METHOD FOR IDENTIFYING PANCREATIC PROGENITOR CELLS

GOVERNMENT RIGHTS

[0001] This invention was made with government support under Grant Nos. R01 DK21344, U 19

DK61245 and 5P30 DK063720 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS-REFERENCING

[0002] This application claims the benefit of U.S. provisional application Serial No. 61/337,755, filed on February 9, 2010, which application is incorporated by reference in its entirety herein.

BACKGROUND

[0003] Diabetes mellitus is the third leading cause of death in the U.S. and the leading cause of

blindness, renal failure, and amputation. Diabetes is also a major cause of premature heart attacks and stroke and accounts for 15% of U.S. health care costs. Approximately 5% of Americans, and as many as 20% of those over the age of 65, have diabetes.

[0004] Diabetes results from the failure of the β-cells in the islets of Langerhans in the endocrine pancreas to produce adequate insulin to meet metabolic needs. Diabetes is categorized into two clinical forms: Type 1 diabetes (or insulin-dependent diabetes) and Type 2 diabetes (or non-insulin-dependent diabetes). Type 1 diabetes is caused by the loss of the insulin-producing β-cells. Type 2 diabetes is a more strongly genetic disease than Type 1 (Zonana & Rimoin, 1976 N. Engl. J. Med. 295:603), usually has its onset later in life, and accounts for approximately 90% of diabetes in the U.S. Affected individuals usually have both a decrease in the capacity of the pancreas to produce insulin and a defect in the ability to utilize the insulin (insulin resistance). Obesity causes insulin resistance, and approximately 80% of individuals with Type 2 diabetes are clinically obese (greater than 20% above ideal body weight). Unfortunately, about one-half of the people in the U.S. affected by Type 2 diabetes are unaware that they have the disease. Clinical symptoms associated with Type 2 diabetes may not become obvious until late in the disease, and the early signs are often misdiagnosed, causing a delay in treatment and increased complications. While the role of genetics in the etiology of type 2 diabetes is clear, the precise genes involved are largely unknown.

[0005] Insulin is made exclusively by the β-cells in the islets of Langerhans in the pancreas. During development, the islet cells, including the β-cells, develop from an undifferentiated precursor within the growing pancreatic bud. As the bud grows, the undifferentiated cells form into ducts, and it is these cells that function as precursors. Duct cells appear to retain the capacity to differentiate into islet cells throughout life, and in some circumstances when the pancreas is damaged, new islet cells can form from the duct cells. Unfortunately, islet cell regeneration does not appear to occur when the islet cells alone are damaged, such as in type 1 diabetes.

[0006] This developmental process is clinically relevant for several reasons. First, the formation of islet cells and especially β-cells is necessary in order to make insulin and control energy metabolism. If the process of β-cell development is in anyway impaired, it predisposes that individual to the later development of diabetes. Therefore genes involved in this process are candidate genes for neonatal diabetes, maturity onset diabetes of the young (MODY) or type 2 diabetes. The sequence of these genes could be used to identify individuals at risk for the development of diabetes, or to develop new pharmacological agents to prevent and treat diabetes.

[0007] Second, as discussed above, insulin production is impaired in individuals with diabetes. In type

1 diabetes the impairment is caused by the destruction of the β-cells, while in type 2 diabetes, insulin production is intact, but inadequate. Treatment of type 1 diabetes, as well as many cases of type 2 diabetes, may involve replacement of the β-cells.

[0008] During embryonic development, the pancreas first appears as clusters of cells on the dorsal and ventral aspects of the gut endoderm. The exocrine and endocrine cells that form the adult pancreas differentiate from this pool of pancreatic progenitors. A single transcription factor, the pro-endocrine bHLH factor Neurogenin3, (also called Neurog3 or Ngn3), is both necessary and sufficient to drive these progenitor cells to differentiate into the endocrine cells that form the islets of Langerhans.

Transient activation of Neurogenin3 expression in scattered progenitor cells initiates expression of additional transcription factors, including NeuroDl , Pax4, Nkx2.2, Nkx6.1 , Arx, and others, which then direct the differentiation of those cells into the distinct islet cell subtypes and the activation of the mature islet transcription factors such as MafA, Pax6 and Isl 1. Mutations in many of these genes can cause diabetes, highlighting the pathway's importance in human beta-cell formation and insulin production.

[0009] The replacement of β-cells may be accomplished in several ways. However, the development of new β-cells from precursor cells, either in culture or in vivo in a patient, would be the most physiologically acceptable method.

SUMMARY

[0010] Provided herein is a method of identifying a pancreatic progenitor cell in a population of isolated cells. In general terms, the method comprises assaying for the expression of Rfx6 by a cell of the population and assaying for the expression of a pancreatic marker by a cell of the population. A pancreatic progenitor cell expresses the pancreatic marker and does not express Rfx6. [0011] In some embodiments, the method comprises assaying a cell in the population for expression of

Rfx6 and a pancreatic marker and identifying the cell as a pancreatic progenitor cell if the cell expresses the pancreatic marker and does not express Rfx6. In certain embodiments, the pancreatic marker is selected from the group consisting of Hnfl b, Nkx6.1 , Sox9, pdx l and Nkx2.2. In particular embodiments, the method may comprises assaying the cell for the expression Rfx6 and at least two pancreatic markers selected from the group consisting of Hnfl b, Nkx6.1 , Sox9, pdx l and Nkx2.2. The assaying may be done by a variety of different methods, including immunohistochemistry.

[0012] The method has a number of different applications. In particular embodiments, the method may be used to stage cells. In this application, the method may further comprise staging the population of isolated cells based on how many pancreatic progenitor cells there are in the population. In a related embodiment, method may also be employed to determine how many non-pancreatic progenitor cells there are in the population.

[0013] If pancreatic progenitor cells are identified in the population, the method may further comprise transferring the isolated cell population onto a medium that induces the development of insulin- producing cells from the pancreatic progenitor cells. This embodiment may further comprise administering the insulin-producing islet cells to a mammalian recipient. Alternatively, if pancreatic progenitor cells are identified in the population, the method may further comprise administering the population to a mammalian recipient.

[0014] Also provided is a method that comprises periodically monitoring a cell culture comprising cells having potential to differentiate into pancreatic progenitor cells for a decrease in expression of Rfx6 by the cells. In this method, a decrease in expression of Rfx6 in the cells indicates that the cell culture contains more pancreatic progenitor cells. This method may further involve periodically monitoring the expression of a pancreatic marker. The cells used in this method may, for example, be derived from embryonic stem cells.

[0015] Also provided is a method of producing a pancreatic progenitor cell. In certain embodiments, this method comprises: administering an agent to a cell having potential to differentiate into a pancreatic progenitor cell to decrease the expression of Rfx6 in the cell and thereby produce a pancreatic progenitor cell. The agent may be, for example, an inhibitory or antisense RNA that downregulates the expression of Rfx6, for example. Examples of cells that can differentiate into a pancreatic progenitor cells include, but are not limited to, definitive endoderm, gut endoderm, prepancreatic endoderm, as well as cell types derived from embryonic stem cells and other pluripotent and multipotent stem cell types. In one embodiment, the pancreatic progenitor cells produced by this method can be assessed by assaying the expression of factors that mark pancreatic progenitor cells, such as Sox9, Pdx l , Hnfl b, Onecut l , or Nkx6.1 , singly or in combination. In other embodiments, the resultant cells can be assessed by testing their ability to generate multiple pancreatic cell types, including pancreatic acinar cells, pancreatic duct cells, and pancreatic islet cells.

[0016] Also provided is a method of producing a definitive endoderm cell. In certain embodiments, this method comprises: administering an agent to a cell having potential to differentiate into a definitive endoderm cell to increase the expression of Rfx6 in the cell and thereby produce a definitive endoderm cell. The agent may be, for example, a virus or isolated DNA capable of generating an mRNA encoding the Rfx6 protein, a synthetic RNA encoding the Rfx6 protein, or a protein or small molecule capable of activating the production of the Rfx6 protein. Examples of cells that can differentiate into a definitive endoderm cell include, but are not limited to, embryonic stem cells, induced pluripotent stem cells (iPS cells), adult stem cells, as well as differentiated cell types susceptible to reprogramming by Rfx6, and cell types derived from embryonic stem cells and other pluripotent stem cell types. In one embodiment, the cells produced by this method can be assessed by assaying the expression of factors that mark definitive endoderm cells, such as Sox 17, singly or in combination with other markers. In other embodiments, the resultant cells can be assessed by testing their ability to generate multiple endoderm cell types, including cells that make up endoderm tissues lung, thymus, thyroid, liver, gut and pancreas.

[0017] Also provided is a method of producing a gut endoderm cell. In certain embodiments, this method comprises: administering an agent to a cell having potential to differentiate into a gut endoderm cell to increase the expression of Rfx6 in the cell and thereby produce a gut endoderm cell. The agent may be, for example, a virus or isolated DNA capable of generating an mRNA encoding the Rfx6 protein, a synthetic RNA encoding the Rfx6 protein, or a protein or small molecule capable of activating the production of the Rfx6 protein. Examples of cells that can differentiate into a gut endoderm cell include, but are not limited to, embryonic stem cells, induced pluripotent stem cells (iPS cells), adult stem cells, definitive endoderm cells, as well as differentiated cell types susceptible to reprogramming by Rfx6, and cell types derived from embryonic stem cells and other pluripotent stem cell types. In this embodiment, Rfx6 may be activated in combination with one or more additional factors involved in the formation of gut endoderm, such as the transcription factors Hnfl b and Foxa2. The cells produced by this method can be assessed by evaluating by assaying the expression of factors that mark gut endoderm cells, such as Hnflb and Foxa2, singly or in combination with other markers. Alternatively, the resultant cells can be assessed by testing the ability of the gut endoderm cell to generate multiple gut endoderm cell types, including cells that make up tissues that originate from gut endoderm, such as lung, liver, and pancreas.

[0018] Also provided is a method of producing an islet progenitor cell or islet cell. In certain

embodiments, this method comprises the following: administering an agent to a cell having potential to differentiate into a islet progenitor cell or islet cell to increase the expression of Rfx6 in the cell and thereby produce a islet progenitor cell or islet cell. The agent may be, for example, a virus or isolated DNA capable of generating an mRNA encoding the Rfx6 protein, a synthetic RNA encoding the Rfx6 protein, or a protein or small molecule capable of activating the production of the Rfx6 protein.

Examples of cells that can differentiate into an islet progenitor cell or islet cell include, but are not limited to, embryonic stem cells, induced pluripotent stem cells (iPS cells), adult stem cells, gut endoderm cells, pancreatic progenitor cells, as well as differentiated cell types susceptible to reprogramming by Rfx6, and cell types derived from embryonic stem cells and other pluripotent stem cell types. In this embodiment, Rfx6 may be activated in combination with one or more additional factors involved in the formation of islet progenitor cells or islet cells, such as, but not limited to, the transcription factors Neurog3, Nkx6.1 , Nkx2.2, NeuroD l , Pax4, Pdx l , and Pax4. In one embodiment, islet progenitor cells produced by this method can be assessed by assaying the expression of factors that mark islet progenitor cells, such as Neurog3, singly or in combination with other markers. In other embodiments, the islet progenitor cells can be assessed by evaluating their ability to generate islet cell types, including the islet cells that produce insulin, glucagon, somatostatin, pancreatic polypeptide or ghrelin. The presence of islet cells can be evaluated by the expression of factors that mark islet cells, such as the hormones insulin, glucagon, somatostatin, pancreatic polypeptide or ghrelin, or the transcription factors, NeuroDl , Pax6, isl l , pdx l or Nkx2.2, singly or in combination with other markers.

[0019] An isolated pancreatic progenitor cell that expresses a pancreatic marker but does not express

Rfx6 is also provided.

[0020] A method of identifying a definitive endoderm cell is also provided. In some embodiments this method may comprise assaying a cell for the expression of Rfx6 and, optionally, at least one other marker. A definitive endoderm cell is Rfx6 ⁺ . This method may be employed to distinguish definitive endoderm cells from extra-embryonic endoderm cells, for example. The method may further comprise assaying the cell for the presence of at least one additional marker, e.g., Sox 17.

[0021] Any of the methods described herein may comprise isolating the cells.

[0022] Also provided is a method of identifying a gut endoderm progenitor cell. In certain

embodiments, this method comprises assaying a cell for the expression of Rfx6. A gut progenitor cell if it is Rfx6 ⁺ . This method may in certain embodiments comprise assaying the cell for at least one additional endoderm marker, e.g., Hnfl b, Foxa2, FoxA l or a cdx protein such as cdx2.

[0023] Also provided is a method of identifying an islet progenitor or mature islet cell. In certain embodiments, this method may in certain embodiments comprise assaying a cell for the expression of Rfx6. An islet progenitor or mature islet cell is Rfx6 ⁺ . This method may further comprising assaying the cell for at least one additional pancreatic marker, e.g., Nkx6.1 , Neurog3, Pax4, Pax6, Isl l , Neurod l , MafA, pdx l or Nkx2.2.

[0024] An isolated population of pancreatic progenitor cells is provided. This population of cells may comprise cells that are Rfx6- and Hnfl b ⁺ , Nkx6. T, Sox9 ⁺ , pdx l ⁺ or Nkx2.2 ⁺ . [0025] An isolated population of islet cell progenitors or islet cells is also provided. The population may comprise cells that are Rfx6 ⁺ and Nkx6.1 ⁺ , Neurog3 ⁺ , Pax4 ⁺ , Pax6 ⁺ , Isl T, Neurod T, MafA ⁺ , pdx l ⁺ or Nkx2.2 ⁺ .

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 Expression of Rfx6 in mice and human tissues. In a, the mRNA for Rfx genes 1 -6 were amplified by RT-PCR from RNA isolated from the pancreas and brain of mouse embryos at el 7.5 and from ΝΓΗ3Τ3 fibroblasts. In b, levels of RFX6 mRNA were determined by real-time PCR of RNA from whole pancreas of human foetuses at the ages shown. n= 5 samples per foetal age group. *p= 0.017, weeks 8- 10 vs. 19-21 , by two-tailed Student's t test. In c, mRNA for RFX6 and control gene Cyclophilin A (PPIA) genes were amplified by RT-PCR from RNA isolated from the human adult tissues shown. In d, levels of Rfx3 and Rfx6 mRNA were determined by real-time RT-PCR (TaqMan) of RNA isolated from the pancreata of wildtype and Neurog3 ^-/- mouse embryos at e l 7.5 values and expressed relative to the level of Gusb. n= 3 samples per group. **p = 0.0025, wildtype vs. mutant, by two-tailed Student's t test.

[0027] FIG. 2 Expression of Rfx6 in mice. Immunofluorescence staining was performed for Rfx6 in mouse embryos. In a - c, at e9, Rfx6 staining overlaps with Foxa2 in the gut epithelium (including foregut, FG) and nascent dorsal pancreatic bud (DP), but Foxa2 is expressed alone in the liver bud (Li) and extraembryonic endoderm (EE). Separate colour channels are shown for a and d and b and e. In d - f, costaining was performed with Pdx l (green) in gut (duodenum, Du), dorsal pancreas (DP) and ventral pancreas (VP) at el O. In g, e l 5.5 pancreas was costained for Neurogenin3 (green). Costaining nuclei appear yellow. In h, el 8.5 pancreas was costained for insulin (green). In i, adult pancreas was costained for insulin (green). Higher resolution photomicrographs from additional dates with additional markers can be found in Figs. 10-16. Scale bars, 25 μηι.

[0028] FIG. 3 Targeting of the Rfx6 gene in mice. In panel a-d, lineage tracing was performed on Rfx6

^+/+ /R26R (left in a and c) or Rfx6 ^+/eCFPcre /R26R (b and d, and right in a and c) mice at el 0.5 (a, b) and e l 2.5 (c, d) by staining for β-galactosidase activity with Xgal (blue). Panel b shows a close-up view of the animal on the right in panel a, and panel d shows a close-up view of the animal on the right in panel c. In panel e, an Rfx6 ^{eGFPcre/eGFPcre} pup at p2 is shown on the right, with a wildtype liter mate on the left. In panel f, the dissected abdominal viscera are shown for wildtype (left) and Rfx6 ^{eGFPcre/eGFPcre} (right) pups at p0.5. Li, liver; Du, duodenum; GB, gall bladder; VD, vitelline duct; Th, thymus; Tr, trachea; Oe, oesophagus; Lu, lung; St, stomach; DP, dorsal pancreas; VP, ventral pancreas; Hg, hindgut.

[0029] FIG. 4 Expression patterns of islet markers in wildtype and Rfx6 ^{eGFPcre/eGFPcre} mice at e 17.5. On pancreas sections from e l 7.5 embryos with the genotypes shown at the left, immunofluorescence costaining was performed for ChromograninA (ChromoA, red, a - f , green k, 1), Synaptophysin (Syn, g, h), insulin (, a, b), glucagon (, c, d), somatostatin (Sst, e, 0. ghrelin (g, h), Nkx6.1 (in i - 1), and pancreatic polypeptide (Ppy, k, 1). Quantification of cells expressing Ppy and Nkx6.1 is shown in Table 3. Scale bars, 25 μπι.

[0030] FIG. 5 Function of the human Rfx6 protein. In a and c, DNA binding of the human in vitro- translated proteins shown above each lane to the double-stranded, radiolabeled oligonucleotide HBV X-box probe ⁴² was tested by electromobility shift assay (EMSA). In a, combined proteins were co- translated, and probe bound by the heterodimer partners has a mobility between that of the two homodimers. Truncated proteins Rfx3Tl and Rfx3T2 have the first 1 19 and 160 amino-terminal amino acids removed respectively, but retain the DNA-binding and dimerization domains. In viiro-translated luciferase is included as a negative control. A close-up view of a longer gel is shown in Fig. 19a. In b, mouse pancreatic ductal mPac L20 cells were co-transfected with a DNA plasmid containing the reporter constructs shown and another expressing the RFX cDNAs shown, luciferase reporter expression was assayed for each combination. *p = 0.0026 vs. "no cDNA", 0.0024 vs. Rfx3 alone, and 0.01 1 vs. Rfx6 alone by two-tailed Student's t test. In c, a schematic shows the proposed interactions, either direct or indirect, of several transcription factors during pancreas development. In d, increasing amounts of the in rro-translated human Rfx6 wild type and R181Q and S217P mutant proteins were assayed for binding to the X-box DNA probe. Efficiency of mutant protein production is demonstrated in Fig. 19b. In e, mutations found in patients are indicated on a map of the RFX6 gene. All mutations were homozygous except for proband 3.

[0031] FIGS. 6 and 7 show co-staining of Rfx6 and Nkx6. 1 at embryonic days 12.5 and 15.5 in mouse, respectively.

[0032] FIG. 8 schematically illustrates the developmental pathway for β-cells.

[0033] FIG. 9 schematically illustrates the developmental pathway for β-cells and the genes expressed in pancreatic progenitor cells.

[0034] FIG. 10 Expression pattern of Rfx6 at e9.5. Immunofluorescence staining was performed for

Rfx6 (panel a) and Nkx2.2 (panel b) on a mouse embryo at e9.5. Rfx6 and Nl x2.2 staining overlaps (panel c) in the nuclei of scattered cells in the dorsal pancreatic bud (DP); Rfx6 is expressed alone in the duodenum (Du). Scale bars, 25 μιτι.

[0035] FIG. 11 shows the expression pattern of Rfx6 at e l 0.5. Immunofluorescence staining was performed for Rfx6 (a and d) with Pdx 1 (b and c) and Neurogenin3 (e and f) on a mouse embryo at e l 0.5. Nuclei are counter stained with 4' , 6-diamidino-2-phenylindole (DAPI). Rfx6 and Pdx l stain the nuclei of distinct cells in the dorsal (DP) and ventral pancreatic bud (VP). Rfx6 and Pdx l expression overlaps in the duodenum (Du) adjacent to the pancreatic buds, but Rfx6 staining extends further rostral and caudal along the gut that Pdx l . Rfx6 and Neurogenin3 staining overlaps (yellow) in the nuclei of scattered cells in the dorsal pancreas in panel f. Scale bars, 25 μπι. [0036] FIG. 12 shows the expression pattern of Rfx6 in the pancreas at e l2.5. Immunofluorescence staining was performed for Rfx6 with glucagon (a) and Pdx l (b) in the embryonic mouse pancreas at el 2.5. The nuclei of the glucagon expressing cells stain for Rfx6, while Pdx l and Rfx6 only overlap in the nuclei of rare cells. Scale bars, 25 μm.

[0037] FIG. 13 shows the expression pattern of Rfx6 in the pancreas at e l 3.5. Immunofluorescence staining was performed for Rfx6 with Neurog3 (a) and eGFP expressed from the Neurog3 locus (b and c) in the embryonic pancreas harvested from Neurog3 ^+/+ (a), Neurog3 ^{+/eG FP} (b), and Neurog3 ^{eG FP/eGFP} (c) mice ⁹ at e l 3.5. Rfx6 nuclear staining overlaps with Neurog3 in the Neurog3 ^+/+ pancreas (a) and with eGFP in the Neurog3 ^+eGFP pancreas (b), but is not detectable in the Neurog3 ^{eGFP/eG FP} pancreas. Scale bars 25 μm.

[0038] FIG. 14 shows the expression pattern of Rfx6 in the pancreas at e l 5.5. Immunofluorescence staining was performed for Rfx6 (a, d and g) with Nkx2.2 (b and c), Pdx 1 (e and 0 and Nkx6. 1 (h and i) in the embryonic mouse pancreas at e l 5.5. Scale bars, 25 μιτι.

[0039] FIG. 15 shows the expression pattern of Rfx6 in the pancreas at e 18.5. Immunofluorescence staining was performed for Rfx6 with insulin (a) glucagon (Geg, b), somatostatin (Sst, c), pancreatic polypeptide (Ppy, d) and ghrelin (e) in the embryonic mouse pancreas at e l 8.5. In panels f - h, confocal imaging was used to localize accurately Rfx6 in the nuclei of cells expressing pancreatic polypeptide (Ppy). Scale bars, 25 μπι.

[0040] FIG. 16 shows the expression pattern of Rfx6 in the adult pancreas. Immunofluorescence staining was performed for Rfx6 (red) with insulin (a) glucagon (Gcg, b), somatostain (Sst, c) and pancreatic polypeptide (Ppy, d) in the adult mouse pancreas. Scale bars, 25 μιτι.

[0041] FIG. 17 schematically shows a method for targeting of the Rfx6 locus in mice. The design is shown for the Rfx6 ^eGFPcre targeting construct. The first 5 exons were replaced with a marker gene encoding an eGFPcre fusion protein.

[0042] FIG. 18 shows lineage tracing of the Rfx6-expressing cells. Lineage tracing was performed on

Rfx6 ^+/+ /R26R (left in panel a, and right in panel b) on Rfx6 ^+/eGFPcre /R26R (right in panel a, and left in panel b) mice at pO by staining for β-galactosidase activity with Xgal (blue). In a, Xgal stains the lungs, but not the heart, which derives from the mesoderm germ layer. In panel b, Xgal stains the entire gut, pancreas and liver, but not the mesoderm-derived spleen.

[0043] FIG. 19 shows that Rfx3 and Rfx6 bind to DNA as heterodimer partners. In a, DNA binding of the human in vitro-translated proteins shown above each lane to the double-stranded, radiolabeled oligonucleotide HBV X-box probe ²¹ was tested by electromobility shift assay (EMSA). With Rfx3 in excess, binding of Rfx6 homodimers (6/6) was completely lost and replaced with the Rfx6/Rfx3 heterodimers in lanes 4 and 5. In b, the efficiency of translation of the wildtype and mutant Rfx6 proteins was compared by including ³⁵ S-labelled methionine in the translation mix and separation by gel electrophoresis of equal amounts of the translation products. [0044] FIG. 20 shows that Rfx6 is not required for the generation of primary cilia. In panels a-d, immunofluorescence staining was performed for chromograninA with acetylated β-tubulin, a marker primary cilia, in pancreas harvested at e l 7.5. Primary cilia appear as long, fine strands linked to chromograninA-expressing cells. Scale bars, 25 μιτι. In e, the mRNA for genes expressed in primary cilia, Ift88 and Dync2lil were amplified by real-time RT-PCR (TaqMan) from RNA isolated from the pancreas at e l 7.5 from mouse embryos with the genotypes shown and expressed relative to the level of Gusb.

DETAILED DESCRIPTION

[0045] Before the present compositions and methods for islet cell and insulin production are

described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0046] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an islet transcription factor" includes a plurality of such islet transcription factors and functional equivalents thereof, and reference to "the polynucleotide" includes reference to one or more polynucleotides and equivalents thereof known to those skilled in the art, and so forth.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[0048] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the cell lines, vectors, and methodologies which are described in the publications which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

[0049] The term "pancreas" is art recognized, and refers generally to a large, elongated, racemose gland situated transversely behind the stomach, between the spleen and duodenum. The pancreatic exocrine function, e.g., external secretion, provides a source of digestive enzymes. Indeed, "pancreatin" refers to a substance from the pancreas containing enzymes, principally amylase, protease, and lipase, which substance is used as a digestive aid. The exocrine portion is composed of several serous cells surrounding a lumen. These cells synthesize and secrete digestive enzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospholipase A ₂ , elastase, and amylase.

[0050] The endocrine portion of the pancreas is composed of the islets of Langerhans. The islets of

Langerhans appear as rounded clusters of cells embedded within the exocrine pancreas. Four different types of cells-ct, β, δ, and φ-have been identified in the islets. The a cells constitute about 20% of the cells found in pancreatic islets and produce the hormone glucagon. Glucagon acts on several tissues to make energy available in the intervals between feeding. In the liver, glucagon causes breakdown of glycogen and promotes gluconeogenesis from amino acid precursors. The δ cells produce somatostatin which acts in the pancreas to inhibit glucagon release and to decrease pancreatic exocrine secretion. The hormone pancreatic polypeptide is produced in the φ cells. This hormone inhibits pancreatic exocrine secretion of bicarbonate and enzymes, causes relaxation of the gallbladder, and decreases bile secretion. The most abundant cell in the islets, constituting 60-80% of the cells, is the β cell, which produces insulin. Insulin is known to cause the storage of excess nutrients arising during and shortly after feeding. The major target organs for insulin are the liver, muscle, and fat-organs specialized for storage of energy.

[0051] By "precursor cell" or "progenitor cell" is meant any cell that is capable of developing into a different cell, either directly or indirectly (i.e., via an intermediate cell type).

[0052] The term "pancreatic progenitor cell" refers to a cell which can differentiate into a cell of pancreatic lineage, e.g. a cell which can produce a hormone or enzyme normally produced by a pancreatic cell. A pancreatic progenitor cell can be caused to differentiate into any epithelial cell of the mature pancreas, including and endocrine, exocrine or duct cells, e.g., into an α, β, δ, or φ islet cell, or a cell of exocrine fate. A pancreatic progenitor cell can also be cultured prior to administration to a subject under conditions which promote cell proliferation and differentiation. These conditions include culturing the cells to allow proliferation and confluence in vitro at which time the cells can be made to form pseudo islet-like aggregates or clusters and secrete insulin, glucagon, and somatostatin. Pancreatic progenitor cells are reviewed in Ku et al (Endocrinology 2008 149:4312-4316), which publication is incorporated by reference for all description of those cells. Such cells may express at least one of the following markers: Nkx6.1 +, Sox9+, pdx l + Hnfl b, Onecut l , Foxa2, Gata4, Gata6, Mnx l/Hb9, Ptfl a, Sox9 and Nkx2.2, as illustrated in Fig. 9. Such cells are Rfx6-. As shown in Fig. 9, some of these markers are seen early but not late in pancreatic progenitor cells, distinguishing between primary and secondary pancreatic progenitors cells.

[0053] Pancreatic progenitor cells are a type of "multipotent" cell, where such a cell can give rise to a limited number of other particular cell types. Multipotent cells are committed to one or more embryonic cell fates, and thus, in contrast to pluripotent cells, cannot give rise to each of the three embryonic cell lineages as well as extraembryonic cells.

[0054] The term "pluripotent stem cell" is used herein to describe a cell that can give rise to each of the three embryonic cell lineages as well as extraembryonic cells. This term includes embryonic stem (ES) cells (for example, human or mice ES cells) that are derived from e.g., the inner cell mass of a blastocyst or primordial germ cells of a post-implantation embryo, as well as induced pluripotent stem (iPS) cells derived from adult tissue.

[0055] The term "substantially pure", with respect to progenitor cells, refers to a population of

progenitor cells that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% pure, with respect to progenitor cells making up a total cell population. Recast, the term "substantially pure" refers to a population of progenitor cells that contain fewer than about 40%, fewer than about 30%, fewer than about 20%, fewer than about 10%, or fewer than about 5%, of lineage committed cells in the original unamplified and isolated population prior to subsequent culturing and amplification.

[0056] As used herein, the term "differentiate" refers to the development of a cell type that is generic to a more specialized cell type. The term "differentiated" as used herein encompasses cell types that are both partially and terminally differentiated. As used herein the term "differentiate" generally refers to a process by which a generic cell develops into a more specialized cell.

[0057] As used herein, "definitive endoderm" or "gut endoderm" refer to multipotent cells that can differentiate into cells of the gut tube or organs derived from the gut tube.

[0058] By "euglycemia" or "euglycemic state" is meant a state associated with a level of blood

glucose that is normal or nearly normal, particularly relative to the levels of blood glucose in a subject having a disease or condition associated with hyperglycemia. In humans, euglycemia correlates with blood glucose levels in the range of 70 mg/dl to 130 mg/dl.

[0059] As used herein, "insulin-producing cell" is a cell that produces insulin. In certain cases, an insulin-producing cell may have the characteristics of a β cell. Insulin production by an insulin- producing cell may or may not be inducible by glucose.

[0060] By "islet cell" is meant a cell having a phenotype similar to the hormone-producing cells

normally comprising the pancreatic islets of Langerhans, and generally characterized by the expression of markers that normally distinguishing the cells in the pancreatic islets of Langerhans from other pancreatic cells, such as insulin, glucagon, somatostatin, pancreatic polypeptide, or islet amyloid polypeptide.

[0061] By "β cell" is meant a pancreatic islet cell having a phenotype characterized by the expression of markers that normally distinguish the beta-cells from the other pancreatic islets cells, such as insulin, Nkx6.1 or glucokinase. [0062] By "a cell" is meant a pancreatic islet cell having a phenotype characterized by the expression of markers that normally distinguish the a-cells from the other pancreatic islets cells, such as proglucagon or glucagon.

[0063] By "subject" or "patient" is meant any mammalian subject for whom therapy is desired. In one embodiment, the subject or patient may be a human. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. Of particular interest are subjects having an insulin- associated disorder that is amenable to treatment (e.g., to mitigate symptoms associated with the disorder) transplantation of cells produced using methods described herein.

[0064] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease, disorder, or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease or disorder and/or any adverse effect attributable to the disease or disorder. "Treatment" as used herein covers any treatment of a disease or disorder in a mammal, particularly a human, and includes: (a) preventing the disease, disorder, or symptom from occurring in a subject which may be predisposed to the disease, disorder or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease, disorder, or symptom, i.e., arresting its development; or relieving the disease, disorder, or symptom, i.e., causing regression of the disease, disorder, or symptom. Thus "treatment of diabetes" encompasses one or more of reduction of blood glucose levels, increase in insulin production, and the like.

[0065] By "insulin-associated disorder" is meant a disease, disorder, or condition that is caused by or involves, either directly or indirectly, a change in level of insulin production or a change in ability of a subject to utilize insulin, for example, to modulate blood glucose levels. Insulin-associated disorders include, but are not limited to, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, hypoglycemia, and the like. Of particular interest are insulin-associated disorder that are amenable to treatment (e.g. , to mitigate symptoms associated with the disorder) by implantation of cells.

[0066] The term "assaying for the expression" and grammatical equivalents thereof includes

measuring the expression of a protein or its encoding mRNA. Assays for the expression of proteins and mRNA are well known in the art and include immunochemistry, RT-PCR and FACS, etc.

[0067] The term "pancreatic endoderm cells" refers to a population of cells that are derived from definitive endoderm and that can develop into all cell types of the pancreas. A population of pancreatic endoderm cells includes pancreatic progenitor cells and their progeny, including differentiated, mature pancreatic cells. The presence or absence of Rfx6 in a cell, in combination with the presence of at least one other pancreatic marker, determines whether the population of cells contain pancreatic progenitor cells. In certain cases pancreatic progenitor cells are produced by culturing definitive endoderm cells on a medium that promotes the differentiation of those cells into pancreatic progenitor cells. In certain cases, the cells may result from the culture of definitive endoderm cells on a medium that contains retinoinic acid.

[0068] The term "pancreatic marker" refers to any marker, now known, or later identified, whose presence in a cell identifies pancreatic cells relative to other cells, e.g., other gut cells. Such markers include Hnfl b, Nkx6. 1 , Sox9, pdx l and Nkx2.2, although others are known and are shown in, e.g., Fig. 9. The term pancreatic marker includes markers that are not truly unique to pancreas tissue, but which identify pancreatic cells when used in combination with other markers. For example, in addition to being expressed in pancreatic cells, Pdx l is also expressed in duodenum and brain. Likewise, Pax9 is also expressed in brain, gut, bone, and other tissues. However, when those markers are used in combination with Rfx6, the combination uniquely identifies pancreas cells.

[0069] The term "does not express", with respect to a particular marker, refers to a level of expression that is undetectable over background. Examples of methods that may be used to detect expression of a marker include, but are not limited to, immunohistochemistry, western blot, northern blot, or RT-PCR. In certain cases, expression over background may be detected if a signal is at least twice that of the background.

[0070] The term "reduced expression", refers to at least a 50% reduction, e.g., at least a 60%, 70%,

75%, 80%, 85%, 90%, 95%, 98% or 99% reduction, relative to a control (e.g., a related cell at a different stage or a cell from a subject with a disorder compared to one from an individual without the disorder).

[0071] As used herein, the term "isolated", with respect to a cell, refers to a cell that is cultured, or otherwise obtained in vitro. If a mammal is described as containing isolated cells, then those isolated cells were obtained in vitro and then implanted into the animal.

[0072] As used herein, the term "implanting" is intended to encompass direct (e.g., injection directly into a region) and indirect (e.g., systemic administration) methods by which cells are placed in a recipient.

[0073] The term "culturing", in the context of culturing one cell type (e.g., an embryonic stem cell) into another cell type (e.g., a pancreatic endoderm cell) may be a multistep process.

[0074] "Monitoring," as used with respect to a culture may be done using either the entire, or a

portion of the culture.

[0075] Cells "having potential to differentiate into a cell of the islet cell lineage" refers to cells that can ultimately differentiate into islet cells. Examples of such cells include, but are not limited to, definitive endoderm, gut endoderm, pancreatic endoderm, pancreatic progenitor, and other cells shown in Figs. 8 and 9 and described above and below. DESCRIPTION OF SPECIFIC EMBODIMENTS

[0076] Provided herein are a number of methods and compositions relating to Rfx6, a protein that can be employed to identify or induce various cell types in the islet cell lineage.

[0077] In some embodiments, Rfx6 can be employed alone or in conjunction with other markers to identify cell types in the islet cell lineage. For example, in one embodiment, Rfx6 can be used alone or in conjunction with Sox 17 to identify definitive endoderm cells. In another embodiment, Rfx6 can be used alone or in conjunction with Hnfl b Foxa2, FoxA l or cdx2, to identify gut endoderm cells. In a further embodiment, Rfx6 can be used alone or in conjunction with other markers, e.g., Hnfl b, Nkx6.1 , Sox9, pdx l or Nkx2.2, to identify pancreatic progenitor cells. Rfx6 can also be used alone or in conjunction with other markers, e.g., Nkx6. 1 , Neurog3, Pax4, Pax6, Isl l , Neurod l , MafA, pdx l or Nkx2.2, to identify islet cell progenitor cells or islet cells. Definitive endoderm cells, gut endoderm cells, islet progenitor cells and islet cells are Rfx6 ⁺ , whereas pancreatic progenitor cells are Rfx6-. This method may further comprise isolating target cells from other cells based on their expression of Rfx6.

[0078] In other embodiments, a first cell type can be induced to be a cell in the islet cell lineage by altering the expression of Rfx6. For example, definitive endoderm cell, gut endoderm cells, islet progenitor cells and islet cells can be induced from cells having potential to differentiate into those cells by either inducing (for definitive endoderm cell, gut endoderm cells, islet progenitor cells and islet cells) or repressing (for pancreatic progenitor cells) the expression of Rfx6. The cells produced by this method can be tested by evaluating the expression of proteins that mark the cell type produced, or by testing the cells to determine which cell types they can differentiate into.

[0079] Also provided are a variety of compositions comprising cells made by any of the methods described above (i.e., made by identify or inducing cells). These cells find use in a variety of different applications. For example, the cells may be administered to a subject to modulate, e.g., increase or decrase,blood glucose levels, or the cells may be induced to another cell in the islet cell lineage by altering the culture medium.

[0080] In one embodiment, a method of identifying a pancreatic progenitor cell is provided. In general terms, the method comprises assaying the expression of Rfx6 and a pancreatic marker by a cell. The method may further comprise identifying the cell as a pancreatic progenitor cell if the cell expresses the pancreatic marker and does not express Rfx6. In particular embodiments, the pancreatic marker may be Hnfl b, Nkx6. 1 , Sox9, pdx l or Nkx2.2, although other markers are known and may be employed. Figs. 8 and 9 shows exemplary markers that may be employed. Also, as shown in Figs. 8 and 9, several cell types, including definitive endoderm, gut endoderm, prepancreatic endoderm, pancreatic progenitors and mature pancreatic cells can be identified by the presence or absence of Rfx6, in combination with one or more (e.g., two, three, four or five or more other markers). For example, pancreatic progenitor cells can be identified by being Rfx67Hnfl b ⁺ , Rfx67Nkx6.1 \ Rfx6 ^" /Sox9 ⁺ , Rfx67pdx l ⁺ or Rfx67Nkx2.2 ⁺ , etc. The expression of further markers may be evaluated in determining whether a cell is a pancreatic precursor cell.

[0081] The method of identifying a pancreatic progenitor cell may be performed using any of a variety of suitable gene expression assays, including, but not limited to, RT-PCR, western blotting, immunohistochemistry, in situ hybridization, FACS, etc., many methods for which are known to one of skill in the art.

[0082] The methods of the invention may further include staging the population of isolated cells based on how many pancreatic progenitor cells there are in the population. As will be described in greater detail below, the method may be employed in an in vitro cell differentiation protocol in which a population of multipotent or pluri potent cells are cultured on a medium that is expected to effect their development into pancreatic endoderm cells. The correct staging of the cells allows the stage with the optimum number of pancreatic progenitor cells to be employed in the next step of the culture method. For example, once a pancreatic progenitor cell is identified in the population, the population may be administered to a mammalian recipient in order to, e.g., modulate blood glucose levels in the recipient.

[0083] In another embodiment, once a pancreatic progenitor cell is identified in the population, the population may be transferred onto a medium that induces the development of insulin-producing cells from the pancreatic progenitor cells. The insulin producing cells may then be administered the mammalian recipient. In certain embodiments, the method may further comprise determining how many non-pancreatic progenitor cells there are in the population, which indicates the proportion of non-effective cells that are being transferred to the next step. In some embodiments, the methods of the invention may involve assaying a first portion of a population of cells and administering a second population of cells that have been cultured along with the first population of cells to the mammalian recipient.

[0084] Also included is a method that comprises periodically monitoring a cell culture comprising cells that have potential to differentiate into pancreatic progenitor cells for a decrease in expression of Rfx6 by the cells. A decrease in the expression of Rfx6 in the cells indicates that the cell culture contains pancreatic progenitor cells. This method may further comprise periodically monitoring the expression of a pancreatic marker, as described above. In particular embodiments, the cells may be derived from a pluripotent cells, e.g., an ES cell.

[0085] The methods of the invention may be employed to isolate a pancreatic progenitor cell that expresses a pancreatic marker but does not express Rfx6. As such, in addition to the above method, an isolated a pancreatic progenitor cell that expresses a pancreatic marker but does not express Rfx6 is also provided. The method may be employed to identify cultures that have a high proportion of pancreatic progenitor cells. An isolated culture of cells that contains at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% pancreatic progenitor cell) is also provided.

[0086] Also provided herein is a method of producing a pancreatic progenitor cell. In general terms, this method includes administering a compound to an isolated precursor of a pancreatic progenitor cell to decrease the expression of Rfx6 in the cell and thereby produce a pancreatic progenitor cell. This method may be done by a variety of methods, e.g., by transient expression of an inhibitory compound, e.g., a dominant negative mutant of Rfx6 or an inhibitory RNA that indicibly reduces the expression of Rfx6 and allows the cell to differentiate into a pancreatic progenitor cell. Other recombinant methods known to one of skill in the art may be used to decrease Rfx6 expression in addition to the methods described above.

[0087] The method described above may be used to more efficiently guide the differentiation of ES cells or cells from other cell sources into cell populations that can be transplanted to a mammalian recipient (e.g., a human) in order to treat the subject for an insulin-related disorder, for example, diabetes. As noted above, the transplanted cells may be a culture of cells containing pancreatic progenitor cells, or insulin-producing cells that are differentiated from pancreatic progenitor cells. The ability to identify pancreatic progenitor cells using the methods of the invention allows one to optimize the efficiency of cell culture methods for making such cells, and to optimize the timing at which a culture is used in the next step of a method. The ability to identify pancreatic progenitor cells also allows one to determine the portion of non-productive cells (i.e., define how "pure" the pancreatic progenitor cells are) that are used in the next step of the method.

[0088] The above-described method for identifying pancreatic progenitor cells may be incorporated into any method for producing insulin-producing cells from a pluripotent or multipotent cell population. Examples of methods for producing insulin-producing cells are described in a variety of publications including, but not limited to: D' Amour et al (Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 2006 24: 1392-1401 ), Cho et al (Betacellulin and nicotinamide sustain PDX l expression and induce pancreatic beta-cell differentiation in human embryonic stem cells. Biochem. Biophys. Res. Commun. 2008 366: 129-134), Jiang et al (Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 2007 25: 1940-1953), Zhang et al (Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res. 2009 19: 429-438), Eshpeter et al (In vivo characterization of transplanted human embryonic stem cell-derived pancreatic endocrine islet cells. Cell Prolif. 2008 41 : 843-858), Phillips et al (Directed differentiation of human embryonic stem cells in to the pancreatic endocrine lineage. Stem Cells Dev. 2007 16: 561-578), Shim et al (Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia 2007 50: 1228-1238), Mao et al (The reversal of hyperglycaemiain diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells. Biomaterials 200930: 1706-171 ), Jiang et al (In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res. 2007 17: 333-344), Kroon et al, (Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo Nature Biotechnology 2008 26, 443- 452), US20090092586, US20090325294, US20090263896, US20090092586, US2009001 1502, US20080268534, US20060275900, US20090004238, Van Hoof et al (Derivation of insulin-producing cells from human embryonic stem cells. Stem Cell Res. 2009 3:73-87), Murtaugh et al (Pancreas and - beta-cell development: from the actual to the possible. Development 2007 134, 427-438), Santana et al (Insulin-producing cells derived from stem cells: recent progress and future directions. J. Cell. Mol. Med. 2006 10:866-83) and Roche et al (Generation of new islets from stem cells. Cell Biochem Biophys. 2004 40: 1 13-24). The methods described in these references are incorporated by reference herein, specifically for disclosure of generic and specific descriptions of those methods, culture media, terminology to describe cells and markers.

[0089] Some of the approaches described above involve passing cells through three key

developmental stages: definitive endoderm induction, pancreas specialization and islet maturation, that mimic the in vivo developmental process of the pancreas.

[0090] Activin A is thought to be a key induction factor for hES differentiation into definitive

endoderm lineages, and has been reported to be indispensable for definitive endoderm differentiation from both mouse and hES cells. hES cells have been shown to differentiate into up to 80% definitive endoderm cells with activin A treatment. Currently, certain methods used for the differentiation of hES cells into pancreatic cells utilize activin A to induce initial definitive endoderm specialization.

However, TGF /activin A is not the only factor used for endoderm induction. Repression of the PI3K pathway may also be essential for definitive endoderm specialization. Wortmannin and activin may be used. In certain cases, the expression of specific endoderm genes may be monitored, and SOX 17 and FOXA2 expression may be increased whereas the expression of OCT4 may be decreased.

Differentiated hES cells may be double-positive for CXCR4 and SOX 17. This step may also include inducing signaling through Wnt3a, Na-butyrate, or BMP4 which have also been utilized. Other methods may be employed in this step.

[0091] The specialization.of pancreatic progenitors from definitive endoderm may be done using RA,

KGF7FGF10, NOGGIN and EGF, among others. Retinoic acid is an important signaling molecule that is involved in the development of the early embryonic pancreas as well as in the induction of ectoderm and mesoderm development. During zebrafish development, increased RA signaling can induce remarkable anterior expansion of the pancreas and liver endoderm. Conversely, inhibition of RA signaling by BMS493 is thought to block early pancreas differentiation from embryonic endoderm. The sequential treatment of activin A and RA, allows mouse ES cells to express pancreatic progenitor markers, such as pdx 1 , hnOp and hnf4a, as well as others. Besides RA, other factors, e.g., FGF4, ' NOGGIN and EGF, may be important for the specialization of pancreatic progenitor cells. This step is generally described in D'Amour et al (Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol, 2006, 24: 1392— 1401 ), Jiang et al. (Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells, 2007, 25: 1940- 1953), Phillips et al. (Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage. Stem Cells Dev, 2007, 16: 561 -578), Shim et al. (Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia, 2007, 50: 1228-1238), Zhang et al. (Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin- producing cells. Cell Res, 2009, 19: 429-438), Shi et al. (Inducing embryonic stem cells to differentiate into pancreatic β cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells, 2005, 23: 656— 662) and roon et al. (Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol, 2008, 26: 443— 452). The above-described method for identifying pancreatic precursor cells may be employed at the end of this step, for example, to identify the optimum time to induce islet cell maturation and/or to optimize growth conditions.

[0092] The final step for the differentiation of hES cells into pancreatic islet cells is to induce

pancreatic islet cell maturation. Several strategies are currently used to promote pancreatic islet cell maturation from hES cell-derived pancreatic progenitors. For example, such cells may be induced using, e.g., insulin-transferrin-selenite (ITS) medium containing fibronectin, fresh RPMI 1640 medium supplemented with 0.5% bovine serum albumin, nicotinamide and insulin-like growth factor (IGF-Π), or RPMI- 1640 medium supplemented with glucose, 5% FBS, HGF, pancreatic polypeptide Y (PYY) and forskolin.DMEM/F12 media supplemented with ITS, bFGF, nicotinamide, exendin-4 and BMP4. The following reference is incorporated by reference in its entirety for all purposes: Soyer et al, (Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development Development. 2010 137:203- 12).

[0093] Also provided is a method of identifying a definitive endoderm cell. This method may

comprise assaying the cell for the expression of Rfx6, where a cell is a definitive endoderm cell if it is Rfx6 ⁺ and expresses at least one additional marker, e.g., Sox 17 or another marker described above or in Figs. 8 or 9, or another phenotypic characteristic. In particular embodiments, the cell may be a definitive endoderm cell if it is Rfx6+ and Sox l 7 ⁺ . This method may distinguish definitive endoderm cells, which develop into, e.g., lung, thymus, thyroid, gut and other internal organs, from extraembryonic endoderm cells, which develop into extra embryonic membranes and tissues.

[0094] Also provided is a method of identifying a gut endoderm progenitor cell comprising: assaying the cell for the expression of Rfx6, where a cell is a gut progenitor cell if it is Rfx6 ⁺ characteristic and expresses at least one additional endoderm markers, e.g., Hnfl b, Foxa2, FoxA l or a cdx protein such as cdx2, or another phenotypic characteristic. Exemplary additional markers include those discussed above and are illustrated in Figs. 8 and 9. Also provided is a method of identifying an islet progenitor or mature islet cell comprising: assaying the cell for the expression of Rfx6, where a cell is an islet progenitor or mature islet cell if it is Rfx6 ⁺ and expresses at least one additional pancreatic marker. Exemplary markers are discussed above and are illustrated in Figs. 8 and 9.

[0095] A composition comprising an isolated population of pancreatic progenitor cells is also

provided, where the cells have a marker profile discussed above. In one embodiment, the population is a) Rfx6- and b) Hnfl b\ Nkx6.1 \ Sox9 ⁺ , pdx l ⁺ or Nkx2.2 ⁺ . Also provided is a composition comprising an isolated population of islet cell progenitors or islet cells, where the population is Rfx6 ⁺ and Nkx6.1 ⁺ , Neurog3 ⁺ , Pax4 ⁺ , Pax6 ⁺ , Isl T, Neurod l ⁺ , MafA\ pdx l ⁺ or Nkx2.2 ⁺ .

[0096] The isolated cells described above may be employed to modulate, for example, lower or raise, blood glucose levels in a subject. As such, a method of lowering blood glucose level in a subject, for example, a mammal, comprising administering to the mammal the composition of: a) pancreatic progenitor cells or b) islet cell progenitors or islet cells, is provided. In particular embodiments, this method may be achieved by directly administering the cells to the mammal, e.g., by direct injection or angiography of a suitable number of cells (e.g., 10 x 10 ⁴ cells to 1 x 10'° cells). In particular embodiments, the cells may be administered directly to the pancreases, liver, or fat pad, or subcutaneously, either as free cells or encapsulated, for example, in a gel, membrane or a device.

EXAMPLES

[0097] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out the invention and is not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

METHODS SUMMARY

[0098] All studies involving mice were approved by the UCSF Institutional Animal Care and Use

Committee. Timed matings were carried out with embryonic day 0.5 being set as midday of the day of discovery of a vaginal plug. The Rfx6 targeting allele (Fig. 17) was generated by recombineering in a modified bacterial artificial chromosome followed by recombination into plasmid DNA by gap-repair. This construct was used by the UCSF DERC Transgenic Core Laboratory to target the Rfx6 allele in 129 (E l 4) mouse embryonic stem cells, which were injected into mouse blastocysts to generate chimeric Rfx6 ^+/,GFPcrr mice. Chimeras and subsequent generations were crossed to C57BL/6 mice. Mouse tissue processing, β-galactosidase detection, immunofluorescence staining, automated cell counting, transfections of mPAC cells, EMSA and mRNA quantification with low density TaqMan arrays were performed using known methods. All antisera used for immunofluorescence studies are listed in Table 4. Sequences of oligonucleotides used for RT-PCR, ESC colony screening, mouse genotyping and EMSA are available on request.

[0099] The human subjects protocol was approved by the IRB of the Montreal Children's Hospital and written informed consent was obtained from all participating families. Clinical findings and case-report references are summarized in the web supplement. For homozygosity mapping, results from the Illumina Hap 550 (proband #1 , call rate 0.99) or 1 M (proband #2, call rate 0.978) microarrays, were used to scan autosomes in 300-SNP windows and HBD was defined as absence of any heterozygous SNP in the proband and presence of at least one in either parent or the unaffected sibling. Long-oligo Nimblegen capture arrays included around 100 bp extension of intronic sequence coverage from the boundary of the exons. The eluted enriched regions from proband #1 were run on the Roche 454 FLX Genome Sequencer. The 1 exons of RFX6 were PCR-amplified manually for Sanger sequencing in the remaining probands. For the long range PCR, we used the Kit # K0182 from Fermentas for fragments ranging from 6 to 17 Kb (Fig 17).

EXAMPLE 1 : EXPRESSION OF RFX6

[00100] In independent screens for genes co-expressed with Neurogenin3 in islet progenitor cells

(Miyatsuka, T., Li, Z. & German, M. S. Chronology of islet differentiation revealed by temporal cell labeling. Diabetes 58, 1863-1868 (2009)), activated by Neurogenin3 (Gasa, R. et al. Induction of pancreatic islet cell differentiation by the neurogenin-neuroD cascade. Differentiation; research in biological diversity 76, 381 -391 (2008)), and uniquely expressed in islets, we identified Rfx6, a member of the RFX (Regulatory Factor X-box binding) family of winged-helix transcription factors (Aftab, S., Semenec, L., Chu, J. S. & Chen, N. Identification and characterization of novel human tissue-specific RFX transcription factors. BMC Evol Biol 8, 226 (2008); Emery, P., Durand, B., Mach, B. & Reith, W. RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic acids research 24, 803-807 ( 1996)). Rfx6 transcripts could be detected in mouse and human embryonic pancreas by RT-PCR (Fig. l a.b), but, unlike all other known islet transcription factors, not in brain (Fig. l a, c), and not in mouse embryonic pancreas lacking Neurogenin3 (Fig. I d). In contrast, mouse Rfx4, the mammalian RFX gene with the highest homology to Rfx6, was amplified from brain and not from pancreas, and the other RFX genes were more widely expressed (Fig. l a).

[00101] To explore the pattern of Rfx6 protein expression, antiserum generated against recombinant

Rfx6 protein was used for immunofluorescence studies. In mice, Rfx6 was detected as early as embryonic day 7.5 (e7.5) throughout the definitive, but not extraembryonic, endoderm and persisted broadly in gut endoderm at e9.0, after which it becomes progressively restricted to the pancreas and scattered cells in the gut (Fig. 2a-c, Fig. 10 and data not shown). At e l 0.0, immunofluorescence staining detected Rfx6 in foregut/midgut epithelium and in scattered cells in the nascent pancreatic buds, as indicated by staining for pancreatic transcription factor Pdx l (Fig. 2d-f). Most of these scattered Rfx6-expressing cells did not co-express Pdx l , but many co-expressed Nkx2.2 and

Neurogenin3 (Figs. 1 1 and 12). By e l 2.5, the Rfx6-expressing cells were generally distinct from the Pdx l -expressing progenitor cells, but most co-expressed glucagon, demonstrating restricted expression of Rfx6 in the endocrine lineage even at this early stage (Fig. 12). In pancreata from Neurog3 ^v- embryos, there were no Rfx6-expressing cells (Fig. 13).

[00102] During the peak of endocrine cell differentiation at el 5.5, Rfx6 co-localized with Neurogenin3 in the nuclei of a subset of the endocrine progenitor cells (Fig 2g), and overlapped with the islet transcription factors Nkx2.2, Nkx6.1 , and Pdxl (Fig. 14). At el 8.5, Rfx6 could be found in the nuclei of cells expressing each of the major pancreatic endocrine hormones (Fig. 2h and Fig. 15). In the adult pancreas, Rfx6 expression was restricted to the islets where it could be detected in all endocrine lineages (Fig. 2i and Fig. 16).

[00103] To generate Rfx6 null mice, homologous recombination was used to replace the first five exons of the Rfx6 gene, including the sequences encoding the DNA-binding domain, with a cassette encoding an eGFP-cre fusion protein (Fig. 17). By crossing mice heterozygous for the mutant allele with mice carrying the marker gene ROSA26 loxP-stop-loxP lacZ (R26R), we generated

Rfx6 ^+/eCFPcre /R26R double heterozygous mice in which Rfx6-expressing cells and their descendents are marked by the expression of β-galactosidase and can be visualized with the X-gal substrate (Fig. 3a-d and Fig. 18). β-galactosidase expression was detected in all embryonic tissue derived from the endoderm germ layer, but not in other embryonic or in extraembryonic tissues, demonstrating that Rfx6 is broadly expressed in and. restricted to the definitive endoderm prior to the formation of the endoderm-derived organs. Taken together, the immunohistochemistry data and lineage tracing demonstrate that Rfx6 is expressed initially broadly in the definitive endoderm after gastrulation, becomes restricted to the gut and pancreatic bud at mid gestation, is reactivated by Neurogenin3 in islet progenitor cells and is ultimately restricted to pancreatic islets in the mature pancreas.

[00104] From heterozygous crosses, homozygous Rfx6 ^{eGFPcre/eGFPcre} _m j _{ce were} born at the expected

Mendelian ratio, but failed to feed normally, exhibited gross bowel distension due to small bowel obstruction (Fig. 3e-f) and died within 2 days post partum. Some, but not all, of the Rfx6 null animals also had reduced pancreas size (data not shown).

[00105] To test for effects on gene expression prior to birth, RNA was harvested from e l 7.5 pancreata, and used low density TaqMan arrays (Miyatsuka, T., Li, Z. & German, M. S. Chronology of islet differentiation revealed by temporal cell labeling. Diabetes 58, 1863- 1868 (2009)) to measure the levels of a set of pancreatic genes (Table 1 ). Rfx6 ^{eGFPcre/eGFPcre} pancreata had almost no expression of the islet hormones genes, except for pancreatic polypeptide (Ppy). Several other islet/p-cell genes, such as the zinc transporter Slc30a8 and G-protein coupled receptor GPR40/Ffar genes, were similarly reduced; but other β-cell genes were more modestly reduced, including the glucose sensing genes Gck, Slc2a2, and Kcnjl l. Immunofluorescence staining with the endocrine markers Chromogranin A or Synaptophysin at e l 7.5 demonstrated that the Rfx6 ^{eGFPcre/eGFPcre} pancreata still contained a large number of endocrine cells, but confirmed that none of these cells expressed insulin, glucagon, somatostatin or ghrelin (Fig. 4). Although the number of Ppy-expressing cells was increased in the Rfx6 ^{eGFPcre/eGFPcre} pancreata, they only accounted for a subset of the endocrine cells (Fig. 4i-l), leaving the identity of the remaining endocrine cells unknown.

[00106] Whether Rfx6 regulates other transcription factor genes was also tested (Table 2). The

absence of Rfx6 did not affect Neurogenin3 expression, and this was confirmed at the protein level (data not shown). In sharp contrast, expression of genes downstream of Neurogenin3 encoding factors involved in alpha-cell development, including Irx2 and Arx, was markedly reduced in Rfx6 ^{eGFPcre/eGFPcre} pancreata. Interestingly, genes encoding several factors involved in insulin gene transcription (Pax6, MafA, NeuroDl and Pdxl) also had reduced expression, but some key genes involved in β-cell specification either did not significantly change {Nkx2.2 and Nkx6.1), or increased (Pax4).

Immunofluorescence staining at e l 7.5 revealed that the field of Nkx6.1 expression expanded from β- cells alone in wildtype pancreata to include all of the Chromogranin A + endocrine cells, including the PP cells in Rfx6 ^{eGFPcre/eGFPcre} pancreata (Fig. 4i-l and Table 3). These studies suggest that while Rfx6 regulates the transcription factors involved in β-cell maturation and function, it restricts the expression of the β-cell differentiation and specification genes, and thus the β-cell fate choice.

[00107] Mice with a targeted disruption of the Rfx3 gene have an islet phenotype that is similar to, but less extreme than, the Rfx6 ^{eGFPcre/eGFPcre} mice, with reductions in the numbers, but not complete loss, of insulin- and glucagon-expressing cells and an increase in pancreatic polypeptide-expressing cells (Ait- Lounis, A. et al. Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas. Diabetes 56, 950-959 (2007)). In a proteome-wide screen of protein-protein interactions, Rfx6 was found to interact with Rfx2 and 3 (Rual, J. F. et al. Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1 173-1 178 (2005)). Since the RFX transcription factors generally bind to their target DNA sites (the "X box") as dimers, we tested whether Rfx6 and Rfx3 form a heterodimeric DNA binding complex in an electromobility shift assay (EMSA). We found that both full length Rfx3, and truncations that retain the DNA-binding and dimerization domains, bound to an X box site together with Rfx6 (Fig. 5a and Fig. 19), and that the two factors cooperated in activating a promoter containing multimers of this X box site (Fig 5b).

[00108] It has been proposed that the islet phenotype of the Rfx3 ^~ ' ^~ mice results from defects in primary cilia formation on islet cells, although islets lacking any primary cilia develop fairly normally. Unlike in the Rfx3 ^-/- mice, we found that primary cilia formation was unaffected in the Rfx6 ^{eGFPcre/eGFPcre} islets (Fig. 20a-d). In addition, expression of the cilia genes Ift88 and Dync2lil, which are reduced in the pancreas of Rfx3 ^~/' mice, was not reduced in the pancreas of Rfx6 ^{eGFPcre/eGFPcre} mice (Fig. 20e). It is concluded that Rfx3 and Rfx6 cooperate in regulating a set of genes involved in islet development but not in cilia formation. The more modest islet phenotype in the Rfx3 ^-/- mice may be due to the ability of Rfx2 to compensate partially for the loss of Rfx3 (Fig. l a), or the ability of Rfx6 to direct gene expression as a homodimer (Fig. 5a, b).

[00109] The data discussed above shows that in the pancreas, Rfx6 acts downstream of the pro- endocrine factor Neurogenin3 (Fig. 5c), and mutation of the two genes give similar but distinct phenotypes.

[00110] In addition, RFX6 mutations cause diabetes at birth, while the reported patients with

homozygous mutations in Neurogenin3 did not develop diabetes until several years later, despite evidence that Neurogenin3 is required for the generation of islet cells and production of insulin in mice. Incomplete loss of function in the reported human NEUROG3 mutations could explain the continued insulin production in these patients. Alternatively, recent evidence that Neurog3 ^~/~ mice still generate a small number of islet cells (Wang, S. et al. Mytl and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation. Dev Biol 317, 531 -540 (2008)) suggests some redundancy of Neurogenin3's pro-endocrine function, possibly due to the presence of related bHLH proteins in the pancreas. In contrast, the above data do not indicate any redundancy of Rfx6 function in endocrine differentiation and demonstrate remarkable conservation of the genetic control of islet development, despite some discrepancy between mouse and human NEUROG3 mutants.

[00111] In summary, a new factor in endoderm and islet development, Rfx6, has been identified that is required for the differentiation of 4 of the 5 islet cell types and for the production of insulin in both humans and mice. In the hierarchy of islet developmental factors, it lies downstream of Neurogenin3 and upstream of many of the other islet transcription factors (Fig. 4d).

Table 1 : Pancrease gene expression*

^♦ Morphometric analyses were done by serially sectioning the entire pancreas in 6 urn sections followed by staining and counting every 15th section. DAPI stained cell nuclei were counted using the cell counting application and cytoplasmic staining was qxiantified using the integrated metamorphic analysis: both in the Metamorph suite of programs (version 7, Molecular Devices: Union City, CA).

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.