# US 2019/0169569 A1 · Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells

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- Number: US 2019/0169569 A1
- Document ID: US20190169569A1
- Status: Published application
- Kind: A1
- Application number: 15/758,314
- Filed: 2016-09-07
- Published: 2019-06-06
- Earliest claimed: 2015-09-08
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Serv; Cellular Dynamics International, Inc.
- Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Serv; Cellular Dynamics International, Inc.
- Inventors: Kapil Bharti; Lucas Chase; Feng Xuezhu; Balendu Shekhar Jha
- Web page: https://patentgrep.com/patent/US20190169569A1

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- C12N 2500/30: Organic components
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/415: Wnt; Frizzeled
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/50: Proteins
- C12N 2533/52: Fibronectin; Laminin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are methods of producing an RPE cell population from a starting cell suspension, such as a single cell suspension, of pluripotent stem cells (PSCs). Such a method may comprise culturing the starting single cell suspension of PSCs in differentiation media to produce human RPE cells.

## Claims

### Claim 1

A method for producing human retinal pigment epithelial (RPE) cells, comprising:
a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) that are dissociated into essentially single cells;
b) culturing the iPSCs in a retinal induction medium to initiate differentiation of the cells into retinal lineage cells;
c) further culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells;
d) culturing the cells in retinal medium to form differentiating RPE cells; and
e) culturing the RPE cells in a RPE maturation medium, thereby producing human RPE cells;
wherein the method does not comprise the formation of embryoid bodies.

### Claim 2 (depends on 1)

The method of claim 1, wherein the iPSCs in step (b) are cultured on a matrix.

### Claim 3 (depends on 2)

The method of claim 2, wherein the matrix comprises at least one recombinant cellular adhesion protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the at least one cellular adhesion protein is laminin, vitronectin or fibronectin.

### Claim 5 (depends on 3)

The method of claim 3, wherein the at least one cellular adhesion protein is human.

### Claim 6 (depends on 1)

The method of claim 1, wherein the retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor and insulin growth factor 1 (IGF1).

### Claim 7 (depends on 1)

The method of claim 1, wherein the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor and IGF1.

### Claim 8 (depends on 1)

The method of claim 1, further comprising following step (e) dissociating the RPE cells, reseeding the RPE cells and culturing the RPE cells in the RPE maturation medium comprising a MEK inhibitor.

### Claim 9 (depends on 8)

The method of claim 8, further comprising dissociating the RPE cells and reseeding the RPE cells on a degradable scaffold in the RPE maturation medium.

### Claim 10 (depends on 1)

The method of claim 1, further comprising culturing the RPE cells in the RPE maturation medium, wherein the RPE maturation medium comprises at least one primary cilium inducer thereby producing mature RPE cells.

### Claim 11 (depends on 10)

The method of claim 10, wherein the at least one primary cilium inducer is prostaglandin E2 (PGE2) or aphidicolin.

### Claim 12 (depends on 1)

The method of claim 1, further comprising culturing the RPE cells in the RPE maturation medium, wherein the RPE maturation medium comprises N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2) and/or 4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide (endo-IWR1).

### Claim 13 (depends on 1)

The method of claim 1, further comprising cryopreserving the RPE cells.

### Claim 14 (depends on 1)

The method of claim 1, wherein the starting population of iPSCs of step (a) are pre-confluent cells that have been dissociated into single cells.

### Claim 15 (depends on 1)

The method of claim 1, wherein the iPSCs of step (b) are cultured mat an initial cell density of about 5,000 to 40,000 cells/cm2; ii) without a feeder layer, iii) in a fully-defined culture medium, and/or iv) in a xeno-free culture medium.

### Claim at position 16

16-18. (canceled)

### Claim 19 (depends on 6)

The method of claim 6, wherein the WNT pathway inhibitor is N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamide dihydrochloride (CKI-7), N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-Phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazide (PNU 74654) 2,4-diamino-quinazoline, quercetin, 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-Dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH 535), N-[4-[2-Ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK 715), Dickkopf-related protein one (DKK1), or Secreted frizzled-related protein (SFRP1) 1.

### Claim 20 (depends on 6)

The method of claim 6, wherein the TGFβ pathway inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-Benzo[1,3]dioxol-5-yl-2-ieri-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, left-right determination factor (Lefty), 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D 4476), 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]quinoline (LY 364847), 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R 268712), or 2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox).

### Claim 21 (depends on 7)

The method of claim 7, wherein the MEK inhibitor N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).

### Claim 22 (depends on 6)

The method of claim 6, wherein the BMP pathway inhibitor is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), or 5-[6-(4-Methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347).

### Claim 23 (depends on 6)

The method of claim 6, wherein the BMP pathway inhibitor is LDN193189 in the retinal induction medium.

### Claim 24 (depends on 7)

The method of claim 7, wherein the BMP pathway inhibitor is LDN193189 and the MEK inhibitor is PD0325901 in the retinal differentiation medium.

### Claim 25 (depends on 1)

The method of claim 1, wherein the starting population of iPSCs are MHC haplotype-matched to a subject in need thereof.

### Claim 26 (depends on 1)

The method of claim 1, wherein the starting population of iPSCs are homozygous for at least one HLA allele.

### Claim 27 (depends on 26)

The method of claim 26, wherein the at least one HLA allele is HLA-A, HLA-B or HLA-DR.

### Claim 28

A method for producing human retinal pigment epithelial (RPE) cells, comprising:
a) obtaining a starting population comprising human induced pluripotent stem cells (iPSCs) that are dissociated into essentially single cells in a fully defined medium;
b) culturing the iPSCs on laminin, vitronectin or a combination thereof, in a retinal induction medium comprising LDN193189, CKI-7, and SB431542 to initiate differentiation of the cells into retinal lineage cells;
c) further culturing the retinal lineage cells in a retinal differentiation medium comprising LDN193189, CKI-7, SB431542, and PD0325901 to further differentiate the retinal lineage cells;
d) culturing the cells in retinal medium comprising nicotinamide and Activin A to form differentiating RPE cells; and
e) culturing the RPE cells in a RPE maturation medium, thereby producing human RPE cells;
wherein the method does not comprise the formation of embryoid bodies.