INDUCED DERIVATION OF SPECIFIC ENDODERM FROM hPS CELL-DERIVED DEFINITIVE ENDODERM
The present invention relates to a method to control differentiation of human pluripotent stem cells, including human balstocyst derived stem (hBS) cells and to obtain specific endoderm cells. In particular, present invention relates to the use of FGF2 as the key factor in a specific concentration to control differentiation of definitive endoderm cells derived from hPS cells to specific endoderm cells. The invention also provides methods of obtaining endoderm cells comprising the use of FGFR and activation of the MAPK signalling pathway.
1 - 33 . (canceled)
34 . A method to control differentiation of definitive endodermal cells derived from human pluripotent stem (hPS) cells comprising:
providing a concentration of fibroblast growth factor 2 (FGF2) to control differentiation of definitive endodermal cells derived from hPS cells to specific endoderm cells.
35 . The method of claim 34 , wherein the hPS cells are human blastocyst derived stem (hBS) cells.
36 . The method of claim 34 , wherein the concentration of FGF2 in a culture medium is less than or equal to 500 ng/ml.
37 . The method of claim 34 , wherein the concentration of FGF2 in a culture medium ranges from about 16 ng/ml to about 150 ng/ml, and wherein the specific endoderm cells are pancreatic endoderm cells.
38 . The method of claim 34 , wherein the concentration of FGF2 in a culture is 64 ng/ml, and wherein the specific endoderm cells are pancreatic endoderm cells.
39 . The method of claim 37 , wherein the pancreatic endoderm cells express PDX1, and one or more of the following markers NGN3, CPA1, SOX9, HNF6, HNF1b, Ecadherin, MNX1, PTF1A and NKX6-1.
40 . The method of claim 39 , wherein the pancreatic endoderm cells express PDX1 and NKX6-1.
41 . The method of claim 37 , wherein the pancreatic endoderm cells express the following markers: SOX9, ONECUT1, and FOXA2.
42 . The method of claim 39 , wherein the pancreatic endoderm cells express the following markers: SOX9, ONECUT1, and FOXA2.
43 . The method of claim 37 , wherein the pancreatic endoderm cells express at least one pancreatic hormone selected from the group consisting of insulin, glucagon, somatostatin, pancreatic polypeptide, and ghrelin.
44 . The method of claim 34 , wherein the differentiation comprises incubation of definitive endoderm cells in a culture medium containing FGF2 in a concentration that is suitable for differentiation in the desired endodermal fate selected from the group consisting of hepatic endoderm cells, pancreatic endoderm cells, intestinal endoderm cells, and lung endoderm cells.
45 . A method for the preparation of pancreatic endodermal cells, the method comprising incubating definitive endodermal cells in a culture medium comprising from about 16 ng/ml to about 150 ng/ml FGF2 for about 2 to about 20 days.
46 . The method of claim 45 , wherein incubating definitive endodermal cells in a culture mediumis occurs for about 6 to about 8 days.
47 . Pancreatic endodermal cells obtainable by the method of claim 45 .
48 . The pancreatic endodermal cells of claim 47 , wherein the pancreatic endoderm cells express PDX1, and one or more of the following markers NGN3, CPA1, SOX9, HNF6, HNF1b, Ecadherin, MNX1, PTF1A and NKX6-1.
49 . The pancreatic endodermal cells of claim 48 , wherein the pancreatic endoderm cells express PDX1 and NKX6-1.
50 . The pancreatic endodermal cells of claim 48 , wherein the pancreatic endoderm cells express the following markers: SOX9, ONECUT1, and FOXA2.
51 . The pancreatic endodermal cells of claim 47 , wherein the pancreatic endoderm cells express the following markers: SOX9, ONECUT1, and FOXA2.
52 . The method of claim 37 , wherein the pancreatic endoderm cells comprises progenitor cells that express at least one marker for proliferation selected from the group consisting of MKI67, PH3, Brdu.