Differentiation of Human Embryonic Stem Cells into Pancreatic Endoderm
First Claim
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1. A method of culturing pluripotent stem cells comprising seeding the pluripotent stem cells on a surface at a seeding density of from about 0.8×
- 105 cells/cm2 to about 3.0×
105 cells/cm2.
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Abstract
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of pancreatic endoderm cells, wherein the initial seeding density of undifferentiated epluripotent cells is defined.
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Citations
53 Claims
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1. A method of culturing pluripotent stem cells comprising seeding the pluripotent stem cells on a surface at a seeding density of from about 0.8×
- 105 cells/cm2 to about 3.0×
105 cells/cm2. - View Dependent Claims (2, 3, 4)
- 105 cells/cm2 to about 3.0×
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5. A method of differentiating pluripotent stem cells comprising seeding the pluripotent stem cells on a surface at a density of from about 0.8×
- 105 cells/cm2 to about 3.0×
105 cells/cm2; and
differentiating the pluripotent stem cells to cells expressing markers indicative of definitive endoderm. - View Dependent Claims (6, 7, 8, 9)
- 105 cells/cm2 to about 3.0×
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10. A method of obtaining cells expressing markers indicative of definitive endoderm comprising differentiating pluripotent stem cells into cells expressing markers indicative of definitive endoderm, wherein the pluripotent stem cells have been seeded on a surface at a density of from about 0.8×
- 105 cells/cm2 to about 3.0×
105 cells/cm2. - View Dependent Claims (11, 12, 13, 14)
- 105 cells/cm2 to about 3.0×
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15. A method of differentiating cells expressing markers indicative of definitive endoderm comprising seeding pluripotent stem cells on a first surface at a seeding density sufficient to maximize differentiation efficiency of the pluripotent stem cells;
- differentiating the pluripotent stem cells into cells expressing markers indicative of definitive endoderm;
seeding the cells expressing markers indicative of definitive endoderm at a seeding density sufficient to maximize differentiation efficiency of the cells expressing markers indicative of definitive endoderm; and
differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endoderm. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24)
- differentiating the pluripotent stem cells into cells expressing markers indicative of definitive endoderm;
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25. A method of differentiating cells expressing markers indicative of definitive endoderm comprising differentiating pluripotent stem cells into cells expressing markers indicative of definitive endoderm;
- and differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endoderm;
wherein the pluripotent stem cells have been seeded on a surface at a seeding density of from about 0.8×
105 cells/cm2 to about 3.0×
105 cells/cm2. - View Dependent Claims (26, 27, 28, 29, 30)
- and differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endoderm;
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31. A method of obtaining cells expressing markers indicative of pancreatic endoderm comprising:
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a) seeding pluripotent stem cells on a surface; b) differentiating the pluripotent stem cells into cells expressing markers indicative of definitive endoderm; and c) differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endoderm. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38)
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39. A method of obtaining cells expressing markers indicative of pancreatic endocrine comprising:
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a) seeding pluripotent stem cells on a surface; b) differentiating the pluripotent stem cells into cells expressing markers indicative of definitive endoderm; and c) differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endocrine. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46)
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47. A method of differentiating cells expressing markers indicative of definitive endoderm comprising seeding cells expressing markers indicative of definitive endoderm on a surface at a seeding density of from about 1.5×
- 105 cells/cm2 to about 5.0×
105 cells/cm2; and
differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endoderm. - View Dependent Claims (48, 49)
- 105 cells/cm2 to about 5.0×
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50. A method of differentiating cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endocrine comprising seeding cells expressing markers indicative of definitive endoderm on a surface at a seeding density of from about 1.5×
- 105 cells/cm2 to about 5.0×
105 cells/cm2; and
differentiating the cells expressing markers indicative of definitive endoderm into cells expressing markers indicative of pancreatic endocrine. - View Dependent Claims (51, 52)
- 105 cells/cm2 to about 5.0×
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53. A population of cells differentiated in vitro from human embryonic stem cells, showing a drop in expression of at least one marker selected from PDX-1, NKX6.1, NGN3, NKX2.2, NeuroD, and insulin, and upregulation of ZIC1 and CDX2 when compared to human embryonic stem cells;
- and wherein the human embryonic stem cells are seeded on a surface at a seeding density of less than 5×
105 cells/cm2.
- and wherein the human embryonic stem cells are seeded on a surface at a seeding density of less than 5×
Specification