctactgc acgggctggg gcgacccgca ctatgtcacc

13501 ttcgacggac tctactacag ctaccagggc aactgcacct acgtgctggt ggaggagatc

13561 agcccctccg tggacaactt cggagtttac atcgacaact accactgcga tcccaacgac

13621 aaggtgtcct gcccccgcac cctcatcgtg cgccacgaga cccaggaggt gctgatcaag

13681 accgtgcata tgatgcccat gcaggtgcag gtgcaggtga acaggcaggc ggtggcactg

13741 ccctacaaga agtacgggct ggaggtgtac cagtctggca tcaactacgt ggtggacatc

13801 cccgagctgg gtgtcctcgt ctcctacaat ggcctgtcct tctccgtcag gctgccctac

13861 caccggtttg gcaacaacac caagggccag tgtggcacct gcaccaacac cacctccgac

13921 gactgcattc tgcccagcgg ggagatcgtc tccaactgtg aggctgcggc tgaccagtgg

13981 ctggtgaacg acccctccaa gccacactgc ccccacagca gctccacgac caagcgcccg

14041 gccgtcactg tgcccggggg cggtaaaacg accccacaca aggactgcac cccatctccc

14101 ctctgccagc tcatcaagga cagcctgttt gcccagtgcc acgcactggt gcccccgcag

14161 cactactacg atgcctgcgt gttcgacagc tgcttcatgc cgggctcgag cctggagtgc

14221 gccagtctgc aggcctacgc agccctctgt gcccagcaga acatctgcct cgactggcgg

14281 aaccacacgc atggggcctg cttggtggag tgcccatctc acagggagta ccaggcctgt

14341 ggccctgcag aagagcccac gtgcaaatcc agctcctccc agcagaacaa cacagtcctg

14401 gtggaaggct gcttctgtcc tgagggcacc atgaactacg ctcctggctt tgatgtctgc

14461 gtgaagacct gcggctgtgt gggacctgac aatgtgccca gagagtttgg ggagcacttc

14521 gagttcgact gcaagaactg tgtctgcctg gagggtggaa gtggcatcat ctgccaaccc

14581 aagaggtgca gccagaagcc cgttacccac tgcgtggaag acggcaccta cctcgccacg

14641 gaggtcaacc ctgccgacac ctgctgcaac attaccgtct gcaagtgcaa caccagcctg

14701 tgcaaagaga agccctccgt gtgcccgctg ggattcgaag tgaagagcaa gatggtgcct

14761 ggaaggtgct gtcctttcta ctggtgtgag tccaaggggg tgtgtgttca cgggaatgct

14821 gagtaccagc ccggttctcc agtttattcc tccaagtgcc aggactgcgt gtgcacggac

14881 aaggtggaca acaacaccct gctcaacgtc atcgcctgca cccacgtgcc ctgcaacacc

14941 tcctgcagcc ctggcttcga actcatggag gcccccgggg agtgctgtaa gaagtgtgaa

15001 cagacgcact gtatcatcaa acggcccgac aaccagcacg tcatcctgaa gcccggggac

15061 ttcaagagcg acccgaagaa caactgcaca ttcttcagct gcgtgaagat ccacaaccag 15121 ctcatctcgt ccgtctccaa catcacctgc cccaactttg atgccagcat ttgcatcccg

15181 ggctccatca cattcatgcc caatggatgc tgcaagacct gcacccctcg caatgagacc

15241 agggtgccct gctccaccgt ccccgtcacc acggaggttt cgtacgccgg ctgcaccaag

15301 accgtcctca tgaatcattg ctccgggtcc tgcgggacat ttgtcatgta ctcggccaag

15361 gcccaggccc tggaccacag ctgctcctgc tgcaaagagg agaaaaccag ccagcgtgag

15421 gtggtcctga gctgccccaa tggcggctcg ctgacacaca cctacaccca catcgagagc

15481 tgccagtgcc aggacaccgt ctgcgggctc cccaccggca cctcccgccg ggcccggcgc

15541 tcccctaggc atctggggag cgggtgagcg gggtgggcac agcccccttc actgccctcg

15601 acagctttac ctcccccgga ccctctgagc ctcctaagct cggcttcctc tcttcagata

15661 tttattgtct gagtctttgt tcagtccttg ctttccaata ataaactcag ggggacatgc

By“NKX2-5 polypeptide” or“human NKX2-5 (hNKX2-5) polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_004378.l (isoform 1), NP_001159647.1 (isoform 2), or NP_001159648.1 (isoform 3) and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_004378.l is shown below:

1 MFPSPALTPT PFSVKDILNL EQQQRSLAAA GELSARLEAT LAPSSCMLAA FKPEAYAGPE

61 AAAPGLPELR AELGRAPSPA KCASAFPAAP AFYPRAYSDP DPAKDPRAEK KELCALQKAV

121 ELEKTEADNA ERPRARRRRK PRVLFSQAQV YELERRFKQQ RYLSAPERDQ LASVLKLTST

181 QVKIWFQNRR YKCKRQRQDQ TLELVGLPPP PPPPARRIAV PVLVRDGKPC LGDSAPYAPA

241 YGVGLNPYGY NAYPAYPGYG GAACSPGYSC TAAYPAGPSP AQPATAAANN NFWFGVGDL

301 NAVQSPGIPQ SNSGVSTLHG IRAW

By“NKX2-5 polynucleotide” is meant a polynucleotide encoding a NKX2-5 polypeptide or fragment thereof. An exemplary NKX2-5 polynucleotide sequence is provided at NCBI Ref: NM_004387.3. The sequence provided at NCBI Ref: NM_004387.3 is reproduced below:

1 gctcctgtca tcgaggcccc tggcccaatg gcaggctgag tccccctcct ctggcctggt 61 cccgcctctc ctgccccttg tgctcagcgc tacctgctgc ccggacacat ccagagctgg 121 ccgacgggtg cgcgggcggg cggcggcacc atgcagggaa gctgccaggg gccgtgggca 181 gcgccgcttt ctgccgccca cctggcgctg tgagactggc gctgccacca tgttccccag 241 ccctgctctc acgcccacgc ccttctcagt caaagacatc ctaaacctgg aacagcagca 301 gcgcagcctg gctgccgccg gagagctctc tgcccgcctg gaggcgaccc tggcgccctc 361 ctcctgcatg ctggccgcct tcaagccaga ggcctacgct gggcccgagg cggctgcgcc 421 gggcctccca gagctgcgcg cagagctggg ccgcgcgcct tcaccggcca agtgtgcgtc 481 tgcctttccc gccgcccccg ccttctatcc acgtgcctac agcgaccccg acccagccaa 541 ggaccctaga gccgaaaaga aagagctgtg cgcgctgcag aaggcggtgg agctggagaa 601 gacagaggcg gacaacgcgg agcggccccg ggcgcgacgg cggaggaagc cgcgcgtgct 661 cttctcgcag gcgcaggtct atgagctgga gcggcgcttc aagcagcagc ggtacctgtc 721 ggcccccgaa cgcgaccagc tggccagcgt gctgaaactc acgtccacgc aggtcaagat 781 ctggttccag aaccggcgct acaagtgcaa gcggcagcgg caggaccaga ctctggagct 841 ggtggggctg cccccgccgc cgccgccgcc tgcccgcagg atcgcggtgc cagtgctggt 901 gcgcgatggc aagccatgcc taggggactc ggcgccctac gcgcctgcct acggcgtggg 961 cctcaatccc tacggttata acgcctaccc cgcctatccg ggttacggcg gcgcggcctg 1021 cagccctggc tacagctgca ctgccgctta ccccgccggg ccttccccag cgcagccggc

1081 cactgccgcc gccaacaaca acttcgtgaa cttcggcgtc ggggacttga atgcggttca

1141 gagccccggg attccgcaga gcaactcggg agtgtccacg ctgcatggta tccgagcctg

1201 gtagggaagg gacccgcgtg gcgcgaccct gaccgatccc acctcaacag ctccctgact

1261 ctcgggggga gaaggggctc ccaacatgac cctgagtccc ctggattttg cattcactcc

1321 tgcggagacc taggaacttt ttctgtccca cgcgcgtttg ttcttgcgca cgggagagtt

1381 tgtggcggcg attatgcagc gtgcaatgag tgatcctgca gcctggtgtc ttagctgtcc

1441 ccccaggagt gccctccgag agtccatggg cacccccggt tggaactggg actgagctcg

1501 ggcacgcagg gcctgagatc tggccgccca ttccgcgagc cagggccggg cgcccgggcc

1561 tttgctatct cgccgtcgcc cgcccacgca cccacccgta tttatgtttt tacctattgc

1621 tgtaagaaat gacgatcccc ttcccattaa agagagtgcg ttgaccccg

By“NEUROD1 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_00249l.2 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_002491.2 is shown below:

1 MTKSYSESGL MGEPQPQGPP SWTDECLSSQ DEEHEADKKE DDLEAMNAEE DSLRNGGEEE

61 DEDEDLEEEE EEEEEDDDQK PKRRGPKKKK MTKARLERFK LRRMKANARE RNRMHGLNAA

121 LDNLRKWPC YSKTQKLSKI ETLRLAKNYI WALSEILRSG KSPDLVSFVQ TLCKGLSQPT

181 TNLVAGCLQL NPRTFLPEQN QDMPPHLPTA SASFPVHPYS YQSPGLPSPP YGTMDSSHVF

241 HVKPPPHAYS AALEPFFESP LTDCTSPSFD GPLSPPLSIN GNFSFKHEPS AEFEKNYAFT

301 MHYPAATLAG AQSHGSIFSG TAAPRCEIPI DNIMSFDSHS HHERVMSAQL NAIFHD

By“NEUROD1 polynucleotide” is meant a polynucleotide encoding a NEURODl polypeptide or fragment thereof. An exemplary NEUROD1 polynucleotide sequence is provided at NCBI Ref: NM_002500.4. The sequence provided at NCBI Ref: NM_002500.4 is reproduced below:

1 ggggaggagg ggagaacggg gagcgcacag cctggacgcg tgcgcaggcg tcaggcgcat 61 agacctgcta gcccctcagc tagcggcccc gcccgcgctt agcatcacta actgggctat 121 ataacctgag cgcccgcgcg gccacgacac gaggaattcg cccacgcagg aggcgcggcg 181 tccggaggcc ccagggttat gagactatca ctgctcagga cctactaaca acaaaggaaa 241 tcgaaacatg accaaatcgt acagcgagag tgggctgatg ggcgagcctc agccccaagg 301 tcctccaagc tggacagacg agtgtctcag ttctcaggac gaggagcacg aggcagacaa 361 gaaggaggac gacctcgaag ccatgaacgc agaggaggac tcactgagga acgggggaga 421 ggaggaggac gaagatgagg acctggaaga ggaggaagaa gaggaagagg aggatgacga 481 tcaaaagccc aagagacgcg gccccaaaaa gaagaagatg actaaggctc gcctggagcg 541 ttttaaattg agacgcatga aggctaacgc ccgggagcgg aaccgcatgc acggactgaa 601 cgcggcgcta gacaacctgc gcaaggtggt gccttgctat tctaagacgc agaagctgtc 661 caaaatcgag actctgcgct tggccaagaa ctacatctgg gctctgtcgg agatcctgcg 721 ctcaggcaaa agcccagacc tggtctcctt cgttcagacg ctttgcaagg gcttatccca 781 acccaccacc aacctggttg cgggctgcct gcaactcaat cctcggactt ttctgcctga 841 gcagaaccag gacatgcccc cccacctgcc gacggccagc gcttccttcc ctgtacaccc 901 ctactcctac cagtcgcctg ggctgcccag tccgccttac ggtaccatgg acagctccca 961 tgtcttccac gttaagcctc cgccgcacgc ctacagcgca gcgctggagc ccttctttga 1021 aagccctctg actgattgca ccagcccttc ctttgatgga cccctcagcc cgccgctcag

1081 catcaatggc aacttctctt tcaaacacga accgtccgcc gagtttgaga aaaattatgc

1141 ctttaccatg cactatcctg cagcgacact ggcaggggcc caaagccacg gatcaatctt

1201 ctcaggcacc gctgcccctc gctgcgagat ccccatagac aatattatgt ccttcgatag

1261 ccattcacat catgagcgag tcatgagtgc ccagctcaat gccatatttc atgattagag

1321 gcacgccagt ttcaccattt ccgggaaacg aacccactgt gcttacagtg actgtcgtgt

1381 ttacaaaagg cagccctttg ggtactactg ctgcaaagtg caaatactcc aagcttcaag

1441 tgatatatgt atttattgtc attactgcct ttggaagaaa caggggatca aagttcctgt

1501 tcaccttatg tattattttc tatagctctt ctatttaaaa aataaaaaaa tacagtaaag

1561 tttaaaaaat acaccacgaa tttggtgtgg ctgtattcag atcgtattaa ttatctgatc

1621 gggataacaa aatcacaagc aataattagg atctatgcaa tttttaaact agtaatgggc

1681 caattaaaat atatataaat atatattttt caaccagcat tttactactt gttacctttc

1741 ccatgctgaa ttattttgtt gtgattttgt acagaatttt taatgacttt ttataatgtg

1801 gatttcctat tttaaaacca tgcagcttca tcaattttta tacatatcag aaaagtagaa

1861 ttatatctaa tttatacaaa ataatttaac taatttaaac cagcagaaaa gtgcttagaa

1921 agttattgtg ttgccttagc acttctttcc tctccaattg taaaaaaaaa aaaaaaaaaa

1981 aaaaaaaaaa aaaaattgca caatttgagc aattcatttc actttaaagt ctttccgtct

2041 ccctaaaata aaaaccagaa tcataatttt caagagaaga aaaaattaag agatacattc

2101 cctatcaaaa catatcaatt caacacatta cttgcacaag cttgtatata catattataa

2161 ataaatgcca acataccctt ctttaaatca aaagctgctt gactatcaca tacaatttgc

2221 actgttactt tttagtcttt tactcctttg cattccatga ttttacagag aatctgaagc

2281 tattgatgtt tccagaaaat ataaatgcat gattttatac atagtcacaa aaatggtggt

2341 ttgtcatata ttcatgtaat aaatctgagc ctaaatctaa tcaggttgtt aatgttggga

2401 tttatatcta tagtagtcaa ttagtacagt agcttaaata aattcaaacc atttaattca

2461 taattagaac aatagctatt gcatgtaaaa tgcagtccag aataagtgct gtttgagatg

2521 tgatgctggt accactggaa tcgatctgta ctgtaatttt gtttgtaatc ctgtatatta

2581 tggtgtaatg cacaatttag aaaacattca tccagttgca ataaaatagt attgaaagtg

2641 agagcaattg ttgcatttct tcttaaaggg attctgtttt tatttttggg gaaagtagtt

2701 gcttttttgc tgagttaaaa aatactaaac actatatgta gaataaaaga aaagaaaaaa

2761 gtttaccttg gcatatgctc ttgtctgttt atcttgcaca gggagtcacc agttctatgt

2821 agataatgaa aagacctaac tgatatttca ttatttggaa tatgggactg gacggcagta

2881 caaacagtgt gtttttttct ttgttttaag tggcttagcc tttaggtttt ttatttccat

2941 ttttaaaaat gattgttaca tgttttcttc tatttctttt tttaaaaggt ggattttaat

3001 aa

By“NKX6-1 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_006l59.2 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_006159.2 is shown below:

1 MLAVGAMEGT RQSAFLLSSP PLAALHSMAE MKTPLYPAAY PPLPAGPPSS SSSSSSSSSP 61 SPPLGTHNPG GLKPPATGGL SSLGSPPQQL SAATPHGIND ILSRPSMPVA SGAALPSASP 121 SGSSSSSSSS ASASSASAAA AAAAAAAAAA SSPAGLLAGL PRFSSLSPPP PPPGLYFSPS 181 AAAVAAVGRY PKPLAELPGR TPIFWPGVMQ SPPWRDARLA CTPHQGSILL DKDGKRKHTR 241 PTFSGQQIFA LEKTFEQTKY LAGPERARLA YSLGMTESQV KVWFQNRRTK WRKKHAAEMA 301 TAKKKQDSET ERLKGASENE EEDDDYNKPL DPNSDDEKIT QLLKKHKSSS GGGGGLLLHA 361 SEPESSS By“NKX6-1 polynucleotide” is meant a polynucleotide encoding a NKX6-1 polypeptide or fragment thereof. An exemplary NKX6-1 polynucleotide sequence is provided at NCBI Ref: NM_006l68.2. The sequence provided at NCBI Ref: NM_006l68.2 is reproduced below:

1 cgtgggatgt tagcggtggg ggcaatggag ggcacccggc agagcgcatt cctgctcagc 61 agccctcccc tggccgccct gcacagcatg gccgagatga agaccccgct gtaccctgcc 121 gcgtatcccc cgctgcctgc cggccccccc tcctcctcgt cctcgtcgtc gtcctcctcg 181 tcgccctccc cgcctctggg cacccacaac ccaggcggcc tgaagccccc ggccacgggg 241 gggctctcat ccctcggcag ccccccgcag cagctctcgg ccgccacccc acacggcatc 301 aacgatatcc tgagccggcc ctccatgccc gtggcctcgg gggccgccct gccctccgcc 361 tcgccctccg gttcctcctc ctcctcttcc tcgtccgcct ctgcctcctc cgcctctgcc 421 gccgccgcgg ctgctgccgc ggccgcagcc gccgcctcat ccccggcggg gctgctggcc 481 ggactgccac gctttagcag cctgagcccg ccgccgccgc cgcccgggct ctacttcagc 541 cccagcgccg cggccgtggc cgccgtgggc cggtacccca agccgctggc tgagctgcct 601 ggccggacgc ccatcttctg gcccggagtg atgcagagcc cgccctggag ggacgcacgc 661 ctggcctgta cccctcatca aggatccatt ttgttggaca aagacgggaa gagaaaacac 721 acgagaccca ctttttccgg acagcagatc ttcgccctgg agaagacttt cgaacaaaca 781 aaatacttgg cggggcccga gagggctcgt ttggcctatt cgttggggat gacagagagt 841 caggtcaagg tctggttcca gaaccgccgg accaagtgga ggaagaagca cgctgccgag 901 atggccacgg ccaagaagaa gcaggactcg gagacagagc gcctcaaggg ggcctcggag 961 aacgaggaag aggacgacga ctacaataag cctctggatc ccaactcgga cgacgagaaa 1021 atcacgcagc tgttgaagaa gcacaagtcc agcagcggcg gcggcggcgg cctcctactg 1081 cacgcgtccg agccggagag ctcatcctga acgccg

By“NDUFA4 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_002480.l and having NADH

dehydrogenase activity and oxidoreductase activity. The amino acid sequence provided at NCBI Accession No. NP_002480.1 is shown below:

1 MAAELAMGAE LPSSPLAIEY WDFDLMKFE VKKEPPEAER FCHRLPPGSL SSTPLSTPCS 61 SVPSSPSFCA PSPGTGGGGG AGGGGGSSQA GGAPGPPSGG PGAVGGTSGK PALEDLYWMS 121 GYQHHLNPEA LNLTPEDAVE ALIGSGHHGA HHGAHHPAAA AAYEAFRGPG FAGGGGADDM 181 GAGHHHGAHH AAHHHHAAHH HHHHHHHHGG AGHGGGAGHH VRLEERFSDD QLVSMSVREL 241 NRQLRGFSKE EVIRLKQKRR TLKNRGYAQS CRFKRVQQRH ILESEKCQLQ SQVEQLKLEV 301 GRLAKERDLY KEKYEKLAGR GGPGSAGGAG FPREPSPPQA GPGGAKGTAD FFL

By“NDUFA4 polynucleotide” is meant a polynucleotide encoding a NDUFA4 polypeptide or fragment thereof. An exemplary NDUFA4 polynucleotide sequence is provided at NCBI Ref: NM_002489.3. The sequence provided at NCBI Ref: NM_002489.3 is reproduced below:

1 gggtccttca ggtaggaggt cctgggtgac tttggaagtc cgtagtgtct cattgcagat

61 aatttttagc ttagggcctg gtggctaggt cggttctctc ctttccagtc ggagacctct

121 gccgcaaaca tgctccgcca gatcatcggt caggccaaga agcatccgag cttgatcccc 181 ctctttgtat ttattggaac tggagctact ggagcaacac tgtatctctt gcgtctggca

241 ttgttcaatc cagatgtttg ttgggacaga aataacccag agccctggaa caaactgggt

301 cccaatgatc aatacaagtt ctactcagtg aatgtggatt acagcaagct gaagaaggaa

361 cgtccagatt tctaaatgaa atgtttcact ataacgctgc tttagaatga aggtcttcca

421 gaagccacat ccgcacaatt ttccacttaa ccaggaaata tttctcctct aaatgcatga

481 aatcatgttg gagatctcta ttgtaatctc tattggagat tacaatgatt aaatcaataa

541 ataactgaaa cttgatatgt gtcacttttt tatgctgaaa gtatgctctg aactttagag

601 tataggaaat taactattag aatttaaaga atttcttgaa tttctgtagt ttgaaaatac

661 gactttaagc tgctttagta aaacacttcc attttgtgta tagactgttg gtaacttcac

721 tagagcatac ataacaactg gaactggaaa ttatacaaaa gtaaattggg aaggatactc

781 cagcatctga cactggcaaa atggaaacct ttgagtttct cttactggct gttgaagtgt

841 gtgcagtttt taacaatggt ttttacttgg catctctttg ttgtgatttt caaggttata

901 agttgctttg gtcctaggat tgaagttgaa atctgagttt atcagtgcta accatggtgc

961 tagtagtcaa gagatcttga gaattttggc tgctgagtct tggtgcaggg tgcaggtttt

1021 cttttctttt ttcttttttt tttttttgag atagtctctg tcacccaggc tggagtgcag

1081 tggtacaaac atggatcact gcagcctcta cctcccgggc ttaagtgatc ctcctgcctc

1141 agcccctaag tagccgggac tacaggtatg tgccaccatg cccagttaat ttttgtaatt

1201 ttttttagag acagggtttt gccatgttgc ccaggctggt ctcaaactct tgagctcaag

1261 cgatccattc tcctcagcct cccagggtgc tgggattaca ggcgtgagcc attgcgctta

1321 gccatggtgc aggttttcaa aggccaggaa gtatattcat aattttaaga tggggaatat

1381 agcaagtttt cacataggtg tgtgtaagtc atcacatcat agaaacttga ggaattcagt

1441 gacattaatt ttggattttc atacgtaagt atacaattaa atgtttacag ggtagtagaa

1501 gcacatttta aatgtcagga actgaactaa gtatttgaat tacgtggatt atctcaaaaa

1561 ttttgaaatt gttaaacgag ttgaattact tgaattcatt ctgttagtca aatggtggat

1621 atttacaccc atgtagtttt gaatttagag tgtgtagagt gttttcagtt accagactcc

1681 atgcttttac ctcctatgtg tcaggtataa tttgaacctc taagaacagg gtttctcaac

1741 cttgccactg ttgactattt ctgaaagaca gtttggttta gcagaccatc ccatgcgctt

1801 tagcttgttt agtagctaac ttgggctctg ccactacaga caaaaagcac tctttccctc

1861 caattcccac aggctatgag aagaatggag acattaccaa atgtccattg gtgggcaaaa

1921 ttgcttcatt cctacctctg ttgagaatta ctctagatcc tttggcacaa attacctcaa

1981 agtttaaaat tgtgtaaaca aacagtgtgt catgtaattg aaaaacatta agcaactcca

2041 aataaatgct acattaag

As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, procuring, deriving, or otherwise acquiring the agent.

By“organ” is meant a collection of cells that perform a biological function. In one embodiment, an organ includes, but is not limited to, bladder, brain, nervous tissue, glial tissue, esophagus, fallopian tube, heart, pancreas, intestines, gallbladder, kidney, liver, lung, ovaries, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, breast, skeletal muscle, skin, bone, and cartilage. The biological function of an organ can be assayed using standard methods known to the skilled artisan.

By“organoid” is meant an in vitro generated body that mimics organ structure and function. “Organoid” and“mini organ” are used interchangeably herein. An“islet-like organoid,”“pancreatic islet organoid,”“pancreatic islet,” or“pancreatic organoid” is an in vitro generated cell cluster that mimics the structure and function of a pancreatic islet.

Exemplary functions of a pancreatic islet include, without limitation, glucose-stimulated insulin secretion (GSIS), potassium chloride (KCl)-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, or glucagon secretion. “Pancreatic islet organoid,”“islet-like organoid,”“pancreatic organoid” and“mini pancreatic islet” are used interchangeably herein. In an embodiment, a“pancreatic organoid” is an in vitro generated body that mimics structure and function of a pancreas. Exemplary functions of a pancreas include, without limitation, endocrine secretion of hormones, such as glucose and glucagon, that regulate glucose metabolism and blood glucose concentration, and exocrine secretion of digestive enzymes that help break down carbohydrates, proteins, and lipids. “Pancreatic organoid” and“mini pancreas” are also used interchangeably herein. In an embodiment, an organoid is a human islet-like organoid (“HILO”) as described herein. In an embodiment, a HILO is generated from induced pluripotent stem cells (iPSCs). In an embodiment, the HILO is functionally mature and contains endocrine-like cell types that, upon transplantation, effectively re-establish glucose homeostasis, e.g., in a diabetic mouse model (NOD-SCID mouse). In an embodiment, the HILO is a WNT4-treated HILO (wHILOs). In an

embodiment, overexpression of the checkpoint protein PD-L1 in HILOs allowed the HILOs to evade an immune reaction or surveillance by T cells such that they were able to maintain glucose homeostasis in immune-competent diabetic mice (NOD-SCID mice) for a long time period, e.g., at least 50 days. In an embodiment, induction of endogenous PD-L1 expression in HILOs following multiple intermittent ex vivo exposures to interferon gamma (IFNy) over a given time period, e.g., at least 24 hours, restricts T cell activation and graft rejection. In embodiments, multiple intermittent exposure of cells or HILOs and the cells therein to IFNy encompasses exposure (e.g., in culture, such as liquid culture or 3D matrix culture) of cells or HILOs and the cells therein to an amount (e.g., low levels) of IFNy for multiple times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, over a given time period, with periods of no IFNy exposure in between. In an embodiment, HILOs that have undergone multiple intermittent exposure to IFNy so as to express PD-L1 polypeptide as described herein may be referred to as immune evasive HILOs, wHILOs or wHILO ^lc herein.

By“PD-L1 polypeptide” (also called CD274) is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at UniProt Accession No. Q9NZQ7-1 and having transcription factor activity. The amino acid sequence is provided at NCBI Accession No. NP_006184.2 is shown below:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEME DKNI IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG ADYKRITVKVNAPYNKINQRILWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT TTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH LVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

By“PD-L1 polynucleotide” is meant a polynucleotide encoding a PD-L1 polypeptide or fragment thereof. An exemplary PD-L1 polynucleotide sequence is provided at NCBI Accession No.: CCDS59118.1. The sequence provided at NCBI Accession No.:

CCDS59118.1 is reproduced below:

Nucleotide Sequence (531 nt):

atgaggatatttgctgtctttatattcatgacctactggcatttgctgaacgcccca tacaacaaaat caaccaaagaattttggttgtggatccagtcacctctgaacatgaactgacatgtcaggc tgagggct accccaaggccgaagtcatctggacaagcagtgaccatcaagtcctgagtggtaagacca ccaccacc aattccaagagagaggagaagcttttcaatgtgaccagcacactgagaatcaacacaaca actaatga gattttctactgcacttttaggagattagatcctgaggaaaaccatacagctgaattggt catcccag aactacctctggcacatcctccaaatgaaaggactcacttggtaattctgggagccatct tattatgc cttggtgtagcactgacattcatcttccgtttaagaaaagggagaatgatggatgtgaaa aaatgtgg catccaagatacaaactcaaagaagcaaagtgatacacatttggaggagacgtaa

By“PAX4 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_006184.2 and having transcription factor activity. The amino acid sequence is provided at NCBI Accession No. NP_006184.2 is shown below:

1 MNQLGGLFW GRPLPLDTRQ QIVRLAVSGM RPCDISRILK VSNGCVSKIL GRYYRTGVLE 61 PKGIGGSKPR LATPPWARI AQLKGECPAL FAWEIQRQLC AEGLCTQDKT PSVSSINRVL 121 RALQEDQGLP CTRLRSPAVL APAVLTPHSG SETPRGTHPG TGHRNRTIFS PSQAEALEKE 181 FQRGQYPDSV ARGKLATATS LPEDTVRVWF SNRRAKWRRQ EKLKWEMQLP GASQGLTVPR 241 VAPGIISAQQ SPGSVPTAAL PALEPLGPSC YQLCWATAPE RCLSDTPPKA CLKPCWGHLP 301 PQPNSLDSGL LCLPCPSSHC HLASLSGSQA LLWPGCPLLY GLE

By“PAX4 polynucleotide” is meant a polynucleotide encoding a PAX4 polypeptide or fragment thereof. An exemplary PAX4 polynucleotide sequence is provided at NCBI Ref: NM_006l93.2. The sequence provided at NCBI Ref: NM_006l93.2 is reproduced below:

1 caaagactca cccgtgagcc agctctcaaa gaaagcagct tgcgttgaca gcctgggggc

61 agcaaggatg cagtctccca ggagaggatg cactcggtgg tgggaagcca ggctggaggg

121 gcctgagtga ccctctccac aggcgggcag ggcagtggga gaggtggtgt gtggatacct

181 ctgtctcacg cccagggatc agcagcatga accagcttgg ggggctcttt gtgaatggcc

241 ggcccctgcc tctggatacc cggcagcaga ttgtgcggct agcagtcagt ggaatgcggc

301 cctgtgacat ctcacggatc cttaaggtat ctaatggctg tgtgagcaag atcctagggc 361 gttactaccg cacaggtgtc ttggagccaa agggcattgg gggaagcaag ccacggctgg

421 ctacaccccc tgtggtggct cgaattgccc agctgaaggg tgagtgtcca gccctctttg

481 cctgggaaat ccaacgccag ctttgtgctg aagggctttg cacccaggac aagactccca

541 gtgtctcctc catcaaccga gtcctgcggg cattacagga ggaccaggga ctaccgtgca

601 cacggctcag gtcaccagct gttttggctc cagctgtcct cactccccat agtggctctg

661 agactccccg gggtacccac ccagggaccg gccaccggaa tcggactatc ttctccccaa

721 gccaagcaga ggcactggag aaagagttcc agcgtgggca gtatcctgat tcagtggccc

781 gtggaaagct ggctactgcc acctctctgc ctgaggacac ggtgagggtc tggttttcca

841 acagaagagc caaatggcgt cggcaagaga agctcaagtg ggaaatgcag ctgccaggtg

901 cttcccaggg gctgactgta ccaagggttg ccccaggaat catctctgca cagcagtccc

961 ctggcagtgt gcccacagca gccctgcctg ccctggaacc actgggtccc tcctgctatc

1021 agctgtgctg ggcaacagca ccagaaaggt gtctgagtga caccccacct aaagcctgtc

1081 tcaagccctg ctggggccac ttgcccccac agccgaattc cctggactca ggactgcttt

1141 gccttccttg cccttcctcc cactgtcacc tggccagtct tagtggctct caggccctgc

1201 tctggcctgg ctgcccacta ctgtatggct tggaatgagg caggagtggg aaggagatgg

1261 catagagaag atctaatacc atcctgccca ttgtccttac cgtcctgccc atacagactg

1321 tggctccttc ctccttcctg tgattgctcc ctcctgtgtg gacgttgcct ggccctgcct

1381 cgatgcctct ctggcgcatc acctgattgg aggggetggt aaagcaacac ccacccactt

1441 ctcacactag ccttaagagg cctccactca gcagtaataa aagctgtttt tattagcagt

1501 agttctgttg tccatcatgt tttccctatg agcaccccta tgcccactct aatattcaac

1561 aattatagac aatttgccct atcatttatt tacatctatg tatctaccat ctaatctatg

1621 catgtatgta ggcaatacat gtatctaaac aatgtatttg tcaatgcatc aatttaccta

1681 ctctatgtat gcatctatat gtgtattatg tatgcgtgca tgcgtgcgcg cacacacaca

1741 cacacacaca cacactgaca ttatatcatg gcattttatt cctaaatctt ccagcatgca

1801 tccccaaaaa acaagaaact tgtcttacat aatcacaata atatatccac atctaagaaa

1861 atttactgta acttcttaat ctaagaaaat tatgtatttt tgtcatatgt attttgtcat

1921 atgtattttg tatttgcata tgtattttgt atttgcatat gtatttttgt catagcagca

1981 aacagagtga aatgccattt ttcatattct

By“PAX6 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NR_001297090.1 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_001297090.1 is shown below:

1 MGADGMYDKL RMLNGQTGSW GTRPGWYPGT SVPGQPTQDG CQQQEGGGEN TNSISSNGED

61 SDEAQMRLQL KRKLQRNRTS FTQEQIEALE KEFERTHYPD VFARERLAAK IDLPEARIQV

121 WFSNRRAKWR REEKLRNQRR QASNTPSHIP ISSSFSTSVY QPIPQPTTPV SSFTSGSMLG

181 RTDTALTNTY SALPPMPSFT MANNLPMQPP VPSQTSSYSC MLPTSPSWG RSYDTYTPPH

241 MQTHMNSQPM GTSGTTSTGL ISPGVSVPVQ VPGSEPDMSQ YWPRLQ

By“PAX6 polynucleotide” is meant a polynucleotide encoding a PAX6 polypeptide or fragment thereof. An exemplary PAX6 polynucleotide sequence is provided at NCBI Ref: NM_00l310161.1. The sequence provided at NCBI Ref: NM_00l310161.1 is reproduced below:

1 cttttcaatt agccttccat gcatgatccg gagcgacttc cgcctatttc cagaaattaa 61 gctcaaactt gacgtgcagc tagttttatt ttaaagacaa atgtcagaga ggctcatcat

121 attttccccc ctcttctata tttggagctt atttattgct aagaagctca ggctcctggc

181 gtcaatttat cagtaggctc caaggagaag agaggagagg agaggagagc tgaacaggga

241 gccacgtctt ttcctgggag ggctgctatc taagtcgggg ctgcaggtca cagcggagtg

301 aatcagctcg gtggtgtctt tgtcaacggg cggccactgc cggactccac ccggcagaag

361 attgtagagc tagctcacag cggggcccgg ccgtgcgaca tttcccgaat tctgcagacc

421 catgcagatg caaaagtcca agtgctggac aatcaaaacg tgtccaacgg atgtgtgagt

481 aaaattctgg gcaggtatta cgagactggc tccatcagac ccagggcaat cggtggtagt

541 aaaccgagag tagcgactcc agaagttgta agcaaaatag cccagtataa gcgggagtgc

601 ccgtccatct ttgcttggga aatccgagac agattactgt ccgagggggt ctgtaccaac

661 gataacatac caagcgtgtc atcaataaac agagttcttc gcaacctggc tagcgaaaag

721 caacagatgg gcgcagacgg catgtatgat aaactaagga tgttgaacgg gcagaccgga

781 agctggggca cccgccctgg ttggtatccg gggacttcgg tgccagggca acctacgcaa

841 gatggctgcc agcaacagga aggaggggga gagaatacca actccatcag ttccaacgga

901 gaagattcag atgaggctca aatgcgactt cagctgaagc ggaagctgca aagaaataga

961 acatccttta cccaagagca aattgaggcc ctggagaaag agtttgagag aacccattat

1021 ccagatgtgt ttgcccgaga aagactagca gccaaaatag atctacctga agcaagaata

1081 caggtatggt tttctaatcg aagggccaaa tggagaagag aagaaaaact gaggaatcag

1141 agaagacagg ccagcaacac acctagtcat attcctatca gcagtagttt cagcaccagt

1201 gtctaccaac caattccaca acccaccaca ccggtttcct ccttcacatc tggctccatg

1261 ttgggccgaa cagacacagc cctcacaaac acctacagcg ctctgccgcc tatgcccagc

1321 ttcaccatgg caaataacct gcctatgcaa cccccagtcc ccagccagac ctcctcatac

1381 tcctgcatgc tgcccaccag cccttcggtg aatgggcgga gttatgatac ctacaccccc

1441 ccacatatgc agacacacat gaacagtcag ccaatgggca cctcgggcac cacttcaaca

1501 ggactcattt cccctggtgt gtcagttcca gttcaagttc ccggaagtga acctgatatg

1561 tctcaatact ggccaagatt acagtaaaaa aaaaaaaaaa aaaaaaaagg aaaggaaata

1621 ttgtgttaat tcagtcagtg actatgggga cacaacagtt gagctttcag gaaagaaaga

1681 aaaatggctg ttagagccgc ttcagttcta caattgtgtc ctgtattgta ccactgggga

1741 aggaatggac ttgaaacaag gacctttgta tacagaaggc acgatatcag ttggaacaaa

1801 tcttcatttt ggtatccaaa cttttattca ttttggtgta ttatttgtaa atgggcattt

1861 gtatgttata atgaaaaaaa gaacaatgta gactggatgg atgtttgatc tgtgttggtc

1921 atgaagttgt tttttttttt tttaaaaaga aaaccatgat caacaagctt tgccacgaat

1981 ttaagagttt tatcaagata tatcgaatac ttctacccat ctgttcatag tttatggact

2041 gatgttccaa gtttgtatca ttcctttgca tataattaaa cctggaacaa catgcactag

2101 atttatgtca gaaatatctg ttggttttcc aaaggttgtt aacagatgaa gtttatgtgc

2161 aaaaaagggt aagatataaa ttcaaggaag aaaaaaagtt gatagctaaa aggtagagtg

2221 tgtcttcgat ataatccaat ttgttttatg tcaaaatgta agtatttgtc ttccctagaa

2281 atcctcagaa tgatttctat aataaagtta atttcattta tatttgacaa gaatatagat

2341 gttttataca cattttcatg caatcatacg tttctttttt ggccagcaaa agttaattgt

2401 tcttagatat agttgtatta ctgttcacgg tccaatcatt ttgtgcatct agagttcatt

2461 cctaatcaat taaaagtgct tgcaagagtt ttaaacttaa gtgttttgaa gttgttcaca

2521 actacatatc aaaattaacc attgttgatt gtaaaaaacc atgccaaagc ctttgtattt

2581 cctttattat acagttttct ttttaacctt atagtgtggt gttacaaatt ttatttccat

2641 gttagatcaa cattctaaac caatggttac tttcacacac actctgtttt acatcctgat

2701 gatccttaaa aaataatcct tatagatacc ataaatcaaa aacgtgttag aaaaaaattc

2761 cacttacagc agggtgtaga tctgtgccca tttataccca caacatatat acaaaatggt

2821 aacatttccc agttagccat ttaattctaa agctcaaagt ctagaaataa tttaaaaatg

2881 caacaagcga ttagctagga attgtttttt gaattaggac tggcattttc aatctgggca

2941 gatttccatt gtcagcctat ttcaacaatg atttcactga agtatattca aaagtagatt

3001 tcttaaagga gactttctga aagctgttgc ctttttcaaa taggccctct cccttttctg

3061 tctccctccc ctttgcacaa gaggcatcat ttcccattga accactacag ctgttcccat 3121 ttgaatcttg ctttctgtgc ggttgtggat ggttggaggg tggagggggg atgttgcatg

3181 tcaaggaata atgagcacag acacatcaac agacaacaac aaagcagact gtgactggcc

3241 ggtgggaatt aaaggccttc agtcattggc agcttaagcc aaacattccc aaatctatga

3301 agcagggccc attgttggtc agttgttatt tgcaatgaag cacagttctg atcatgttta

3361 aagtggaggc acgcagggca ggagtgcttg agcccaagca aaggatggaa aaaaataagc

3421 ctttgttggg taaaaaagga ctgtctgaga ctttcatttg ttctgtgcaa catataagtc

3481 aatacagata agtcttcctc tgcaaacttc actaaaaagc ctgggggttc tggcagtcta

3541 gattaaaatg cttgcacatg cagaaacctc tggggacaaa gacacacttc cactgaatta

3601 tactctgctt taaaaaaatc cccaaaagca aatgatcaga aatgtagaaa ttaatggaag

3661 gatttaaaca tgaccttctc gttcaatatc tactgttttt tagttaagga attacttgtg

3721 aacagataat tgagattcat tgctccggca tgaaatatac taataatttt attccaccag

3781 agttgctgca catttggaga caccttccta agttgcagtt tttgtatgtg tgcatgtagt

3841 tttgttcagt gtcagcctgc actgcacagc agcacatttc tgcaggggag tgagcacaca

3901 tacgcactgt tggtacaatt gccggtgcag acatttctac ctcctgacat tttgcagcct

3961 acattccctg agggctgtgt gctgagggaa ctgtcagaga agggctatgt gggagtgcat

4021 gccacagctg ctggctggct tacttcttcc ttctcgctgg ctgtaatttc caccacggtc

4081 aggcagccag ttccggccca cggttctgtt gtgtagacag cagagacttt ggagacccgg

4141 atgtcgcacg ccaggtgcaa gaggtgggaa tgggagaaaa ggagtgacgt gggagcggag

4201 ggtctgtatg tgtgcacttg ggcacgtata tgtgtgctct gaaggtcagg attgccaggg

4261 caaagtagca cagtctggta tagtctgaag aagcggctgc tcagctgcag aagccctctg

4321 gtccggcagg atgggaacgg ctgccttgcc ttctgcccac accctaggga catgagctgt

4381 ccttccaaac agagctccag gcactctctt ggggacagca tggcaggctc tgtgtggtag

4441 cagtgcctgg gagttggcct tttactcatt gttgaaataa tttttgttta ttatttattt

4501 aacgatacat atatttatat atttatcaat ggggtatctg cagggatgtt ttgacaccat

4561 cttccaggat ggagattatt tgtgaagact tcagtagaat cccaggacta aacgtctaaa

4621 ttttttctcc aaacttgact gacttgggaa aaccaggtga atagaataag agctgaatgt

4681 tttaagtaat aaacgttcaa actgctctaa gtaaaaaaat gcattttact gcaatgaatt

4741 tctagaatat ttttccccca aagctatgcc tcctaaccct taaatggtga acaactggtt

4801 tcttgctaca gctcactgcc atttcttctt actatcatca ctaggtttcc taagattcac

4861 tcatacagta ttatttgaag attcagcttt gttctgtgaa tgtcatctta ggattgtgtc

4921 tatattcttt tgcttatttc tttttactct gggcctctca tactagtaag attttaaaaa

4981 gccttttctt ctctgtatgt ttggctcacc aaggcgaaat atatattctt ctctttttca

5041 tttctcaaga ataaacctca tctgcttttt tgtttttctg tgttttggct tggtactgaa

5101 tgactcaact gctcggtttt aaagttcaaa gtgtaagtac ttagggttag tactgcttat

5161 ttcaataatg ttgacggtga ctatctttgg aaagcagtaa catgctgtct tagaaatgac

5221 attaataatg ggcttaaaca aatgaatagg ggggtccccc cactctcctt ttgtatgcct

5281 atgtgtgtct gatttgttaa aagatggaca gggaattgat tgcagagtgt cgcttccttc

5341 taaagtagtt ttattttgtc tactgttagt atttaaagat cctggaggtg gacataagga

5401 ataaatggaa gagaaaagta gatattgtat ggtggctact aaaaggaaat tcaaaaagtc

5461 ttagaacccg agcacctgag caaactgcag tagtcaaaat atttatctca tgttaaagaa

5521 aggcaaatct agtgtaagaa atgagtacca tatagggttt tgaagttcat atactagaaa

5581 cacttaaaag atatcatttc agatattacg tttggcattg ttcttaagta tttatatctt

5641 tgagtcaagc tgataattaa aaaaaatctg ttaatggagt gtatatttca taatgtatca

5701 aaatggtgtc tatacctaag gtagcattat tgaagagaga tatgtttatg tagtaagtta

5761 ttaacataat gagtaacaaa taatgtttcc agaagaaagg aaaacacatt ttcagagtgc

5821 gtttttatca gaggaagaca aaaatacaca cccctctcca gtagcttatt tttacaaagc

5881 cggcccagtg aattagaaaa acaaagcact tggatatgat ttttggaaag cccaggtaca

5941 cttattattc aaaatgcact tttactgagt ttgaaaagtt tcttttatat ttaaaataag

6001 ggttcaaata tgcatattca atttttatag tagttatcta tttgcaaagc atatattaac

6061 tagtaattgg ctgttaattt tatagacatg gtagccaggg aagtatatca atgacctatt

6121 aagtattttg acaagcaatt tacatatctg atgacctcgt atctcttttt cagcaagtca 6181 aatgctatgt aattgttcca ttgtgtgttg tataaaatga atcaacacgg taagaaaaag

6241 gttagagtta ttaaaataat aaactgacta aaatactcat ttgaatttat tcagaatgtt

6301 cataatgctt tcaaaggaca tagcagagct tttgtggagt atccgcacaa cattatttat

6361 tatctatgga ctaaatcaat tttttgaagt tgctttaaaa tttaaaagca cctttgctta

6421 atataaagcc ctttaatttt aactgacaga tcaattctga aactttattt tgaaaagaaa

6481 atggggaaga atctgtgtct ttagaattaa aagaaatgaa aaaaataaac ccgacattct

6541 aaaaaaatag aataagaaac ctgattttta gtactaatga aatagcgggt gacaaaatag

6601 ttgtcttttt gattttgatc acaaaaaata aactggtagt gacaggatat gatggagaga

6661 tttgacatcc tggcaaatca ctgtcattga ttcaattatt ctaattctga ataaaagctg

6721 tatacagtaa aa

By“PDX1 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_000200.1 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_000200.l is shown below:

1 MNGEEQYYAA TQLYKDPCAF QRGPAPEFSA SPPACLYMGR QPPPPPPHPF PGALGALEQG 61 SPPDISPYEV PPLADDPAVA HLHHHLPAQL ALPHPPAGPF PEGAEPGVLE EPNRVQLPFP 121 WMKSTKAHAW KGQWAGGAYA AEPEENKRTR TAYTRAQLLE LEKEFLFNKY ISRPRRVELA 181 VMLNLTERHI KIWFQNRRMK WKKEEDKKRG GGTAVGGGGV AEPEQDCAVT SGEELLALPP 241 PPPPGGAVPP AAPVAAREGR LPPGLSASPQ PSSVAPRRPQ EPR

By“PDX1 polynucleotide” is meant a polynucleotide encoding a PDX1 polypeptide or fragment thereof. An exemplary PDX1 polynucleotide sequence is provided at NCBI Ref:

NM_000209.3. The sequence provided at NCBI Ref: NM_000209.3 is reproduced below:

1 gggtggcgcc gggagtggga acgccacaca gtgccaaatc cccggctcca gctcccgact

61 cccggctccc ggctcccggc tcccggtgcc caatcccggg ccgcagccat gaacggcgag

121 gagcagtact acgcggccac gcagctttac aaggacccat gcgcgttcca gcgaggcccg

181 gcgccggagt tcagcgccag cccccctgcg tgcctgtaca tgggccgcca gcccccgccg

241 ccgccgccgc acccgttccc tggcgccctg ggcgcgctgg agcagggcag ccccccggac

301 atctccccgt acgaggtgcc ccccctcgcc gacgaccccg cggtggcgca ccttcaccac

361 cacctcccgg ctcagctcgc gctcccccac ccgcccgccg ggcccttccc ggagggagcc

421 gagccgggcg tcctggagga gcccaaccgc gtccagctgc ctttcccatg gatgaagtct

481 accaaagctc acgcgtggaa aggccagtgg gcaggcggcg cctacgctgc ggagccggag

541 gagaacaagc ggacgcgcac ggcctacacg cgcgcacagc tgctagagct ggagaaggag

601 ttcctattca acaagtacat ctcacggccg cgccgggtgg agctggctgt catgttgaac

661 ttgaccgaga gacacatcaa gatctggttc caaaaccgcc gcatgaagtg gaaaaaggag

721 gaggacaaga agcgcggcgg cgggacagct gtcgggggtg gcggggtcgc ggagcctgag

781 caggactgcg ccgtgacctc cggcgaggag cttctggcgc tgccgccgcc gccgcccccc

841 ggaggtgctg tgccgcccgc tgcccccgtt gccgcccgag agggccgcct gccgcctggc

901 cttagcgcgt cgccacagcc ctccagcgtc gcgcctcggc ggccgcagga accacgatga

961 gaggcaggag ctgctcctgg ctgaggggct tcaaccactc gccgaggagg agcagagggc

1021 ctaggaggac cccgggcgtg gaccacccgc cctggcagtt gaatggggcg gcaattgcgg

1081 ggcccacctt agaccgaagg ggaaaacccg ctctctcagg cgcatgtgcc agttggggcc

1141 ccgcgggtag atgccggcag gccttccgga agaaaaagag ccattggttt ttgtagtatt

1201 ggggccctct tttagtgata ctggattggc gttgtttgtg gctgttgcgc acatccctgc 1261 cctcctacag cactccacct tgggacctgt ttagagaagc cggctcttca aagacaatgg

1321 aaactgtacc atacacattg gaaggctccc taacacacac agcggggaag ctgggccgag

1381 taccttaatc tgccataaag ccattcttac tcgggcgacc cctttaagtt tagaaataat

1441 tgaaaggaaa tgtttgagtt ttcaaagatc ccgtgaaatt gatgccagtg gaatacagtg

1501 agtcctcctc ttcctcctcc tcctcttccc cctccccttc ctcctcctcc tcttcttttc

1561 cctcctcttc ctcttcctcc tgctctcctt tcctccccct cctcttttcc ctcctcttcc

1621 tcttcctcct gctctccttt cctccccctc ctctttctcc tcctcctcct cttcttcccc

1681 ctcctctccc tcctcctctt cttccccctc ctctccctcc tcctcttctt ctccctcctc

1741 ttcctcttcc tcctcttcca cgtgctctcc tttcctcccc ctcctcttgc tccccttctt

1801 ccccgtcctc ttcctcctcc tcctcttctt ctccctcctc ttcctcctcc tctttcttcc

1861 tgacctcttt ctttctcctc ctcctccttc tacctcccct tctcatccct cctcttcctc

1921 ttctctagct gcacacttca ctactgcaca tcttataact tgcacccctt tcttctgagg

1981 aagagaacat cttgcaaggc agggcgagca gcggcagggc tggcttagga gcagtgcaag

2041 agtccctgtg ctccagttcc acactgctgg cagggaaggc aaggggggac gggcctggat

2101 ctgggggtga gggagaaaga tggacccctg ggtgaccact aaaccaaaga tattcggaac

2161 tttctattta ggatgtggac gtaattcctg ttccgaggta gaggctgtgc tgaagacaag

2221 cacagtggcc tggtgcgcct tggaaaccaa caactattca cgagccagta tgaccttcac

2281 atctttagaa attatgaaaa cgtatgtgat tggagggttt ggaaaaccag ttatcttatt

2341 taacatttta aaaattacct aacagttatt tacaaacagg tctgtgcatc ccaggtctgt

2401 cttcttttca aggtctgggc cttgtgctcg ggttatgttt gtgggaaatg cttaataaat

2461 actgataata tgggaagaga tgaaaactga ttctcctcac tttgtttcaa acctttctgg

2521 cagtgggatg attcgaattc acttttaaaa ttaaattagc gtgttttgtt ttg

By“PTF1 polypeptide” is meant a protein or fragment thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence provided at NCBI Accession No. NP_835455.1 and having transcription factor activity. The amino acid sequence provided at NCBI Accession No. NP_835455.l is shown below:

1 MDAVLLEHFP GGLDAFPSSY FDEDDFFTDQ SSRDPLEDGD ELLADEQAEV EFLSHQLHEY 61 CYRDGACLLL QPAPPAAPLA LAPPSSGGLG EPDDGGGGGY CCETGAPPGG FPYSPGSPPS 121 CLAYPCAGAA VLSPGARLRG LSGAAAAAAR RRRRVRSEAE LQQLRQAANV RERRRMQSIN 181 DAFEGLRSHI PTLPYEKRLS KVDTLRLAIG YINFLSELVQ ADLPLRGGGA GGCGGPGGGG 241 RLGGDSPGSQ AQKVIICHRG TRSPSPSDPD YGLPPLAGHS LSWTDEKQLK EQNIIRTAKV 301 WTPEDPRKLN SKSSFNNIEN EPPFEFVS

By“PTF1 polynucleotide” is meant a polynucleotide encoding a PTF1 polypeptide or fragment thereof. An exemplary PTF1 polynucleotide sequence is provided at NCBI Ref: NM_178161.2. The sequence provided at NCBI Ref: NM_178161.2 is reproduced below:

1 atggacgcgg tgttgctgga gcacttcccc gggggcctag acgcctttcc ttcttcgtac

61 ttcgacgagg acgacttctt caccgaccag tcttcacggg accccctgga ggacggcgat

121 gagctgctgg cggacgagca ggccgaggtg gagttcctta gccaccagct ccacgagtac

181 tgctaccgcg acggggcgtg cctgctgctg cagcccgcgc ccccggccgc cccgctagcg

241 ctcgccccgc cgtcctcggg gggcctcggt gagccagacg acggcggcgg cggcggctac

301 tgctgcgaga cgggggcgcc cccaggcggc ttcccctact cgcccggctc gccgccctcg

361 tgcctggcct acccgtgcgc cggggcggca gtactgtctc ccggggcgcg gctgcgcggc

421 ctgagcggag cggcggctgc ggcggcgcgg cgccggcggc gggtgcgctc cgaggcggag 481 ctgcagcagc tgcggcaggc ggccaacgtg cgcgagcggc ggcgcatgca gtccatcaac

541 gacgccttcg aggggetgcg ctcgcacatc cccacgctgc cctacgagaa gcgcctctcc

601 aaggtggaca cgctgcgcct ggccatcggc tacatcaact tcctcagcga gctcgtgcag

661 gccgacctgc ccttgcgcgg cggtggcgcg ggcggctgcg gggggeeggg cggcggcggg

721 cgcctgggcg gggacagccc gggcagccag gcccagaagg tcatcatctg ccatcggggc

781 acccggtccc cctcccccag cgaccctgat tatggcctcc ctcccctagc aggacactct

841 ctctcatgga ctgatgaaaa acaactcaag gaacaaaata ttatccgaac agccaaagtc

901 tggaccccag aggaccccag aaaactcaac agcaaatctt ccttcaacaa catagaaaac

961 gaaccaccat ttgagtttgt gtcctgagaa gtcccagact cggctgaaga tctgattatg

1021 tctctgtgca tattgtacat gtaaatatct ataatgtaaa tgtaatttaa gaatcaaatt

1081 tttcgaatgg caatcaactg tttattattt atctatttat tatcctgttg agttgatgaa

1141 atagatgatt tctttttaaa tatataattt atataactta tcctgatttt ctgaaaatat

1201 gcaatagcct atgattttcc tgaactctgt gttgttggga gaactctggc cagaaaacgt

1261 cctgcttatt tattgccaga tatggtttat ttctaagcgt tgtcaataaa tgctatttac

1321 accttttcct gaaaaaaaa

By“Wnt3a polynucleotide” is meant a polynucleotide encoding a Wnt3a polypeptide or a fragment thereof. An exemplary human Wnt3a polynucleotide sequence is provided at NCBI GenBank Accession No. AB060284.1. The polynucleotide sequence provided at NCBI GenBank Accession No. AB060284.1 is reproduced below:

1 cggcgatggc cccactcgga tacttcttac tcctctgcag cctgaagcag gctctgggca

61 gctacccgat ctggtggtcg ctggctgttg ggccacagta ttcctccctg ggctcgcagc

121 ccatcctgtg tgccagcatc ccgggcctgg tccccaagca gctccgcttc tgcaggaact

181 acgtggagat catgcccagc gtggccgagg gcatcaagat tggcatccag gagtgccagc

241 accagttccg cggccgccgg tggaactgca ccaccgtcca cgacagcctg gccatcttcg

301 ggcccgtgct ggacaaagct accagggagt cggcctttgt ccacgccatt gcctcagccg

361 gtgtggcctt tgcagtgaca cgctcatgtg cagaaggcac ggccgccatc tgtggctgca

421 gcagccgcca ccagggctca ccaggcaagg gctggaagtg gggtggctgt agcgaggaca

481 tcgagtttgg tgggatggtg tctcgggagt tcgccgacgc ccgggagaac cggccagatg

541 cccgctcagc catgaaccgc cacaacaacg aggctgggcg ccaggccatc gccagccaca

601 tgcacctcaa gtgcaagtgc cacgggctgt cgggcagctg cgaggtgaag acatgctggt

661 ggtcgcaacc cgacttccgc gccatcggtg acttcctcaa ggacaagtac gacagcgcct

721 cggagatggt ggtggagaag caccgggagt cccgcggctg ggtggagacc ctgcggccgc

781 gctacaccta cttcaaggtg cccacggagc gcgacctggt ctactacgag gcctcgccca

841 acttctgcga gcccaaccct gagacgggct ccttcggcac gcgcgaccgc acctgcaacg

901 tcagctcgca cggcatcgac ggctgcgacc tgctgtgctg cggccgcggc cacaacgcgc

961 gagcggagcg gcgccgggag aagtgccgct gcgtgttcca ctggtgctgc tacgtcagct

1021 gccaggagtg cacgcgcgtc tacgacgtgc acacctgcaa gtaggcaccg gccgcggctc

1081 cccctggacg gggcgggccc tgcctgaggg tgggcttttc cctgggtgga gcaggactcc

1141 cacctaaacg gggcagtact cctccctggg ggcgggactc ctccctgggg gtggggctcc

1201 tacctggggg cagaactcct acctgaaggc agggctcctc cctggagcta gtgtctcctc

1261 tctggtggct gggctgctcc tgaatgaggc ggagctccag gatggggagg ggctctgcgt

1321 tggcttctcc ctggggacgg ggctcccctg gacagaggcg gggctacaga ttgggcgggg

1381 cttctcttgg gtgggacagg gcttctcctg cgggggcgag gcccctccca gtaagggcgt 1441 ggctctgggt gggcggggca ctaggtaggc ttctacctgc aggcggggct cctcctgaag 1501 gaggcggggc tctaggatgg ggcacggctc tggggtaggc tgctccctga gggcg

By“Wnt3a polypeptide” is meant a Wnt3a polypeptide or a fragment thereof, or a polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the human Wnt3a polypeptide sequence. An exemplary human Wnt3a polypeptide sequence is provided at NCBI GenBank: AAI03924.1. The sequence provided at GenBank: AAI03924.1 is reproduced below:

1 MAPLGYFLLL CSLKQALGSY PIWWSLAVGP QYSSLGSQPI LCASIPGLVP KQLRFCRNYV

61 EIMPSVAEGI KIGIQECQHQ FRGRRWNCTT VHDSLAIFGP VLDKATRESA FVHAIASAGV

121 AFAVTRSCAE GTAAICGCSS RHQGSPGKGW KWGGCSEDIE FGGMVSREFA DARENRPDAR

181 SAMNRHNNEA GRQAIASHMH LKCKCHGLSG SCEVKTCWWS QPDFRAIGDF LKDKYDSASE

241 MWEKHRESR GWVETLRPRY TYFKVPTERD LVYYEASPNF CEPNPETGSF GTRDRTCNVS

301 SHGIDGCDLL CCGRGHNARA ERRREKCRCV FHWCCYVSCQ ECTRVYDVHT CKNPGSRAGN

361 SAHQPPHPQP PVRFHPPLRR AGKVP

By“Wnt4 polynucleotide” is meant a polynucleotide encoding Wnt4 polypeptide or a fragment thereof. An exemplary human Wnt4 polynucleotide sequence is provided at NCBI GenBank Accession No. AY009398.1. Accession number NCBI Ref NG_008974. l is a reference standard Wnt4a polynucleotide sequence. The polynucleotide sequence provided at NCBI GenBank Accession No. AY009398.1 is reproduced below:

1 atgagtcccc gctcgtgcct gcgttcgctg cgcctcctcg tcttcgccgt cttctcagcc

61 gccgcgagca actggctgta cctggccaag ctgtcgtcgg tggggagcat ctcagaggag

121 gagacgtgcg agaaactcaa gggcctgatc cagaggcagg tgcagatgtg caagcggaac

181 ctggaagtca tggactcggt gcgccgcggt gcccagctgg ccattgagga gtgccagtac

241 cagttccgga accggcgctg gaactgctcc acactcgact ccttgcccgt cttcggcaag

301 gtggtgacgc aagggattcg ggaggcggcc ttggtgtacg ccatctcttc ggcaggtgtg

361 gcctttgcag tgacgcgggc gtgcagcagt ggggagctgg agaagtgcgg ctgtgacagg

421 acagtgcatg gggtcagccc acagggcttc cagtggtcag gatgctctga caacatcgcc

481 tacggtgtgg ccttctcaca gtcgtttgtg gatgtgcggg agagaagcaa gggggcctcg

541 tccagcagag ccctcatgaa cctccacaac aatgaggccg gcaggaaggc catcctgaca

601 cacatgcggg tggaatgcaa gtgccacggg gtgtcaggct cctgtgaggt aaagacgtgc

661 tggcgagccg tgccgccctt ccgccaggtg ggtcacgcac tgaaggagaa gtttgatggt

721 gccactgagg tggagccacg ccgcgtgggc tcctccaggg cactggtgcc acgcaacgca

781 cagttcaagc cgcacacaga tgaggacttg gtgtacttgg agcctagccc cgacttctgt

841 gagcaggaca tgcgcagcgg cgtgctgggc acgaggggcc gcacatgcaa caagacgtcc

901 aaggccatcg acggctgtga gctgctgtgc tgtggccgcg gcttccacac ggcgcaggtg

961 gagctggctg aacgctgcag ctgcaaattc cactggtgct gcttcgtcaa gtgccggcag

1021 tgccagcggc tcgtggagtt gcacacgtgc cgatga By“Wnt4 polypeptide” is meant a Wnt4 polypeptide or a fragment thereof, or a polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the human Wnt4 polypeptide sequence. An exemplary human Wnt4 polypeptide sequence is provided at NCBI GenBank Accession No.:

AAG38658.1. The sequence provided at GenBank Accession No.: AAG38658.1 is reproduced below:

1 MSPRSCLRSL RLLVFAVFSA AASNWLYLAK LSSVGSISEE ETCEKLKGLI QRQVQMCKRN 61 LEVMDSVRRG AQLAIEECQY QFRNRRWNCS TLDSLPVFGK WTQGIREAA LVYAISSAGV 121 AFAVTRACSS GELEKCGCDR TVHGVSPQGF QWSGCSDNIA YGVAFSQSFV DVRERSKGAS 181 SSRALMNLHN NEAGRKAILT HMRVECKCHG VSGSCEVKTC WRAVPPFRQV GHALKEKFDG 241 ATEVEPRRVG SSRALVPRNA QFKPHTDEDL VYLEPSPDFC EQDMRSGVLG TRGRTCNKTS 301 KAIDGCELLC CGRGFHTAQV ELAERCSCKF HWCCFVKCRQ CQRLVELHTC R

By“Wnt5a polynucleotide” is meant a polynucleotide encoding Wnt5a polypeptide or a fragment thereof. An exemplary polynucleotide sequence coding for human Wnt5a is provided at NCBI Ref: GenBank NM_003392, a reference standard sequence. Nucleotides 658-1800 of the Wnt5a genomic sequence having 6194 nucleotides codes for a human Wnt5a polypeptide. The polynucleotide sequence of the human Wnt5a coding sequence provided at bases 658-1800 of NCBI Ref: GenBank M_003392 is reproduced below:

658 atg

661 aagaagtcca ttggaatatt aagcccagga gttgctttgg ggatggctgg aagtgcaatg

721 tcttccaagt tcttcctagt ggctttggcc atatttttct ccttcgccca ggttgtaatt

781 gaagccaatt cttggtggtc gctaggtatg aataaccctg ttcagatgtc agaagtatat

841 attataggag cacagcctct ctgcagccaa ctggcaggac tttctcaagg acagaagaaa

901 ctgtgccact tgtatcagga ccacatgcag tacatcggag aaggcgcgaa gacaggcatc

961 aaagaatgcc agtatcaatt ccgacatcga aggtggaact gcagcactgt ggataacacc

1021 tctgtttttg gcagggtgat gcagataggc agccgcgaga cggccttcac atacgcggtg

1081 agcgcagcag gggtggtgaa cgccatgagc cgggcgtgcc gcgagggcga gctgtccacc

1141 tgcggctgca gccgcgccgc gcgccccaag gacctgccgc gggactggct ctggggcggc

1201 tgcggcgaca acatcgacta tggctaccgc tttgccaagg agttcgtgga cgcccgcgag

1261 cgggagcgca tccacgccaa gggctcctac gagagtgctc gcatcctcat gaacctgcac

1321 aacaacgagg ccggccgcag gacggtgtac aacctggctg atgtggcctg caagtgccat

1381 ggggtgtccg gctcatgtag cctgaagaca tgctggctgc agctggcaga cttccgcaag

1441 gtgggtgatg ccctgaagga gaagtacgac agcgcggcgg ccatgcggct caacagccgg

1501 ggcaagttgg tacaggtcaa cagccgcttc aactcgccca ccacacaaga cctggtctac

1561 atcgacccca gccctgacta ctgcgtgcgc aatgagagca ccggctcgct gggcacgcag 1621 ggccgcctgt gcaacaagac gtcggagggc atggatggct gcgagctcat gtgctgcggc

1681 cgtggctacg accagttcaa gaccgtgcag acggagcgct gccactgcaa gttccactgg

1741 tgctgctacg tcaagtgcaa gaagtgcacg gagatcgtgg accagtttgt gtgcaagtag

By“Wnt5a polypeptide” is meant a Wnt5a polypeptide or a fragment thereof, or a polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the human Wnt5a polypeptide sequence. An exemplary human Wnt5a (isoform 1) polypeptide sequence is provided at UniProtKB Identifier:

P41221-1. The sequence provided at UniProtKB Identifier: P41221-1 is reproduced below:

1 MKKSIGILSP GVALGMAGSA MSSKFFLVAL AIFFSFAQVV IEANSWWSLG

51 MNNPVQMSEV YIIGAQPLCS QLAGLSQGQK KLCHLYQDHM QYIGEGAKTG

101 IKECQYQFRH RRWNCSTVDN TSVFGRVMQI GSRETAFTYA VSAAGVVNAM

151 SRACREGELS TCGCSRAARP KDLPRDWLWG GCGDNIDYGY RFAKEFVDAR

201 ERERIHAKGS YESARILMNL HNNEAGRRTV YNLADVACKC HGVSGSCSLK

251 TCWLQLADFR KVGDALKEKY DSAAAMRLNS RGKLVQVNSR FNSPTTQDLV

301 YIDPSPDYCV RNESTGSLGT QGRLCNKTSE GMDGCELMCC GRGYDQFKTV

351 QTERCHCKFH WCCYVKCKKC TEIVDQFVCK

An“immune checkpoint protein or molecule” or“immune checkpoint” refers to a specific subtype of transmembrane protein molecule that provides fine-tuning of the immune response. In normal tissues, immune checkpoints are inhibitory signals and play an important role in immune cell function by preventing autoimmunity. In a subject with a tumor or cancer, up-regulation of immune checkpoint proteins on the tumor or cancer cells allows tumors and cancers to escape immune surveillance and evade anti-tumor immunity.

Nonlimiting examples of immune checkpoint proteins that have been the focus of clinical immunotherapeutics are cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4),

programmed cell death protein 1 (PD-l), and programmed cell death protein ligand 1 (PD- Ll). CTLA-4, also known as CD152, is essential for the activation of CD4 ⁺ T cells and the priming phase of the immune response. PD-l, also known as CD279 and formerly as B7.1, is a key immune checkpoint receptor expressed by activated T cells, B cells and myeloid cells, and mediates immunosuppression. PD-L1, also known as CD274 and formerly as B7-H1, is an immune regulatory protein that plays a significant role in suppressing the immune system during certain disease states, including cancer and autoimmune disease. PD-L1 is the cognate ligand that binds to PD-l to modulate activation or inhibition of immune cells.

Under normal circumstances, the immune system reacts to foreign antigens that are associated with exogenous or endogenous agents, e.g., microorganisms or cells, which triggers the proliferation of antigen-specific cytotoxic CD8+ T cells and/or CD4+ helper T cells. The binding of PD-L1 to PD-l transmits an inhibitory signal that reduces the proliferation of the antigen-specific T cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).

The K _d (dissociation constant), which reflects the binding affinity between PD-L1 and PD-l, is 770 nM. PD-L1 also has an appreciable affinity for the costimulatory molecule CD80 (B7-1), but not for CD86 (B7-2). The affinity of PD-L1 of CD80 is 1.4 mM, which is a value that is intermediate between the affinity of PD-L1 for CD28 and CTLA-4 (4.0 mM and 400 nM, respectively). The related molecule PD-L2 does not have affinity for CD80 or CD86, but shares PD-l as a receptor (with a stronger K _d of 140hM). PD-l is up-regulated on activated CD4 T-cells and can bind to PD-L 1 -expressing monocytes to induce the production of IL-10. (E.A. Said et al., 2010, Nature Medicine, l6(4):452-459). The interaction of PD- Ll with its receptor PD-l on T cells delivers a signal that inhibits T cell receptor (TCR)- mediated activation of IL-2 production and T cell proliferation. The PD-1/PD-L1 interaction has been implicated in autoimmunity. By way of example, NOD mice, an animal model for autoimmunity, exhibit a susceptibility to spontaneous development of type I diabetes and other autoimmune diseases and have been shown to develop a precipitated onset of diabetes from the blockade of PD-l or PD-L1 (but not PD-L2), (M.J. Ansari et al., 2003, J Exp. Med., l98(l):63-69).

By“immune surveillance” or“immunological surveillance” is meant a monitoring process by cells of the immune system to detect and destroy cells that are recognized as non- self, other, or allogeneic in the tissues and organs of the body. For example, such non-self cells may be virally-infected, mutated, neoplastically transformed, or may express a cell surface molecule that is not recognized as a self or autologous molecule by cells of the immune system.

By“progenitor cell” is meant a cell that a multipotent stem cell that is capable of generating (e.g., by differentiation or division) an endothelial cell. A progenitor cell that is capable of generating an endothelial cell may express this capability when grown under appropriate in vitro or in vivo conditions, such as those described herein. By“progeny” is meant a cell derived from a multipotent stem cell of the invention. Progeny include without limitation progenitor cells, differentiated cells, and terminally differentiated cells.

By“derived from” is meant“obtained from” or the process of obtaining a progeny cell.

By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By“reference” or“control” is meant a standard condition. For example, an untreated or healthy (nondiseased) cell, tissue, or organ that is used as a reference.

A "reference sequence" is a defined sequence used as a basis for sequence

comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, or at least about 25 amino acids. The length of the reference polypeptide sequence can be about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, or at least about 75 nucleotides. The length of the reference nucleic acid sequence can be about 100 nucleotides, about 300 nucleotides or any integer thereabout or therebetween.

A“somatic” cell refers to a cell that is obtained from a tissue of a subject. Such subjects are at a post-natal stage of development (e.g., adult, infant, child). In contrast, an “embryonic cell” or“embryonic stem cell” is derived from an embryo at a pre-natal stage of development.

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, and at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In yet another embodiment,

hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. ETseful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, at least about 42° C, and at least about 68° C. In one embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, at least 80%, at least 85%, at least 90%, at least 95% or even at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,

BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence. The term“self-renewal” as used herein refers to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells with development potentials that are indistinguishable from those of the mother cell. Self renewal involves both proliferation and the maintenance of an undifferentiated state.

The term“stem cell” is meant a pluripotent cell or multipotent stem cell having the capacity to self-renew and to differentiate into multiple cell lineages.

By "subject" is meant a mammal, including, but not limited to, a human or non human mammal, such as a non-human primate, bovine, equine, canine, ovine, rodent, or feline. In a particular embodiment, a subject is a human subject, such as a human patient.

Ranges provided herein are understood to be shorthand for all of the values within the range, inclusive of the first and last values. By way of nonlimiting example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, or 50.

By“tissue” is meant a collection of cells having a similar morphology and function.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

By“vascularized” is meant having a blood vessel. In some embodiments, the pancreatic islet organoid or pancreatic organoid is vascularized.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided and described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G provide images, a schematic diagram, and graphs related to

enhancement of the functionality of hiPSC-derived b-like cells via cellular crosstalk in polymer-based cultures. FIG. 1A (top) shows the results of a Principal Component analysis of transcriptomes from human iPSCs (hiPSCs), primary human pancreatic epithelial cells

(hPanc Epithelial), human adipose-derived stem cells (hADSCs), human pancreatic fibroblasts (hPanc Fibroblast), human umbilical vein endothelial cells (FtUVECs) and human pancreatic microvascular endothelial cells (hPanc Endothelial). FIG. 1A (bottom) shows a time course of human adipose-derived stem cell (hADSC) culture in Matrigel (1 : 1 dilution in hADSC medium, 2 million cells in 300m1) showing intrinsic self-organization (Scale bar lmm). FIG. IB shows a schematic of the generation of multicellular islet-like spheroids

(MCS) and islet-like spheroid (IS). hiPSC-derived endocrine progenitor cells (EP) were co- cultured with hADSC and endothelial cells (ECs, FtUVECs) in gellan gum-based 3D culture system (left). EPs are multipotent cells that differentiate into endocrine cells including a, b, d, e, pancreatic polypeptide and G cells, as defined by the expression of neurogenin 3, neurodl, Nkx2.2 and Pax4 biomarkers (Rezania, A. et ak, 2014, Nature Biotechnology ,

32: 1121-1133). MCS generated in the matrigel environment show the incorporation of ECs

(mCherry expression) and insulin expression as detected by Green Fluorescent Protein (GFP) expression, right). (Scale bar 100 pm). FIG. 1C illustrates multicellular islet-like spheroids

(MCS) cultured in the 3D gellan gum system showing insulin expression (GFP, upper panel).

Electron microscopy images of MCS showing insulin granules (lower right) and lipid droplets in hADSC (lower right). FIG. ID presents graphs of gene expression in sorted insulin-expressing cells (GFP ⁺ ) in islet-like spheroids (IS; hiPSC derived b-like cells generated in the absence of hADSCs and ECs), MCSs, or human islets (hislets). FIG. IE presents a graph demonstrating human c-peptide secretion in response to 3mM (G3) or 20 mM (G20) glucose from IS, MCS and hislets. FIG. IF presents a graph demonstrating random fed blood glucose levels in STZ-induced diabetic NOD-SCID mice after sham treatment or transplantation of MCS (500) or human islets. FIG. 1G presents a graph demonstrating serum human c-peptide levels during feeding, fasting, and refeeding cycles in mice from 4 weeks after transplantation. Error bars represent ± SEM. *p<0.05, **p<0.0l, ***p<0.00l.

FIGS. 2A-2F provide a heat map, graphs and plots demonstrating the expression of non-canonical Wnts in endocrine and supportive cells in human islets. FIG. 2A presents a heatmap of expression changes during hADSC culture in Matrigel. A significantly affected gene ontology category is presented at the right, namely, Wnt5a and downstream signaling (5.le -03). FIG. 2B presents a graph showing tSNE clustering of temporal expression of WNTs during hADSC self organization as shown in FIG. 2A. FIG. 2C presents a graph and heatmap showing relative expression of WNTs in human islets (n=5). FIG. 2D shows t-SNE clustering of human islet single cell transcriptomes (n= 3245). Annotated cell types are assigned based on known marker gene expression. FIGS. 2E and 2F show a single cell plot and violin plots, respectively, of WNT2B, WNT4, WNT5A , WNT7A, WNT7B and WNT9A expression in human islets. Error bar represents ± SEM.

FIGS. 3A-3K provide schematics, images, heatmaps and graphs related to the generation of human islets like organoids (HILOs) and the induction of functional maturation of HILOs by WNT4. FIG. 3A presents a schematic of human islet-like organoid (HILO) generation. FIG. 3B shows representative images of HILOs in 3D culture (left) and insulin expression (human insulin promoter driven GFP (right, scale bar lOOpm). FIG. 3C depicts electron microscopy images showing insulin and glucagon granules in b and a cells, respectively, in WNT4-treated HILOs (“wHILOs”) and human islets. Scale bar, 1 pm FIG. 3D-1 presents a heatmap of relative expression of key islets genes in hiPSCs, HILOs treated with PBS (P) or WNT4 (W), and in human islets (log ₂ expression with Z-score). FIG. 3D-2 presents plots showing the relative expression of I SLR SYT4, PDX1, GCK , NEUROD1, NKX2-2 , INSULIN NKC6-1, MAFA , MAFB and UCN3 in wHILOs and human islets as determined by qPCR (n=8 per sample type). FIG. 3E is a gene ontology map of genes that are up- and down-regulated in HILOs by treatment with WNT4 (lOOng/ml from day26 to day33). FIG. 3F shows the relative expression of ERRy , NDUFA7 and COX7A2 in HILOs treated with increasing concentrations of WNT4 (0, 10, 25, 50, 200ng/ml) for 5 days. FIG. 3G presents a heatmap of relative expressions of oxidative phosphorylation genes in 3D cultured hiPSCs, HILOs with PBS and HILOs with WNT4 treatment (wHILOs), and human islets (Z-Score). FIG. 3H is a graph demonstrating oxygen consumption rates (OCRs) measured in hiPSC spheroids on day 0 (upside down triangle), PBS treated HILOs (upright triangle), WNT4 treated HILOs (square) and human islets (circle). FIG. 31 presents a graph showing in vitro human c-peptide secretion in response to 3mM (G3) or 20 mM (G20) glucose or 20mM KC1 (K20) from HILOs generated with and without WNT4 treatment.

FIG. 3J presents a cartoon schematic depicting culture conditions for commercially available hiPSC-derived b-like cells (left) and light microscopy images of cultured cells (right). FIG. 3K presents a bar graph showing in vitro c-peptide secretion in response to 3 mM (G3) and 20 mM (G20) glucose from cultures described in FIG. 7D-2.

FIGS. 4A-4M provide plots, graphs, a microscopy image, flow cytometry results and a schematic related to studies of PD-L1 -expressing wHILOs extended functionality and glucose control in immune competent mice and immune profiling of wHILO grafts in C57BL6J mice. FIG. 4A shows tSNE clustering of single cell transcriptomes from WNT4 treated HILOs (wHILOs, n=4840). FIG. 4B is a graph showing relative cell type populations in HILOs and human islets. FIG. 4C presents a graph demonstrating random fed blood glucose levels after transplantation of wHILOs with or without PD-L1 expression (in kidney/kidney capsule of induced diabetic C57BL6J mice (n=l l and 9 mice, respectively). The top plot on the graph represents wHILOs (-); the middle plot on the graph represents wHILOs (PD-L1 expression); the bottom plot on the graph represents mislets. FIG. 4D presents flow cytometric analysis of insulin-expressing and mouse immune (CD45 ⁺ ) cells recovered from kidney capsule grafts 27 days after transplantation of wHILOs with and without PD-L1 expression. Grafts containing HILOs expressing PD-L1, which can potentially bind to PD-l on T cells (e.g., CD45+ cells), thereby suppressing T cell activation and killing activity, show fewer infiltrating CD45+ T cells compared with grafts containing HILOs that do not express PD-L1. FIG. 4E shows the quantification of the analysis of blood glucose levels in STZ-induced diabetic mice after transplantation of wHILOs with or without PD-L1 expression, as shown in FIG. 4D (Error bars represent ± SEM. *p<0.05, **p<0.0l, ***p<0.00l). FIG. 4F presents a flow cytometry analysis of insulin expressing and mouse immune (CD45 ⁺ ) cells recovered from kidney capsule grafts 27 days after transplantation of wHILOs with and without PD-L1 expression. CD45 ⁺ cells were further categorized as B cells (CDl9 ⁺ ), T cells (CD3 ⁺ ) and NK cells (NKl. l ⁺ ). FIG. 4G shows dot plots of the quantification of the analysis described for FIG. 4F (n=6 and 6). FIG. 4H shows an image of wHILO (PD-L1) cells in a kidney graft 27 days after transplantation (insulin promoter driven GFP expression). Scale bar, 100 pm Error bars represent ± SEM. *p<0.05. FIG. 41 presents a schematic showing transplantation of wHILOs with and without PD-L1 overexpression (500 HILOs per mouse) into multi low dose streptozotocin (MLD-STZ, 50mg/kg/day for 5 days) induced diabetic Hu-PBMC-NSG mice. FIG. 4J presents a flow cytometric analysis of human T cells (CD4 ⁺ and CD8 ⁺ cells in CD45 ⁺ /CD3 ⁺ population) in PBMC from Hu-PBMC-NSG mice (n=l5 mice) 3 weeks after human PBMC transplantation. FIG. 4K shows a graph of random fed blood glucose levels in MLD-STZ induced diabetic Hu-PBMC-NSG mice after transplantation of wHILOs with or without PD-L1 expression (n=6 and 6 mice). FIG. 4L shows a graph of serum human c-peptide levels in mice described in FIG. 4K. FIG. 4M presents a flow cytometric analysis of insulin-expressing and human CD45 ⁺ immune cells recovered from kidney capsule grafts 27 days after transplantation of wHILOs, with and without PD-L1 expression. FIG. 4N presents dot plot graphs that quantify the results of analyses shown in FIG. 4M. (Error bars represent ± SEM. *p<0.05, **p<0.0l, ***p<0.00l).

FIGS. 5A-5K provide graphs and schematic diagrams demonstrating that immune tolerance is induced by epigenetic memory. FIG. 5A presents a graph showing PD-L1 expression in islet (wHILOs) cells sorted by flow cytometry based on insulin expression (GFP+ and GFP-, respectively) after IFNy treatment (10 ng/ml, 12 hours). The GFP+ cells comprise b-like cells; the GFP- cells comprise alpha (a), delta (d) and epsilon (e) cells. FIG. 5B presents a graph showing temporal PD-L1 expression in wHILOs after a single IFNy treatment (10 ng/ml, 2 hours). FIG. 5C is a schematic illustrating IFNy (10 ng/ml) pulse treatment of wHILOs. (MPS treatment). FIG. 5D presents a graph showing PD-L1 expression induced by indicated cycles of IFNy treatment, 7 days after last treatment. FIG. 5E presents a graph of PD-L1 protein levels 1 and 7 days after indicated IFNy (10 ng/ml) treatments. PD-L1 overexpressing wHILOs (PDLlOE) and a single 12 h exposure to IFNy was used as a positive control. FIG. 5F presents a dot plot showing human c-peptide secretion from IFNy treated wHILOs in response to 3mM (G3) or 20mM (G20) glucose.

FIG. 5G is a schematic illustrating IFNy treatment in combination with an IL- 1 b treatment challenge (lOng/ml for 24 hours) to induce b cell dedifferentiation. FIG. 5H presents a graph showing INS and UCN3 expression after the indicated IFNy and IL- 1 b treatments (lOng/ml, 24 hours) of wHILOs. FIG. 51 presents a schematic of an experimental protocol for in vivo transplantation of wHILOs into induced diabetic animals. High dose streptozotocin (HD- STZ, l80mg/kg) induced diabetic C57BL6J mice received transplants of wHILOs that had or had not been subjected to the IFNy treatment protocol shown in FIG. 5C, ( n=6 and 6, 500 wHILOs/mouse). FIG. 5J presents a graph showing blood glucose levels in recipient mice (STZ-treated (l80mg/kg) diabetic C57BL6J mice) at day 17 following kidney capsule transplantation of wHILOs and IFNy pulse stimulated wHILO (“immune evasive wHILOs” or“wHILO ^lc ”) FIG. 5K presents a graph showing serum human c-peptide levels in mice treated as described in FIG. 51. Error bars represent ± SEM. *p<0.05, **p<0.0l.

FIGS. 6A-6F provide images, graphs and results related to multicellular spheroids (MCSs) as described herein. FIG. 6A shows a 3D gellan gum suspension of multicellular spheroids (MCS, top), light microscopy images of single MCS (lower left) and hislets (lower right). FIG. 6B shows images of insulin promoter driven GPF expression, and endothelial cells (EC, marked by mCherry expression) in MCS. FIG. 6C presents images showing the progressive development of vascular-like structures in MCSs that were cultured with endothelial growth media in the Matrigel system. FIG. 6D is a schematic for single cell RNA-seq analyses. FIG. 6E presents a heatmap of expression of the top 10 signature genes in human islet cell clusters from FIG. 2D. FIG. 6F present plots showing t-SNE_2 single cell expression of signature hormonal and cell type specific genes in human islets. Relative expression scale: low (0.5, least intense), to high (5, most intense).

FIGS. 7A-7F provide a schematic, graphs, images, and data related to the

characterization of mature HILOs. FIG. 7A depicts a diagram of CRISPR-Cas9-based knockin for human insulin promoter driven GFP expression in hiPSC. FIG. 7B presents a differential interference contrast (DIC) image of wHILOs with insulin-GFP and UCN3-RFP expression (scale bar, lOOpm). FIG. 7C presents a Seahorse analysis of extracellular acidification rate (ECAR) measured in day 0 hiPSC spheroids (open square), HILOs (Vehicle/PBS-treated, filled triangle), wHILOs (Wnt4 treated, filled circle) and human islets (open circle). 20 mM glucose (Glu), oligomycin (Olig), Fccp, antimycin + Rotenon

(Ant+Rot) were treated in order. FIG. 7D-1 presents a graph showing the kinetics of human c-peptide secretion from WNT4 treated HILOs in response to progressive exposure of the HILOs to 3 mM glucose, 20 mM glucose, 20 mM glucose + 100 mM GLP-l, 3 mM glucose, and 3 mM glucose + 20 mM KC1 over time. FIG. 7D-2 presents a bar graph showing glucose stimulated human c-peptide secretion from wHILOs treated with and without XAV939 to promote b-catenin degradation (XAV939, ImM for 3 days). FIG. 7E presents data illustrating motif enrichment in chromatin regions with enhanced accessibility upon WNT4 treatment. FIG. 7F depicts chromatin accessibility at ERR target genes (determined by ATAC-Seq) in insulin expressing cells sorted from HILO treated with PBS or WNT4 for 7 days (fold change>l.5).

FIGS. 8A-8H provide images, graphs, a schematic and a diagram showing results related to WNT4 mediated insulin-GFP expression and WNT4-driven metabolic maturation. FIG. 8A presents representative images of mitochondrial content, indicated by MitoTracker (red) staining, in PBS and WNT4 treated HILOs (scale bar, lOOpm). FIG. 8B presents graphs of flow cytometry quantification of insulin expression (GFP) and mitochondrial content in HILOs treated with recombinant human WNT4 (rhWNT4), WNT5A (rhWNT5A), or conditioned medium (CM) from control or WNT5A overexpressing fibroblasts (n= 3). Error bars represent ± SEM. *p<0.05. FIG. 8C presents a gene ontology of transcriptional changes induced by WNT4 treatment (lOOng/ml WNT4 from day26 to day33) in HILOs.

FIG. 8D presents a graph demonstrating blood glucose levels in STZ-induced diabetic NOD- SCID mice after transplantation (TP) of 500 wHILOs or hislets, or sham surgery was performed at day 3 (n = 7, wHILOs; n =6, hislets; n = 3, Sham). Error bars represent ± SEM. *p<0.05. FIG. 8E presents a Venn diagram showing overlap between WNT4-induced increases in chromatin accessibility in GFP ⁺ cells and increases in HILO gene expression (upper panel), and gene ontology pathways enriched in the intersection gene set. FIG. 8F shows motifs that are enriched in the intersection gene set shown in FIG. 8E. FIGS. 8G and 8H demonstrate the results of experiments in which postnatal islets (day Pl 1-14) from WT and b cell specific ERRyKO mice were cultured with or without rhWNT4 (lOOng/ml) for >5 days. FIG. 8G shows relative gene expression measured by qPCR, and FIG. 8H shows insulin secretion in response to 3mM and 20mM glucose. *p<0.05, ***p<0.00l. For FIGS. 8G and 8H, postnatal islets (day Pl 1-14) from WT and b cell specific ERRyKO mice were cultured with or without rhWNT4 (lOOng/ml) for >5 days.

FIGS. 9A-9M provide microscopy (confocal) images, plots, heatmaps and graphs demonstrating immunofluorescent characterization of wHILOs, flow cytometry analysis of HILOs, and single cell analysis of wHILOs. FIGS. 9AB, 9C and 9D present confocal images of wHILOs stained for C-peptide. FIG. 9A shows representative immunofluorescent staining results for glucagon, somatostatin and pancreatic polypeptide (PP) in wHILOs. FIG. 9B presents confocal images of wHILOs stained for C-peptide. FIG. 9C presents confocal images of wHILOs stained for b cell enriched markers NKX2-2, NKX6-1, MAFA, MAFB, PDX1. Images are representative of three independent experiments. FIG. 9D presents confocal images of wHILOs stained for endocrine markers chromogranin A (CHGA), Synaptophysin (red, middle images) with Insulin-GFP (green, left images) visualization. Hoechst nuclei staining is shown in the right (Merge) panels. Scale bar, 100 pm. Images are representative of three independent experiments. FIG. 9E shows representative flow cytometry results for b cell and endocrine marker co-staining in HILOs with and without WNT4 treatment. FIG. 9F graphically depicts the quantification of results presented in FIG. 9E (n=6 and 6). FIG. 9G shows tSNE clustering of single cell transcriptomes from WNT4 treated HILOs (wHILOs, n=4840). FIGS. 9H and 91 show Violin Plots (9H) and single cell expression (91) of INS, CHGA , SOX9 , HES1 in wHILOs. FIG. 9J shows expression of b cell-enriched (INS, PDX1, NKX6-1, NKX2-2 , NEUROD1 , NPTX2, ITGA1, PCSK1 , MAFA , MAFB , UCN3, CHGA), a cell-enriched ( GCG , ARX) and d cell-enriched genes (AST, RBP4) overlaid on tSNE clustering. FIG. 9K presents a heatmap of the top 10 differentially- expressed genes in each cell cluster. FIG. 9L presents tSNE clusters according to cell type (Pane P = pancreatic progenitor, Rep = replicating, UK = unknown). FIG. 9M presents tSNE clustering of combined HILOs and wHILO single cell data sets (right panel) and clustering analysis-defined cell types.

FIGS. 10A-10C provide plots showing quality analyses of scRNA-seq. FIG. 10A shows plots illustrating a correlation of number of detected genes and UMIs in HILO, wHILO and human islets. FIG. 10B presents tSNE clustering of combined wHILO (blue dots, n=4840) and human islet (red dots, n= 3245) single cell transcriptomes (left panel) and clustering analysis-defined cell types (left). FIG. 10C shows the expression of endocrine specific genes (INS, NKX2-2, GCG, SST, PPY), duct marker ( KRT19 ) and stellate cell marker (ACTA2) in tSNE visualization of merged single cell data sets for wHILO and hislets.

FIGS. 11A-11D provide a schematic depiction, graphs and plots related to plate based scRNA-seq analysis. FIG. 11A is a scheme of plate based scRNA-seq. Dissociated single cells from wHILO were sorted by FACS into 96 well tissue culture plate (microplate). FIGS. 11B and 11C: A box plot showing average gene counts per cells (FIG. 11B) and

identification of 45 single cells with high quality gene detection (FIG. 11C). FIG. 11D illustrates that single cell RNA-seq revealed single hormone expressing insulin, glucagon, somatostain cells in wHILOs. FIGS. 12A-12F provide graphs and images related to PD-L1 gene and protein expression in b cells and HILOs. FIG. 12A (left) shows tSNE endogenous expression of PD- L1 in human islet cells (b cells are outlined), and (right) a heatmap of the top differentially expressed genes between PD-L1+ and PD-L1- b cells. FIG. 12B presents

immunohistochemistry results overlap of lentiviral-driven PD-L1 expression and insulin promoter-driven GFP expression in wHILOs (scale bar, lOOpm). FIG. 12C presents bar graphs showing human PD-L1 expression (left) and human insulin expression (right) in wHILOs, with and without lentiviral PD-L1 overexpression, as measured by qPCR. FIG.

12D (top) presents a schematic depiction of an in vivo experimental study conducted in induced diabetic C57BL6J mice. High dose streptozotocin (HD-STZ, l80mg/kg) induced diabetic C57BL6J mice received transplants of wHILOs with and without PD-L1

overexpression (n=500), or mouse islets; FIG. 12D (bottom) shows results following transplantation of PD-L1 -overexpressing wHILOs into the kidney capsule of STZ-induced diabetic mice. FIG. 12E presents a bar graph showing PD-L1 expression in wHILOs 12 hours after indicated IFNy stimulation. Error bars represent ± SEM. ***p<0.00l. FIG. 12F presents a bar graph showing PD-L1 gene expression in human islets 12 hours after INFy (ng/ml) stimulation. Error bars represent ± SEM. ***p<0.00l.

FIG. 13 provides a schematic diagram of the strategy for generation of mature, immune evasive wHILOs (wHILO ^ie s).

FIGS. 14A-14D present a Venn diagram, heatmap, gene ontology chart and browser track related to studies investigating IFNy-induced changes in wHILOs. FIG. 14A shows a Venn diagram of differentially regulated genes upon acute (l2h at 10 ng/ml) and multi pulse stimulated (MPS), (2h at 10 ng/ml for 3 days) IFNy treatment of wHILOs. In the diagram, the leftmost circle represents“MPS IFNy treatment” and the rightmost circle represents “acute IFNy treatment.” FIG. 14B shows a heatmap of differentially expressed genes upon acute and MPS IFNy stimulation. Sustainable PD-L1 genes expression by MPS are highlighted. FIG. 14C shows gene ontology of selectively regulated genes upon MPS- IFNy (top panel) and acute IFNy (bottom panel) treatments. FIG. 14D shows panels of browser tracks indicating chromatin accessibility at selected genes 7days after the last IFNy treatment in the MPS method, or 12 hours after acute IFNy stimulation in wHILOs.

FIGS. 15A-15C present a schematic, graph and flow cytometry plots related to studies demonstrating the immune evasiveness of wHILOs by enhanced endogenous PD-L1 expression. FIG. 15A shows a schematic of a treatment regimen involving multi low dose streptozotocin treatment (MLD-STZ, 50mg/kg/day for 5 days) of Hu-PBMC-NSG mice to produce an immune competent diabetic animal model. MPS induced PD-L1 expressed wHILOs (n=500) were transplanted under kidney capsule. FIG. 15B shows a graph of random fed blood glucose levels in STZ-induced diabetic Hu-PBMC-NSG mice after transplantation of wHILOs that had undergone or had not undergone MPS (n=6 mice, respectively). wHILOs (-) data from FIG. 4K and FIG. 4G are replicated, since those experiments were performed parallely. FIG. 15C shows a flow cytometry analysis of insulin-expressing and human immune (CD45 ⁺ ) cells recovered from kidney capsule grafts 27 days after transplantation of wHILOs with or without MPS. Error bars represent ± SEM. *p<0.05, **p<0.0l, ***p<0.00l.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Featured herein are methods and systems for the generation and utilization of stem cell-derived human islets and human islet-like organoids, which provide a promising strategy for the therapeutic treatment of diseases and pathologies, such as pancreatic diseases and insulin dependent diabetes, a disease caused by the loss of endogenous insulin-producing b cells. Advantageously, the methods and systems as described can generate biological products, e.g., cells, human islet-like organoids and cells thereof, as therapeutics that can alleviate the shortage of donor-matched cadaveric human islets, which are currently being used to treat patients.

As described herein, functional human islet-like organoids (HILOs) are generated from human pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). In an embodiment, a culture system which allows for non-canonical WNT4 signaling is employed to generate HILOs. Without wishing to be bound by theory, WNT4 signaling in cells such as iPSCs, human islet and HILO cells drives metabolic maturation necessary for robust glucose stimulated insulin secretion (GSIS). The stem-cell derived islets and HILOs as described herein achieve functional maturity and exhibit robust, glucose-stimulated insulin secretion (GSIS) through enhanced glucose-responsive oxidative capacity, which is regulated by the WNT4-ERR (Estrogen-Related Receptor) metabolic pathway. The functionally mature HILOs contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic NOD-SCID mice (e.g., Examples 4 and 5). In an embodiment and as described herein, the HILOs and cells thereof avoid rejection by immune cells under immune-competent conditions.

In an aspect, single cell RNA (scRNA)-sequencing analysis of functional HILOs, as well as human cadaveric islets, revealed transcriptional heterogeneity of HILO -derived cells, including a small population of immune-evasive b cells. As described in an aspect herein, HILOs were molecularly engineered to express a checkpoint protein, e.g., PD-L1, in order to mimic the transcriptional program of immune-evasive b cells. When the PD-L1 -expressing HILOs were assessed, it was found that PD-L1 expression overcame autoimmune rejection of the HILOs, which had been transplanted in immune-competent mice with type 1 diabetes. Thus, the generation, in a scalable fashion, of functional b cells and HILOs that can avoid immune detection, autoimmune activation, and transplant or implant rejection afford advantageous and beneficial treatments and therapies for diabetes, in particular, type 1 diabetes and late stage type 2 diabetes. In an embodiment, b cells, human HILOs and human islets are molecularly engineered (e.g., transduced or transfected) to express a checkpoint protein such as PD-L1. In an embodiment, b cells, human HILOs and human islets are induced to express the PD-L1 protein as described herein.

Methods of protecting islets, organoids and the cells therein from immune surveillance and immune cell killing and clearance

In an aspect, methods, particularly in vitro or ex vivo methods, are provided for generating islets and organoids, including the cells therein, (e.g., donor cells, islet and organoid cells) that survive, have reduced cell death and/or can better evade immune detection by cells of the immune system, especially after transplantation, implantation, or transfer into a subject, such as a recipient individual. In an embodiment, the transplantation, implantation, or transfer involves allogeneic cells, islets, and/or organoids that survive and have reduced killing and detection by immune cells, e.g., T cells, B cells, monocytes, macrophages and the like, subsequent to the practice of the methods described herein.

In an aspect, the expression (or upregulated expression) of a checkpoint protein encoding gene and/or its encoded product, in particular, PD-L1 and/or the PD-L1 protein, in or by IFNy receptor-expressing islets, organoids (e.g., HILOs), or cells (e.g., b cells of HILOs) following multiple intermittent exposures to interferon gamma (IFNy) over a given time period (such as at least 24 hours) allows the HILOs to maintain glucose homeostasis, e.g., in immune-competent diabetic mice for a long time period, e.g., at least 50 days, as well as to evade an immune response by activated T cells and/or graft rejection. In an embodiment, the islets, organoids, or cells are human islets, organoids, or cells. In embodiments, such islets, organoids, or cells express IFNy receptors and/or are responsive to treatment with IFN g. In an embodiment, the islets, organoids, or cells naturally express IFNy receptors. In an embodiment, IFNy receptors may be introduced into the islets, organoids, or cells, for example, without limitation, by recombinant, viral, or molecular biology techniques as known and practiced in the art. In an embodiment, PD-L1 gene and/or protein expression (or upregulated expression) in the IFNy receptor-expressing islets, organoids, and cells constitutes a detectable marker, which is indicative of the response of the islets, organoids, and cells to IFNy exposure. PD-L1 expression or upregulated expression of PD-L1 as a marker of IFNy responsiveness following exposure of islets, organoids, and cells to IFNy may be assayed by polynucleotide and/or protein detection methods routinely used and known in the art, and are not intended to be limiting.

In embodiments, the method comprises stimulating the cells with interferon gamma (IFNY) in low amounts or doses, e.g., 0.5-100 ng/ml, 1-50 ng/ml, 1-25 ng/ml, 1-20 ng/ml, 1- 10 ng/ml, 10 ng/ml or 20 ng/ml. In an embodiment, this is achieved by subjecting the islets, organoids, and/or cells, e.g., HILOs, to IFNy for discrete time periods, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 hours, or more, in particular, for about or equal to 2 hours or 12 hours, for example, multiple times, e.g., 2 times, 3 times, 4 times, 5 times, 6 times or more, over a given time period. In some embodiments, the multiple exposures or pulses are performed over at least a 24-hour period of time (about 1 day), a 48-hour period, a 72-hour period, or over the course of 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the cells are exposed to IFNy for a total of 0.5-3 hours, 0.5-4 hours, 0.5-5 hours, 0.5-6 hours, 0.5-7 hours, or 0.5-10 hours. Between IFNy exposures or pulses the cells are allowed to‘rest,’ e.g., in culture medium or 3D matrix, in the absence of IFNy between the time periods of exposure to IFNy. In some embodiments, the cells are allowed to‘rest’ in the absence of IFNy for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours between exposure to IFNy. In other embodiments, the cells are allowed to‘rest’ in the absence of IFNy for about 1, 2, 3, 4 or 5 days. In one embodiment, the IFNy treatment causes a constitutive (prolonged) upregulation and expression (and maintenance) of PD-L1 expression in the islets, organoids, and/or cells, e.g., HILOs. This procedure involves multiple pulse stimulation (MPS), also referred to as intermittent exposure, of cells, islets, organoids, e.g., HILOs or islets and the cells therein, to IFNy Expression of PD-L1 by the cells, islets, and/or organoids, is long-lasting following MPS, particularly, if the islets, organoids, and/or cells (e.g., HILOs) experience at least 3 pulses or intermittent exposures to IFNy (e.g., 10 ng/ml) for about or equal to a 2-hour time period per pulse of IFNy For example, by this regimen, sustained expression of PD-L1 is found in the islets, organoids and/or cells, e.g., HILOs, for at least 7 days following subjecting the islets, organoids and/or cells, e.g., HILOs, to the MPS procedure. In an embodiment, islets, organoids, (e.g., HILOs), or cells generated by the method survive in a recipient subject following transplantation, implantation, or transfer for at least about or equal to 50 days.

Without wishing or intending to be bound by theory, the MPS IFNy exposure procedure results in PD-L1 expression (or upregulation of PD-L1 expression) in islets, organoids and/or cells (e.g., HILOs and the cells therein (e.g., b cells)), which involves a mechanism of transcriptional memory. The described procedure comprising MPS IFNy exposure of cells, islets, and/or organoids may stimulate or create an intracellular signaling cascade in which the de-differentiation of the cells, islets and/or organoids is inhibited or blocked. The short pulses of IFNy (MPS IFNy) to which the cells, islets or organoids are exposed in the methods may ultimately involve an alteration of chromatin structure, thereby protecting the cells, islets or organoids from de-differentiation and affording the MPS IFNy exposed cells, islets, or organoids, with the ability to survive (e.g. by reduced cell death by cells of the immune system), as well as to be immune to the effects of inflammatory cytokines and chemokines, e.g., Interleukin- 1B (IL-1B) as described infra, so as to provide an anti-inflammatory effect for the cells, islets, or organoids. The absence or reduction of inflammation associated with MPS IFNy exposed cells, islets, or organoids generated from the described methods may enhance their potential for survival and reduction in killing by immune cells post transplantation, implantation, or transfer into a subject. The described methods thus generate donor cells, islets and organoids that have improved survival and retain their functionalities following transplant, implant, or transfer into a subject and offer a number of beneficial advantages in their use as therapeutics.

In a particular embodiment, a method is provided for generating human islets, organoids (e.g., HILOs) and various primary or differentiated cells (of different lineages) that survive, have reduced cell death, and can better evade immune detection or autoimmunity in which the method involves (a) contacting the human islets, organoids (e.g., HILOs), or cells with interferon gamma (IFNy) for greater than one hour at a predetermined time point;

repeating step (a) at least about two times during a given time period, e.g., a time period of about or equal to 72-hours; wherein the human islets, organoids (e.g., HILOs), or cells are maintained in the absence of IFNy between times of contact with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in the human islets, organoids (e.g., HILOs), or cells. In an embodiment of the method, the human islets, organoids (e.g., HILOs), or cells are contacted with IFNy for a time period of about or equal to at least 1 hour, or at least 2 hours, or more than 2 hours in step (a). In a particular embodiment of the method, the human islets, organoids (e.g., HILOs), or cells are contacted with IFNy for a time period of about or equal to 2 hours or about or equal to 12 hours in step (a). In another particular embodiment of the method, step (a) is repeated three times for at least about or equal to 2 hours each time in the given time period, e.g., an about or equal to 72-hour time period. In another embodiment of the method, the human islets, organoids (e.g., HILOs), or cells are washed to remove the presence of IFNy between step (a) and step (b). In another embodiment of the method, IFNy is used in an amount of 1-25 ng/ml. In another embodiment of the method, IFNy is used in an amount of 10 ng/ml. In another embodiment of the method, PD-L1 expression in the human islets, organoids (e.g., HILOs), or cells is maintained following step (b) for greater than about or equal to 7 days. In an embodiment, the method generates human cadaveric islets (e.g., syngeneic or allogeneic) that are protected from destruction or clearance by the immune system.

In another particular aspect, a method of generating various cells, islets, or organoids (e.g., HILOs), including human cells, islets, or organoids, that survive, have reduced cell death, and/or evade immune detection or autoimmunity is provided in which the method involves (a) contacting the cells, human islets, or organoids (e.g., HILOs) with interferon gamma (IFNy) in an amount of about 1 ng/ml to 25 ng/ml for greater than 1 hour at a first time point during a given time period, e.g., a time period of about or equal to 24-hours; and (b) contacting the cells, human islets, or HILOs with IFNy in an amount of about 1 ng/ml to 25 ng/ml for greater than about or equal to 0.5 hours or more, or about or equal to 1 hour at at least two additional time points during a following time period, e.g., a 48-hour time period, following step (a); wherein the islets or organoids (e.g., HILOs) are washed and rested in medium in the absence of IFNy between being contacted with IFNy; and wherein steps (a) and (b) induce sustained expression of PD-L1 in the islets or organoids (e.g., HILOs). In a particular embodiment of the method, the cells, islets, or organoids (e.g., HILOs) are contacted with IFNy in an amount of 10 ng/ml for at least 2 hours in step (a) and step (b). In another particular embodiment of the method, the cells, islets, or organoids (e.g., HILOs) are contacted with IFNy for at least about or equal to 2 hours at 3 time points (different time points) during a 72-hour time period.

The practice of the above-described methods for immune evasion of IFNy receptor expressing islets, organoids, and cells provide advantages for such islets, organoids and cells, particularly, human cells, islets and organoids, used for transplants, implants, or transfer from one subject to another as therapeutics and therapeutic treatment of diseases, disorders and pathologies. The practice of the described methods provides immunoprotection and enhanced survival of islets, organoids and cells that are transplanted, implant, or transferred into a recipient subject (e.g., an adoptive recipient, transplant recipient, and the like), such that the transplanted, implanted, or transferred islets, organoids, or cells are maintained and are functional in the recipient for several days, or weeks, or longer, for example, for about 2 days or longer to 1, 2, 3, 4, or more weeks, or longer.

The methods and systems described herein are suitable for use with a variety of cells and cell types, or donor cells for transplantation, particularly, IFNy receptor-expressing cells, derived from different lineages, as well as islets, and organoids, e.g., to provide immune protection after transplant, implant, administration or transfer into a recipient subject. In general, by way of nonlimiting example, stem cells, primary cells, differentiated cells of various lineages and types, or cells of one type derived from cells of a different source may be used. In embodiments, such suitable cells express IFNy receptors and/or are responsive to treatment with IFN /may be used in accordance with the above-described methods.

Responsiveness to IFNy treatment in the described methods may be determined or identified by assaying for detectable expression of PD-L1 or the PD-L1 protein by the IFNy receptor expressing cells, islets, or organoids (and cells therein).

By way of particular, yet nonlimting, example, the methods described herein, which involve induction of sustained PD-L1 expression by IFNy MPS, may be suitable or applicable for use with a variety of cells and cell types, or donor cells for transplantation, including, without limitation, cardiac cells, colon cells, kidney cells, bladder cells, liver cells (hepatocytes), gastrointestinal cells, gastric (stomach) cells, lung cells, ovarian cells, cervical cells, uterine cells, testicular cells, pancreatic cells, pancreatic b cells, muscle cells, hematopoietic cells, immune cells (B cells, T cells), retinal cells, corneal cells, brain cells, chimeric antigen receptor-T cells (CAR-T cells), bone marrow cells, e.g., mononuclear cells, neurons, neuronal cells, insulin-producing pancreatic b cells derived from human skin cells (e.g., as reported by Li, K. et al., 2014, Cell Stem Cell, l4(2):228- 236); umbilical cord blood (UCB) cells, adipose derived mesenchymal stromal (stem) cells, cardiac stem cells, colon stem cells, kidney stem cells, liver (hepatocyte) stem cells, gastrointestinal stem cells, gastric (stomach) stem cells, lung stem cells, pancreatic stem cells, pancreatic b stem cells, muscle stem cells, hematopoietic stem cells, immune cell (T cell or B cell) stem cells, bone marrow stem cells, CD133+ stem cells, CD34+

hematopoietic cells, CD34+ stem cells, mesenchymal stem cells, umbilical cord

mesenchymal stem cells, retinal stem cells, neuronal stem cells, and the like, as well as islets and organoids generated from or containing such cells. By way of example, the following types of organoids are suitable for use in the methods: intestinal organoids, hepatic organoids, neural organoids, pulmonary organoids, for example, as may be produced using art-described procedures, or commercially available, e.g., Stemcell™

Technology, Cambridge, MA.

Other suitable cells are those derived from embryonic stem cells which give rise to various differentiated cell types, for example, ectoderm-derived cells, such as neuronal cells, dopaminergic neuronal cells (e.g., immortalized dopaminergic neuronal precursor cells (LUHMES) commercially available from abm, Vancouver, British Columbia); corneal- derived cells (e.g., normal human corneal epithelial cells, commercially available from LifeLine Cell Technology, Oceanside, CA); endoderm-derived cells, such as liver cells (e.g., human hepatocytes wild type, available from DefmiGEN, Cambridge, UK); and mesoderm- derived cells, such as muscle cells, bone marrow cells, kidney cells and skeletal muscle cells (e.g., human skeletal muscle cells (skMDC), commercially available from Cook MyoSite®, Pittsburgh, PA). Nonlimiting examples of b cells (e.g., having pancreatic b-cell

characteristics/function) or islets which may be used in the described methods may be found, for example, in WO 2016/100898, WO 2016/100909, WO 2016/100921, WO 2016/100925, WO 2016/100930, WO 2014/145625.

Accordingly, the methods, systems and compositions as featured and described herein are useful and applicable for generating cells, tissues and organoids, which exhibit long- lasting viability and functional activity following administration, e.g., via transplantation, implantation, injection, and the like, to a subject in need thereof, based on the sustained expression of a checkpoint protein, such as PD-L1 by the cells, tissues and organoids, and their resultant evasion of and protection from immune surveillance and destruction by cells of the immune system, e.g., as occurs in graft versus host reaction.

In a particular aspect, the methods, systems and compositions as featured and described herein are useful for generating in vitro scalable, functional, vascularized organoids, particularly human pancreatic or pancreatic islet organoids (HILOs), that can evade immune detection following transplantation or implantation. In an embodiment, the culturing of iPSC-derived beta-like cells, which express an immune checkpoint protein, with human adipose-derived stem cells (hADSC) and human umbilical vein endothelial cells (Huvec) in a three-dimensional matrix containing gellan gum generated functional pancreatic and pancreatic islet organoids is also provided.

The HILOs generated in accordance with the described methods were vascularized and exhibited functional properties, such as glucose-stimulated insulin secretion (GSIS). While recent studies have reported the possibility of generating glucose-responsive, insulin- producing, beta-like cells from human Pluripotent Stem Cells (PSCs), the generation of functional, vascularized pancreatic islets organoids from PSCs that secrete insulin, glucagon and somatostatin in response to nutrients and that are capable of evading immune detection and graft or transplantation or implantation rejection by cells of the immune system for substantial periods of time is advantageously provided herein.

As described herein, the self-organizing function of human adipose-derived stem cells (hADSC), HUVEC, and human iPSC-derived beta-like cells allows for the in vitro generation of glucose-responsive insulin secreting islet-like organoids with the ability to form functional vasculature. In addition, successful scaling of islet-like organoids production through the use of Gellan gum based 3D culture systems is achieved. Using a Gaussia luciferase reporter to measure insulin secretion, the functional heterogeneity in hiPSC-derived islet-like organoids was characterized. Without intending to be bound by theory, results herein suggest that the human islet-like organoids (HILOs) which express a checkpoint protein may offer a beneficial therapeutic treatment for diabetes and a new treatment for organ failure, as well as a platform for drug screening, genome editing, and the modeling of organogenesis and pathogenesis of diabetes. Immune checkpoint proteins

Maintaining immune homeostasis is critical for host survival. Overt or uncontrolled immune responses to pathogens or to mutated, modified, or over-expressed self-antigens can cause inflammatory tissue damage and autoimmune diseases. To prevent this, the breadth and magnitude of the immune response is regulated by a balance between co-stimulatory and inhibitory signals. These signals are collectively referred to as immune checkpoints, which are necessary for maintaining self-tolerance and protecting a subject from tissue damage.

Activated T cells are the primary mediators of immune effector functions and as such, they express multiple co-inhibitory receptors such as, e.g., lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-l) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). These immune checkpoint molecules have been shown to modulate T cell responses to‘self proteins, as well as to chronic infections and tumor antigens. Of note, the pathways utilized by these checkpoint proteins are unique and non-redundant, thus, reflecting the important role of immune checkpoints in regulating immune homeostasis,

As noted supra , an immune checkpoint protein” or“immune checkpoint molecule,” or simply,“checkpoint protein or molecule” is a protein or molecule that regulates the immune system and frequently binds to or interact with ligands (cognate ligands), which may cause a given effect, e.g., cell stimulation, anergy, or apoptosis. In an embodiment, the immune checkpoint protein is one that binds a cognate ligand (e.g., a receptor ligand) on the membrane surface of an immune cell, e.g., a T cell surface receptor. In a specific

embodiment, an immune checkpoint protein is PD-L1 or a binding portion thereof, where the cognate ligand of PD-L1 is PD-l, e.g., as expressed on the surface of T cells. In an embodiment, the checkpoint protein is the extracellular domain of the protein.

In an aspect, a checkpoint protein binds to its cognate ligand, which may also be a checkpoint protein receptor on an immune cell, such as a T cell, and blocks or interrupts signaling, activity, or function of the cell that expresses the cognate ligand or receptor.

Alternatively, immune checkpoint inhibitors, which include antibodies and fragments of the antibodies that retain binding to checkpoint proteins, can bind to checkpoint proteins on cells, such as immune cells (e.g., effector T cells) and block or interrupt signaling, activity, or function of the cell. The binding of a checkpoint protein inhibitor to a checkpoint protein expressed on a cell can cause inactivation of the normal activity of the cell expressing the checkpoint protein. In embodiments, a checkpoint protein inhibitor is an antibody, such as a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, etc., or a fragment thereof that binds to a checkpoint protein (cognate ligand).

Nonlimiting examples of immune checkpoint proteins, or cognate ligand binding portions thereof, that may be expressed in a cell, an iPSC, beta-cell, and the like, or an organoid, e.g., HILOs and other organoids as described herein, include PD-l, programmed cell-death protein 1, PD-L1, programmed cell-death ligand 1, which is the cognate binding ligand of PD-l; PD-L2, programmed cell-death ligand 2, which also binds PD-l; CTLA-4 (cytotoxic T-lymphocyte protein 4, also called CD152); LAG-3, lymphocyte activation gene 3 protein; KIR, killer cell immunoglobulin-like receptor; IDOl, indoleamine 2,3-dioxygenase 1; 4-1BB, a tumor necrosis factor receptor superfamily member 9, (also known as CD137); 4- 1BBL (binds to 4-1BB); GITR,“glucocorticoid-induced TNFR family related gene; TIM-3, “T-cell immunoglobulin domain and mucin domain;” 0X40, tumor necrosis factor receptor superfamily member 4, (also known as CD 134); OX40L (binds to 0X40), CD40, CD40L, A2AR, adenosine A2A receptor; B7-H3 (also called CD276); B7-H4 (also called VTCN1); B7-1/B7-2; BTLA (also called CD272); VISTA,“V-domain Ig suppressor of T cell activation;” and the like.

In embodiments, the immune checkpoint protein molecule is, without limitation, PD- Ll or the extracellular domain of PD-L1, which binds to PD-l expressed by T cells. In an embodiment, a polynucleotide encoding an immune checkpoint protein is utilized to molecularly engineer a cell to express a checkpoint protein, or one or more checkpoint proteins, such as by infecting the cell with a viral or bacterial vector containing the checkpoint protein-encoding polynucleotide. In some embodiments, a cell (e.g., a beta-cell, or HILO cell) expresses more than one immune checkpoint protein, or a ligand binding portion thereof. In some embodiments, the cell is molecularly engineered to contain one, or more than one immune checkpoint protein, or ligand binding portion thereof, which is expressed by the cell. In an embodiment, the cell is infected with a viral vector, e.g., a lentiviral vector or adeno-associated viral vector, or more than one viral vector, that contains one or more polynucleotide(s) that encode(s) one or more immune checkpoint proteins or a ligand binding portion thereof, using procedures and methods that are well-known in the art. In an embodiment, the cell is transformed or transfected with a plasmid vector, or more than one plasmid vector, that contains one or more polynucleotide(s) that encode(s) one or more immune checkpoint proteins or a ligand binding portion thereof, using procedures and methods that are well-known in the art.

PD-l, the Programmed Death 1 (PD-l) protein, is a key immune checkpoint protein (receptor protein) that is expressed by activated T cells, as well as B cells, antigen presenting cells (APCs) and natural killer cells (NK cells) and mediates immunosuppression. PD-l functions mainly in peripheral tissues where T cells may encounter the immunosuppressive PD-l ligands PD-L1 (B7-H1) and PD-L2 that are expressed by other cells, such as cells molecularly engineered to express PD-L1, as well as, e.g., tumor cells, stromal cells, or both. Without intending to be limited by theory and by way of particular, nonlimiting example, PD- Ll expressed by transplanted, implanted, or engrafted beta(P)-cells, organoid cells, including HILO cells as described herein, binds to PD-l expressed by effector T cells, thus effectively suppressing a T cell response directed against the beta-cells, organoid cells, or HILO cells and mediating the normal T cell response so as to tamp down or block autoimmunity and inactivate the immune response against the beta-cells, organoid cells, or HILOs. In an embodiment, the beta-cells, organoid cells, or HILOs express the immune checkpoint protein in situ , in the localized area of a transplant, implant, or graft; therefore, the ability of the cells and HILOs to evade autoimmunity occurs in and around the localized area of the transplant, implant, or graft and results in less risk of a systemic or more widespread modulation of immune cell activity in a recipient subject.

Pancreas

In some aspects, a pancreatic organoid or a pancreatic islet organoid, also called a human islet-like organoid, or HILO, herein, is provided. The pancreas is an organ that lies in the abdomen and has endocrine and exocrine functions. The portion of the pancreas having an endocrine role are cell clusters called“pancreatic islets” (also known as islets of

Langerhans). Pancreatic endocrine secretions include hormones that regulate glucose metabolism and blood glucose concentration. Four main cell types are present in the islets: alpha cells, which secrete glucagon (a hormone that increases blood glucose concentration); beta cells, which secrete insulin (a hormone that decreases blood glucose concentration); delta cells, which secrete somatostatin (a hormone that regulates alpha and beta cells), and gamma cells, which secrete pancreatic polypeptide.

The portion of the pancreas that has an exocrine role is referred to as the exocrine component. The exocrine pancreatic secretions contain digestive enzymes that pass into the small intestine and help break down carbohydrates, proteins, and lipids. The exocrine component has ducts arranged in clusters called pancreatic acini. Pancreatic exocrine secretions are secreted into the lumen of the acinus; the secretions accumulate and drain into the pancreatic duct and duodenum.

Pancreatic islet organoids, pancreatic organoids and HILOs as described herein mimic the structure of a pancreatic islet and a pancreas, respectively. In some embodiments, the pancreatic islet organoid or pancreatic organoid contains any one or more of the following cells: an iPSC-derived beta-like cell, an iPSC-derived alpha-like cell, an iPSC derived delta- like cell, and an iPSC-derived duct-like cell. In some embodiments, the pancreatic organoid contains an iPSC-derived exocrine component. In some embodiments, the iPSC is a human iPSC (hiPSC). Human embryonic stem cells and human induced pluripotent stem cells are commercially available (e.g., from WiCell, which provides iPS(IMR-90)-l, iPS(IMR-90)-4 and iPS(Foreskin)-l). Human induced pluripotent stem cells can also be generated using methods known in the art from a variety of somatic cell types (Yu, J., K. Hu, et al. (2009). "Human induced pluripotent stem cells free of vector and transgene sequences." Science , 324(5928): 797-801).

Pancreatic islet organoids, pancreatic organoids and HILOs as described herein also exhibit function(s) of a pancreatic islet and a pancreas. In certain embodiments, the pancreatic islet organoid or pancreatic organoid exhibits any one or more of the following functions: glucose-stimulated insulin secretion (GSIS), KCl-stimulated insulin secretion, GLP-l stimulated insulin secretion, somatostatin secretion, and glucagon secretion. In some embodiments, the pancreatic islet or pancreatic organoid expresses any one or more of the transcription factors Pdxl, MafA, Pax4, Pax6, NeuroDl, Nkx6-l, Gata6, and Foxa2. In some embodiments, the HILOs express a checkpoint protein, or a functional portion thereof, that functions to allow the HILOs to evade immune detection and destruction by cells of the immune system. In some embodiments, the HILOs express more than one type of checkpoint protein or molecule, or a functional portion thereof.

Generation of Pancreatic and Pancreatic Islet Organoids

In other aspects, methods of generating a pancreatic or pancreatic islet organoid are described. Recent studies have shown that while it was possible to generate glucose- responsive, insulin-producing, beta-like cells, efforts to generate pancreatic islets which are capable of secreting insulin, glucagon and somatostatin in response to nutrients, as well as efforts to obtain vascularization from stem cells, have not succeeded. Described herein are results demonstrating that using the self-organizing function of human adipose-derived stem cells (hADSCs), human umbilical vein endothelial cells (HUVECs), and human iPSC-derived beta-like cells, glucose responsive insulin secreting islet-like organoids (HILOs) capable of functional vascularization are successfully generated in vitro. Further, islet-like organoid generation methods were successfully scaled up using gellan gum based 3D culture systems. The functional heterogeneity in hiPSC-derived human islet-like organoids was also investigated using a Gaussia luciferase reporter to measure insulin secretion.

Generation of functional human organs provides new therapeutic strategies in drug- screening, disease modeling and inhibiting or preventing end point organ failure. Efficient stepwise differentiation methods from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) to insulin producing b-like cells have been

demonstrated. For example, D’Amour et al. and Kroon E. et al. reported the efficient differentiation of hESCs into insulin producing cells which, after 4 to 5 months of in vivo maturation, were able to secrete insulin in response to glucose (D'Amour et al., 2006, Nature Biotechnology , 24, 1392-1401; Kroon et al., 2008, Nature Biotechnology, 26, 443-452). Recently, Rezania et al. and Pagliuca et al. reported in vitro differentiation methods that induced the formation of mature human beta-like cells that expressed the terminal b-cell markers MAFA and Nkx6-l, and exhibited partial functionality (e.g., insulin secretion) (Rezania et al., 2014, Nature Biotechnology, 32(11): 1121-33; Pagliuca et al., 2014, Cell, 159, 428-439). However, in contrast to cadaveric human islets, those beta-like cells required in vivo functional maturation for a few months, and lacked the functionality provided by the other pancreatic islet cell types, such as glycemic control by a-cells (glucagon secretion) and d-cells (somatostatin secretion). Further, the beta-like cells lacked both a mesenchyme and vascularized endothelial cells, which human islets naturally have. These crucial differences between hPSCs derived beta-like cells and human islets may compromise the ability of hPSCs-based therapies to treat insulin dependent diabetes (such as type 1 or late stage type 2 diabetes).

Previously, it was identified that a metabolic transition occurs during the neonatal to adult maturation of b-cells in which the orphan nuclear receptor Estrogen-related receptor g (ERRy) regulates an increase in oxidative metabolism required for fully functional b cells. Consistent with this result, human iPSC-derived b like cells expressing insulin, MAFA, and Nkx6-l can be metabolically matured through the overexpression of ERR to increase their oxidative metabolism and thereby enhance their glucose stimulated insulin secretion (GSIS) functionality. These results indicated that, in addition to the expression of lineage determination factors such as PDX1, MAFA, Nkx6-l and insulin, further cellular signaling which mature the b-cells’ metabolism is required to generate fully functional b-cells. (FIG. 13)

During early pancreas organogenesis, newly specified pancreatic cells originate from the foregut endodermal sheet and form a pancreatic bud, a condensed tissue mass that is soon vascularized. A similar progression has been observed in liver organogenesis as well. Such large-scale morphogenetic changes depend on the exquisite orchestration of signals between endodermal epithelial, mesenchymal, and endothelial progenitors before blood perfusion. Takebe et al. successfully generated hepatic organ buds by culturing hepatic endoderm cells with endothelial and mesenchymal linages which rapidly vascularized and functionally matured in vivo (Takebe et al., 2013, Nature , 499:481-484).

Previous work did not reveal the possibility of generating in vitro other organoid tissue types, such as pancreas organoids, which were mature, functional, and vascularized. Further, previous work showed a lack of scalability because the organoids were generated using MATRIGEL® matrix, which is not efficient to use for scaled-up production.

Described herein are studies demonstrating successful large-scale generation of human islet-like organoids (HILOs) that can secrete insulin and are vascularized, as seen in human islets, and that express one or more immune checkpoint proteins, thus affording the HILOs the ability to evade autoimmunity or immune detection by surveilling immune cells, e.g., T cells. It is demonstrated herein that (1) human adipose derived mesenchymal stem cells (hADSCs) have a self-organizing capacity (FIGS. 1A and IB); (2) late stage pancreatic progenitors are capable of forming an islet-like cluster (organ buds) when co-cultured with HUVECs and hADSCs with comparable efficiency to beta-like cells; (FIGS. 1A-1C, FIG. IE and FIGS. 3A-3C); (3) human islet-like organoids had improved expression of lineage determination factors, as well as metabolic regulatory genes including ERR ; (4) islet insulin secretion assays revealed that human islet-like organoids contain functional cells capable of secreting insulin in response to glucose (e.g., Example 8); (5) human islet-like organoids (HILOs) exhibited vascularization (FIG. 6C); (6) human islet-like organoids derived from hiPSC as described herein recaptured human islet organogenesis and pathogenesis of type 1 and type 2 diabetes in a dish; (7) human islet-like organoids derived from hiPSC as described herein offered a new replaceable resource for human islet transplantation to treat type 1 and type 2 diabetes; (8) human islet-like organoids transplanted into an STZ-induced NODSCID mouse model of type 1 diabetes ameliorated type 1 diabetes in the recipient animals (FIGS. IF and 1G); and (9) Wnt4 and Wnt5a increased the number of mitochondria-enriched b cells in HILOs (FIGS. 8A-8D), thus suggesting that both Wnt4 and Wnt5a (derived from pancreatic endocrine cells and supportive cells, respectively) enhance mitochondrial metabolic function to promote b cell maturation and sustainable GSIS function.

Also described herein are studies in which the role of certain Wnt (also“WNT” herein) proteins was assessed in developing human islet-like organoids which are capable of secreting insulin and which are vascularized, as seen in human islets. The WNT gene family consists of structurally related genes that encode secreted signaling proteins, which have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis. Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a variety of cell biological and

developmental processes. Wnt proteins undergo a complex set of posttranslational modifications involving several highly specialized processing enzymes. Upon release from the cell, the Wnt proteins interact with a number of molecules in the extracellular

environment, such as glycans, protein-binding partners (e.g., WIF, Sfrp) and cell surface receptors. (Willert, K. et ah, 2012, Cold Spring Harbor, Perspectives in Biology, 2012).

From studies described herein, Wnt5a is the predominant Wnt protein that induces the self- organization of hADSCs; (2) Wnt5a, as well as Wnt4, activate the ERRy-mitochondrial metabolic pathway; (3) Wnt4 is sufficient to induce in vitro functional maturation of hiPSC- derived islet-like organoids in the absence of additional cell types such as hADSC and HUVECs.

Generation of mature HILOs that evade immune detection

In vivo , b cells become functionally mature via a long, postnatal maturation process. To date, human induced pluripotent stem cells (hiPSCs) have not been successfully transformed into fully functional b cells by duplicating this process in vitro. Moreover, even though b cells derived from hiPSCs are immune-matched to the patient, life-long immune suppression may still be required to protect against transplant rejection after b cells are transplanted into a patient, particularly, patients with type 1 diabetes who generally have a hyper-reactive immune system. Thus, the generation of universal PSCs that resist immune rejection by expressing one or more checkpoint molecules is highly beneficial, as this would obviate a need for costly personalized therapies.

A self-organized, three-dimensional (3D) tissue architecture is required for organ formation and the terminal differentiation of organ-specific cell types. As described herein, 3D structured organoids comprising human pancreatic islet tissue were generated. The production of functional b cells requires cellular diversity within the developing islet, as well as cellular interactions that may influence the functional differentiation of islets from hiPSCs.

As described herein, a method for the scalable generation of human islet-like organoids (HILOs) from hiPSC is provided. The method utilizes a differentiation pathway that results in enhanced functional maturation and endows the resulting HILOs with immune evasive function. Advantageously, the described method does not require the use of instruments, such as a magnetic spinner or an air-liquid surface, thereby resulting in a simplified and highly reproducible procedure. The scalability of the system allows for both large- and small-scale production of mature HILOs. Tissue maturity is critical for recapitulating all aspects of pancreatic islet function. Since hiPSC-derived pancreatic progenitors or b-like cells reach functional maturation with physiological levels of insulin secretion in vivo within a few months, the in vitro differentiated b-like cells have the potential to be fully functional, mature b-like cells.

The scalable process for generating islet-like organoids from hiPSCs as described herein includes effective signals for functional maturation of the cells, and cellular heterogeneity. In an aspect, a functional, polymer-based, 3 -dimensional (3D) culture system and activation of non-canonical Wnt (e.g., Wnt4) signaling are provided to generate 3D structured human islet-like organoids (HILOs) that contain critical pancreatic islet cell types, including beta (b) cells (insulin), alpha (a) cells (glucagon), delta (d) cells (somatostatin), gamma (g) cells (PPY), and e cells (ghrelin (GHRL)).

The scalable, 3D system for generating mature human islet-like organoids (HILOs) involves stimulating the non-canonical Wnt pathway to achieve mitochondrial OxPhos function and functional insulin secretion as described herein provides medically useful, therapeutic biological material for the treatment of diseases, such as diabetes. As described herein, the stem cell derived, mature islets or HILOs can express an immune check point molecule; therefore, they are capable of evading allogenic immune rejection and thus provide a fundamental cure for insulin dependent diabetes, without resorting to immunosuppressants. Such HILOs may serve as universal (allogeneic) pancreatic islets, instead of patient-specific or autologous islets, leading to greater availability of therapeutic biological materials and cost reductions in the treatment of insulin dependent diabetes.

As described herein, the IFNy pathway was assessed for the ability to minimize host immune responses against transplanted or implanted wHILOs. Following a short exposure of wHILOs to IFNy stimulation, it was found that IFNy rapidly and robustly induced PD-L1 expression in wHILOs (FIGS. 12E and 12F). Notably, IFNy induced PD-L1 expression to levels similar to those in both insulin-expressing and insulin non-expressing cells (GFP+ and GFP- cells, respectively), (FIGS. 5 A and 5B). Repeated exposure of HILOs to IFNy (IFNy stimulation) induced a similar effect in wHILOs, specifically, a sustained induction of PD-L1 in the HILOs. In an aspect, repeated short exposures to IFNy (multiple pulse stimulation, MPS) led to sustained PD-L1 expression and concomitant increases in PD-L1 protein levels (FIGS. 5C, 5D and 5E). In embodiments, human islets or HILOs, e.g., mature islets or HILOs are exposed to (contacted with) IFNy for at least 0.5-5 hours, at least 1-5 hours, at least 1-3 hours, at least 1-2.5 hours, or at least 1-2 hours. In particular embodiments, human islets or HILOs, e.g., mature islets or HILOs are exposed to (contacted with) IFNy for greater than 1 hour, greater than 2 hours, for 1 hour, for 2 hours, or for 3 hours, prior to washing the islets or HILOs and allowing them to rest in medium without IFNy. In embodiments, each exposure of the human islets or HILOs to IFNy is termed a“pulse.” In embodiments, the human islets or HILOs are exposed to, contacted or pulsed with IFNy at least one time, at least two times, at least three times, at least four times, at least five times, etc., or 1, 2, 3, 4, or 5 times, in a one-day or a multi -day (e.g., over a 72 hour time period, or a longer time period) protocol in which cells are allowed to recover (e.g., in medium or matrix without IFNy) between IFNy pulses for about 24 hours. In a particular embodiment, the human islets or HILOs are pulsed with IFNy three times over 3 days, (72 hours), for 2 hours per pulse period, to achieve a constitutive level of PD-L1 expression in the islets or HILOs. Following this IFNy MPS regimen, the IFNy-stimulated human islets or HILOs showed high levels of PD- Ll protein expression at 7 days post MPS. In embodiments, the human islets or HILOs are exposed to (contacted or pulsed with) IFNy in an amount of 1-100 ng/ml, 1-50 ng/ml, 1-25 ng/ml, 1-20 ng/ml, 1-10 ng/ml, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/ml. In a particular embodiment, IFNy is in an amount of 10 ng/ml or 20 ng/ml for each exposure or pulse period. In a particular

embodiment, the human islets or HILOs, including mature human islets or HILOs, are exposed to, contacted or pulsed with 2 pulses of IFNy for 2 hours per pulse in a 2-day period. In a particular embodiment, the human islets or HILOs, including mature human islets or HILOs, are exposed to, contacted or pulsed with 3 pulses of IFNy for 2 hours per pulse over a 3 -day (day3) period.

GSIS functionality was not compromised by exposure of the wHILOs to MPS by IFNy (FIG. 5F). Furthermore, IFNy-treated wHILOs were protected against IL- 1 b-induced b cell dedifferentiation, as revealed by the expression of the b cell identity markers INS and UCN3 (FIG. 5H).

Normal, in utero development of a human pancreas takes more than 280 days, and full functional maturity is not reached until a few years after birth; therefore, gaining a complete understanding of the complex pathways involved in the development and maturation of human islets is a necessary step toward generating functional islets in vitro. A pivotal aspect for functional maturity of b cells is the activation of the mitochondrial metabolic pathway, which occurs naturally in postnatal maturation and is required for functional b cells nutritional sensing insulin secretion function. For HILOs, sustainable mitochondrial activation may be achieved through Wnt4 driven mitochondria metabolic regulation.

In an aspect, enhancing the ability of transplanted b cells to evade immune detection as described herein provides an alternative or adjunct strategy to MHC matching (A.

Morizane et ak, 2017, Nature communications , 8:385) for reducing the risk of autoimmune rejection of transplanted islet cells, pancreatic islets, organoids and HILOS. Stem cell-, islets- and organoid-based treatments for diabetes must achieve protection of the transplanted cells, islets and organoids from autoimmune rejection, in addition to their functional maturity. When PD-L1 negative mature HILOs were transplanted into diabetic immune-competent C57BL/6J mice, the xenograft was rejected and failed to produce detectable amounts of human c-peptide. In contrast, mature HILOs that expressed PD-L1 (either via molecular engineering or induction of expression of PD-L1 in organoid cells as described herein), successfully survived more than 50 days following transplantation into immune competent animals. (FIGS. 4D-4E and FIGS. 12A-12C) Moreover, acquisition of immune tolerance did not require the presence of Tregs. Thus, in an aspect, additional immune protection may be achieved by co-culturing Tregs in the gel-based system used to produce mature HILOs. During antigen presentation, interactions between cytotoxic T-lymphocyte anti gen -4 (CTLA- 4) and B7 molecules, as well as programed death 1 (PD-l) protein and its ligand PD-L1, negatively regulate immune responses in a non-redundant manner. As described herein, PD- Ll negative, control HILOs were rejected in T and B cell competent C57BL6J mice, but were not rejected in T and B cell-deficient NOD-SCID mice (e.g., Example 8), suggesting that allogenic rejection for PD-L1 negative control mature HILOs were mainly through T cells and B cells reaction in vivo.

The generation of iPSCs by somatic cell reprogramming provides a source of patient- specific cells (e.g., autologous cells) that may be differentiated into any lineage. Moreover, generating insulin-producing cells from iPSCs provides an invaluable tool for autologous transplantation, which would greatly reduce the risk for autoimmune rejection. While allogenic transplantation of MHC -matching grafts has proven effective in reducing immune responses and is useful, this technique may not result in complete evasion of the immune system and immune surveillance, even in less immunological sites, such as the brain. Thus, a combination of MHC matching and the induction of immune tolerance may provide a further approach to controlling immune responses against transplanted stem cells, islets and organoids. In some cases, such procedures may obviate a need for immunosuppressive drugs.

Because ongoing autoimmunity in patients with type 1 diabetes could still result in immunogenicity when patient-specific, stem cell-derived islets are transplanted, or stem cell- based islet cell replacement approaches are used, employing allogeneic hiPSCs together with immunosuppressive or tolerogenic treatments (for controlling both alloreactivity and autoreactivity) provide advantageous therapies for patients with type 1 diabetes. In addition, co-stimulation blockade procedures involving the expression of one or more checkpoint inhibitor molecules as well as a checkpoint protein to evade immune surveillance, e.g., CTLA4Ig- and PD-L 1 -expressing human stem cells, b cells, islets cells, or organoid cells, may provide clinically relevant materials for successful transplantation/implantation in subjects for diabetes treatment. By protecting HILOs via PD-L1 expression to promote graft/transplant/implant survival, HILO allografts can experience reduced immune cell infiltration, in the absence of immunosuppressive drugs. However, it will be appreciated that one or more immunosuppressive may be used if medically required or desired.

Methods of Treatment Islet transplantation is a therapy for treating insulin deficient diabetes such as type 1 and late stage type 2 diabetes. Thus, in an aspect, a method of treating a pancreatic disease such as type 1 or type 2 diabetes are provided, in which the method comprises administering a pancreatic or pancreatic islet organoid, in particular, a HILO expressing a checkpoint protein as described, to a subject (e.g., a mammalian subject, such as a human or human patient) by transplantation (or implantation). In an embodiment, the method treats a subject suffering from, susceptible to, or at risk of having, a pancreatic disease (e.g., type 1 diabetes), disorder, or symptom thereof. The method includes the step of transplanting a pancreatic or pancreatic islet organoid (HILO) in the mammal sufficient to treat the disease, disorder, or symptom thereof, under conditions such that the disease, disorder, or symptom is treated.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing, diminishing, ameliorating, abrogating, or alleviating a disease, disorder and/or the symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease, disorder, condition, or symptom thereof does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prop hylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of, or susceptible to, developing or having a disorder or condition.

The therapeutic methods (which include prophylactic treatment) generally comprise administration, in particular, transplantation or implantation, of an effective amount of a pancreatic islet or pancreatic islet organoid (e.g., a HILO) to a subject (e.g., animal, mammal, human) in need thereof, including a mammal, particularly a human. In particular, the pancreatic islet or pancreatic islet organoid (e.g., HILO) is molecularly engineered to express one or more checkpoint proteins. In an embodiment, the checkpoint protein is PD-L1. In an embodiment, a cell, islet, or organoid is subjected to multiple intermittent exposures to interferon gamma (IFNy), (multiple pulse stimulation or MPS), according to the methods described herein. The MPS methods yield cells, islets, or organoids in which the expression of a checkpoint protein such as PD-L1 is sustained over long time periods following transplantation or administration to a subject, thereby allowing the transplanted or

administered cells, islets, or organoids to function while avoiding autoimmunity or immune detection. In an embodiment, the administration of a pancreatic islet or pancreatic islet organoid (e.g., HILO) may be by any suitable means that results in an amount of the organoid that, combined with other components, is effective in ameliorating, reducing, abrogating, diminishing, or stabilizing a pancreatic disease such as type 1 or type 2 diabetes.

In certain aspects, the subject may be further administered an immunosuppressant.

The immunosuppressant can be administered to the subject before, during, or after the subject is administered (e.g., transplanted or implanted) with the organoid. The immunosuppressive agent can be an agent that inhibits or prevents rejection (e.g., acute rejection) of the transplanted organoid upon transplantation, or an agent that maintains immunosuppression after the transplantation. Immunosuppressants include, but are not limited to, basilizimab, antithymocyte globulin, alemtuzumab, prednisone, azathioprine, my cophenol ate,

cyclosporine, sirolimus, and tacrolimus.

In some embodiments, at least about 100,000, at least about 200,000, at least about 300,000, at least about 400,000, at least about 500,000, at least about 600,000, at least about 700,000, at least about 800,000, at least about 900,000 or at least about 1 million pancreatic islet organoids (HILOs) are transplanted or implanted into the subject. In some

embodiments, islets of the subject are removed prior to transplanting or implanting the organoids of the invention. In some other embodiments, pancreatic islet organoids (HILOs) are transplanted or implanted into a subject by injection into the upper abdomen of the subjects. In some embodiments, the pancreatic islet organoids (HILOs) are injected into the liver. The pancreatic islet organoids can be injected into the subject using a catheter. In some other embodiments, the pancreatic organoid or pancreatic islet organoid (HILO) is administered to the subject by surgery, e.g., transplant surgery. In another embodiment, pancreatic islet organoids (HILOs) are transplanted onto the omentum. For omentum transplantation, a layering technique can be used in which the islet organoid (or cells thereof) are combined with autologous plasma and are laparoscopically layered onto the omentum. A solution (20 ml) containing recombinant thrombin (1000 U/ml) is next layered over the islet organoid, followed by another layer of autologous plasma to produce a biodegradable biologic scaffold that can survive and function in the patient for at least a year (See, e.g., Baidal, D. et al., 2017, N. Engl. J. Med., 376: 19). In another embodiment, hydrogel biomaterials that mitigate an immune response by the recipient can be used for islet organoid transplantation. (See, e.g., Vegas, A. et al., 2016, Nature Biotechnology, 34:345-352). While organoids, pancreatic organoids, or pancreatic islet organoids (e.g., HILOs) are preferably engineered to express one or more checkpoint proteins as described herein, an immune reaction to the transplanted organoid (e.g., HILO) may be further reduced in the subject by encapsulating the organoid, pancreatic organoid, or pancreatic islet organoid (HILO) in a hydrogel prior to transplanting in the subject. Such methods of transplantation are further described in Vegas et al., 2016, Nature Medicine . doi: l0.l038/nm.4030; Vegas et al., 2016, Nature Biotechnology , doi:l0. l038/nbt.3462. In some embodiments, the hydrogel contains an alginate or alginate derivative (e.g., triazole-thiomorpholine dioxide). Various modifications of alginate hydrogels that substantially reduce inflammatory or fibrotic effects of alginate hydrogels have also been identified (Vegas et al., 2016, Nature Biotechnology , doi: l0.l038/nbt.3462). Thus, in some other embodiments, the hydrogel contains a chemical modification that reduces an inflammatory effect of the transplanted organoid in the subject.

Screening Assays

Pancreatic islet organoids and pancreatic organoids (HILOs) as described herein can be employed to model diseases of the pancreas in vitro or in vivo. Such pancreas disease models can identify drugs that are useful for treatment of a pancreatic disease. Thus, in some aspects, the invention provides methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, polynucleotides, small molecules or other drugs) that can treat a pancreatic disease, particularly type 2 diabetes and/or pancreatic cancer. In one embodiment, the compound or agent modulates an activity of a pancreatic organoid or pancreatic islet organoid (HILO) as described herein.

The test compounds or agents can be obtained singly or using any of the numerous approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic

degradation and remain bioactive; see, e.g., Zuckermann, R.N. et al., 1994, ./. Med. Chem., 37:2678-85; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer DrugDes., 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA, 91 : 11422; Zuckermann et al., 1994, ./. Med. Chem., 37:2678; Cho et al., 1993 , Science, 261 : 1303; Carrell et al., 1994 , Angew. Chem. Int. Ud. Ungl., 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl., 33:2061; and Gallop et al., 1994, ./. Med. Chem., 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992,

Biotechniques, 13:412-421), or on beads (Lam, 1991, Nature, 354:82-84), chips (Fodor,

1993, Nature, 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al., 1992, Proc Natl Acad Sci USA, 89: 1865- 1869) or on phage (Scott and Smith, 1990, Science, 249:386-390; Devlin, 1990, Science, 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA, 87:6378-6382; Felici, 1991, J. Mol. Biol., 222:301-310; and Ladner, Ibid., supra).

Chemical compounds to be used as test agents (i.e., potential inhibitors, antagonists, agonists) can be obtained from commercial sources or can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, 1989, Comprehensive Organic Transformations, VCH Publishers; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fie ser's Reagents for Organic

Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Combinations of substituents and variables in compounds encompassed by these methods are only those that result in the formation of stable compounds. The term“stable”, as used herein, refers to compounds that possess stability sufficient to allow manufacture and that maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., transport, storage, assaying, activity, therapeutic administration to a subject).

The compounds described herein can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the described methods. The compounds described herein can also be represented in multiple tautomeric forms, all of which are included herein. The compounds can also occur in cis- or trans- or E- or Z-double bond isomeric forms. All such isomeric forms of such compounds are expressly included.

Test agents, molecules and compounds can also be peptides (e.g., growth factors, cytokines, receptor ligands) or polynucleotides encoding such peptides, and the like.

Screening methods identify agents that increase or decrease a biological activity of pancreatic organoids and pancreatic islet organoids (e.g., EULOs) as described herein. In some embodiments, a pancreatic disease, such as diabetes, (e.g., type 2 diabetes) or pancreatic cancer, is induced or mimicked in the pancreatic islet organoid (e.g., HILO) or pancreatic organoid. Type 2 diabetes in the pancreatic organoid or pancreatic islet organoid (e.g., HILO) can be induced, for example, by contacting the organoid with free fatty acids (FFAs), glucose, and cytokines (in particular, high levels of glucose and/or high levels of FFAs). In one embodiment, a pancreatic organoid or pancreatic islet organoid (e.g., HILO) is co-cultured with pancreatic cancer cells, stellate cells and immune cells to create a human pancreatic cancer microenvironment in vitro.

In some embodiments, the organoid is contacted with a candidate agent, molecule, or compound, and an effect of the candidate agent, molecule, or compound on a biological activity, function, or event is assayed. In some embodiments, the candidate agent, molecule, or compound is a drug approved by the Food and Drug Administration (FDA). For example, biological activities of a pancreatic organoid or pancreatic islet organoid (e.g., HILO) assayed in the screening methods include insulin secretion (e.g., glucose-stimulated insulin secretion (GSIS)), beta cell apoptosis, LDHA activity, K(ATP) channel activity, mitochondrial function, level or activity of NDUFA4, ESRRG, KCNK3, or MAFA polypeptides or encoding polynucleotides, cell death, cell growth, and metastasis. In some embodiments, the agent, molecule, or compound increases GSIS.

In other embodiments, pancreatic islet cells, pancreatic organoid, or pancreatic islet organoid (e.g., HILO) is transplanted or implanted into a host to model pancreatic disease, such as type 2 diabetes or pancreatic cancer, in vivo. Methods of transplanting or implanting an organ or organoid are known in the art. The host can be any non-human mammal, such as a rat or mouse. In addition to the expression of a checkpoint protein in cells, islets, organoids, pancreatic islet cells, pancreatic organoids, or pancreatic islet organoids (e.g., HILOs) for evading autoimmunity and immune detection, a recipient’s immune reaction to the transplanted biological material, such as an organoid (e.g., HILO), can be further reduced, if desired, by encapsulating the organoid (e.g., HILO) in a hydrogel and then transplanting the encapsulated organoid (e.g., HILO) in the animal. Such methods of transplantation are described in Vegas et al., 2016, Nature Medicine, doi:l0. l038/nm.4030; and Vegas et al., 2016, Nature Biotechnology , doi: 10. l038/nbt.3462. In some embodiments, the hydrogel contains an alginate or alginate derivative (e.g., triazole-thiomorpholine dioxide). Various modifications of alginate hydrogels that substantially reduce inflammatory or fibrotic effects of alginate hydrogels have also been identified (Vegas et al., 2016, Nature Biotechnology, Ibid). In still other embodiments, the hydrogel contains a chemical modification that reduces an inflammatory effect of the transplanted organoid in the host.

In some embodiments, a pancreatic organoid or pancreatic islet organoid (e.g., HILO) and liver organoid are co-transplanted or implanted in the animal. The liver is a major target organ for metastasis of pancreatic cancer. In mice, in vivo endothelial cells in the mini pancreas and in the mini liver are connected to each other and create a pancreas-liver vasculature network for pancreatic cancer metastasis. Therefore, an animal co-transplanted with a a pancreatic organoid or pancreatic islet organoid (e.g., HILO) and a liver organoid can be useful for studies of human pancreatic cancer metastasis into human liver. In some embodiments, the co-transplanted organoids are subjected to multiple intermittent exposures to IFNy (MPS procedure) according to the methods as described herein.

In some embodiments, an animal transplanted with an organoid (e.g., HILO) as described herein is administered an environmental stress (e.g., a high fat/high glucose diet or is administered pancreatic cancer cells) to induce or mimic pancreatic disease in the animal.

In some other embodiments, the animal is transplanted with a pancreatic islet, pancreatic organoid, or pancreatic islet organoid (e.g., HILO) and/or a liver organoid in which a disease (e.g., type 2 diabetes or pancreatic cancer) has been induced.

In some embodiments, a candidate agent, molecule, or compound is administered to an animal. In certain embodiments, the candidate agent, molecule, or compound is a drug approved by the Food and Drug Administration (FDA). In some embodiments, an effect of the candidate agent, molecule, or compound on a phenotype in the animal (such as biological activity or function associated with the pancreas, or activities associated with a disease such as pancreatic disease) is assayed. Exemplary, yet nonlimiting, biological activities include one or more of insulin secretion (e.g., glucose-stimulated insulin secretion (GSIS)), beta cell apoptosis, lactate dehydrogenase (LDHA) activity, K(ATP) channel activity, mitochondrial function, level or activity of NDUFA4 (Cytochrome c oxidase subunit NDUFA4), ESRRG, or MAFA (musculoaponeurotic fibrosarcoma oncogene family, protein A) polypeptide or encoding polynucleotide, cell death, cell growth, and metastasis. In some embodiments, the candidate agent, molecule, or compound increases GSIS.

In any one of the embodiments herein, the effect of the candidate agent, molecule, or compound (i.e., ability to modulate a pancreatic activity or function) is measured relative to a reference or control. The reference can be, for example, an untreated pancreatic organoid or pancreatic islet organoid. In some embodiments, the reference is a host transplanted with an organoid (e.g, HILO) as described herein, where the host is not administered a candidate agent, molecule, or compound.

Agents, molecules, or compounds useful in the methods as described herein can also be detected by identifying an increase in expression of a desirable marker (e.g., MAFA as a beta cell fate marker). The level of expression can be measured in a number of ways, including, but not limited to, measuring the mRNA encoded by the genetic markers;

measuring the amount of protein encoded by the genetic markers; or measuring the activity of the protein encoded by the genetic markers.

The level of mRNA corresponding to a marker can be determined both by in situ and by in vitro formats. The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.

In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. The skilled practitioner can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers described herein.

The level of mRNA in a sample can be evaluated with nucleic acid amplification, e.g., by rtPCR (C. Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88: 189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology, 6: 1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5’ or 3’ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule (polynucleotide) comprising the nucleotide sequence flanked by the primers.

Kits

Also provided are kits containing an immunoprotected cell, human islet-like organoid or pancreatic islet organoid as described herein, or a pharmaceutically acceptable

composition (therapeutic composition) containing the immunoprotected cell, human islet-like organoid or pancreatic islet organoid and a pharmaceutically acceptable carrier, diluent, or excipient, for administering to, or transplanting into, a subject in need thereof. As will be appreciated by the skilled practitioner in the art, such a kit comprises a sterile container which contains the therapeutic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, or other suitable container forms known in the art. The containers can be made of plastic, glass, or other materials suitable for holding biological medicaments. In some embodiments, a kit may include multiple containers that house the immunoprotected cell, human islet-like organoid or pancreatic islet organoid, a composition thereof, diluents, vehicles, or excipients, as necessary, and instructions for use. The instructions will generally include information about the use of the immunoprotected cell, human islet-like organoid or pancreatic islet organoid or composition thereof for treating a disease, such as a pancreatic disease or diabetes. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent (immunoprotected cell, human islet-like organoid or pancreatic islet organoid); dosage schedule and administration for treatment of the disease, or transplantation; precautions; warnings; indications; counter-indications;

overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Advantages and Applicability of the Embodiments

A combination of genetic and environmental factors underlies the autoimmune destruction of B cells, and while exogenous insulin provides glycemic control, the long-term complications associated with Type 1 diabetes are a continuing concern. Thus, the ability to generate B cells suitable for transplantation has the potential to significantly improve patients' lives. While cadaveric islet cell transplantation offers one mode of therapy, alternative stem cell-based approaches continue to face numerous challenges in generating GSIS competent B cells on a large-scale and protecting transplanted cells from auto-immunity and allogenic rejection. For the latter, it is generally considered that self-contained transplantation devices, immune suppressive therapies, or both are required.

The methods and systems described herein provide useful protocols, such as 3D culturing conditions that systematically drive the differentiation of pluripotent stem cells (e.g., hiPSCs), stem cells, or embryonic stem (ES) cells, into insulin-positive, glucose- sensitive B-like cells, and lead to the generation of metabolically mature, immune evasive human islet-like organoids (wHILO ^lc ) capable of secreting insulin in response to a glucose challenge. Furthermore, these functionally mature HILOs rapidly reestablish glucose homeostasis upon transplantation into diabetic, immune-competent mice. A feature of the described protocols furthers the inventors’ discoveries that oxidative mitochondrial metabolism was central for postnatal B cell maturation and that the transcription factor ERR was necessary and sufficient for this metabolic program. The identification of WNT4 as a potent maturation factor for inducing both ERR expression and for enhancing mitochondrial oxidative phosphorylation allowed for the production of wHILOs in fully chemically defined medium (FIGS. 3F and 3H).

As would be appreciated by the skilled practitioner, challenges for stem cell-based therapeutics include autoimmune rejection of transplanted cells, in addition to metabolic and functional maturity of the cells. However, the methods, systems, and biological products generated and provided herein provide advantageous solutions to such challenges. By way of example, the finding that wHILOs maintained functionality in NOD-SCID but not in

C57BL6J mice implicates T cells and B cells in the xenograft rejection (FIG. 3K and FIG. 7C). During antigen presentation, interactions between cytotoxic T-lymphocyte antigen-4 (CTLA-4) and B7 molecules, as well as programmed cell death protein 1 (PD1) receptor and its ligand PD-L1, negatively regulate immune responses in a non-redundant manner. As described and exemplified herein, HILOs, such as wHILOs, overexpressing PD-L1 are protected from xenograft (FIG. 4C) and allogenic (FIG. 4K) rejection. As further described and exemplified herein, methods and systems were developed in which multiple, repeated exposures to limited IFNy concentrations (IFNy MPS treatment method) over period of time led to sustained, endogenous PD-L1 expression without compromising the GSIS activity of the cells (e.g., B-cells), HILOs and the cells therein. Notably, the resultant immune evasive HILOs maintained glucose homeostasis in immune-competent as well as in humanized diabetic mice in the absence of a transplantation device.

The generation of iPSCs by somatic cell reprogramming provides a source of patient- specific syngeneic or autologous cells that can potentially be differentiated into any lineage. Thus, generating insulin-producing cells from iPSCs for autologous transplantation might dramatically reduce the risk for autoimmune rejection. However, in practical terms, generating clinical-grade autologous transplants that meet manufacturing standards, quality assurance, and regulatory compliance involves expensive and time-consuming procedures. Although the allogenic transplantation of MHC -matching grafts has proven effective in reducing immune responses, this technique generally does not result in complete evasion of the immune system, even in less immunological sites such as the brain. Furthermore, the possible destruction of the transplanted insulin-producing cells by autoreactive T cells remains. Thus, the present methods and their resulting cells and products (e.g., immune evasive HILOs and cells) provide beneficial and long-lasting therapeutics that maintain function (e.g., GSIS) and integrity for significant time periods after transplantation or administration to a subject in need. In embodiments, MHC matching and/or the induction of immune tolerance may further be employed to control immune responses, optimally without immunosuppressive drugs.

Provided and described in an embodiment herein are advantageous methods and culture systems (e.g., a 3D culture system) for the generation of human islet-like organoids (HILOs). The methods and systems incorporate non-canonical WNT signaling to promote metabolic maturation and glucose-sensitive insulin secretion in HILOs and the cells therein, and limited IFNy exposure, namely, multiple pulse stimulation with IFNy, to drive the sustained expression of endogenous PD-L1 in the HILOs and cells therein. The ability to generate functional immune evasive HILOs, e.g., wHILO ¹⁶ , that are capable of avoiding immune detection over a significant period of time (over 50 days or longer) represents a major advance that offers a viable alternative to current cadaveric islet use or device- dependent technologies.

The practice of the methods and protocols described herein employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989), as well as subsequent editions;“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);

“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides described herein, and, as such, may be considered and employed in making and practicing the invention.

Particularly useful techniques for particular embodiments are discussed in the following examples, which are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the products, assays, procedures, screening, and therapeutic methods as described, without intending to limit the description and disclosure herein.

EXAMPLES

Example 1: Generation and characterization of pancreatic and pancreatic islet organoids

Although an animal disease model can yield insight into the pathogenesis of diseases, drugs identified from screens using animal models often fail to be adopted in human patients. Generation of functional human organoids provides a new therapeutic strategy in drug- screening and disease modeling. Described herein is a technique to generate a 3D human pancreatic mini-organ, or organoid (e.g., HILO), in a dish. Using this technique, diseases such as human type 2 diabetes can be modeled in vitro to find effective drugs in genetic, patient or environmental specific diseases such as human type 2 diabetes.

Developing Gellan gum based 3D culture system for b-like cells differentiation

It is known that 3 dimensional (3D) culture systems contribute to facilitating self- organization and integration of cells. Therefore, MATRIGEL® matrix containing

extracellular matrix components such as collagen and fibronectin is often used as the basement of a 3D culture system. However, MATRIGEL® matrix-based 3D culture systems are not ideal for large-scale human organoid generation because of their cost and difficulties in scale up. Described hereinbelow are Gellan-gum based 3D culture systems and methods for b-like cell differentiation, which are cost effective and easily scalable. In an embodiment, using a fully chemically-defined stepwise differentiation protocol, human pluripotent cells (hPSCs) are differentiated into insulin producing islet-like spherical cell clusters with high efficiency and reproducibility in Gellan-gum based 3D culture systems. Single dissociated pluripotent stem cells (PSCs) successfully formed into spheres within 5 days in Gellan gum containing STEMCELL™ TeSR™ media. Fifteen (15) to 21 days after differentiation in Gellan gum-containing Custom TeSR™ with defined small molecule stimulation, insulin positive GFP clusters were observed. Global transcriptome analysis by RNA-seq revealed the stepwise differentiation of hiPSCs into insulin positive cells expressing b cell lineage specific marker genes including Pdxl, Nkx6-l, GATA6 and MAFB. The differentiation of hiPSCs, as well as the human ESC lines HuES8 and H1ES, into islet-like cell clusters was further confirmed by the progressive loss of the pluripotent marker Nanog, the induction of the b cell specific marker Nkx6-l, and the progressive induction of the endocrine hormones insulin, somatostatin and glucagon, as determined by qPCR. These results demonstrated that the Gellan-gum based 3D culture systems is suitable for the generation of large-scale islet like organoids from hPSCs.

Generation of scalable, human islet-like organoids in vitro b-like cells derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC) have limited functionality and lack the morphological and functional feature of human islets. Previous studies revealed that co-culturing hiPSC derived hepatocyte with human umbilical vein endothelial cells (HUVECs) and human bone marrow- derived mesenchymal stem cells (hMSC) generates self-organized 3D liver-bud spheres in matrigel (Takebe et al., 2013, Nature, 499:481-484). This study found that the liver “organoids” had superior expression of lineage determinant factors compared to the differentiation of isolated hepatocytes and that these organoids rapidly vascularized and functionally matured in vivo.

Studies have found that hiPSC-derived pancreatic progenitor cells (hiPSC-PP) generated using a 2D differentiation protocol (Yoshihara et al, 2016, CellMetab. 23, 622- 634) did not self-organize in 3D MATRIGEL® matrix. (See, e.g., WO 2017/205511). In contrast, HUVEC cells rapidly formed a vasculature-like structure while human adipocyte- derived stem cells (hADSCs) self-organized in 3D MATRIGEL® matrix. In MATRIGEL® matrix, dispersed hADSC cells projected processes within 4 hours, formed a cloth-like wrapper within 12 hours, and adopted a sphere-like formation within 24 to 48 hours.

Furthermore, a minimum cell density for self- organization was identified (i.e., -10,000- 20,000 cells in 300 mΐ of MATRIGEL® matrix in ~2cm ² well. RNA-seq analysis identified dynamic transcriptional changes during hADSC 3D self-organization, suggesting that the ability to self-organize under 3D culture conditions is an inherent feature of naive hADSCs. These results identify the mesenchymal hADSC as a resource for generating self-organizing organoids.

To explore pancreatic organogenesis, hiPSC-PP (lxlO ⁶ cells) cells were co-cultured with EtUVECs (7xl0 ⁵ cells) and hADSCs (1-2 xlO ⁵ cells) (FIGS. 1A and IB) in Matrigel matrix. This co-culture yielded macroscopically visible 3D cell clusters 48 hours after seeding. Furthermore, insulin expression, based on the expression of a GFP reporter, was detected 5 days after seeding and increased with time in culture in the human islet-like organoids. In addition, HUVECs-based endothelial cells are integrated inside the organoids as shown by fluorescence-labeled (mCherry) FtUVECs. The limitations of MATRIGEL® matrix for organoid production include high cost, difficult organoid recovery, scaling restrictions, and batch to batch variabilities.

Methods to generate morphologically identical human islet-like organoids using gellan gum based 3D cultures are described herein below and in WO 2017/205511. Human induced pluripotent stem cells derived pancreatic progenitors (hiPSC-PPs) (lxlO ⁸ cells) were cultivated with a stromal cell population such as human umbilical vein endothelial cells (FtUVECs) (2-7xl0 ⁶ cells) and human adipose-derived stem cells (hADSCs) (2-7 xlO ⁶ ) in 50 ml of gellan gum based 3D culture media. HiPSC-PP rapidly formed isle-like sphere formation with HUVECs and hADSCs within 5 days after seeding into the gellan gum based 3D culture media. Human islets like mini-organs expressed human insulin GFP reporter in 5 days after seeding with gradually enhancing GFP intensity. Co-culturing hiPSC-PP, hADSCs, and HUVECs according to this method, generated human islet-like organoids with high reproducibility that were morphologically similar to human islets. In addition, the generated human islet-like organoids contained insulin granules in b-like cells. Gene expression analyses revealed increased expression of b cell fate determinant genes (Insulin, Nkx6-l, PCSK1 and UCN3) and mitochondrial related metabolic genes (Esrrg, Ndufal,

Ndufa 12, Cox7a2. Atp5b) in the insulin expressing cell population (GFP enriched (GFP +)) in islet-like organoids compared to those prepared without hADSC and HUVEC co-culture. Glucose-stimulated human c-peptide secretion assay revealed that islet-like organoids generated by this method are able to secrete human c-peptide in response to high (20mM) glucose.

An in vitro functional vascularization test was performed. Islet-like mini organs generated in gellan gum were transferred to MATRIGEL® matrix and cultured in endothelial growth media (EGM). Green fluorescence indicates expression of insulin genes. Within 24 hours to 48 hours after stimulation by EGM, the outgrowth of HUVEC cells was observed, indicating that human islet-like organoids generated by the method possessed the ability to form vascular structures.

Establishment of single islet insulin secretion assay using Proinsulin-NanoLuc Gaussia Luciferase assay system

It was previously published that a reporter construct, in which the Gaussia luciferase is placed within the c-peptide portion of proinsulin accurately measures insulin secretion without affecting b-cell function (Bums et ak, 2015, Cell metabolism , 21, 126-137). Using a lentiviral system, INS-l cells stably expressing this Gaussia luciferase were generated.

Luciferase secretion from INS-l cells stably expressing Proinsulin-NanoLuc increased with high-glucose (20mM), high glucose with Exendin-4 (G20mM+Ex4), and the depolarizing agent, potassium chloride, confirming the utility of this reporter system. Next, the usefulness of this reporter to measure insulin secretion in mouse or human islets transiently infected with the Proinsulin-NanoLuc reporter was evaluated. Luciferase secretion in response to 20mM high glucose was detected in both transiently infected mouse and human islets were detected. Importantly, the assay sensitivity was sufficient that insulin secretion could be qualified at the level of single islets. These results indicate that the Proinsulin-NanoLuc luciferase reporter based insulin secretion assay is applicable to not only the rat beta cell line INS-l cells, but also to primary mouse and human primary b cells. (See, e.g., WO 2017/205511).

Establishment of hiPSC and hESC cells incorporating dual lineage and functional reporters

Human iPSCs and hESCs stably expressing reporters for peel 1 lineage (human insulin reporter) and b cell function (proinsulin-NanoLuc reporter) were generated, hiPSC ^{hlNS GKP/Scc Luc} and ESC ^{hINS GFP/Sec Luc} , respectively. First, a neomycin resistant construct of human insulin GFP reporter was generated by inserting human insulin promoter sequence of pGreenZeo lenti-reporter (SR10028PA-1, System Bioscience) into pGreenFire Lenti- Reporter plasmid (TR019PA-1, System Bioscience) (named as hINS-GFP-EFla-Neo).

hINS-GFP-EFla-Neo lenti virus was infected into hiPSC and hESC by spin fection (800g, 1 hour, 37°C) followed by a medium changed to fresh STEMCELL™ TeSR™ medium. Three (3) days after the first infection, the cells were treated with 100pg/ml G418 in STEMCELL™ TeSR™ medium for 7 days. Selected hiPSC and hESC cells stably expressing hlNS-GFP- EFla-Neo were subsequently infected with the Proinsulin-NanoLuc (Addgene, Plasmid #62057) lenti-virus by spin fection (800g, 1 hour, 37°C) followed by a medium change to fresh STEMCELL™ TeSR™ medium. Three (3) days after the second infection, the cells were treated with 5pg/ml blasticysin and l00pg/ml G418 in STEMCELL™ TeSR™ medium for 7 days. Subsequently, cells were maintained in STEMCELL™ TeSR™ medium. The generated stable cell lines incorporating the dual reporters maintained self-renewal and pluripotency capabilities, as well as the capacity to differentiate into insulin producing b like cells (see, e.g, WO 2017/205511).

Pooled human islet-like organoid cultures display consistent insulin secretion despite variable functionality seen in individual organoids

Recent studies have reported the generation of insulin producing b-like cells from hESC and hiPSC capable of secreting insulin in response to glucose (Pagliuca et ak, 2014, Cell , 159, 428-439; Rezania et ak, 2014, Nature Biotechnology, 32(11): 1121-33; Russ et ak, 2015, EMBO Journal , 34: 1759-1772). However, fully functional human islet-like clusters able to appropriately secrete insulin in response to nutritional signals including glucose, amino acids, fatty acids and incretins such as GLP-l have yet to be demonstrated. To date, efforts have focused on the independent generation of insulin producing b-like cells, glucagon producing a-like cells, and somatostatin producing d-like cells from hPSC. However, these approaches lack the supporting cells important for regulation, such as mesenchymal cells, adipose cells, and vasculature cells. Since the 3D structure of islets naturally enhances their function, these missing cellular components may compromise the functionality of islet-like cells clusters. In addition, organogenesis of pancreatic islets involves clonal expansion of b-cells, suggesting that these cells may have multiple functions in islet-like organoids. To test this idea, single organoid proinsulin secretion assays were performed. Human islet-like organoids generated by methods described herein are morphologically identical with human islet. However, significant variability was seen in the glucose-stimulated insulin secretion (GSIS) capabilities of individual human islet-like organoids compared to human islets, as measured by proinsulin luciferase secretion assay. Consistent GSIS functionality was demonstrated in pooled organoids (10 to 100 organoids for assay). Furthermore, pooled human islet like organoids demonstrate enhanced GSIS when co-stimulation with GLP-l, as well as robust KCl-stimulated insulin secretion.

In vitro cultured iPSC-derived human pancreatic islet-like organoids generated herein retained their ability to respond to glucose, GLP1 and KC1 after extended time (133 days) in culture.

Example 2: Transplantation of functional pancreatic islet organoids rescued type 1 diabetic mice

Expression of specific functional islets markers such as MAFA, UCN3 and mitochondrial oxidative genes such as ERR (Esrrg), Ndufa 1, Ndufa 12, Cox7a2 and Atp5b in hiPSC-derived human islet-like organoids was observed, as further described in the below Examples. Notably, these islet-like organoids recapture in a dish both human islets development as well as the pathogenesis of diabetes. Transplantation of these functional islet-like organoids rescue type 1 diabetic mice with long survival, rapid vascularization, and reduced immune rejection.

Example 3: Wnt proteins in the metabolic maturation of iPSC-derived islet organoids

Fltp and Esrrg genes were found to be expressed in iPSC-derived islet organoids (day 21, generated without co-culture with hADSCs or HUVECs) after treatment with PBS, WNT3a (500 ng/ml), recombinant human (rh)WNT4 (100 ng/ml), or rhWNT5a (400 ng/ml) for 5 days. Esrrg gene expression was induced in hiPSC-derived islet organoids that were generated in the absence of supporting hADSC or HUVECs, in response to increasing doses of rhWNT4 (0, 10, 25, 50, 100, 200 ng/ml) and rhWNT5a (0, 25, 50, 100, 200, 400 ng/ml). In addition, mitochondrial genes involved in oxidative phosphorylation (Cox7a2, Ndufal, Ndufa7), lactate dehydrogenase (Ldha) and Fltp (a Wnt/planar cell polarity (PCP) effector and reporter gene) were induced in hiPSC-derived islet organoids that were generated in the absence of supporting hADSC or HUVECs, in response to increasing doses of rhWNT4 (0,

10, 25, 50, 100, 200ng/ml) and rhWNT5a (0, 25, 50, 100, 200, 400ng/ml). Mitochondrial (Mitotracker; Mito-Red) and insulin (Insulin-GFP) levels were increased in hiPSC-derived islet organoids (day 27) after 8 days treatment with PBS or WNT4 (100 ng/ml). Human iPSC-derived islet organoids (day 27) were generated after 8 days treatment with PBS or WNT4 (100 ng/ml). Insulin production was found in hiPSC-derived islet organoids (day 27) after 8 days treatment with rhWNT4 (100 ng/ml), rhWNT5a (400 ng/ml), or WNT5a secreting fibroblast conditioned media (50%), compared with PBS and control fibroblast conditioned media (50%). Human iPSC (hiPSC)-derived islet organoids (day 22) treated with rhWnt4 (100 ng/ml) for 12 days showed functional maturation based on their secretion of human c-peptide, as measured in response to low glucose (3mM,“G3mM”), high glucose (20mM,“G20mM”), or high KC1 levels (20 mM,“KCL20mM”), (see, e.g., WO

2017/205511).

Example 4: Generation of functional human islet-like organoids (HILOs) from induced pluripotent stem cells (iPSC) using a functional polymer-based 3D culture system

Stem cell-derived human islets hold promise as a therapy for insulin dependent diabetes. This Example describes the generation of human islet-like organoids (HILOs) from induced pluripotent stem cells (iPSCs) and shows that activation of the non-canonical WNT pathway drives a metabolic maturation step necessary for robust glucose-stimulated insulin secretion. These functionally mature HILOs containing multiple endocrine cell types maintain glucose homeostasis upon transplantation into diabetic NOD-SCID mice.

Furthermore, overexpression of PD-L1 generated immune evasive, immunologically protected HILOs that maintained glucose homeostasis in immune-competent type 1 diabetic mice for at least 50 days. The ability to generate, in a scalable fashion, functional islet-like organoids that avoid immune detection provides an advantageous and beneficial new therapy for diabetes.

Islet transplantation provides superior long-term blood glucose control for type 1 and late-stage type 2 diabetics; however, the availability and quality of cadaveric islets is currently limiting. While the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing b-like cells represents an advance in the field, the methods for generating functional b-like cells appropriate for human therapy and treatment provided herein provide biologically functional cell and HILO products suitable for use as therapeutics and in transplantation.

As described, an ERRy-driven, postnatal metabolic maturation step is necessary for b cell glucose stimulated insulin secretion (GSIS). In addition, ERRy overexpression in iPSC- derived b-like cells was sufficient for in vitro and in vivo functionality. To generate functional cells suitable for transplantation, culture conditions that replicate the cellular architecture, as well as the cell type complexity of islets, were developed. Accordingly, as transcriptionally-similar models of pancreatic fibroblast and epithelial cells, human adipose derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) were used for their cell-intrinsic abilities of to form organ-like and vascular structures,

respectively, when grown in 3 dimensional (3D) Matrigel cultures (FIG. 1A). Incorporating hADSCs and HUVECs during the differentiation of human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors (EPs) in a 3-dimensional polysaccharide based gel (gellan gum) led to the formation of multicellular spheroids (MCSs), comparable in size to human islets. (FIG. IB; FIGS. 6A-6F) These MCSs contain insulin-producing cells, as seen from the expression of GFP driven by the insulin promoter and the presence of insulin granules (FIG. 1C); the incorporation of hADSCs was confirmed by the presence of cells containing lipids in droplet-like structures. (FIG. IE). Compared to endocrine progenitors (EPs) differentiated in the absence of hADSCs and HUVECS (IS), the expression of ERRy and the mitochondrial genes NDUFA1 and COX7A2 were increased in MCSs, consistent with functional metabolic maturation (FIG. ID). Consistent with their functional maturation, the MCSs displayed improved insulin secretion in response to a glucose challenge (measured by c-peptide secretion), (FIG. IE). In addition, MCSs developed vascular-like structures when stimulated with endothelial growth media, suggesting the possibility of extended in vivo functionality (FIG. 6C). Indeed, MCSs transplanted into the kidney capsule were able to maintain glucose homeostasis for approximately 40 days in STZ-induced diabetic NOD- SCID mice (diabetic mouse model), displaying similar efficacy to human islet

transplantations (FIG. IF). Furthermore, transplanted MCSs remained glucose responsive, appropriately regulating insulin secretion in the fed, fasted, and refed states as indicated by c- peptide levels (FIG. 1G); (mouse insulin levels were <0.2 ng/ml, not shown). The results obtained support the role of 3D multicellular interactions in organogenesis, as previously shown for liver organoids. The transcriptional changes induced during the initial 48 hours of hADSC single cell type 3D culture were assessed to understand the molecular signals driving the cell-intrinsic ability to self-assemble (FIG. 2A). Gene ontology analysis identified metabolic and cytokine signaling pathways, as well as WNT signaling, enriched in the altered transcripts (FIG. 2A). Consistent with this, the temporal expressions of WNTs during hADSC self-assembly revealed a transient, approximately 2-fold increase in WNT5a expression that coincided with the initial cell-cell interactions observed in three dimensional (3D) cultures (FIG. 2B).

Example 5: The non-canonical Wnt pathway regulates gene expression to enable oxidative phosphorylation and maturation of HILOs

The non-canonical WNT pathway is a marker for non-proliferative, mature b cells, and WNT4 expression is enhanced during the postnatal functional maturation of mouse islets. In experimental studies using human islets, WNT4 was discovered to be highly expressed in the human islets (FIG. 2C), in agreement with these findings. Moreover, single cell sequencing of human islets identified widespread expression of WNT4 in b and a cells, along with more restricted WNT5A expression predominantly in stellate cells (FIGS. 2D, 2E, 2F; FIGS. 6D-6F). To demonstrate that non-canonical WNT signaling was sufficient for the functional maturation of iPSC-derived b cells or b-like cells, CRISPR-Cas9 genome editing was used to insert the GFP coding sequences downstream of the insulin promoter in hiPSCs (FIG. 7A), to generate a reporter for endogenous insulin promoter activity and to allow endogenous insulin promoter activity to be visualized. These engineered hiPSCs were subsequently differentiated in a fully chemically-defined 3D culture system that incorporated WNT4 in the final endocrine progenitor (EP) maturation step (FIG. 3A). This optimized 3D differentiation protocol led to the formation of human islet-like organoids (HILOs) that expressed insulin (FIGS. 3A and 3B). In addition, expression of Urocortin-3, secreted from b cells to regulate d (delta) cell somatostatin secretion, co-localized with insulin in HILOs (FIG. 2B). The analysis of the HILOs by electron microscopy revealed structural similarity to human islets, most notably, by the presence of insulin and glucagon granules in the HILOs (FIG. 3C).

Comparative transcriptional analyses confirmed the induction of key islet cell markers in WNT4-treated HILOs (wHILOs) to levels comparable to those seen in human islets, including b cell specific genes (. NKX2-2 , NEUR0D1, RFX6 , GCK ) and a cell-specific genes ( ARX ), (FIGS. 3D-1 and 3D-2). Importantly, the expression of b cell lineage specification markers, including INS, NKX6-1, UCN3 , MAFB and SYT4 , was not affected by the addition of WNT4, thus indicating that this non-canonical WNT signaling was not affecting cell fate determination. In contrast, WNT4 dose-dependently increased the expression of ERRy (encoded by ESRRG ), as well as components of the mitochondrial respiratory chain NDUFA7 and COX7A2 in HILOs (FIG. 3F). Consistent with these inductions, HILOs generated in the presence of WNT4 displayed increased oxidative metabolism, as measured by an increase in oxygen consumption rate (OCR) and decreased extracellular acidification rate (ECAR), replicating the metabolic characteristics of healthy human islets (FIG. 3H and FIG. 7C). WNT4 treated HILOs showed improved in vitro GSIS; an effect that was not blocked by the b-catenin inhibitor XAV939 (FIG. 31; FIGS. 7D-1 and 7D-2). Similarly, culturing commercially-available hiPSC-derived b like cells in 3D differentiation medium containing WNT4 promoted pseudo-islet formation and GSIS functionality. (FIG. 3J and FIG. 3K). Importantly, wHILOs (i.e., HILOs cultured in culture or differentiation medium containing WNT4) restored glycemic control upon transplantation into STZ-induced NOD-SCID diabetic mice and maintained glucose homeostasis for more than 6 weeks (FIG. 8D). In combination, these results indicate that non-canonical WNT signaling is sufficient to induce a metabolic maturation of HILOs needed for robust GSIS, in a manner that mimics the postnatal maturation of human islets. Accordingly, culturing stem cells (e.g., hiPSCs, PSCs, or embryonic stem (ES) cells) in medium containing WNT (e.g., WNT4) generates islets and islet like organoids (wHILOs) which are functionally mature and islet-like and which express more mature B-cell markers and produce insulin.

To understand the molecular transformations driving the maturation of HILOs, the transcriptional changes induced by WNT4 treatment of HILOs were assessed. The expression of 1581 and 1354 genes were increased and decreased, respectively, by WNT4 treatment (100 ng/ml for days 26-33). Gene ontology analysis identified metabolic pathways, most notably oxidative phosphorylation, enriched in this gene set FIG. 3E. Genes associated with the ribosome include mitochondrial translation and elongation gene clusters, as determined by GOTERM BP analysis by DAVID, FIG. 8C). Consistent with an effect on cellular metabolism, WNT4 treatment comprehensively increased the expression of OxPhos genes in HILOs to levels similar to those seen in human islets, and increased mitochondrial number (FIG. 3G and FIG. 8A).

To examine the specific effects on the b-like cell population, insulin-expressing cells were sorted based on GFP expression from HILOs with and without WNT4 or WNT5a treatment. The proportion of insulin expressing cells was not affected by WNT treatment, in agreement with the invariant b cell lineage marker expression during HILO maturation (FIG. 8B). However, WNT4 and WNT5a treatment increased the mitochondrial content of the insulin-expressing cells, supporting the notion of a metabolic maturation of b cells (FIG. 8B). To identify genetic effectors of this maturation step, the WNT4-induced changes in chromatin accessibility were mapped in the sorted, GFP+ cells by ATAC-Seq. Widespread alterations in chromatin accessibility were seen with WNT4 treatment, in agreement with the extent of transcriptional changes. An overlap of the regions with increased chromatin accessibility with the HILO genes induced by WNT4 treatment identified 123 genes (FIG. 8E). Gene ontology identified metabolic pathways, including oxidative phosphorylation, enriched in this gene set. Furthermore, motif analysis in genes where increased chromatin accessibility corresponded with increased gene expression identified b cell maturation factors including Foxa2 and ERRs. (FIG. 8F). Consistent with this, WNT4-induced increases in chromatin accessibility were seen at oxidative phosphorylation genes including ERRy target genes NDUFA4 , NDUFA 7 and ATP5E (FIG. 7F). Further supporting the essential role of ERRy signaling, WNT4 (lOOng/ml for 5 days) induced the expression of mitochondrial metabolic genes and improved GSIS function in isolated neonatal islets from WT, but not from ERRy b cell specific knockout (KO) mice (ERRyKO mice), (FIG. 8G and FIG. 8H). Without wishing to be bound by theory, these results, taken together, support the concept that non-canonical WNT4 signaling enhances mitochondrial function, in large part through the induction of ERRy , to drive the metabolic maturation of b-like cells.

Example 6: Cellular complexity of mature HILOs

Immunohistochemical and flow cytometric analyses revealed that approximately 50- 60% of wHILO cells co-expressed insulin and b cell markers, as well as low levels of additional endocrine cells (glucagon ⁺ , somatostatin ⁺ , pancreatic polypeptide ⁺ (PP ⁺ )) (FIGS. 9A-9F). In agreement with the transcriptional comparisons, the cellular composition of HILOs was not altered by WNT4 treatment (FIG. 9F). To comprehensively characterize the cellular complexity of metabolically mature HILOs and gain insight into the in vitro maturation program, the single cell transcriptomes of HILOs (PBS-treated, n=4078) and wHILOs (WNT4-treated, n=4840) were compared with those of human islets (n=3245)

(Table 1). Cellular transcriptomes in each analysis were clustered by principal component analysis of read counts with dimensionality reduction using /-distributed stochastic neighbor embedding (t-SNE). Clustering of wHILOs revealed populations enriched in b cell markers, as well as in Sox9 ⁺ HESl ⁺ pancreatic progenitor clusters (FIGS. 9G-9J). Signature gene expression analyses further distinguished non-replicating and replicating ductal-endocrine bipotent cells (+/-TOP2A), hormone positive endocrine enriched cells (GCG ⁺ , SST ⁺ ), ductal- like cells (KRTl9 ⁺ ) and a small population of cells with unknown function (UK). (FIG. 9K and FIG. 9L) Co-clustering of HILO and wHILO data sets provided additional evidence for the presence of multiple endocrine-like cell types (based on the highly expressed genes in each cluster) that were largely independent of WNT4 treatment (FIG. 9M). To confirm the presence of multiple endocrine-like cell types, an integrated analysis of the combined wHILO and human islet single cell data sets was performed (FIGS. 10A-10C). While differences were evident, wHILO cells were found clustering with islet endocrine cells including b, a, d and g cells, indicating transcriptional similarities (FIG. 10B). Notably, a functional classification based on co-clustering with islet cell types revealed a predominance of b- and a-like cells in wHILOs (FIG. 10B).

Table 1

Example 7: PD-L1 provides immune protection for HILOs

The clinical utility of transplanted islets is limited by both allogenic and autoimmune responses. Given the ability of checkpoint molecules to suppress immune responses, the endogenous expression of immune checkpoint proteins in human islets was investigated. A small subset of b cells in healthy islets showed a unique gene expression signature that included PD-L1 expression (FIG. 12A), a determinant of immune tolerance in b cells. To create wHILOs that exhibited exogenous PD-L1 expression to thereby protect them upon transplantation, PD-L1 -expressing hiPSC clones were generated using a lentiviral system and subsequently differentiated into metabolically mature wHILOs, as delineated in FIG. 3A. PD-L1 over-expression in the HILOs did not affect insulin expression (FIGS. 12B and 12C). PD-L1 -expressing wHILOs and those that did not express PD-L1 were transplanted into the kidney capsules of immune competent diabetic mice (STZ-treated C57BL6J mice), (FIG. 12D). wHILOs with and without PD-L1 overexpression were able to restore glycemic control within days of transplantation with similar efficacy (FIG. 4C). However, the functionality of wHILOs lacking PD-L1 expression was progressively lost over a period of weeks, as monitored by the increases in blood glucose levels. By contrast, the PD-Ll ⁺ wHILOs were able to maintain glucose homeostasis for >50 days in the absence of immunosuppressive drugs (FIG. 4C).

To confirm the immune-suppressive actions of PD-L1, transplanted wHILOs were recovered from recipient mice 27 days after transplantation, and the cellular compositions were compared by flow cytometry. The infiltration of CD45 ⁺ immune cells, including T and NKT cells, was markedly decreased in grafts that had received wHILOs that expressed PD- Ll (FIGS. 4D-4G). Furthermore, negligible numbers of insulin-expressing cells were found in grafts that had received wHILOs lacking PD-L1 expression, in agreement with the largely unregulated blood glucose levels observed 27 days after transplantation (FIG. 4D, FIG. 4F and FIG. 4H)

The persistence of wHILO (PD-L1) as xenografts led to an assessment of their functionality in a model incorporating a reconstituted human T cell repertoire. After confirming the presence of human T cells, HuPBMS-NSG-SGM3 mice were rendered diabetic by multi low dose STZ treatment (50 mg/kg/day for 5 days, MLD-STZ) and were subsequently transplanted with wHILO (FIG. 41 and FIG. 4J). Transplanted wHILOs (PD- Ll) provided sustained blood glucose control compared to those lacking PD-L1 expression, with human c-peptide levels correlating with the extent of glycemic control (FIG. 4K and FIG. 4L). The rapid development of hyperglycemia upon surgical removal of the transplanted kidneys implicated graft-derived insulin as the primary effector (FIG. 4K). Subsequent analysis of the recovered grafts revealed a marked reduction in the number of insulin expressing cells in wHILOs and a corresponding increase in human lymphocytes (FIG. 4E and FIG. 4M)

Example 8: Epigenetic memory drives immune tolerant wHILOs

PD-L1 expression is induced by IFNy stimulation in multiple cancers; however, extended exposure to cytokines, including IFNy, has been found to induce b-cell death and/or de-differentiation. In this Example, experiments were performed to assess whether the IFNy pathway was capable of minimizing host immune responses against transplanted wHILOs. Following exposure of wHILOs to IFNy stimulation, it was found that IFNy rapidly and robustly induced PD-L1 expression in wHILOs (FIGS. 12E and 12F). In particular, an approximately 20-fold increase in PD-L1 expression was observed 12 hours after IFNy treatment. (FIG. 12F). Notably, IFNy induced PD-L1 expression in wHILOs to similar levels in both insulin-expressing and insulin non-expressing cells (GFP+ and GFP- cells, respectively), (FIG. 5A). Subsequent dose-escalating studies in wHILOs identified maximum PD-L1 induction after a 2-hour, 10 ng/ml IFNy exposure. (FIG. 12E). However, the induction was transient, with PD-L1 expression rapidly decreasing in the days following exposure to IFNy (FIG. 5B). Because tolerance to inflammatory stimuli such as

lipopolysaccharide has been associated with epigenetic changes, experiments were performed to investigate whether sequential IFNy stimulation induced longer term or sustained effects in wHILOs, specifically, a sustained induction of PD-L1 in the HILOs. Indeed, it was discovered that repeated short exposures (intermittent exposure) to IFNy (multiple pulse stimulation,“MPS”) led to sustained PD-L1 expression and concomitant increases in PD-L1 protein levels (FIGS. 5C, 5D and 5E). Importantly, GSIS functionality was not

compromised by exposure of the wHILOs to MPS IFNy (FIG. 5F). Furthermore, MPS IFNy-treated wHILOs were protected against IL- 1 b-induced b cell dedifferentiation, as revealed by the expression of the b cell identity markers INS and UCN3 (FIG. 5G and FIG. 5H).

ATAC-Seq was used in studies to provide mechanistic insight into the IFNy-driven changes in wHILOs. As measured by ATAC-Seq, the genome-wide transcriptional changes induced by acute (l2h exposure) and MPS treatments were associated with alterations in chromatin accessibility. Largely overlapping gene sets were induced by the IFNy treatments that included PD-L1, while approximately half of the downregulated genes were commonly affected (FIG. 14A and FIG. 14B) Gene ontology of the commonly upregulated gene set identified IFNy pathways (not shown). In contrast, pathways that reflect the cell

inflammation status including negative regulation of IL-1B production and inflammatory pathways were identified only in the MPS -upregulated gene set, while positive regulation of NFAB signaling and apoptosis were found selectively in the MPS -downregulated gene set (FIG. 14C). Overlaying changes in chromatin accessibility revealed persistent increases at gene loci including PD-LJ IRF9, JUNB , and JUND after MPS IFNy treatment, in agreement with the sustained increases in gene transcript levels. In contrast, while increased

accessibility was seen at known IFNy-responsive genes, including IRF1 and STAT1 , after the acute treatment, these increases were not sustained (FIG. 14D).

To confirm that IFNy treatment generated immune evasive wHILOs (wHILO ^lc ), the ability of wHILO ^lc to provide long term glucose regulation in immune competent mice was assessed. Transplantation of wHILO ¹⁶ into STZ-induced diabetic C56BL6J mice lowered blood glucose levels in the mice within days and maintained reduced levels for >40 days (FIG. 51, FIG. 5J). In contrast, the efficacy of transplanted, naive wHILOs (no IFNy exposure) progressively decreased, which was consistent with the reduced levels of human c- peptide observed in the serum of recipient mice (FIG. 5K). Similar results were found with transplantation into humanized diabetic mice. Notably, the reduced glucose levels achieved with wHILO (MPS treated) transplantation were lost upon surgical removal of the recipient kidney (FIGS. 15A and 15B). As support for the immunosuppressive role of IFNy-induced PD-L1 in the transplanted wHILOs, reduced lymphocyte infiltration, as well as a decrease in the relative number of activated T helper cells (CD4 ⁺ CD3 ⁺ ), were observed in the recovered grafts. Moreover, the number of insulin expressing cells was markedly increased in wHILO (MPS treated) grafts (FIG. 15C).

Without intending to be bound by theory, the results described herein suggest that prior IFNy stimulation, namely, exposure of cells, such as wHILOs, to the MPS IFNy protocol, induces an epigenetic memory that leads to cytokine tolerance and sustained de novo PD-L1 expression in wHILOs. Such IFNy stimulated wHILOs (wHILO ¹⁶ ) offer utility of as a therapy to alleviate diseases, such as pancreatic diseases, or insulin dependent diabetes, for example, type 1 or type 2 diabetes.

The findings, based on the above-described experiments, that wHILOs maintained functionality in NOD-SCID but not in C57BL6J mice implicates T cells and B cells in their allogenic rejection. During antigen presentation, interactions between cytotoxic T- lymphocyte antigen-4 (CTLA-4) and B7 molecules, as well as programmed cell death protein 1 (PD1) and its ligand PD-L1, negatively regulate immune responses in a non-redundant manner. The results of the experiments demonstrate that wHILOs that express PD-L1, such as by induction or overexpression as described herein, are protected from allogenic rejection. Furthermore, as described supra , a protocol is provided in which repeated exposure to limited IFNy concentrations leads to sustained, endogenous PD-L1 expression without compromising glucose stimulated insulin secretion (GSIS) activity. Of note and unexpectedly, the resultant immune evasive HILOs described herein were able to maintain glucose homeostasis in immune-competent type 1 diabetic mice for ~50 days in the absence of a transplantation device. The immune evasive cells (such as in HILOs) that result from IFNy exposure according to the method described herein not only exhibt metabolic and functional maturity, but they overcome autoimmune rejection of transplanted cells, which provides a solution to a general problem that exists for other stem cell-based therapeutics.

Example 9: Methods used in the above-described examples

Maintenance of Mouse lines

Animals were maintained in a specific pathogen-free animal facility on a 12 hour light-dark cycle at an ambient temperature of 23°C. Water and food were provided ad libitum. Animal experiments used age- and background-matched male C57BL6J (Stock No 000664), NOD-SCID mice (NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ , Stock No 005557), b cell specific ERR knockout mice (Yoshihara, E. et al., 2016, Cell metabolism 23, 622- 634, doi: l0.l0l6/j.cmet.20l6.03.005), hu-PBMC-SGM3 mice, called‘humanized mice’. Female NSG™ mice were injected with human peripheral blood mononuclear cells (PBMCs) in NSG-SGM3 (Jackson 013062) strain) All procedures involving animals were performed in accordance with protocols approved by the IACETC and Animal Resources Department of the Salk Institute for Biological Studies.

Generation of human insulin reporter and PD-L1 overexpressing human iPSC lines

To mark b cell specification, human induced pluripotent stem cells (hiPSCs) derived from HUVECs were infected with a human insulin GFP reporter, as described by E.

Yoshihara et al. (2016, Cell metabolism , 23:622-634). To visualize endogenous insulin promoter activity, CRISPR/Cas9 genome editing was used to knockin GFP into the insulin promoter (Tables 1 and 2).

Table 2

Species*: hu: human; mo: mouse

Table 3

PD-L1 expressing hiPSCs were generated by infecting hiPSCs with a lentivirus (abm, LV113090) encoding human CD274 (PD-L1) with puromycin selection (Table 4). The human UCN3 proximal promoter sequence (-1298/+103) was introduced by In-Fusion cloning (Clonetech) into the promoterless pLV-Cherry-Pickerl backbone (Clontech, 632574) using the Apal/Notl restriction enzyme sites. Primer sequences for PCR amplification of the promoter sequence from genomic DNA were 5’-GTCCATGCTGATCCATCCTT-3’ (forward) and 5’-TGCTTCTCCGGTATTGTTCC-3’ (reverse). A dual reporter line for human UCN3 mcherry and human insulin GFP (hINS-GFP-EFla-Neo), Yoshihara et ak, Ibid., was generated in hiPSC.

Table 4

Plasmid Information

Virus Production

Lentiviruses were produced using second- or third-generation lentiviral systems in HEK293T cell line using methods as described herein (e.g., Example 10 methods) and as known and practiced by those skilled in the art.

3D Getan Gum (3DKG) culture medium

Aqueous solutions of low acyl gellan gum (Kelcogel F GG-LA), (Modernist pantry), 0.3% w/v, were sterilized by autoclaving prior to dilution in mTeSRl or Custom TeSR medium (StemCell Technologies, final concentration 0.015%) and the addition of methylcellulose (R&D systems, final concentration 0.3%) and penicillin/streptozocin.

More specifically, by way of example, Kelcogel F low acyl GG GG-LA (Modernist pantry) was suspended in pure water 0.3% (w/v) and dissolved by stirring at 90°C or by microwave. The aqueous solution was sterilized at l2l°C for 20 minutes in an autoclave. The solution was added to TeSR or Custam TeSR at a final concentration of 0.015%.

Methylcellulose (MC) stock solution was added to a final concentration of 0.3% (R&D systems) (e.g., 0.3% Kelcogel stock; Kelcogel F low acyl GG GG-LA 300mg+MilliQ water lOOml: 3DKG Stem TeSR Base Medium; Stem TeSR 95ml + 0.3%Kelcogel 5ml+ MC stock solution 300m1. A 1% final concentration of Penicillin/streptozocin was added for 3DKG Stem TeSR.

Human multicellular spheroids (MCSs)

Pancreatic endocrine (PE) cells were prepared from human iPSC as described in the publication of Yoshihara, E. et al. (2016, Cell Metabolism, 23(4):622-634). In brief, HUVEC-derived hiPSC, obtained from the Salk Stem Cell Core Facility, were maintained on matrigel (BD)-coated dishes in complete Stem TeSR Medium at 37°C in a humidified 5% C0 ₂ incubator. Prior to pancreatic differentiation, hiPSC were infected with a human insulin reporter lentivirus (pGreenZero lenti reporter human insulin, System Biosciences) by Spinfection (800g, 1 hour), and then the cell medium was changed to lOOng/ml human Activin (R&D Systems), 3mM CHIR99021 (Selleckchem) in differentiation medium (800ml DMEM/F12, l3.28g BSA, lOml Glutamax, 560mg NaHCO ₃ , 33 Omg thiamine, lOOmg reduced glutathione, 3300 mg Vitamin C, 14pg Selenium, lOml NEAA, 2ml Trace Element B, lml Trace Element C, 7m 1 b-ME, 2ml DLC, 2ml GABA, 2ml LiCl, 129.7pg PA, Insulin 2mg, made up to 1000ml) for 2 days, and then the cells were maintained in lOOng/ml human Activin in differentiation medium for another 2 days (Stage 1, Pancreatic Endoderm).

Subsequently, this medium was replaced with differentiation medium containing ImM dorsomorphin (Calbiochem), 2mM Retinoic Acid (Sigma), 10mM SB431542 and 1% of B27 supplement for 7 days (Stage 2). The medium was then replaced with differentiation medium containing 10mM forskolin (Sigma), 10 mM dexamethasone (Stemgent), 10mM TGFp RI Kinase inhibitor II/A11<5 inhibitor II (Calbiochem or Enzo), 10mM Nicotinamide (Sigma), ImM S^^S-Triiodo-L-thyronine sodium salt (T3) and 1% of B27 supplement for 4-5 days (dayl5-dayl9, Pancreatic endocrine progenitors developed). The medium was replaced every day (stage 1), and then every other day (stage 2 and stage 3).

Primary EtUVEC cells and human adipose-derived stem cells (hADSC) (Invitrogen or PromoCell) were cultured in l5cm dishes with EBM Media (Lonza, CC-3121) or

MesenProRS Media (GIBCO, 12747-010 or Preadipocyte Growth Medium Kit, C-27417), respectively, at 37°C in a humidified 5% C0 ₂ incubator. For co-culturing experiments, pancreatic endocrine progenitors derived from human iPSC were treated with Accutase, while FtUVECs and hADSC were treated with TrypLE (GIBCO, 12604-013). Cells were collected into 50ml tubes. hiPSC-EP (lxlO ⁶ cells), FtUVECs (7xl0 ⁶ cells) and hADSCs (1- 2xl0 ⁵ cells) were co-cultured in a single well of a 24 well plate with 300m1 of matrigel.

For MCS generation, hiPSC-EP (dayl5-day2l, lxlO ⁶ cells), FtUVECs (7xl0 ⁶ cells) and hADSCs (l-2xl0 ⁵ cells) were co-cultured in 3D Kelco Gel Custom TeSR with 10mM forskolin (Sigma), 10mM dexamethasone (Stemgent), 10mM TGFP RI Kinase inhibitor IEAlk5 inhibitor II (Calbiochem or Enzo), 10mM Nicotinamide (Sigma), I mM 3,3’,5-Triiodo- L-thyronine sodium salt (T3) and 1% of B27 supplement, R428 (2mM), Zinc sulfate (10mM) and N-Cys (lmM). The medium was changed every other day, and islet-like clusters formed within a few days. (FIGS. 6A-6F).

Human pancreatic islet-like organoid (HILO) cultures

hiPSCs were cultured in matrigel -coated plates. Single cell suspensions were prepared using Accutase, washed in PBS, and collected by centrifugation (l000-l300rpm for 5 min). Cells were re-suspended with 3D Kelco Gel Stem TeSR™ Base Medium in the presence of the ROCK inhibitor (10mM Y-27632, StemCell) for 5 to 7 days until spheroids reached 50-100 pm diameter. The medium was then replaced with 0.015% Kelco gel containing 0.3% methyl cellulose and supplemented with 100 ng/ml human Activin A (R&D Systems), 3mM CHIR99021 (Axon or Selleckchem) in differentiation medium (Sl) for 1 day, and then lOOng/ml human Activin in differentiation medium (Sl) for another 2 days (Stage 1, Definitive Endoderm). Subsequently, the medium was replaced with differentiation medium (S2) with 50ng/ml FGF7 (R&D Systems) for 2 days, differentiation medium (S3) with 50ng/ml FGF7, 0.25mM SANT-l (Sigma), 1 mM Retinoic Acid (Sigma), 100hM LDN193189, 10mM Alk5 inhibitor II and 200nM of the B- Amyloid Precursor Protein modulator TPB for 3 days, then 50ng/ml FGF7, 0.25mM SANT-l (Sigma), ImM Retinoic Acid (Sigma), 100hM LDN193189, 10mM Alk5 inhibitor II and 100hM of the B- Amyloid Precursor Protein modulator TPB for 2 days. Subsequently the medium was replaced with differentiation medium (S4) with 0.25 mM SANT-l, 50nM retinoic acid, 100hM LDN193189, 10mM Alk5 inhibitor II, ImM T3 for 3 days. Subsequently, the medium was replaced with differentiation medium (S5) with 100hM LDN193189, 100hM g-secretase inhibitor XX (GSiXX, Millipore), 10mM Alk5 inhibitor II, ImM T3 for 7 days. Subsequently, the medium was replaced with differentiation media (S5) with 10mM Trolox (Calbiochem), 2mM R428 (Selleckchem), lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 for an additional 7 to 20 days. After confirmation of insulin expression by qPCR or reporter activity (typically days 20-30), the medium was changed to differentiation medium (S5) with 10mM Trolox (Calbiochem), 2mM R428 (Selleckchem), lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 and lOOng/ml rhWnt4 (R&D Systems) with or without the addition of laminins (LM-511/521 and LM- 411/421) for 5-10 days.

WNT5A Conditional Medium

WNT5A-producing fibroblasts (ATCC CRL-2814) and control fibroblasts (ATCC CRL-2648) were cultured in DMEM containing 10% FBS and 1% penicillin/Streptomycin (Complete Medium). ETpon reaching confluency, cells were washed with PBS prior to incubation in Complete Medium for one week. Conditioned medium was subsequently collected, filtered through a 0.2pm sterile filter, and frozen at -80 °C in 50ml aliquots.

Conditioned medium was mixed with Differentiation Mediim (S5 with IOmM Trolox, 2mM R428, lmM N-acetyl cysteine, IOmM Alk5 inhibitor II, ImM T3) at a 1 : 1 ratio, and then was used to treat HILOs for 5-10 days. PD-L1 induction in human islets and wHILOs

PD-L1 expression was induced by recombinant human IFNy (R&D Systems, 285-IF, 2-12 hours treatment at l-50ng/ml final concentration). For acute treatment, wHILOs were treated with lOng/ml IFNy in the differentiation medium (S5 with 10mM Trolox, 2mM R428, lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 and lOOng/ml rhWnt4

(recombinant human Wnt4)) for 2 hours. Cells were then washed twice with PBS prior to culturing in differentiation medium (S5 with 10mM Trolox, 2mM R428, lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 and lOOng/ml rhWnt4) (single pulse stimulation). IFNy exposure was repeated 3 times with washing and 24 hours resting time in differentiation medium (S5 with 10mM Trolox, 2mM R428, lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 and lOOng/ml rhWnt4) between each IFNy exposure (MPS stimulation) to generate wHILO ¹⁶ . After the final IFNy pulse, cells were cultured in the tissue culture incubator for a week prior to the RNA-seq analyses (FIGS. 14A-14C), ATAC-seq analyses (FIGS. 14D) and transplantation into STZ-induced diabetic C57BL6J mice (FIG. 5J) or humanized mice (FIG. 15B).

Isolation of pancreatic islets

Mouse pancreatic islets were isolated as previously described by E. Yoshihara et ah, 2010, Nature communications , 1 : 127, with slight modifications. Briefly, 0.5 mg/ml collagenase P (Roche REF 11213873001, diluted in HBSS buffer, GIBCO, 14170-112) was injected through the common bile duct, and the perfused pancreas was dissected and incubated at 37°C for 21 minutes. Digested exocrine cells and intact islets were separated via centrifugation over Hi stopaque- 1077 (Sigma, H8889) at 900xg for 15 minutes, and intact islets were manually selected. Human islets were provided by the Integrated Islets

Distribution Program under an approved protocol.

Insulin/c-peptide secretion assays

Insulin release from intact islets was monitored using batch incubation methods as reported by E. Yoshihara et ah, 2016, Cell metabolism , 23:622-634. Briefly, overnight- cultured, isolated pancreatic islets (RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) Antibiotic-Antimycotic (Gibco)) were pre-cultured at 37°C for 30 minutes in Krebs-Ringer bicarbonate buffer (KRBB) containing 129.4 mM NaCl, 3.7 mM KC1, 2.7 mM CaCh, 1.3 mM KH ₂ P0 ₄ , 1.3 mM MgS0 ₄ , 24.8 mM NaHCCb (equilibrated with 5% C0 ₂ , 95% O2, pH 7.4), lOmM HEPES and 0.2% (v/v) BSA (fraction V, Sigma) (KRBH) with 3 mM glucose). Pancreatic islets were incubated in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (500 m1/10 islets) with 3 mM or 20 mM glucose for 30 minutes to determine insulin secretion levels. After 30 minutes, the islets were pelleted by

centrifugation and secreted insulin levels were determined in the medium by Enzyme Linked Immunosorbent Assay (ELISA), (Rat/mouse Insulin ELISA KIT (Millipore) and Human Insulin ELISA KIT or ultrasensitive human c-peptide ELISA Kit (Millipore) for mouse and human islets, respectively). For human iPSC derived cells, the cells (lxlO ⁶ cells/well in 24 well culture plates) were pre-cultured in 3mM glucose KRBH buffer (500 mΐ/well). The cells were then incubated in KRBB (200 pl/well) with 3 mM or 20 mM glucose for 30 minutes to determine c-peptide secretion levels as an indicator of insulin secretion levels. After 30 minutes, the cells were pelleted by centrifugation and c-peptide levels were determined in the supernatant medium using the human c-peptide ELISA KIT (Millipore). (e.g., FIGS. 7D-1 and 7D-2).

Oxygen Consumption and Extracellular Acidification Rates

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (e.g., of islets) were recorded in 24-well plates using an XF24 sea horse (Seahorse Biosciences).

(FIG. 7C). Briefly, 70 size matched, human islets, hiPSC spheroids, or HILOs were pre- cultured in 3 mM glucose XF DMEM medium (pH 7.4) supplemented to contain lmM sodium pyruvate (Base Medium) for 1 hour prior to transfer to XF24 islet culture plates in Base Medium. OCRs (reported as percent change compared to 3 mM glucose) were recorded during the incremental addition of glucose, up to a final concentration of 20 mM glucose. Subsequently, mitochondrial stress reagents (oligomycin, Fccp, Rotenone, and Antimycin A), were added as instructed in the Mitostress Kit (Seahorse Biosciences).

Islet and HILO Transplantation Studies

Immunodeficient NOD-SCID, C57BL6J and Hu-PBMC-SGM3 mice were purchased from Jackson Laboratory and maintained in autoclaved cages in a SPF facility at the Salk Institute. Mice were rendered diabetic by a single high dose (l80mg/kg) injection or 5 times with a multi low dose (MLD, 50mg/kg) injection of streptozotocin (STZ; i.p., Sigma S0130- 500MG). One week after the STZ injection, mice with blood glucose levels higher than 300 mg/dl were used as transplant recipients. Human and mouse islets (200-500 islets or 500- 1,000 IEQ for mouse islets, 500-1,000 islets or 1,000-2,000 IEQ for human islets per animal) or HILOs (500 clusters) were resuspended in 200 mΐ RPMI-1640 medium, loaded into laboratory tubing (SiLastic, 508-004), and centrifuged (400 x g for 1-2 minutes) to generate cell clusters in the center of the tubing. Cell clusters were transplanted (approximately 30-50 mΐ) under the kidney capsules in 8 to 16-week-old STZ-injected diabetic mice. Ketamine (80 mg/kg) and xylazine (10 mg/kg) were used as surgical anesthetics, and mice were placed on 37°C heating pads to recover. Blood glucose levels were monitored by using a commercially available blood glucose/ketone monitor (Nova Max Plus). Nephrectomy (Nx) for graft removal experiments were carried out to confirm the efficacy for glucose regulation in the transplanted wHILOs. The kidney with graft was ligated at the renal hilum using 4-0 silk (LOOK, SP116), and then was resected. Removed grafts were processed for analyses of immune profiling.

ATAC-seq

ATAC-seq was performed on 5 x 10 ⁴ GFP-positive (GFP+) cells isolated using Fluorescence Activated Cell Sorting (FACS) from HILOs treated with PBS or with lOOng/ml rhWnt4 from day 27 to day 34 as described in J.D. Buenrostro et ak, 2015, Current Protocols in Molecular Biology , 109:21-29. Reads were aligned by Bowtie to hgl9, and peaks were called by HOMER using default settings. Differential peaks and motif analyses from 2 biological duplicates were identified using HOMER essentially as instructed (see, e.g, S. Heinz et ak, 2010, J Mol. Cell , 38:576-589). Detailed methods for HOMER are freely available, e.g., at http:// http://homer.salk.edu/homer/. Briefly, the program searches against the target and background sequences for enrichment of known motifs, and returns motifs enriched with a threshold of 1.5-fold change and a p-value of less than 0.05. Promoter regions, defined as 1 kilobase (kB) upstream from the transcription start site, of genes with enhanced chromatin accessibility upon Wnt4 treatment, were interrogated for enriched motifs of 8-16 bp using HOMER motif analysis.

Bulk RNA-Seq library generation

Total RNA was isolated from cell pellets treated with RNAlater (Invitrogen) using the RNeasy micro kit (Qiagen) and treated with DNasel (Qiagen) for 30 minutes at room temperature. Sequencing libraries were prepared from l00-500ng total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the manufacturer’s protocol. Briefly, mRNA was purified, fragmented, and used for first- and second-strand cDNA synthesis followed by adenylation of 3 ' ends. Samples were ligated to unique adapters and PCR amplified. Libraries were then validated using the 2100 BioAnalyzer (Agilent), normalized and pooled for sequencing.

High-throughput sequencing and analysis

RNA-Seq libraries prepared from 3 biological replicates for each experimental condition were sequenced on the Illumina HiSeq 2500 using bar-coded multiplexing and a 100 bp read length. Image analysis and base calling were automatically generated with the Illumina HiSeq Real-Time Analysis Software. This yielded a median of 29.9M usable reads per sample. Short read sequences were mapped to a UCSC hgl9 reference sequence using the RNA-Seq aligner STAR (A. Dobin et al., 2013, Bioinformatics , 29: 15-21). Known splice junctions from hgl9 were supplied to the aligner and de novo junction discovery was also permitted. Differential gene expression analysis, statistical testing and annotation were performed using Cuffdiff 2 (C. Trapnell et al., 2013, Nature Biotechnology , 31 :46-53).

Transcript expression was calculated as gene-level relative abundance in fragments per kilobase of exon model per million (fpkm) mapped fragments and employed correction for transcript abundance bias (A. Roberts et al., 2011, Bioinformatics, 27:2325-2329). RNA-Seq results for genes of interest were also explored visually using the UCSC Genome Browser. Heatmaps were generated by R-Script with heatmap.2 (gplot) software or Cluster with Javatree view software. Scale of heatmaps was determined by Z-score (FIG. 2A, FIG. 3D and FIG. 3G)

Droplet-based single-cell RNA sequence

Three biological replicates (200 clusters per replicate) of hiPSC-derived endocrine progenitor cells (dayl5), HILOs, and WNT4-treated HILOs (lOOng/ml rhWNT4 for 5 days), as well as human islets (IIDP donor ID 1874), were dissociated into single cell suspensions using TrypLE. Single cells were processed through the Chromium Single Cell Platform using the GemCode Gel Bead, Chip and Library Kits (10X Genomics) as per the

manufacturer’s protocol. In brief, 8,800 single cells were sorted into 0.4% BSA in PBS for a targeted 5000 cell recovery. Cells were transferred into Gel Beads (Chromium Single Cell 3” v2) in Emulsion in the Chromium instrument, where cell lysis and barcoded reverse transcription of RNA was carried out, followed by amplification, shearing and 5' adaptor and sample index attachment. Libraries were sequenced on an Illumina HiSeq 4000 instrument. scRNA-seq data analysis

Initial data processing, including de-multiplexing, alignment to the GRCh38 transcriptome and unique molecular identifier (UMI)-collapsing, were performed using Cell Ranger software (lOx Genomics, ver2.0.2). An overview of single cell sample information was generated from the results of Cell Ranger pipelines. R studio (https:www.rstudio.com), Cell Ranger R Kit, Seurat, monocle and other custom R scripts were used. For the identification of cell types, the cluster cell function of monocle was used. (FIG. 4B).

Clustering of cells was performed using the Seurat R package in two iterative rounds of principal component analysis.

Cells having unique gene counts less than 200 were removed (FilterCells function) prior to normalization of digital gene expression matrices by total expression, multipled by a scale factor (default setting of 10,000) and log-transformed (NormalizeData function). A set of variable genes was then identified by binning the average expression of all genes and dispersion (variance divided by the mean) for each gene, placing these genes into bins, and then calculating z-score for dispersion within each bin (FindValiableGenes Function). Linear dimensional reduction was performed using the default setting of RunPCA, and the principal components were evaluated for statistically significant gene expression signals using the Jackstraw method (JackStraw function, not shown). At most, 12 principal components were used in this second round of clustering t-distributed stochastic neighbor embedding (t-SNE) mapping was used to visualize scRNA-seq results.

Clustered cell populations were classified, and the top 10 differentially expressed genes were identified (FindAllMarkers function). Cell types within the clustered cell populations were verified by examining the expression of canonical marker genes, including insulin (b-cells), glucagon (a-cells), somatostain (d-cells), pancreatic polypeptide (g-cells), ghrelin (e-cells), Prssl (aciner cells), Krtl9 (duct cells) and Acta2 (stellate cells). (FIGS. 2D, 2E, FIG. 4A and FIGS. 6D-6F)

scRNA-seq data from WNT4-treated HILOs (4,840 cells) and human islets (7,248 cells) were combined in 1 Seurat object, and the highly variable genes were identified as described above. Cell types within the clustered populations were identified by reference to differentially expressed genes in human islet cells. The b-cell populations identified in WNT4-treated HILOs and human islets were compared to identify differentially expressed genes. (FIGS. 10A-10C; FIGS. 11A-11D)

Software and program for bioinformatics analysis

The following software or programs were used for genomic data analysis: R studio (https://www.rstudio.com/); Cell Ranger R Kit (https ://support. lOxgenomics.com/single-cell- gene-expression/ software/pipelines/latest/rkit); Seurat (https :// satij alab . org / seurat/); Monocle (http://cole-trapnell-lab.github.io/monocle-release/); DAVID

(https://david.ncifcrf.gov/home.jsp); GOplot (https://wencke.github.io); UCSC genome browser (http://genome.ucsc.edu); and Homer (http://homer.ucsd.edu/homer/).

Immunohistochemistry ( IHC)

Immunohistochemistry (IHC) of frozen or paraffin-embedded sections of pancreas and human islets or ipeta cells in the kidney capsule (4% PFA-fixed cells) was performed using antibodies to insulin (anti-insulin antibody, 1/100, Abeam ab7842)), c-peptide (anti-c- peptide antibody, 1/100, Abeam ab30477), glucagon (anti -glucagon antibody, 1/100, Abeam abl0988), somatostatin (anti-somatostatin antibody, 1/100, Abeam abl03790), pancreatic polypeptide (anti-pancreatic polypeptide antibody, 1/100, Abeam, abl 13694), NKX2-2 (anti- NKX2-2 antibody, 1/100, DSHB, 74.5A5), NKX6-1 (anti-NKX6-l antibody, 1/100, DSHB, F55A12), MAFA (anti-MAFA antibody, 1/100, Abeam, ab26405), MAFB (anti-MAFB antibody, 1/100, Abeam, ab26405), PDX-l (anti-PDX-l antibody, 1/100, R&D, AF2419), CHGA (anti-CHGA antibody, 1/100, Abeam, abl5l60), Synaptophysin (anti-Synaptophysin antibody, 1/100, Biogenex, MU363-UC) and PD-Ll (anti-PD-Ll antibody, 1/100, Abeam, ab20592), (Table 5). Secondary antibodies were coupled to Alexa 568, 647 (Life

Technologies), and IHC staining was visualized by confocal microscopy (ZEISS) or fluorescence microscopy. Hoechst 33342 (Thermo Scientific, 62249, lpg/ml final concentration) was used for nuclear staining.

Table 5

Antibody (Ab) Source/

Name Species* Host Ab Type Applications Company Catalog ID Antibody (Ab) Source/

Name Species* Host Ab Type Applications Company Catalog ID

Flow cytometry

Clusters at indicated stages were dissociated with TrypLE (GIBCO) with 20ug/ml DNase for 12 minutes at 37°C and then were fixed with 4% PFA for 10 minutes at room temperature. Clusters were then permeabilized with 0.2 % Triton X for 10 min, blocking with 10% goat serum for 30 min and stained for various intracellular markers with antibodies, c-peptide, (1/100, abeam, ab30477), PDX-l (1/100, BD, 562161), NKX6-1 (1/100, BD, 563338), Chromogranin A (1/100, BD, 564583), MAFA (1/100, abeam, ab264583), MAFB (1/100, abeam, ab66506), Glucagon (1/100, abeam, ab82270), Somatostatin (1/100, abeam, 108456) for analysis on a BD Biosciences LSRII instrument. Data were analysed by FlowJo software. Secondary antibodies for c-peptide, Glucagon and Somatostatin were coupled to Alexa 647 (Life Technologies).

Electron Microscopy (EM) analysis

Human islets and HILOs in suspension were pelleted in 2% low melting point agarose and subsequently fixed in 2.5% glutaraldehyde with 2% paraformaldehyde in 0.15M cacodylate buffer containing 2mM calcium chloride (pH 7.4) for one hour at 4°C. Excess agarose was removed, and the pellet was washed in buffer prior to secondary fixing in 1% osmium tetroxide/0.3% potassium ferrocyanide in buffer. After washing in water, the pellet was en bloc stained with 2% uranyl acetate, followed by graded dehydration in ethanol (35%, 50%, 70%, 90%, 100%, 100%). Samples were then rapidly infiltrated in Spurr’s resin using a Pelco BioWave microwave processing unit (Ted Pella, Redding, CA), embedded in Pelco Pyramid tip mold (Ted Pella, Redding, CA), and cured at 60°C overnight. 70nm ultrathin sections were cut on a Leica ETC7 ultramicrotome (Leica, Vienna) and examined on a

Libral20 (Zeiss, Oberkochen, Germany) at 120V.

Immune profiling of transplanted HILOs

Transplanted HILOs were harvested at day 26 after transplantation and were dissociated into single cells using TrypLE. After blocking a common epitope found in extracellular regions of mouse Fc-receptors by Fc block (Anti-mouse CD16/CD32 (Fc Shield) (70-0161-U500) staining, antibodies (1 : 100 dilution) to the cell surface markers CD19 (PerCP/Cy5.5 anti-mouse CD19, BioLegend, 115533), Nkl. l (anti-mouse Nkl.l PE, eBioscience, 12-5941-81), CD45 (brilliant viol et510 anti-mouse CD45, BioLegend, 103138), CD3 (brilliant violet650 anti-mouse CD3, BioLegend, 100229), Cdl lb (anti-human/mouse APC-cyanine, TONBO, 25-0112U100) were used for FACS-based immune profiling. For flow cytometry analyses, data were collected using a BD Biosciences LSRII. For cell sorting, a BD Influx was used (100 micron nozzle tip and lx PBS sheath fluid with sheath pressure set to 18.5PSI) with sample and collection cooling set to 4 degrees C. Viable (Zombie-UV dye negative) single cells were selected for FACS or analyses using Forward scatter (FSC) and Side scatter (SSC) gating, followed by pulse-width discrimination for FSC and SSC.

The described protocol assays infiltration of lymphocytes (T cells, B cells) into an organ or tissue, e.g., kidney or kidney capsule, following transplant, implant, or transfer of donor cells, islets, organoids (and cells therein). The reduced numbers of CD45+ T cells that infiltrate into tissue such as kidney following transplantation of insulin-producing PD-L1+ wHILOs versus insulin-producing PD-L1- wHILOs demonstrates that the HILOs (and cells therein) expressing PD-L1 are protected from recognition as foreign by T cells and from T cell killing after transplantation (e.g., 27 days after transplantation), (FIGS. 4D and 4E).

Detecting irnmu n oprotected cells, islets, or organoids (and cells therein) following transplant, implant, or transfer into a recipient subject

Primary human ceils, islets, and/or organoids derived from human tissues are labeled via infection with a leniiviral-mediated TYF-C V-eGFP (green fluorescent protein), (Mao, Y. et ah, 201 5, International Journal of Medical Sciences , 12(5), 407-15.

doi: l0.7l50/ijms.11270), which has been shown to produce sustained, high GFP expression. GFP-expressing cells/islets/organoids are then exposed to 2-3 IFNy treatments (e.g., MPS IFNy expose; ear described supra), and the subsequent induction of PDL-l expression is confirmed by qPCR. IFNy-exposed cells, islets and/or organoids are transplanted into the kidney capsule of an immune-competent mouse, with naive celis/islets//organoids (i.e , no IFNy exposure) transplanted into the ipsi lateral kidney capsule as controls. Mice are sacrificed 2-3 weeks after transplantatio and kidney resident GFP-positive ceils are quantified by fluorescence activated cell sorting (FACS) analysis. Increases in

cells/islets/organoids that survive following IFNy exposure are determined quantitatively, based on the numbers of GFP ⁺ cells in each kidney as determined from individual mice.

Quantitative RT-PCR analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy KIT (Qiagen). Reverse transcription was performed with a Superscript III First-Strand Synthesis System kit (Invitrogen) or PrimeScript RT reagent kit (TAKARA). Real time quantitative RT-PCR (qPCR) was performed using SYBR Green (Bio-Rad). Primer information is listed in Table 2.

In vitro vascularization

Human multicellular spheroids (MCSs) were embedded in 300 mΐ of Matrigel with EBM medium (Ronza, cc-3121) in 24 well tissue culture plates. Vascularization was observed over the following 24-72 hours.

Statistical Methods

Results were expressed as the mean ± SEM. Statistical comparisons were made using Student’s t test. Statistically significant differences are indicated as *p < 0.05, **p<0.0l, ***p<0.00l.

Example 10: Human islet-like organoids

The generation of functional human organs according to methods described herein provides new strategies for drug-screening and disease modeling. Specifically, functional organoids can be used as models of type 2 diabetes for drug screening. Human islet-like organoids responded to amyloid polypeptide (hlAPP) toxicity, an inducer of b cell loss in type 2 diabetic patients and islet dysfunction after transplantation in hyperglycemic patients, hlAPP dose-dependently induced G0/G1 arrest in 24 hours in human islet-like organoids (See, e.g., WO 2017/205511). Such human-like organoids may also be induced to express PD-L1 according to the methods and systems described herein, so as to avoid immune detection and destruction when used for transplantation, implantation, or administration to a subject in need thereof.

In an exemplary assay, 3D mini organs are exposed to stressors that induce type 2 diabetes, such as high levels of free fatty acids (FFAs) and/or, glucose and selected cytokines. The stressed 3D mini organs are then treated with various drugs. In some embodiments, the drug is approved by the Food and Drug Administration (FDA).

As output, the following are assayed in human pancreatic islet organoids: insulin secretion, beta cell apoptosis (PI stain), lactate dehydrogenase A (LDHA) expression via a luciferase reporter, and changes in expression of marker genes including NDEIFA4

(Mitochondrial oxidative phosphorylation), ESRRG (Mitochondrial function), KCNK3 (Katp channel activity) and MAFA (beta cell fate marker). For the human pancreas organoid, amylase secretion and apoptosis of exocrine cells (PI stain) are assayed.

In an exemplary assay for modeling human pancreatic cancer turn origenesis and metastasis in a dish and the potential to screen for drugs that target those diseases, a 3D mini human pancreas is co-cultured with pancreatic cancer cells, stellate cells, and immune cells to create human pancreatic cancer microenvironment in a dish. Various drugs (e.g., FDA- approved drugs) are then screened to find compounds which effectively suppress pancreatic cancer growth or metastasis in a mini human pancreas microenvironment. As output, the following are measured for the pancreas organoid: apoptosis of exocrine cells (PI stain), collagen synthesis (Trichrome stain) and stellate cells activation (GFAP -reporter). Potential candidate drugs identified in these assays are tested in pancreatic cancer tumorigenesis and metastasis mouse models. Genes expression and morphology as well as the degree of cell death, cell growth, and metastasis are investigated.

In an exemplary assay for modeling of human Type 2 diabetes in mice, human islet organoids and/or human liver organoids are transplanted into mice. The mice are then administered various stressors that induce type 2 diabetes, such as a high fat diet (HFD) or cytokines injection. The potential candidate drugs identified in this assay are further tested in human type 2 diabetic mouse model. Genes expression and morphology as well as the degree of diabetes are investigated.

In an exemplary assay for modeling of human pancreatic cancer tumorigenesis and metastasis in mice, human pancreas organoids and/or human liver organoids are transplanted into mice. Mice transplanted with a mini pancreas are used to study human pancreatic cancer growth in human pancreas microenvironment. In another exemplary assay, a mini pancreas and mini liver are co-transplanted in mice. The liver is a major site for metastasis of pancreatic cancer. In vivo , endothelial cells in the mini pancreas and in the mini liver create a pancreas-liver vasculature network for pancreatic cancer metastasis. Thus, mice co transplanted with a mini pancreas and mini liver are used to study the metastasis of human pancreatic cancer into the human liver. The generation of functional organ-like clusters from pluripotent stem cells (PSC) and human islets and HILOs as described herein provides insight into the mechanisms underlying human diseases, as well as biological therapeutics that function following introduction or transplant into a recipient subject.

The results hereinabove were obtained using the following materials and methods: 31) KELCOGEL® (3DKG) culture medium

KELCOGEL® F low acyl gellan gum (GG-LA) obtained from Modernist Pantry was suspended in pure water 0.3% (w/v) and dissolved by stirring at 90°C or by microwave. The aqueous solution was sterilized at l2l°C for 20 minutes in an autoclave. The solution was added to TeSR™ medium (Ludwid et al., Nature Methods, 3, 637-646) or custom TeSR™ medium (800ml DMEM/F12, l3.28g BSA. lOml Glutamax, 560mg NaHCO _3, 330mg thiamine, lOOmg reduced glutathione, 3300 mg Vitamin C, l4pg Selenium, lOml NEAA,

2ml Trace Element B, lml Trace Element C, 7m1 b-ME, 2ml DLC, 2ml GABA, 2ml LiCl, 129.7pg pipecolic acid, Insulin 2mg up to lOOOml) at a final concentration of 0.015%.

Methylcellulose (MC) stock solution was added to a final concentration of 0.3% (R&D systems) (e.g., 0.3% KELCOGEL® stock: KELCOGEL® F low acyl GG-LA 300mg+MilliQ water lOOml; 3D-KELCOGEL® (3DKG) Stem TeSR™ Base Medium: STEMCELL™ TeSR™ 95ml + 0.3% KELCOGEL® stock 5ml+ MC stock solution 300ul; 3 DKG Custom TeSR™ Base Medium: custom TeSR™ media 95ml + 0.3% KELCOGEL® stock 5ml + MC stock solution 300ul; 1% final concentration of Penicillin/streptozocin was added for 3DKG medium.

Preparation of human pancreatic endocrine progenitors and b-like cells in vitro

Pancreatic endocrine cells (hiPSC-PEs) were prepared from human iPSC using differentiation methods as previously described. Briefly, human induced pluripotent stem cells (hiPSC) derived from FtUVECs were obtained from the Stem Cell Core (Salk Institute). Cells were maintained on MATRIGEL® (BD)-coated dishes in complete STEMCELL™ TeSR™ medium at 37°C in a humidified 5% C0 ₂ incubator. For pancreatic differentiation, hiPSC were infected with a human insulin reporter lentivirus (pGreenZero lenti reporter human insulin, System Biosciences) by Spinfection (800g, 1 hour). Methods 1 : Medium was changed to 100 ng/ml human Activin (R&D Systems), 25ng/ml recombinant human Wnt3a (R&D Systems) in custom TeSR™ medium (800ml DMEM/F12, l3.28g BSA, lOml Glutamax, 560 mg NaHC0 ₃ , 330 mg thiamine, lOOmg reduced glutathione, 3300 mg Vitamin C, l4pg Selenium, lOml NEAA, 2ml Trace Element B, lml Trace Element C, 7m1 b-ME, 2ml DLC, 2ml GABA, 2ml LiCl, 129.7pg PA, Insulin 2mg up to lOOOml) for 2 days and then lOOng/ml human Activin in differentiation medium for another 2 days (Stage 1, Pancreatic Endoderm). Subsequently, the medium was replaced with custom TeSR™ medium with ImM dorsomorphin (Calbiochem), 2mM Retinoic Acid (Sigma), 10mM SB431542 and 1% of B27 supplement for 7 days (Stage 2). Medium was then replaced with custom TeSR™ medium with lOuM forskolin (Sigma), 10 mM dexamethasone (Stemgent), 10mM TGFp RI Kinase inhibitor II/Alk5 inhibitor II (Calbiochem or Enzo), 10mM Nicotinamide (Sigma), ImM 3,3’,5-Triiodo-L-thyronine sodium salt (T3) and 1% of B27 supplement for 4-5 days (day 15-day 21, Pancreatic endocrine progenitors). Medium was replaced every day (stage 1) or every other day (stage 2 & stage 3).

Methods 2: Medium was changed to 100 ng/ml human Activin (R&D Systems), 25ng/ml recombinant human Wnt3a (R&D Systems) or 3mM CHIR99021 (Axon or

Selleckchem) in differentiation medium (Sl) for 1 day and then lOOng/ml human Activin in differentiation medium (Sl) for another 2 days (Stage 1, Pancreatic Endoderm).

Subsequently, medium was replaced with differentiation medium (S2) with 50ng/ml FGF7 (R&D Systems) for 2 days and then differentiation medium (S3) with 50ng/ml FGF7,

0.25 mM SANT-l (Sigma), ImM Retinoic Acid (Sigma), 100hM LDN193189 and 100hM a- Amyloid Precursor Protein Modulator TPB for 3 days. Subsequently, medium was replaced with differentiation medium (S4) with 0.25mM SANT-l, 50nM Retinoic Acid, 10mM Alk5 inhibitor II, ImM T3 for 3 days. Subsequently, medium was replaced with differentiation medium (S5) with 100hM LDN193189, 100hM Gamma Secretase inhibitor XX GSiXX (Millipore), 10mM Alk5 inhibitor II, ImM T3 for 7 days. Subsequently, medium was replaced with differentiation medium (S5) with 10mM Trolox (Calbiochem), 2mM R428 (Selleckchem), lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 for additional 7 to 20 days.

Sl Medium (MCDB131 Medium, 8mM glucose, 2.46g/L NaHC0 ₃ , 2% Fatty acid free BSA, 0.25mM L- Ascorbic acid 0.002% Insulin-Transferrin-Selenium ITS-X (GIBCO), 2mM Glutamax, 1% Penicillin-Streptomycin), S2 Medium (MCDB131 Medium, 8mM glucose, l.23g/L NaHC0 ₃ , 2% Fatty acid free BSA, 0.25mM L- Ascorbic acid, 0.002% Insulin-Transferrin-Selenium ITS-X (GIBCO), 2mM Glutamax, 1% Penicillin- Streptomycin), S3 Medium (MCDB131 Medium, 8mM glucose, l.23g/L NaHC0 ₃ , 2% Fatty acid free BSA, 0.25mM L-Ascorbic acid, 0.5% Insulin-Transferrin-Selenium ITS-X

(GIBCO), 2mM Glutamax, 1% Penicillin-Streptomycin), S4 Medium (MCDB131 Medium, 8 mM glucose, l.23g/L NaHC03, 2% Fatty acid free BSA, 0.25mM L-Ascorbic acid, 0.002% Insulin-Transferrin-Selenium ITS-X (GIBCO), 2mM Glutamax, 1% Penicillin-Streptomycin, 1 Opg/ml Heparin, IOmM Zinc Sulfate), S5 Medium (MCDB131 Medium or BLAR Medium, 20mM glucose, l.754g/L NaHC03, 2% Fatty acid free BSA, 0.25mM L- Ascorbic acid, 0.002% Insulin-Transferrin-Selenium ITS-X (GIBCO), 2mM Glutamax, 1% Penicillin- Streptomycin). For 3-dimensional (3D) culture, hiPSC or hESC were cultured in 3DKG Stem TeSR™ Base Medium with 10mM Y-27632 for 5 to 7 days and then the medium was replaced each Differentiation medium with 0.015% Kelcogel and 0.3% Methylcellulose.

Generation of three-dimensional pancreatic islet bud in vitro: Islet-like organoids in Matrigel through co-culture with hADSCs and HUVECs

Primary HUVECs and human Adipose-derived stem cells (hADSC) (Invitrogen or PromoCell) were cultured in l5cm dish with EBM Medium (Ronza, cc-3121) or

MesenProRS™ Medium (GIBCO, 12747-010 or Preadipocyte Growth Medium Kit, C- 27417), respectively, at 37°C in a humidified 5% C0 ₂ incubator. For co-culturing experiments, pancreatic endocrine progenitors derived from human iPSC were treated with Accutase, while HUVECs and hADSC were treated with TrypLE (GIBCO, 12604-013) and cells collected into a 50 ml tube, respectively. After the cells were counted, lxlO ⁶ cells of hiPS-PP, 7xl0 ⁶ cells of HUVEC and l-2xl0 ⁵ cells of hADSC were co-cultured in 1 well of 24 well with 300ul of MATRIGEL® matrix. For the purpose of scalable generation of human islets like organoids, lxlO ⁶ cells of hiPS-PP (day l5-day 21), 7xl0 ⁶ cells of HUVEC and l-2xl0 ⁵ cells of hADSC were co-cultured in 3DKG Custom TeSR® media with 10mM forskolin (Sigma), 10 mM dexamethasone (Stemgent), 10mM TGFP RI Kinase inhibitor IEAlk5 inhibitor II (Calbiochem or Enzo), 10mM Nicotinamide (Sigma), luM 3,3’,5-Triiodo- L-thyronine sodium salt (T3) and 1% of B27 supplement, R428 (2mM), Zinc sulfate (10mM) and N-Cys (lmM). (Methods 1) or co-cultured in differentiation medium (S5) with 100hM LDN193189, 100hM Gamma Secretase inhibitor XX GSiXX (Millipore), 10mM Alk5 inhibitor II, ImM T3 for 7 days. Subsequently, medium was replaced with differentiation medium (S5) with 10mM Trolox (Calbiochem), 2mM R428 (Selleckchem), lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 for an additional 7 to 20 days (Methods 2). Mixed cells formed spherical, islet-like clusters within a few days. The medium was changed every other day. Generation of 3D (three-dimensional) pancreatic islet buds in vitro: Islet-like organoids in Scalable Gellan Gum through co-culture with hADSCs and HUVECs

Cells were prepared as described above. Briefly, lxlO ⁸ cells of hiPS-PP, 2-7xl0 ⁷ cells of HUVECs and 5-7xl0 ⁶ cells of hADSC were co-cultured in 60-l00ml of 3DKG Custom TeSR™ with 1 OmM forskolin (Sigma), 10 mM dexamethasone (Stemgent), 1 OmM TGFp RI Kinase inhibitor II/A11<5 inhibitor II (Calbiochem or Enzo), 10mM Nicotinamide (Sigma), ImM 3,3’,5-Triiodo-L-thyronine sodium salt (T3) and 1% of B27 supplement, R428 (2mM), Zinc sulfate (10mM) and N-Cys (lmM) (Methods 1) or co-cultured in differentiation media (S5) with 100hM LDN193189, 100hM Gamma Secretase inhibitor XX GSiXX

(Millipore), 10mM Alk5 inhibitor II, ImM T3 for 7 days. Subsequently, media was replaced with differentiation media (S5) with 10mM Trolox (Calbiochem), 2mM R428 (Selleckchem), lmM N-acetyl cysteine, 10mM Alk5 inhibitor II, ImM T3 for additional 7 to 20 days

(Methods 2). Mixed cells formed spherical, islet-like clusters within a few days. Media was changed every day or every other day.

Generation of 3D (three-dimensional) pancreatic islets bud in vitro: Islet-like organoids in Scalable Gellan Gum 3D culture methods without (w/o) using hADSC and HUVECs

Human PSCs, including iPSC or ESC, were initially cultured in matrigel -coated plates (2 dimensional (2D) cultures. Cells were then treated with Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to generate a single cell suspension, washed with PBS and centrifuged at 1000-1300 rpm for 5 minutes to pellet cells. Cells were resuspended with 3 DKG Stem TeSR™ Base Medium (Stemcell Technologies, Cambridge, MA) with 10mM Y- 27632 (a RHO/ROCK pathway inhibitor compound) and cultured for an additional for 5 to 7 days until PSC sphere growth reached 50-100 pm diameter. Media was then replaced with differentiation media supplemented with 0.015% Kelcogel and 0.3% Methylcellulose. The culture medium was changed to differentiation medium (Sl) containing 100 ng/ml human Activin (R&D Systems), 25ng/ml recombinant human Wnt3a (R&D Systems) or 3mM CHIR99021, a glycogen synthase kinase GSK-3 inhibitor (Axon Medchem, Reston, VA; or Selleckchem) for 1 day and then to differentiation medium (Sl) containing 100 ng/ml human Activin for another 2 days (Stage 1, Pancreatic Endoderm). Subsequently, the medium was replaced with differentiation medium (S2) containing 50 ng/ml FGF7 (R&D Systems) for 2 days, and then with differentiation medium (S3) containing 50 ng/ml FGF7, 0.25uM SANT-l (Sigma), 1 mM Retinoic Acid (Sigma), 100 nM LDN193189 (an ALK2 and ALK3 inhibitor, Sigma) and 100 nM a- Amyloid Precursor Protein Modulator TPB for 3 days. Subsequently, this medium was replaced with differentiation medium (S4) containing 0.25 mIUI SANT-l, 50 nM Retinoic Acid, 10 mM Alk5 inhibitor II, 1 mM T3 for 3 days. Subsequently, the medium was replaced with differentiation medium (S5) containing 100 nM LDN193189, 100 nM Gamma Secretase inhibitor XX GSiXX (Millipore) 10 mM Alk5 inhibitor II, ImM T3 for 7 days. Subsequently, the medium was replaced with differentiation medium (S5) containing 10mM Trolox (Calbiochem), 2 pM R428 (Selleckchem), 1 mM N-acetyl cysteine, 10 mM Alk5 inhibitor II, 1 mM T3 for an additional 7 to 20 days.

After confirmation of the insulin gene expression by either reporter expression or qPCR (typically on day 20-30), the medium was changed to differentiation medium (S5) containing 10 mM Trolox (Calbiochem), 2 pM R428 (Selleckchem), 1 mM N-acetyl cysteine, 10 mM Alk5 inhibitor II, 1 mM T3 and 100 ng/ml recombinant human (rh)Wnt4 (R&D Systems), 400 ng/ml rhWnt5a, or 50% Wnt5a conditioned medium for 1-20 days. Wnt5a conditioned medium was prepared by culturing an L-Wnt5a cell line (ATCC, CRL-2814) in DMEM with 10% FBS, 1% Penicillin-streptomycin for 4 days after cells had reached 70- 100% confluence in T175-T225 cell culture flasks.

Generation of 3D (three-dimensional) liver bud in vitro: Organ buds

Hepatocyte cells (hiPSC-HEs) from human iPSC were prepared using differentiation methods as previously described. Briefly, hiPSCs were maintained on MATRIGEL® (BD)- coated dishes in complete STEMCELL™ TeSR™ medium at 37°C in a humidified 5% C0 ₂ incubator. For hepatic differentiation, hiPSC (90% confluence in 6 well) were cultured with lOOng/ml human Activin (Sigma) and 25ng/ml recombinant human Wnt3a (R&D systems) or 3mM CHIR99021 and 1% B27 supplement minus Insulin in RPMI-1640 medium for 1 day and then lOOng/ml human Activin and 1% B27 supplement minus Insulin in RPMI medium for another 4 days (Stage 1 Hepatic-Endoderm). Subsequently, the medium was replaced with differentiation medium with lOng/ml bFGF, 20ng/ml BMP4 and 1% of B27 supplement in RPMI-1640 medium for 3 days (Stage 2). The medium was then replaced with

differentiation medium with 0.1 mM Dexamethasone, 20ng/ml OncostatinM (R&D Systems) and l0-20ng/ml Hepatic Growth Factor (HGF, R&D Systems) and 1% of B27 supplement in Hepatocyte Culture Media (Lonza, MD, CC-3198, withdraw EGF and

Gentamicin/ Amphotericin-B) for 4-22 days (dayl 5-dayl9, Pancreatic endocrine progenitors). The medium was replaced every day (stage 1) or every other day (stage 2 & stage 3).

Primary HUVECs cells and human Adipose-derived stem cells (hADSC) (InVitrogen or PromoCell) were cultured in l5cm dish with EBM Medium (Ronza, cc-3121) or MesenProRS Medium (GIBCO, 12747-010 or Preadipocyte Growth Medium Kit, C-27417), respectively, at 37°C in a humidified 5% C0 ₂ incubator. For co-culturing experiments, day lO-hepatocytes derived from human iPSC were treated with Accutase, while FtUVECs and hADSC were treated with TrypLE (GIBCO, 12604-013) and cells were collected into 50ml tubes, respectively. After the cells were counted, lxlO ⁶ cells of hiPS-PP, 7xl0 ⁶ cells of HUVEC and l-2xl0 ⁵ cells of hADSC were co-cultured in 1 well of 24 well with 300ul of matrigel. Liver-like organoids were formed within 1 to 2 days. Then, liver-like organoids were taken out from MATRIGEL® matrix and cultured in in 3DKG Custom TeSR™. In an embodiment, cells (hepatocytes) of the liver-like organoids were molecularly engineered to express one or more checkpoint proteins.

Generation of 3D (three-dimensional) heart bud in vitro: Organ buds

Cardiomyocyte cells (hiPSC-CDs) were prepared from human iPSC using

differentiation methods as previously described. Briefly, hiPSCs were maintained on MATRIGEL® (BD)-coated dishes in complete Stemcell™ TeSR™ media at 37°C in a humidified 5% C0 ₂ incubator. For cardiac differentiation, hiPSC (90% confluence in 6 well) were cultured with lOOng/ml human Activin (R&D Systems) and 1 OmM CHIR99021 and 1% B27 supplement minus Insulin in RPMI1640 media for 1 days and then 1% B27 supplement minus Insulin in RPMI media for another 2 days (Stage 1 cardiac-Mesoderm). Subsequently, medium was replaced with RPMI1640 with 5mM IWP-2 and 1% B27 supplement minus Insulin in RPMI medium for 1 days (Stage 2). The medium was then replaced with 1% B27 supplement minus Insulin in RPMI Medium for 6 days or more (Stage 3). Cardiac contraction started around day 13. The medium was replaced every day (stage 1) or every other day (stage 2 & stage 3). Primary FtUVECs cells and human Adipose-derived stem cells (hADSC) (Invitrogen or PromoCell) were cultured in l5cm dish with EBM Medium (Ronza, cc-3121) or MesenProRS™ Media (GIBCO, 12747-010 or Preadipocyte Growth Medium Kit, C-27417), respectively, at 37°C in a humidified 5% C0 ₂ incubator. For co-culturing experiments, day 13 to day 15 cardiomyocytes derived from human iPSC were treated with Dispase, while FtUVECs and hADSC were treated with TrypLE (GIBCO, 12604-013) and cells collected into 50ml tubes, respectively. After the cells were counted, lxlO ⁶ cells of hiPS-PP, 7xl0 ⁶ cells of HUVEC and l-2xl0 ⁵ cells of hADSC were co-cultured in 3DKG Custom TeSR™ medium. Mini heart like organs capable of contracting were formed within a few days. In an embodiment, cells (cardiomyocytes) of the mini-heart-like organoids were molecularly engineered to express one or more checkpoint proteins.

Generation of 3D (three-dimensional) intestine bud in vitro: Organ buds

Intestinal cells (hiPSC-ITs) were prepared from human iPSC using differentiation methods as previously described. Briefly, hiPSCs were maintained on Matrigel® (BD)- coated dishes in complete Stemcell™ TeSR™ Medium at 37°C in a humidified 5% C0 ₂ incubator. For intestinal cell differentiation, hiPSC (90% confluence in 6 well plates) were cultured with lOOng/ml human Activin (R&D Systems), 3mM CHIR99021, 2mM Glutamax and 1% B27 supplement minus Insulin in RPMI1640 medium for 1 day and then lOOng/ml human Activin (R&D Systems), 2mM Glutamax and 1% B27 supplement minus Insulin in RPMI1640 medium for another 3 days (Stage 1 Forgut-Endoderm). Subsequently, medium was replaced with 500ng/ml Wnt3a, 500ng/ml FGF4 and 1% B27 supplement in RPMI 1640 medium for 4 days (Stage 2). Cells were transferred to Matrigel® matrix and then a 3D- spheroid Matrigel® dorm was made in the bottom of 24 well. The medium was then replaced with l% B27 supplement, l% N2 supplement, 500ng/ml R-spondin, lOOng/ml Noggin, 50ng/ml EGF, 2mM Glutamax™ supplement, 10mM HEPES in DMEM/F12 Medium for 7 days or more (stage3). Intestinal-like organoid spheroids were observed within a week. The medium was replaced every day (stage 1) and every other day (stage 2 & stage 3). Primary FtUVECs cells and human Adipose-derived stem cells (hADSC) (Invitrogen or PromoCell) were cultured in a l5cm dish with EBM Media (Ronza, cc-3121) or

MesenProRS™ Medium (GIBCO®, 12747-010 or Preadipocyte Growth Medium Kit, C- 27417), respectively, at 37°C in a humidified 5% C0 ₂ incubator. For co-culturing experiments, intestinal progenitors (day 7) derived from human iPSC were treated with Accutase, while FtUVECs and hADSC were treated with TrypLE (GIBCO®, 12604-013) and cells were collected into 50ml tubes, respectively. After counting the cells, lxlO ⁶ cells of hiPS-PP, 7xl0 ⁶ HUVEC cells and l-2xl0 ⁵ hADSC cells were co-cultured in 3DKG Custom TeSR™ medium. In an embodiment, intestinal cells of the intestine-like organoids were molecularly engineered to express one or more checkpoint proteins.

Insulin secretion assay (primary mouse and human pancreatic islets and human iPSC- derived cells)

Insulin release from intact islets was monitored using batch incubation methods (Yoshihara et al., 2010, Nat. Commun. 1 : 127). Briefly, overnight-cultured isolated pancreatic islets (RPMI-1640 supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) Antibiotic- Antimycotic (Gibco)) were pre-cultured at 37°C for 30 min (Krebs-Ringer bicarbonate buffer (KRBB) containing 129.4 mM NaCl, 3.7 mM KC1, 2.7 mM CaCh, 1.3 mM KH ₂ P0 ₄ , 1.3 mM MgS0 ₄ , 24.8 mM NaHC0 ₃ (equilibrated with 5% C0 ₂ , 95% 0 ₂ , pH7.4), lOmM HEPES and 0.2% (v/v) BSA (fraction V, Sigma) (KRBH) with 3 mM glucose). Pancreatic islets were then incubated in KRBH buffer (500 m1/10 islets) with 3 mM or 20 mM glucose to determine insulin secretion levels. After 30 min, islets were pelleted by centrifugation and insulin levels determined by ELISA (Rat/mouse Insulin ELISA KIT (Millipore) and Human Insulin ELISA KIT (Millipore) for mouse and human islets, respectively). For human iPSC derived cells, the cells (lxlO ⁶ cells/well in 24 well) were pre-cultured in 3mM glucose KRBH buffer (500 mΐ/well). The cells were then incubated in KRBB (200 mΐ/well) with 3 mM or 20 mM glucose to determine c-peptide secretion levels as indicator of insulin secretion levels. After 30 min, the cells were pelleted by centrifugation and c-peptide levels were determined by human c-peptide ELISA KIT (Millipore).

Example 10 Methods

Quantitative RT-PCR analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy KIT

(Qiagen). Reverse transcription was performed with a Superscript III First-Strand Synthesis System kit (Invitrogen) or PrimeScript RT reagent kit (TAKARA). Real time quantitative RT-PCR (qPCR) was performed using SYBR Green (Bio-Rad).

Lentivirus Production for Proinsulin-NanoLuc

Proinsulin-NanoLuc in pLX304 (Addgene, #62057) was obtained from Addgene. Proinsulin-NanoLuc lentivirus was produced using a second-generation viral packaging system. Briefly, 14 pg of Proinsulin-NanoLuc, 6.6 pg of PsPAX2 packaging plasmid (Addgene 12260), 5.4pg of pMD2.G envelope plasmid (Addgene 12259) and 54m1

Lipofectamin2000 (Invitrogen) were used to transfect a T75 flask of HEK293LTV packaging cells. Twenty-four (24) hours after transfection, media was changed to fresh DMEM with 10% FBS and 1% Penicillin/Streptozocine. Forty-eight (48) hours and 96 hours after transfection, viruses were collected as day 1 and day 3, respectively and passed through 0.2pm cellulose acetate filters (VWR). Viruses were aliquoted and frozen at -80°C until use. Gaussia Luciferase assay for insulin secretion measurement

Mouse islets, human islets and human islets like organoids were plated in their respective growth media with l0pg/ml Polybrene® polymer (Santacruz). Viruses were then added. After overnight culture, cells were placed in fresh growth media. Forty-eight (48) to 72 hours after infection, mouse islets, human islets and human islet-like organoids were picked up by hand and then placed into 96 wells with single islet or organoid. Then, insulin secretion assays were performed. Briefly, a single islet or organoid was pre-incubated with 3mM glucose KRBB at 37°C for 30 min to 1 hour. The cells were then incubated in KRBB (100 mΐ/well) with 3 mM for 30min and then sequentially incubated with 20 mM glucose with or without 100hM Exendin-4 or 3mM glucose with 20mM KC1 (100 mΐ/well). To determine Gaussia Luciferase activity as indicator of insulin secretion levels, 10m1 of samples are used for Luciferase assay using Pierce Gaussia Luciferase Flash Assay Kit (Prod# 16159, Thermo Scientific).

INS-l cells were infected with the virus by spinfection (800g, 1 hour at 37°C and then changed to fresh INS-l growth media. Seventy-two (72) hours after transfection, INS-l cells were treated with 5pg/ml Blasticidin (Invitrogen) for 7 days to select for Proinsulin-NanoLuc expressing cells. For insulin secretion assay, the cells (5xl0 ⁴ -lxl0 ⁵ cells/well in 96 well) were pre-cultured in 3mM glucose KRBB (100 mΐ/well). The cells were then incubated in KRBB (100 mΐ/well) with 3 mM and then sequentially incubated with 20 mM glucose with or without 100hM Exendine-4 or 3mM glucose with 20mM KC1 (100 pl/well). To determine Gaussia Luciferase activity as indicator of insulin secretion levels, lOpl of samples are used for Luciferase assay using Pierce Gaussia Luciferase Flash Assay Kit (Prod# 16159, Thermo Scientific).

Vascularization test in vitro

Human islet-like organoids were embedded in 1 well of 24 well plate with 300pl of Matrigel® matrix with EBM Media (Ronza, cc-3121). Vascularization was observed within 24-72 hours.

31) culture of hADSCs and WNT protein expression

hADSCs undergo changes in the expression of Wnt genes, in particular genes in the Wnt5a pathway, during the spontaneous self-organization that occurs in 3D culture. Wnt5a was found to be the predominant protein expressed among the Wnt proteins in hADSC 3D culture over time.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.