IP Library Granted Patent US 12,385,008
Granted Patent B2
US 12,385,008 · App. 15/758,255 · Granted Aug 12, 2025

Method for producing retinal tissue

Inventors: Satoshi Ando (Kobe, JP); Takao Kuroda (Kobe, JP); Yoshiki Sasai (Kobe, JP)
Assignees: SUMITOMO PHARMA CO., LTD.; RIKEN
C12N5/0621A61K35/30C12N5/10C12Q1/02G01N33/5014G01N33/5058A61K35/545C12N2500/90C12N2501/115C12N2501/155C12N2501/41C12N2501/727C12N2506/45C12N2513/00
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Quick Facts
Patent No.
US 12,385,008
App. No.
15/758,255
Granted
Aug 12, 2025
Kind
B2
Abstract

The present invention provides a production method of a retinal cell or retinal tissue, including the following steps: (1) a first step of culturing mammalian pluripotent stem cells in the absence of a feeder cell for a period not exceeding 30 days in a medium comprising 1) a factor for maintaining an undifferentiated state and 2) an MEK inhibitor, (2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a BMP signal transduction pathway activating substance to obtain an aggregate containing retinal cells or a retinal tissue.

Claims (30)

1. A method for producing a retinal cell or a retinal tissue, comprising the following steps:

(1) a first step of maintaining and/or expanding mammalian pluripotent stem cells comprising culturing the mammalian pluripotent stem cells in the absence of a feeder cell in a medium comprising a factor for maintaining an undifferentiated state, wherein the medium does not contain a mitogen-activated protein kinase kinase (MEK) inhibitor,

(2) a second step of culturing the mammalian pluripotent stem cells obtained in the first step in the absence of a feeder cell for a period not less than 4 days and not exceeding 30 days in a medium comprising (a) a factor for maintaining an undifferentiated state and (b) a MEK inhibitor, wherein the medium does not contain a transforming growth factor (TGF)-β family signal transduction pathway inhibitor,

(3) a third step of dispersing cells obtained in the second step and culturing the dispersed cells in suspension to form aggregates, wherein the aggregates are formed within 72 hours after the cells obtained in the second step are dispersed, and wherein six days after the start of the third step, at least 70% of the aggregates are uncollapsed or non-cystic, and

(4) a fourth step of culturing the aggregates obtained in the third step in suspension in the presence of a bone morphogenetic protein (BMP) signal transduction pathway activating substance in the absence of a basement membrane preparation to obtain aggregates containing a retinal cell or a retinal tissue, wherein the BMP signal transduction pathway activating substance is added to the medium at a point between day 1 and day 9 from the start of the third step.

2. The production method according to claim 1 , wherein the medium in the second step further comprises a protein kinase C (PKC) inhibitor or a B-Raf inhibitor.

3. The production method according to claim 2 , wherein the PKC inhibitor is Go6983.

4. The production method according to claim 2 , wherein the B-Raf inhibitor is SB590885.

5. The production method according to claim 1 , wherein the second step is performed under serum-free conditions.

6. The production method according to claim 1 , wherein the culture period is 4 days-6 days in the second step.

7. The production method according to claim 1 , wherein the second step is performed with an adhesion culture method.

8. The production method according to claim 1 , wherein the factor for maintaining an undifferentiated state in the first step and/or the second step is a fibroblast growth factor (FGF) signal transduction pathway activating substance.

9. The production method according to claim 8 , wherein the FGF signal transduction pathway activating substance is bFGF.

10. The production method according to claim 1 , wherein the MEK inhibitor is PD0325901.

11. The production method according to claim 1 , wherein the mammalian pluripotent stem cells are cultured in the presence of a Rho-associated coiled-coil kinase (ROCK) inhibitor in the second step.

12. The production method according to claim 11 , wherein the ROCK inhibitor is Y-27632.

13. The production method according to claim 1 , wherein the cells are cultured in suspension in a serum-free medium containing a Sonic hedgehog signal transduction pathway activating substance in the third step.

14. The production method according to claim 13 , wherein the Sonic hedgehog signal transduction pathway activating substance is Smoothened Agonist (SAG).

15. The production method according to claim 1 , wherein, in the third step, the concentration of the Sonic hedgehog signal transduction pathway activating substance in the medium is a concentration corresponding to Sonic hedgehog signal transduction activity of SAG at 10 nM to 700 nM.

16. The production method according to claim 1 , wherein the BMP signal transduction pathway activating substance is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and Growth Differentiation Factor 7 (GDF7).

17. The production method according to claim 16 , wherein the BMP signal transduction pathway activating substance is BMP4.

18. The production method according to claim 13 , wherein, in the fourth step, the BMP signal transduction pathway activating substance is added to the medium at a point between day 3 and day 6 from the start of the third step.

19. The production method according to claim 1 , wherein the pluripotent stem cell is an induced pluripotent stem cell.

20. The production method according to claim 19 , wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.

21. The production method according to claim 1 , wherein the medium in the second step does not further contain a Sonic hedgehog signal transduction pathway activating substance.

22. The production method according to claim 1 , wherein, in the fourth step, the aggregates are cultured in suspension in the presence of a BMP signal transduction pathway activating substance until a cell expressing a Rx gene, Pax6 gene, and/or Chx10 gene appears.

23. The production method according to claim 1 , wherein the cells obtained in the second step contain cells maintaining pluripotent-like properties.

24. A method for producing a retinal tissue comprising:

(1) producing cell aggregates comprising a retinal tissue by the production method according to claims 1 , and

(2) cutting out the retinal tissue from the cell aggregates.

Assignments (4)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 30, 2025
From: SUMITOMO PHARMA CO., LTD.
To: RACTHERA CO., LTD.
Reel/Frame 072825/0888 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 4, 2024
From: SUMITOMO CHEMICAL COMPANY, LIMITED
To: SUMITOMO PHARMA CO., LTD.
Reel/Frame 068799/0134 →
NAME AND ADDRESS CHANGE Recorded May 4, 2022
From: SUMITOMO DAINIPPON PHARMA CO., LTD.
To: SUMITOMO PHARMA CO., LTD.
Reel/Frame 059855/0333 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 7, 2018
From: ANDO, SATOSHI; KURODA, TAKAO; SASAI, MIYUKI (LEGAL REPRESENTATIVE OF DECEASED INVENTOR YOSHIKI SASAI)
To: SUMITOMO DAINIPPON PHARMA CO., LTD.; SUMITOMO CHEMICAL COMPANY, LIMITED; RIKEN
Reel/Frame 045136/0719 →
Priority Claims (1)
JP 2015-176897 · Sep 8, 2015 · national
Continuity (1)
Related Publication 20180245039A1 · Aug 30, 2018
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