# US 10,501,724 B2 · Method for producing retinal tissue and retina-related cells

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- Number: US 10,501,724 B2
- Document ID: US10501724B2
- Status: Granted
- Kind: B2
- Application number: 14/913,628
- Filed: 2014-08-22
- Published: 2016-09-01
- Granted: 2019-12-10
- Earliest claimed: 2013-08-23
- Assignees: Sumitomo Chemical Company, Limited; Riken
- Applicants: Sumitomo Chemical Company, Limited; Riken
- Inventors: Tokushige Nakano; Yoshiki Sasai; Chikafumi Ozone
- Web page: https://patentgrep.com/patent/US10501724B2

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- 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 27/3804: {characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/3895: {using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions}
- A61P 27/02: Ophthalmic agents
- A61L 2430/16: for reconstruction of eye parts, e.g. intraocular lens, cornea
- C12N 2500/33: other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
- C12N 2500/44: Thiols, e.g. mercaptoethanol
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/19: Growth and differentiation factors [GDF]
- 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 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- 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

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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).

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

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

### Claim 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.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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

### Claim 11 (depends on 8)

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

### Claim 12 (depends on 8)

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

### Claim 13 (depends on 8)

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.

### Claim 14 (depends on 8)

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).

### Claim 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.