Endocrine precursor cells, pancreatic hormone-expressing cells, and methods for producing them

Abstract

To provide glucose responsive-insulin producing cells derived from human embryonic stem cells in vitro, and reliable methods for producing such cells.SOLUTION: The invention provides cell populations that express markers characteristic of pancreatic endoderm cell lines and express pancreatic duodenal homeobox 1 (PDX1) or NKX6 transcription factor related locus 1 (NKX6.1).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of medicine and cell biology. In particular, the present invention relates to compositions comprising mammalian endocrine precursor cells and compositions comprising pancreatic hormone-expressing cells, as well as methods of making and using such cells.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Patent Application No. 60 / 852,878, entitled "ENRICHMENT OF ENDOCRINE PRECURSOR CELLS, IMMATURE PANCREATIC ISLET CELLS AND MATURE PANCREATIC ISLET CELLS USING NCAM," filed October 18, 2006, under 35 U.S.C. §119(e). No. 60 / 833,633, entitled "INSULIN-PRODUCING CELLS AND METHOD OF PRODUCTION," filed July 26, 2006, and claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60 / 778,649, entitled "INSULIN-PRODUCING CELLS AND METHOD OF PRODUCTION," filed March 2, 2006. The entire disclosure of each of the above-listed priority applications is incorporated herein by reference. [Background technology]

[0003] Human embryonic stem cells (hESCs) have the potential to generate differentiated cell types that encompass all human somatic tissues and organs. Of paramount importance for the cell therapy treatment of insulin-dependent diabetes is the unlimited production of pancreatic endocrine cells that function similarly to pancreatic islets in response to glucose-stimulated insulin release. Thus, there is a need for glucose-responsive insulin-producing cells derived from human embryonic stem cells in vitro, as well as a reliable method for producing such cells. DISCLOSURE OF THEINVENTION [Means for solving the problem]

[0004] Some embodiments of the present invention relate to compositions, such as cell cultures, that include human pancreatic islet hormone-expressing cells. In such embodiments, the amount of human pancreatic islet hormone-expressing cells may range from about 2% to about 80% of the human cells present in the cell culture. In some embodiments of the present invention, the pancreatic islet hormone-expressing cells may be either mature pancreatic islet hormone-expressing cells, immature pancreatic islet hormone-expressing cells, or a combination of mature and immature pancreatic islet hormone-expressing cells. In certain embodiments, the human pancreatic islet hormone-expressing cells express one or more hormones selected from the group consisting of ghrelin, insulin, somatostatin, and glucagon. In some embodiments, the pancreatic islet hormone-expressing cells express insulin in response to glucose stimulation.

[0005] Other embodiments relate to cell cultures comprising both human pancreatic islet hormone-expressing cells and human endocrine precursor cells. In such embodiments, the amount of human endocrine precursor cells can range from about 5% to about 80% of the cells present in the cell culture. In some embodiments, the cell cultures comprise primarily immature pancreatic islet hormone-expressing cells and endocrine precursor cells. In other embodiments, the cell cultures comprise both mature and immature pancreatic islet hormone-expressing cells, as well as endocrine precursor cells.

[0006] Some embodiments described herein include compositions, such as cell cultures, that contain human endocrine precursor cells but that do not contain significant amounts of human pancreatic islet hormone-expressing cells. In such embodiments, the amount of human endocrine precursor cells is about 5% of the human cells present in the cell culture. In certain embodiments, the human endocrine precursor cells express a marker selected from the group consisting of neurogenin 3 (NEUROG3 or NGN3), paired box 4 (PAX4) and NKX2 transcription factor associated locus 2 (NKX2.2).

[0007] Other embodiments relate to cell cultures comprising both human endocrine precursor cells and human PDX1-positive pancreatic endoderm cells (PDX1-positive foregut endoderm cells), which are PDX1-expressing pluripotent cells derived from the anterior portion of the intestinal tube that can differentiate into cells, tissues, or organs. In such embodiments, the human endocrine precursor cells can range from about 5% to about 95% of the cells present in the cell culture. In some embodiments, the amount of human PDX1-positive pancreatic endoderm cells can range from about 5% to about 95% of the cells present in the cell culture.

[0008] Yet further embodiments of the present invention relate to methods of producing human mature pancreatic islet hormone-expressing cells, human immature pancreatic islet hormone-expressing cells, and human endocrine precursor cells. In some embodiments, the human mature pancreatic islet hormone-expressing cells are produced from human immature pancreatic islet hormone-expressing cells. In some embodiments, the human immature pancreatic islet hormone-expressing cells are produced from human endocrine precursor cells. In some embodiments, the human endocrine precursor cells are produced from human PDX1 positive pancreatic endoderm cells.

[0009] Another embodiment of the present invention relates to a method for producing human pancreatic islet hormone-expressing cells from human embryonic stem cells (hESCs) or other human pluripotent cells. In such an embodiment, hESCs or other human pluripotent cells are first differentiated into human definitive endoderm cells. Definitive endoderm cells are pluripotent cells that can differentiate into cells of the gut tube or organs derived therefrom. Human definitive endoderm cells and their production are described in U.S. Patent Application No. 11 / 021618, filed December 23, 2004, the entire disclosure of which is incorporated by reference. The definitive endoderm cells are then differentiated into foregut endoderm. Human foregut endoderm cells are pluripotent cells derived from the anterior part of the gut tube that can differentiate into cells, tissues or organs. Foregut endoderm cells and their production are described in U.S. Provisional Patent Application No. 60 / 730917, filed October 27, 2005, the entire disclosure of which is incorporated by reference. The foregut endoderm cells then differentiate into PDX1-positive pancreatic endoderm cells (PDX1-positive foregut endoderm). Human PDX1-positive pancreatic endoderm cells are pluripotent cells derived from the anterior part of the gut tube and capable of differentiating into cells, tissues or organs. PDX1-positive pancreatic endoderm cells and their production are described in U.S. Patent Application No. 11 / 115868, filed April 26, 2005, and U.S. Provisional Patent Application No. 60 / 730917, filed October 27, 2005, the entire disclosures of which are incorporated herein by reference. As described in U.S. Provisional Patent Application No. 60 / 833,633, filed July 26, 2006, the entire disclosures of which are incorporated herein by reference, and in the methods described herein, PDX1-positive pancreatic endoderm cells differentiate into endocrine precursor cells, immature and then finally mature pancreatic islet hormone expressing cells.

[0010] Other embodiments described herein relate to methods of producing a cell population enriched in human endocrine precursor cells and methods of producing a cell population enriched in human immature pancreatic islet hormone-expressing cells. In some embodiments, the method of producing a cell population enriched in endocrine precursor cells involves providing a reagent that binds neural cell adhesion molecule (NCAM) to a cell population comprising human endocrine precursor cells, and separating the human endocrine precursor cells bound to the reagent from cells that do not bind the reagent. Similarly, in some embodiments, the method of producing a cell population enriched in human immature pancreatic islet hormone-expressing cells involves providing a reagent that binds NCAM to a cell population comprising human immature pancreatic islet hormone-expressing cells, and separating the human immature pancreatic islet hormone-expressing cells bound to the reagent from cells that do not bind the reagent. In some embodiments, In this case, further enrichment of immature pancreatic islet hormone-expressing cells can be achieved by contacting the NCAM-positive cell population with a second reagent that binds CD133, and then removing cells that bind the second reagent from the cell population.

[0011] In some embodiments of the present invention, the cell population comprising human pancreatic islet hormone-expressing cells produced by the methods described herein can be derived from human endocrine precursor cells. In certain embodiments of the methods of producing a cell population enriched for human endocrine precursor cells, the endocrine precursor cells can be derived from human PDX1-positive pancreatic endoderm cells. In yet further embodiments, the human PDX1-positive pancreatic endoderm cells are derived from human foregut endoderm cells. In yet further embodiments, the human foregut endoderm cells are derived from human definitive endoderm cells. In yet further embodiments, the human definitive endoderm cells are derived from human embryonic stem cells.

[0012] Other embodiments of the invention relate to cell populations enriched for human endocrine precursor cells. In certain embodiments, the cell population enriched for human endocrine precursor cells comprises greater than about 5% human endocrine precursor cells that express neurogenin 3 (NGN3) but do not substantially express a marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, NFM, INS, GCG, SST, and GHRL. In some embodiments, the cell population enriched for human endocrine precursor cells is obtained using the methods described herein for producing a cell population enriched for human endocrine precursor cells.

[0013] Yet other embodiments of the present invention relate to cell populations enriched for human immature pancreatic islet hormone-expressing cells. The enriched cell populations can be obtained by the methods described herein, including providing a reagent that binds NCAM to a cell population comprising immature pancreatic islet hormone-expressing cells, and separating the cells that bind the reagent from cells that do not bind the reagent. In certain embodiments, the cell population comprises at least about 25% to at least about 90% immature pancreatic hormone-expressing cells that express MAFB but do not substantially express MAFA and / or NGN3. In some embodiments, the enriched cell population comprises at least about 50% immature pancreatic islet hormone-expressing cells that express MAFB but do not substantially express MAFA and / or NGN3.

[0014] Yet another embodiment of the present invention relates to a cell population enriched for human mature pancreatic islet hormone-expressing cells derived in vitro from human pluripotent cells. The enriched cell population can be obtained by the methods described herein, for example, by providing a reagent that binds NCAM to a cell population containing pancreatic islet hormone-expressing cells produced in vitro from human pluripotent cells, and separating cells that bind the reagent from cells that do not. In certain embodiments, the cell population comprises at least about 25% to at least about 90% pancreatic hormone-expressing cells that express at least one marker selected from the group consisting of GHRL, IAPP, INS, GCG, NKX6.1, SST, and PP, but do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC, and NFM. In some embodiments, the enriched cell population comprises at least about 50% immature pancreatic islet hormone-expressing cells that express GHRL, IAPP, INS, GCG, NKX6.1, SST and PP, but do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC and NFM.

[0015] Further embodiments of the invention relate to ex vivo reagent-cell complexes comprising an NCAM-binding reagent and human endocrine precursor cells expressing NCAM, human immature pancreatic islet hormone-expressing cells expressing NCAM, or human mature pancreatic islet hormone-expressing cells expressing NCAM. In certain embodiments, the endocrine precursor cells, immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells are derived in vitro from human pluripotent cells. The reagents of the conjugate can include molecules such as anti-NCAM antibodies, anti-NCAM antibody fragments, or NCAM ligands.

[0016] Another aspect of the invention is the production of in vitro fertilization (IFR) vectors as described herein that did not differentiate in the presence of sodium butyrate or other histone deacetylase inhibitors during any stage of their development. Other embodiments encompassed herein relate to methods of producing endocrine precursor cell cultures or populations, and / or pancreatic hormone-expressing cell cultures or populations in the absence of sodium butyrate or other histone deacetylase inhibitors. In such embodiments, hESCs are differentiated into definitive endoderm cells, and cell types derived from definitive endoderm, such as endocrine precursor cells and pancreatic hormone-expressing cells, in the absence of sodium butyrate or other histone deacetylase inhibitors.

[0017] Yet another aspect of the invention relates to cell cultures and cell populations comprising non-recombinant or non-genetically engineered human endocrine precursor cells and / or human pancreatic hormone-expressing cells. In some embodiments, the non-recombinant human endocrine precursor cells and / or human pancreatic hormone-expressing cells of the cell cultures and / or cell populations are differentiated from non-recombinant hESCs. In some embodiments, the non-recombinant hESCs are differentiated into definitive endoderm cells and cell types derived from definitive endoderm, such as endocrine precursor cells and pancreatic hormone-expressing cells.

[0018] In a particular jurisdiction, there may be no universally accepted definition of the term "comprising." As described herein, the term "comprising" is intended to represent "open" language that allows for the inclusion of any additional elements. With this in mind, further embodiments of the present invention are described with reference to the following numbered paragraphs:

[0019] 1. An in vitro cell culture comprising human cells, at least about 2% of the human cells being pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of ghrelin, insulin, somatostatin and glucagon, wherein the pancreatic islet hormone-expressing cells are derived in vitro from human pluripotent cells.

[0020] 2. The in vitro cell culture of paragraph 1, wherein at least about 5% of the human cells are pancreatic islet hormone-expressing cells.

[0021] 3. The method of paragraph 1, wherein at least about 10% of the human cells are pancreatic islet hormone-expressing cells. vitro cell culture.

[0022] 4. The in vitro cell culture of any of paragraphs 1-3, wherein at least about 10% of the human cells are human endocrine precursor cells that express neurogenin 3 (NEUROG3).

[0023] 5. The in vitro cell culture of paragraph 4, wherein the human endocrine precursor cells express a marker selected from the group consisting of paired box 4 (PAX4) and NKX2 transcription factor associated locus 2 (NKX2.2).

[0024] 6. The in vitro cell culture of any of paragraphs 1-3, wherein at least about 50% of the human cells are human endocrine precursor cells that express neurogenin 3 (NEUROG3).

[0025] 7. The in vitro cell culture of paragraph 6, wherein the human endocrine precursor cells express a marker selected from the group consisting of paired box 4 (PAX4) and NKX2 transcription factor associated locus 2 (NKX2.2).

[0026] 8. The in vitro cell culture of paragraph 1, wherein said pancreatic islet hormone-expressing cells express at least two hormones selected from the group consisting of ghrelin, insulin, somatostatin and glucagon.

[0027] 9. The in vitro cell culture of paragraph 1, wherein the pancreatic islet hormone-expressing cells express ghrelin, insulin, somatostatin, and glucagon.

[0028] 10. The in vitro cell culture of paragraph 1, wherein at least about 5% of said pancreatic islet hormone-expressing cells express insulin, but do not significantly express ghrelin, somatostatin, or glucagon.

[0029] 11. The in vitro cell culture of paragraph 1, wherein at least about 10% of said pancreatic islet hormone-expressing cells express insulin, but do not significantly express ghrelin, somatostatin, or glucagon.

[0030] 12. The in vitro cell culture of paragraph 1, wherein at least about 20% of said pancreatic islet hormone-expressing cells express insulin, but do not significantly express ghrelin, somatostatin, or glucagon.

[0031] 13. The in vitro cell culture of paragraph 1, wherein at least about 30% of the pancreatic islet hormone-expressing cells express insulin, but do not significantly express ghrelin, somatostatin, or glucagon.

[0032] 14. The in vitro cell culture of any one of paragraphs 10-13, wherein insulin is secreted in response to glucose stimulation.

[0033] 15. The in vitro cell culture of any one of paragraphs 10-13, wherein C-peptide is secreted in response to glucose stimulation.

[0034] 16. The in vitro cell culture of paragraph 1, wherein at least 10% of the islet cells are present in islet cell clusters.

[0035] 17. The in vitro cell culture of paragraph 1, wherein the pancreatic islet hormone-expressing cells further express a marker selected from the group consisting of pancreatic duodenal homeobox 1 (PDX1), islet amyloid polypeptide (IAPP), pancreatic polypeptide (PP), ISL1 transcription factor (ISL1), NKX6 transcription factor-related locus 1 (NKX6.1) and paired box 6 (PAX6).

[0036] 18. The in vitro cell culture of paragraph 17, wherein said pancreatic islet hormone-expressing cells do not substantially express a marker selected from the group consisting of neurogenin 3 (NEUROG3) and paired box gene 4 (PAX4).

[0037] 19. The in vitro cell culture of paragraph 1, wherein at least about 1 pancreatic islet hormone-expressing cell is present for about every 10 endocrine precursor cells in said cell culture.

[0038] 20. The in vitro cell culture of paragraph 1, wherein at least about 1 pancreatic islet hormone-expressing cell is present for about every 5 endocrine precursor cells in said cell culture.

[0039] 21. The in vitro cell culture of paragraph 1, wherein at least about 1 pancreatic islet hormone-expressing cell is present for about every 2 endocrine precursor cells in said cell culture.

[0040] 22. The in vitro cell culture of paragraph 1, wherein the pancreatic islet hormone-expressing cells are non-recombinant cells.

[0041] 23. The in vitro cell culture of paragraph 1, further comprising a medium comprising a factor selected from the group consisting of nicotinamide (NIC), exendin 4 (Ex4), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and combinations thereof.

[0042] 24. The in vitro cell culture of paragraph 1, further comprising a medium comprising a factor selected from the group consisting of exendin 4 (Ex4), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1), and combinations thereof.

[0043] 25. The in vitro cell culture of paragraph 1, further comprising a medium containing nicotinamide (NIC) at a concentration of about 10 mM.

[0044] 26. The in vitro cell culture of paragraph 1, further comprising medium containing exendin 4 (Ex4) at a concentration of about 40 ng / ml.

[0045] 27. The in vitro cell culture of paragraph 1, further comprising a medium containing hepatocyte growth factor (HGF) at a concentration of about 25 ng / ml.

[0046] 28. The in vitro cell culture of paragraph 1, further comprising a medium containing insulin-like growth factor 1 (IGF1) at a concentration of about 50 ng / ml.

[0047] 29. An in vitro cell culture comprising human cells, wherein at least about 5% of the human cells are endocrine precursor cells that express neurogenin 3 (NEUROG3), and the endocrine precursor cells are pluripotent cells that can differentiate into pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of insulin, somatostatin, and glucagon.

[0048] 30. The in vitro cell culture of paragraph 29, wherein at least about 10% of the human cells are endocrine precursor cells.

[0049] 31. The in vitro cell culture of paragraph 29, wherein at least about 25% of the human cells are endocrine precursor cells.

[0050] 32. The in vitro cell culture of paragraph 29, wherein at least about 50% of the human cells are endocrine precursor cells.

[0051] 33. The in vitro cell culture of any of paragraphs 29-32, wherein at least about 10% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0052] 34. The in vitro cell culture of any of paragraphs 29-32, wherein at least about 25% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0053] 35. The in vitro cell culture of any of paragraphs 29-32, wherein at least about 50% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0054] 36. The in vitro cell culture of any of paragraphs 29 to 32, wherein the in vitro cell culture is substantially devoid of human pancreatic islet hormone-expressing cells.

[0055] 37. The in vitro cell culture of paragraph 36, wherein at least about 10% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0056] 38. The in vitro cell culture of paragraph 36, wherein at least about 25% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0057] 39. The in vitro cell culture of paragraph 36, wherein at least about 50% of the human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

[0058] 40. The in vitro cell culture of paragraph 29, wherein said endocrine precursor cells express a marker selected from the group consisting of paired box 4 (PAX4) and NKX2 transcription factor associated locus 2 (NKX2.2).

[0059] 41. The in vitro cell culture of paragraph 29, wherein at least about 1 endocrine precursor cell is present for about every 10 PDX1-positive pancreatic endoderm cells in said cell culture.

[0060] 42. The in vitro cell culture of paragraph 29, wherein at least about 1 endocrine precursor cell is present for about every 5 PDX1-positive pancreatic endoderm cells in said cell culture.

[0061] 43. The in vitro cell culture of paragraph 29, wherein at least about 1 endocrine precursor cell is present for about every 2 PDX1-positive pancreatic endoderm cells in said cell culture.

[0062] 44. The in vitro cell culture of paragraph 29, wherein said endocrine precursor cells are non-recombinant cells.

[0063] 45. The in vitro cell culture of paragraph 29, further comprising a medium comprising N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0064] 46. ​​The in vitro cell culture of paragraph 45, wherein the concentration of DAPT is at least about 1 μM.

[0065] 47. The in vitro cell culture of paragraph 45, wherein the concentration of DAPT is about 3 μM.

[0066] 48. The in vitro cell culture of paragraph 45, further comprising a factor selected from retinoic acid (RA) and exendin 4 (Ex4).

[0067] 49. The in vitro cell culture of paragraph 45, wherein the medium is CMRL.

[0068] 50. A method for producing human pancreatic islet hormone-expressing cells, comprising the steps of obtaining a cell population comprising human endocrine precursor cells, which are pluripotent cells capable of differentiating into human pancreatic islet hormone-expressing cells, and incubating the human endocrine precursor cells in a culture medium for a period of time sufficient to form human pancreatic islet hormone-expressing cells, as determined by detecting the presence of human pancreatic hormone-expressing cells in the cell population.

[0069] 51. The method of paragraph 50, wherein at least about 2% of the human cells in said cell population differentiate into human pancreatic islet hormone-expressing cells.

[0070] 52. The method of paragraph 50, wherein at least about 5% of the human cells in said cell population differentiate into human pancreatic islet hormone-expressing cells.

[0071] 53. The method of paragraph 50, wherein at least about 10% of the human cells in said cell population differentiate into human pancreatic islet hormone-expressing cells.

[0072] 54. The method of paragraph 50, further comprising supplying to the human pancreatic endocrine cells a factor selected from the group consisting of nicotinamide (NIC), exendin 4 (Ex4), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), and combinations thereof, in an amount sufficient to further promote differentiation of the human endocrine precursor cells into human pancreatic islet hormone-expressing cells, wherein the human pancreatic islet hormone-expressing cells express at least one pancreatic hormone selected from the group consisting of insulin, somatostatin, and glucagon.

[0073] 55. The method of paragraph 54, wherein the factor is selected from the group consisting of Ex4, HGF and IGF1.

[0074] 56. The method of paragraph 54, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration in the range of about 10 ng / ml to about 100 ng / ml.

[0075] 57. The method of paragraph 54, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration of about 40 ng / ml.

[0076] 58. The method of paragraph 54, wherein the factor is IGF1.

[0077] 59. The method of paragraph 58, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration ranging from about 10 ng / ml to about 1000 ng / ml.

[0078] 60. The method of paragraph 58, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration in the range of about 10 ng / ml to about 100 ng / ml.

[0079] 61. The method of paragraph 58, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration ranging from about 25 ng / ml to about 75 ng / ml.

[0080] 62. The method of paragraph 58, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration of about 50 ng / ml.

[0081] 63. The method of paragraph 50, wherein detecting the presence of human pancreatic islet hormone-expressing cells in the cell population comprises detecting expression of at least one marker selected from the group consisting of pancreatic duodenal homeobox 1 (PDX1), ghrelin (GHRL), islet amyloid polypeptide (IAPP), pancreatic polypeptide (PP), ISL1 transcription factor (ISL1), NKX6 transcription factor-related locus 1 (NKX6.1), and paired box 6 (PAX6) in cells of the cell population.

[0082] 64. The method of paragraph 63, wherein expression of at least one of said markers is determined by Q-PCR.

[0083] 65. The method according to claim 1, wherein the expression of at least one of the markers is determined by immunocytochemistry. How to fall 63.

[0084] 66. The step of obtaining a cell population containing human endocrine precursor cells comprises: Obtaining a population of human PDX1-positive pancreatic endoderm cells, which are pluripotent cells derived from the anterior part of the intestine and can differentiate into cells, tissues or organs; The method of paragraph 50, comprising the step of providing a gamma secretase inhibitor to said population of human PDX1-positive pancreatic endoderm cells, thereby producing a population of human endocrine precursor cells.

[0085] 67. The method of paragraph 66, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0086] 68. The method of paragraph 67, wherein DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 1 μM to about 10 μM.

[0087] 69. The method of paragraph 67, wherein DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 3 μM.

[0088] 70. The method of paragraph 66, further comprising providing exendin 4 (Ex4) to said population of human PDX1-positive pancreatic endoderm cells.

[0089] 71. The method of paragraph 70, wherein Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0090] 72. The method of paragraph 70, wherein Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 40 ng / ml.

[0091] 73. The step of obtaining a population of human PDX1-positive pancreatic endoderm cells comprises: Obtaining a population of human foregut endoderm cells that are PDX1-negative pluripotent cells derived from the anterior part of the gut and capable of differentiating into cells, tissues or organs; The method of paragraph 70, comprising the step of providing a retinoid to said population of human foregut endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells.

[0092] 74. The method of paragraph 73, wherein the retinoid is retinoic acid (RA).

[0093] 75. The method of paragraph 74, wherein RA is provided to said population of human foregut endoderm cells at a concentration ranging from about 1 nM to about 10 μM.

[0094] 76. The process for obtaining a population of human foregut endoderm cells comprises: Obtaining a population of human definitive endoderm cells, which are pluripotent cells capable of differentiating into cells of the intestinal tract or into organs derived therefrom; The method of paragraph 73, comprising the step of providing fibroblast growth factor 10 (FGF-10) and a Hedgehog pathway inhibitor to said population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells.

[0095] 77. The method of paragraph 76, further comprising recovering any growth factor of the TGF-β superfamily that may be present in said population of definitive endoderm cells.

[0096] 78. The method of paragraph 77, wherein said growth factor of the TGF-β superfamily is selected from the group consisting of Nodal, Activin A, Activin B, and combinations thereof.

[0097] 79. The method of paragraph 77, wherein the growth factor of the TGF-β superfamily is activin A.

[0098] 80. The method of paragraph 76, wherein the hedgehog inhibitor comprises KAAD-cyclopamine.

[0099] 81. The method of paragraph 80, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration ranging from about 0.01 μM to about 1 μM.

[0100] 82. The method of paragraph 80, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration of about 0.2 μM.

[0101] 83. The method of paragraph 76, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 1 ng / ml to about 1000 ng / ml.

[0102] 84. The method of paragraph 76, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0103] 85. The method of paragraph 76, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration of about 50 ng / ml.

[0104] 86. The method of paragraph 76, wherein obtaining a population of human definitive endoderm cells comprises obtaining a population of pluripotent human embryonic stem cells and providing said population of pluripotent human embryonic stem cells with at least one growth factor of the TGF-β superfamily.

[0105] 87. The method of paragraph 86, wherein the at least one growth factor is Nodal.

[0106] 88. The method of paragraph 86, wherein the at least one growth factor is activin A.

[0107] 89. The method of paragraph 86, wherein the at least one growth factor is activin B.

[0108] 90. The method of paragraph 86, further comprising providing said population of pluripotent human embryonic stem cells with wingless MMTV integration site family member 3A (Wnt3A).

[0109] 91. The method of paragraph 86, wherein a plurality of growth factors of the TGFβ superfamily are provided.

[0110] 92. The method of paragraph 91, wherein Wnt3A is also provided.

[0111] 93. The method of paragraph 86, wherein the at least one growth factor is provided at a concentration of at least about 10 ng / ml.

[0112] 94. The method of paragraph 86, wherein the at least one growth factor is provided at a concentration of at least about 100 ng / ml.

[0113] 95. The method of paragraph 86, wherein the at least one growth factor is provided at a concentration of at least about 500 ng / ml.

[0114] 96. The method of paragraph 86, wherein the at least one growth factor is provided at a concentration of at least about 1000 ng / ml.

[0115] 97. The method of paragraph 86, wherein the at least one growth factor is provided at a concentration of at least about 5000 ng / ml.

[0116] 98. The method of paragraph 86, wherein said pluripotent human embryonic stem cells are differentiated into human definitive endoderm cells in a medium containing less than about 2% serum.

[0117] 99. The method of paragraph 86, wherein said pluripotent human embryonic stem cells are derived from a tissue selected from the group consisting of the morula, the ICM of an embryo, and the gonadal ridges of an embryo.

[0118] 100. A human pancreatic islet hormone-expressing cell produced by the method of paragraph 86.

[0119] 101. A method for producing human pancreatic islet hormone-expressing cells, comprising: (a) obtaining a population of pluripotent human embryonic stem cells; (b) providing at least one growth factor of the TGF-β superfamily to said population of pluripotent human embryonic stem cells, thereby producing a population of human definitive endoderm cells; (c) providing at least one fibroblast growth factor to said population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells; (d) providing a retinoid to said population of human foregut endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells; (e) providing a γ-secretase inhibitor to said population of human PDX1-positive pancreatic endoderm cells, thereby producing a population comprising human endocrine precursor cells; (f) incubating said population of human endocrine precursor cells in culture medium for a period of time sufficient to form human pancreatic islet hormone-expressing cells; A method for producing human pancreatic islet hormone-expressing cells.

[0120] 102. The method of paragraph 101, wherein step (b) further comprises providing a hedgehog pathway inhibitor.

[0121] 103. The method of paragraph 101, wherein the fibroblast growth factor is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0122] 104. The method of paragraph 101, wherein the fibroblast growth factor comprises FGF10.

[0123] 105. The method of paragraph 101, wherein step (d) further comprises providing insulin or an insulin-like growth factor.

[0124] 106. The method of paragraph 101, further comprising substantially recovering said at least one growth factor of the TGF-β superfamily.

[0125] 107. The method of paragraph 101, wherein said retinoid and said gamma secretase inhibitor are provided at about the same time.

[0126] 108. The method of paragraph 101, wherein the foregut endoderm cells are capable of further differentiating into pancreatic cells.

[0127] 109. A method for producing human pancreatic islet hormone-expressing cells, comprising: (a) obtaining a population of pluripotent human embryonic stem cells; (b) providing at least one growth factor of the TGF-β superfamily to said population of pluripotent human embryonic stem cells, thereby producing a population of human definitive endoderm cells; (c) providing a retinoid to said population of human definitive endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells; (d) a method of producing human pancreatic islet hormone-expressing cells, comprising the step of incubating said population of human PDX1-positive pancreatic endoderm cells in the presence of a retinoid for a period of time sufficient for human pancreatic islet hormone-expressing cells to be formed.

[0128] 110. The method of paragraph 109, further comprising the step of providing said population of human definitive endoderm cells with a fibroblast family growth factor.

[0129] 111. The method of paragraph 110, wherein the fibroblast family growth factor comprises FGF10 or FGF7.

[0130] 112. The method of paragraph 109, further comprising the step of providing said population of human definitive endoderm cells with a Hedgehog pathway inhibitor.

[0131] 113. The method of paragraph 112, wherein the hedgehog pathway inhibitor is KAAD-cyclopamine.

[0132] 114. The method of paragraph 109, wherein the retinoid is retinoic acid.

[0133] 115. The method of paragraph 109, further comprising the step of providing a γ-secretase inhibitor to said population of human PDX1-positive pancreatic endoderm cells.

[0134] 116. The method of paragraph 115, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0135] 117. A method for producing a cell population enriched in human endocrine precursor cells, comprising the steps of providing a reagent that binds to neural cell adhesion molecule (NCAM) to a cell population containing human endocrine precursor cells, and separating human endocrine precursor cells that bind to the reagent from cells that do not bind to the reagent, thereby producing a cell population enriched in human endocrine precursor cells.

[0136] 118. The method of paragraph 117, wherein the human endocrine precursor cells are derived in vitro from human pluripotent cells.

[0137] 119. The method of paragraph 117, wherein the human endocrine precursor cells express neurogenin 3 (NGN3) and do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, NFM, insulin (INS), glucagon (GCG), somatostatin (SST) and ghrelin (GHRL).

[0138] 120. The method of paragraph 119, wherein the human endocrine precursor cells express paired box gene 4 (PAX4).

[0139] 121. The method of paragraph 119, wherein the human endocrine precursor cells do not substantially express paired box 6 transcription factor (PAX6).

[0140] 122. The method of paragraph 117, wherein the reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment, and an NCAM ligand.

[0141] 123. The method of paragraph 122, wherein the NCAM ligand is NCAM binding protein 10 (NBP10).

[0142] 124. The method of paragraph 122, wherein the anti-NCAM antibody is labeled.

[0143] 125. The method of paragraph 124, wherein the anti-NCAM antibody is fluorescently labeled.

[0144] 126. The method of paragraph 117, further comprising providing to the cell population and the reagent a secondary reagent that binds to the reagent.

[0145] 127. The method of paragraph 126, wherein the reagent comprises an anti-NCAM antibody and the secondary reagent is fluorescently labeled.

[0146] 128. The method of paragraphs 125 or 127, wherein the separating step comprises separating the endocrine precursor cells that bind the anti-NCAM antibody from those cells that do not bind the anti-NCAM antibody using fluorescence-activated cell sorting (FACS).

[0147] 129. The method of paragraph 117, further comprising the step of disaggregating the cells in said cell population comprising human endocrine precursor cells prior to providing said cell population with said agent that binds NCAM.

[0148] 130. The method of paragraph 117, further comprising the steps of obtaining a cell population comprising a population of human PDX1-positive pancreatic endoderm cells, which are pluripotent cells derived from the anterior part of the intestinal tract and capable of differentiating into cells, tissues or organs, and providing a γ-secretase inhibitor to the population of human PDX1-positive pancreatic endoderm cells, thereby producing a population of human endocrine precursor cells.

[0149] 131. The method of paragraph 130, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0150] 132. The method of paragraph 131, wherein the DAPT is provided to the population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 1 μM to about 10 μM.

[0151] 133. The method of paragraph 131, wherein said DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 3 μM.

[0152] 134. The method of paragraph 130, further comprising providing exendin 4 (Ex4) to said population of human PDX1-positive pancreatic endoderm cells.

[0153] 135. The method of paragraph 134, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0154] 136. The method of paragraph 134, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 40 ng / ml.

[0155] 137. The method of obtaining a population of human PDX1-positive pancreatic endoderm cells, comprising the steps of obtaining a population of human foregut endoderm cells (the human foregut endoderm cells are derived from the anterior part of the intestinal tract, a cell, tissue or are PDX1-negative pluripotent cells capable of differentiating into an organ; and providing a retinoid to said population of human foregut endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells.

[0156] 138. The method of paragraph 137, wherein the retinoid is retinoic acid (RA).

[0157] 139. The method of paragraph 138, wherein RA is provided to said population of human foregut endoderm cells at a concentration ranging from about 1 nM to about 10 μM.

[0158] 140. The method of paragraph 137, wherein obtaining a population of human foregut endoderm cells comprises obtaining a population of human definitive endoderm cells, the human definitive endoderm cells being pluripotent cells capable of differentiating into cells of the intestinal tube or organs derived therefrom, and providing fibroblast growth factor 10 (FGF-10) and a Hedgehog pathway inhibitor to the population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells.

[0159] 141. The method of paragraph 140, further comprising withdrawing an exogenously added factor of the TGF-β superfamily that may be present in said population of definitive endoderm cells.

[0160] 142. The method of paragraph 141, wherein said growth factor of the TGF-β superfamily is selected from the group consisting of Nodal, Activin A, Activin B, and combinations thereof.

[0161] 143. The method of paragraph 142, wherein the growth factor of the TGF-β superfamily is activin A.

[0162] 144. The method of paragraph 140, wherein the hedgehog inhibitor comprises KAAD-cyclopamine.

[0163] 145. The method of paragraph 144, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration ranging from about 0.01 μM to about 1 μM.

[0164] 146. The method of paragraph 145, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration of about 0.2 μM.

[0165] 147. The method of paragraph 140, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 10 ng / ml to about 1000 ng / ml.

[0166] 148. The method of paragraph 140, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 1 ng / ml to about 100 ng / ml.

[0167] 149. The method of paragraph 140, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration of about 50 ng / ml.

[0168] 150. The method of paragraph 140, wherein obtaining a population of human definitive endoderm cells comprises obtaining a population of human pluripotent cells and providing said population of human pluripotent cells with at least one growth factor of the TGF-β superfamily.

[0169] 151. The method of paragraph 150, wherein the at least one growth factor is Nodal.

[0170] 152. The method of paragraph 150, wherein the at least one growth factor is activin A.

[0171] 153. The method of paragraph 150, wherein the at least one growth factor is activin B.

[0172] 154. The method of paragraph 150, further comprising providing wingless MMTV integration site family member 3A (Wnt3A) to said population of human pluripotent cells.

[0173] 155. The method of paragraph 150, wherein a plurality of growth factors of the TGFβ superfamily are provided.

[0174] 156. The method of paragraph 155, wherein Wnt3A is also provided.

[0175] 157. The method of paragraph 150, wherein the at least one growth factor is provided at a concentration of at least about 10 ng / ml.

[0176] 158. The method of paragraph 150, wherein the at least one growth factor is provided at a concentration of at least about 100 ng / ml.

[0177] 159. The method of paragraph 150, wherein the at least one growth factor is provided at a concentration of at least about 500 ng / ml.

[0178] 160. The method of paragraph 150, wherein the at least one growth factor is provided at a concentration of at least about 1000 ng / ml.

[0179] 161. The method of paragraph 150, wherein the at least one growth factor is provided at a concentration of at least about 5000 ng / ml.

[0180] 162. The method of paragraph 150, wherein said human pluripotent cells are differentiated into human definitive endoderm cells in a medium containing less than about 2% serum.

[0181] 163. The method of paragraph 150, wherein said human pluripotent cells are human embryonic stem cells derived from a tissue selected from the group consisting of the morula, the ICM of an embryo, and the gonadal ridges of an embryo.

[0182] 164. An enriched in vitro human endocrine precursor cell population, wherein the human endocrine precursor cells express NGN3 and do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, NFM, INS, GCG, SST and GHRL.

[0183] 165. An enriched in vitro human endocrine precursor cell population of paragraph 164, derived in vitro from human pluripotent cells.

[0184] 166. An enriched in vitro human endocrine precursor cell population of paragraph 164 produced by the method of paragraph 117.

[0185] 167. An enriched in vitro human endocrine precursor cell population of paragraph 164 produced by the method of paragraph 150.

[0186] 168. The enriched in vitro human endocrine precursor cell population of paragraph 164, wherein at least about 5% of the enriched human cell population comprises human endocrine precursor cells that express neurogenin 3 (NGN3) and do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, NFM, INS, GCG, SST, and GHRL.

[0187] 169. The method of paragraph 168, wherein the human endocrine precursor cells express PAX4. In vitro human endocrine precursor cell populations.

[0188] 170. The enriched in vitro human endocrine precursor cell population of paragraph 164, wherein said endocrine precursor cells are derived in vitro from human PDX1-positive pancreatic endoderm.

[0189] 171. The enriched in vitro human endocrine precursor cell population of paragraph 170, wherein said human PDX1-positive pancreatic endoderm cells are derived in vitro from human foregut endoderm cells.

[0190] 172. The enriched in vitro human endocrine precursor cell population of paragraph 171, wherein said human foregut endoderm cells are derived in vitro from definitive endoderm cells.

[0191] 173. The enriched in vitro human endocrine precursor cell population of paragraph 172, wherein said definitive endoderm cells are derived in vitro from human embryonic stem cells (hESCs).

[0192] 174. A method for producing a cell population enriched in human immature pancreatic islet hormone-expressing cells, comprising the steps of: providing a reagent that binds to neural cell adhesion molecule (NCAM) to a cell population containing human immature pancreatic islet hormone-expressing cells; and separating human immature pancreatic islet hormone-expressing cells that bind to the reagent from cells that do not bind to the reagent, thereby producing a cell population enriched in human immature pancreatic islet hormone-expressing cells.

[0193] 175. The method of paragraph 174, wherein said human immature pancreatic islet hormone-expressing cells are derived in vitro from human pluripotent cells.

[0194] 176. The method of paragraph 174, wherein said human immature pancreatic islet hormone-expressing cells express MAFB and do not substantially express a marker selected from the group consisting of NGN3 and MAFA.

[0195] 177. The method of paragraph 176, wherein said human immature pancreatic islet hormone-expressing cells do not substantially express at least one marker selected from the group consisting of MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and NFM.

[0196] 178. The method of paragraph 176, wherein said human immature pancreatic islet hormone-expressing cells express at least one marker selected from the group consisting of synaptophysin (SYP), chromogranin A (CHGA), NKX2.2, Islet1 (ISL1), paired box gene 6 (PAX6), and neurodevelopment differentiation 1 (NEUROD), PDX1, and HB9.

[0197] 179. The method of paragraph 174, wherein said human immature pancreatic islet hormone-expressing cells process less than about 98% of the insulin produced by said immature pancreatic islet hormone-expressing cells.

[0198] 180. The method of paragraph 174, wherein said human immature pancreatic islet hormone-expressing cells process less than about 70% of the insulin produced by said immature pancreatic islet hormone-expressing cells.

[0199] 181. The method of paragraph 179 or 180, wherein said insulin processing is measured by C-peptide release.

[0200] 182. The method of paragraph 174, wherein the reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment, and an NCAM ligand.

[0201] 183. The method of paragraph 182, wherein the NCAM ligand is NCAM binding protein 10 (NBP10).

[0202] 184. The method of paragraph 182, wherein the anti-NCAM antibody is labeled.

[0203] 185. The method of paragraph 184, wherein the anti-NCAM antibody is fluorescently labeled.

[0204] 186. The method of paragraph 174, further comprising providing to said cell population a secondary reagent that binds to said reagent.

[0205] 187. The method of paragraph 186, wherein the reagent comprises an anti-NCAM antibody, and the secondary reagent that binds to the anti-NCAM antibody is fluorescently labeled.

[0206] 188. The method of paragraphs 185 or 187, wherein the separating step comprises separating the human immature pancreatic islet hormone-expressing cells that bind to the anti-NCAM antibody from those cells that do not bind to the anti-NCAM antibody using fluorescence-activated cell sorting (FACS).

[0207] 189. The method of paragraph 174, further comprising the steps of providing said human immature pancreatic islet hormone-expressing cells with a second reagent that binds CD133, and separating said human immature pancreatic islet hormone-expressing cells from cells that bind said second reagent.

[0208] 190. The method of paragraph 174, further comprising the step of dissociating the cell population prior to providing said cell population with said agent that binds NCAM.

[0209] 191. The method of paragraph 174, further comprising obtaining a cell population comprising human endocrine precursor cells, the human endocrine precursor cells being pluripotent cells capable of differentiating into human immature pancreatic islet hormone-expressing cells, and incubating the human endocrine precursor cells in culture medium for a period of time sufficient for human immature pancreatic islet hormone-expressing cells to be formed.

[0210] 192. The method of paragraph 191, further comprising providing said human endocrine precursor cells with a factor selected from the group consisting of nicotinamide (NIC), exendin 4 (Ex4), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), glucose-dependent insulinotropic polypeptide (GIP), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and combinations thereof, in an amount sufficient to further promote differentiation of said human endocrine precursor cells into human immature pancreatic islet hormone-expressing cells.

[0211] 193. The method of paragraph 192, wherein the factor is selected from the group consisting of Ex4, HGF, and IGF1.

[0212] 194. The method of paragraph 193, wherein the factor is Ex4.

[0213] 195. The method of paragraph 194, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration in the range of about 10 ng / ml to about 100 ng / ml.

[0214] 196. The method of paragraph 194, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration of about 40 ng / ml.

[0215] 197. The method of paragraph 193, wherein the factor is IGF1.

[0216] 198. IGF1 is secreted at concentrations ranging from about 10 ng / ml to about 1000 ng / ml. The method of paragraph 197, wherein said cell population is provided with progenitor cells.

[0217] 199. The method of paragraph 197, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration in the range of about 10 ng / ml to about 100 ng / ml.

[0218] 200. The method of paragraph 197, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration ranging from about 25 ng / ml to about 75 ng / ml.

[0219] 201. The method of paragraph 197, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration of about 50 ng / ml.

[0220] 202. The method of paragraph 191, further comprising the steps of obtaining a cell population comprising a population of human PDX1-positive pancreatic endoderm cells, which are pluripotent cells derived from the anterior part of the intestinal tract and capable of differentiating into a cell, tissue or organ, and providing a gamma-secretase inhibitor to the population of human PDX1-positive pancreatic endoderm cells, thereby producing a population of human endocrine precursor cells.

[0221] 203. The method of paragraph 202, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0222] 204. The method of paragraph 203, wherein the DAPT is provided to the population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 1 μM to about 10 μM.

[0223] 205. The method of paragraph 203, wherein said DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 3 μM.

[0224] 206. The method of paragraph 202, further comprising providing exendin 4 (Ex4) to said population of human PDX1-positive pancreatic endoderm cells.

[0225] 207. The method of paragraph 206, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0226] 208. The method of paragraph 206, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 40 ng / ml.

[0227] 209. The method of paragraph 202, wherein the step of obtaining a population of human PDX1-positive pancreatic endoderm cells comprises the steps of obtaining a population of human foregut endoderm cells that are PDX1-negative pluripotent cells derived from the anterior part of the intestinal tube and capable of differentiating into cells, tissues or organs, and providing a retinoid to the population of human foregut endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells.

[0228] 210. The method of paragraph 209, wherein the retinoid is retinoic acid (RA).

[0229] 211. The method of paragraph 210, wherein RA is provided to said population of human foregut endoderm cells at a concentration ranging from about 1 nM to about 10 μM.

[0230] 212. The step of obtaining a population of human foregut endoderm cells includes the steps of obtaining a population of human definitive endoderm cells, which are pluripotent cells capable of differentiating into cells of the intestinal tract or organs derived therefrom, and providing fibroblast growth factor 10 (FGF-10) and a Hedgehog pathway inhibitor to the population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells. , Method of paragraph 209.

[0231] 213. The method of paragraph 212, further comprising withdrawing exogenously added factors of the TGF-β superfamily that may be present in said population of definitive endoderm cells.

[0232] 214. The method of paragraph 213, wherein said growth factor of the TGF-β superfamily is selected from the group consisting of Nodal, Activin A, Activin B, and combinations thereof.

[0233] 215. The method of paragraph 214, wherein the growth factor of the TGF-β superfamily is activin A.

[0234] 216. The method of paragraph 212, wherein the hedgehog inhibitor comprises KAAD-cyclopamine.

[0235] 217. The method of paragraph 216, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration ranging from about 0.01 μM to about 1 μM.

[0236] 218. The method of paragraph 216, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration of about 0.2 μM.

[0237] 219. The method of paragraph 212, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 1 ng / ml to about 1000 ng / ml.

[0238] 220. The method of paragraph 212, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0239] 221. The method of paragraph 212, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration of about 50 ng / ml.

[0240] 222. The method of paragraph 212, wherein obtaining a population of human definitive endoderm cells comprises obtaining a population of human pluripotent cells and providing said population of human pluripotent cells with at least one growth factor of the TGF-β superfamily.

[0241] 223. The method of paragraph 222, wherein the at least one growth factor is Nodal.

[0242] 224. The method of paragraph 222, wherein the at least one growth factor is activin A.

[0243] 225. The method of paragraph 222, wherein the at least one growth factor is activin B.

[0244] 226. The method of paragraph 222, further comprising providing wingless MMTV integration site family member 3A (Wnt3A) to said population of human pluripotent cells.

[0245] 227. The method of paragraph 222, wherein a plurality of growth factors of the TGFβ superfamily are provided.

[0246] 228. The method of paragraph 227, wherein Wnt3A is also provided.

[0247] 229. The method of paragraph 222, wherein the at least one growth factor is provided at a concentration of at least about 10 ng / ml.

[0248] 230. The method of paragraph 222, wherein the at least one growth factor is provided at a concentration of at least about 100 ng / ml.

[0249] 231. The method of paragraph 222, wherein the at least one growth factor is provided at a concentration of at least about 500 ng / ml.

[0250] 232. The method of paragraph 222, wherein the at least one growth factor is provided at a concentration of at least about 1000 ng / ml.

[0251] 233. The method of paragraph 222, wherein the at least one growth factor is provided at a concentration of at least about 5000 ng / ml.

[0252] 234. The method of paragraph 222, wherein the human pluripotent cells are differentiated into human definitive endoderm cells in a medium containing less than about 2% serum.

[0253] 235. The method of paragraph 222, wherein said human pluripotent cells are human embryonic stem cells derived from a tissue selected from the group consisting of the morula, the ICM of an embryo, and the gonadal ridges of an embryo.

[0254] 236. An enriched in vitro human immature pancreatic islet hormone-expressing cell population, wherein the human immature pancreatic islet hormone-expressing cells express MAFB and do not substantially express NGN3 and MAFA.

[0255] 237. An enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, derived in vitro from human pluripotent cells.

[0256] 238. An enriched in vitro human immature pancreatic islet hormone-expressing cell population of Paragraph 236, produced by the method of Paragraph 174.

[0257] 239. An enriched in vitro human immature pancreatic islet hormone-expressing cell population of Paragraph 236, produced by the method of Paragraph 222.

[0258] 240. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 238 or 239, wherein at least about 25% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0259] 241. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 238 or 239, wherein at least about 50% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0260] 242. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 238 or 239, wherein at least about 70% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0261] 243. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 238 or 239, wherein at least about 90% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0262] 244. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein at least about 25% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0263] 245. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein at least about 50% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0264] 246. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein at least about 70% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0265] 247. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein at least about 90% of the enriched human cell population comprises human immature pancreatic islet hormone-expressing cells that express MAFB and do not substantially express NGN3 and MAFA.

[0266] 248. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein said human immature pancreatic islet hormone-expressing cells do not substantially express at least one marker selected from the group consisting of MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and NFM.

[0267] 249. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein said human immature pancreatic islet hormone-expressing cells express at least one marker selected from the group consisting of synaptophysin (SYP), chromogranin A (CHGA), NKX2.2, Islet1 (ISL1), paired box gene 6 (PAX6), and neurodevelopment and differentiation 1 (NEUROD), PDX1 and HB9.

[0268] 250. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein said human immature pancreatic islet hormone-expressing cells process less than about 98% of the insulin produced by said immature pancreatic islet hormone-expressing cells.

[0269] 251. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 236, wherein said human immature pancreatic islet hormone-expressing cells process less than about 70% of the insulin produced by said immature pancreatic islet hormone-expressing cells.

[0270] 252. The enriched in vitro human immature pancreatic islet hormone-expressing cell population of paragraph 250 or 251, wherein said insulin processing is measured by C-peptide release.

[0271] 253. A method for producing a cell population enriched in human pancreatic islet hormone-expressing cells, the human pancreatic islet hormone-expressing cells being derived in vitro from human pluripotent cells, the method comprising the steps of providing a reagent that binds neural cell adhesion molecule (NCAM) to a cell population comprising human pancreatic islet hormone-expressing cells, and separating human endocrine precursor cells that bind the reagent from cells that do not bind the reagent, thereby producing a cell population enriched in human pancreatic islet hormone-expressing cells.

[0272] 254. The method of paragraph 253, wherein the reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment, and an NCAM ligand.

[0273] 255. The method of paragraph 254, wherein the NCAM ligand is NCAM binding protein 10 (NBP10).

[0274] 256. The method of paragraph 254, wherein the anti-NCAM antibody is labeled.

[0275] 257. The method of paragraph 256, wherein the anti-NCAM antibody is fluorescently labeled.

[0276] 258. The method of paragraph 254, further comprising providing to said cell population a secondary reagent that binds to said reagent.

[0277] 259. The method of paragraph 258, wherein the reagent comprises an anti-NCAM antibody, and the secondary reagent that binds to the anti-NCAM antibody is fluorescently labeled.

[0278] 260. The method of paragraph 257 or 259, wherein said separating step comprises separating said pancreatic islet hormone-expressing cells that bind said anti-NCAM antibody from said cells that do not bind said anti-NCAM antibody using fluorescence-activated cell sorting (FACS).

[0279] 261. The method of paragraph 253, further comprising the steps of providing said human immature pancreatic islet hormone-expressing cells with a second reagent that binds CD133, and separating said human immature pancreatic islet hormone-expressing cells from cells that bind said second reagent.

[0280] 262. The method of paragraph 253, further comprising the step of dissociating the cell population prior to providing said cell population with said agent that binds NCAM.

[0281] 263. The method of paragraph 253, further comprising obtaining a cell population comprising human endocrine precursor cells, the human endocrine precursor cells being pluripotent cells capable of differentiating into human pancreatic islet hormone-expressing cells, and incubating the human endocrine precursor cells in culture medium for a period of time sufficient for human pancreatic islet hormone-expressing cells to be formed.

[0282] 264. The method of paragraph 263, further comprising providing said human endocrine precursor cells with a factor selected from the group consisting of nicotinamide (NIC), exendin 4 (Ex4), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), glucose-dependent insulinotropic polypeptide (GIP), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and combinations thereof, in an amount sufficient to further promote differentiation of said human endocrine precursor cells into human pancreatic islet hormone-expressing cells, wherein said human pancreatic islet hormone-expressing cells express at least one pancreatic hormone selected from the group consisting of insulin, somatostatin, and glucagon.

[0283] 265. The method of paragraph 264, wherein the factor is selected from the group consisting of Ex4, HGF, and IGF1.

[0284] 266. The method of paragraph 265, wherein the factor is Ex4.

[0285] 267. The method of paragraph 266, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration in the range of about 10 ng / ml to about 100 ng / ml.

[0286] 268. The method of paragraph 266, wherein Ex4 is provided to said cell population of endocrine precursor cells at a concentration of about 40 ng / ml.

[0287] 269. The method of paragraph 265, wherein the factor is IGF1.

[0288] 270. The method of paragraph 269, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration ranging from about 10 ng / ml to about 1000 ng / ml.

[0289] 271. IGF1 is a pro-endocrine hormone at concentrations ranging from about 10 ng / ml to about 100 ng / ml. The method of Paragraph 269, wherein said cell population of progenitor cells is provided.

[0290] 272. The method of paragraph 269, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration ranging from about 25 ng / ml to about 75 ng / ml.

[0291] 273. The method of paragraph 269, wherein IGF1 is provided to said cell population of endocrine precursor cells at a concentration of about 50 ng / ml.

[0292] 274. The method of paragraph 263, further comprising the steps of obtaining a cell population comprising a population of human PDX1-positive pancreatic endoderm cells, which are pluripotent cells derived from the anterior part of the intestinal tract and capable of differentiating into a cell, tissue or organ, and providing a gamma-secretase inhibitor to the population of human PDX1-positive pancreatic endoderm cells, thereby producing a population of human endocrine precursor cells.

[0293] 275. The method of paragraph 274, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0294] 276. The method of paragraph 275, wherein said DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 1 μM to about 10 μM.

[0295] 277. The method of paragraph 275, wherein said DAPT is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 3 μM.

[0296] 278. The method of paragraph 274, further comprising providing exendin 4 (Ex4) to said population of human PDX1-positive pancreatic endoderm cells.

[0297] 279. The method of paragraph 278, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0298] 280. The method of paragraph 278, wherein said Ex4 is provided to said population of human PDX1-positive pancreatic endoderm cells at a concentration of about 40 ng / ml.

[0299] 281. The method of paragraph 274, wherein obtaining a population of human PDX1-positive pancreatic endoderm cells comprises obtaining a population of human foregut endoderm cells that are PDX1-negative pluripotent cells derived from the anterior part of the intestinal tube and capable of differentiating into cells, tissues or organs, and providing a retinoid to the population of human foregut endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells.

[0300] 282. The method of paragraph 281, wherein the retinoid is retinoic acid (RA).

[0301] 283. The method of paragraph 282, wherein RA is provided to said population of human foregut endoderm cells at a concentration ranging from about 1 nM to about 10 μM.

[0302] 284. The method of paragraph 281, wherein obtaining a population of human foregut endoderm cells comprises obtaining a population of human definitive endoderm cells, the cells being pluripotent cells capable of differentiating into cells of the intestinal tube or into organs derived therefrom, and providing fibroblast growth factor 10 (FGF-10) and a Hedgehog pathway inhibitor to the population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells.

[0303] 285. The TGF-β superfamily member that may be present in the population of definitive endoderm cells. The method of paragraph 284, further comprising recovering the exogenously added factor.

[0304] 286. The method of paragraph 285, wherein said growth factor of the TGF-β superfamily is selected from the group consisting of Nodal, Activin A, Activin B, and combinations thereof.

[0305] 287. The method of paragraph 286, wherein the growth factor of the TGF-β superfamily is activin A.

[0306] 288. The method of paragraph 284, wherein the hedgehog inhibitor comprises KAAD-cyclopamine.

[0307] 289. The method of paragraph 288, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration ranging from about 0.01 μM to about 1 μM.

[0308] 290. The method of paragraph 288, wherein KAAD-cyclopamine is provided to said population of human definitive endoderm cells at a concentration of about 0.2 μM.

[0309] 291. The method of paragraph 284, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 1 ng / ml to about 1000 ng / ml.

[0310] 292. The method of paragraph 284, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration ranging from about 10 ng / ml to about 100 ng / ml.

[0311] 293. The method of paragraph 284, wherein FGF-10 is provided to said population of human definitive endoderm cells at a concentration of about 50 ng / ml.

[0312] 294. The method of paragraph 284, wherein obtaining a population of human definitive endoderm cells comprises obtaining a population of human pluripotent cells and providing said population of human pluripotent cells with at least one growth factor of the TGF-β superfamily.

[0313] 295. The method of paragraph 294, wherein the at least one growth factor is Nodal.

[0314] 296. The method of paragraph 294, wherein the at least one growth factor is activin A.

[0315] 297. The method of paragraph 294, wherein the at least one growth factor is activin B.

[0316] 298. The method of paragraph 294, further comprising providing said population of human pluripotent cells with wingless MMTV integration site family member 3A (Wnt3A).

[0317] 299. The method of paragraph 294, wherein a plurality of growth factors of the TGFβ superfamily are provided.

[0318] 300. The method of paragraph 299, wherein Wnt3A is also provided.

[0319] 301. The method of paragraph 294, wherein the at least one growth factor is provided at a concentration of at least about 10 ng / ml.

[0320] 302. The method of paragraph 294, wherein the at least one growth factor is provided at a concentration of at least about 100 ng / ml.

[0321] 303. The method of paragraph 294, wherein the at least one growth factor is provided at a concentration of at least about 500 ng / ml.

[0322] 304. The method of paragraph 294, wherein the at least one growth factor is provided at a concentration of at least about 1000 ng / ml.

[0323] 305. The method of paragraph 294, wherein the at least one growth factor is provided at a concentration of at least about 5000 ng / ml.

[0324] 306. The method of paragraph 294, wherein said human pluripotent cells are differentiated into human definitive endoderm cells in a medium containing less than about 2% serum.

[0325] 307. The method of paragraph 294, wherein said human pluripotent cells are human embryonic stem cells derived from a tissue selected from the group consisting of the morula, the ICM of an embryo, and the gonadal ridges of an embryo.

[0326] 308.Enriched in vitro human pancreatic islet hormone-expressing cell populations derived in vitro from human pluripotent cells.

[0327] 309. An enriched in vitro human pancreatic islet hormone-expressing cell population of Paragraph 308, produced by the method of Paragraph 253.

[0328] 310. An enriched in vitro human pancreatic islet hormone-expressing cell population of Paragraph 308 produced by the method of Paragraph 294.

[0329] 311. The enriched in vitro human islet hormone-expressing cell population of paragraphs 309 or 310, wherein at least about 25% of the enriched human cell population comprises human islet hormone-expressing cells that express at least one marker selected from the group consisting of ghrelin, islet amyloid polypeptide (IAPP), insulin (INS), glucagon (GCG), NKX6 transcription factor-related locus 1 (NKX6.1), somatostatin (SOM), and pancreatic polypeptide (PP), and do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, and NFM.

[0330] 312. The enriched in vitro human islet hormone-expressing cell population of paragraph 311, wherein at least about 50% of said enriched human cell population comprises human islet hormone-expressing cells that express at least one marker selected from the group consisting of ghrelin, islet amyloid polypeptide (IAPP), insulin (INS), glucagon (GCG), NKX6 transcription factor associated locus 1 (NKX6.1), somatostatin (SOM), and pancreatic polypeptide (PP), and that do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, and NFM.

[0331] 313. The enriched in vitro human islet hormone-expressing cell population of paragraph 311, wherein at least about 90% of said enriched human cell population comprises human islet hormone-expressing cells that express at least one marker selected from the group consisting of ghrelin, islet amyloid polypeptide (IAPP), insulin (INS), glucagon (GCG), NKX6 transcription factor-related locus 1 (NKX6.1), somatostatin (SOM), and pancreatic polypeptide (PP), and do not substantially express at least one marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1, and NFM.

[0332] 314. An ex vivo reagent-cell complex comprising human endocrine precursor cells expressing NCAM, the endocrine precursor cells being capable of differentiating into human pancreatic islet hormone-expressing cells. An ex vivo reagent-cell complex comprising a pluripotent cell and a human endocrine precursor cell expressing NCAM, the reagent binding to said NCAM.

[0333] 315. The ex vivo reagent-cell complex of Paragraph 314, wherein the reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment, and an NCAM ligand.

[0334] 316. The ex vivo reagent-cell complex of paragraph 315, wherein the NCAM ligand is NCAM-binding protein 10 (NBP10).

[0335] 317. The ex vivo reagent-cell complex of Paragraph 315, wherein the reagent is an anti-NCAM antibody.

[0336] 318. The ex vivo reagent-cell complex of paragraph 317, wherein the anti-NCAM antibody is labeled.

[0337] 319. The ex vivo reagent-cell complex of paragraph 318, wherein the anti-NCAM antibody is fluorescently labeled.

[0338] 320. The ex vivo reagent-cell complex of paragraph 314, further comprising a secondary reagent that binds to the reagent.

[0339] 321. The ex vivo reagent-cell complex of Paragraph 320, wherein the reagent comprises an anti-NCAM antibody, and the secondary reagent that binds to the anti-NCAM antibody is fluorescently labeled.

[0340] 322. An ex vivo reagent-cell complex comprising human immature pancreatic islet hormone-expressing cells expressing NCAM, wherein the human immature pancreatic islet hormone-expressing cells are pluripotent cells capable of differentiating into human pancreatic islet hormone-expressing cells, and a reagent that binds to the NCAM.

[0341] 323. The ex vivo reagent-cell complex of Paragraph 322, wherein the reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment, and an NCAM ligand.

[0342] 324. The ex vivo reagent-cell complex of paragraph 323, wherein the NCAM ligand is NCAM-binding protein 10 (NBP10).

[0343] 325. The ex vivo reagent-cell complex of Paragraph 323, wherein the reagent is an anti-NCAM antibody.

[0344] 326. The ex vivo reagent-cell complex of Paragraph 325, wherein the anti-NCAM antibody is labeled.

[0345] 327. The ex vivo reagent-cell complex of Paragraph 326, wherein the anti-NCAM antibody is fluorescently labeled.

[0346] 328. The ex vivo reagent-cell complex of paragraph 322, further comprising a secondary reagent that binds to the reagent.

[0347] 329. The ex vivo reagent-cell complex of Paragraph 328, wherein the reagent comprises an anti-NCAM antibody, and the secondary reagent that binds to the anti-NCAM antibody is fluorescently labeled.

[0348] 330. The method of any of paragraphs 86, 150, 222 or 294, wherein the human pluripotent cells are human embryonic stem cells derived from a preimplantation embryo.

[0349] 331. A method for producing human pancreatic hormone-expressing cells, comprising: 1. A method for producing human pancreatic hormone-expressing cells, comprising: (a) providing at least one growth factor of the TGF-β superfamily to a population of pluripotent human embryonic stem cells (hESCs), thereby producing a population of human definitive endoderm cells; (b) providing at least one fibroblast growth factor to the population of human definitive endoderm cells, thereby producing a population of human foregut endoderm cells; (c) providing Noggin to the population of human foregut endoderm cells, thereby producing a population comprising human endocrine precursor cells; and (d) incubating the population of human endocrine precursor cells in culture medium for a time sufficient to form human pancreatic islet hormone-expressing cells, wherein the time sufficient to form human pancreatic islet hormone-expressing cells is determined by detecting the presence of human pancreatic hormone-expressing cells in the cell population.

[0350] 332. The method of paragraph 331, wherein at least about 2% of said human cells in said cell population differentiate into human pancreatic hormone-expressing cells.

[0351] 333. The method of paragraph 331, wherein at least about 5% of said human cells in said cell population differentiate into human pancreatic hormone-expressing cells.

[0352] 334. The method of paragraph 331, wherein at least about 10% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0353] 335. The method of paragraph 331, wherein at least about 20% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0354] 336. The method of paragraph 331, wherein at least about 40% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0355] 337. The method of paragraph 331, wherein at least about 50% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0356] 338. The method of paragraph 331, wherein at least about 70% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0357] 339. The method of paragraph 331, wherein at least about 90% of said human cells in said cell population are differentiated into human pancreatic hormone-expressing cells.

[0358] 340. The method of paragraph 331, wherein detecting the presence of human pancreatic islet hormone-expressing cells in the cell population comprises detecting expression of at least one marker selected from the group consisting of pancreatic duodenal homeobox 1 (PDX1), ghrelin (GHRL), insulin (INS), islet amyloid polypeptide (IAPP), pancreatic polypeptide (PP), ISL1 transcription factor (ISL1), NKX6 transcription factor-related locus 1 (NKX6.1), and paired box 6 (PAX6) in cells of the cell population.

[0359] 341. The method of paragraph 340, wherein expression of at least one of said markers is determined by Q-PCR.

[0360] 342. The expression of at least one of the markers is determined by immunocytochemistry; Method of paragraph 340.

[0361] 343. The method of paragraph 331, further comprising providing to said cell population a gamma-secretase inhibitor.

[0362] 344. The method of paragraph 343, wherein the gamma-secretase inhibitor comprises N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0363] 345. The method of paragraph 343, wherein said gamma secretase inhibitor is provided to said cell population at about the same time as providing Noggin or after providing Noggin.

[0364] 346. The method of paragraph 344, wherein the gamma-secretase inhibitor is provided to the cell population at a concentration ranging from about 0.1 μM to about 10 μM.

[0365] 347. The method of paragraph 331, wherein the at least one fibroblast growth factor is fibroblast growth factor 7 (FGF-7).

[0366] 348. The method of paragraph 347, wherein the FGF-7 is provided to the cell culture at a concentration ranging from about 1 ng / ml to about 1000 ng / ml.

[0367] 349. The method of paragraph 331, further comprising providing a hedgehog inhibitor to said cell population at about the same time as adding said at least one fibroblast growth factor.

[0368] 350. The method of paragraph 349, wherein the hedgehog inhibitor comprises KAAD-cyclopamine.

[0369] 351. The method of paragraph 350, wherein the KAAD-cyclopamine is provided to the cell population at a concentration ranging from about 0.01 μM to about 10 μM.

[0370] 352. The method of paragraph 331, further comprising recovering any growth factor of the TGF-β superfamily that may be present in said population of definitive endoderm cells.

[0371] 353. The method of paragraph 331, wherein said growth factor of the TGF-β superfamily is selected from the group consisting of Nodal, Activin A, Activin B, and combinations thereof.

[0372] 354. The method of paragraph 353, wherein the growth factor of the TGF-β superfamily comprises activin A.

[0373] 355. The method of paragraph 354, wherein the activin A is provided to the hESCs at a concentration ranging from about 10 ng / ml to about 1000 ng / ml.

[0374] 356. The method of paragraph 331, further comprising providing wingless MMTV integration site family member 3A (Wnt3A) to said hESCs.

[0375] 357. The method of paragraph 356, wherein the Wnt3A is provided at a concentration ranging from about 1 ng / ml to about 1000 ng / ml.

[0376] 358. The method of paragraph 331, wherein the hESCs are differentiated into human definitive endoderm cells in a medium containing less than about 2% serum.

[0377] 359. The method of paragraph 331, wherein said hESCs are derived from a tissue selected from the group consisting of the morula, an embryonic ICM, and an embryonic gonadal ridge.

[0378] 360. The method of paragraph 331, further comprising providing a retinoid to said cell population at about the same time as providing Noggin.

[0379] 361. The method of paragraph 331, further comprising providing a retinoid to said cell population about simultaneously with or after adding at least one fibroblast growth factor.

[0380] 362. The method of paragraph 360 or 361, wherein the retinoid is retinol.

[0381] 363. The method of paragraph 360 or 361, wherein the retinoid is retinoic acid.

[0382] 364. The method of paragraph 363, wherein the retinoic acid is provided at a concentration ranging from about 0.01 μM to about 10 μM.

[0383] 365. The cell culture or cell population of any of paragraphs 1-49, 164-173, 236-252, or 308-313, wherein at least some of the cells are non-recombinant cells.

[0384] 366. The cell culture or cell population of any of paragraphs 1-49, 164-173, 236-252 or 308-313, wherein the cells are non-recombinant cells.

[0385] 367. The cell culture or cell population of any of paragraphs 1-49, 164-173, 236-252 or 308-313, wherein the cells are not cultured in the presence of a histone deacetylase inhibitor.

[0386] 368. The cell culture or cell population of paragraph 367, wherein the histone deacetylase inhibitor comprises sodium butyrate.

[0387] 369. The method of any of paragraphs 50-163, 174-235, 253-307, or 331-364, wherein at least some of the cells are non-recombinant cells.

[0388] 370. The method of any of paragraphs 50-163, 174-235, 253-307, or 331-364, wherein the cell is a non-recombinant cell.

[0389] 371. The method of any of paragraphs 50-163, 174-235, 253-307, or 331-364, wherein the cells are not cultured in the presence of a histone deacetylase inhibitor.

[0390] 372. The method of paragraph 371, wherein the histone deacetylase inhibitor comprises sodium butyrate.

[0391] It is understood that the above methods and compositions relate to cells cultured in vitro, however, the in vitro differentiated cell compositions may be used for in vivo applications, such as cell replacement therapy.

[0392] Further embodiments of the present invention are described in U.S. Provisional Patent Application No. 60 / 532,004, entitled "DEFINITIVE ENDODERM," filed December 23, 2003; U.S. Provisional Patent Application No. No. 60 / 566,293 (titled "PDX1 EXPRESSING ENDODERM") No. 60 / 586,566 (entitled "CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM," filed July 9, 2004), U.S. Provisional Patent Application No. 60 / 587,942 (entitled "CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM," filed July 14, 2004), U.S. Patent Application No. 11 / 021,618 (entitled "DEFINITIVE ENDODERM," filed December 23, 2004), U.S. Patent Application No. No. 1 / 115,868 (entitled "PDX1 EXPRESSING ENDODERM," filed April 26, 2005), U.S. patent application Ser. No. 11 / 165,305 (entitled "METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM," filed June 23, 2005), U.S. Provisional Patent Application No. No. 60 / 730,917 (entitled "PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM," filed October 27, 2005), U.S. Provisional Patent Application No. 60 / 736,598 (entitled "MARKERS OF DEFINITIVE ENDODERM," filed November 14, 2005), U.S. Provisional Patent Application No. 60 / 778,649 (entitled "INSULIN-PRODUCING CELLS AND METHOD OF PRODUCTION," filed March 2, 2006), U.S. Provisional Patent Application No. 60 / 833,633 (entitled "INSULIN-PRODUCING CELLS AND METHOD OF PRODUCTION," filed March 2, 2006), No. 60 / 852,878, entitled "ENRICHMENT OF ENDOCRINE PRECURSOR CELLS, IMMATURE PANCREATIC ISLET CELLS AND MATURE PANCREATIC ISLET CELLS USING NCAM," filed July 26, 2006, and U.S. Provisional Patent Application No. 60 / 852,878, entitled "ENRICHMENT OF ENDOCRINE PRECURSOR CELLS, IMMATURE PANCREATIC ISLET CELLS AND MATURE PANCREATIC ISLET CELLS USING NCAM," filed October 18, 2006. Application No. 2005 / 0133991, the disclosures of which are incorporated herein by reference in their entireties. [Brief description of the drawings]

[0393] [Figure 1]Schematic diagram showing steps in in vitro differentiation of hESCs to pancreatic islet cells that correspond to intermediate stages observed during pancreatic development in vivo. Sequential treatment with various growth factor / media combinations that are used to induce this stepwise differentiation of hESCs through each intermediate is shown. Conditions and cell characteristics are shown in the boxes. Below the boxes are hours (h) or days (d) in hours, providing an exemplary timeline showing a typical differentiation of human embryonic stem cells (hESCs) to pancreatic islet hormone-expressing cells. Below each intermediate is a list of genes whose expression is specific to this intermediate, but not necessarily exclusive. Monitoring the expression of one or more genes for each intermediate along this development strongly indicates the occurrence of each transition in vitro. Abbreviations are as follows: ESC - embryonic stem cell, ME - mesoderm, Ant.DE - pre-definitive endoderm. [Figure 2-1] 1 is a bar graph showing the relative expression of various markers during the 21 day differentiation protocol. The markers that resulted in substantial differences in differentiation as a result of the three conditions used in step 1 of differentiation are: (A) SOX17, (B) CXCR4, (C) SOX7, (D) ISL1, (E) SOX1, and (F) PAX6. [Figure 2-2] 1 is a bar graph showing the relative expression of various markers during the 21 day differentiation protocol. Panels (G) PDX1, (H) NGN3, (I) NKX2.2 and (J) NKX6.1 show the relative expression of markers associated with differentiation of hESCs into pancreatic endoderm and endocrine progenitor cells. Panels (K) insulin, (L) glucagon, (M) ghrelin and (N) SOM show the relative expression of the islet hormones insulin, glucagon, ghrelin and somatostatin towards the end of the differentiation process. [Diagram 3] Bar graphs showing the relative expression of (A) FOXA1, (B) HNF1b, (C) HNF6, (D) PDX1, (E) NGN3, (F) PAX4, (G) NKX2.2, (H) NKX6.1, (I) ghrelin, (J) glucagon, (K) insulin and (L) IAPP on days 0-16 of the differentiation protocol. [Figure 4]Figure 4A shows a Western blot analysis of PDX1 protein expression in cells subjected to various media conditions. Abbreviations: MS1-PDX1 - protein lysate from MS1 cells transfected with PDX1 (positive control), A100 - 100 ng / ml activin A, A25R2 - 25 ng / ml activin A and 2 μM RA, RP - RPMI medium, CM - CMRL medium, Ex - 40 ng / ml exendin 4. Figure 4B is a bar graph showing the relative expression of PDX1 mRNA in the 7th, 8th and 9th day cultures described in Figure 4A. [Diagram 5] 1 is a bar graph showing the relative expression of (A) HB9, (B) PDX1, (C) NGN3, and (D) NKX2.2 after 13 days of differentiation in the presence or absence of retinoic acid, and (E) PDX1, (F) NKX2.2, (G) insulin, and (H) glucagon after 17 days of differentiation. [Figure 6] 14 is a bar graph showing the relative expression of (A) NGN3, (B) NKX2.2, (C) insulin, (D) glucagon, (E) ghrelin, and (F) somatostatin (SOM) after 19 days of differentiation exposed to three different concentrations of the γ-secretase inhibitor DAPT or no DAPT at all. [Figure 7] (A) Micrographs of insulin, (B) glucagon, and (C) somatostatin (SOM) immunoreactive cells. A merge of these three images is shown in (D), with triple-labeled cells indicated by arrows. [Figure 8] (A) Micrographs showing immunoreactivity for insulin and (B) PAX6. These micrographs show that insulin-positive cells are also PAX6 positive. (C) Micrographs showing immunoreactivity for insulin and (D) ISL1 show that insulin-positive cells are also ISL1 positive. There are also many ISL1 cells that are negative for insulin immunoreactivity (compare C with D). [Figure 9]1 is a bar graph showing that insulin mRNA detection (Panel A) correlates with the ability to measure C-peptide released into the culture medium (Panel B). Abbreviations are as follows: A100 - 100 ng / ml activin A, 2NF - 2% fetal bovine serum (FBS) and no factors, Fstnog - 50 ng / ml holstatin and 100 ng / ml noggin, "B" - cultures receiving A100 for days 1-5, "C" - cultures receiving 2% FBS and no factors for days 1-5, and "D" - cultures receiving 50 ng / ml holstatin and 100 ng / ml noggin for days 1-5. [Figure 10] FIG. 1 is a bar graph showing conditions that showed strong insulin mRNA detection (Panel A) and glucose stimulated C-peptide secretion (Panel B). Abbreviations are as follows: g50 - 1.6 mM glucose, g400 - 16 mM glucose. [Figure 11] 1 is a bar graph showing that hESC lines BG01 and BG02 can differentiate into pancreatic islet hormone-expressing cells. Panels A and B show upregulation of PDX1 mRNA for BG01 (A) and BG02 (B), panels C and D show upregulation of NGN3 mRNA for BG01 (C) and BG02 (D), and panels E and F show upregulation of insulin mRNA for BG01 (E) and BG02 (F). [Figure 12] 12A-12D are photomicrographs showing immunoreactivity of hESC-derived cells treated to differentiate into early pancreatic islet hormone-expressing cells for NCAM (FIG. 12C) and NKX2.2 (FIG. 12B). The total cell population is stained with DAPI (FIG. 12A). These photomicrographs show that NKX2.2-positive cells are also NCAM-positive (FIG. 12D). [Figure 13] 13A-13D are photomicrographs showing immunoreactivity for NCAM (FIG. 13C) and insulin (FIG. 13B) of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. The total cell population is stained with DAPI (FIG. 13A). These photomicrographs show that insulin-positive cells are also NCAM-positive (FIG. 13D). [Figure 14]14A-14C are photomicrographs showing immunoreactivity of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells for NCAM (FIG. 14E), INS (FIG. 14F), and PAX6 (FIG. 14D). The total cell population is stained with DAPI (FIG. 14A). These photomicrographs show that PAX6-positive cells are also NCAM-positive (FIG. 14B), and INS-positive cells are also NCAM-positive (FIG. 14C). [Figure 15] 15A and 15B are photomicrographs showing the immunoreactivity of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells for NKX2.2 and synaptophysin. These photomicrographs show that synaptophysin positive cells are also NKX2.2 positive (FIGS. 15A and 15B). [Figure 16] 16A and 16B are photomicrographs showing the immunoreactivity of hESC-derived cells treated to differentiate into endocrine precursor cells for MAFB and INS. FIG. 16A and FIG. 16B show that MAFB and INS are co-expressed in hESC-derived endocrine precursor cells. FIG. 16C and FIG. 16D show the immunoreactivity of 13.5 week old human fetal pancreas for MAFB and INS. MAFB and INS are co-expressed in fetal pancreas. [Figure 17] 17A-B are flow cytometry dot plots showing the co-isolation of immature pancreatic islet hormone-expressing cells expressing synaptophysin and NCAM (FIG. 17A) and the co-isolation of immature pancreatic islet hormone-expressing cells expressing INS and NCAM (FIG. 17B). [Figure 18]18A is a flow cytometry dot plot of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. FIG. 18A is a flow cytometry dot plot showing labeling of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells with anti-NCAM antibody. FIG. 18B is a flow cytometry dot plot showing the distribution of hESC-derived cells positive or negative for NCAM and SYP. FIG. 18C is a flow cytometry dot plot showing the distribution of hESC-derived NCAM-positive cells of FIG. 18A reanalyzed by FACS for both NCAM and SYP. The dot plot shows the distribution of these cells positive or negative for both NCAM and SYP. FIG. 18D is a flow cytometry dot plot showing the distribution of hESC-derived NCAM-negative cells of FIG. 18A reanalyzed by FACS for both NCAM and SYP. The dot plot shows the distribution of these cells positive or negative for both NCAM and SYP. [Figure 19] 19A and 19B show the distribution of cells positive and negative for both NCAM and SYP. FIG. 19C and FIG. 19D show the distribution of cells positive and negative for both NCAM and INS. [Figure 20] 20A and 20B are flow cytometry dot plots of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. Figure 20A shows a small subset of cell lines that showed fluorescence for NCAM. Figure 20B shows a small subset of cell lines that showed fluorescence for SYP. Figure 20C shows that when the NCAM-fluorescent cells of Figure 20A are harvested and analyzed for SYP, a very high percentage of hESC-derived cells are SYP positive. [Figure 21]21A-C are flow cytometry dot plots of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. Figure 21A shows the distribution of hESC-derived cells stained for NCAM. Figure 21B shows that a subpopulation of hESC cells treated to differentiate into immature pancreatic islet hormone-expressing cells are NCAM positive and CD133 negative. Figure 21C shows the distribution of NCAM positive / CD133 negative cells that are SYP positive and SYP negative. [Figure 22-1] 22A-22C are bar graphs showing mRNA levels of certain markers, as detected by QPCR, in hESC-derived cells treated to differentiate into endocrine precursor cells ("early") or treated to differentiate into immature pancreatic islet hormone-expressing cells ("mid-stage" and "late-stage"). Data labeled "before sorting" represent mRNA levels of the marker in cells that have not been processed and sorted by the FACS instrument. Data labeled "NCAM fluorescence" represent mRNA levels of the marker in cells that are NCAM positive. Data labeled "NCAM non-fluorescent (dim)" represent mRNA levels of the marker in cells that are NCAM negative. Specifically, the mRNA levels of NGN3 (FIG. 22A), PAX4 (FIG. 22B), INS (FIG. 22C), pancreatic polypeptide (FIG. 22D), PAX6 (FIG. 22E), and GCG (FIG. 22F) are shown. [Figure 22-2] 22A-22C are bar graphs showing the mRNA levels of certain markers, as detected by QPCR, in hESC-derived cells treated to differentiate into endocrine precursor cells ("early") or treated to differentiate into immature pancreatic islet hormone-expressing cells ("mid-stage" and "late-stage"). Data labeled "before sorting" represent the mRNA levels of the marker in cells that have not been processed and sorted by the FACS instrument. Data labeled "NCAM fluorescence" represent the mRNA levels of the marker in cells that are NCAM positive. Data labeled "NCAM non-fluorescent (dim)" represent the mRNA levels of the marker in cells that are NCAM negative. Specifically, the mRNA levels of GHRL (FIG. 22G), GCK (FIG. 22H), SST (FIG. 22I), NXK2.2 (FIG. 22J), and SYP (FIG. 22K) are shown. [Diagram 23] 23 is a bar graph showing the mRNA levels of certain markers as detected by QPCR in hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells (day 19). Data labeled "Before sorting" represents the mRNA levels of the marker in cells that have not been treated and sorted by the FACS machine. Data labeled "NCAM fluorescence" represents the mRNA levels of the marker in cells that are NCAM positive. Data labeled "NCAM non-fluorescence" represents the mRNA levels of the marker in cells that are NCAM negative. Specifically, the mRNA levels of NEUROD (FIG. 23A), ISL1 (FIG. 23B), GAS (FIG. 23C), KIR6.2 (FIG. 23D), and SUR1 (FIG. 23E) are shown. [Figure 24-1] 24A-24C are bar graphs showing mRNA levels of certain markers as detected by QPCR in hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells (day 19). Data labeled "Before sorting" represent the mRNA levels of the marker in cells that have not been treated and sorted by the FACS machine (gated live cells). Data labeled "NCAM fluorescence" represent the mRNA levels of the marker in cells that are NCAM positive. Specifically, the mRNA levels of NCAM1 (FIG. 24A), NKX2.2 (FIG. 24B), SYP (FIG. 24C), PAX6 (FIG. 24D), NEUROD (FIG. 24E), and ISL1 (FIG. 24F) are shown. [Figure 24-2] 24A-24C are bar graphs showing the mRNA levels of certain markers as detected by QPCR in hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells (day 19). Data labeled "Before sorting" represent the mRNA levels of the marker in cells that have not been treated and sorted by the FACS machine (gated live cells). Data labeled "NCAM fluorescence" represent the mRNA levels of the marker in cells that are NCAM positive. Specifically, the mRNA levels of INS (FIG. 24G), GCG (FIG. 24H), GHRL (FIG. 24I), SST (FIG. 24J), and PP (FIG. 24K) are shown. [Diagram 25]25A, 25C, and 25E are flow cytometry dot plots of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. Figures 25A, 25C, and 25E show the percentage of NCAM-positive cells that are SYP, CHGA, and INS positive, respectively, in the cell population before sorting the cell population for NCAM-positive cells. Figures 25B, 25D, and 25F show the percentage of SYP, CHGA, and INS positive, respectively, after sorting the cells for cells that are positive for NCAM expression. [Figure 26] Flow cytometry dot plots of hESC-derived cells treated to differentiate into immature pancreatic islet hormone-expressing cells. Figure 26A shows the percentage of NCAM-positive cells that are SYP positive. Figure 26B shows the percentage of NCAM-positive cells that are CD133 negative. Figure 26C shows the percentage of cells that are SYP positive and sorted for NCAM-positive / CD133-negative cells. [Figure 27] Photomicrographs showing immunoreactivity of immature pancreatic islet hormone-expressing cells for INS (FIG. 27D) and PAX6 (FIG. 27C). The entire cell population is stained with DAPI (FIG. 27B). The cells are hESC-derived stem cells that were treated to differentiate into immature pancreatic islet hormone-expressing cells and sorted using fluorescence-activated cell sorting techniques. The cells represented in this photomicrograph were also fluorescently stained for NCAM. Some NCAM-positive hESC-derived cells co-express PAX6 and INS (FIG. 27A). [Figure 28] 28C and 28D are photomicrographs showing immunoreactivity of immature pancreatic islet hormone-expressing cells expressing INS (FIG. 28C) or GCG (FIG. 28D). The cells are hESC-derived stem cells that were treated to differentiate into immature pancreatic islet hormone-expressing cells and sorted using fluorescence-activated cell sorting techniques. The cells represented in this photomicrograph also fluorescently stained for NCAM. FIG. 28A shows the overlap of cells expressing both INS and GCG. [Figure 29]29A-29F are bar graphs showing mRNA levels of certain markers as detected by QPCR in hESC-derived cells treated to differentiate into pancreatic insulin-expressing cells (day 19). Specifically, mRNA levels of PDX1 (FIG. 29A), NGN3 (FIG. 29B), INS (FIG. 29C), SST (FIG. 29D), GCG (FIG. 29E), and GHRL (FIG. 29F) are shown. Abbreviations are indicated as follows: A100 - 100 ng / ml activin A, KC - 50 ng / ml KGF and 0.25 μM KAAD cyclopamine, and nog - noggin. [Diagram 30] 3 is a bar graph showing the mRNA levels of certain markers as detected by QPCR in hESC-derived cells treated to differentiate into hormone-expressing cells (day 11). Specifically, the mRNA levels of PDX1 (FIG. 30A), NGN3 (FIG. 30B), PTF1A (FIG. 30C), NKX6.1 (FIG. 30D), INS (FIG. 30E), and GCG (FIG. 30F) are shown. Abbreviations are indicated as follows: A100 - 100ng / ml activin A, KC - 50ng / ml KGF and 0.25μM KAAD cyclopamine, N - Noggin, CR0.1 - 0.25μM KAAD cyclopamine and 0.1μM retinoic acid, CR2 - 0.25μM KAAD cyclopamine and 2μM retinoic acid, "A" - 0ng / ml Noggin and 0.1μM retinoic acid, "B" - 30ng / ml Noggin and 0.1μM retinoic acid, "C" - 100ng / ml Noggin and 0.1μM retinoic acid, "D" - 0ng / ml Noggin and 2μM retinoic acid, "E" - 30ng / ml Noggin and 2μM retinoic acid, and "F" - 100ng / ml noggin and 2μM retinoic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0394] Described herein is a process progression that converts undifferentiated hESCs into endocrine precursor cells and immature pancreatic islet hormone-expressing cells, and ultimately into mature pancreatic endocrine cells (mature pancreatic islet hormone-expressing cells) capable of synthesizing insulin, glucagon, somatostatin, pancreatic polypeptide, PPY, and ghrelin in vitro. This process progression involves sequential differentiation of hESCs through intermediates that are currently known to occur during pancreatic development in vivo. A common method for producing hESC-derived pancreatic endocrine cells begins with the production of definitive endoderm (DE), followed by a DE patterning step in which TGF-β signaling is altered and fibroblast growth factor or a ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb) is provided. The PDX1-positive pre-patterned endoderm is further recruited to the pancreatic endocrine lineage by primary exposure to retinoic acid and γ-secretase inhibitors, which then give rise to pancreatic endocrine hormone-producing cells.

[0395] No. 11 / 021,618 (titled "DEFINITIVE ENDODERM") As previously shown in "Definitive Endoderm (DE) Production in Somatic Cells," filed Dec. 23, 2004 and D'Amour et al. Nat. Biotech. 23, 1534-1541, (2005), the disclosures of which are incorporated herein by reference in their entireties, the present inventors have developed a robust method for producing definitive endoderm (DE) from somatic germ layers. In vivo, the DE lineage arises during the gastrula stage of embryonic development in a region called the primitive streak. The emergence of DE is required for the later specification of tissues and organs such as the small intestine, stomach, lung, thymus, endocrine pancreas, parathyroid gland, thyroid gland, and pancreas. This is a necessary condition.

[0396] In humans and most other vertebrates, the pancreas is derived from the foregut endoderm at the foregut-midgut junction as both a ventral and a dorsal pancreatic bud. In humans, the ventral and dorsal pancreatic buds form what are called the posterior head and uncinate processes of the pancreas at approximately 41-45 days post conception (pc). The ventral bud fuses with the smaller ventral bud to form the region that is the primary site of islet cell proliferation (Bocian-Sobkowska, J., et al. Histochem. Cell Biol. 112, 147-153, (1999)). In humans, this region primarily produces islet cells. It consists of pancreatic polypeptide, which is involved in the function of the pancreatic duct (Rahier J., et al., Cell Tissue Res. 200 (3), 359-366, (1979); Malaisse-Langae F., et al., Diabetologia 17(6), 361-365, (1979 ), Fiocca R., et al., Histochemistry 77(4), 511-523,(1983), Stefan Y., et al., Diabetologica 23(2), 141-142,(1982)). The dorsal pancreatic bud is located in the anterior part of the pancreatic head in humans. The dorsal blastema cells form the pancreatic body and tail, which are the source of all the hormone-producing cells of the pancreas. In frogs (Xenopus) and fish (zebrafish), only the dorsal blastema cells generate insulin-producing islet cells (Kelly, OG and Melton, DA, Dev. Dyn. 218, 615-627,(2000); Chen, Y., et al., Dev. Biol. 271(1), 144-160,(2004); Field, HA, et al., Dev. Biol. 263, 197- 208(2003) Similarly, in humans, the ventral bud is thought to generate pancreatic polypeptide-expressing islet cells primarily for the clearance of insulin (Rahier J., et al., Cell Tissue Res. 200 (3), 359-366,(1979); Malaisse-Langae F., et al., Diabetologia 17(6), 361-365,(1979) ), Fiocca R., et al., Histochemistry 77(4), 511-523, (1983), Stefan Y., et al., Diabetologica 23(2), 141-142, (1982)). In contrast, rabbit and mouse In the mouse, both the ventral and dorsal buds give rise to insulin-producing cells (Spooner, BS, et al., J. Cell Biology, 47, 235- 246, (1970); Li, H., et al., Nature 23, 67-70, (1999)).

[0397] As shown in FIG. 1, pancreatic endocrine cells can be efficiently generated from hESCs in a series of developmental stages. The first stage is the formation of mesoendoderm (ME), characterized by transitional expression of the T-box gene brachyury. As hESCs differentiate into the DE, they downregulate E-cadherin and transition from an epithelial ectodermal state to mesenchymal DE cells (D'Amour et al. Nat. Biotech. 23, 1534-1541, (2005)). The main markers that define early DE cells are FOXA2, GSC, N-cadherin, CXCR4, and SOX17. As described in our previous U.S. patent application Ser. No. 11 / 021,618, DE is further characterized by the lack of significant expression of certain other markers, such as SOX1, SOX7, thrombomodulin (TM), SPARC, and alpha-fetoprotein (AFP). The nascent DE is further patterned from its strong anterior characteristics to a more posterior, but still foregut endoderm, by removal of Activin signaling. Such foregut endoderm is characterized by expression of the HNF1b and FOXA1 gene markers. This endoderm expands, Exposure to FGF10, retinoic acid, and cyclopamine (a Sonic Hedgehog (SHH) inhibitor) appears to result in a more dorsal phenotype. Posterior foregut (posterior region of the foregut) patterning cells express PDX1, PTF1a, HNF1b, Onecut1 / 2, and HB9. These pancreatic endoderm cells, which represent endocrine precursor cells, are preferentially recruited to the endocrine lineage by modulation of γ-secretase signaling (potentially through inhibition of Notch pathway signaling) as shown by the transient expression of NGN3. hESC-derived endocrine precursor cells also express paired box gene 4 (PAX4) and NKX2.2. Further incubation of the endocrine precursor cells gives rise to immature islet hormone-expressing cells. Immature islet hormone-expressing cells express V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) as well as NKX2.2, whereas islet hormone-expressing cells express NKX2.2. Finally, further incubation of the immature islet hormone-expressing cells results in the transition from immature cells to mature islet hormone-expressing cells capable of expressing endocrine hormones insulin, glucagon, somatostatin, -PPY, ghrelin, and pancreatic transcription factors NKX2.2 / 6.1, PAX6, NEUROD1, PDX1, ISL1, as well as V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA).

[0398] definition It is understood that the numerical ranges expressed herein are inclusive of the recited endpoints and recite all integers between the endpoints of the stated numerical range.

[0399] As used herein, "pancreatic islet hormone-expressing cells" refer to cells derived in vitro from human pluripotent cells, expressing one or more pancreatic hormones, and having at least some of the functions of human pancreatic islet cells. Pancreatic islet hormone-expressing cells can be mature or immature. Immature pancreatic islet hormone-expressing cells can be distinguished from mature pancreatic islet hormone-expressing cells based on differential expression of certain markers. As used herein, "pancreatic hormone-expressing cells" are used interchangeably with "pancreatic islet hormone-expressing cells."

[0400] As used herein, "endocrine precursor cells" refer to pluripotent cells of the definitive endoderm lineage that express neurogenin 3 (NEUROG3) and can further differentiate into cells of the endocrine system, including (but not limited to) pancreatic islet hormone-expressing cells. Endocrine precursor cells cannot differentiate into as many different types of cells, tissues and / or organs as less clearly differentiated definitive endoderm lineage cells, such as PDX1-positive pancreatic endoderm cells.

[0401] As used herein, "PDX1-positive pancreatic endoderm cells" and "PDX1-positive foregut endoderm cells" refer to pluripotent cells of the definitive endoderm lineage that express pancreatic and duodenal homeobox gene 1 (PDX1) and can further differentiate into cells derived from the foregut, including, but not limited to, endocrine precursors and pancreatic islet hormone-expressing cells. PDX1-positive pancreatic endoderm cells cannot differentiate into as many different types of cells, tissues and / or organs as definitive endoderm cells.

[0402] As used herein, "multipotent" or "pluripotent cells" refer to cell types that can give rise to a limited number of other specific cell types. Pluripotent cells are committed to one or more embryonic cell fates and thus, in contrast to pluripotent cells, are unable to give rise to each of the three embryonic cell lineages and extraembryonic cells.

[0403] In some embodiments, "pluripotent cells" are used as starting material for differentiation of pancreatic islet hormone-expressing cells. By "pluripotent" we mean that the cells can generate each of the three embryonic cell lineages as well as extraembryonic cells. However, pluripotent cells are not capable of producing an entire organism.

[0404] In certain embodiments, the pluripotent cells used as starting material are stem cells, including human embryonic stem cells. As used herein, "embryonic" refers to the sequence of developmental stages of an organism that begins with a single zygote and ends with a multicellular structure that no longer contains pluripotent or totipotent cells other than the developing gamete cells. In addition to embryos derived by gamete fusion, the term "embryonic" refers to embryos derived by nuclear transfer of somatic cells.

[0405] "Conditioned medium" refers to a medium that is altered compared to a basal medium. For example, by conditioning the medium, molecules such as nutrients and / or growth factors may be added or depleted from the original levels found in the basal medium. In some embodiments, certain types of cells are cultured in a medium. A medium is conditioned by culturing or maintaining hESCs in a medium of defined composition for a specified time under specified conditions. For example, a medium can be conditioned by growing, differentiating or maintaining hESCs in a medium of defined composition for a specified time at a specified temperature. As will be appreciated by one of skill in the art, numerous combinations of cells, medium types, durations and environmental conditions can be used to create a nearly infinite array of conditioned media.

[0406] When used in reference to cell cultures and / or cell populations, the term "portion" The term refers to any non-zero amount of a cell culture or cell population, ranging from a single cell to an entire cell culture or cell population. In a preferred embodiment, the term "portion" refers to at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89 " at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%.

[0407] With respect to cells in a cell culture or cell population, the term "substantially free" refers to The term "abundance of" refers to the abundance of a particular cell type that is not present in a cell culture or cell population. By this definition, we mean less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells present in the cell culture or cell population.

[0408] As used herein, compounds such as growth factors and differentiation factors that are "exogenously added" in a culture or conditioned medium refer to growth factors that are added to the culture or medium to supplement any compounds or growth factors that may already be present in the culture or medium. For example, in some embodiments of the invention, the cell cultures and / or cell populations do not contain exogenously added retinoids.

[0409] As used herein, "generated from hESCs," "derived from hESCs," "differentiated from hESCs," and equivalent expressions refer to the production of differentiated cell types from hESCs in vitro rather than in vivo.

[0410] In some embodiments, hESCs can be derived from a "preimplantation embryo." As used herein, a "preimplantation embryo" refers to an embryo between the fertilization and implantation stages. Thus, a preimplantation embryo typically does not progress beyond the blastocyst stage. Implantation is usually a blastocyst stage. This occurs 7 to 8 days after fertilization. However, implantation can also occur at about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more than about 14 days after fertilization.

[0411] As used herein, a "hedgehog inhibitor" or "hedgehog pathway inhibitor" refers to any molecule that inhibits any member of the hedgehog signaling pathway. Exemplary hedgehog pathway inhibitors include, but are not limited to, KAAD-cyclopamine, cyclopamine analogs, jervine, jervine analogs, hedgehog pathway blocking antibodies, and any other inhibitors of hedgehog pathway function known to one of skill in the art.

[0412] As used herein, "γ-secretase inhibitor" refers to any molecule that inhibits γ-secretase or the signaling events caused by the activity of γ-secretase. Exemplary γ-secretase inhibitors include N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-bromoglycerin, and the like. Examples of inhibitors of gamma-secretase function include, but are not limited to, diethyl ester (DAPT), F-box protein SEL-10, gamma-secretase blocking antibodies, and any other inhibitor of gamma-secretase function known to those skilled in the art. In some embodiments, the gamma-secretase inhibitor inhibits the Notch signaling pathway. In some embodiments, a Notch pathway inhibitor or a Notch specific inhibitor can be used instead of a gamma-secretase inhibitor.

[0413] As used herein, "retinoid" refers to retinol, retinal, or retinoic acid, as well as derivatives of any of these compounds. In a preferred embodiment, the retinoid is retinoic acid.

[0414] By "FGF family growth factor", "fibroblast growth factor" or "fibroblast growth factor family member" is meant an FGF selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23. In some embodiments, by "FGF family growth factor", "fibroblast growth factor" or "fibroblast growth factor family member" is meant any growth factor having similar homology and / or function to a known member of the fibroblast growth factor family.

[0415] As used herein, "expression" refers to the production of a material or substance, and the level or amount of production of a material or substance. Thus, determining the expression of a particular marker refers to either detecting the relative or absolute amount of the marker that is expressed, or simply detecting the presence or absence of the marker.

[0416] As used herein, a "marker" refers to any molecule that can be observed or detected. For example, markers include, but are not limited to, nucleic acids, such as the transcription product of a particular gene, the polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein, or a small molecule (e.g., a molecule with a molecular weight of less than 10,000 amu).

[0417] For most markers described herein, a Human Genetics Organization (HUGO) gene symbol is provided. Such symbols, created by the HUGO Gene Nomenclature Committee, provide unique abbreviations for named human genes and gene products. These gene symbols are easily recognized by those of skill in the art and can be easily associated with the corresponding unique human gene and / or protein sequence.

[0418] In accordance with the HUGO nomenclature, the following gene symbols are defined as follows: GHRL - ghrelin; IAPP - islet amyloid polypeptide; INS - insulin; GCG - glucagon; ISL1 - ISL1 transcription factor; PAX6 - paired box gene 6; PAX4 - paired box gene 4; NEUROG3 - neurogenin 3 (NGN3); NKX2-2 - NKX2 transcription factor related, locus 2 (NKX2.2); NKX6-1 - NKX6 transcription factor related, locus 1 (NKX6.1); IPF1 - insulin promoter factor 1 (PDX1); ONECUT1 - one-cut domain, family member 1 (HNF6); HLXB9 - homeobox B9 (HB9); TCF2 - transcription factor 2, liver (HNF1b); FOXA1 - forkhead box A1; HGF - hepatocyte growth factor; IGF1 - Insulin-like growth factor 1;POU5F1 - POU domain, group 5, transcription factor 1 (OCT4);NANOG - Nanog homeobox;SOX2 - SRY-box 2;CDH1 - E-cadherin, type 1 (ECAD);T - brachyury homolog (BRACH);FGF4 - fibroblast growth factor 4;WNT3 - wingless MMTV integration site family, member 3;SOX17 - SRY-box 17;GSC - goosecoid;CER1 - cerberus 1, cysteine ​​knot superfamily, homolog (CER);CXCR4 - chemokine (CXC motif) receptor 4;FGF17 - fibroblast growth factor 17;FOXA2 - forkhead box A2;SOX7 - SRY-box 7;SOX1 - SRY (Sex determining region Y)-box 1; AFP - alpha-fetoprotein; SPARC - secreted protein, acidic, cysteine ​​rich (osteonectin); and THBD - thrombomodulin (TM), NCAM - neural cell adhesion molecule; SYP - synaptophysin; ZIC1 - Zic family member 1; NEF3 - SST - somatostatin; MAFA - v-maf musculoaponeurotic fibrosarcoma oncogene homolog A; MAFB - v-maf musculoaponeurotic fibrosarcoma oncogene homolog B; SYP - synaptophysin; CHGA - chromogranin A (parathyroid secretory protein 1).

[0419] The following are full gene names corresponding to non-HUGO gene symbols and other abbreviations that may be used herein: SS - somatostatin (SOM); PP - pancreatic polypeptide; C-peptide - connecting peptide; Ex4 - exendin 4; NIC - Nicotinamide and DAPT - N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; RA - retinoic acid; RPMI - Roswell Park Memorial Institute medium ;CMRL - ​​Connaught Medical Research Labs medium;FB S - fetal bovine serum; NBP10 - NCAM-binding protein 10; PTF1a - Pancreas-specific transcription factor 1a.

[0420] The terms fibroblast growth factor 7 (FGF7) and keratinocyte growth factor (KGF) are synonymous.

[0421] As used herein, the term "label" refers to a tag or label, e.g., radioactive, fluorescent, biological or enzymatic, as standard in the art. The label can be conjugated or otherwise associated with a nucleic acid, a polypeptide, e.g., an antibody, or a small molecule. For example, oligonucleotides of the invention can be labeled after synthesis by incorporation into biotinylated dNTPs or rNTPs, or by some similar means (e.g., photocrosslinking a psoralen derivative of biotin with RNA), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the like. Alternatively, when fluorescently labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus ), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and FluorX (Amersham), and the like, can be attached to the nucleic acid. Non-limiting examples of detectable labels that can be conjugated to a polypeptide, such as an antibody, include radioactive labels, e.g. 3 H, 11 C. 14 C. 18 F, 32 P, 35 S, 64 Cu, 76 Br, 86 Y, 99 Tc, 111 In, 123 I, 125 I, or 177 These include, but are not limited to, Lu, enzymes such as horseradish peroxidase, fluorophores, chromophores, chemiluminescent agents, chelate complexes, dyes, colloidal gold or latex particles.

[0422] Human Embryonic Stem Cells A preferred method for deriving definitive endoderm cells, and ultimately endocrine precursor cells and / or pancreatic islet hormone-expressing cells, utilizes human embryonic stem cells as starting material. Such pluripotent cells can be cells derived from the morula, the inner cell mass of an embryo, or cells obtained from the gonadal ridges of an embryo. Human embryonic stem cells can be maintained in culture in a pluripotent state without substantial differentiation using methods known in the art. Such methods are described, for example, in U.S. Patent Nos. 5,453,357, 5,670,372, 5,690,926, 5,843,780, 6,200,806, and 6,251,671, the disclosures of which are incorporated herein by reference in their entirety.

[0423] In some processes, hESCs are maintained on a feeder layer. In such processes, any feeder layer that allows hESCs to be maintained in a pluripotent state can be used. One feeder layer that is commonly used for culturing human embryonic stem cells is a layer of mouse fibroblasts. More recently, human fibroblast feeder layers have been developed for use in culturing hESCs (see U.S. Patent Application No. 2002 / 0072117, the entire disclosure of which is incorporated herein by reference). Alternative processes allow for the maintenance of pluripotent hESCs without the use of a feeder layer. A method for maintaining pluripotent hESCs under feeder-free conditions is described in U.S. Patent Application No. 2003 / 0175956, the entire disclosure of which is incorporated herein by reference.

[0424] Human embryonic stem cells used herein can be maintained in cultures that contain or do not contain serum. Some embryonic stem cell maintenance procedures use serum replacements. Others use serum-free culture techniques, such as those described in U.S. Patent Application No. 2003 / 0190748 (the entire disclosure of which is incorporated herein by reference).

[0425] Stem cells are maintained in culture in a pluripotent state by routine passage until required to differentiate into definitive endoderm and then ultimately into endocrine precursor cells and / or pancreatic islet hormone-expressing cells.

[0426] Production of definitive endoderm In some processes, differentiation into definitive endoderm is achieved by providing the stem cell culture with a growth factor of the TGFβ superfamily in an amount sufficient to promote differentiation into definitive endoderm. Growth factors of the TGFβ superfamily that are useful for producing definitive endoderm are selected from the Nodal / Activin or BMP subgroups. In some preferred differentiation processes, the growth factor is selected from the group consisting of Nodal, Activin A, Activin B and BMP4. Growth factor Wnt3a and other Wnt family members are also useful for producing definitive endoderm cells. In certain differentiation processes, any combination of the above growth factors may be used.

[0427] For some processes for differentiation of pluripotent stem cells into definitive endoderm cells, growth factors are cultured at concentrations sufficient to promote differentiation of at least a portion of the stem cells into definitive endoderm cells. In some processes, the growth factors are provided to the cells so that they are present in the cell culture. In some processes, the growth factors are present in the cell culture at a concentration of at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, at least about 1000 ng / ml, at least about 2000 ng / ml, at least about 3000 ng / ml, at least about 4000 ng / ml, at least about 5000 ng / ml or greater than about 5000 ng / ml.

[0428] In certain processes for differentiation of pluripotent stem cells into definitive endoderm cells, the above-mentioned growth factors are removed from the cell culture after their addition.For example, the growth factors can be removed within 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 after their addition.In a preferred process, the growth factors are removed about 4 days after their addition.

[0429] Cultures of definitive endoderm cells can be produced from embryonic stem cells in low-serum or serum-free medium. Under certain culture conditions, serum concentrations can range from about 0.05% (v / v) to about 20% (v / v). For example, in some differentiation processes, the serum concentration of the medium can be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). % (v / v), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v), or less than about 20% (v / v). In some processes, the definitive endoderm cells are grown without serum or without serum replacement. In still other processes, the definitive endoderm cells are grown in the presence of B27. In such processes, the concentration of B27 supplement can range from about 0.1% (v / v) to about 20% (v / v). In other embodiments, the definitive endoderm cells are grown in the absence of B27.

[0430] In some processes of differentiating human definitive endoderm cells from hESCs, differentiation is initiated in the absence of serum and in the absence of insulin and / or insulin-like growth factors. During differentiation, serum concentration can be gradually increased to promote proper cell survival. In a preferred embodiment, differentiation of hESCs into definitive endoderm cells is initiated in the absence of serum and in the absence of any supplements, including insulin or insulin-like growth factors. The absence of serum and the absence of supplements, including insulin or insulin-like growth factors, is maintained for about 1 to about 2 days, after which serum is gradually added to the cell culture being differentiated during differentiation. In a preferred embodiment, serum concentration does not exceed about 2% during differentiation.

[0431] Definitive endoderm cell cultures and cell populations, as well as detailed processes for producing definitive endoderm from embryonic stem cells, are set forth in U.S. Patent Application Serial No. 11 / 021,618, entitled "DEFINITIVE ENDODERM," filed December 23, 2004, the entire disclosure of which is incorporated herein by reference. (which is incorporated herein by reference).

[0432] Enrichment, isolation and / or purification of definitive endoderm In some embodiments of the processes described herein, the definitive endoderm cells are enriched, isolated and / or purified prior to further separation. In such embodiments, the definitive endoderm cells may be enriched, isolated and / or purified using any known method. In a preferred embodiment, the definitive endoderm cells are enriched, isolated and / or purified using any method known in the art. Title: "DEFINITIVE ENDODERM" (submitted December 23, 2004) and the United States The cells are enriched, isolated and / or purified using one or more of the methods described in Provisional Patent Application No. 60 / 736,598 (entitled "MARKERS OF DEFINITIVE ENDODERM," filed November 14, 2005), the disclosures of which are incorporated herein by reference in their entireties.

[0433] Compositions Comprising Definitive Endoderm Cells The cell compositions produced by the methods include cell cultures containing definitive endoderm cells and cell populations enriched for definitive endoderm cells. For example, cell cultures and / or cell populations containing definitive endoderm cells can be produced, where at least about 50%-99% of the cells in the cell cultures and / or cell populations are definitive endoderm cells. Because the efficiency of the differentiation process can be adjusted by modifying certain parameters, including (but not limited to) cell growth conditions, growth factor concentrations, and timing of the culture steps, the differentiation procedures described herein can convert about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or more than about 99% of pluripotent cells into definitive endoderm. In processes where isolation of definitive endoderm cells is utilized, a substantially pure population of definitive endoderm cells can be recovered, for example, by using an affinity reagent that binds to the CXCR4 receptor. In embodiments where the cell culture or cell population comprises human feeder cells, the percentages are calculated without regard to the human feeder cells in the cell culture or cell population.

[0434] Generation of PDX1-positive foregut endoderm The definitive endoderm cells can be specified to pancreatic differentiation by further differentiation of these cells to produce PDX1-positive foregut endoderm cells. In some differentiation processes described herein, the cell cultures and enriched or purified cell populations comprising definitive endoderm cells can be used for further differentiation into cell cultures and / or enriched cell populations comprising PDX1-positive foregut endoderm cells.

[0435] Typically, definitive endoderm cells are differentiated into PDX1-positive foregut endoderm cells by supplying a retinoid, such as retinoic acid (RA), to a cell culture comprising SOX17-positive definitive endoderm cells. Also, in some differentiation processes, a member of the fibroblast growth factor family is supplied to the definitive endoderm cells in the medium before or at about the same time as the addition of RA. A preferred fibroblast growth factor is FGF-10. In another preferred process, the fibroblast growth factor includes any fibroblast growth factor or ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb)). In an even more preferred process, an FGF family growth factor is used in conjunction with a hedgehog pathway inhibitor. A preferred hedgehog pathway inhibitor is KAAD-cyclopamine. In a particularly preferred differentiation process, FGF-10 and / or KAAD-cyclopamine are supplied to a cell culture comprising PDX1-negative definitive endoderm cells in the presence of RA. In certain processes, BMP4 may be included with FGF-10 and / or KAAD-cyclopamine in the presence of RA. In some processes, retinoids are used in conjunction with members of the TGFβ superfamily of growth factors, and / or Connaught Medical Research Labs medium (CRML medium) (Invitrogen, Carlsbad, Calif.). can be.

[0436] For some embodiments of the differentiation processes described herein, a retinoid and / or combination of the differentiation factors described above is provided to the cells such that these factors are present in the cell cultures or cell populations at concentrations sufficient to promote differentiation of at least a portion of the definitive endoderm cell cultures or cell populations into PDX1-positive foregut endoderm cells.

[0437] In some processes, the retinoid is at least about 1 nM, at least about 0.01 μM, at least about 0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, at least about 2.0 μM, at least about 2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3.0 μM, at least about 3.1 μM, at least about 3.2 μM, at least about 3.3 μM, at least about 3.4 μM, at least about 3.5 μM, at least about 3.6 μM, at least about 3.7 μM, at least about 3.8 μM, at least about 3.9 μM, at least about 3.1 μM, at least about 3.2 μM, at least about 3.3 μM, at least about 3.4 μM, at least about 3.5 μM, at least about 3.6 μM, at least In some embodiments, the antibody is provided to cells of the cell culture such that the antibody is present at a concentration of at least about 1.5 μM, at least about 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about 2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, or at least about 50 μM.

[0438] In other processes, FGF-10 is provided to cells of the cell culture such that FGF-10 is present at a concentration of at least about 1 ng / ml, at least about 2 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml. In other embodiments, when used alone or with FGF-10, KAAD-cyclopamine has a concentration of at least about 0.01 μM, at least about 0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, at least about 2.0 μM, at least about 2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3.0 μM, at least about 3.1 μM, at least about 3.2 μM, at least about 3.3 μM, at least about 3.4 μM, at least about 3.5 μM, at least about 3.6 μM, at least about 3.7 μM, at least about 3.8 μM, at least about 3.9 μM, at least about 4.0 μM, at least about 4.0 μM, at least about 4.0 μM, at least about 4.0 μM, at least about 4.0 μM, at least about The fibroblast growth factor or ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb)) may be provided at a concentration of about 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about 2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, or at least about 50 μM. In some embodiments of the invention, a fibroblast growth factor or ligand that stimulates or otherwise interacts with fibroblast growth factor 2 receptor IIIb (FGFR2(IIIb)) is provided either alone or in combination with a hedgehog pathway inhibitor.

[0439] In a preferred process for producing PDX1-positive foregut endoderm cells from definitive endoderm cells, a cell culture or enriched cell population of definitive endoderm cells is provided with 50 ng / ml FGF-10 and 0.2 μM KAAD-cyclopamine in CMRL medium in the presence of 2 μM RA.

[0440] In some processes described herein, activin A and / or activin B are provided to the cell culture together with a retinoid and / or a fibroblast growth factor and a hedgehog inhibitor. For example, in such processes, activin A and / or activin B are provided at a concentration of at least about 5 ng / ml, at least about 10 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml, at least about 100 ng / ml, at least about 15 ng / ml, at least about 100 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 15 ... Provided to the cell culture at a concentration of about 25 ng / ml, at least about 50 ng / ml, at least about 75 ng / ml, at least about 100 ng / ml, at least about 200 ng / ml, at least about 300 ng / ml, at least about 400 ng / ml, at least about 500 ng / ml, or at least about 1000 ng / ml.

[0441] In some processes, the differentiation factors and / or CRML medium are provided to the definitive endoderm cells about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more than about days after initiation of differentiation from hESCs. In a preferred process, the differentiation factors and / or CRML medium are provided to the definitive endoderm cells about 5 days after initiation of differentiation from hESCs.

[0442] In certain processes described herein, the differentiation factors are removed from the cell culture after their addition. For example, the differentiation factors can be removed within 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 after their addition.

[0443] Cultures of PDX1-positive foregut endoderm cells may be differentiated and further expanded in low-serum or serum-free medium. The serum concentration may range from about 0.05% (v / v) to about 20% (v / v). In some processes, dorsal PDX1-positive foregut endoderm cells are expanded with serum replacement. For example, in certain processes, the serum concentration of the medium is less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). (v / v), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v), or less than about 20% (v / v). In certain processes described herein, the differentiation medium does not contain serum, serum replacements, or any supplements, including insulin or insulin-like growth factors.

[0444] In certain processes, PDX1-positive foregut endoderm cells are grown in the presence of B27. In such differentiation processes, B27 can be provided in the culture medium at a concentration ranging from about 0.1% (v / v) to about 20% (v / v), or at a concentration of more than about 20% (v / v). In certain processes, the concentration of B27 in the medium is about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), about 5% (v / v), about 6% (v / v), about 7% (v / v), about 8% (v / v), about 9% (v / v), about 10% (v / v), about 15% (v / v), or about 20% (v / v). Alternatively, the concentration of B27 supplement added can be measured in multiples of the concentration of a commercially available B27 stock solution. For example, B27 is available from Invitrogen (Carlsbad, CA) as a 50× stock solution. A sufficient amount of this stock solution is added to a sufficient volume of growth medium to obtain a medium supplemented with the desired amount of B27. For example, adding 10 ml of 50× B27 stock solution to 90 ml of growth medium will obtain a growth medium supplemented with 5× B27. The concentration of B27 supplement in the medium can be about 0.1x, about 0.2x, about 0.3x, about 0.4x, about 0.5x, about 0.6x, about 0.7x, about 0.8x, about 0.9x, about 1x, about 1.1x, about 1.2x, about 1.3x, about 1.4x, about 1.5x, about 1.6x, about 1.7x, about 1.8x, about 1.9x, about 2x, about 2.5x, about 3x, about 3.5x, about 4x, about 4.5x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 17x, about 18x, about 19x, about 20x, and greater than about 20x.

[0445] In some processes for differentiating PDX1-positive foregut endoderm cells from definitive endoderm cells, U.S. Provisional Patent Application No. 60 / 730,917 (entitled "PDX1-EXPRESSING DORSA L AND VENTRAL FOREGUT ENDODERM"), 2005 Definitive endoderm cells are differentiated for further differentiation into either the ventral or dorsal pancreatic bud, as described in U.S. Pat. No. 6,393,311, filed Oct. 27, the disclosure of which is incorporated herein by reference in its entirety. Further detailed methods for producing PDX1-positive foregut endoderm cells can be found in U.S. Patent Application No. 11 / 115,868, entitled "PDX1 EXPRESSING ENDODERM," filed Apr. 26, 2005, the disclosure of which is incorporated herein by reference in its entirety.

[0446] Enrichment, isolation and / or purification of PDX1-positive foregut endoderm In some embodiments of the processes described herein, the PDX1-positive foregut endoderm cells are enriched, isolated and / or purified prior to further separation. In such embodiments, the PDX1-positive foregut endoderm cells can be enriched, isolated and / or purified using any known method. In a preferred embodiment, the PDX1-positive foregut endoderm cells are enriched, isolated and / or purified using any known method. In a preferred embodiment, the PDX1-positive foregut endoderm cells are enriched, isolated and / or purified using any known method, as described in U.S. Patent Application No. 11 / 115,868 (entitled "PDX1 EXPRESSING ENDODERM," filed April 26, 2005) and U.S. Provisional Patent Application No. 60 / 730,917 (entitled "PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM," filed October 27, 2005) (the entire disclosures of which are incorporated herein by reference). The protein is enriched, isolated and / or purified using one or more of the methods described in the above, which are incorporated herein by reference.

[0447] Composition comprising PDX1-positive foregut endoderm cells The cell compositions produced by the above-mentioned methods include cell cultures containing PDX1-positive foregut endoderm cells and cell populations enriched for PDX1-positive foregut endoderm cells. For example, cell cultures and / or cell populations containing PDX1-positive foregut endoderm cells can be produced, and at least about 50% to 99% of the cells in the cell cultures and / or cell populations are PDX1-positive foregut endoderm cells. Because the efficiency of the differentiation process can be adjusted by modifying certain parameters, including but not limited to cell growth conditions, growth factor concentrations, and timing of the culture steps, the differentiation procedures described herein can convert about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or more than about 99% of pluripotent cells into PDX1-positive foregut endoderm. In processes that utilize isolation of PDX1-positive foregut endoderm cells, a substantially pure population of PDX1-positive foregut endoderm cells can be recovered. In embodiments in which the cell culture or cell population comprises human feeder cells, the percentage is calculated without considering the human feeder cells in the cell culture or cell population.

[0448] Endocrine precursor cell production Some embodiments of the present invention relate to a method for producing endocrine precursor cells starting from hESCs. As mentioned above, endocrine precursor cells can be produced by first differentiating hESCs to produce definitive endoderm cells, and then further differentiating the definitive endoderm cells to produce PDX1-positive foregut endoderm cells. In such embodiments, the PDX1-positive foregut endoderm cells are further differentiated into multipotent endocrine precursor cells, which can be differentiated into human pancreatic islet hormone-expressing cells.

[0449] In one embodiment of the present invention, PDX1-positive foregut endoderm cells are differentiated into endocrine precursor cells by continuing to incubate the PDX1-positive foregut endoderm cells in the presence of a retinoid, e.g., retinoic acid, for a period of time sufficient to produce endocrine precursor cells. In some embodiments, the time sufficient to produce endocrine precursor cells ranges from about 1 hour to about 10 days after expression of the PDX1 marker in a portion of the cells in the cell culture. In some embodiments, the retinoid is maintained in the cell culture for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, 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, about 10 days, or more than about 10 days after expression of the PDX1 marker in a portion of the cells in the cell culture.

[0450] In some processes described herein, the retinoid concentrations used to differentiate PDX1-positive foregut endoderm cells in the cell cultures or cell populations into endocrine precursor cells range from about 1 nM to about 100 μM. In some processes, the retinoid is at least about 1 nM, at least about 0.01 μM, at least about 0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at least about 1. The antibody is provided to cells of the cell culture such that the antibody is present at a concentration of at least about 8 μM, at least about 1.9 μM, at least about 2 μM, at least about 2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 75 μM, or at least about 100 μM.

[0451] In some preferred embodiments of the present invention, differentiation of PDX1-positive foregut endoderm cells to pancreatic endocrine precursor cells is mediated by providing a γ-secretase inhibitor to a cell culture or cell population comprising human PDX1-positive foregut endoderm cells. In a preferred embodiment, the γ-secretase inhibitor is N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester ( DAPT).

[0452] In another embodiment of the present invention, the gamma-secretase inhibitor is provided at the beginning of the differentiation process, for example at the hESC stage, and remains in the cell culture during differentiation into pancreatic islet hormone-expressing cells.In yet another embodiment, the gamma-secretase inhibitor is added after the start of differentiation, but before differentiation into PDX1-positive foregut endoderm stage.In a preferred embodiment, the gamma-secretase inhibitor is provided to the cell culture or cell population at about the same time that the differentiation factor that promotes the conversion of definitive endoderm into PDX1-positive endoderm is provided.In another preferred embodiment, the gamma-secretase inhibitor is provided to the cell culture or cell population after the majority of cells in the cell culture or cell population have differentiated into PDX1-positive foregut endoderm cells.

[0453] For some embodiments of differentiation of PDX1-positive foregut endoderm cells to endocrine precursor cells, a γ-secretase inhibitor is provided to the cells such that the γ-secretase inhibitor is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least some of the PDX1-positive cells to endocrine precursor cells. In some embodiments, the γ-secretase inhibitor is present in the cell culture or cell population at a concentration ranging from about 0.01 μM to about 1000 μM. In preferred embodiments, the γ-secretase inhibitor is present in the cell culture or cell population at a concentration ranging from about 0.1 μM to about 100 μM. In more preferred embodiments, the γ-secretase inhibitor is is present in the cell culture or cell population at a concentration ranging from about 1 μM to about 10 μM. In other embodiments, the γ-secretase inhibitor is at least about 0.01 μM, at least about 0.02 μM, at least about 0.04 μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about 2.1 μM, at least about In some embodiments, the antibody is present in the cell culture or cell population at a concentration of about 2.2 μM, at least about 2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM, at least about 20 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 250 μM, at least about 500 μM, at least about 750 μM, or at least about 1000 μM.

[0454] In certain embodiments of the process for producing endocrine precursor cells described herein, the gamma-secretase inhibitor is provided after one or more differentiation factors that have been previously provided are removed from the cell culture.For example, the one or more differentiation factors that have been previously provided can be removed 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, about 10 days, or more than about 10 days before adding the gamma-secretase inhibitor.In other embodiments, the gamma-secretase inhibitor is provided to a cell culture or cell population that includes one or more differentiation factors that have been previously provided or that have been provided at about the same time as the gamma-secretase inhibitor.In a preferred embodiment, the differentiation factors that have been previously provided or that have been provided at about the same time as the gamma-secretase inhibitor include, but are not limited to, FGF-10, KAAD-cyclopamine, activin A, activin B, BMP4 and / or RA.

[0455] In some embodiments of the invention described herein, exendin 4 is provided to the cell culture or cell population to be differentiated at about the same time as the gamma secretase inhibitor. In certain embodiments, exendin 4 is provided at a concentration of at least about 0.1 ng / ml, at least about 0.2 ng / ml, at least about 0.3 ng / ml, at least about 0.4 ng / ml, at least about 0.5 ng / ml, at least about 0.6 ng / ml, at least about 0.7 ng / ml, at least about 0.8 ng / ml, at least about 0.9 ng / ml, at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml, at least about 100 ng / ml, at least about 150 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 100 ng / ml, at least about 150 ng / ml, at least about 150 ng / ml, at least about 150 ng / ml, at least about At least about 65 ng / ml, at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, at least about 100 ng / ml, at least about 150 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 550 ng / ml, at least about 600 ng / ml, at least about 650 ng / ml, at least about 700 ng / ml, at least about 750 ng / ml, at least about 800 ng / ml, at least about 850 ng / ml, at least about The antibody may be provided so as to be present in the cell culture or cell population at a concentration of at least about 900 ng / ml, at least about 950 ng / ml, or at least about 1000 ng / ml.

[0456] In a preferred process for producing endocrine precursor cells from PDX1-positive foregut endoderm cells, the cell culture or cell population of PDX1-positive foregut endoderm cells is provided with 3 μM DAPT and 40 ng / ml exendin 4. In a particularly preferred embodiment, the cells are differentiated in CMRL. In another particularly preferred process for producing endocrine precursor cells from PDX1-positive foregut endoderm cells, the cell culture or cell population of PDX1-positive foregut endoderm cells is provided with 3 μM DAPT and 40 ng / ml exendin 4 in the presence of 2 μM RA.

[0457] As described herein, in certain processes for producing endocrine precursor cells, the differentiation factors are removed from the cell culture or cell population after their addition. For example, the gamma-secretase inhibitor and / or exendin 4 can be removed within 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 after their addition. In some embodiments, the differentiation factors are not removed from the cell culture.

[0458] Cultures of endocrine precursor cells can be produced in low-serum or serum-free medium. Under certain culture conditions, serum concentrations can range from about 0.05% (v / v) to about 20% (v / v). For example, in some differentiation processes, the serum concentration of the medium can be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). % (v / v), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v), or less than about 20% (v / v). In some processes, the endocrine precursor cells are grown without serum, without serum replacement, and / or without any supplements containing insulin or insulin-like growth factors. In still other processes, the endocrine precursor cells are grown in the presence of B27. In such processes, the concentration of the B27 supplement can range from about 0.1% (v / v) to about 20% (v / v). In other embodiments, the endocrine precursor cells are grown in the absence of B27.

[0459] Monitoring the differentiation of PDX1-positive cells into endocrine precursor cells The development of PDX1-positive endoderm cells into endocrine precursor cells can be monitored by determining the expression of markers characteristic of endocrine precursor cells. In some processes, the expression of a particular marker is determined by detecting the presence or absence of the marker. Alternatively, the expression of a particular marker can be determined by measuring the level of the marker present in the cells of the cell culture or cell population. In such processes, the measurement of marker expression can be qualitative or quantitative. One method of quantifying the expression of markers produced by marker genes is by using quantitative PCR (Q-PCR). Methods for performing Q-PCR are known in the art. Other methods known in the art can also be used to quantify the expression of marker genes. For example, the expression of marker gene products can be detected by using antibodies specific to the marker gene products of interest. Certain processes require expression of marker genes characteristic of endocrine precursor cells and loss of significant expression of marker genes characteristic of hESCs, definitive endoderm, PDX1-positive foregut endoderm, extraembryonic endoderm, mesoderm, ectoderm, immature or mature pancreatic islet hormone-expressing cells, and / or other cell types.

[0460] As described further in the Examples below, a reliable marker for endocrine precursor cells is NG NGN3 gene. Thus, the endocrine precursor cells produced by the process described herein express the NGN3 marker gene, thereby producing the NGN3 gene product. Other markers for endocrine precursor cells are NKX2.2 and PAX4.

[0461] In some processes, the expression of genes indicative of hESCs, definitive endoderm cells, and / or PDX1 positive foregut endoderm cells is also monitored. For example, in some processes, the expression of AFP, SOX7, SOX1, ZIC1, and NFM is monitored. In some processes, the expression of genes indicative of immature pancreatic islet hormone expressing cells and / or mature pancreatic islet hormone expressing cells is also monitored. For example, in some embodiments, the expression of MAFB, SYP, CHGA, INS, GCG, SST, GHRL, and PAX6 is monitored.

[0462] It is understood that the expression of NGN3, NKX2.2 and / or PAX4 markers is induced in endocrine precursor cells by differentiation conditions over a range of different levels.Thus, in some embodiments described herein, the expression of NGN3, NKX2.2 and / or PAX4 markers in endocrine precursor cells or cell populations is at least about 2-fold to at least about 10,000-fold higher than the expression of NGN3, NKX2.2 and / or PAX4 markers in non-endocrine precursor cells or cell populations, such as pluripotent stem cells, definitive endoderm cells, PDX1-positive foregut endoderm cells, immature pancreatic islet hormone-expressing cells, mature pancreatic islet hormone-expressing cells, extraembryonic endoderm cells, mesoderm cells and / or ectoderm cells. In other embodiments, expression of the NGN3, NKX2.2 and / or PAX4 markers in an endocrine precursor cell or cell population is at least about 4 fold, at least about 6 fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 40 fold, at least about 80 fold, at least about 100 fold, at least about 150 fold, at least about 200 fold, at least about 500 fold, at least about 750 fold, at least about 1000 fold, at least about 2500 fold, at least about 5000 fold, at least about 7500 fold, or at least about 10000 fold higher than expression of the NGN3, NKX2.2 and / or PAX4 markers in a non-endocrine precursor cell or cell population (e.g., pluripotent stem cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and / or ectoderm cells). In some embodiments, expression of the NGN3, NKX2.2 and / or PAX4 markers in an endocrine precursor cell or cell population is much higher than expression of the NGN3, NKX2.2 and / or PAX4 markers in a non-endocrine precursor cell or cell population, such as pluripotent stem cells, definitive endoderm cells, PDX1 positive foregut endoderm cells, immature pancreatic islet hormone expressing cells, mature pancreatic islet hormone expressing cells, extraembryonic endoderm cells, mesoderm cells and / or ectodermal cells.

[0463] Enrichment, isolation and / or purification of endocrine precursor cells Regarding further aspects of the invention, endocrine precursor cells can be enriched, isolated and / or purified. In some embodiments of the invention, cell populations enriched, isolated and / or purified for endocrine precursor cells are produced by isolating such cells from cell culture.

[0464] Endocrine precursor cells produced by any of the processes described herein can be enriched, isolated and / or purified by using affinity tags specific for such cells. Examples of affinity tags specific for endocrine precursor cells are antibodies, antibody fragments, ligands, or other binding agents specific for marker molecules, such as polypeptides, that are present on the cell surface of endocrine precursor cells but are substantially absent from other cell types found in the cell cultures produced by the methods described herein. In some processes, an antibody that binds to NCAM is used as an affinity tag to enrich, isolate or purify endocrine precursor cells. In other processes, an antibody that binds to NCAM ligand NBP10, or other currently known or future discovered affinity tags, is used as an affinity tag to enrich, isolate or purify endocrine precursor cells. Any other NCAM ligand that can be used as an affinity tag can also be used. See, for example, Ronn, L. (2002) Eur J Neurosci., 16:1720-30, the entire disclosure of which is incorporated herein by reference. (see, e.g., J. Immunol. 1999, 144:1311-1315, which is incorporated by reference in its entirety). Such molecules include NBP10 fusions and NBP10 mimetics.

[0465] Methods for making antibodies and using them for cell isolation are known in the art, and such methods can be performed for use with the antibodies and endocrine precursor cells described herein. In one process, an antibody that binds to NCAM is attached to magnetic beads and then bound to endocrine precursor cells in a cell culture that has been enzymatically treated to reduce cell-cell and substrate adhesion. The cell / antibody / beads complex is then exposed to a movable magnetic field that is used to isolate the bead-bound endocrine precursor cells from unbound cells. Once the endocrine precursor cells are physically isolated from other cells in the culture, the antibody binding is disrupted and the cells are re-plated in appropriate tissue culture medium. If necessary, the isolated cell composition can be further purified by alternative affinity-based methods or by a further series of enrichments using the same or different markers specific for endocrine precursor cells.

[0466] Additional methods of obtaining enriched, isolated or purified endocrine precursor cell cultures or endocrine precursor cell populations may also be used. For example, in some embodiments, NCAM antibodies are incubated with endocrine precursor cell-containing cell cultures that have been treated to reduce cell-cell and substrate adhesion. (We do not use a secondary antibody. The NCAM antibody is directly conjugated to either PE, APC, or FITC.) The cells are then washed, centrifuged, and resuspended. The cell suspension is then incubated with a secondary antibody, such as a FITC-conjugated antibody, capable of binding to the primary antibody. The cells are then washed, centrifuged, and resuspended in buffer. The cell suspension is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). NCAM-positive cells are collected separately from NCAM-negative cells, thereby isolating such cell types. If necessary, the isolated cell composition may be further purified by using alternative affinity-based methods or by a further series of sorts using the same or different markers specific for endocrine precursor cells. Alternatively, the isolated cell composition can be further purified by negatively selecting for markers that are present on most cells in the cell population other than endocrine precursor cells.

[0467] In yet another process, endocrine precursor cells are enriched, isolated and / or purified using a ligand or other molecule that binds to NCAM, in some processes this molecule is NBP10 or a fragment thereof, a fusion thereof, or a mimetic thereof.

[0468] In some embodiments of the processes described herein, a nucleic acid encoding green fluorescent protein (GFP) or another nucleic acid encoding an expressible fluorescent marker gene (e.g., yellow fluorescent protein (YFP) or luciferase) is used to label NCAM-positive cells. For example, in some embodiments, at least one copy of a nucleic acid encoding GFP or a biologically active fragment thereof downstream of the NCAM promoter, NGN3 promoter, PAX4 promoter, or the promoter of any endocrine precursor cell-specific gene is introduced into pluripotent cells, preferably human embryonic stem cells, such that expression of the GFP gene product or a biologically active fragment thereof is under the control of the NCAM promoter, NGN3 promoter, or PAX4 promoter. In some embodiments, the entire coding region of the nucleic acid encoding NCAM, NGN3, or PAX4 is replaced with a nucleic acid encoding GFP or a biologically active fragment thereof. In other embodiments, a nucleic acid encoding GFP or a biologically active fragment thereof is fused in frame to at least a portion of a nucleic acid encoding NCAM, NGN3, or PAX4, thereby generating a fusion protein. In such an embodiment, the fusion protein has a fluorescent activity comparable to that of GFP. Maintain sexuality.

[0469] The fluorescently labeled cells, e.g., the pluripotent cells, are differentiated into endocrine precursor cells as described herein. Since endocrine precursor cells express the fluorescent marker gene, whereas other cell types do not, endocrine precursor cells can be isolated from other cell types. In some embodiments, the cell suspension containing a mixture of fluorescently labeled endocrine precursor cells and unlabeled non-endocrine precursor cells is sorted using FACS. The endocrine precursor cells are collected separately from the non-fluorescent cells, thereby isolating the endocrine precursors. If necessary, the isolated cell composition can be further purified by additional rounds of sorting using the same or different markers specific for endocrine precursor cells.

[0470] In a preferred process, after endoderm cell cultures have been induced to differentiate into the endocrine precursor lineage, the endocrine precursor cells are enriched, isolated and / or purified from other non-endocrine precursor cells. The enrichment, isolation and purification procedures described above can be used with such cultures at any stage of differentiation.

[0471] In addition to the above procedures, endocrine precursor cells can be isolated by other techniques for cell isolation. Additionally, endocrine precursor cells can be enriched or isolated by serial subculture methods under growth conditions that promote the selective survival or selective proliferation of endocrine precursor cells.

[0472] Using the methods described herein, enriched, isolated and / or purified populations of endocrine precursor cells and / or tissues can be produced from pluripotent cell cultures or cell populations, such as stem cell cultures or populations, that have been differentiated at least some times in vitro. In some methods, the cells are randomly differentiated. However, in preferred methods, the cells are directed to differentiate primarily into endocrine precursor cells. Some preferred enrichment, isolation and / or purification methods involve the in vitro production of endocrine precursor cells from human embryonic stem cells.

[0473] Using the methods described herein, cell populations or cell cultures can be enriched at least about 2-fold to about 1000-fold in endocrine precursor cell content compared to untreated cell populations or cell cultures. In some embodiments, endocrine precursor cells can be enriched at least about 5-fold to about 500-fold compared to untreated cell populations or cell cultures. In other embodiments, endocrine precursor cells can be enriched at least about 10-fold to about 200-fold compared to untreated cell populations or cell cultures. In still other embodiments, endocrine precursor cells can be enriched at least about 20-fold to about 100-fold compared to untreated cell populations or cell cultures. In still other embodiments, endocrine precursor cells can be enriched at least about 40-fold to about 80-fold compared to untreated cell populations or cell cultures. In certain embodiments, endocrine precursor cells can be enriched at least about 2-fold to about 20-fold compared to untreated cell populations or cell cultures.

[0474] Compositions Comprising Endocrine Precursor Cells Some embodiments of the present invention relate to cell compositions, such as cell cultures or cell populations, comprising endocrine precursor cells, which are pluripotent cells capable of differentiating into cells of the endocrine system, such as pancreatic islet hormone-expressing cells. According to certain embodiments, the endocrine precursor cells are mammalian cells, and in preferred embodiments, such cells are human cells.

[0475] Other embodiments of the invention relate to compositions, such as cell cultures or cell populations, that include endocrine precursor cells and cells that are less specifically differentiated than endocrine precursor cells. In such embodiments, the cells that are less specifically differentiated than endocrine precursor cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 3 ... Less than 0%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%.

[0476] Other embodiments relate to compositions, such as cell cultures or cell populations, that include endocrine precursor cells and cells that are more specifically differentiated than endocrine precursor cells, such as immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells. In such embodiments, the cells that are more specifically differentiated than endocrine precursor cells comprise less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.

[0477] Certain other embodiments of the invention relate to compositions, such as cell cultures or cell populations, that include endocrine precursor cells and cells of one or more cell types selected from the group consisting of hESCs, preprimitive streak cells, endodermal mesoderm cells, definitive endoderm cells, PDX1-negative foregut endoderm cells, PDX1-positive foregut endoderm cells (PDX1-positive pancreatic endoderm cells), and mesoderm cells. In some embodiments, hESCs represent less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In some embodiments, preprimitive streak cells represent less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In other embodiments, definitive endoderm cells represent less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In still other embodiments, definitive endoderm cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In still other embodiments, PDX1-negative foregut endoderm cells are less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.In certain embodiments, PDX1-positive foregut endoderm cells are less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In still other embodiments, mesodermal cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.

[0478] Certain other embodiments of the invention relate to compositions, such as cell cultures or cell populations, that include endocrine precursor cells and cells of one or more cell types selected from the group consisting of immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells. In some embodiments, the immature pancreatic islet hormone-expressing cells comprise less than about 25%, less than about 20%, less than about 10%, or less than about 20% of the total cells in the culture. Less than about 5%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In certain embodiments, mature pancreatic islet hormone-expressing cells are less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.

[0479] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, produced by the processes described herein and comprising endocrine precursor cells as the majority cell type. In some embodiments, the processes described herein produce endocrine precursor cells that are at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about 89%, at least about 98%, at least about 9 ... In a preferred embodiment, the cells of the cell culture or cell population comprise at least about 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% human cells.In other embodiments, the processes described herein produce cell cultures and / or cell populations that comprise at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% endocrine precursor cells. In a preferred embodiment, the cells of the cell cultures or cell populations comprise human cells. In some embodiments, the percentage of endocrine precursor cells in a cell culture or cell population is calculated without taking into account any feeder cells remaining in the culture.

[0480] Yet another embodiment of the present invention relates to compositions such as cell cultures or cell populations that comprise a mixture of endocrine precursor cells and PDX1-positive foregut endoderm cells.For example, cell cultures or cell populations can be produced that comprise at least about 5 endocrine precursor cells for every about 95 PDX1-positive foregut endoderm cells.In other embodiments, cell cultures or cell populations can be produced that comprise at least about 95 endocrine precursor cells for every about 5 PDX1-positive foregut endoderm cells.Furthermore, cell cultures or cell populations that comprise other ratios of endocrine precursor cells and PDX1-positive foregut endoderm cells are contemplated. For example, at least about 1 endocrine precursor cell for every about 1,000,000 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 100,000 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 10,000 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 1,000 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 500 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 100 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 10 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 5 PDX1-positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 4 PDX1-positive foregut endoderm cells progenitor cells, at least about 1 endocrine precursor cell for every about 2 PDX1 positive foregut endoderm cells, at least about 1 endocrine precursor cell for every about 1 PDX1 positive foregut endoderm cell, at least about 2 endocrine precursor cells for every about 1 PDX1 positive foregut endoderm cell, at least about 4 endocrine precursor cells for every about 1 PDX1 positive foregut endoderm cell, at least about 5 endocrine precursor cells for every about 1 PDX1 positive foregut endoderm cell, at least about 10 endocrine precursor cells for every about 1 PDX1 positive foregut endoderm cell, at least about 20 endocrine precursor cells for every about 1 PDX1 positive foregut endoderm cell Compositions comprising endocrine precursor cells, at least about 50 endocrine precursor cells for about every PDX1 positive foregut endoderm cell, at least about 100 endocrine precursor cells for about every PDX1 positive foregut endoderm cell, at least about 1000 endocrine precursor cells for about every PDX1 positive foregut endoderm cell, at least about 10,000 endocrine precursor cells for about every PDX1 positive foregut endoderm cell, at least about 100,000 endocrine precursor cells for about every PDX1 positive foregut endoderm cell, and at least about 1,000,000 endocrine precursor cells for about every PDX1 positive foregut endoderm cell are contemplated.

[0481] Yet another embodiment of the present invention relates to compositions such as cell cultures or cell populations that include a mixture of endocrine precursor cells and immature and / or mature pancreatic islet hormone-expressing cells. For example, cell cultures or cell populations can be produced that include at least about 5 endocrine precursor cells for every about 95 immature and / or mature pancreatic islet hormone-expressing cells. In other embodiments, cell cultures or cell populations can be produced that include at least about 95 endocrine precursor cells for every about 5 immature and / or mature pancreatic islet hormone-expressing cells. Additionally, cell cultures or cell populations that include other ratios of endocrine precursor cells to immature and / or mature pancreatic islet hormone-expressing cells are contemplated. For example, at least about 1 endocrine precursor cell for about 1,000,000 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 100,000 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 10,000 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 1,000 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 1 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 100 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 100 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells 1 endocrine precursor cell, at least about 1 endocrine precursor cell for about 10 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 5 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 4 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 2 immature pancreatic islet hormone-expressing cells and / or mature pancreatic islet hormone-expressing cells, at least about 1 endocrine precursor cell for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 2 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell,At least about 4 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 5 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 10 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 20 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 50 endocrine precursor cells for each immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 100 endocrine precursor cells for each immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 1000 endocrine precursor cells for each immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 10,000 endocrine precursor cells for each immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, at least about 100,000 endocrine precursor cells for each immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell, endocrine precursor cells, as well as compositions comprising at least about 1,000,000 endocrine precursor cells for about 1 immature pancreatic islet hormone-expressing cell and / or mature pancreatic islet hormone-expressing cell are contemplated.

[0482] In some embodiments of the present invention, the PDX1 positive foregut endoderm cells from which endocrine precursor cells are produced are derived from human pluripotent cells, such as human pluripotent stem cells. In certain embodiments, the human pluripotent cells are derived from the morula, the inner cell mass of an embryo, or the gonadal ridges of an embryo. In certain other embodiments, the human pluripotent cells are derived from gonad tissue or embryo tissue that has a multicellular structure and is generated after the embryonic stage.

[0483] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, comprising human cells (e.g., human endocrine precursor cells), wherein expression of the NGN3 marker is greater than expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers in at least about 2% of the human cells. In other embodiments, expression of the NGN3 marker is greater than expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and / or PAX6 markers in at least about 5% of the human cells, at least about 10% of the human cells, at least about 15% of the human cells, at least about 20% of the human cells, at least about 25% of the human cells, at least about 30% of the human cells, at least about 35% of the human cells, at least about 40% of the human cells, at least about 45% of the human cells, at least about 50% of the human cells, at least about 55% of the human cells, at least about 60% of the human cells, at least about 65% of the human cells, at least about 70% of the human cells, at least about 75% of the human cells, at least about 80% of the human cells, at least about 85% of the human cells, at least about 90% of the human cells, at least about 95% of the human cells, or at least about 98% of the human cells. In some embodiments, the percentage of human cells in a cell culture or cell population in which expression of NGN3 is greater than expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers is calculated without taking into account feeder cells.

[0484] Some embodiments of the present invention are understood to relate to compositions, such as cell cultures or cell populations, comprising human endocrine precursor cells, wherein expression of NKX2.2 and PAX4 is greater than expression of AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers in at least about 2% to more than about 98% of the human cells. In some embodiments, expression of NKX2.2 and / or PAX4 is greater than expression of AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers in at least about 5% of the human cells, at least about 10% of the human cells, at least about 15% of the human cells, at least about 20% of the human cells, at least about 25% of the human cells, at least about 30% of the human cells, at least about 35% of the human cells, at least about 40% of the human cells, at least about 45% of the human cells, at least about 50% of the human cells, at least about 5 ... In at least about 60%, in at least about 65%, in at least about 70%, in at least about 75%, in at least about 80%, in at least about 85%, in at least about 90%, in at least about 95%, or in at least about 98% of the human cells, expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and / or PAX6 markers is greater. In some embodiments, the percentage of human cells in a cell culture or cell population in which expression of NKX2.2 and / or PAX4 is greater than expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL, and / or PAX6 markers is calculated without taking into account feeder cells.

[0485] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, comprising mammalian cells differentiated in vitro from definitive endoderm, e.g., human cells differentiated in vitro from definitive endoderm, wherein expression of the NGN3, NKX2.2 and / or PAX4 markers is greater than expression of the AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers in at least about 2% of the cells differentiated in vitro from definitive endoderm.In other embodiments, expression of the NGN3, NKX2.2 and / or PAX4 markers is in at least about 5% of cells differentiated from definitive endoderm in vitro, in at least about 10% of cells differentiated from definitive endoderm in vitro, in at least about 15% of cells differentiated from definitive endoderm in vitro, in at least about 20% of cells differentiated from definitive endoderm in vitro, in at least about 25% of cells differentiated from definitive endoderm in vitro, in at least about 30% of cells differentiated from definitive endoderm in vitro, in at least about 35% of cells differentiated from definitive endoderm in vitro, in at least about 40% of cells differentiated from definitive endoderm in vitro, in at least about 45% of cells differentiated from definitive endoderm in vitro, in at least about 50% of cells differentiated from definitive endoderm in vitro, in at least about 55% of cells differentiated from definitive endoderm in vitro, in at least about 60% of cells differentiated from definitive endoderm in vitro, in greater than expression of AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers in at least about 65% of cells differentiated in vitro from definitive endoderm, in at least about 70% of cells differentiated in vitro from definitive endoderm, in at least about 75% of cells differentiated in vitro from definitive endoderm, in at least about 80% of cells differentiated in vitro from definitive endoderm, in at least about 85% of cells differentiated in vitro from definitive endoderm, in at least about 90% of cells differentiated in vitro from definitive endoderm, in at least about 95% of cells differentiated in vitro from definitive endoderm, or in at least about 98% of cells differentiated in vitro from definitive endoderm.

[0486] In preferred embodiments of the invention, the cell cultures and / or cell populations of endocrine precursor cells comprise human endocrine precursor cells that are non-recombinant cells. In such embodiments, the cell cultures and / or cell populations are devoid of or substantially free of recombinant human endocrine precursor cells.

[0487] In some embodiments of the invention, the cell cultures and / or cell populations comprising endocrine precursor cells also comprise a medium comprising a gamma-secretase inhibitor. In preferred embodiments, the gamma-secretase inhibitor is N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). In some preferred embodiments, the DAPT concentration is at least about 1 μM. In more preferred embodiments, the DAPT concentration is at least about 3 μM. In some embodiments, the medium also comprises a factor selected from retinoic acid (RA) and exendin 4 (Ex4). In some embodiments, the medium is CMRL.

[0488] The processes described herein can be used to produce compositions comprising endocrine precursor cells substantially free of other cell types. In some embodiments of the invention, the endocrine precursor cell populations or cell cultures produced by the methods described herein are substantially free of cells that significantly express AFP, SOX7, SOX1, ZIC1 and / or NFM markers. In some embodiments, the endocrine precursor cell populations of the cell cultures produced by the methods described herein are substantially free of cells that significantly express AFP, SOX7, SOX1, ZIC1, NFM, MAFA, SYP, CHGA, INS, GCG, SST, GHRL and / or PAX6 markers.

[0489] In one embodiment of the present invention, endocrine precursor cells based on expression of markers The description is NGN3 high, NKX2.2 high, PAX4 high, AFP low, SOX7 low, SOX1 low, ZIC1 low, NFM low, MAFA low, SYP low, CHGA low, INS low, GCG low, SST low, GHRL low and / or PAX6 low.

[0490] Pancreatic endocrine precursor cell screening Certain screening methods described herein relate to methods for identifying at least one differentiation factor capable of promoting differentiation of endocrine precursor cells.

[0491] In some embodiments of these differentiation screening methods, a cell population is obtained, including endocrine precursor cells, for example human endocrine precursor cells. A candidate differentiation factor is then provided to the cell population. At a first time point, prior to or approximately simultaneous with providing the candidate differentiation factor, expression of a marker is determined. Alternatively, expression of the marker can be determined after providing the candidate differentiation factor. At a second time point after the first time point and after providing the cell population with the candidate differentiation factor, expression of the same marker is again determined. Whether the candidate differentiation factor is capable of promoting differentiation of endocrine precursor cells is determined by comparing expression of the marker at the first time point with expression of the marker at the second time point. If expression of the marker at the second time point is increased or decreased compared to expression of the marker at the first time point, the candidate differentiation factor is capable of promoting differentiation of endocrine precursor cells.

[0492] Some embodiments of the screening methods described herein utilize a cell population or cell culture comprising human endocrine precursor cells. For example, the cell population can be a substantially purified population of endocrine precursor cells. Alternatively, the cell population can be a population enriched for human endocrine precursor cells, in which at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, or at least more than about 97% of the human cells in the cell population are human endocrine precursor cells. In other embodiments described herein, the cell population comprises human cells, where at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least more than about 85% of the human cells are human endocrine precursor cells. In some embodiments, the cell population comprises non-human cells, e.g., non-human feeder cells. In other embodiments, the cell population comprises human feeder cells. In such embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least more than about 95% of the human cells other than the feeder cells are human endocrine precursor cells.

[0493] In embodiments of the screening methods described herein, a cell population is contacted or otherwise provided with a candidate (test) compound. The candidate differentiation factor may include any molecule that may have the ability to promote differentiation of human endocrine precursor cells. In some embodiments described herein, the candidate differentiation factor includes a molecule known to be a differentiation factor for one or more types of cells. In alternative embodiments, the candidate differentiation factor includes a molecule that is not known to promote cell differentiation. In a preferred embodiment, the candidate differentiation factor includes a molecule that is not known to promote differentiation of human endocrine precursor cells.

[0494] In some embodiments of the screening methods described herein, the candidate differentiation factor comprises a small molecule. In a preferred embodiment, the small molecule is a molecule with a molecular weight of about 10,000 amu or less.

[0495] In other embodiments described herein, the candidate differentiation factor comprises a polypeptide. The polypeptide can be any polypeptide, such as, but not limited to, a glycoprotein, a lipoprotein, an extracellular matrix protein, a cytokine, a chemokine, a peptide hormone, an interleukin, or a growth factor. Preferred polypeptides include growth factors.

[0496] In some embodiments of the screening methods described herein, the candidate differentiation factor is amphiregulin, B-lymphocyte stimulating factor, IL-16, thymopoietin, TRAIL / Apo-2, pre-B cell colony-enhancing factor, endothelial differentiation-associated factor 1 (EDF1), endothelial monocyte-activating polypeptide II, macrophage migration inhibitory factor (MIF), natural killer cell-enhancing factor (NKEFA), bone morphogenetic protein 2, bone morphogenetic protein 8 (osteogeneic protein 2), bone morphogenetic protein 6, bone morphogenetic protein 7, binding protein 9, or the like. CTGF, CGI-149 protein (neuroendocrine differentiation factor), cytokine A3 (macrophage inflammatory protein 1-α), gliablastoma cell differentiation-related protein (GBDR1), hepatocellular carcinoma-derived growth factor, neuromedin U-25 precursor, vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B), T cell-specific RANTES precursor, thymic dendritic cell-derived factor 1, transferrin, interleukin-1 (IL1), interleukin-2 (IL2), interleukin-3 (IL3), interleukin -4 (IL4), interleukin-5 (IL5), interleukin-6 (IL6), interleukin-7 (IL7), interleukin-8 (IL8), interleukin-9 (IL9), interleukin-10 (IL10), interleukin-11 (IL11), interleukin-12 (IL12), interleukin-13 (IL13), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, thrombopoietin, vitamin D 3 , epidermal growth factor (EGF), brain-derived neurotrophic factor, leukemia inhibitory factor, thyroid hormone, basic fibroblast growth factor (bFGF), aFGF, FGF-4, FGF-6, FGF-7 / keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), platelet-derived growth factor-BB, beta nerve growth factor, activin A, transforming growth factor beta 1 (TGF-beta 1), interferon-alpha, interferon-beta, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta, burst promoting activity (BPA), erythroid promoting activity (EPA), PGE 2, insulin growth factor-1 (IGF-1), IGF-II, neurotrophin growth factor (NGF), neurotrophin-3, neurotrophin 4 / 5, ciliary neurotrophic factor, glial-derived nexin, dexamethasone, β-mercaptoethanol, retinoic acid, butylated hydroxyanisole, 5-azacytidine, amphotericin B, ascorbic acid, ascorbate, isobutylxanthine, indomethacin, β-glycerol phosphate, nicotinamide, DMSO, thiazolidinediones, TWS119, oxytocin, vasopressin, melanocyte-stimulating hormone, corticotropin, lipotropin, thyrotropin tropin, growth hormone, prolactin, luteinizing hormone, human chorionic gonadotropin, follicle-stimulating hormone, corticotropin-releasing factor, gonadotropin-releasing factor, prolactin-releasing factor, prolactin inhibitory factor, growth hormone-releasing factor, somatostatin, thyrotropin-releasing factor, calcitonin gene-related peptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, gastrin, secretin, cholecystokinin, motilin, vasoactive intestinal peptide, substance P, pancreatic polypeptide, peptide tyrosine, neuropeptide tyrosine, insulin, glucagon, placental lactogen, relaxin, angiotensin II, calcitonin calctriol, atrial natriuretic peptide, and melatonin, thyroxine In some embodiments, the growth factor may be selected from the group consisting of acetylcholine, triiodothyronine, calcitonin, estradiol, estrone, progesterone, testosterone, cortisol, corticosterone, aldosterone, epinephrine, norepinephrine, androstiene, calcitriol, collagen, dexamethasone, β-mercaptoethanol, retinoic acid, butylated hydroxyanisole, 5-azacytidine, amphotericin B, ascorbic acid, ascorbate, isobutylxanthine, indomethacin, β-glycerol phosphate, nicotinamide, DMSO, thiazolidinediones, and TWS119.

[0497] In some embodiments of the screening methods described herein, the candidate differentiation factor is provided to the cell population at one or more concentrations. In some embodiments, the candidate differentiation factor is provided to the cell population such that the concentration of the candidate differentiation factor in the medium surrounding the cells ranges from about 0.1 ng / ml to about 10 mg / ml. In some embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells ranges from about 1 ng / ml to about 1 mg / ml. In other embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells ranges from about 10 ng / ml to about 100 μg / ml. In yet other embodiments, the concentration of the candidate differentiation factor in the medium surrounding the cells ranges from about 100 ng / ml to about 10 μg / ml.In a preferred embodiment, the concentration of the candidate differentiation factor in the medium surrounding the cells is about 5 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, about 200 ng / ml, about 225 ng / ml, about 250 ng / ml, about 275 ng / ml, about 300 ng / ml, about 325 ng / ml, about 350 ng / ml, about 375 ng / ml, about 400 ng / ml, about 425 ng / ml, about 450 ng / ml, about 50 ng / ml, about 55 ng / ml, about 56 ng / ml, about 57 ng / ml, about 58 ng / ml, about 59 ng / ml, about 60 ng / ml, about 61 ng / ml, about 62 ng / ml, about 63 ng / ml, about 64 ng / ml, about 65 ng / ml, about 66 ng / ml, about 67 ng / ml, about 68 ng / ml, about 69 ng / ml, about 70 ng / ml, about 72 ng / ml, about 75 ng / ml, about 76 ng / ml, about 77 ng / ml, about 78 ng / ml, about 79 ng / ml, about 80 ng / ml, about 82 ng / ml, about 85 ng / ml, about 86 ng / ml, about 87 ng / ml, about 88 ng / ml, about 89 ng / ml, about 90 ng / ml, about 91 ng / ml, about 92 ng / ml, about 93 ng / ml, about g / ml, approximately 475ng / ml, approximately 500ng / ml, approximately 525ng / ml, approximately 550ng / ml, approximately 575ng / ml, approximately 600ng / ml, approximately 625ng / ml, approximately 650ng / ml, approximately 675ng / ml, approximately 700ng / ml, approximately 7 25ng / ml, approximately 750ng / ml, approximately 775ng / ml, approximately 800ng / ml, approximately 825ng / ml, approximately 850ng / ml, approximately 875ng / ml, approximately 900ng / ml, approximately 925ng / ml, approximately 950ng / ml, approximately 975ng / ml , about 1μg / ml, about 2μg / ml, about 3μg / ml, about 4μg / ml, about 5μg / ml, about 6μg / ml, about 7μg / ml, about 8μg / ml, about 9μg / ml, about 10μg / ml, about 11μg / ml, about 12μg / ml, about 13μg / ml ml, about 14μg / ml, about 15μg / ml, about 16μg / ml, about 17μg / ml, about 18μg / ml, about 19μg / ml, about 20μg / ml, about 25μg / ml, about 50μg / ml, about 75μg / ml, about 100μg / ml, about 125 μg / ml, approximately 150 μg / ml, approximately 175 μg / ml, approximately 200 μg / ml, approximately 250 μg / ml, approximately 300 μg / ml, approximately 350 μg / ml, approximately 400 μg / ml, approximately 450 μg / ml, approximately 500 μg / ml, approximately 550 μg / ml, approximately 600 μg / ml, about 650 μg / ml, about 700 μg / ml, about 750 μg / ml, about 800 μg / ml, about 850 μg / ml, about 900 μg / ml, about 950 μg / ml, about 1000 μg / ml, or more than about 1000 μg / ml.

[0498] In some embodiments, the steps of the screening methods described herein include determining the expression of at least one marker at a first time point and at a second time point. In some of these embodiments, the first time point can be before or about the same time as providing the cell population with the candidate differentiation factor. Alternatively, in some embodiments, the first time point is after providing the cell population with the candidate differentiation factor. In some embodiments, the expression of multiple markers is determined at the first time point.

[0499] Some preferred markers for use in the above embodiments include one or more markers selected from the group consisting of NGN3, NKX2.2 and PAX4.

[0500] In addition to determining the expression of at least one marker at a first time point, some embodiments of the screening methods described herein consider determining the expression of at least one marker at a second time point after the first time point and after providing the cell population with a candidate differentiation factor. In such embodiments, the expression of the same marker is determined at both the first time point and the second time point. In some embodiments, the expression of multiple markers is determined at both the first time point and the second time point. In such embodiments, the expression of the same multiple markers is determined at both the first time point and the second time point. In some embodiments, the marker expression is determined at multiple time points, each time point after the first time point and each time point after providing the cell population with a candidate differentiation factor. In certain embodiments, the marker expression is determined by Q-PCR. In other embodiments, the marker expression is determined by immunocytochemistry.

[0501] In certain embodiments of the screening method described herein, the marker having its expression at the first time point and the second time point is a marker related to the differentiation of endocrine precursor cells into cells that are precursors of terminally differentiated cells that make pancreatic islet tissue. Such cells can include immature pancreatic islet hormone-expressing cells. In some embodiments, the marker indicates endocrine precursor cells. In a preferred embodiment, the marker is NGN3, NKX2.2, NKX6.1, PAX4, PDX1, insulin, ghrelin and / or glucagon.

[0502] In some embodiments of the screening methods described herein, a sufficient amount of time elapses between providing the cell population with the candidate differentiation factor and determining marker expression at a second time point. The sufficient amount of time between providing the cell population with the candidate differentiation factor and determining marker expression at a second time point can be as little as about 1 hour to as much as about 10 days. In some embodiments, expression of at least one marker is determined multiple times after providing the cell population with the candidate differentiation factor. In some embodiments, a sufficient time is at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 54 hours, at least about 60 hours, at least about 66 hours, at least about 72 hours, at least about 78 hours, at least about 84 hours, at least about 90 hours, at least about 96 hours, at least about 102 hours, at least about 108 hours, at least about 114 hours, at least about 120 hours, at least about 126 hours, at least about 132 hours, at least about 148 hours, at least about 152 hours, at least about 162 hours, at least about 172 hours, at least about 182 hours, at least about 192 hours, at least about 204 hours, at least about 216 hours, at least about 220 hours, at least about 232 hours, at least about 240 hours, at least about 250 hours, at least about 260 hours, at least about 270 hours, at least about 280 hours, at least about 290 hours, at least about 300 hours, at least about 310 hours, at least about 320 hours, at least about 330 hours, at least about 340 hours, at least about 350 hours, at least about 360 hours, at least about 370 hours, at least about 380 hours, at least about 390 hours, at least about 400 hours, at least about 410 hours, at least about 420 hours, at least about 430 hours, at least about 440 hours, at least about 450 hours, at least about 460 hours, at least about 470 hours, at least about 480 hours, at least about 138 hours, at least about 144 hours, at least about 150 hours, at least about 156 hours, at least about 162 hours, at least about 168 hours, at least about 174 hours, at least about 180 hours, at least about 186 hours, at least about 192 hours, at least about 198 hours, at least about 204 hours, at least about 210 hours, at least about 216 hours, at least about 222 hours, at least about 228 hours, at least about 234 hours, at least about 240 hours, at least about 246 hours, at least about 252 hours, at least about 258 hours, at least about 264 hours, or at least about 270 hours.

[0503] In some embodiments of the methods described herein, it is further determined whether the expression of the marker at the second time point is increased or decreased compared to the expression of the marker at the first time point. Increased or decreased expression of at least one marker indicates that the candidate differentiation factor can promote differentiation of endocrine precursor cells. Similarly, when the expression of multiple markers is determined, it is further determined whether the expression of the multiple markers at the second time point is increased or decreased compared to the expression of the multiple markers at the first time point. Increased or decreased marker expression can be determined by measuring or otherwise evaluating the amount, level or activity of the marker in the cell population at the first time point and the second time point. Such a determination can be relative to the expression of other markers, e.g., housekeeping genes, or absolute. Marker Expression In certain embodiments where marker expression is increased at the second time point compared to the first time point, the increase is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or at least more than about 100-fold. In some embodiments, the increase is less than 2-fold. In embodiments where marker expression is decreased at the second time point compared to the first time point, the decrease is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or at least more than about 100-fold. In some embodiments, the decrease is less than 2-fold.

[0504] Generation of immature pancreatic islet hormone-expressing cells The present invention relates to a method for producing immature pancreatic islet hormone-expressing cells starting from hESC.As mentioned above, immature pancreatic islet hormone-expressing cells can be produced by first differentiating hESC to produce definitive endoderm cells, differentiating definitive endoderm cells to produce foregut endoderm cells, differentiating foregut endoderm to produce PDX1-positive foregut endoderm cells, and then further differentiating PDX1-positive foregut endoderm cells to produce endocrine precursor cells.In some embodiments, this process continues by further differentiating endocrine precursor cells into immature pancreatic islet hormone-expressing cells.

[0505] In some embodiments of the invention, differentiation of endocrine precursor cells into immature pancreatic islet hormone-expressing cells proceeds by continuing incubation of the culture of endocrine precursor cells with a gamma-secretase inhibitor for a sufficient time for the cells to substantially cease expression of NGN3, begin expression of PAX6, and become competent for the cells to express at least one pancreatic islet cell hormone. In some embodiments, the gamma-secretase inhibitor is removed 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, about 10 days, or more than about 10 days after incubation of the endocrine precursor cells. In a preferred embodiment, the gamma-secretase inhibitor is N-[N-(3,5-difluorophenacetyl) )-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT).

[0506] Certain processes for producing immature pancreatic islet hormone-expressing cells disclosed herein are mediated by providing one or more factors selected from the group consisting of nicotinamide, exendin 4, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1) to a cell culture or cell population comprising human endocrine precursor cells. In some embodiments, all four of the factors are provided simultaneously. In some embodiments, one or more of the factors are provided to the cell culture prior to differentiation of endocrine precursor cells and remain present in the cell culture during differentiation of at least some of the cells in the cell culture into endocrine precursor cells. In other embodiments, one or more of the factors are provided to the cell culture at about the same time as differentiation of the majority of the cells into endocrine precursor cells and remain present in the cell culture until at least the majority of the cells have differentiated into immature pancreatic islet hormone-expressing cells. In some embodiments of the invention, one or more of the factors are provided at the beginning of the differentiation process, for example at the hESC stage, and are maintained in the cell culture during differentiation into immature pancreatic islet hormone-expressing cells.

[0507] In some of the processes for producing immature pancreatic islet hormone-expressing cells disclosed herein, nicotinamide, nicotinamide adenine dinucleotide (NAD) or nicotinic acid is provided to the cells so that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, nicotinamide is provided at a concentration of at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, present in the cell culture or cell population at a concentration of at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM, or at least about 1000 mM.

[0508] In other processes for producing immature pancreatic islet hormone-expressing cells disclosed herein, exendin 4 is provided to cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, exendin 4 is provided to cells at a concentration of at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml. at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, at least about 100 ng / ml, at least about 15 ... or at least about 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml.

[0509] In yet another process for producing immature pancreatic islet hormone-expressing cells disclosed herein, HGF is provided to cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, HGF is provided at a concentration of at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml, at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, at least about 100 ng / ml, at least about 15 ... or at least about 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml in the cell culture or cell population. exist.

[0510] In yet another process for producing immature pancreatic islet hormone-expressing cells disclosed herein, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. In some embodiments, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells into immature pancreatic islet hormone-expressing cells. The antibody is present in the cell culture or cell population at a concentration of 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml.

[0511] In certain embodiments of the process for producing immature pancreatic islet hormone-expressing cells as described in the present invention, one or more of nicotinamide, exendin 4, HGF and IGF1 are supplied after removing one or more pre-supplied differentiation factors from the cell culture. In other embodiments, one or more of nicotinamide, exendin 4, HGF and IGF1 are supplied to a cell culture or cell population that includes one or more differentiation factors that have been pre-supplied or that have been supplied approximately simultaneously with one or more of nicotinamide, exendin 4, HGF and IGF1. In a preferred embodiment, the differentiation factors that have been pre-supplied or that have been supplied approximately simultaneously with one or more of nicotinamide, exendin 4, HGF and IGF1 include, but are not limited to, DAPT, FGF-10, KAAD-cyclopamine, activin A, activin B, BMP4 and / or RA.

[0512] In one process for producing immature pancreatic islet hormone-expressing cells from endocrine precursor cells, a cell culture or population of endocrine precursor cells is fed with 10 mM nicotinamide, 40 ng / ml exendin 4, 25 ng / ml HGF, and 50 ng / ml IGF1. In a preferred process, the cells are differentiated in Dulbecco's Modified Eagle's Medium (DMEM).

[0513] As described herein, in certain processes for producing immature pancreatic islet hormone-expressing cells, one or more of the differentiation factors are removed from the cell culture or cell population after their addition.For example, nicotinamide can be removed within 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 after addition.In some embodiments, differentiation factors are not removed from the cell culture.

[0514] Cultures of immature pancreatic islet hormone-expressing cells can be produced in low-serum or serum-free medium. Under certain culture conditions, serum concentrations can range from about 0.05% (v / v) to about 20% (v / v). For example, in some differentiation processes, the serum concentration of the medium can be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). ), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v), or less than about 20% (v / v). In some processes, the immature pancreatic islet hormone-expressing cells are grown without serum, without serum replacement, and / or without any supplements containing insulin or insulin-like growth factors.

[0515] In yet other processes, immature pancreatic islet hormone-expressing cells are grown in the presence of B27. In such processes, the concentration of B27 supplement can range from about 0.1% (v / v) to about 20% (v / v), or at concentrations greater than about 20% (v / v). In certain processes, the concentration of B27 in the medium is about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), about 5% (v / v), about 6% (v / v), about 7% (v / v), about 8% (v / v), about 9% (v / v), about 10% (v / v), about 15% (v / v), or about 20% (v / v). Alternatively, the concentration of B27 supplement added can be measured in multiples of the concentration of a commercially available B27 stock solution. For example, B27 is available from Invitrogen (Carlsbad, CA) as a 50× stock solution. A sufficient amount of this stock solution is added to a sufficient volume of growth medium to obtain a medium supplemented with the desired amount of B27. For example, adding 10 ml of 50× B27 stock solution to 90 ml of growth medium will obtain a growth medium supplemented with 5× B27. The concentration of B27 supplement in the medium can be about 0.1x, about 0.2x, about 0.3x, about 0.4x, about 0.5x, about 0.6x, about 0.7x, about 0.8x, about 0.9x, about 1x, about 1.1x, about 1.2x, about 1.3x, about 1.4x, about 1.5x, about 1.6x, about 1.7x, about 1.8x, about 1.9x, about 2x, about 2.5x, about 3x, about 3.5x, about 4x, about 4.5x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 11x, about 12x, about 13x, about 14x, about 15x, about 16x, about 17x, about 18x, about 19x, about 20x, and greater than about 20x.

[0516] Monitoring the production of immature pancreatic islet hormone-expressing cells The development of endocrine precursor cells into immature pancreatic islet hormone-expressing cells can be monitored by determining the expression of markers characteristic of immature pancreatic islet hormone-expressing cells, including genetic and phenotypic markers, such as expression of pancreatic islet hormones and the processing of proinsulin into insulin and C-peptide. In some processes, the expression of a particular marker is determined by detecting the presence or absence of a marker. Alternatively, the expression of a particular marker can be determined by measuring the level of the marker present in the cells of the cell culture or cell population. In certain processes, the expression of markers characteristic of immature pancreatic islet hormone-expressing cells and the lack of significant expression of markers characteristic of hESC, definitive endoderm, foregut endoderm, PDX1-positive foregut endoderm, endocrine precursors, extraembryonic endoderm, mesoderm, ectoderm, mature pancreatic islet hormone-expressing cells and / or other cell types is determined.

[0517] As described in connection with monitoring the production of other less differentiated cell types of the definitive endoderm lineage, quantitative or semi-quantitative techniques, such as blot transfer methods, may be used. ) and immunocytochemistry can be used to measure the expression of the marker. Alternatively, the expression of the marker can be accurately quantified by using techniques such as Q-PCR. Furthermore, at the polypeptide level, it is understood that many markers of pancreatic islet hormone-expressing cells are secreted proteins. Thus, techniques for measuring extracellular marker content, such as ELISA, can be utilized.

[0518] As described in the Examples below, markers for immature pancreatic islet hormone-expressing cells include M The immature pancreatic islet hormone-expressing cells produced by the process described herein express one or more of the above-listed markers to produce the corresponding gene products. However, it is understood that immature pancreatic islet hormone-expressing cells do not need to express all of the above markers. For example, pancreatic islet hormone-expressing cells differentiated from hESC do not simultaneously express INS and GHRL.

[0519] Because pancreatic islet hormone-expressing cells do not substantially express the endocrine precursor cell markers NGN3 and PAX4, transition of endocrine precursor cells to immature pancreatic islet hormone-expressing cells can be demonstrated by monitoring the increased expression of one or more of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX6.1, SST, PP, CHGA, SYP, and / or C-peptide while monitoring the decreased expression of NGN3 and PAX4. In some processes, in addition to monitoring the increased and / or decreased expression of one or more of the above markers, the expression of genes indicative of hESCs, definitive endoderm cells, foregut endoderm cells, PDX1 positive foregut endoderm cells, and / or endocrine precursor cells is also monitored.

[0520] It is understood that expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX6.1, SST, PP, CHGA, SYP and / or C-peptide markers is induced to a range of different levels in immature pancreatic islet hormone-expressing cells depending on the differentiation conditions. Thus, in some embodiments described herein, expression of the MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX6.1, SST, PP, CHGA, SYP, and / or C-peptide markers in a pancreatic islet hormone-expressing cell or cell population is at least about 2-fold to at least about 10,000-fold greater than expression of the MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX6.1, SST, PP, CHGA, SYP, and / or C-peptide markers in a non-immature pancreatic islet hormone-expressing cell or cell population, such as a pluripotent stem cell, a definitive endoderm cell, a foregut endoderm cell, a PDX1 positive foregut endoderm cell, an endocrine precursor cell, an extraembryonic endoderm cell, a mesoderm cell, and / or an ectodermal cell. In other embodiments, expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX6.1, SST, PP, CHGA, SYP and / or C-peptide markers in immature pancreatic islet hormone-expressing cells or cell populations is related to expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX2.2, SST, PP, CHGA, SYP and / or C-peptide markers in non-immature pancreatic islet hormone-expressing cells or cell populations, such as pluripotent stem cells, definitive endoderm cells, foregut endoderm cells, PDX1 positive foregut endoderm cells, endocrine precursor cells, extraembryonic endoderm cells, mesoderm cells and / or ectoderm cells. , SST, PP, CHGA, SYP and / or C-peptide marker expression is at least about 4 fold, at least about 6 fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 40 fold, at least about 80 fold, at least about 100 fold, at least about 150 fold, at least about 200 fold, at least about 500 fold, at least about 750 fold, at least about 1000 fold, at least about 2500 fold, at least about 5000 fold, at least about 7500 fold, or at least about 10000 fold higher than the expression of the markers.In some embodiments, expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX2.2, SST, PP, CHGA, SYP and / or C-peptide markers in an immature pancreatic islet hormone-expressing cell or cell population is much higher than expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX2.2, SST, PP, CHGA, SYP and / or C-peptide markers in a non-immature pancreatic islet hormone-expressing cell or cell population, such as a pluripotent stem cell, a definitive endoderm cell, a foregut endoderm cell, a PDX1 positive foregut endoderm cell, an endocrine precursor cell, an extraembryonic endoderm cell, a mesoderm cell and / or an ectodermal cell.

[0521] Expression of NGN3 and / or PAX4 markers may be involved in the differentiation of immature islets and their hormone production. It is understood that expression of the NGN3 and / or PAX4 markers in immature pancreatic islet hormone-expressing cells or cell populations is reduced to a range of different levels in endocrine precursor cells. Thus, in some embodiments described herein, expression of the NGN3 and / or PAX4 markers in immature pancreatic islet hormone-expressing cells or cell populations is at least about 2-fold to at least about 10,000-fold lower than expression of the NGN3 and / or PAX4 markers in endocrine precursor cells. In other embodiments, expression of the NGN3 and / or PAX4 markers in the immature pancreatic islet hormone-expressing cells or cell population is at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 40-fold, at least about 80-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, at least about 2500-fold, at least about 5000-fold, at least about 7500-fold, or at least about 10000-fold lower than expression of the NGN3 and / or PAX4 markers in the endocrine precursor cells. In some embodiments, the NGN3 and / or PAX4 markers are not substantially expressed in the immature pancreatic islet hormone-expressing cells or cell population.

[0522] In some embodiments of the processes described herein, the amount of hormone release from cells and / or cell populations can be determined. For example, the amount of insulin release, glucagon release, somatostatin release, and / or ghrelin release can be monitored. In a preferred embodiment, the amount of glucose-stimulated insulin secretion (GSIS) is measured. In yet another embodiment, the secreted breakdown or by-products produced by immature pancreatic islet hormone-expressing cells, such as C-peptide and islet amyloid protein, can be monitored.

[0523] It is understood that methods for measuring the expression of secreted proteins are known in the art. For example, antibodies against one or more hormones produced by pancreatic islet cells can be used in an ELISA assay.

[0524] In some embodiments of the invention, insulin release by immature pancreatic islet hormone-expressing cells is measured by measuring C-peptide release. C-peptide is a breakdown product produced in equimolar amounts to insulin during the maturation of proinsulin. Measurement of C-peptide is advantageous because it has a longer half-life than insulin. Methods for measuring C-peptide release are known in the art (e.g., ELISA using anti-C-peptide monoclonal antibodies (Linco Research, St. Louis, Missouri)). ... In some embodiments, the immature pancreatic islet hormone-expressing cells produced from hESCs contain at least about 50 pmol C-peptide (insulin) / μg cellular DNA, at least about 100 pmol C-peptide (insulin) / μg cellular DNA, at least about 150 pmol C-peptide (insulin) / μg cellular DNA, at least about 200 pmol C-peptide (insulin) / μg cellular DNA, at least about 300 pmol C-peptide (insulin) / μg cellular DNA, at least about 400 pmol C-peptide (insulin) / μg cellular DNA, at least about 50 pmol C-peptide (insulin) / μg cellular DNA, at least about 600 pmol C-peptide (insulin) / μg cellular DNA, at least about 700 pmol C-peptide (insulin) / μg cellular DNA, at least about 800 pmol C-peptide (insulin) / μg cellular DNA, at least about 900 pmol C-peptide (insulin) / μg cellular DNA, at least about 100 pmol C-peptide (insulin) / μg cellular DNA, at least about 150 pmol C-peptide (insulin) / μg cellular DNA, at least about 200 pmol C-peptide (insulin) / μg cellular DNA, at least about 1 ... At least about 250 pmol / μg cellular DNA, at least about 300 pmol / μg cellular DNA of C-peptide(insulin), at least about 350 pmol / μg cellular DNA of C-peptide(insulin), at least about 400 pmol / μg cellular DNA of C-peptide(insulin), at least about 450 pmol / μg cellular DNA of C-peptide(insulin), at least about 500 pmol / μg cellular DNA of C-peptide(insulin), at least about 550 pmol / μg cellular DNA of C-peptide(insulin), at least about 600 pmol / μg cellular DNA of C-peptide(insulin), at least about 650 pmol / μg cellular DNA of C-peptide(insulin), at least about 700 pmol / μg cellular DNA of C-peptide(insulin), at least about 750 pmol / cellular DNA of C-peptide(insulin). 1 μg, C-peptide (insulin) at least about 800 pmol / cell DNA 1 μg, C-peptide (insulin) at least about 850 pmol / cell DNA 1 μg, C-peptide (insulin) at least about 900 pmol / cell DNA 1 μg, C -peptide (insulin) at least about 950 pmol / μg of cell DNA, or C-peptide (insulin) at least about 1000 pmol / μg of cell DNA. In preferred embodiments, the immature pancreatic islet hormone-expressing cells are cells that secrete a single type of pancreatic islet cell hormone (e.g., cells that secrete only insulin). In certain preferred embodiments, insulin is secreted in response to glucose. In other embodiments, the immature pancreatic islet hormone-expressing cells are cells that secrete insulin in addition to one or more pancreatic islet cell hormones, such as somatostatin, glucagon, and / or ghrelin.

[0525] In some embodiments of the invention, the immature pancreatic islet hormone-expressing cells process less than about 98% of the insulin produced by the immature pancreatic islet hormone-expressing cells, hi other embodiments, the immature pancreatic islet hormone-expressing cells process less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, or less than about 30% of the insulin produced by the immature pancreatic islet hormone-expressing cells.

[0526] In other embodiments of the invention, immature pancreatic islet hormone-expressing cells produced from hESCs contain at least about 50 pmol glucagon / μg cell DNA, at least about 100 pmol glucagon / μg cell DNA, at least about 150 pmol glucagon / μg cell DNA, at least about 200 pmol glucagon / μg cell DNA, at least about 250 pmol glucagon / μg cell DNA, at least about 300 pmol glucagon / μg cell DNA, at least about 350 pmol glucagon / μg cell DNA, at least about 400 pmol glucagon / μg cell DNA, at least about 450 pmol glucagon / μg cell DNA, at least about 500 pmol glucagon / μg cell DNA, at least about 550 pmol glucagon / μg cell DNA, at least about 600 pmol glucagon / μg cell DNA. The immature pancreatic islet hormone-expressing cells secrete at least about 1 μg of glucagon, at least about 650 pmol / 1 μg of glucagon, at least about 700 pmol / 1 μg of glucagon, at least about 750 pmol / 1 μg of glucagon, at least about 800 pmol / 1 μg of glucagon, at least about 850 pmol / 1 μg of glucagon, at least about 900 pmol / 1 μg of glucagon, at least about 950 pmol / 1 μg of glucagon, or at least about 1000 pmol / 1 μg of glucagon. In a preferred embodiment, the immature pancreatic islet hormone-expressing cells are cells that secrete a single type of pancreatic islet cell hormone (e.g., cells that secrete only glucagon). In another embodiment, the immature pancreatic islet hormone-expressing cells are cells that secrete glucagon in addition to one or more pancreatic islet cell hormones, such as ghrelin, somatostatin, and insulin.

[0527] In yet another embodiment of the invention, the immature pancreatic islet hormone-expressing cells produced from hESCs contain at least about 50 pmol somatostatin / μg cell DNA, at least about 100 pmol somatostatin / μg cell DNA, at least about 150 pmol somatostatin / μg cell DNA, at least about 200 pmol somatostatin / μg cell DNA, at least about 250 pmol somatostatin / μg cell DNA, at least about 300 pmol somatostatin / μg cell DNA, at least about 350 pmol somatostatin / μg cell DNA, at least about 400 pmol somatostatin / μg cell DNA, at least about 450 pmol somatostatin / μg cell DNA, at least about 500 pmol somatostatin / μg cell DNA, at least about 550 pmol somatostatin / μg cell DNA. 1μg, Somatostat The immature pancreatic islet hormone-expressing cells secrete at least about 600 pmol somatostatin / μg of cell DNA, at least about 650 pmol somatostatin / μg of cell DNA, at least about 700 pmol somatostatin / μg of cell DNA, at least about 750 pmol somatostatin / μg of cell DNA, at least about 800 pmol somatostatin / μg of cell DNA, at least about 850 pmol somatostatin / μg of cell DNA, at least about 900 pmol somatostatin / μg of cell DNA, at least about 950 pmol somatostatin / μg of cell DNA, or at least about 1000 pmol somatostatin / μg of cell DNA. In a preferred embodiment, the immature pancreatic islet hormone-expressing cells are cells that secrete a single type of pancreatic islet cell hormone (e.g., cells that secrete only somatostatin). In other embodiments, the immature pancreatic islet hormone-expressing cells are cells that secrete somatostatin in addition to one or more pancreatic islet cell hormones, eg, ghrelin, glucagon, and insulin.

[0528] In other embodiments of the invention, immature pancreatic islet hormone-expressing cells produced from hESCs contain at least about 50 pmol ghrelin / μg cellular DNA, at least about 100 pmol ghrelin / μg cellular DNA, at least about 150 pmol ghrelin / μg cellular DNA, at least about 200 pmol ghrelin / μg cellular DNA, at least about 300 pmol ghrelin / μg cellular DNA, at least about 400 pmol ghrelin / μg cellular DNA, at least about 50 pmol ghrelin / μg cellular DNA, at least about 600 pmol ghrelin / μg cellular DNA, at least about 700 pmol ghrelin / μg cellular DNA, at least about 800 pmol ghrelin / μg cellular DNA, at least about 900 pmol ghrelin / μg cellular DNA, at least about 100 pmol at least about 250 pmol / μg cellular DNA, at least about 300 pmol / μg ghrelin, at least about 350 pmol / μg ghrelin, at least about 400 pmol / μg ghrelin, at least about 450 pmol / μg ghrelin, at least about 500 pmol / μg ghrelin, at least about 550 pmol / μg ghrelin, at least about 600 pmol / μg ghrelin, at least about 650 pmol / μg ghrelin, at least about 700 pmol / μg ghrelin, at least about 750 pmol / μg ghrelin, at least about 800 pmol / μg ghrelin, at least about 850 pmol / μg ghrelin, at least about 900 pmol / μg ghrelin 1 μg of ghrelin, at least about 950 pmol ghrelin / μg of cell DNA, or at least about 1000 pmol ghrelin / μg of cell DNA. In a preferred embodiment, the immature pancreatic islet hormone-expressing cells are cells that secrete a single type of pancreatic islet cell hormone (e.g., cells that secrete only ghrelin). In another embodiment, the immature pancreatic islet hormone-expressing cells are cells that secrete ghrelin in addition to one or more pancreatic islet cell hormones.

[0529] Enrichment, isolation and / or purification of immature pancreatic islet hormone-expressing cells Immature pancreatic islet hormone-expressing cells produced by any of the above processes can be enriched, isolated and / or purified by using affinity tags specific for such cells using the methods described in connection with the enrichment, isolation and / or purification of endocrine precursor cells. Examples of affinity tags specific for immature pancreatic islet hormone-expressing cells are antibodies, ligands or other binding agents specific for marker molecules, e.g., polypeptides, present on the cell surface of immature pancreatic islet hormone-expressing cells but substantially absent from other cell types found in the cell cultures produced by the methods described herein. A preferred example of an affinity tag for enrichment, isolation and / or purification of immature pancreatic islet hormone-expressing cells is an antibody against NCAM. Anti-NCAM antibodies are commercially available, e.g., from Abcam (Cambridge, MA). Another example of an affinity tag for enrichment, isolation and / or purification of immature pancreatic islet hormone-expressing cells is an antibody against synaptophysin (SYP). Anti-synaptophysin antibodies are available from Dako (Glostrup, Denmark). It is commercially available. In other processes, the NCAM ligand NBP10 or any other NCAM ligand now known or discovered in the future can be used to bind the affinity tag (Ronn, L., 2002). Such molecules include, but are not limited to, NBP10 fusions and NBP10 mimetics.

[0530] Additional methods for obtaining enriched, isolated or purified immature pancreatic islet hormone-expressing cell cultures or populations can also be used. For example, in some embodiments, a reagent, such as an NCAM antibody, is incubated with a cell culture containing immature pancreatic islet hormone-expressing cells, and the cell culture has been treated to reduce cell-cell and substrate adhesion. The cells are then washed, centrifuged and resuspended. The cell suspension is then incubated with a secondary antibody, such as an FITC-conjugated antibody that can bind to the primary antibody. The cells are then washed, centrifuged and resuspended in a buffer. The cell suspension is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). The antibody-bound fluorescent cells are collected separately from the unbound non-fluorescent cells, thereby isolating such cell types.

[0531] In preferred embodiments of the process described herein, the isolated cell composition can be further purified by using alternative affinity-based methods or by additional rounds of sorting using the same or different markers specific for immature pancreatic islet hormone-expressing cells. For example, in some embodiments, FACS sorting is used to isolate NCAM-positive immature pancreatic hormone-expressing cells from NCAM-negative cells from a cell population that initially contains immature pancreatic hormone-expressing cells. Those skilled in the art will appreciate that other conventional marker-based cell sorting methods can be used in the methods described herein, including, but not limited to, various magnetic bead sorting or panning methods. FACS is again used to isolate cells that are NCAM-positive, thereby enriching the cell population for immature pancreatic hormone-expressing cells that express markers characteristic of this cell type, including SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, or HB9. In other embodiments, FACS sorting is used to separate cells by negatively selecting for markers present in most cells in the cell population other than immature pancreatic islet hormone-expressing cells. An example of such negative selection is the use of CD133, a marker that is not substantially expressed on the surface of immature pancreatic islet hormone-expressing cells in the NCAM-positive cell population after the first round of enrichment, but is expressed by many other NCAM-positive cells in this cell population.

[0532] In some embodiments of the processes described herein, immature pancreatic islet hormone-expressing cells are fluorescently labeled without antibodies and then isolated from unlabeled cells by using a fluorescence-activated cell sorter (FACS) method similar to that described for enrichment, isolation and / or purification of endocrine precursor cells. For example, in some embodiments, nucleic acids encoding GFP, YFP, luciferase, or biologically active fragments thereof can be introduced into pluripotent cells downstream of the promoter of a marker useful for identifying immature pancreatic islet hormone-expressing cells, such as those described above, e.g., SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, or HB9. Thereby, expression of the GFP gene product or biologically active fragments thereof is under the control of the immature pancreatic islet hormone-expressing cell marker. As described for enrichment, isolation and / or purification of endocrine precursor cells, the fluorescently labeled cells can be differentiated into immature pancreatic islet hormone-expressing cells and separated from other cell types, thereby producing an enriched or purified population of immature pancreatic islet hormone-expressing cells.

[0533] In addition to the above procedures, it is understood that immature pancreatic islet hormone-expressing cells can be isolated by other techniques for cell isolation. Furthermore, immature pancreatic islet hormone-expressing cells can also be enriched or isolated by serial subculture methods under growth conditions that promote the selective survival or selective expression of immature pancreatic islet hormone-expressing cells.

[0534] The methods described herein can be used to produce enriched, isolated and / or purified populations and / or tissues of immature pancreatic islet hormone-expressing cells from pluripotent cell cultures or populations, e.g., stem cell cultures or populations, that have undergone sufficient differentiation in vitro to produce at least some immature pancreatic islet hormone-expressing cells. In a preferred method, the cells are primarily are directed to differentiation into immature pancreatic islet hormone-expressing cells. Some preferred enrichment, isolation and / or purification methods involve the in vitro production of immature pancreatic islet hormone-expressing cells from human embryonic stem cells.

[0535] Using the methods described herein, a cell population or cell culture can be enriched for immature pancreatic islet hormone-expressing cell content by at least about 2-fold to about 1000-fold compared to an untreated or less specifically differentiated cell population or cell culture. In some embodiments, the immature pancreatic islet hormone-expressing cells can be enriched by at least about 5-fold to about 500-fold compared to an untreated or less specifically differentiated cell population or cell culture. In other embodiments, the immature pancreatic islet hormone-expressing cells can be enriched by at least about 10-fold to about 200-fold compared to an untreated or less specifically differentiated cell population or cell culture. In yet another embodiment, the immature pancreatic islet hormone-expressing cells can be enriched by at least about 20-fold to about 100-fold compared to an untreated or less specifically differentiated cell population or cell culture. In yet another embodiment, the immature pancreatic islet hormone-expressing cells can be enriched by at least about 40-fold to about 80-fold compared to an untreated or less specifically differentiated cell population or cell culture. In certain embodiments, immature pancreatic islet hormone-expressing cells can be enriched at least about 2-fold to about 20-fold relative to untreated or less specifically differentiated cell populations or cell cultures.

[0536] Compositions Comprising Immature Pancreatic Islet Hormone-Expressing Cells - Patent application Some embodiments of the present invention relate to cell compositions, such as cell cultures or cell populations, comprising immature pancreatic islet hormone-expressing cells, which are derived in vitro from human pluripotent cells, express one or more pancreatic hormones, and have at least some of the functions of human pancreatic islet cells. According to certain embodiments, the immature pancreatic islet hormone-expressing cells are mammalian cells, and in preferred embodiments, such cells are human cells.

[0537] Other embodiments of the invention relate to compositions, such as cell cultures or cell populations, that include immature pancreatic islet hormone-expressing cells and cells that are less specifically differentiated than the immature pancreatic islet hormone-expressing cells. In such embodiments, the cells that are less specifically differentiated than the immature pancreatic islet hormone-expressing cells comprise less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.

[0538] Certain other embodiments of the invention relate to compositions, such as cell cultures or cell populations, comprising immature pancreatic islet hormone-expressing cells and one or more cell types selected from the group consisting of hESCs, preprimitive streak cells, mesoendoderm cells, definitive endoderm cells, PDX1-negative foregut endoderm cells, PDX1-positive foregut endoderm cells (PDX1-positive pancreatic endoderm cells), endocrine precursor cells, and mesoderm cells. In some embodiments, hESCs are less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In certain embodiments, preprimitive streak cells are less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In other embodiments, the definitive endoderm cells represent less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In still other embodiments, the definitive endoderm cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In still other embodiments, PDX1-negative foregut endoderm cells are less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In certain embodiments, PDX1-positive foregut endoderm cells are less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In other embodiments, endocrine precursor cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture. In still other embodiments, mesodermal cells represent less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the culture.

[0539] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, produced by the processes described herein and comprising immature pancreatic islet hormone-expressing cells as the predominant cell type. In some embodiments, the processes described herein produce immature pancreatic islet hormone-expressing cells that are at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 89%, at least about 98%, at least about 9 ... In a preferred embodiment, the cells of the cell culture or cell population comprise human cells. In other embodiments, the processes described herein reduce the number of immature pancreatic islet hormone-expressing cells to at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 9%, at least about 10 ... In some embodiments, the cells of the cell culture or cell population comprise at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of immature pancreatic islet hormone-expressing cells. In preferred embodiments, the cells of the cell culture or cell population comprise human cells. In some embodiments, the percentage of immature pancreatic islet hormone-expressing cells in the cell culture or population is calculated without taking into account any feeder cells remaining in the culture.

[0540] Yet another embodiment of the present invention relates to a composition, such as a cell culture or cell population, that comprises a mixture of immature pancreatic islet hormone-expressing cells and endocrine precursor cells.For example, a cell culture or cell population can be produced that comprises at least about 5 immature pancreatic islet hormone-expressing cells for every about 95 endocrine precursor cells.In other embodiments, a cell culture or cell population can be produced that comprises at least about 95 immature pancreatic islet hormone-expressing cells for every about 5 endocrine precursor cells.Furthermore, cell cultures or cell populations that comprise other ratios of immature pancreatic islet hormone-expressing cells and endocrine precursor cells are contemplated.For example, at least about 1 immature pancreatic islet hormone-expressing cell for about every 1,000,000 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for about every 100,000 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for about every 10,000 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for about every 1,000 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for about every 500 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for about every 100 endocrine precursor cells, At least about 1 immature pancreatic islet hormone-expressing cell for every progenitor cell, at least about 1 immature pancreatic islet hormone-expressing cell for every 10 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for every 5 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for every 4 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for every 2 endocrine precursor cells, at least about 1 immature pancreatic islet hormone-expressing cell for every 1 endocrine precursor cell, at least about 1 immature pancreatic islet hormone-expressing cell for every 1 endocrine precursor cell, at least about 2 immature pancreatic islet hormone-expressing cells for every about secretory precursor cell, at least about 4 immature pancreatic islet hormone-expressing cells for every about endocrine precursor cell, at least about 5 immature pancreatic islet hormone-expressing cells for every about endocrine precursor cell, at least about 10 immature pancreatic islet hormone-expressing cells for every about endocrine precursor cell, at least about 20 immature pancreatic islet hormone-expressing cells for every about endocrine precursor cell, at least about 50 immature pancreatic islet hormone-expressing cells for every about endocrine precursor cell, Compositions comprising at least about 100 immature pancreatic islet hormone-expressing cells for about every endocrine precursor cell, at least about 1000 immature pancreatic islet hormone-expressing cells for about every endocrine precursor cell, at least about 10,000 immature pancreatic islet hormone-expressing cells for about every endocrine precursor cell, at least about 100,000 immature pancreatic islet hormone-expressing cells for about every endocrine precursor cell, and at least about 1,000,000 immature pancreatic islet hormone-expressing cells for about every endocrine precursor cell are contemplated.

[0541] In some embodiments of the invention, the immature pancreatic islet hormone-expressing cells produced are derived from human pluripotent cells, such as human pluripotent stem cells. In certain embodiments, the human pluripotent cells are derived from the morula, the inner cell mass of an embryo, or the gonadal ridges of an embryo. In certain embodiments, the human pluripotent cells are derived from gonadal tissue or embryonic tissue, which are multicellular structures that have developed beyond the embryonic stage.

[0542] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, comprising human cells (e.g., human immature pancreatic islet hormone-expressing cells), wherein expression of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP and / or C-peptide markers is greater than expression of NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers in at least about 2% of the human cells. In other embodiments, expression of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP and / or C-peptide markers is greater than expression of NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers in at least about 5% of the human cells. , greater than expression of NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers in at least about 10% of the human cells, at least about 15% of the human cells, at least about 20% of the human cells, at least about 25% of the human cells, at least about 30% of the human cells, at least about 35% of the human cells, at least about 40% of the human cells, at least about 45% of the human cells, at least about 50% of the human cells, at least about 55% of the human cells, at least about 60% of the human cells, at least about 65% of the human cells, at least about 70% of the human cells, at least about 75% of the human cells, at least about 80% of the human cells, at least about 85% of the human cells, at least about 90% of the human cells, at least about 95% of the human cells, or at least about 98% of the human cells. In some embodiments, the percentage of human cells in a cell culture or cell population having greater expression of the MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP and / or C-peptide markers than expression of the NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers is calculated without taking into account feeder cells.

[0543] Further embodiments of the invention relate to compositions, such as cell cultures or cell populations, comprising mammalian cells differentiated in vitro from definitive endoderm, e.g., human cells differentiated in vitro from definitive endoderm, wherein expression of the MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP and / or C-peptide markers is greater than expression of the NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers in at least about 2% of the cells differentiated in vitro from definitive endoderm. In other embodiments, expression of MAFB, PAX6, GHRL, IAPP, INS, GCG, NKX2.2, SST, PP, CHGA and / or C-peptide markers is in at least about 5% of cells differentiated from definitive endoderm in vitro, in at least about 10% of cells differentiated from definitive endoderm in vitro, in at least about 15% of cells differentiated from definitive endoderm in vitro, in at least about 20% of cells differentiated from definitive endoderm in vitro, in at least about 25% of cells differentiated from definitive endoderm in vitro, in at least about 30% of cells differentiated from definitive endoderm in vitro, in at least about 35% of cells differentiated from definitive endoderm in vitro, in at least about 40% of cells differentiated from definitive endoderm in vitro, in at least about 45% of cells differentiated from definitive endoderm in vitro, in at least about 50% of cells differentiated from definitive endoderm in vitro, in at least about 55% of cells differentiated from definitive endoderm in vitro, in At least about 60% of the cells differentiated in vitro from definitive endoderm, at least about 65% of the cells differentiated in vitro from definitive endoderm, at least about 70% of the cells differentiated in vitro from definitive endoderm, at least about 75% of the cells differentiated in vitro from definitive endoderm, Greater than expression of NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers in at least about 80% of cells differentiated in vitro from definitive endoderm, in at least about 85% of cells differentiated in vitro from definitive endoderm, in at least about 90% of cells differentiated in vitro from definitive endoderm, in at least about 95% of cells differentiated in vitro from definitive endoderm, or in at least about 98% of cells differentiated in vitro from definitive endoderm.

[0544] In preferred embodiments of the invention, the cell cultures and / or cell populations of immature pancreatic islet hormone-expressing cells comprise human immature pancreatic islet hormone-expressing cells that are non-recombinant cells. In such embodiments, the cell cultures and / or cell populations are devoid of or substantially free of recombinant human immature pancreatic islet hormone-expressing cells.

[0545] In some embodiments of the invention, the cell cultures and / or cell populations comprising immature pancreatic islet hormone-expressing cells also comprise a medium comprising one or more factors selected from nicotinamide, exendin 4, HGF and / or IGF1. In some preferred embodiments, the nicotinamide concentration is at least about 10 mM, the exendin 4 concentration is at least about 40 ng / ml, the HGF concentration is at least about 25 ng / ml, and the IGF1 concentration is at least about 50 ng / ml. In some embodiments, the medium is DMEM.

[0546] In certain embodiments of the invention, the cell cultures and / or cell populations comprising immature pancreatic islet hormone-expressing cells also comprise a medium comprising one or more secreted hormones selected from ghrelin, insulin, somatostatin and / or glucagon. In other embodiments, the medium comprises C-peptide. In preferred embodiments, the concentration of one or more secreted hormones or C-peptide in the medium ranges from at least about 1 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA to at least about 1000 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA. In a further preferred embodiment, the concentration of one or more secreted hormones or C-peptide in the medium is at least about 1 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, at least about 10 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, at least about 25 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, at least about 50 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, at least about 75 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, at least about 100 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg of cellular DNA, At least about 150 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 200 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 250 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 300 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 350 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 400 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 450 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 500 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 550 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 600 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 650 pmol / μg cellular DNA, ghrelin, insulin, somatostatin, glucagon or C-peptide At least about 700 pmol / μg of cellular DNA, ghrelin, insulin, somatostatin, glucagon, or C-peptide At least about 750 pmol / μg of cellular DNA, ghrelin, insulin, somatostatin, glucagon, or C-peptide At least about 800 pmol / μg of cellular DNA, ghrelin, insulin, somatostatin, glucagon, or C-peptide At least about 850 pmol / μg of cellular DNA, ghrelin, insulin, somatostatin, glucagon, or C-peptide At least about 900 pmol / μg of cellular DNA, ghrelin, insulin, somatostatin, glucagon, or C-peptide At least about 950 pmol / cell DNA NA 1 μg, or at least about 1000 pmol ghrelin, insulin, somatostatin, glucagon or C-peptide per μg cellular DNA.

[0547] In some embodiments of the cell cultures and / or cell populations described herein, the immature pancreatic islet hormone-expressing cells secrete two or more pancreatic hormones. In other embodiments of the cell cultures and / or cell populations described herein, the immature pancreatic islet hormone-expressing cells secrete a single pancreatic hormone. In a preferred embodiment, the hormone is insulin. In an even more preferred embodiment, the pancreatic islet insulin-expressing cells are responsive to glucose. In other embodiments, the in vitro differentiated human pancreatic islet insulin-expressing cells secrete an amount of insulin that is similar to or exceeds the amount of insulin secreted by pancreatic β cells of the human pancreas in vivo.

[0548] The processes described herein can be used to produce compositions comprising immature pancreatic islet hormone-expressing cells that are substantially free of other cell types. In some embodiments of the present invention, the immature pancreatic islet hormone-expressing cell populations or cell cultures produced by the methods described herein are substantially free of cells that significantly express NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers. In some embodiments of the immature pancreatic islet hormone-expressing cell populations or cell cultures produced by the methods described herein, expression of one or more markers selected from the group consisting of MAFB, SYP, CHGA, NKX2.2, ISL1, PAX6, NEUROD, PDX1, HB9, GHRL, IAPP, INS, GCG, SST, PP, and / or C-peptide is greater than expression of one or more markers selected from the group consisting of NGN3, MAFA, MOX1, CER, POU5F1, AFP, SOX7, SOX1, ZIC1 and / or NFM markers.

[0549] In one embodiment of the invention, the description of immature pancreatic islet hormone-expressing cells based on marker expression is MAFB high, PAX6 high, NKX2.2 high, SYP high, PP high, CHGA high, NGN3 low, PAX4 low, and MAFA low.

[0550] Generation of mature pancreatic islet hormone-expressing cells The present invention relates to a method for producing mature pancreatic islet hormone-expressing cells starting from hESCs. As described above, pancreatic islet hormone-expressing cells can be produced by first differentiating hESCs to produce definitive endoderm cells, differentiating the definitive endoderm cells to produce PDX1-positive foregut endoderm cells, differentiating the PDX1-positive foregut endoderm cells to produce endocrine precursor cells, and then further differentiating the endocrine precursor cells to produce immature pancreatic islet hormone-expressing cells. In some embodiments, the process is completed by further differentiating the immature pancreatic islet hormone-expressing cells into mature pancreatic islet hormone-expressing cells.

[0551] In some embodiments of the present invention, differentiation of immature pancreatic islet hormone-expressing cells to mature pancreatic islet hormone-expressing cells proceeds by continuing to incubate the culture of immature pancreatic islet hormone-expressing cells with a gamma-secretase inhibitor for a sufficient time for the cells to become capable of expressing at least one mature pancreatic islet cell hormone. In some embodiments, the gamma-secretase inhibitor is removed 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, about 10 days, or more than about 10 days after induction of endocrine precursor cells. In a preferred embodiment, the gamma-secretase inhibitor is N-[N-(3,5-Diflurophenacetyl-L-alanyl)]-S-phenylglycine t- butyl ester (DAPT).

[0552] Certain processes for producing mature pancreatic islet hormone-expressing cells described herein involve the administration of nicotinamide, exendin 4, hepatocyte growth factor (HGF), insulin-like growth factor-1 (insulin-like growth factor-1), and / or acetylcholinesterase inhibitors. (IGF1) to a cell culture or cell population comprising human endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells. In some embodiments, all four of the factors are provided simultaneously. In some embodiments, one or more of the factors are provided to the cell culture prior to differentiation of the endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells and remain present in the cell culture during differentiation of at least some of the cells in the cell culture into mature pancreatic islet hormone-expressing cells. In other embodiments, one or more of the factors are provided to the cell culture at about the same time as differentiation of the majority of the cells into endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells and remain present in the cell culture until at least the majority of the cells have differentiated into mature pancreatic islet hormone-expressing cells. In some embodiments of the invention, one or more of the factors are provided at the beginning of the differentiation process, for example at the hESC stage, and are maintained in the cell culture during differentiation into mature pancreatic islet hormone-expressing cells.

[0553] In some of the processes for producing mature pancreatic islet hormone-expressing cells disclosed herein, nicotinamide is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells to mature pancreatic islet hormone-expressing cells. In some embodiments, nicotinamide is provided at a concentration of at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 21 mM, at least about 22 mM, at least about 23 mM, at least about 24 mM, at least about 25 mM, at least about 26 mM, at least about 27 mM, at least about 28 mM, at least about 29 mM, at least about 30 mM, at least about 31 mM, at least about 32 mM, at least about 33 mM, at least about 34 mM, at least about 35 mM, at least about 36 mM, at least about 37 mM, at least about 38 mM, at least about 39 mM, at least about 40 mM, at least about 40 mM, at least about 41 mM, at least about 42 mM, at least about 43 mM, at least about 44 mM, at least about 45 mM, at least about 46 mM, at least about 47 mM, at least about 48 mM, at least about At least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM, or at least about 1000 mM.

[0554] In other processes for producing mature pancreatic islet hormone-expressing cells disclosed herein, exendin 4 is provided to cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells into pancreatic islet hormone-expressing cells. In some embodiments, exendin 4 is provided to cells at a concentration of at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml. at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, at least about 100 ng / ml, at least about 15 ... or at least about 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml.

[0555] In yet another process for producing mature pancreatic islet hormone-expressing cells disclosed herein, HGF is provided to cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells into pancreatic islet hormone-expressing cells. In some embodiments, HGF is provided at a concentration of at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml, at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 85 ng / ml, at least about 90 ng / ml, at least about 95 ng / ml, at least about The antibody is present in the cell culture or cell population at a concentration of 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml.

[0556] In yet another process for producing mature pancreatic islet hormone-expressing cells disclosed herein, IGF1 is provided to the cells such that it is present in the cell culture or cell population at a concentration sufficient to promote differentiation of at least a portion of the endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells into pancreatic islet hormone-expressing cells. In some embodiments ... The antibody is present in the cell culture or cell population at a concentration of 100 ng / ml, at least about 110 ng / ml, at least about 120 ng / ml, at least about 130 ng / ml, at least about 140 ng / ml, at least about 150 ng / ml, at least about 160 ng / ml, at least about 170 ng / ml, at least about 180 ng / ml, at least about 190 ng / ml, at least about 200 ng / ml, at least about 250 ng / ml, at least about 300 ng / ml, at least about 350 ng / ml, at least about 400 ng / ml, at least about 450 ng / ml, at least about 500 ng / ml, at least about 750 ng / ml, or at least about 1000 ng / ml.

[0557] In certain embodiments of the process for producing mature pancreatic islet hormone-expressing cells as described herein, one or more of nicotinamide, exendin 4, HGF and IGF1 are provided after one or more pre-supplied differentiation factors are removed from the cell culture.In other embodiments, one or more of nicotinamide, exendin 4, HGF and IGF1 are provided to a cell culture or cell population that includes one or more differentiation factors that are pre-supplied or are provided approximately simultaneously with one or more of nicotinamide, exendin 4, HGF and IGF1.In a preferred embodiment, the differentiation factors that are pre-supplied or are provided approximately simultaneously with one or more of nicotinamide, exendin 4, HGF and IGF1 include, but are not limited to, DAPT, FGF-10, KAAD-cyclopamine, activin A, activin B, BMP4 and / or RA.

[0558] In one process for producing mature pancreatic islet hormone-expressing cells from endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells, a cell culture or population of endocrine precursor cells and / or immature pancreatic islet hormone-expressing cells is provided with 10 mM nicotinamide, 40 ng / ml exendin 4, 25 ng / ml HGF and 50 ng / ml IGF1. In a preferred process, the cells are differentiated in Dulbecco's Modified Eagle's Medium (DMEM).

[0559] As described herein, in certain processes for producing mature pancreatic islet hormone-expressing cells, one or more of the differentiation factors are removed from the cell culture or cell population after their addition.For example, nicotinamide can be removed within 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 after addition.In some embodiments, the differentiation factor is not removed from the cell culture.

[0560] Cultures of mature pancreatic islet hormone-expressing cells can be produced in low-serum or serum-free medium. Under certain culture conditions, serum concentrations can range from about 0.05% (v / v) to about 20% (v / v). For example, in some differentiation processes, the serum concentration of the medium can be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). (v / v), less than about 1% (v / v), less than about 2% (v / v), less ...

Claims

1. 1. A method for producing human pancreatic islet hormone-expressing cells, comprising: (a) obtaining a population of pluripotent human stem cells; (b) providing said population of pluripotent human stem cells with a growth factor of the TGF-β superfamily selected from the group consisting of activin A and activin B, thereby producing a population of human definitive endoderm cells; (c) providing a retinoid to said population of human definitive endoderm cells, thereby producing a population of human PDX1-positive pancreatic endoderm cells; (d) incubating said population of human PDX1-positive pancreatic endoderm cells in the presence of a retinoid for a period of time sufficient to form human pancreatic islet hormone-expressing cells; The method of producing human pancreatic islet hormone-expressing cells further comprising the step of providing DAPT to said population of human PDX1-positive pancreatic endoderm cells.

2. 10. The method of claim 1, further comprising the step of providing said population of human definitive endoderm cells with a growth factor of the fibroblast growth factor family.

3. 3. The method of claim 1 or 2, further comprising the step of providing B27 to said population of pluripotent human stem cells.

4. The method of any one of claims 1 to 3, further comprising the step of providing said population of pluripotent human stem cells with a Wnt family member.

5. A method according to any one of claims 1 to 4, comprising removing a growth factor of the TGF-β superfamily selected from the group consisting of activin A and activin B from the population of human definitive endoderm cells in step (b).

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