Method for producing retinal pigment epithelial cells
The present invention provides a method for more efficiently producing retinal pigment epithelial cells from pluripotent stem cells. The method of the present invention for producing retinal pigment epithelial cells includes the following steps: (1) a first step for culturing a pluripotent stem cell in a medium comprising an FGF receptor inhibitor and/or an MEK inhibitor for a period of not more than 30 days, and (2) a second step for culturing the cell obtained in the first step in the presence of a Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form a retinal pigment epithelial cell.
1. A production method of a human retinal pigment epithelial cell comprising the following steps:
(1) a maintenance and/or expanding step of maintaining and/or expanding human pluripotent stem cells comprising culturing the human pluripotent stem cells in the absence of a feeder cell in a medium comprising a factor for maintaining an undifferentiated state,
(2) a first step for culturing the maintained and/or expanded human pluripotent stem cells in a medium comprising an FGF receptor inhibitor in the absence of feeder cells for a period of not less than 2 days and not more than 30 days, wherein the culture condition in the first step is a condition sufficient for inducing gene expression of at least one eye field transcription factor, wherein the first step excludes the presence of a MEK inhibitor, and
(3) a second step for culturing the cells obtained in the first step in the presence of a Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form a retinal pigment epithelial cell.
2. The production method according to claim 1 , wherein the first step is performed in serum-free conditions.
3. The production method according to claim 1 , wherein the maintenance and/or expanding step, the first step, and/or the second step is performed under adherent conditions.
4. The production method according to claim 1 , wherein the medium in the first step further comprises a factor for maintaining undifferentiated state.
5. The production method according to claim 4 , wherein the factor for maintaining undifferentiated state is an FGF signal transduction pathway agonist.
6. The production method according to claim 5 , wherein the FGF signal transduction pathway agonist is bFGF.
7. The production method according to claim 1 , wherein the FGF receptor inhibitor is at least one kind selected from the group consisting of PD173074 and SU5402.
8. The production method according claim 1 , wherein the Nodal signal transduction pathway inhibitor is an Alk4, 5 or 7 inhibitor.
9. The production method according to claim 8 , wherein the ALK4, 5 or 7 inhibitor is SB431542.
10. The production method according to claim 1 , wherein the Wnt signal transduction pathway inhibitor is CKI-7.
11. The production method according to claim 1 , wherein the medium in the first step is a medium further comprising a BMP receptor inhibitor.
12. The production method according to claim 11 , wherein the BMP receptor inhibitor is an ALK2/3 inhibitor.
13. The production method according to claim 12 , wherein the ALK2/3 inhibitor is LDN193189.
14. The production method according to claim 1 , wherein the medium in the first step further comprises a Sonic hedgehog signal transduction pathway agonist.
15. The production method according to claim 14 , wherein the Sonic hedgehog signal transduction pathway agonist is SAG.
16. The production method according to claim 1 , wherein the medium in the first step further comprises a PKC inhibitor.
17. The production method according to claim 16 , wherein the PKC inhibitor is Go6983.
18. The production method according to claim 1 , wherein the culture period in the first step is for 2 days to 13 days.
19. The production method according to claim 1 , wherein the culture period in the first step is for 4 days to 6 days.
20. A method of evaluating toxicity or efficacy of a test substance comprising contacting a retinal pigment epithelial cell produced by the production method according to claim 1 with the substance, and assaying the effect of the substance on the cells.
21. The production method according to claim 1 , wherein the culture period in the first step is a period sufficient for inducing gene expression of at least one of PAX6, LHX2, and SIX3.
22. The production method according to claim 1 , wherein the first step further excludes the presence of a ROCK inhibitor.
23. The production method according to claim 1 , wherein the factor for maintaining undifferentiated state is an FGF signal transduction pathway agonist.
24. The production method according to claim 23 , wherein the FGF signal transduction pathway agonist is bFGF.