EFFICIENT INDUCTION OF DEFINITIVE ENDODERM FROM PLURIPOTENT STEM CELLS
The present invention relates to a method to differentiate pluripotent stem cells to a primitive streak cell population, in a stepwise manner for further maturation to definitive endoderm.
1 - 15 . (canceled)
16 . A method for differentiation of stem cells into definitive endoderm comprising the steps of:
a. incubating stem cells in a medium comprising at least 2 μM CHIR, wherein activin A is not present; and
b. subsequently incubating stem cells in a medium comprising activin A.
17 . The method according to claim 16 , wherein said medium is RPMI-1640.
18 . The method according to claim 16 , wherein said stem cells are embryonic stem cells or induced pluripotent stem cells.
19 . The method according to claim 16 , wherein the concentration of CHIR is at least about 2.5 μM.
20 . The method according to claim 16 , wherein the concentration of CHIR is at least about 3.1 μM.
21 . The method according to claim 16 , wherein the concentration of CHIR is in a range selected from the group consisting of about 2.5-15 μM, about 3.1-15 μM, about 3.1-7 μM, 3.5-7 μM, about 3.5-6 μM, and about 3.5-5 μM.
22 . The method according to claim 16 , wherein the concentration of CHIR is at least about 3.5 μM.
23 . The method according to claim 16 , wherein said incubation with CHIR is at least 24 hours.
24 . The method according to claim 16 , wherein said incubation with activin A is at least 24 hours.
25 . The method according to claim 16 , wherein said incubation with activin A is 48 to 72 hours.
26 . The method according to claim 16 , wherein endodermal cells are obtained from said definitive endoderm cells.
27 . The method according to claim 26 , wherein said endoderm cells are pancreatic endoderm cells.
28 . Pancreatic endoderm cells obtainable by the method of claim 27 .
29 . The method according to claim 16 , wherein the concentration of CHIR in the culture medium is at least 3 μM.
30 . The method according to claim 16 , wherein the concentration of CHIR in the culture medium is in a range selected from the group consisting of about 2-15 μM, about 3-15 μM, about 3.1-15 μM, about 3.1-7 μM, about 3.5-15 μM, and about 3.5-7 μM.
31 . A method for inducing primitive streak cells from embryonic stem cells by using CHIR in a concentration of at least 2 μM in the culture medium.
32 . A method for inducing definitive endoderm cells from embryonic stem cells by using CHIR in a concentration in the range of 3.5-7 μM.