阅读说明：本技术 用于治疗糖尿病的胰腺细胞及其生成方法 (Pancreatic cells for treating diabetes and methods of producing the same ) 是由 W·L·鲁斯特 于 2019-02-08 设计创作，主要内容包括：本公开提供了用于治疗糖尿病的基于细胞的组合物、用于鉴定优先分化成内胚层细胞的细胞的方法、用于制备胰岛素产生胰腺细胞的方法以及用于治疗与胰岛素缺乏相关的疾病的相关方法。(The present disclosure provides cell-based compositions for treating diabetes, methods for identifying cells that preferentially differentiate into endodermal cells, methods for making insulin-producing pancreatic cells, and related methods for treating diseases associated with insulin deficiency.)

1. A method of producing a mammalian insulin secreting cell comprising:

a. culturing mammalian stem cells adherently, thereby allowing the mammalian stem cells to spontaneously form a three-dimensional structure; and

b. culturing the three-dimensional structure in suspension;

wherein the culturing step comprises exposure to retinoic acid and cyclopamine for at least 20 days and does not comprise exposure of said stem cells in a three-dimensional structure to Wnt 3A.

2. The method of claim 1, wherein the mammalian stem cells are human stem cells.

3. The method of claim 1, wherein the mammalian stem cells are non-human primate stem cells.

4. The method of claim 1, wherein the mammalian stem cells are derived from a cell line.

5. A method of producing an insulin-secreting cell comprising:

a. culturing mammalian stem cells on a viscous substrate in a first medium comprising activin-a and wortmannin, wherein the mammalian stem cells are not exposed to Wnt3 a;

b. further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and

c. transferring the cells into a suspension culture while the cells form a three-dimensional cell structure;

wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

6. The method of claim 5, wherein the mammalian stem cells form a three-dimensional structure when cultured on the viscous substrate.

7. The method of claim 5, wherein the mammalian stem cells are human stem cells.

8. The method of claim 5, wherein the mammalian stem cells are non-human primate stem cells.

9. A method of producing a mammalian insulin secreting cell comprising:

a. culturing mammalian stem cells in a first culture medium comprising an endodermal induction factor, thereby differentiating said mammalian stem cells into endodermal cells; and

b. culturing the endoderm cells from (a) in a second medium comprising endocrine inducing factors, thereby differentiating the endoderm cells into endocrine cells;

wherein the mammalian stem cells are not exposed to Keratinocyte Growth Factor (KGF) prior to differentiation into endodermal cells.

10. The method of claim 9, wherein the mammalian stem cell is a human stem cell.

11. The method of claim 9, wherein the mammalian stem cells are non-human primate stem cells.

12. The method of any of claims 9-11, wherein the endoderm induction factor comprises activin-a.

13. The method of any one of claims 9-12, wherein the first medium comprises wortmannin.

14. The method of any one of claims 9-13, wherein the first medium does not comprise Wnt 3A.

15. The method of any one of claims 9-14, wherein the cells are cultured in the first medium for 1-3 days.

16. The method of any one of claims 9-15, wherein the endocrine inducing factor comprises retinoic acid and cyclopamine.

17. The method of any one of claims 9-16, wherein the second medium comprises noggin.

18. The method according to any one of claims 9-17, wherein the second medium comprises KGF.

19. The method of any one of claims 9-18, wherein the cells are cultured in the second medium for 1-4 days.

20. The method of any one of claims 9-19, further comprising culturing the endocrine cells in a third medium comprising KGF, thereby differentiating the endocrine cells into pancreatic progenitor cells.

21. The method of claim 20, wherein the third medium comprises noggin and Epidermal Growth Factor (EGF).

22. The method according to claim 20 or claim 21, wherein the third medium comprises retinoic acid and cyclopamine.

23. The method of any one of claims 20-22, wherein the cells are cultured in the third medium for 1-4 days.

24. The method of claim 20, further comprising culturing the pancreatic progenitor cells in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XXI, and Alk5 iiii.

25. The method according to claim 24, wherein the fourth medium comprises retinoic acid and cyclopamine.

26. The method of claim 24 or claim 25, wherein the cells are cultured in the fourth medium for 1-4 days.

27. The method of claim 24, further comprising culturing the pancreatic progenitor cells in a fifth medium comprising Alk5i II and retinoic acid.

28. The method of claim 27, wherein the fifth medium comprises cyclopamine.

29. The method of claim 27 or claim 28, wherein the cells are cultured in the fifth medium for 1-5 days.

30. The method of claim 27, further comprising culturing the pancreatic progenitor cells in a sixth medium comprising Alk5I II, nicotinamide and insulin-like growth factor (IGF) -I.

31. The method according to claim 30, wherein the sixth medium comprises retinoic acid and cyclopamine.

32. The method of claim 30 or claim 31, wherein the cells are cultured in the sixth medium for 1-9 days.

33. The method of any one of claims 9-32, wherein the mammalian stem cells are derived from pancreatic primary tissue.

34. The method of any one of claims 9-32, wherein the mammalian stem cells are human embryonic stem cells.

35. The method of any one of claims 9-32, wherein the mammalian stem cell is an induced pluripotent stem cell.

36. The method of any one of claims 9-32, wherein the mammalian stem cell is a non-pluripotent reprogrammed cell.

37. The method of claim 36, wherein the reprogrammed cell is derived from pancreatic primary tissue.

38. The method of claim 36 or claim 37, wherein the reprogrammed cell is reprogrammed by expressing a reprogramming gene without incorporating the reprogramming gene into the genome of the cell.

39. The method of claim 38, wherein the reprogramming genes are encoded on at least one episomal expression plasmid.

40. The method of claim 38 or claim 39, wherein the reprogramming genes comprise Oct4, Sox2, Klf4, and L-Myc.

41. The method of any one of claims 9-40, wherein the cells are cultured for 30 days or less.

42. A method of producing an insulin-secreting cell comprising:

a. culturing human stem cells in a first medium comprising activin-A and wortmannin, thereby differentiating said human stem cells into endoderm cells, wherein said human stem cells have not been exposed to Keratinocyte Growth Factor (KGF) prior to differentiation into said endoderm cells; and

b. culturing the endoderm cells from (a) in a second medium comprising retinoic acid and cyclopamine, thereby differentiating the endoderm cells into endocrine cells;

c. culturing the endocrine cells from (b) in a third medium comprising KGF, noggin, and EGF, thereby differentiating the endocrine cells into pancreatic progenitor cells;

d. culturing the pancreatic progenitor cells from (c) in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XXI, and Alk5i II, thereby differentiating the pancreatic progenitor cells into insulin producing cells.

43. The method of claim 42, wherein the second medium further comprises KGF.

44. The method according to claim 42 or claim 43, wherein the third and fourth media further comprise retinoic acid and cyclopamine.

45. The method according to any one of claims 42-44, wherein the insulin producing cells from (d) are further cultured in a fifth medium comprising Alk5III and retinoic acid and optionally cyclopamine.

46. The method of claim 45, further comprising further culturing the cells in a sixth medium comprising Alk5I II, nicotinamide, IGF-I, and optionally retinoic acid and cyclopamine.

47. The method of any one of claims 42-46, wherein the total culture time is less than 30 days.

48. The method of any one of claims 42-47, wherein the human stem cells are derived from pancreatic primary tissue.

49. A cell-based composition for treating diabetes comprising a population of surrogate pancreatic cells and a suitable carrier for implantation into a human subject in need thereof, wherein at least 66% of the cells are insulin producing pancreatic cells.

50. The cell-based composition of claim 49, wherein at least 66% of the surrogate pancreatic cells express NeuroD 1.

51. The cell-based composition of claim 49 or claim 50, wherein at least 68% of the surrogate pancreatic cells express Nkx6.1.

52. The cell-based composition of any one of claims 49-51, wherein the insulin-producing pancreatic cells are obtained according to a method comprising:

a. culturing a population of human stem cells on a adherent substrate in a first culture medium comprising endodermal induction factors, wherein the mammalian stem cells are not exposed to Wnt3 a;

b. further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine;

c. transferring the cells into a suspension culture while the cells form a three-dimensional cell structure;

wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days,

wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

53. The cell-based composition of claim 52, wherein the endoderm induction factors comprise activin-A and wortmannin.

54. The cell-based composition of any one of claims 49-53, wherein the human stem cells are derived from pancreatic primary tissue.

55. The cell-based composition of any one of claims 49-53, wherein the human stem cells are human embryonic stem cells.

56. The cell-based composition of any one of claims 49-53, wherein the human stem cells are induced pluripotent stem cells.

57. The cell-based composition of any one of claims 49-53, wherein the human stem cells are non-pluripotent reprogrammed cells.

58. The cell-based composition of claim 57, wherein the reprogrammed cell is reprogrammed by expressing a reprogramming gene without incorporating the reprogramming gene into the genome of the cell.

59. The cell-based composition of claim 58, wherein the reprogramming genes are encoded on at least one episomal expression plasmid.

60. The cell-based composition of claim 59, wherein the reprogramming genes comprise Oct4, Sox2, Klf4, and L-Myc.

61. The cell-based composition of any one of claims 49-60, wherein the carrier comprises a macrocapsule.

62. The cell-based composition of claim 61, wherein the macrocapsule comprises alginate, cellulose sulfate, glucomannan, or a combination thereof.

63. A method of identifying an undifferentiated cell that preferentially differentiates into an endodermal lineage, comprising assessing expression of BHMT2 and NAP1L1 in the undifferentiated cell, and identifying the cell as having a preference for differentiation into an endodermal lineage if expression of BHMT2 is down-regulated relative to a control cell and expression of NAP1L1 is up-regulated relative to a control cell.

64. The method of claim 63, further comprising assessing expression of Cox7A1 and HSPB2 in the undifferentiated cells, and identifying the cells as having a preference for differentiation into an endodermal lineage if expression of Cox7A1 and HSPB2 are both down-regulated relative to control cells.

65. The method of claim 63 or 64, wherein the control cell is a pluripotent cell that does not exhibit preferential differentiation to the endodermal lineage or is substantially unable to differentiate to the mesodermal lineage.

66. The method of claim 63, wherein expression of BHMT2 is downregulated by at least 2 logs relative to the control cell, and expression of NAP1L1 is upregulated by at least 2 logs relative to the control cell.

67. The method of claim 66, wherein expression of Cox7A1 and HSPB2 are each down-regulated by at least 2 log relative to the control cells.

68. The method of claim 63 or 64, further comprising assessing expression of GLIS2, CCDC58, MTX3, and C7orf 29.

69. The method of claim 68, wherein expression of GLIS2, CCDC58, and MTX3 is up-regulated relative to the control cell and expression of C7orf29 is down-regulated relative to the control cell.

70. The method of claim 63 or 64, wherein the level of expression is assessed by Q-PCR.

71. The method of claim 63 or 64, wherein the level of expression is assessed by microarray analysis.

Technical Field

The present disclosure relates generally to the fields of cell biology, stem cells, and cell differentiation. More specifically, the disclosure provides methods for generating pancreatic cells, methods of identifying cells for cell-based therapies, and related methods for treating diabetes.

Background

The following discussion is provided to aid the reader in understanding the present disclosure and is not admitted to describe or constitute prior art to the present disclosure.

Diabetes and insulin

Diabetes mellitus (diabetes) is a disease in which the body's ability to produce or respond to insulin hormones is impaired, resulting in abnormal metabolism of carbohydrates and elevated glucose levels in the blood and urine. The disease is subdivided into several subtypes, alternatively described as type 1 diabetes, Insulin Dependent Diabetes Mellitus (IDDM), adult onset diabetes in young people (MODY), Latent Adult Diabetes (LADA), fragile-onset diabetes, wasting diabetes, type 1.5 diabetes, type 2 diabetes, type 3 diabetes, obesity-related diabetes, gestational diabetes, and other nomenclature accepted in the art.

Typically, a subject with insulin-dependent diabetes mellitus needs to be administered exogenous insulin to sufficiently lower blood glucose. Non-insulin dependent subjects can be sufficiently hypoglycemic by pharmaceutical intervention that includes a class of drugs that enhance sensitivity to insulin or glucose excretion. A subject with insulin-dependent diabetes may benefit from cell replacement therapy in which insulin producing cells are implanted into the subject, whether or not the disease is labeled as type 1 diabetes, MODY, LADA, fragile-onset diabetes, wasting diabetes, type 1.5 diabetes, type 2 diabetes, type 3 diabetes, obesity-related diabetes, or any combination thereof.

Type I diabetes is commonly diagnosed in children and young adults and has previously been referred to as juvenile onset diabetes. Only 5-10% of people with diabetes have this form of disease. Adult onset diabetes is the most common form of the disease, and it results from either damage or destruction of insulin-producing beta cells, the development of insulin resistance, or both damage to insulin-producing beta cells and the development of insulin resistance. Diabetes can occur in non-obese adults and children due to a combination of genetic and environmental factors. In obese adults and children, the pancreas may attempt to make additional insulin in order to control blood glucose, but over time it cannot maintain and maintain blood glucose at normal levels. The body may also become less sensitive to the insulin produced. Prolonged overactivity of insulin-secreting beta cells can lead to beta cell dysfunction and death.

The symptoms of diabetes vary according to how much the subject's blood glucose fluctuates. Some people, especially those with prediabetes or non-insulin dependent diabetes mellitus, may not initially develop symptoms. In type I diabetes, symptoms tend to appear very quickly and are more severe.

Some signs and symptoms of type I diabetes and type II diabetes include, but are not limited to, increased thirst; the frequency of urination; extreme starvation; unexplained weight loss; the presence of ketones in urine (ketones are by-products of muscle and fat breakdown that occurs when there is insufficient insulin available); fatigue; irritability; blurred vision; slow wound healing; frequent infections such as gingival or skin infections and vaginal infections.

Cell-based therapies for treating diabetes

Insulin-dependent diabetic patients can potentially be cured by transplantation of new insulin producing cells, but this approach has been limited to date because these cells are difficult to obtain in sufficient quantity and quality. See, e.g., Pagluca FW, et al. cell,154(2): 428-. Thus, the goal of biomedical research has long been to produce insulin-producing beta cells from human stem cells in a more efficient and predictable manner. To achieve this goal, protocols must be established that produce a uniform population of beta cells that produce insulin when exposed to glucose. However, such a solution is still difficult to achieve. Various protocols have been proposed by many groups which produce different results using different cell lines. This inconsistent generation of functional beta cells increases the total cell dose required to obtain therapeutic benefit, thus increasing the cost of potential therapies and limiting clinical applicability due to variable outcomes.

Most established protocols for the production of insulin-producing beta cells from human stem cells produce highly variable cell populations. See, e.g., Pagluca FW, et al. cell,154(2): 428-. Indeed, it is common practice in the art to tailor a given differentiation protocol to a particular cell line, thereby preventing any standardization of differentiation in the art. Methods for increasing beta cell yield from various differentiation protocols typically involve testing a combination of many factors that affect the differentiation pathway in a trial and error type of approach. See, e.g., Pagluca FW, et al. cell,154(2): 428-; rezenia A.et al.Nat.Biotech.,32:1121-33 (2014); schulz TC et al plos One,7: e37004(2012). Chetty S.et al nat methods,10:553-556 (2012). Thus, a particular protocol for preparing beta cells from one starting stem cell population may be ineffective in differentiating between different starting populations.

Furthermore, inconsistencies in the resulting differentiated cell population have affected the clinical application of any proposed therapy, as researchers in this field have been struggling to obtain differentiated populations with a consistently high percentage of insulin producing cells. This not only reduces the potential efficacy of cell-based therapies for treating diabetes, but also raises concerns about the tumorigenic potential of cell transplants containing heterogeneous cell populations. Furthermore, low reproducibility between cell batches directly affects the cost of producing cells and limits the conversion of this protocol to the clinic.

Some of this variability in employing different differentiation protocols can be traced back to the starting cell line. For example, it has been shown that pluripotent cell lines can vary greatly in their ability to differentiate into certain lineages. See Bock C et al, cell,144:439-452 (2011); lim H et al.J.Vis.Exp., (90): e51755 (2014); osafune Ket al. Nat. Biotechnol.,26:313-315 (2008). This diverse ability of individual human stem cells in their response to currently used differentiation protocols has proven to be a formidable hurdle as researchers have no indication of the potential of a given cell line or starting cell to ultimately produce therapeutic cells.

Furthermore, the guidance provided by the prior art may in fact be counterproductive when using cell lines with slightly different genetic backgrounds. For example, a consistent feature of established protocols for generating insulin producing cells from pluripotent stem cells is the limitation of exposure to retinoic acid and cyclopamine. See, Nostro et al, stem Cell Reports,4:1-14 (2015). Another consistent guideline from the prior art is that for proper maturation of pancreatic cells, differentiation needs to be initiated in a suspended three-dimensional culture. See Pagliuca FW, et al cell,154(2): 428-. As discussed in more detail below, following such guidance can actually inhibit the differentiation of insulin-secreting cells from various stem cell lines.

Thus, there remains a need for improved and predictable methods of generating therapeutic insulin producing cells for the treatment of diabetes. The present disclosure satisfies these needs.

Disclosure of Invention

Cells and cell compositions that produce insulin and that can be used to treat diabetes, and methods for their preparation and identification are described herein.

In one aspect, the present disclosure provides a method of producing a mammalian insulin secreting cell comprising: culturing the mammalian stem cells in an adherent manner, thereby allowing the mammalian stem cells to spontaneously form a three-dimensional structure; and culturing the three-dimensional structure in suspension; wherein the culturing step comprises exposure to retinoic acid and cyclopamine for at least 20 days and does not comprise exposure of the three-dimensional structure of the stem cells to Wnt 3A.

In another aspect, the present disclosure provides a method of producing an insulin-secreting cell comprising: culturing mammalian stem cells on a viscous substrate in a first medium comprising activin-a and wortmannin, wherein the mammalian stem cells are not exposed to Wnt3 a; further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and transferring the cells into suspension culture while the cells form a three-dimensional cell structure; wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

In some embodiments of the foregoing aspect, the mammalian stem cells can be human stem cells, and in some embodiments, the mammalian stem cells can be non-human primate stem cells. In some embodiments of the foregoing aspect, the mammalian stem cell is derived from a cell line.

In one aspect, the present disclosure provides a method of producing a mammalian insulin-secreting cell comprising culturing a mammalian stem cell in a first culture medium comprising an endodermal induction factor, thereby differentiating the mammalian stem cell into an endodermal cell; and culturing the endoderm cells in a second medium comprising an endocrine inducing factor, thereby differentiating the endoderm cells into endocrine cells; wherein the mammalian stem cells are not exposed to Keratinocyte Growth Factor (KGF) prior to differentiation into endodermal cells.

In some embodiments, the mammalian stem cell can be a human stem cell, a non-human primate stem cell, or a stem cell derived from another mammal (including but not limited to a pig, cow, sheep, horse, dog, or cat).

In some embodiments, the endoderm induction factors include activin-a, retinoic acid, and/or cyclopamine. In some embodiments, the first medium can further comprise wortmannin. In some embodiments, the first medium does not include Wnt 3A. In some embodiments, the cells are cultured in the first medium for 1-3 days.

In some embodiments, the second medium may comprise noggin and/or KGF. In some embodiments, the second medium may include retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the second medium for 1-4 days.

Some embodiments of this aspect may include further culturing the endocrine cells in a third medium comprising KGF, thereby differentiating the endocrine cells into pancreatic progenitor cells. In some embodiments, the third medium comprises noggin and/or Epidermal Growth Factor (EGF). In some embodiments, the third medium comprises retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the third medium for 1-4 days.

Some embodiments of this aspect may include further culturing the pancreatic progenitor cells in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XXI, and/or Alk5i II. In some embodiments, the fourth medium may comprise T3. In some embodiments, the fourth medium may comprise retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the fourth medium for 1-4 days.

Some embodiments of this aspect may include further culturing pancreatic progenitor cells in a fifth medium comprising Alk5i II and/or retinoic acid. In some embodiments, the fifth medium may include T3. In some embodiments, the fifth medium may include retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the fifth medium for 1-5 days.

Some embodiments of this aspect may include further culturing the pancreatic progenitor cells in a sixth medium comprising Alk5I II, nicotinamide, and/or insulin-like growth factor (IGF) -I. In some embodiments, the sixth medium may include T3 and/or BMP 4. In some embodiments, the sixth medium may include retinoic acid and cyclopamine. In some embodiments, the sixth medium can include glucagon. In some embodiments, the cells are cultured in the sixth medium for 1-9 days.

In another aspect, the present disclosure provides a method of producing insulin-secreting pancreatic cells, comprising culturing human stem cells in a first medium comprising activin-a and wortmannin, thereby differentiating the human stem cells into endoderm cells, wherein the human stem cells have not been exposed to Keratinocyte Growth Factor (KGF) prior to differentiation into endoderm cells; and culturing the endoderm cells in a second medium comprising retinoic acid and cyclopamine, thereby differentiating the endoderm cells into endocrine cells; culturing the endocrine cells in a third medium comprising KGF, noggin and EGF, thereby differentiating the endocrine cells into pancreatic progenitor cells; and culturing the pancreatic progenitor cells in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XXI, and Alk5i II, thereby differentiating the pancreatic progenitor cells into insulin-producing pancreatic cells.

In some embodiments of this aspect, the second medium further comprises KGF. In some embodiments, the human stem cells are derived from pancreatic primary tissue. In some embodiments, the fourth medium may include a thyroid hormone, such as T3.

In some embodiments of this aspect, both the third medium and the fourth medium can include retinoic acid and cyclopamine.

Some embodiments of this aspect may include further culturing the cells in a fifth medium and/or a sixth medium, wherein the fifth medium includes Alk5I II and retinoic acid and optionally cyclopamine, and wherein the sixth medium includes Alk5I II, nicotinic amine, IGF-I and optionally retinoic acid and cyclopamine. In some embodiments, the sixth medium can include glucagon.

In some embodiments of the foregoing aspect, the cells can be cultured for 30 days or less.

In another aspect, the present disclosure provides a cell-based composition for treating diabetes comprising a population of surrogate pancreatic cells and a suitable carrier for implantation into a human subject in need thereof, wherein at least 66% of the surrogate pancreatic cells are insulin producing pancreatic cells.

In some embodiments, at least 66% of the surrogate pancreatic cells express NeuroD1, and in some embodiments, at least 68% of the surrogate pancreatic cells express nkx 6.1.

In some embodiments, the insulin-producing pancreatic cells are obtained according to a method comprising culturing a population of human stem cells on a viscous substrate in a first culture medium comprising endodermal induction factors, wherein the mammalian stem cells are not exposed to Wnt3 a; further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and transferring the cells into suspension culture while the cells form a three-dimensional cell structure; wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

In some embodiments, the endoderm induction factor comprises activin-a and/or wortmannin. In some embodiments, the at least one additional culture medium may comprise KGF, noggin, EGF, and/or thyroid hormones (such as T3).

In some embodiments, the surrogate pancreatic cells may be encapsulated in a macrocapsule. For example, the cells may be encapsulated in macrocapsules comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.

In some embodiments of the foregoing aspects, the human stem cells are derived from pancreatic primary tissue, are human embryonic stem cells, are induced pluripotent stem cells, or are non-pluripotent reprogrammed cells. In some embodiments, the reprogrammed cell is derived from pancreatic primary tissue, e.g., by expressing a reprogramming gene without incorporating the reprogramming gene into the genome of the cell. In some embodiments, the reprogramming genes may be encoded on at least one episomal expression plasmid that is not incorporated into the genome. In some embodiments, the reprogramming genes include Oct4, Sox2, Klf4, and L-Myc.

In another aspect, the disclosure provides a method of identifying an undifferentiated cell that preferentially differentiates into an endodermal lineage, comprising assessing expression of BHMT2 and NAP1L1 in the undifferentiated cell, and identifying the cell as having a preference for differentiation into an endodermal lineage if expression of BHMT2 is down-regulated relative to a control cell and expression of NAP1L1 is up-regulated relative to a control cell.

Some embodiments of this aspect further comprise assessing expression of Cox7a1 and HSPB2 in the undifferentiated cell, and identifying the cell as having a preference for differentiation into an endodermal lineage if expression of both Cox7a1 and HSPB2 is down-regulated relative to a control cell. In some embodiments, the control cells can be pluripotent cells that do not exhibit preferential differentiation to an endodermal lineage or are substantially unable to differentiate to a mesodermal lineage.

In some embodiments, when the undifferentiated cells evaluated preferentially differentiate into endodermal lineages, expression of BHMT2 is downregulated by at least 2 logs relative to control cells, and expression of NAP1L1 is upregulated by at least 2 logs relative to control cells. In some embodiments, when the undifferentiated cells evaluated differentiate preferentially into endodermal lineages, expression of both Cox7a1 and HSPB2 is down-regulated by at least 2 logs relative to control cells.

Some embodiments of this aspect further comprise assessing expression of GLIS2, CCDC58, MTX3, and C7orf 29. For example, in some embodiments, when the undifferentiated cells evaluated differentiate preferentially into endodermal lineages, expression of GLIS2, CCDC58, and MTX3 can be upregulated relative to control cells, and expression of C7orf29 can be downregulated relative to control cells.

In some embodiments, the level of expression is assessed by Q-PCR and/or by microarray analysis.

The following detailed description is exemplary and explanatory and is intended to provide further explanation of the invention.

Drawings

FIGS. 1A-1C show the selection of Induced Pluripotent Stem Cell (iPSC) line SR1423 from Langerhans island. Fig. 1A shows an alternative to SR 1423. Figure 1B represents immunostaining followed by fluorescent microscopy showing SR1423 expression of endoderm markers Sox17 (left) and HNF3 β (middle) at day 3 of differentiation. Figure 1C shows immunostaining of pancreatic markers Pdx1 (left) and nkx6.1 (middle) at day 11 of SR1423 cell differentiation. The merged images show co-staining of the marker in combination with nuclear staining. These images were taken at 40 times magnification.

Figure 2 shows that SR1423 cells poorly differentiated into the mesodermal lineage. SR1423 immunostains Sox17 (endoderm), Brachyury (mesoderm) or OTX2 (ectoderm). Nuclear staining (evident in the mesodermal framework) showed total cell number. This figure shows that SR1423 cells have the ability to become endoderm and ectoderm (as indicated by the nearly identical markers of Sox17 and Otx 2), but poorly differentiate into mesoderm as indicated by the inability to express Brachyury.

FIGS. 3A-3D show that SR1423 cells have the typical characteristics of pluripotent stem cells. FIG. 3A shows that SR1423 cells express the pluripotency markers Oct4, Tra-1-81, Tra-1-60, Sox2, SSEA. Figure 3B shows that after 40 passages in culture, the karyotype of SR1423 cells is normal. Fig. 3C shows DNA fingerprints as assessed by single tandem repeat analysis (STR). FIG. 3D shows SR1423 cell doubling time.

FIGS. 4A-4B show that SR1423 cells have a gene expression pattern associated with preferential differentiation into endodermal lineages. FIG. 4A shows the gene expression profiling cluster analysis of SR1423, B, C, and D. The correlation of the expression profiles between the two lines showing preferential differentiation towards the endoderm (SR1423 and B) and the two lines not showing preferential differentiation (C, D) was demonstrated by unsupervised hierarchical cluster analysis. Up-regulated genes are shown in red and down-regulated genes in green. A subset of differentially expressed genes is selected from the cluster analysis based on an intensity filter that identifies genes with large differences in expression between conditions. The 250 genes with the largest expression differences are represented. Figure 4B qRT shows PCR validation of a subset of up-and down-regulated genes identified in a.

FIGS. 5A-5B show that SR1423 cells produce a robust population of hormone secreting cells of the pancreas. Figure 5A shows the differentiation of SR1423 after 28 days, where Pdx (left), nkx6.1 (middle) and pooling (right) were immunostained in the upper row. The lower row shows immunostaining for insulin (left), glucagon (center) and pooled (right). The "merged" image contains nuclear staining. All images were taken at 40 x original magnification (n-10) after 28 days of differentiation. Fig. 5B shows quantification of the average purity of pancreatic cells in the population. It was found that 68% of the cells expressed nkx6.1 and 66.5% of the cells expressed insulin.

Fig. 6A-6C show that by depleting KGF, differentiation can be improved across multiple cell lines. Fig. 6A shows immunofluorescence staining of Pdx (left) and nkx6.1 (middle) of SR1423 cells with or without KGF after differentiation. Fig. 6B shows immunofluorescence staining of BGO1V cells with or without KGF after differentiation for Pdx (left) and Nkx6 (middle). Fig. 6C compares the disclosed protocol to published protocols for exposing early endoderm cells to KGF in HDC57 and BGO1V cells. Expression levels were measured by the raw incorporation density (n ═ 10) and compared to the-KGF and + KGF protocols. Images were collected at 40 x magnification. Error bars represent mean SD; p < 0.05; p < 0.0001; ns, meaningless.

Figure 7 shows that SR1423 differentiates to produce cultures with high levels of hormone secretion. This was determined by comparing the insulin and glucagon secretion in response to glucose by SR1423 cells and HDC57 cells. The levels of insulin and glucagon were assessed by C-peptide (as a surrogate for insulin) or glucagon ELISA.

Figure 8 shows reversal of diabetes in animal models. The implanted cells regulate blood glucose after implantation into streptozotocin-induced normal mice. Encapsulated cells were implanted on day 0. Results shown are the average of three mice. Error bars are standard deviations.

FIGS. 9A-9F show representative examples of stem cell lines derived from non-human primate (NHP) tissue. The undifferentiated line A1.3 from NHP donor A expressed the pluripotency markers Oct4, SSEA4, Tra-1-80 and Tra-1-60 (A-D). These cells expressed the endoderm markers Sox17 and HNF2 β (E) on day 4 of differentiation and the pancreatic markers Pdx1 and nkx6.1(F) on day 12. The nuclei were stained.

Figures 10A-B show that exposure to glucagon reduces the amount of cells co-expressing insulin and glucagon.

Detailed Description

Described herein are improved methods of insulin secreting cells that can be used to treat diabetes and pure therapeutic cell populations that produce beta cells that produce human insulin. More specifically, the disclosure provides methods of producing mammalian insulin-secreting cells comprising starting with a stem cell line that has a preference or propensity to differentiate towards the endodermal lineage, such that a simple differentiation protocol can produce a pure population of insulin-secreting cells that exhibit a mature phenotype that secretes insulin in response to glucose. In other steps, the disclosed regimens may comprise exposing stem cells, particularly stem cells exhibiting a preference or predisposition to the endodermal lineage, to retinoic acid and cyclopamine (or a chemical analog) for an extended period of time (e.g., at least 20 days). The culture can be initiated in adhesion, allowing the cells to naturally and spontaneously form a three-dimensional structure; the three-dimensional structure is then transferred to a suspension culture. During culture, cells may not be exposed to Wnt3a, whether grown in adhesion or suspension. In addition, the disclosure also provides methods for identifying cell populations suitable for cell therapy and having a propensity to differentiate towards the endodermal lineage.

I.Definition of

As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of that term given the context in which it is used is not clear to one of ordinary skill in the art, "about" will mean up to plus or minus 10% of that particular term.

As used herein, the term "substantially free" will refer to an agent that is substantially free of added composition, but does not preclude the presence of trace amounts of the agent.

As used herein, the term "islet cells" refers to terminally differentiated pancreatic endocrine cells as well as any precursor cells committed to the formation of progeny that are generally classified as pancreatic endocrine. Islet cells exhibit some morphological features and phenotypic markers typical of the islet cell lineage (for example, as follows). Mature α cells secrete glucagon; mature beta cells secrete insulin; mature cells secrete somatostatin; PP cells secrete pancreatic polypeptides.

As used herein, "pancreatic progenitor cells," "pancreatic precursor cells," or "pancreatic stem cells" are pancreatic cells or islet cells that are unable to meaningfully secrete endocrine hormones, but these cells can proliferate and produce terminally differentiated cells capable of secreting endocrine hormones (e.g., insulin). Early pancreatic progenitor cells are pluripotent, meaning that they are capable of forming at least pancreatic endocrine and pancreatic exocrine cells.

As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of differentiating into a specialized cell (e.g., an insulin-producing pancreatic cell). For the purposes of this application, the term "stem cell" may encompass pluripotent cells derived from post-fertilization pre-embryonic, embryonic or fetal tissue, which are capable of producing progenitor cells of all three germ layers (i.e., endoderm, mesoderm and ectoderm); induced pluripotent cells (i.e., cells that have been transduced with a reprogramming gene and are capable of producing progenitor cells of all three germ layers); and pluripotent cells, such as reprogrammed cells (i.e., cells that have been transduced by a reprogramming gene) that can differentiate into only one or two germ layers, or preferentially differentiate into a particular germ layer (e.g., reprogrammed cells that preferentially differentiate into ectodermal or endodermal cell types). The term encompasses two established lines of various stem cells (including cells obtained from original tissue) that are pluripotent (pluratent) or multipotent in the manner described.

As used herein, the term "induced pluripotent Cell" or "induced pluripotent stem Cell" ("iPS Cell") refers to a pluripotent Cell obtained by reprogramming adult somatic cells, germ cells, pluripotent cells, or other Cell types following art-recognized standard methods (e.g., somatic Cell nuclear transfer, transduction with reprogramming genes, chemical induction (see De Los et al, Cell Research,23: 1337-. The term encompasses both established induced pluripotent stem cells and cells obtained from primary tissue in the manner described.

As used herein, the term "non-pluripotent reprogrammed cell" or "pluripotent reprogrammed cell" refers to a cell obtained by reprogramming an adult somatic cell, germ cell, pluripotent cell or other cell type with known reprogramming methods (such as transduction/expression of reprogramming genes and other methods described above). Unlike induced pluripotent cells, "non-pluripotent reprogrammed cells" or "pluripotent reprogrammed cells" may differentiate into only one or two germ layers, or have a preference to differentiate into a particular germ layer (e.g., reprogrammed cells that preferentially differentiate into ectodermal or endodermal cell types, but fail to differentiate efficiently into mesodermal cells). The term encompasses both established induced pluripotent cells (e.g., SR1423) and cells obtained from primary tissues reprogrammed to be pluripotent in the manner described.

As used herein, the term "reprogramming genes" refers to known genes and transcription factors commonly used in the art to induce pluripotency (pluripotency) or pluripotency (multipotency) of differentiated cells. Exemplary reprogramming genes include, but are not limited to, Oct4 (i.e., Oct-3/4 or Pou5f 1); sox family transcription factors such as Sox1, Sox2, Sox3, Sox15 and Sox 18; klf family transcription factors such as Klf4, Klf1, Klf2, and Klf 5; myc family transcription factors such as C-Myc, N-Myc and L-Myc; nanog; LIN28 and Glis 1. One skilled in the art will appreciate that the disclosed reprogramming genes, as well as other reprogramming genes known in the art, may be combined in various ways in order to induce pluripotency (pluripotency) or pluripotency (multipotency). For example, Yu et al, Science,318(5858):1917-20(2007) demonstrated that the combination of LIN28, Oct4, Sox2 and Nanog can be used to generate iPS cells, while Maekawa et al, Nature,474(7350):225-29(2011) demonstrated that the combination of Glis1, Oct-3/4, Sox2 and Klf4 can be used to generate iPS cells.

As used herein, the term "differentiation" or "differentiation" refers to the change in cell type from a less specific cell to a more specific cell. For example, any cell that has left the pluripotent state and progressed to a defined germ line along the developmental pathway undergoes differentiation. The term "differentiated" is a relative term, and thus a cell undergoing differentiation may be at different stages during its pathway to mature, functional cell types. Thus, cells in the late stages of the developmental process can be said to be better differentiated than cells in the early stages.

As used herein, "differentiation-inducing factor" as used in the present disclosure refers to one of a collection of compounds used in the culture system of the present invention to induce stem cells to differentiate into differentiated cells of the islet lineage (including precursor cells and terminally differentiated cells). There is no limitation on the mode of action of the compounds. For example, an agent may assist in the differentiation process by inducing or facilitating a change in phenotype, promoting the growth of cells with a particular phenotype, or delaying the growth of other cells. It may also act as an inhibitor of other factors that may be present in the culture medium or synthesized by the cell population that would otherwise lead to direct differentiation along the pathway into unwanted cell types. Within the category of "differentiation inducing factors", one of ordinary skill in the art will appreciate that certain factors are known to induce certain steps throughout the differentiation process. For example, one of ordinary skill in the art will appreciate that "endoderm induction factors" can include, but are not limited to, activin-a and/or wortmannin, alone or in combination. Similarly, "endocrine inducing factors" may include, but are not limited to, retinoic acid and/or cyclopamine, alone or in combination.

As used herein, "long-term" when used in connection with the survival and function of exogenous therapeutic cells used in cell-based therapies/implants refers to a period of at least six months or more.

As used herein, the phrase "therapeutically effective amount" refers to the amount of encapsulated cells transplanted into a subject that provides the specific pharmacological effects of the transplanted cells, i.e., the production of insulin and the regulation of blood glucose. It is emphasized that a therapeutically effective amount of encapsulated cells is not always effective in treating diabetes in a given subject, even though such a concentration is considered by those skilled in the art to be a therapeutically effective amount. The following exemplary quantities are provided for convenience only.

Such amounts can be adjusted by one skilled in the art according to standard practice required to treat a particular subject. The therapeutically effective amount may vary depending on the site of implantation, the age and weight of the subject, and/or the condition of the subject, including the severity of the subject's disease, the diet of the subject, and/or the overall health of the subject.

The term "treatment" or "treating" as used herein with respect to diabetes refers to one or more of the following: reducing, ameliorating or eliminating one or more symptoms or complications of diabetes, such as hyperglycemia and hypoglycemia, heart disease, kidney disease, liver disease, retinopathy, neuropathy, nonhealing ulcers, periodontal disease; reducing the subject's dependence on exogenous insulin to regulate blood glucose, regulating the subject's blood glucose without the use of exogenous insulin; reducing the percentage of glycated hemoglobin or HbA1C levels in the subject; and/or reducing the dependence of the subject on other drug interventions, such as insulin sensitizers, glucose excretion enhancers, and other treatment modalities known in the art.

The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any individual mammalian subject, e.g., a non-human primate, porcine, bovine, canine, feline, equine, or human.

II.Identification of cells for cell-based therapy

One limitation of conventional cell-based therapies is that different cells have different tendencies to differentiate into mature cell types. For example, it has been reported that epigenetic characteristics of the starting cell population can persist in reprogrammed cells, a phenomenon known as "epigenetic memory". As a result, iPS cells and other reprogrammed cells may preferentially differentiate into cells belonging to the same germ layer from which they were derived. Thus, in some embodiments, the stem cells used in the disclosed methods for producing insulin-producing cells can be derived from mature endodermal cells that have been reprogrammed to be pluripotent (pluripotent) or multipotent stem cells. In some embodiments, the stem cells used in the disclosed methods can be derived from human pancreatic cells that have been reprogrammed. Such donor pancreatic cells can be from a subject undergoing treatment for diabetes (i.e., an autologous donor), or from a human not undergoing treatment for diabetes (i.e., an allogeneic donor). In some embodiments, the stem cells used in the disclosed methods can be reprogrammed primary cells from the langerhans islets of the pancreas of consenting healthy adult donors (see, e.g., fig. 1A).

Over time, primary cells grown in cell culture can become homologous and lose the property of functional maturation, which may be the result of adaptation to artificial culture conditions or genetic drift. Thus, when primary cells are used as the starting cell population, it may be advantageous to reprogram the primary cells within, for example, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of cell harvest or isolation. For example, isolated primary cells may be transduced with reprogramming genes within 5 days of cell harvest.

One of ordinary skill in the art will appreciate that when a primary cell is reprogrammed using a reprogramming gene, there are many combinations of reprogramming genes that can be used. In some embodiments, the primary cell can be reprogrammed by transduction with Oct4, Sox2, Klf4, and L-Myc. In some embodiments, the primary cells can be reprogrammed by transduction with Oct4, Sox2, Klf4, and C-Myc. In some embodiments, primary cells can be reprogrammed by transduction with LIN28, Oct4, Sox2, and Nanog. In some embodiments, primary cells can be reprogrammed by transduction with Glis1, Oct-3/4, Sox2, and Klf 4. These exemplary combinations are not intended to be limiting, as other combinations of reprogramming genes are known in the art and can be used for the purposes of the disclosed methods.

In some embodiments, the cells used in the disclosed differentiation and treatment methods may have a preference to differentiate towards one germline rather than another. For example, in some embodiments, primary cells or stem cells (e.g., SR1423) can differentiate efficiently to an ectodermal or endodermal lineage, but are substantially unable to differentiate to a mesodermal lineage. This can be determined, for example, by pushing stem cells to a particular germ line using a differentiation protocol or kit, but failing to detect a germ line marker (e.g., OTX2 for ectoderm, Sox17 for existing endoderm, or Brachyury for mesoderm).

In some embodiments, stem cells or primary cells that preferentially differentiate along the endodermal lineage can be identified by certain molecular markers. For example, stem cells or primary cells that differentiate preferentially along the endodermal lineage can express typical markers of pluripotency (see, e.g., fig. 3A) and normal karyotypes (see, e.g., fig. 3B), whereas even if typical markers of pluripotency are expressed, stem cells can be pluripotent or totipotent and thus do not meet accepted criteria for pluripotency.

Stem cells or primary cells that differentiate preferentially along the endodermal lineage can also have unique gene expression profiles. For example, in some embodiments, a stem cell or primary cell that preferentially differentiates along the endoderm lineage can down-regulate expression of BHMT2, Cox7a1, and HSPB2 relative to a control level or control cell. In some embodiments, stem cells or primary cells that preferentially differentiate along the endodermal lineage can up-regulate expression of NAP1L1 relative to control levels or control cells. Furthermore, cells preferentially differentiated along the endoderm lineage can up-regulate expression of GLIS2, CCDC58, and MTX3, and down-regulate expression of C7orf29, relative to control cells. The expression level may be determined by any means known in the art, such as qRT-PCR or microarray analysis, and the control cells used as comparative standards may comprise pluripotent cells that do not exhibit preferential differentiation to an endodermal lineage or are substantially unable to differentiate into a mesodermal lineage, such as the standard embryonic stem cell lines found in NIH registrations. While not being bound by theory, it is believed that at least BHMT2 and NAP1L1 play a role in DNA modification and may contribute to epigenetic memory.

In some embodiments, the differential expression of BHMT2, Cox7a1, HSPB2, and/or NAP1L1 may be at least about a1 log, at least about a 2 log, or at least about A3 log increase (for BHMT2, Cox7a1, and HSPB2) or decrease (for NAP1L1) relative to pluripotent cells that do not exhibit preferential differentiation to the endodermal lineage and are substantially unable to differentiate to the mesodermal lineage, or stem cells that meet the criteria for pluripotency.

The disclosed expression profiles were used to identify stem cells that demonstrated a preference for differentiation into endodermal lineages and then into insulin producing cells. Direct testing of differentiation preference to specific reproductive layers increases the efficiency of generating cell lines that are inclined to a particular fate and is therefore suitable for cell-based therapies.

III.Protocols for generating insulin producing beta cells

It has been shown that mammalian stem cells (e.g., iPS cells, embryonic stem cells, and reprogrammed cells) can be differentiated into insulin-producing beta cells by mimicking embryonic pancreatic development. See, e.g., Borowiak M.et al.Curr Opincell biol.,21:727-32 (2009). Pluripotent cells differentiate into pancreatic cells in stages. The first stage is differentiation into the endodermal lineage. The endoderm cells further differentiate into pluripotent pancreatic progenitor cells, which can then be differentiated into islet cells, and the islet cells can then be differentiated into beta cells, which produce insulin upon glucose exposure. Current differentiation protocols attempt to mimic these stages in vitro; however, the efficiency and success of clinical adaptation of these differentiation protocols vary widely. See, e.g., Pagluca FW, et al. cell,154(2): 428-. In fact, current differentiation protocols must often be adapted to the individual cells to which they are applied. To date, this has prevented the establishment of a widespread, standardized differentiation protocol. Indeed, some consistent guidelines from current differentiation protocols may inhibit differentiation of stem cell lines from different genetic backgrounds. The improved method disclosed herein addresses this deficiency in the art. For example, in some embodiments, the disclosed protocols can robustly generate insulin-producing beta cells from a variety of cell sources as compared to other differentiation protocols that may be cell-specific.

Conventional means of generating insulin producing cells include culturing and differentiating stem cells. For the disclosed protocols, cell sources can include, but are not limited to, human embryonic stem cells, induced pluripotent stem cells, non-pluripotent reprogrammed cells (e.g., SR1423), and other conventional cell sources known in the art.

The disclosed methods of generating insulin producing cells from stem cells include a multi-step process in which endodermal differentiation is initiated first, followed by differentiation to the pancreatic lineage, followed by differentiation to the endocrine lineage, and finally the maturation process of the insulin producing cells. Endodermal differentiation is typically initiated by contacting the stem cells with an endodermal inducer such as activin-a or wortmannin, or a combination thereof. When a sufficient number of endoderm cells have been reached, the cells are contacted with an endocrine inducer, such as retinoic acid or cyclopamine, or a combination thereof, to further differentiate the cells into pancreatic progenitor cells. By exposure to further differentiation factors (which will be discussed in more detail below), pancreatic progenitor cells can mature into insulin-producing cells, which can be used in cell-based therapies to treat diabetes.

In some embodiments, the stem cells are cultured in a first medium comprising an endoderm inducer. In some embodiments, the endoderm inducers comprise at least activin-a. In some embodiments, the endoderm inducers comprise activin-a and wortmannin. In some embodiments, the disclosed methods do not use or comprise the use of an activator of Wnt signaling, such as CHIR-99021 (a small molecule activator of Wnt signaling) and/or the growth factor Wnt 3A. Exposure of stem cells to endodermal inducers results in differentiation of the cells into endodermal cells.

In contrast to conventional methods of obtaining insulin producing cells, in some embodiments, the disclosed differentiation methods do not use activators of Wnt signaling. In contrast to conventional methods, in some embodiments, the disclosed differentiation methods expose cells to Retinoic Acid (RA) for long periods of time. In contrast to conventional methods, in some embodiments, the disclosed differentiation protocols expose cells to cyclopamine or a chemical analog for long periods of time. In contrast to conventional methods, in some embodiments, the disclosed differentiation protocols do not employ the generation of three-dimensional suspension cultures by the isolation of adherent cultures and the re-aggregation of suspension cells.

In some embodiments, the disclosed differentiation methods do not expose stem cells to Keratinocyte Growth Factor (KGF). KFG has long been recognized as an essential component for promoting differentiation of pancreatic progenitor cells into beta cells. See, e.g., Movasssatj., Diabetologia,46: 822-. KGF has been reported to promote differentiation of beta cells in vivo, particularly in fetal pancreatic tissue, where pancreatic duct cells are formed in the presence of KGF and noggin. Therefore, the application of KGF during the early steps of endoderm differentiation in culture is a reasonable hypothesis in the development of previous protocols. In some embodiments, the disclosed differentiation methods may comprise exposing stem cells to KGF.

In this context, however, the inventors have surprisingly found that for KGF to be effective, pancreatic progenitor cells must have been established by Retinoic Acid (RA) signaling. In fact, it has been determined that treatment with KGF negatively affects beta cell differentiation without other factors. Also, the present disclosure demonstrates that the exclusion of KGF from the early endoderm stage and the addition of KGF at the late pancreatic progenitor stage increases beta cell yield from a variety of cell sources and results in a culture that produces nearly homogeneous insulin producing cells. Thus, in one aspect, the present disclosure provides a novel differentiation method for obtaining insulin producing cells, wherein the stem cells are not contacted with KGF prior to or simultaneously with the endoderm inducer. In contrast, cells were only exposed to KGF at a later stage of differentiation. For example, KGF may be introduced into the cells at the same time as the cells are contacted with RA or in a subsequent culturing step, but not previously introduced into the cells.

Thus, in some embodiments, the differentiating cells are contacted with KGF only at a late stage of endodermal differentiation (such as after the cells have differentiated into endocrine cells). Before this stage, the stem cells should not be contacted with KGF. Thus, the medium used to differentiate stem cells into endodermal cells can comprise activin-a and/or wortmannin, but it should not comprise KGF. In some embodiments, the medium used to differentiate stem cells into endoderm cells can include activin-a, wortmannin, and/or an activator of Wnt signaling, such as CHIR-99021 (a small molecule activator of Wnt signaling), and/or the growth factor Wnt3A, and combinations thereof, but it should not contain KGF.

In some embodiments, the step of differentiating the stem cells into endoderm cells can comprise culturing the cells in a medium comprising activin-a, wortmannin, and combinations thereof, with or without KGF for 1-4 days. For example, stem cells can be cultured in the presence of these endoderm inducers for about 1 day, about 2 days, about 3 days, or about 4 days, thereby differentiating the stem cells into endoderm cells.

In some embodiments, the stem cells differentiate into endodermal cells in the presence of activin A at a concentration of about 1 to about 200ng/mL, about 25 to about 175ng/mL, about 50 to about 150ng/mL, or about 75 to about 125 ng/mL. For example, the concentration of activin A can be about 1ng/mL, about 10ng/mL, about 20ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 110ng/mL, about 120ng/mL, about 130ng/mL, about 140ng/mL, about 150ng/mL, about 160ng/mL, about 170ng/mL, about 180ng/mL, about 190ng/mL, or about 200 ng/mL.

In some embodiments, the stem cells differentiate into endodermal cells in the presence of wortmannin at a concentration of about 0.1 to about 2.0 μ Μ, about 0.25 to about 1.75 μ Μ, about 0.5 to about 1.5 μ Μ or about 0.75 to about 1.25 μ Μ. For example, the concentration of wortmannin may be about 0.1 μ M, about 0.5 μ M, about 1.0 μ M, about 1.5 μ M, or about 2.0 μ M.

In some embodiments, the medium used to differentiate endoderm cells into endocrine cells can include KGF, but in some embodiments, the medium used to differentiate endoderm cells into endocrine cells can include retinoic acid, Noggin, or cyclopamine, and combinations thereof, without KGF.

In some embodiments, the step of differentiating endoderm cells to endocrine cells can comprise culturing the cells in a medium comprising retinoic acid, cyclopamine, and/or noggin, with or without KGF, for 1-5 days. For example, endoderm cells can be cultured in the presence of these endocrine inducers for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days to differentiate into endocrine cells.

In some embodiments, the cells are differentiated in the presence of retinoic acid at a concentration of about 0.05 μ M, about 0.1 μ M, about 0.5 μ M, about 1.0 μ M, about 1.5 μ M, or about 2.0 μ M for at least twenty (20) days.

In some embodiments, the cells are differentiated in the presence of cyclopropylamine at a concentration of about 0.05 μ M, about 0.1 μ M, about 0.25 μ M, or about 0.5 μ M for at least twenty (20) days.

In some embodiments, the cells are differentiated in the presence of the chemical analog of cyclopamine, SANT-1 ((4-benzyl-piperazin-1-yl) - (3, 5-dimethyl-1-phenyl-1H-pyrazol-4-ylmethylene) -amine), at a concentration of about 0.05 μ Μ, about 0.1 μ Μ, about 0.25 μ Μ, or about 0.5 μ Μ.

In some embodiments, the endoderm cells are differentiated into endocrine cells in the presence of retinoic acid at a concentration of from about 1.0 to about 10.0 μ Μ, from about 2.0 to about 8.0 μ Μ, or from about 3.0 to about 5.0 μ Μ. For example, the concentration of retinoic acid may be about 1.0 μ M, about 1.5 μ M, about 2.0 μ M, about 2.5 μ M, about 3.0 μ M, about 3.5 μ M, about 4.0 μ M, about 4.5 μ M, about 5.0 μ M, about 5.5 μ M, about 6.0 μ M, about 6.5 μ M, about 7.0 μ M, about 7.5 μ M, about 8.0 μ M, about 8.5 μ M, about 9.0 μ M, about 9.5 μ M, or about 10.0 μ M.

In some embodiments, the endoderm cells are differentiated into endocrine cells in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 μ Μ, or about 0.25 to about 0.75 μ Μ. For example, the concentration of cyclopamine may be about 0.1 μ M, about 0.2 μ M, about 0.25 μ M, about 0.3 μ M, about 0.4 μ M, about 0.45 μ M, about 0.5 μ M, about 0.55 μ M, about 0.6 μ M, about 0.7 μ M, about 0.75 μ M, about 0.8 μ M, about 0.9 μ M, or about 1.0 μ M.

In some embodiments, the endoderm cells are differentiated into endocrine cells in the presence of Noggin at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of Noggin may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the endoderm cells are differentiated into endocrine cells in the presence of KGF at a concentration of 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of KGF can be about 1ng/mL, about 10ng/mL, about 20ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, or about 100 ng/mL. In some embodiments, the cells are not exposed to KGF until after the cells differentiate from endoderm cells to endocrine cells.

In some embodiments, the endocrine cells may be further cultured in the presence of additional growth factors and/or hormones in order to differentiate the endocrine cells into pancreatic progenitor cells, and then ultimately into insulin producing cells. In some embodiments, endocrine cells can be cultured in a medium comprising KGF after exposure to an endocrine inducing agent such as retinoic acid and/or cyclopamine, thereby differentiating the endocrine cells into pancreatic progenitor cells. In some embodiments, the endocrine cells may be cultured in a medium comprising KGF, Noggin, and/or Epidermal Growth Factor (EGF), or a combination thereof, following exposure to an endocrine inducing agent such as retinoic acid and/or cyclopamine.

In some embodiments, the step of differentiating the endocrine cells into pancreatic progenitor cells may comprise culturing the cells in a medium comprising KGF, Noggin, and/or Epidermal Growth Factor (EGF), or a combination thereof, for 1-5 days. In some embodiments, the step of differentiating the endocrine cells into pancreatic progenitor cells can comprise culturing the cells in a medium comprising KGF, Noggin, and/or Epidermal Growth Factor (EGF), or a combination thereof, and further comprising retinoic acid and/or cyclopamine (e.g., cyclopamine KAAD) for 1-5 days. For example, endoderm cells can be cultured in the presence of these agents for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, thereby differentiating the endocrine cells into pancreatic progenitor cells.

In some embodiments, the endocrine cells are differentiated into pancreatic progenitor cells in the presence of KGF at a concentration of 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of KGF can be about 1ng/mL, about 10ng/mL, about 20ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, or about 100 ng/mL.

In some embodiments, the endocrine cells are differentiated into pancreatic progenitor cells in the presence of Noggin at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of Noggin may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the endocrine cells are differentiated into pancreatic progenitor cells in the presence of EGF at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of EGF may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the endocrine cells are differentiated into pancreatic progenitor cells in the presence of retinoic acid at a concentration from about 1 to about 200ng/mL, from about 50 to about 200ng/mL, or from about 75 to about 125 ng/mL. For example, the concentration of retinoic acid may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, about 110ng/mL, about 115ng/mL, about 120ng/mL, about 125ng/mL, about 130ng/mL, about 135ng/mL, about 140ng/mL, about 145ng/mL, about 150ng/mL, about, About 155ng/mL, about 160ng/mL, about 165ng/mL, about 170ng/mL, about 175ng/mL, about 180ng/mL, about 185ng/mL, about 190ng/mL, about 195ng/mL, or about 200 ng/mL.

In some embodiments, the endocrine cells are differentiated into pancreatic progenitor cells in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 μ Μ or about 0.25 to about 0.75 μ Μ. For example, the concentration of cyclopamine may be about 0.1 μ M, about 0.2 μ M, about 0.25 μ M, about 0.3 μ M, about 0.4 μ M, about 0.45 μ M, about 0.5 μ M, about 0.55 μ M, about 0.6 μ M, about 0.7 μ M, about 0.75 μ M, about 0.8 μ M, about 0.9 μ M, or about 1.0 μ M.

In some embodiments, the pancreatic progenitor cells may be further cultured in the presence of additional growth factors and/or hormones in order to differentiate the pancreatic progenitor cells into the pancreatic lineage, and ultimately into insulin producing cells. In some embodiments, pancreatic progenitor cells can be cultured in media comprising Noggin, EGF, gamma-secretase inhibitor XXI, Alk5i II, and/or T3, and combinations thereof. In some embodiments, pancreatic progenitor cells can be cultured in a medium comprising Noggin, EGF, gamma-secretase inhibitor XXI, Alk5iII and/or T3, and combinations thereof, and further comprising retinoic acid and/or cyclopamine (e.g., cyclopamine KAAD). In some embodiments, T3 may not be included in the culture medium at this stage of differentiation. For example, pancreatic progenitor cells can be cultured in the presence of these agents for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, thereby differentiating the pancreatic progenitor cells into the pancreatic lineage.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of Noggin at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of Noggin may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of EGF at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of EGF may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of gamma-secretase inhibitor XXI at a concentration of about 0.1 to about 2.0 μ Μ, about 0.25 to about 1.75 μ Μ, about 0.5 to about 1.5 μ Μ or about 0.75 to about 1.25 μ Μ. For example, the concentration of the gamma-secretase inhibitor XXI can be about 0.1. mu.M, about 0.5. mu.M, about 1.0. mu.M, about 1.5. mu.M, or about 2.0. mu.M.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of Alk5i II at a concentration of about 1.0 to about 50.0 μ Μ, about 5 to about 25 μ Μ or about 10 to about 20 μ Μ. For example, the concentration of Alk5i II may be about 0.1. mu.M, about 1.0. mu.M, about 5.0. mu.M, about 10. mu.M, about 20. mu.M, about 30. mu.M, about 40. mu.M, or about 50. mu.M.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of T3 at a concentration of about 0.1 to about 2.0 μ M, about 0.25 to about 1.75 μ M, about 0.5 to about 1.5 μ M, or about 0.75 to about 1.25 μ M. For example, the concentration of T3 may be about 0.1. mu.M, about 0.5. mu.M, about 1.0. mu.M, about 1.5. mu.M, or about 2.0. mu.M.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of retinoic acid at a concentration of from about 1 to about 200ng/mL, from about 50 to about 200ng/mL, or from about 75 to about 125 ng/mL. For example, the concentration of retinoic acid may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, about 110ng/mL, about 115ng/mL, about 120ng/mL, about 125ng/mL, about 130ng/mL, about 135ng/mL, about 140ng/mL, about 145ng/mL, about 150ng/mL, about, About 155ng/mL, about 160ng/mL, about 165ng/mL, about 170ng/mL, about 175ng/mL, about 180ng/mL, about 185ng/mL, about 190ng/mL, about 195ng/mL, or about 200 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 μ Μ or about 0.25 to about 0.75 μ Μ. For example, the concentration of cyclopamine may be about 0.1 μ M, about 0.2 μ M, about 0.25 μ M, about 0.3 μ M, about 0.4 μ M, about 0.45 μ M, about 0.5 μ M, about 0.55 μ M, about 0.6 μ M, about 0.7 μ M, about 0.75 μ M, about 0.8 μ M, about 0.9 μ M, or about 1.0 μ M.

In some embodiments, the pancreatic cells may be further cultured in the presence of additional growth factors and/or hormones in order to ultimately differentiate the cells into insulin producing cells. In some embodiments, pancreatic progenitor cells can be cultured in media comprising Alk5i II, T3, and/or retinoic acid, and combinations thereof. In some embodiments, pancreatic progenitor cells can be cultured in a medium comprising Alk5i II, T3, and/or retinoic acid and combinations thereof and further comprising cyclopamine (e.g., cyclopamine KAAD). In some embodiments, T3 may not be included in the culture medium at this stage of differentiation. For example, pancreatic progenitor cells can be cultured in the presence of these agents for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days, thereby differentiating pancreatic cells into insulin producing cell types.

In some embodiments, the pancreatic cells are further cultured in the presence of Alk5i II at a concentration of about 1.0 to about 50.0 μ Μ, about 5 to about 25 μ Μ or about 10 to about 20 μ Μ. For example, the concentration of Alk5i II may be about 0.1. mu.M, about 1.0. mu.M, about 5.0. mu.M, about 10. mu.M, about 20. mu.M, about 30. mu.M, about 40. mu.M, or about 50. mu.M.

In some embodiments, the pancreatic cells are further cultured in the presence of T3 at a concentration of about 0.1 to about 2.0 μ M, about 0.25 to about 1.75 μ M, about 0.5 to about 1.5 μ M, or about 0.75 to about 1.25 μ M. For example, the concentration of T3 may be about 0.1. mu.M, about 0.5. mu.M, about 1.0. mu.M, about 1.5. mu.M, or about 2.0. mu.M.

In some embodiments, the pancreatic cells are further cultured in the presence of retinoic acid at a concentration of from about 1 to about 200 μ M, from about 25 to about 175 μ M, from about 50 to about 150 μ M, or from about 75 to about 125 μ M. For example, the concentration of retinoic acid may be about 1 μ M, about 10 μ M, about 20 μ M, about 40 μ M, about 50 μ M, about 60 μ M, about 70 μ M, about 80 μ M, about 90 μ M, about 100 μ M, about 110 μ M, about 120 μ M, about 130 μ M, about 140 μ M, about 150 μ M, about 160 μ M, about 170 μ M, about 180 μ M, about 190 μ M, or about 200 μ M. In some embodiments, the pancreatic progenitor cells are further cultured in the presence of retinoic acid at a concentration of from about 1 to about 200ng/mL, from about 50 to about 200ng/mL, or from about 75 to about 125 ng/mL. For example, the concentration of retinoic acid may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, about 110ng/mL, about 115ng/mL, about 120ng/mL, about 125ng/mL, about 130ng/mL, about 135ng/mL, about 140ng/mL, about 145ng/mL, about 150ng/mL, about, About 155ng/mL, about 160ng/mL, about 165ng/mL, about 170ng/mL, about 175ng/mL, about 180ng/mL, about 185ng/mL, about 190ng/mL, about 195ng/mL, or about 200 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 μ Μ or about 0.25 to about 0.75 μ Μ. For example, the concentration of cyclopamine may be about 0.1 μ M, about 0.2 μ M, about 0.25 μ M, about 0.3 μ M, about 0.4 μ M, about 0.45 μ M, about 0.5 μ M, about 0.55 μ M, about 0.6 μ M, about 0.7 μ M, about 0.75 μ M, about 0.8 μ M, about 0.9 μ M, or about 1.0 μ M.

In some embodiments, the pancreatic cells may be further cultured in the presence of additional growth factors and/or hormones in order to ultimately differentiate the cells into insulin producing cells. In some embodiments, pancreatic progenitor cells can be cultured in a medium comprising Alk5I II, T3, nicotinamide, insulin-like growth factor (IGF) -I, and/or BMP4, and combinations thereof. In some embodiments, pancreatic progenitor cells can be cultured in a medium comprising Alk5I II, T3, nicotinamide, insulin-like growth factor (IGF) -I, and/or BMP4, and combinations thereof, and further comprising retinoic acid and/or cyclopamine (e.g., cyclopamine KAAD). In some embodiments, T3 and/or BMP4 may not be included in the culture medium at this stage of differentiation. For example, pancreatic progenitor cells can be cultured in the presence of these agents for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, thereby differentiating pancreatic cells into insulin-producing cells.

In some embodiments, the pancreatic cells are further cultured in the presence of Alk5i II at a concentration of about 1.0 to about 50.0 μ Μ, about 5 to about 25 μ Μ or about 10 to about 20 μ Μ. For example, the concentration of Alk5i II may be about 0.1. mu.M, about 1.0. mu.M, about 5.0. mu.M, about 10. mu.M, about 20. mu.M, about 30. mu.M, about 40. mu.M, or about 50. mu.M.

In some embodiments, the pancreatic cells are further cultured in the presence of T3 at a concentration of about 0.1 to about 2.0 μ M, about 0.25 to about 1.75 μ M, about 0.5 to about 1.5 μ M, or about 0.75 to about 1.25 μ M. For example, the concentration of T3 may be about 0.1. mu.M, about 0.5. mu.M, about 1.0. mu.M, about 1.5. mu.M, or about 2.0. mu.M.

In some embodiments, the pancreatic cells are further cultured in the presence of nicotinamide at a concentration of about 1.0 to about 50.0mM, about 5 to about 25mM, or about 10 to about 20 mM. For example, the concentration of nicotinamide can be about 0.1mM, about 1.0mM, about 5.0mM, about 10mM, about 20mM, about 30mM, about 40mM, or about 50 mM.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of IGF-I at a concentration of about 1 to about 100ng/mL, about 25 to about 75ng/mL, or about 60 to about 70 ng/mL. For example, the concentration of IGF-I may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100 ng/mL.

In some embodiments, the pancreatic cells are further cultured in the presence of BMP4 at a concentration of from about 1.0 to about 50.0ng/mL, from about 5 to about 25ng/mL, or from about 10 to about 20 ng/mL. For example, the concentration of BMP4 may be about 0.1ng/mL, about 1.0ng/mL, about 5.0ng/mL, about 10ng/mL, about 20ng/mL, about 30ng/mL, about 40ng/mL, or about 50 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of retinoic acid at a concentration of from about 1 to about 200ng/mL, from about 50 to about 200ng/mL, or from about 75 to about 125 ng/mL. For example, the concentration of retinoic acid may be about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, about 110ng/mL, about 115ng/mL, about 120ng/mL, about 125ng/mL, about 130ng/mL, about 135ng/mL, about 140ng/mL, about 145ng/mL, about 150ng/mL, about, About 155ng/mL, about 160ng/mL, about 165ng/mL, about 170ng/mL, about 175ng/mL, about 180ng/mL, about 185ng/mL, about 190ng/mL, about 195ng/mL, or about 200 ng/mL.

In some embodiments, the pancreatic progenitor cells are further cultured in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 μ Μ or about 0.25 to about 0.75 μ Μ. For example, the concentration of cyclopamine may be about 0.1 μ M, about 0.2 μ M, about 0.25 μ M, about 0.3 μ M, about 0.4 μ M, about 0.45 μ M, about 0.5 μ M, about 0.55 μ M, about 0.6 μ M, about 0.7 μ M, about 0.75 μ M, about 0.8 μ M, about 0.9 μ M, or about 1.0 μ M.

In some embodiments, the cells differentiate on a viscous substrate consisting of vitronectin and/or laminin and/or collagen. In some embodiments, cells that spontaneously and naturally form three-dimensional structures are collected and transferred to suspension culture.

In some embodiments, the cells are encapsulated in a hydrogel, and differentiation may proceed further when the cells are encapsulated in the hydrogel. In these embodiments, the differentiation protocol is the same as for unencapsulated cells (i.e., the differentiation protocol may include the same reagents and incubation times as for the disclosed differentiation methods). That is, even after being encapsulated in a hydrogel, the cells can still be incubated in the disclosed media to produce insulin producing cells. In some embodiments, the hydrogel encapsulating the cells may include or consist of sodium alginate. In some embodiments, the cells are encapsulated in the hydrogel around day 12 of differentiation. For example, cells may be encapsulated in a hydrogel on day 8, 9, 10, 11, 12, 13, 14, or 15 of differentiation. Thus, after the cells have been incubated in a medium comprising noggin and/or EGF (i.e. the "third medium" as disclosed herein), the cells may be encapsulated in the hydrogel. In some embodiments, the cells are encapsulated in the hydrogel at a late stage of differentiation around day 14, around day 16, around day 18, around day 20, around day 22, around day 24, around day 26, or around day 28.

In some embodiments, the stem cells used in the disclosed differentiation methods are from pancreatic primary tissue. In some embodiments, the stem cells used in the disclosed differentiation methods are embryonic stem cells. In some embodiments, the stem cells used in the disclosed differentiation methods are induced pluripotent stem cells. In some embodiments, the stem cells used in the disclosed differentiation methods are non-pluripotent reprogrammed cells. In some embodiments, the stem cell is a human stem cell.

For the purposes of the present disclosure, it may be desirable to reprogram a cell by expressing the reprogramming gene in the cell and thus not incorporating the reprogramming gene into the genome of the cell. One of ordinary skill in the art will recognize that the transduced genes can be expressed in cells without the need to incorporate these genes into the genome using, for example, episomal expression plasmids. The reprogramming genes may be expressed on at least 1, at least 2, at least 3, or at least 4 or more episomal expression plasmids. As described above, multiple reprogramming genes are known in the art and may be used for the purposes of the disclosed methods, but in some embodiments, the reprogramming genes include Oct4, Sox2, Klf4, and L-Myc.

In some embodiments, the total culture time required to differentiate a cell from a stem cell into an insulin producing cell may be about 30 days or less. For example, the cells may be cultured for about 30 days, about 29 days, about 28 days, about 27 days, about 26 days, about 25 days, or less.

One skilled in the art will also appreciate that the total culture time in each differentiation step may vary. Thus, in some embodiments, the present disclosure provides a method of producing insulin-secreting pancreatic cells comprising (a) culturing human stem cells in a first medium comprising activin-a and wortmannin, wherein the human cells were not exposed to Wnt3a, and optionally to Keratinocyte Growth Factor (KGF), prior to differentiation into endodermal cells, thereby differentiating the human stem cells into endodermal cells; (b) culturing endoderm cells from (a) in a second medium comprising retinoic acid and cyclopamine, and optionally comprising KGF, thereby differentiating the endoderm cells into endocrine cells; (c) culturing the endocrine cells from (b) in a third medium comprising KGF, thereby differentiating the endocrine cells into pancreatic progenitor cells; (d) culturing pancreatic progenitor cells from (c) in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XXI, Alk5i II, and T3; (e) culturing the cells from (d) in a fifth medium comprising Alk5i II, T3, and retinoic acid; and (f) culturing in a sixth medium comprising Alk5I II, T3, nicotinamide, insulin-like growth factor (IGF) -I, and BMP 4.

In some embodiments, the present disclosure provides a method of producing insulin-secreting pancreatic cells comprising (a) culturing human stem cells in a first medium comprising activin-a and wortmannin, wherein the human cells were not exposed to Wnt3a, and optionally to Keratinocyte Growth Factor (KGF), prior to differentiation into endodermal cells, thereby differentiating the human stem cells into endodermal cells; (b) culturing endoderm cells from (a) in a second medium comprising retinoic acid, noggin, and cyclopamine, and optionally comprising KGF, thereby differentiating the endoderm cells into endocrine cells; (c) culturing the endocrine cells from (b) in a third medium comprising KGF, noggin, retinoic acid, and cyclopamine, thereby differentiating the endocrine cells into pancreatic progenitor cells; (d) culturing pancreatic progenitor cells from (c) in a fourth medium comprising noggin, EGF, gamma-secretase inhibitor XII, Alk5i II, retinoic acid, and cyclopamine; (e) culturing the cells from (d) in a fifth medium comprising Alk5i II, T3, retinoic acid, and cyclopamine; and (f) culturing in a sixth medium comprising Alk5I II, nicotinamide, IGF-I, retinoic acid, and cyclopamine.

In some embodiments, the sixth medium may include glucagon, which has a beneficial effect in reducing the proportion of endocrine cells that co-express insulin and glucagon. The concentration of glucagon may be, for example, about 40ng/L, about 70ng/L, about 110ng/L, or about 140 ng/L.

In some embodiments, the total incubation time of steps (a) - (f) may be 30 days or less. For example, the cells may be cultured for about 30 days, about 29 days, about 28 days, about 27 days, about 26 days, about 25 days, or less. In some embodiments, step (a) may comprise days 1-3 of culture, step (b) may comprise days 4-7 of culture, step (c) may comprise days 8-11 of culture, step (d) may comprise days 12-15 of culture, step (e) may comprise days 16-19 of culture, and step (f) may comprise days 20-28 of culture. Thus, in some embodiments, step (a) may comprise 1-4 days of culture, step (b) may comprise 1-5 days of culture, step (c) may comprise 1-5 days of culture, step (d) may comprise 1-5 days of culture, step (e) may comprise 1-5 days of culture, and step (f) may comprise 1-10 days of culture.

As disclosed herein, exposure of differentiated cells to KGF at the earlier endodermal stage may hinder the production of insulin producing beta cells, and thus the addition of this component is optional and may vary according to the precise protocol. For the purpose of producing insulin producing cells, it may therefore be beneficial in some embodiments to exclude KGF from the early stages of endodermal differentiation or until at least the time of culture in which the differentiating cells have been exposed to retinoic acid.

In some embodiments, the present disclosure provides a method of producing an insulin-secreting pancreatic cell comprising (a) culturing a human stem cell in a first medium comprising activin-a and wortmannin, wherein the human cell is not exposed to Wnt3a, thereby differentiating the human stem cell into a endodermal cell; (b) exposing the cells to retinoic acid for at least twenty (20) days during a subsequent culturing step; (c) exposing the cells to cyclopamine or a chemical analog for at least twenty (20) days during a subsequent culturing step; (d) initiating cell culture on the adhesive substrate; and (e) transferring the cells that naturally and spontaneously form the three-dimensional structure into a suspension culture; and optionally (f) culturing the cells in the presence of glucagon.

In some embodiments, the present disclosure provides a method of producing a mammalian insulin-secreting cell comprising: culturing the mammalian stem cells adherently, thereby allowing the mammalian stem cells to spontaneously form a three-dimensional structure; and suspension culturing the three-dimensional structure; wherein the culturing step comprises exposure to retinoic acid and cyclopamine for at least 20 days and does not comprise exposure of the three-dimensional structure of the stem cells to Wnt 3A.

In some embodiments, the present disclosure provides a method of producing an insulin-secreting cell comprising: culturing mammalian stem cells on a viscous substrate in a first medium comprising activin-a and wortmannin, wherein the mammalian stem cells are not exposed to Wnt3 a; further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and transferring the cells into suspension culture while the cells form a three-dimensional cell structure; wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

In some embodiments, it may be preferable to select starting stem cells or non-pluripotent progenitor cells that preferentially differentiate or tend to differentiate into endodermal lineages. This may allow for simpler differentiation and may result in a culture of purer and more mature insulin secreting cells compared to traditional methods.

For the purposes of the presently disclosed methods, it has been determined that the production of insulin-secreting cells is optimal when the cells are initially grown in adhesion or cultured on a viscous substrate (e.g., a positively charged surface or a surface coated with vitronectin or a matrigel), thus allowing the cells to naturally and spontaneously form three-dimensional structures (e.g., aggregates of cells). These three-dimensional structures consisting of adhered cells can then be cultured in suspension for the duration of the disclosed method.

Thus, in some embodiments, the initial stem cell population is grown adherently, while the later stages of culturing are performed in suspension. For the purposes of this disclosure, the phrase "adherent growth" or "adherent culture" refers to a standard cell culture in which cells adhere to the surface of a culture dish. In some cases, the culture dish may be coated with a substrate to promote adhesion, and in some cases, the culture dish may be given a net positive charge to promote adhesion. In general, the culture of stem cells (such as iPS cells) requires a viscous substrate, and various substrates that promote adhesion are known in the art. For example, a vitronectin or matrix gel may be applied to cell culture flasks to promote adhesion, but matrix gels are harvested from mouse sarcomas cells and are therefore not preferred for clinical use. In some embodiments, differentiation is initiated by culturing a starting stem cell population with endoderm induction medium and adherent growth. As differentiation proceeds, formation of 3D structures (e.g., aggregates of differentiated/differentiating cells) on the substrate/plate may occur gradually. By about day 15, the 3D structures begin to detach from the plate, and these 3D structures can be transferred to blood vessels that are not coated with an adhesive substrate (e.g., vitronectin), such that the 3D structures are cultured in a free-floating suspension. This transition from adherent to suspension culture is novel and allows for more natural differentiation into insulin-secreting cells.

It has also been determined that, contrary to conventional practice, the stem cells used to start the culture do not need to be contacted with Wnt3a in order to promote differentiation. Indeed, Wnt3a is not required at any time in the disclosed methods.

Finally, it has been determined that even when various differentiation media are exchanged throughout the process of producing insulin-secreting cells, differentiation appears to be most effective when the cells are maintained in contact with at least certain concentrations of retinoic acid and cyclopamine for at least about 20 days. For example, in some embodiments, the cells are preferably exposed to retinoic acid and cyclopamine for at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, or at least about 28 days. In some embodiments, such sustained exposure to retinoic acid and cyclopamine can begin after the starting stem cell population has been forced into transfer to the endodermal lineage (e.g., after the starting stem cell population has been cultured in the presence of activin a and wortmannin for about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days).

One skilled in the art will appreciate that the disclosed methods can be applied to mammalian stem cells in general, such as human stem cells and non-human primate stem cells. However, additional mammalian cells (such as porcine, bovine, equine, ovine, canine, or feline stem cells) can also be differentiated according to the disclosed methods. Furthermore, one skilled in the art will recognize that the disclosed methods may employ various forms of cell cultures, including, for example, adherent cultures and/or suspension cultures.

The disclosed protocol for generating insulin producing cells improves the yield of insulin producing beta cells from both human embryonic stem cells and reprogrammed pancreatic tissue. In contrast to conventional methods of producing insulin producing cells, the protocols disclosed herein produce a population of nearly homogeneous insulin producing cells. The generation of a homogenous cell population is important not only for therapeutic efficacy, but also for safety, since stem cells can form teratomas upon transplantation and have tumorigenic potential. The high degree of differentiation and homogeneity provided by the disclosed differentiation method means fewer cells with tumorigenic potential, which is essential in developing useful cell therapies.

Prior to using the disclosed differentiation methods, stem cells that preferentially differentiate into endoderm cells can be identified according to the methods disclosed in section II of this application. This may improve the overall efficiency of the differentiation process as well as increase the yield of insulin producing cells.

IV.Cell-based compositions and methods of treatment

The insulin producing cells disclosed herein can be used to treat diabetes in a subject in need thereof. In some embodiments, the subject in need of treatment is a mammal, e.g., a human subject with insulin-dependent diabetes mellitus.

The present disclosure provides methods for producing a substantially homogeneous population of insulin producing cells that can be incorporated into a cell-based composition for the treatment of diabetes. Accordingly, provided herein is a cell-based composition for treating diabetes comprising a population of surrogate pancreatic cells and a suitable carrier for implantation into a human subject in need thereof, wherein at least 66% of the cells are insulin producing pancreatic cells. In some embodiments, the cell-based composition can include at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the insulin-producing pancreatic cells.

Suitable carriers for implanting therapeutic cells are known in the art and may include, but are not limited to, hydrogels, natural and synthetic polymer scaffolds, extracellular matrices (which may include, for example, collagen, laminin, fibronectin, and the like), hyaluronic acid, biomimetic scaffolds, Polylactide (PLA) scaffolds, Polyglycolide (PGA) scaffolds, PLA-PGA copolymer (PLGA) scaffolds, as well as hydroxyapatite scaffolds and macroporous cryogels. In some embodiments, a carrier suitable for transplantation may include encapsulating insulin producing cells in a macrocapsule, such as a macrocapsule comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.

In some embodiments, at least 66% of the surrogate pancreatic cells express NeuroD 1. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the surrogate pancreatic cells express NeuroD 1.

In some embodiments, at least 68% of the surrogate pancreatic cells express nkx 6.1. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the surrogate pancreatic cells express nkx 6.1.

Cell-based compositions for treating diabetes can be prepared according to the methods disclosed herein. For example, insulin-producing pancreatic cells of a cell-based composition can be obtained, for example, according to a method comprising: (a) culturing a population of human stem cells in a first culture medium comprising an endoderm inducing factor, thereby differentiating the human stem cells into endoderm cells, wherein the human stem cells have not been exposed to Keratinocyte Growth Factor (KGF) prior to differentiation into endoderm cells; (b) culturing the endoderm cells from (a) in a second medium comprising an endocrine inducing factor, thereby differentiating the endoderm cells into endocrine cells; (c) culturing the endocrine cells from (b) in a third medium comprising KGF, thereby differentiating the endocrine cells into pancreatic progenitor cells; and (d) culturing the pancreatic progenitor cells from (c) in a fourth medium comprising thyroid hormone, thereby differentiating the pancreatic progenitor cells into insulin-producing pancreatic cells.

In some embodiments, the insulin-producing pancreatic cells of the cell-based composition can be obtained, for example, according to a method comprising: (a) culturing a population of human stem cells in a first culture medium comprising a endodermal induction factor, thereby differentiating the human stem cells into endodermal cells, wherein the human stem cells are not exposed to Wnt3 a; (b) exposing the cells to retinoic acid for at least twenty (20) days during a subsequent culturing step; (c) exposing the cells to cyclopamine or a chemical analog for at least twenty (20) days during a subsequent culturing step; (d) initiating cell culture on the adhesive substrate; and (e) transferring the cells that naturally and spontaneously form the three-dimensional structure into a suspension culture.

In some embodiments, the insulin-producing pancreatic cells of the cell-based composition can be obtained, for example, according to a method comprising: culturing a population of human stem cells on a adherent substrate in a first culture medium comprising endodermal induction factors, wherein the mammalian stem cells are not exposed to Wnt3 a; and further culturing the cells in suspension in at least one additional medium comprising retinoic acid and cyclopamine, wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days. In some embodiments, the endoderm induction factor comprises activin-a and/or wortmannin. In some embodiments, the at least one additional culture medium may comprise KGF, noggin, EGF, and/or thyroid hormones (such as T3).

Various endoderm inducing factors are known in the art and include, but are not limited to, activin-a and wortmannin. Likewise, various endocrine inducing factors are known in the art, including but not limited to retinoic acid and cyclopamine.

In some embodiments, the second medium comprises KGF, and in some embodiments, the cells are not contacted with KGF until after step (b). In some embodiments, KGF may be included in the medium of step (a).

In some embodiments, the third medium comprises noggin and/or Epidermal Growth Factor (EGF). In some embodiments, the third medium comprises retinoic acid and/or cyclopamine. In some embodiments, the thyroid hormone may be T3.

The source of the stem cells used to prepare the disclosed cell-based compositions is not particularly limited; however, as disclosed herein, selection of cells/cell lines that preferentially differentiate into endodermal lineages can increase the yield of insulin producing cells and increase the efficiency of differentiation. Thus, in some embodiments, the stem cells used in the disclosed differentiation methods for preparing cell-based compositions are derived from pancreatic primary tissue. In some embodiments, the stem cells used in the disclosed differentiation methods are embryonic stem cells. In some embodiments, the stem cells used in the disclosed differentiation methods are induced pluripotent stem cells. In some embodiments, the stem cells used in the disclosed differentiation methods are non-pluripotent reprogrammed cells. In some embodiments, the stem cell is a human stem cell.

For the purposes of the present disclosure, when preparing insulin producing cells for incorporation into cell-based compositions for the treatment of diabetes, it may be desirable to reprogram the cells by expressing the reprogramming genes in the cells without incorporating the reprogramming genes into the genome of the cells. One of ordinary skill in the art will recognize that the transduced genes can be expressed in cells without the need to incorporate these genes into the genome using, for example, episomal expression plasmids. The reprogramming genes may be expressed on at least 1, at least 2, at least 3, or at least 4 or more episomal expression plasmids. As described above, multiple reprogramming genes are known in the art and may be used for the purposes of the disclosed methods, but in some embodiments, the reprogramming genes include Oct4, Sox2, Klf4, and L-Myc.

In some embodiments, the cell-based composition is encapsulated, for example, in a microcapsule or macrocapsule.

The present disclosure also provides methods of treating diabetes using the disclosed cell-based compositions. Methods of treating diabetes generally comprise implanting a therapeutically effective amount of encapsulated insulin producing cells into a subject in need thereof. The therapeutically effective amount of insulin producing cells may be in the form of a cell-based composition, such as a population of microencapsulated or large-encapsulated surrogate pancreatic cells.

Thus, in some embodiments, the method comprises implanting a therapeutically effective amount of insulin producing cells encapsulated in a macrocapsule into an individual in need thereof. There are no particular limitations on the composition of the macrocapsule, and those skilled in the art will appreciate that various materials may be used to encapsulate the insulin producing cells. For example, the capsule may comprise alginate, cellulose sulfate, glucomannan, or a combination thereof. In some embodiments, the macrocapsule may include at least one barrier, wherein the outer barrier is comprised of cellulose sulfate and glucomannan. In some embodiments, macrocapsules may be formed in the shape of a cylindrical tube, which includes an inner capsule of alginate and an outer capsule of cellulose sulfate and glucomannan.

In some embodiments, the method comprises implanting into an individual in need thereof a therapeutically effective amount of insulin producing cells encapsulated in the disclosed macrocapsules about once a year, once every two years, once every three years, once every four years, once every five years, or more. In some embodiments, the implanted cells will survive at least six months after implantation. Thus, in some embodiments, a subject may only require one implant. In some embodiments, the cell-based composition may need to be replaced every 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months, every 1 year, 2 years, 3 years, 4 years, or 5 years or more, or until the subject has recurrent hyperglycemia or reverts to a diabetic state.

In some embodiments, the cell-based composition may be implanted into the larger omentum of the subject. The larger omentum (also known as the greater omentum (great omentum), omentum maju, gastro-colonic omentum, greater omentum (epiplon) or greater omentum (cau)) is a large apron-like fold of the visceral peritoneum that hangs down from the stomach and extends posteriorly from the greater curvature of the stomach and ascends to the transverse colon and then to the posterior abdominal wall. Thus, the cell-based composition may be implanted into a pocket formed by omentum surgery.

In some embodiments, the cell-based composition is implanted into the peritoneal cavity. In some embodiments, the cell-based composition is implanted into the peritoneal cavity and anchored to the omentum. In some embodiments, the cell-based composition is implanted into the omentum pocket.

Exemplary dosages of insulin producing cells may vary depending on the size and health of the individual to be treated. For example, in some embodiments, an exemplary implant of cells encapsulated in the disclosed cell-based composition can include 500 to 1000 ten thousand cells per kilogram body weight.

In addition, the disclosed methods of treatment may additionally include administration of a second therapeutic agent in addition to the encapsulated therapeutic cells. For example, in some embodiments, the additional therapeutic compound may include, but is not limited to, insulin injections, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, and SGLT2 inhibitors.

A particular treatment regimen comprising implantation of a cell-based composition comprising insulin-producing cells can be evaluated according to whether it will improve the outcome of a given patient, meaning that it will help stabilize or normalize the subject's blood glucose levels or reduce the risk or occurrence of symptoms or complications associated with diabetes, including but not limited to hypoglycemic episodes, elevated glycated hemoglobin levels (HbA1C levels), heart disease, retinopathy, neuropathy, nephropathy, liver disease, periodontal disease and nonhealing ulcers. In some embodiments, the cell-based composition will be encapsulated in, for example, a capsule comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.

Thus, for purposes of this disclosure, a subject is treated if one or more beneficial or desired results (including a desired clinical result) are obtained. For example, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by diabetes, improving the quality of life of patients with diabetes, reducing the dosage of other drugs required to treat diabetes, delaying or preventing complications associated with diabetes, and/or prolonging the survival of individuals.

Furthermore, although the subject of the method is typically a subject suffering from diabetes, the age of the patient is not limited. The disclosed methods can be used to treat diabetes across all age groups and cohorts. Thus, in some embodiments, the subject may be a pediatric subject, while in other embodiments, the subject may be an adult subject.

Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are intended to be included within the spirit of the present disclosure. The following examples are given to illustrate the invention. It is to be understood, however, that the invention is not limited to the specific conditions or details of these examples.

Examples of the invention

Example 1-materials and methods

And (3) islet harvesting: the entire human pancreas was obtained with appropriate consent and anonymity from the enrolled organ donations. The leaflets were injected with collagenase P (Roche # 1129002001) and collagen P was resuspended in 1.4mg/ml islet isolation solution (containing 0.35g NaHCO3/L and 1% human serum albumin (Roche A9731) hanks Balanced salt solution (Invitrogen # 14065-056)). The swollen leaflets are incubated at 37 ℃ for 15-25 minutes with gentle agitation. The digest was diluted with cold islet isolation solution and centrifuged at 1500RPM for 5 minutes. The supernatant was discarded and the pellet washed in cold islet isolation solution under vigorous trituration. The solution was filtered through a 420 μm sieve (Bellco Glass, Inc, Cat # 1985-. The pellet was resuspended in 1.100g/ml Histopaque (Sigma #10771, Sigma #11191) and centrifuged at 1200RPM for 30 minutes. The supernatant was collected, diluted 2-fold in islet isolation solution, and centrifuged at 1500RPM for 5 minutes. The pellet was washed in islet isolation solution, centrifuged, and incubated at 37 ℃ and 5% CO₂Cultured in E8 medium (Gibco # A1517001). The following day, islets were centrifuged at 1500RPM for 5 minutes and resuspended in undiluted TryplE Select 10X (life technologies # a12177) and incubated at 37 ℃ for 10 minutes. The isolated islets were diluted in E8 medium, centrifuged, and resuspended in E8 supplemented with 100ng/mL hydrocortisone (Sigma # H0135), 1U/mL thrombin (Sigma # T9326), and 100ng/mL EGF (Sigma E5036). Cells were cultured on vitronectin (Life technologies # A14700) coated dishes according to the manufacturer's instructions。

Reprogramming: cells were washed with PBS (Gibco #14190144) and incubated in TryplE select 1X for 5 min at 37 ℃. Digestion was stopped with E8 medium and cells were centrifuged at 1000RPM for 5 minutes. Cells were resuspended at 2E6 cells/200. mu.l in BTX electroporation solution (VWR #89130-542) and added to an electroporation cuvette containing 20. mu.g of reprogramming plasmid. Internally, 2 reprogramming plasmids were constructed, which included the EBNA episome expression sequence, ampicillin resistance, and the reprogramming genes Oct4, Sox2, Klf4, and L-Myc under the control of the CMV promoter. The electroporation cuvette was pulsed using a gene pulser XL (Bio-Rad). Cells were transferred to vitronectin coated dishes in E6 medium (Life Technologies # a1516401) supplemented with 100ng/mL bFGF (Life Technologies # PHG6015) and 1. mu.M hydrocortisone. Cells were incubated with 5% CO₂The culture was carried out at 37 ℃ in a humidified incubator. After 24 hours, the medium was replaced with E6 supplemented with 100ng/mL bFGF, and 1 μ M hydrocortisone and 100 μ M sodium butyrate (Sigma # P1269), and every other day. The stem cell colonies were isolated manually and transferred to vitronectin coated dishes in E8 medium. The 73 lineages generated from primary tissues of both donors were initially screened for the ability to express endoderm markers after 4 days of exposure to endoderm inducer activator-a and wortmannin. Selecting the culture with the highest proportion of cells expressing endoderm markers. The twenty-four cell lines that underwent the first screen were then screened for the ability to express pancreatic markers after exposure to a12 day pancreatic differentiation protocol. The cell line that consistently produced the highest proportion of pancreatic cells was designated SR1423, stored and used in all subsequent experiments.

Cell line characterization: SR1423 expresses markers typical of pluripotent cells (fig. 3A) and has a normal karyotype (fig. 3B). DNA STR profiling of SR1423 confirmed that it is a single cell line matched to donor tissue (fig. 3C). In addition, SR1423 grew at a rate typical of pluripotent cell lines (fig. 3D). It was observed that other induced pluripotent stem cell (hereinafter referred to as "iPSC") lines and reprogramming experiments from the same donor also showed preferential differentiation. iPSC lineage "B" also differentiated well into endoderm, while lineages "C" and "D" had no preference for differentiation of endoderm lineages (data not shown). To determine whether there was a correlation between gene expression profiles and the inability of ipscs to differentiate into specific lineages, genome-wide microarray analysis was performed on SR1423 and the expressed genes of lines B, C and D. See, e.g., Koyanagi-Aoi m.et al.proc.natl.acad.sci.110 (2013). Unsupervised hierarchical clustering analysis based on fold-change expression of at least Log2 showed that SR1423 was clustered with cell line B, but not with cell lines C and D. This identifies gene expression patterns associated with robust and preferential differentiation of endoderm lineages (fig. 4A). Of the 10 most differentially expressed genes, BHMT2, Cox7a1, HSPB2, and NAP1L1 were significantly correlated with the ability to form endoderm as measured using qRT-PCR (fig. 4B).

Stem cell culture: undifferentiated iPS cells were stored as described by the manufacturer, either coated with vitronectin XF (StemCell Technologies #07180) or 17. mu.g/cm²Geltrex (Life Technologies # A1413301) in 6-well tissue culture plates (Greiner Bio-One #657160) and fed daily with E8 medium. Cultures were subcultured every 3-5 days with 0.5mM EDTA (Life Technologies #15575) at 75-85% confluency and 7X 10³Individual cell/cm²And (4) inoculating.

Differentiation: first batch of SR1423 cells at 2.3 × 10⁴Individual cell/cm²Inoculated and allowed to grow for 18-24 hours. Then with dPBS (-Mg)²⁺/-Ca²⁺) Cells were washed and media changed according to a 28 day schedule including the following 6 media formulations: day 1, day 2, day 3: DMEM/F-12 medium (Life Technologies #10565018), 0.2% HSA, 1XB27 supplement (Life Technologies # A1486701), 100ng/mL activin A and 1 μ M wortmannin; day 4, day 5, day 6, day 7: DMEM (Life Technologies #10567014), 0.2% HSA, 1XB27 supplement, 4 μ M retinoic acid, 50ng/ml KGF, 50ng/ml Noggin, 0.25 μ M cyclopamine KAAD; day 8, day 10: DMEM, 0.2% HSA, 1XB27 supplement, KGF at 50ng/ml, Noggin at 50ng/ml, EGF at 50 ng/ml; day 12, day 14: DMEM, 0.2% HSA, 1XB27 supplement, 50ng/ml Noggin, 50ng/mlEGF, 1 μ M γ -secretase inhibitor XXI, 10 μ M Alk5i II, 1 μ M T3; day 16, day 18: DMEM, 0.2% HSA, 1XB27 supplement, 10 μ M Alk5i II, 1 μ M T3, 100nM retinoic acid; day 20, day 22, day 24, day 26, day 28: CMRL (Life Technologies #11530037), 0.2% HSA, 1X B27 supplement, 1 Xglutamax (Life Technologies #35050061), 10 μ M Alk5I II, 1 μ M T3, 10mM nicotinamide, 50ng/ml IGF-I, 10ng/ml BMP 4. For differentiation with KGF by addition of the second phase, media with DMEM, 0.2% BSA, 1XB27 supplement and 50ng/ml KGF on day 4, day 5, day 6 were inserted into the schedule, and the remaining phase was transferred after three days. The protocol of this example produced a population of highly pure endocrine pancreatic cells on day 28 of differentiation (fig. 5A). Quantification of representative images revealed a population consisting of cells, of which 68% expressed the endocrine pancreatic marker nkx6.1 (fig. 5A, quantified in 5E), 66.8% expressed the late pancreatic marker NeuroD1 (fig. 5B, quantified in 5E), and 66.5% expressed insulin (fig. 5D, quantified in 5E). This method of depleting KGF from differentiating endoderm cells increases the yield of both pancreas-derived stem cells and established human embryonic stem cells. Fig. 6.

As described above, the second batch of SR1423 cells was cultured at 6.3X 10⁴Inoculated and allowed to grow for 18-24 hours. Then with dPBS (-Mg)²⁺/-Ca²⁺) Cells were washed and media changed according to a 28 day schedule including the following 6 media formulations: day 1, day 2, day 3: DMEM/F-12 medium (Life Technologies #10565018), 0.2% HSA, 1XB27 supplement (Life Technologies # A1486701), 100ng/mL activin A (Pepro technology # AF-120-14E), and 1 μ M wortmannin (Sigma # W3144); day 4, day 5, day 6, day 7: DMEM (Life technologies #10567014), 0.2% HSA, 1XB27 supplement, 2 μ M retinoic acid (Sigma # R2625), 50ng/ml KGF (PeproTech # AF-100-19), 50ng/ml Noggin (PeproTech #120-10C), 0.25 μ M cyclopamine KAAD (Millipore # 239804); day 8, day 10: DMEM, 0.2% HSA, 1XB27 supplement, KGF at 50ng/mL, Noggin at 50ng/mL, EGF at 50ng/mL (PeproTec)h # AF-100-15), 100nM retinoic acid (Sigma # R2625), 0.25. mu.M cyclopamine KAAD (Millipore # 239804); day 12, day 14: DMEM, 0.2% HSA, 1XB27 supplement, 50ng/mL Noggin, 50ng/mL EGF, 1. mu.M gamma-secretase inhibitor XXI (Millipore #565790), 10. mu.M Alk5i II (Axxora, # ALX-270-; day 16, day 18: DMEM, 0.2% HSA, 1XB27 supplement, 10 μ M Alk5i II, 100nM retinoic acid, 0.25 μ M cyclopamine KAAD (Millipore # 239804); day 20, day 22, day 24, day 26, day 28: CMRL (Life Technologies #11530037), 0.2% HSA, 1X B27 supplement, 1 Xglutamax (Life Technologies #35050061), 10. mu.M Alk5I II, 10mM nicotinamide (Sigma # N0636), 50ng/mL IGF-I (PeproTech #100-11), 100nM retinoic acid (Sigma # R2625), 0.25. mu.M cyclopamine KAAD (Millipore #239804), and glucagon (Sigma # G2044).

Testing of glucose stimulated insulin secretion: prior to exposure to the glucose solution, cells were cultured for two hours in CMRL with 1g/dL glucose, 10 μ M Alk5I II +1 μ M T3+10mM nicotinamide +50ng/ml IGF-I +10ng/ml BMP4, 0.2% HSA, 1X B27 supplement, 1 Xglutamax (Life Technologies # 35050061). Cells were incubated in KREBS (Alfa Aesar # J67591-AP) at 2mM glucose for 30 min and the supernatant was collected. The buffer was changed to 20mM glucose in KREBS for 30 min and the supernatant was collected. The buffer was changed to 20mM glucose, 30mM KCl in KREBS for 30 min and the supernatant was collected. The concentration of C peptide in each supernatant was determined using a hypersensitivity C peptide or glucagon ELISA (Mercodia #10-1141-01) and a GENios microplate reader (TECAN). Absorbance readings were measured in duplicate using Magellan software (TECAN). Following this procedure, it was observed that insulin was secreted from SR1423 differentiated cells using C peptide as a surrogate for insulin (fig. 7) and glucagon (not shown).

Example 2 results

Cell line derivation. The iPSC line was generated by introducing reprogramming genes into the nucleus of mature cells. These genes (typically OCT4, Sox2, KLF4, and c-Myc) induce a part of the cell to exploit the gene expression patterns, morphology and behavior of embryonic stem cells. It has been reported that the epigenetic characteristics of the starting cell population persist in the reprogrammed cell, a phenomenon known as "epigenetic memory", although the duration of this effect is unknown. To maximize the potential for generating iPSC lines that differentiated efficiently into the pancreatic lineage, primary cells were selected from the langerhans islets of the pancreas of consenting healthy adult donors (fig. 1A) for reprogramming.

Over time, primary cells grown in cell culture may become homologous and lose the property of functional maturation, possibly as a result of adaptation to artificial culture conditions. To avoid loss of genetic diversity in the starting cell population, reprogramming genes were completed within five days after cell harvest. The reprogramming genes Oct4, Sox2, Klf4, and L-Myc were introduced into primary cells by electroporation of two episomal expression plasmids. L-Myc is selected relative to C-Myc to reduce the likelihood of oncogene introduction. The 73 lineages generated from primary tissues of two donors were initially screened for the ability to express endoderm markers after 4 days of exposure to endoderm inducer activator-a and wortmannin. Selecting the culture with the highest proportion of cells expressing endoderm markers. The twenty-four cell lines that underwent the first screen were then screened for the ability to express pancreatic markers after exposure to a12 day pancreatic differentiation protocol. The cell line that consistently produced the highest proportion of pancreatic cells was designated SR1423, stored and used in all subsequent experiments. This cell line produced nearly homogeneous cultures of definitive endoderm (FIG. 1B) and pancreatic progenitor cells (FIG. 1C). Notably, SR1423 showed a strong ability to differentiate towards ectoderm and endoderm (indicated by OTX2 and Sox17, respectively), but failed to express the mesoderm marker Brachyury when differentiated using a commercially available kit (fig. 2). Since all three germ layers are not reached, SR1423 does not meet the accepted criteria for pluripotency of ipscs, and instead can be considered pluripotency or non-pluripotency.

And (5) cell line characterization. SR1423 expresses markers typical of pluripotent cells (fig. 3A) and has a normal karyotype (fig. 3B). Its DNA STR profile confirmed a single cell line matched to the donor tissue (fig. 3C) and was unique among all fingerprints in NIH, ATCC and DSMZ databases. In addition, SR1423 grew at a rate typical of pluripotent cell lines (fig. 3D). It was observed that other iPSC cell lines from the same donor and reprogramming experiments also showed preferential differentiation. The iPSC lineage "B" also differentiated well into endoderm, whereas iPSC lineages "C" and "D" showed no preference for differentiation into endoderm lineages (data not shown). Whole genome microarray analysis of expressed genes was performed on SR1423 and lines B, C and D. By this comparison, differences in gene expression due to the donor or reprogramming method are eliminated. Unsupervised hierarchical clustering analysis based on fold-change expression of at least Log2 showed SR1423 to be clustered with cell line B, but not C and D. This identified gene expression patterns associated with robust and preferential differentiation into the endodermal lineage (fig. 4A). Of the 10 most differentially expressed genes, BHMT2, Cox7a1, HSPB2, and NAP1L1 were significantly correlated with the ability to form endoderm as measured using qRT-PCR (fig. 4B). These results indicate that gene expression of defined subsets of genes can be used to identify specific iPSC lines with therapeutic use.

And (4) cell differentiation. Other groups that report the generation of pancreatic cells from pluripotent stem cell populations use unique cell culture protocols in conjunction with unique stem cell populations. This means that each protocol is tailored to a specific starting cell population, which confirms the concept that the starting cell population is the primary determinant of differentiation potential.

In most methods, the production of beta cells occurs through the progressive differentiation of pluripotent cells through a known stage of embryonic pancreatic development. This progression begins with the formation of definitive endoderm, followed by a transition to pancreatic progenitor cells, endocrine pancreas, and finally pancreatic cells that express hormones. Traditionally, the production of definitive endoderm cells expressing Sox17 and HNF3 β was achieved by exposure to activin a and Wnt3a, signaling molecules involved in endoderm patterning in mammals. Pancreatic progenitor cells, identified by expression of the pancreatic duodenal homeobox 1(Pdx1), appeared after activation of the HOX gene with retinoic acid, while cyclopamine inhibited hedgehog signaling. Endocrine cells expressing both Pdx1 and nkx6.1 are formed from pancreatic progenitor cells by activating KGF signaling, which is involved in the formation of pancreatic ductal cells, in the presence of the patterned protein noggin. Maturation into the hormone expression phenotype is promoted by thyroid hormones. Great efforts have been made to replace growth factors and hormones used in differentiation protocols with small molecules.

The disclosed methods are capable of driving the differentiation of SR1423 and other stem cells to a beta cell phenotype. The disclosed protocol produced a population of highly pure endocrine pancreatic cells on day 28 of differentiation (fig. 5A). Quantification of representative images revealed a population consisting of cells in which 68% expressed the endocrine pancreatic marker nkx6.1, 66.8% expressed the late pancreatic marker NeuroD1, and 66.5% expressed insulin (fig. 5B).

The protocol does not provide for exposure of definitive endoderm cells to Wnt3A, and optionally does not expose definitive endoderm to KGF (FGF7), with or without inhibition of TGF β RI kinase inhibition on days 4-7 of culture. The effect of KGF in the early stage of differentiation was examined and showed a significant decrease in the yield of pancreas and insulin producing cells (fig. 6A). To determine whether the results were limited to SR1423 cell line only, the disclosed protocol was compared to the known protocol using a reference embryonic stem cell line BGO1V with matching results (fig. 6B, quantified in 6C). Used side-by-side, the disclosed protocol generates more insulin producing cells in these cell lines.

Insulin (fig. 7) and glucagon (not shown) were secreted into the culture medium from SR1423 differentiated cells according to the disclosed protocol. Sequential differentiation of SR1423 showed consistent, reproducible high level detection of C peptide (fig. 7), and higher levels when differentiated with our protocol. These cells may secrete insulin in a glucose responsive manner (not shown). Thus, these hormone secreting cells may be ideal candidates for cell replacement therapy. Furthermore, by adding glucagon to the culture medium, an optimal balance of mature insulin and glucagon expressing cells can be achieved, which has the benefit of reducing the amount of cells co-expressing insulin and glucagon (fig. 10A-C).

Example 3-treatment of diabetes with the disclosed therapeutic cells in animal models

Alginate encapsulation: the differentiation plane cultures of SR1423 were released manually using a cell lifter and shaken overnight at 95RPM in 6-well suspension culture dishes. The clusters formed were washed in 130mM NaCl, 10mM MOPS pH 7.4 and resuspended at a density of 2E6 cells/ml in 2% Pronova UP MVG alginate (Novamatrix). The alginate/cell mixture was loaded into a syringe and fed through a 0.24 μm nozzle at 4 ml/min and 7kV by a Nisco electrostatic droplet generator, or manually dropped into 20mM BaCl₂130mM NaCl and 10mM MOPS. The beads were washed four times and returned to differentiation medium until transplantation.

Induction of diabetes in mice: 8-10 week-old immunocompetent CD1 mice were used to induce diabetes with streptozotocin (STZ, VWR # 102515-. STZ was injected intraperitoneally (200mg/kg) into mice. STZ-induced diabetes was confirmed by measuring blood glucose levels.

Transplantation of alginate-encapsulated differentiated SR1423 cells: STZ-induced diabetic mice were anesthetized with 20mg/kg tribromoethanol (Sigma #776557888), and their abdomens were shaved and sterilized with isopropanol. A vertical incision is made centrally in the abdomen below the sternum. Alginate beads were implanted into the peritoneum and the incision was closed with sutures. After surgery, ketoprofen (2.5mg/Kg, ThermoFisher # P08D009) was administered to the mice for 3 days. Mice were observed regularly after transplantation. Blood glucose levels were monitored twice weekly by taking a small drop of blood from the tail vein using a commercial glucometer. Lower blood glucose was evident within 48 hours of transplantation and was maintained for a period of weeks (fig. 8).

Reversal of diabetes in animal models. A common method for immune protection of pancreatic islet cells for transplantation is to embed the cells in alginate-containing microbeads. Surrogate pancreatic cells embedded within alginate and implanted in the peritoneum may show short-term reversal of diabetes and provide a good basis for proof of concept. Microbeads formed from modified alginate with a lower tendency to stimulate fibrosis are able to reverse diabetes in normal rodents for up to 6 months. To demonstrate the ability of SR 1423-generated cells to reverse diabetes in an immunoprotection device, we embedded differentiated cells in alginate beads and implanted them into the peritoneum of normal mice with chemically induced diabetes. Lower blood glucose was evident within 48 hours of transplantation and was maintained for a period of weeks (fig. 8).

Example 4 culturing non-human primate cells Using the disclosed methods

The disclosed methods for isolating pluripotent stem cells and efficiently differentiating the stem cells into insulin-producing pancreatic lineages are also effective when starting with non-human tissue and when starting with non-pancreatic tissue. Fibroblasts were harvested from skin biopsies of three non-human primates (NHPs) of the rhesus species and cultured in the same manner as described for human cells (i.e., the culture and differentiation steps of example 1). The reprogramming genes Oct4, Sox2, Klf4, and L-Myc were introduced into primary fibroblasts by electroporation of two episomal expression plasmids. Stem cell lines generated from primary tissues of NHP donors express the pluripotency markers Oct4, SSEA4, Tra-1-80 and Tra-1-60. These cell lines were screened for the ability to express endoderm markers after 4 days of exposure to endoderm inducer activator-a and wortmannin. Cultures with the highest proportion of endoderm marker expressing cells were selected from each NHP donor and subsequently screened for the ability to express pancreatic markers after exposure to a12 day pancreatic differentiation protocol. Tissues from all three NHP donors generated at least one stem cell line that was effective to generate pancreatic endoderm cells upon exposure to the 12-day differentiation protocol. Figure 9 shows a representative example of one of these lines derived from NHP donors.

EXAMPLE 5 treatment of adult diabetes with the disclosed therapeutic cells

This example illustrates a method of using the disclosed protocol to generate therapeutic cells to treat type I diabetes in adults.

An adult subject with insulin-dependent diabetes mellitus receives a graft comprising a therapeutically effective amount of a composition comprising the disclosed large encapsulated insulin producing cells into the omentum pocket or peritoneal cavity of the subject. The subject is evaluated for blood glucose levels. After implantation of a therapeutically effective number of large encapsulated cells, the subject is monitored to ensure that the subject's blood glucose level has stabilized. Subjects were further screened for glycated hemoglobin, and for complications of diabetes over time.

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