Method for manufacturing ciliary marginal zone-like structure

Patent metadata

Field	Value
Publication number	US-2016376554-A1
Application number	US-201415103162-A
Country	US
Kind code	A1
Filing date	Oct 16, 2014
Priority date	Dec 11, 2013
Publication date	Dec 29, 2016
Grant date	—

Abstract

The present invention provides a method for producing a cell aggregate containing a ciliary marginal zone-like structure, including a step of culturing a cell aggregate containing a retinal tissue in which Chx10 positive cells are present in a proportion of 20% or more and 100% or less of the tissue in a serum-free medium or serum-containing medium each containing a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway for only a period before the appearance of a RPE65 gene expressing cell, followed by culturing the “cell aggregate in which a RPE65 gene-expressing cell does not appear” thus obtained in a serum-free medium or serum-containing medium each free of a substance acting on the Wnt signal pathway and so on. According to the production method of the present invention, a ciliary marginal zone-like structure can be produced with high efficiency.

First claim

1 . A method for producing a cell aggregate comprising a ciliary marginal zone-like structure, comprising a step of culturing a cell aggregate comprising a retinal tissue in which Chx10 positive cells are present in a proportion of 20% or more and 100% or less of the tissue in a serum-free medium or serum-containing medium each containing a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway for only a period before the appearance of a RPE65 gene-expressing cell, followed by culturing the resulting “cell aggregate in which a RPE65 gene-expressing cell does not appear” in a serum-free medium or serum-containing medium each free of a substance acting on the Wnt signal pathway.

2 . The production method according to claim 1 , wherein the cell aggregate in which a RPE65 gene-expressing cell does not appear is a cell aggregate in which Chx10 positive cells are present in the retinal tissue in a proportion of within 30% to 0% of the tissue.

3 . The production method according to claim 1 , wherein the resulting “cell aggregate in which a RPE65 gene-expressing cell does not appear” is cultured in a serum-free medium or serum-containing medium each free of a substance acting on the Wnt signal pathway until the proportion of Chx10 positive cells present in the retinal tissue reaches 30% or more of the tissue.

4 . The production method according to claim 1 , wherein the retinal tissue is derived from a human pluripotent stem cell.

5 . Use of a cell aggregate comprising a ciliary marginal zone-like structure produced by the production method according to claim 1 , as a reagent for evaluating toxicity or drug efficacy.

6 . A method for evaluating toxicity or drug efficacy of a test substance, comprising contacting the substance with a cell aggregate comprising a ciliary marginal zone-like structure produced by the production method according to claim 1 , and assaying an influence of the substance on the structure or the aggregate.

7 . Use of a cell aggregate comprising a ciliary marginal zone-like structure produced by the production method according to claim 1 , as a biological material for transplantation.

8 . A method of treating a disease due to a disorder of retinal tissue, comprising transplanting an effective amount of cell aggregate comprising a ciliary marginal zone-like structure produced by the production method according to claim 1 to a subject in need of the transplantation.

9 . A cell aggregate comprising a ciliary marginal zone-like structure produced by the production method according to claim 1 , which is for use for the treatment of a disease due to a disorder of the ciliary marginal zone-like structure.

Assignees

• Sumitomo Chemical Co
• Riken

Inventors

• Kuwahara Atsushi
• Sasai Yoshiki

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N2513/00

  3D culture · CPC title

• A61L2300/64

  Animal cells · CPC title