Method for producing retinal tissue and retina-related cells

Patent metadata

Field	Value
Publication number	US-10501724-B2
Application number	US-201414913628-A
Country	US
Kind code	B2
Filing date	Aug 22, 2014
Priority date	Aug 23, 2013
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

Abstract

The present invention provides a method for producing a retinal progenitor cell, including
(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and (2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog signal transduction pathway but containing a substance acting on the BMP signal transduction pathway, thereby obtaining an aggregate containing retinal progenitor cells.

First claim

The invention claimed is:

1. A method for producing a retinal progenitor cell, comprising
(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and
(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells.

2. The method according to claim 1 , wherein the pluripotent stem cells are primate pluripotent stem cells.

3. The method according to claim 1 , wherein the step (1) and step (2) are performed in the presence of a serum replacement.

4. The method according to claim 1 , wherein the floating culture is performed in the absence of a basement membrane preparation.

5. The method according to claim 1 , wherein the substance acting on the BMP signal transduction pathway is added to the medium between day 1 and day 15 from the start of the floating culture in step (1).

6. The method according to claim 1 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is BMP4.

7. The method according to claim 6 , wherein BMP4 is at a concentration of about 1.5 nM.

8. A method for producing a retinal tissue, comprising
(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells,
(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells, and
(3) a third step of subjecting the aggregate formed in step (2) to floating culture in a serum-free medium or serum-containing medium each being free of any of a substance acting on the Shh signal transduction pathway that can enhance signal transduction mediated by Shh, a substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP and a substance acting on the Wingless-related integration site (Wnt) signal pathway that can enhance signal transduction mediated by Wnt, thereby obtaining an aggregate containing retinal tissues and being substantially free of non-neural head ectoderm.

9. The method according to claim 8 , wherein the pluripotent stem cells are primate pluripotent stem cells.

10. The method according to claim 8 , wherein the pluripotent stem cells are human pluripotent stem cells.

11. The method according to claim 8 , wherein the step (1) and step (2) are performed in the presence of a serum replacement.

12. The method according to claim 8 , wherein the floating culture is performed in the absence of a basement membrane preparation.

13. The method according to claim 8 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.

14. The method according to claim 8 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is added to the medium between day 1 and day 15 from the start of the floating culture in step (1).

15. A method for producing a retinal layer-specific neural cell, comprising
(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells,
(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells, and
(3) a third step of subjecting the aggregate formed in step (2) to floating culture in a serum-free medium or serum-containing medium each being free of any of a substance acting on the Shh signal transduction pathway that can enhance signal transduction mediated by Shh, a substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP and a substance acting on the Wingless-related integration site (Wnt) signal pathway that can enhance signal transduction mediated by Wnt until the intended retinal layer-specific neural cells appear, thereby obtaining an aggregate containing retinal tissues containing the intended retinal layer-specific neural cells and being substantially free of non-neural head ectoderm.

Assignees

• Sumitomo Chemical Co
• Riken

Inventors

• Nakano Tokushige
• Sasai Yoshiki
• Ozone Chikafumi

Classifications

• A61P27/02

  Ophthalmic agents · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2501/19

  Growth and differentiation factors [GDF] · CPC title

• C12N2500/44

  Thiols, e.g. mercaptoethanol · CPC title

• C12N2500/33

  other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine · CPC title