Method for producing retinal pigment epithelial cells

Patent metadata

Field	Value
Publication number	US-12221625-B2
Application number	US-202318295428-A
Country	US
Kind code	B2
Filing date	Apr 4, 2023
Priority date	Sep 8, 2015
Publication date	Feb 11, 2025
Grant date	Feb 11, 2025

Abstract

The invention provides a method for producing a retinal pigment epithelial cell by (1) 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 a MEK 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, 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.

First claim

The invention claimed is:

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 a MEK 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, 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 factor for maintaining undifferentiated state is an FGF signal transduction pathway agonist.

8. The production method according to claim 7 , wherein the FGF signal transduction pathway agonist is bFGF.

9. 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.

10. The production method according to claim 1 , wherein the MEK inhibitor is at least one kind selected from the group consisting of PD0325901, PD184352, U0126, TAK-733, and AZD-8330.

11. The production method according claim 1 , wherein the Nodal signal transduction pathway inhibitor is an Alk4, 5, or 7 inhibitor.

12. The production method according to claim 11 , wherein the ALK4, 5, or 7 inhibitor is SB431542.

13. The production method according to claim 1 , wherein the Wnt signal transduction pathway inhibitor is CKI-7.

14. The production method according to claim 1 , wherein the medium in the first step is a medium further comprising a BMP receptor inhibitor.

15. The production method according to claim 14 , wherein the BMP receptor inhibitor is an ALK2/3 inhibitor.

16. The production method according to claim 15 , wherein the ALK2/3 inhibitor is LDN193189.

17. The production method according to claim 1 , wherein the medium in the first step further comprises a Sonic hedgehog signal transduction pathway agonist.

18. The production method according to claim 17 , wherein the Sonic hedgehog signal transduction pathway agonist is SAG.

19. The production method according to claim 1 , wherein the medium in the first step further comprises a PKC inhibitor.

20. The production method according to claim 19 , wherein the PKC inhibitor is Go6983.

21. The production method according to claim 1 , wherein the culture period in the first step is for 2 days to 13 days.

22. The production method according to claim 1 , wherein the culture period in the first step is for 4 days to 6 days.

23. The production method according to claim 1 , wherein the first step further excludes the presence of a ROCK inhibitor.

24. 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.

Assignees

• Sumitomo Pharma Co Ltd
• Healios Kk

Inventors

• Ando Satoshi
• Kuroda Takao

Classifications

• C12N2510/00

  Genetically modified cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2500/90

  Serum-free medium, which may still contain naturally-sourced components · CPC title

• C12N2501/41

  Hedgehog proteins; Cyclopamine (inhibitor) · CPC title