# US 11,111,476 B2 · Method for manufacturing ciliary marginal zone-like structure

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- Number: US 11,111,476 B2
- Document ID: US11111476B2
- Status: Granted
- Kind: B2
- Application number: 16/267,948
- Filed: 2019-02-05
- Published: 2019-06-06
- Granted: 2021-09-07
- Earliest claimed: 2013-12-11
- Assignees: Sumitomo Chemical Company, Limited; Riken
- Applicants: Sumitomo Chemical Company, Limited; Riken
- Inventors: Atsushi Kuwahara; Yoshiki Sasai
- Web page: https://patentgrep.com/patent/US11111476B2

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- A61K 9/0051: {Ocular inserts or implants}
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61L 27/3641: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)}
- A61L 27/3687: {characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents}
- A61L 27/3804: {characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells}
- A61P 27/02: Ophthalmic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/5014: {for testing toxicity}
- A61L 2300/64: Animal cells
- A61L 2430/16: for reconstruction of eye parts, e.g. intraocular lens, cornea
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2513/00: 3D culture
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 1

A method for producing a retinal pigment epithelium tissue comprising the steps:
(a) preparing a cell aggregate from a pluripotent stem cell,
(b) culturing the cell aggregate to form a retinal tissue comprising 20% or more and 100% or less of cells positive for Chx10,
(c) culturing the retinal tissue of step (b) in a serum-free medium comprising a substance that enhances signal transduction mediated by Wnt and a substance inhibiting the FGF signal pathway, thereby obtaining a retinal tissue having a decreased proportion of Chx10 positive cells that is about 3% or less of the tissue and comprising MITF positive cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the decreased proportion of Chx10 positive cells in step (c) is within 3% to 0% of the tissue.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the MITF positive cells are about 80% of the tissue.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the pluripotent stem cell is a human pluripotent stem cell.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the culturing of the cell aggregate in step (b) forms a retinal tissue comprising 40% or more of cells positive for Chx10.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the culturing of the cell aggregate in step (b) forms a retinal tissue comprising 60% or more of cells positive for Chx10.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the culturing of the retinal tissue in step (c) is for 3 to 6 days.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the substance that enhances signal transduction mediated by Wnt is a GSK3β inhibitor.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the substance that enhances signal transduction mediated by Wnt is CHIR99021.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the serum-free medium in step (c) contains about 1 μM to about 30 μM CHIR99021.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the substance inhibiting the FGF signal pathway is a FGF receptor inhibitor.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the substance inhibiting the FGF signal pathway is SU-5402.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the serum-free medium in step (c) contains about 1 μM to about 30 μM SU-5402.

### Claim 14

A method of treating a disease due to a disorder of retinal tissue, comprising the steps:
(a) preparing a cell aggregate from a pluripotent stem cell,
(b) culturing the cell aggregate to form a retinal tissue comprising 20% or more and 100% or less of cells positive for Chx10,
(c) culturing the retinal tissue of step (b) in a serum-free medium comprising a substance that enhances signal transduction mediated by Wnt and a substance inhibiting the FGF signal pathway, thereby obtaining a retinal tissue having a decreased proportion of Chx10 positive cells that is about 3% or less of the tissue, and comprising MITF positive cells, and
(d) transplanting an effective amount of the retinal tissue comprising MITF positive cells of step (c) to a subject in need of the transplantation.