Pluripotent cells from rat and other species
View Patent ↗Pluripotent cells are derived and maintained in a self-renewing state in serum-free culture medium comprising a MEK inhibitor, a GSK3 inhibitor and an antagonist of an FGF receptor.
1. A method of deriving a pluripotent rat cell from a blastocyst, comprising:
(1) culturing a blastocyst in the presence of a MEK1 inhibitor at a concentration ranging from 0.1 μM to 5 μM, a GSK3 inhibitor at a concentration ranging from 0.1 μM to 20 μM, and LIF, to obtain an inner cell mass;
(2) isolating and dissociating the primary outgrowths of the inner cell mass;
(3) isolating a cell or cells from the dissociated primary outgrowths of the inner cell mass; and
(4) culturing the isolated cell or cells in the presence of a MEK1 inhibitor at a concentration ranging from 0.1 μM to 5 μM, a GSK3 inhibitor at a concentration ranging from 0.1 μM to 20 μM, an antagonist of an FGF receptor at a concentration ranging from 0.5 μM to 10 μM, and LIF.
2. The method of claim 1 , comprising culturing the blastocyst in the presence of the MEK inhibitor, the GSK3 inhibitor, LIF, and an antagonist of an FGF receptor.
3. The method of claim 1 , wherein the pluripotent cell expresses one or more of Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase.
4. The method of claim 1 , wherein the pluripotent cell expresses any two or more of Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase.
5. The method of claim 1 , wherein the pluripotent cell expresses Nanog, Oct4 and Sox-2.
6. The method of claim 5 , wherein the pluripotent cell further expresses alkaline phosphatase.
7. The method of claim 1 , wherein the pluripotent cell expresses Rex 1, Stella, FGF4 and Sox-2.
8. The method of claim 1 , wherein the pluripotent cell does not express FGF5.
9. The method of claim 1 , wherein the pluripotent cell is morphologically undifferentiated in culture.
10. The method of claim 1 , wherein the pluripotent cell is capable of being maintained in culture for at least two weeks.
11. The method of claim 10 , wherein the progeny of the pluripotent cell retain the characteristics of the original pluripotent cell after being maintained in culture.
12. The method of claim 1 , wherein the pluripotent cell is capable of contributing to a chimera.
13. The method of claim 12 , wherein all cells of the chimera are cells of the same species as the pluripotent cell.
14. The method of claim 12 , wherein the pluripotent cell is capable of contributing to the germ line of a chimera.
15. The method of claim 1 , wherein the pluripotent cell is capable of forming a teratoma or teratocarcinoma in which differentiated cells from all three germ layers are present.
16. The method of claim 1 , wherein the pluripotent cell is capable of growth and/or proliferation as a single cell in culture.
17. The method of claim 1 , wherein the pluripotent cell is induced to differentiate or fails to grow in the presence of activin and/or FGF.
18. The method of claim 1 , wherein differentiation of the pluripotent cell is not induced by activin receptor blockade.
19. The method of claim 1 , wherein growth or proliferation of the pluripotent cell is supported by the presence of a MEK1 inhibitor at a concentration ranging from 0.1 μM to 5 μM, a GSK3 inhibitor at a concentration ranging from 0.1 μM to 20 μM and an antagonist of an FGF receptor at a concentration ranging from 0.5 μM to 10 μM.
20. The method of claim 1 , wherein the pluripotent cell is an ES cell.
21. The method of claim 1 , wherein the GSK3 inhibitor is a GSK3β inhibitor.
22. The method of claim 1 , wherein the antagonist of an FGF receptor is an antagonist of FGFR1.
23. The method of claim 1 , wherein the MEK1 inhibitor is present at a concentration ranging from 0.2 μM to 2 μM.
24. The method of claim 1 , wherein the GSK3 inhibitor is present at a concentration ranging from 0.3 μM to 10 μM.
25. The method of claim 1 , wherein the antagonist of an FGF receptor is present at a concentration ranging from 1 μM to 5 μM.
26. The method of claim 1 , wherein the MEK1 inhibitor is selected from the group consisting of PD184362, PD98059, U0126, and SL327.
27. The method of claim 1 , wherein the GSK3 inhibitor is selected from the group consisting of CHIR98014, CHIR99021, AR-AO144-18, TDZ-8, SB216763, and SB415286.
28. The method of claim 1 , wherein the antagonist of an FGF receptor is selected from the group consisting of SU5402, and PD173074.
29. The method of claim 1 , wherein the MEK1 inhibitor is PD184632, the GSK3 inhibitor is CHIR99021 and the antagonist of an FGF receptor is SU5402.
30. The method of claim 19 , wherein the GSK3 inhibitor is a GSK3β inhibitor.
31. The method of claim 19 , wherein the antagonist of an FGF receptor is an antagonist of FGFR1.
32. The method of claim 19 , wherein the MEK1 inhibitor is present at a concentration ranging from 0.2 μM to 2 μM.
33. The method of claim 19 , wherein the GSK3 inhibitor is present at a concentration ranging from 0.3 μM to 10 μM.
34. The method of claim 19 , wherein the antagonist of an FGF receptor is present at a concentration ranging from 1 μM to 5 μM.
35. The method of claim 19 , wherein the MEK1 inhibitor is selected from the group consisting of PD184362, PD98059, U0126, and SL327.
36. The method of claim 19 , wherein the GSK3 inhibitor is selected from the group consisting of CHIR98014, CHIR99021, AR-AO144-18, TDZ-8, SB216763, and SB415286.
37. The method of claim 19 , wherein the antagonist of an FGF receptor is selected from the group consisting of SU5402, and PD173074.
38. The method of claim 19 , wherein the MEK1 inhibitor is PD184632, the GSK3 inhibitor is CHIR99021 and the antagonist of an FGF receptor is SU5402.
39. A method of deriving a pluripotent rat cell from a blastocyst, comprising:
(1) culturing the blastocyst in the presence of PD184352 at a concentration of 0.8 μM, CHIR99021 at a concentration of 3 μM, and LIF in N2B27 medium, to obtain an inner cell mass;
(2) isolating and dissociating the primary outgrowths of the inner cell mass;
(3) isolating a cell or cells from the dissociated primary outgrowths of the inner cell mass; and
(4) culturing the isolated cell or cells in the presence of PD184352 at a concentration of 0.8 μM, CHIR99021 at a concentration of 3 μM, SU5402 at a concentration of 2 μM, and LIF in N2B27 medium.