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Title:
NEURAL CREST CELLS TO REVITALIZE CRANIAL ALLOGRAFTS
Document Type and Number:
WIPO Patent Application WO/2019/113522
Kind Code:
A1
Abstract:
Described herein are methods and compositions related to use of cranium-specific induced pluripotent stem cell (iPSC)-derived cells that are demonstrated as capable of revitalizing structural allografts. Specifically, iPSC-derived neural crest cells (iNCCs) can be differentiated into mesenchymal stem cells (iNCC-MSCs) that cells are seeded on allografts. Coating cranial allografts with mesenchymal stem cells derived from induced neural crest cells, provides a new effective therapeutic avenue for cranial defects. This approach improves allograft function by combination with a reproducible and inexhaustible source of cranium-specific MSCs. The results shown herein demonstrate improved integration and revitalization of induced neural crest (iNCC-MSC)-coated allografts compared to bone marrow (BM)-MSE-coated allografts, both applied in combination with intermittent parathyroid hormone (PTH) therapy in a calavarial defect model.

Inventors:
SHEYN DMITRIY (US)
GLAESER JULIANE (US)
GAZIT ZULMA (US)
TAWACKOLI WAFA (US)
Application Number:
PCT/US2018/064583
Publication Date:
June 13, 2019
Filing Date:
December 07, 2018
Export Citation:
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Assignee:
CEDARS SINAI MEDICAL CENTER (US)
International Classes:
A01N63/00; A61K35/12; A61K35/28; A61K35/32; A61K35/34; A61K38/00
Foreign References:
US20050249731A12005-11-10
US20150110747A12015-04-23
US20160237405A12016-08-18
US20170119823A12017-05-04
US20170035935A12017-02-09
US20160310540A12016-10-27
US20030109934A12003-06-12
Other References:
KALLAI ET AL.: "Quantitative, Structural and Image-based Mechanical Analysis of Nonunion Fracture Repaired by Genetically Engineered Mesenchymal Stem Cells", JOURNAL OF BIOMECHANICS, vol. 43, no. 12, 26 August 2010 (2010-08-26), pages 2315 - 2320, XP027206856
LIU ET AL.: "Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell -Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats", TISSUE ENGINEERING: PART A, vol. 23, no. 11-12, 10 March 2017 (2017-03-10), pages 546 - 555, XP055616104
Attorney, Agent or Firm:
CHEN, Stephen W. et al. (US)
Download PDF:
Claims:
nil CLAIMS

1. A method for treating a cranial bone defect, comprising:

transplanting in a subject, a graft comprising a quantity of mesenchymal stem cells (MSCs), wherein the graft treats a cranial bone defect in the subject.

2. The method of claim 1, wherein the MSCs are derived from neural crest cells (NCCs).

3. The method of claim 2, wherein the NCCs are derived from induced pluripotent stem cells (iPSCs).

4. The method of claim 1, comprising administration of PTH.

5. The method of claim 4, wherein the quantity of PTH comprises 0.1 to 1, 1-10, 10-20, 20-30, 30-40, or at least 40 ug/kg.

6. The method of claim 1, wherein the quantity of MSCs comprises at least 0.2xl06, 0.5xl06, lxlO6, 2xl06, 3xl06, 4xl06 or 5xl06 cells.

7. The method of claim 1, wherein treating a cranial bone defect comprises one or more of osteogenesis, osteoconduction, osteoinduction, bone volume increase, and bone graft incorporation.

8. The method of claim 1, wherein the graft is from cranium.

9. The method of claim 1, wherein the graft is from a long bone.

10. The method of claim 1, wherein the graft is an allograft.

11. The method of claim 1, wherein the grafts is an autograft

12. A composition comprising:

a graft comprising a quantity of mesenchymal stem cells (MSCs).

13. The composition of claim 12, wherein the MSCs are derived from neural crest cells (NCCs).

14. The composition of claim 12, wherein the NCCs are derived from induced pluripotent stem cells (iPSCs).

15. The composition of 12, wherein the graft is an allograft.

16. The composition of claim 12, wherein the graft is from cranium.

17. The composition of claim 12, wherein the graft is from a long bone.

18. The composition of claim 12, wherein the MSCs are coated on the surface of the graft.

Description:
NEURAL CREST CELLS TO REVITALIZE CRANIAL ALLOGRAFTS

FIELD OF THE INVENTION

Described herein are methods and compositions for use with cranial allografts, including grafts coated with mesenchymal stem cells (MSCs) from neural crest cells (NCCs) produced from induced pluripotent stem cell (iPSCs).

BACKGROUND

Cranial bone loss due to trauma or tumor resection continues to present a major clinical challenge, affecting more than 100,000 Americans each year. Bone grafts are typically used to repair such defects. While the use of autografts is associated with donor site morbidity, allografts consist of nonviable tissue and relies on the invasion of host cells and tissues. Revitalization of cranial allografts is challenging due to the limited reservoir of resident stem cells in the membranous bones of the craniofacial complex. Intermittent Parathyroid hormone (PTH) therapy enhances revitalization of structural allografts in vivo, although host cell engraftment and integration of the allograft is found to be partial. Currently employed stem cell therapies to revitalize cranial allografts include the use of allogenic bone marrow-derived mesenchymal stem cells (BM-MSCs), which are harvested from long bones or iliac crest and of mesodermal origin. While the parietal bone in the mammalians is mesodermal, the frontal bones originate in the neural crest cells (NCCs). NCCs generate cranial skeleton during embryogenesis, differentiate into mesenchymal stem cells (MSCs) and are therefore a potential source to augment cranial regeneration. However, these cells are extremely rare in adults.

Induced pluripotent stem cells (iPSCs) provide a potentially inexhaustible source of patient-specific autologous or allogeneic cells, can be reprogrammed to induced neural crest cells (iNCCs), subsequently to MSCs, and might therefore, resolve the unmet need for cranium- specific MSCs. In this study, the Inventors aimed to demonstrate a successful differentiation of iPSCs into iNCC-MSCs and to evaluate the impact of iNCC-MSC seeding onto allografts in combination with intermittent PTH therapy on the graft integration and revitalization compared to a BM-MSC/allograft and allograft only treatment in a mouse calvarial defect model.

Described herein are methods and compositions related to use of cranium-specific iPSC-derived stem cells that are demonstrated as capable of revitalizing structural allografts. These cells are seeded on the allografts and coating cranial allografts with mesenchymal stem cells derived from induced neural crest cells, provides a new, effective therapeutic avenue for cranial defects. This approach improves allograft function by combination with a reproducible and inexhaustible source of cranium-specific MSCs. The results shown herein demonstrate improved integration and revitalization of induced neural crest (iNCC)-MSC-coated allografts compared to bone marrow (BM)-MSC-coated allografts, both applied in combination with intermittent parathyroid hormone (PTH) therapy in a calavarial defect model.

SUMMARY OF THE INVENTION

Described herein is a method for treating a cranial bone defect, including transplanting in a subject, a graft including a quantity of mesenchymal stem cells (MSCs), wherein the graft treats a cranial bone defect in the subject. In other embodiments, the MSCs are derived from neural crest cells (NCCs). In other embodiments, the NCCs are derived from induced pluripotent stem cells (iPSCs). In other embodiments, the method includes administration of PTH. In other embodiments, the quantity of PTH includes 0.1 to 1, 1-10, 10-20, 20-30, 30-40, or at least 40ug/kg. In other embodiments, the quantity of MSCs includes at least 0.2xl0 6 , 0.5xl0 6 , lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 or 5xl0 6 cells. In other embodiments, treating a cranial bone defect includes one or more of osteogenesis, osteoconduction, osteoinduction, bone volume increase, and bone graft incorporation. In other embodiments, the graft is from cranium. In other embodiments, the graft is from a long bone. In other embodiments, the grafts are allografts. In other embodiments, the grafts are autografts

Further described herein is a composition including a graft including a quantity of mesenchymal stem cells (MSCs). In other embodiments, the MSCs are derived from neural crest cells (NCCs). In other embodiments, the NCCs are derived from induced pluripotent stem cells (iPSCs). In other embodiments, the graft is an allograft. In other embodiments, the graft is from cranium. In other embodiments, the graft is from a long bone. In other embodiments, the MSCs are coated on the surface of the graft.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : The potential of allografts to regenerate calvarial defects coated with iNCC- MSC+PTH is higher than with BM-MSC+PTH. (A) A higher bone volume in the iNCC- MSC+PTH group was detected by pCT, DBU was calculated as BY (week 3)-BV(day 1) in each mouse; (B) H&E staining shows an improved integration of iNCC-MSC+PTH allograft compared to controls (Top). Immunostaining of the allograft-host junction shows an increased expression of osteocalcin (OC) and bone sialoprotein (BSP) of Dil labeled cells in the iNCC- MSC+PTH group (Bottom). Yellow arrows: allograft-host junction site indicate significance: p<0.05.

Figure. 2. iNCC-MSCs present MSC phenotype and show similar cell viability in vivo. (A)The expression of MSC surface consensus markers in iNCC-MSC was tested using flow cytometry and found similar to BM-MSCs. (B) BLI imaging of cell-seeded allografts post- surgery showed that both BM-MSCs and iNCC-MSCs proliferated during the first two weeks and survived on the allograft for at least 6 weeks.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and Practice of Pharmacy 22 nd ed. , Pharmaceutical Press (September 15, 2012); Homyak et al, Introduction to Nanoscience and Nanotechnology , CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 rd ed. , revised ed. , J. Wiley & Sons (New Y ork, NY 2006); Smith, March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary ofDNA and Genome Technology 3 rd ed., Wiley- Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed. , Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Mil stein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7): 511 -9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al. , Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6l62):323-7.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. As described, bone grafts are typically used to repair cranial loss defects due to trauma or tumor resection. Autografts are associated with donor site morbidity and allografts consist of nonviable tissue with limited function as an osteoconductive scaffold not capable of stimulate new bone formation. In fact, allograft healing relies on the invasion of host cells and tissue and occurs at an extremely slow rate. Revitalization is difficult due to the limited reservoir of resident stem cells in the membranous bone of the craniofacial complex. While intermittent PTH therapy can enhance revitalization of structural allografts in vivo, host cell engraftment and integration of the allograft is only partial. While regenerative medicine approaches using stem cells therapies to revitalize cranial allografts include the use of allogenic bone marrow derived mesenchymal stem cells (BM-MSCs), these cells are harvested from long bones or iliac crest and of mesodermal origin. While parietal bones in mammals is of mesodermal origin, the front bones originate in the ectodermal neural crest cells (NCCs). During embryogenesis, it is ectodermal germ layer NCCs that generate cranial skeleton, differentiate into MSCs.

Specifically, MSCs and neural crest cells (NCCs) are both used in various approaches in craniofacial biology because of their developmental similarities. However, as described, the craniofacial mesenchyme developmentally originates from ectodermal germ layer NCCs. The rarity and difficulty of isolating NCCs has prevented feasibility of direct use NCCs in patients. Easily accessible MSCs are readily available in adult tissues such as bone marrow and fat tissues. As a result, they have been a leading choice for regenerative medicine application. Despite a variety of similarities, including similar self-renewal and differentiation potential between MSCs and NCCs, development of MSCs of similar ectodermal developmental origin would likely spur improved engraftment, graft function, and augment regeneration in cranial locations.

Although extremely rare in adults, NCCs can be derived from induced pluripotent stem cells (iPSCs). The source material of iPSCs provide a potentially inexhaustible source of patient-specific cells that can be reprogrammed to iPSC-derived NCCs and subsequently to MSCs and might therefore resolve the unmet need for cranium-specific MSCs.

A variety of methods have been reported to generate iNCCs. Such techniques typically rely on the activation of canonical Wnt signaling and the prevention of TGFP signaling to obtain a highly enriched population of CD27l(+) -iNCCs. Importantly, iNCCs can be expanded for long term under conditions of bFGF supplementation and TGFP inhibition. Such iNCCs can be cryopreserved, imparting significant advantages for future clinical use. Although it is generally understood that iPSC-derived NCCs can possess osteogenic and chondrogenic potential in vitro after mesenchymal induction, there are little or no description of use of iNCC derived MSCs for bone or cartilage repair in vivo. One study studying MSC-like cells from iNCCs attempted to investigate cartilage and bone repair in vivo using an athymic nude rat osteochondral defect model. However, the MSC-like cells were reported as not affecting regenerative repair of osteochondral defects in vivo. While iPS- derived MSCs would be an attractive source of cells for regenerative therapy applications for osteochondral repair, it is clear that development of an effective local delivery system, would be required to fulfill their potential for future therapeutic use in vivo.

Described herein is a method for treating a cranial bone defect including transplanting in a subject, a graft including a quantity of mesenchymal stem cells (MSCs), wherein the graft treats a cranial bone defect in the subject. In other embodiments, the MSCs are derived from neural crest cells (NCCs) (i.e., neural crest cell-derived MSCs (NCC-MSCs), and induced pluripotent stem cell neural crest cell-derived mesenchymal stem cells (iNCC-MSCs). In other embodiments, the NCCs are derived from induced pluripotent stem cells (iPSCs) (i.e, induced pluripotent stem cell derived neural crest cells (iNCCs). Derivation of iNCCs and iNCC-MSCs can be by a variety of techniques known in the art. For example, NCCs can be generated from iPSCs using a modified stem cell maintenance medium by including Fgf2 (8ng/mL), optionally including Igf-l (200ng/mL) and small molecules such as GSK3 inhibitor IX (BIO) (2-4 mM) and SB431542 (20mM). NCCs can also be generated using sonic hedgehog (200ng/mL,) FGF8 (lOOng/mL), brain-derived neurotrophic factor (BDNF) (20ng/mL). For example, MSCs can be generated from iNCCs by culturing iNCCs in an exemplary MSC medium, such as alpha- MEM (aMEM), 10% fetal bovine serum and 5ng/mL bFGF.

In other embodiments, the method includes administration of PTH. In other embodiments, the quantity of PTH includes 0.01 to 0.1, 0.1 to 1, 1-10, 10-20, 20-30, 30-40, or at least 40ug/kg. In other embodiments, the quantity of MSCs includes at least 0.2xl0 6 , 0.5xl0 6 , lxl0 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 or 5xl0 6 cells. In other embodiments, the MSCs express one or more of CD73+, CD44+, Cdl3+, PDGFRcr+ Sca-l+ and Gli-l+. In other embodiments, the MSCs do not express one or more of CD45- Terl 19- In other embodiments, NCCs express one or more of p75+, Hnkl+, AP2+ and FoxD3+. In other embodiments, NCCs do not express one or more of Sox2-, Oct4-, Nanog- and Pax6-. In various embodiments, the NCCs express one or more of: CD29, CD57, CD73, CD271, TFAP2A, SoxlO, Pax3, and nestin. In various embodiments, MSCs, including iNCC-MSCs, express one or more of: CD29, CD 105, CD90 and CD44. In other embodiments, treating a cranial bone defect includes one or more of osteogenesis, osteoconduction, osteoinduction, bone volume increase, and bone graft incorporation. In other embodiments, the graft is from a flat bone. In other embodiments, the graft is from cranium. In other embodiments, the graft is from a long bone. Examples of these sources include calavarial, iliac crest, chin, tibial, rib, and resected bone section. In other embodiments, the grafts are allografts. In other embodiments, the grafts are autografts

In various embodiments, the iPSCs are generated from ceils reprogrammed from a blood draw from a subject, including for example, blood cell derived iPSCs (BC-iPSCs). In various embodiments, the transplant subject is the same as the donor subject for a blood draw from which BC-iPSCs are derived.

Also described herein is a composition including a graft including a quantity of mesenchymal stem cells (MSCs). In other embodiments, the MSCs are derived from neural crest cells (NCCs). In other embodiments, the NCCs are derived from induced pluripotent stem cells (iPSCs), such cells described herein as induced pluripotent stem cell neural crest cells (iNCCs), and if differentiated to mesenchymal stem cells are described herein as iNCC-MSCs. In other embodiments, the graft is an allograft. In other embodiments, the graft is from a flat bone. In other embodiments, the graft is from cranium. In other embodiments, the graft is from a long bone. In other embodiments, the MSCs are coated on the surface of the graft. In some embodiments, the composition includes contacting a graft with MSCs, including iNCC-MSCs, optionally including culturing of the MSCs and graft. In some embodiments, contacting a graft with MSCs and/or culturing of the MSCs includes use of low or non-adherent culture substrates. In other embodiments, the MSCs express one or more of CD73+, CD44+, Cdl3+, PDGFRaN Sca-l+ and GH-1+. In other embodiments, the MSCs do not express one or more of CD45- Terl 19- In other embodiments, NCCs express one or more of p75+, Hnkl+, AP2+ and FoxD3+. In other embodiments, NCCs do not express one or more of Sox2-, Oct4-, Nanog- and Pax6-. In various embodiments, the NCCs express one or more of: CD29, CD57, CD73, CD271, TFAP2A, SoxlO, Pax3, and nestin. In various embodiments, MSCs, including iNCC- MSCs, express one or more of: CD29, CD105, CD90 and CD44.

In various embodiments, the iPSCs are obtained from a subject including cells reprogrammed from a blood draw. In various embodiments, cells reprogrammed from a blood draw are made by a method including contacting a quantity of blood cells with one or more vectors encoding a reprogramming factor, and delivering a quantity of reprogramming factors into the blood cells, culturing the blood cells in a reprogramming media, and further wherein delivering the reprogramming factors, and culturing in a reprogramming media generates blood cell derived induced piuripotent stem cells (iPSCs). Further information on iPSC reprogramming is found in U.S. App. No. 15/184,241 and PCX App. No. PCT/US2017/038041 , each of which is fully incorporated by reference herein. In various embodiments, iPSCs obtained from a subject include cells reprogrammed lymphoblastoid cells or lymphoblast cell lines (LCLs). Further information on iPSC reprogramming is found in Barrett, R. et al. Reliable Generation of Induced Pluripotent Stem Cells from Human Lymphoblastoid Cell Lines. Stem Cells Transl Med. 2014 Dec;3(l2): 1429-34, which is fully incorporated by reference herein.

In various embodiments, generating iPSCs includes providing a quantity of cells, delivering a quantity of reprogramming factors into the cells, culturing the cells in a reprogramming media for at least 4 days, wherein delivering the reprogramming factors, and culturing generates induced pluripotent stem cells. In certain embodiments, the cells are primary culture cells. In other embodiments, the cells are blood cells (BCs). In certain embodiments, the blood cells are T-cells. In other embodiments, the blood cells are non-T- cells. In other embodiments, the cells are mononuclear cells (MNCs), including for example peripheral blood mononuclear cells (PBMCs). In other embodiments, the cells are primary granulocytes, monocytes and B-lymphocytes.

In certain embodiments, the reprogramming factors are Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA- p53”). In other embodiments, these reprogramming factors are encoded in a combination of vectors including pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNAl . This includes, for example, using about 0.5 - l.Oug pCXLE-hOCT3/4- shp53, 0.5 - l .Oug pCXLE-hSK, 0.5 - l .Oug pCXLE-UL, about 0.25 - 0.75ug pCXWB- EBNA1 and 0.5 - l .Oug pEP4 E02S ET2K. This includes, for example, using 0.83ug pCXLE- hOCT3/4-shp53, 0.83ug pCXLE-hSK, 0.83ug pCXLE-UL, 0.5ug pCXWB-EBNAl and 0.83ug pEP4 E02S ET2K, wherein the stoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) and EBNA-l (encoded in pCXWB-EBNAl) supports the reprogramming of non-T cell component of blood, including peripheral blood mononuclear cells. In various embodiments, the reprogramming media is embryonic stem cell (ESC) media. In various embodiments, the reprogramming media includes bFGF. In various embodiments, the reprogramming media is E7 media. In various embodiments, the reprogramming E7 media includes L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF. In different embodiments, the reprogramming media comprises at least one small chemical induction molecule. In certain other embodiments, the reprogramming media includes PD0325901, CHIR99021, HA-100, and A-83-01. In other embodiments, the culturing the blood cells in a reprogramming media is for 4-30 days.

In various embodiments, the iPSCs are capable of serial passaging as a cell line. In various embodiments, the iPSCs possess genomic stability. Genomic stability can be ascertained by various techniques known in the art. For example, G-band karyotyping can identify abnormal cells lacking genomic stability, wherein abnormal cells possess about 10% or more mosaicism, or one or more balanced translocations of greater than about 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomic stability can be measured using comparative genomic hybridization (aCGH) microarray, comparing for example, iPSCs against iPSCs from a non blood cell source such as fibroblasts. Genomic stability can include copy number variants (CNVs), duplications/deletions, and unbalanced translocations. In various embodiments, iPSCs exhibit no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mb average size of amplification and deletion. In various embodiments, BC-iPSCs exhibit no more than about 20-30 Mb average size of amplification and deletion. In various embodiments, iPSCs exhibit no more than about 30-40 Mb average size of amplification and deletion. In various embodiments, iPSCs exhibit no more than about 40-50 Mb average size of amplification and deletion. In various embodiments, the average number of acquired de novo amplification and deletions in iPSCs is less than about 5, 4, 3, 2, or 1. For example, de novo amplification and deletions in fib-iPSCs are at least two-fold greater than in PBMC -iPSCs. In various embodiments, the methods produce iPSC cell lines collectively exhibiting about 20%, 15%, 10%, 5% or less abnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or more passages when serially passaged as a cell line.

In other embodiments, the reprogramming factors are delivered by techniques known in the art, such as nucleofection, transfection, transduction, electrofusion, electroporation, microinjection, cell fusion, among others. In other embodiments, the reprogramming factors are provided as RNA, linear DNA, peptides or proteins, or a cellular extract of a pluripotent stem cell. In certain embodiments, the cells are treated with sodium butyrate prior to delivery of the reprogramming factors. In other embodiments, the cells are incubated or 1, 2, 3, 4, or more days on a tissue culture surface before further culturing. This can include, for example, incubation on a Matrigel coated tissue culture surface. In other embodiments, the reprogramming conditions include application of norm-oxygen conditions, such as 5% O2, which is less than atmospheric 21% O2. In various embodiments, the reprogramming media is embryonic stem cell (ESC) media. In various embodiments, the reprogramming media includes bFGF. In various embodiments, the reprogramming media is E7 media. In various embodiments, the reprogramming E7 media includes L- Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF. In different embodiments, the reprogramming media comprises at least one small chemical induction molecule. In different embodiments, the at least one small chemical induction molecule comprises PD0325901, CHIR99021, HA-100, A-83-01, valproic acid (VP A), SB431542, Y-27632 or thiazovivin (“Tzv”). In different embodiments, culturing the BCs in a reprogramming media is for at least 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 days.

Further described herein is a composition of mesenchymal stem cells (MSCs), inducing MSCs derived from neural crest cells (NCCs), further including NCCs derived from induced pluripotent stem cells (iNCCs), further including induced pluripotent stem cells derived from blood cells (BCs-iPSCs). Further described here in a graft including one or more the aforementioned cell types. Further described herein is a method of using a composition including a graft including the quantity of mesenchymal stem cells (MSCs). In various embodiments, this includes a graft including induced pluripotent stem cell-derived neural crest cell derived mesenchymal stem cells (iNCC-MSCs). In various embodiments, the iPSCs are generated from cells reprogrammed from a blood draw from a subject, including for example, blood cell derived iPSCs (BC-iPSCs). In various embodiments, the method promotes one or more of osteogenesis, osteoconduction, osteoinduction, bone volume increase, and bone graft incorporation. In other embodiments, the method includes transplanting the graft into a subject with a cranial bone defect. In various embodiments, the transplant subject is the same as the donor subject for a blood draw from which BC-iPSCs are derived.

Example 1

Generation of iNCC-MSCs

For generation of iNCC-MSCs, human iPSCs were reprogramed by the Cedars-Sinai iPSC Core Facility from healthy human fibroblasts, which were nucleofected with episomal plasmid vectors. The iPSC lines were expanded and differentiated to iNCCs. NCC phenotype was verified using immunofluorescent staining and flow cytometry for NC markers. Differentiation of iNCCs to MSCs was performed by culturing the cells in standard media and passing. As reference, BM-MSCs were isolated from whole bone marrow aspirates using standard plastic adherence. Osteogenic differentiation of iNCC-MSCs and BM-MSCs was shown in terms of a quantitative alkaline phosphatase (ALP) assay. The iNCC-MSCs’ adipogenic differentiation potential was analyzed using Oil Red O staining. The tumorigenic potential of the iNCC-MSCs was determined using the soft agar assay in vitro and teratoma formation assay in vivo. To exclude teratoma formation, both iNCCs and iNCC-MSCs were injected intramuscularly into NOD/SCID mice.

Example 2

iNCC-MSCs and Calavarial Allografts

To analyze the impact of iNCC-MSCs on calvarial allograft integration, structural calvarial allografts were harvested from FVB/N mice and decellularized chemically and enzymatically to exclude cell remnants. Both BM-MSCs and iNCC-MSCs were transduced with a lentiviral vector encoding for Luciferase reporter gene under constitutive ubiquitin promoter. Per allograft, 10 5 transduced cells were seeded using non-attachment culture plates. Unattached cells were washed out and counted. A calvarial defect (5mm in diameter) was created in NOD/SCID mice and implanted with allografts, with or without cell coating. PTH treatment for applied for 3 weeks. Luciferin was injected intraperitoneally. Cell survival was tracked with Bioluminescence (BLI). To evaluate bone volume and union ratio post-treatment, pCT analysis was performed. Allograft integration and cell differentiation in vivo was analyzed via H&E staining, and immunostaining. For statistical analysis, ANOVA was used. A critical significance level of 5% was used for all statistical tests.

Example 3

Properties of iNCC-MSCs

NGFR-P75 and HNK1 neural crest marker expression via immunofluorescent staining and flow cytometry indicated the successful differentiation of iPSCs into iNCCs. Further differentiation of iNCCs into MSCs demonstrated by the expression of all five consensus MSC markers, tested by flow cytometry. Differentiation of iNCC-MSCs into the osteogenic and adipogenic lineages were shown via ALP activity after 14 days of exposure to osteogenic media, which was comparable between iNCC-MSCs and BM-MSCs. Quantification of fat vacuoles via Oil Red O staining revealed a similar uptake of the stain by both BM-MSCs and iNCC-MSCs. No higher tumorigenic potential of iNCC-MSCs was detected compared to BM- MSCs, tested in week 1, 2 and 4. No teratoma formation was detected after 8 weeks (10 6 cells per injection, n=5).

Example 4

Allograft Improvements

BLI imaging of BM-MSCs and iNCC-MSCs seeded allografts post-surgery showed that

both BM-MSCs and iNCC-MSCs survived on the allograft for at least 6 weeks. Micro CT analysis showed a significant increase in bone volume in the allograft+iNCC-MSC+PTH group compared to“allograft only” and“allograft+BM-MSC+PTH” groups on week 3 post-surgery (p<0.05) (Fig. 1A). The union ratio followed the same trend. H&E staining showed an improved integration of iNCC-MSC+PTH allograft compared to BM-MSCs and allograft only controls. An increased expression of OC and BSP of Dil-labeled iNCC-MSCs and BM-MSCs was shown compared to allograft only controls (Fig. 1B).

Example 5

Discussion

These results indicate that the iNCC-MSCs are multipotential and can respond to osteogenic signals comparable to BM-MCSs in vitro in our calvarial defect model, an improved integration and revitalization of iNCC-MSC-coated allografts compared to BM- MSC-coated allografts was shown, which both were applied in combination with intermittent PTH therapy. However, whether the observed improved integration iNCC-MSC-coated allografts results from an increased response of iNCC-MSCs to the cranial environment and/or whether it results from an increased responsiveness to PTH is unclear to date and requires further investigation. Our study demonstrates the potential of iNCC-MSC-coated allografts applied in combination with intermittent PTH therapy to efficiently revitalize cranial allografts.

Example 6

Further Characterization of iNCC-MSCs

As shown in Fig. 2, iNCC-MSCs can be generated to present MSC phenotype, including markers such as CD29, CD 105, CD90, and CD44 (Fig. 2A). Imaging of cell-seeded allografts post-surgery showed that both BM-MSCs and iNCC-MSCs proliferated during the first two weeks and survived on the allograft for at least 6 weeks (Fig. 2B). Example 7

Blood Cell Derived iPSCs as Cell Source

iPSCs can be obtained from a subject including cells reprogrammed from a blood draw, such cells are described as possess reduced mutational load and genomic stability when compared to other cell sources such as fibroblasts. Thereafter, blood cell derived iPSCs (BC- iPSCs) are differentiated into neural crest cells in accordance with methods descried herein. Graft recipient subjects can be the same as the blood cell donor subject, thereby providing patient-specific immunocompatability.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are techniques and compositions for generating induced pluripotent stem cell (iPSC) derived neural crest cell (iNCCs), mesenchymal stem cell (MSCs) derived from iNCCs (iNCC-MSCs), manipulation of any of the aforementioned cell types, transplant techniques including use with autografts and allografts, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term“about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms“a” and“an” and“the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.