# US 11,441,184 B2 · Method for generating retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (IPSCs)

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- Number: US 11,441,184 B2
- Document ID: US11441184B2
- Status: Granted
- Kind: B2
- Application number: 16/586,656
- Filed: 2019-09-27
- Published: 2020-03-26
- Granted: 2022-09-13
- Earliest claimed: 2013-02-01
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Services
- Inventors: Kapil Bharti; Janine Davis; Arvydas Maminishkis; Sheldon S. Miller
- Web page: https://patentgrep.com/patent/US11441184B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- G01N 33/5058: {Neurological cells}
- G01N 33/56966: {Animal cells}
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q 2600/178: miRNA, siRNA or ncRNA
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

High efficiency methods for producing retinal pigment epithelial cells from induced pluripotent stem cells (iPSCs) are disclosed herein. The iPSCs are produced from somatic cells, including retinal pigment epithelial (RPE) cells, such as fetal RPE stem cells. In some embodiments, the iPSC include a tyrosinase promoter operably linked to a marker. Methods are disclosed for using the RPE cells, such as for treatment. Methods for screening for agents that affect RPE differentiation are also disclosed.

## Claims

### Claim 1

A method for producing human retinal pigment epithelial (RPE) cells, comprising:
(a) culturing human induced pluripotent stem cells (hiPSCs) in a human embryonic stem cell culture medium comprising human basic fibroblast growth factor (bFGF) and not containing ingredients obtained from non-human animals to produce small embryoid bodies of 200-500 cells;
(b) culturing the small embryoid bodies from step (a) in a first medium comprising retinal induction medium (RIM) and rock inhibitor (RI), wherein the RIM comprises a Wnt inhibitor, a Nodal pathway inhibitor, Noggin, and 1 to 3% v/v knockout serum replacement, wherein the Nodal pathway inhibitor is SB-431542 or SB-505124, to form embryoid bodies that have increased efficiency of RPE differentiation;
(c) culturing the embryoid bodies that have increased efficiency of RPE differentiation from step (b) on a matrigel coated tissue culture substrate-in a second medium comprising a retinal differentiation medium that does not comprise basic fibroblast growth factor (bFGF) and comprises Dickkopf-related protein 1 (DKK1), the Wnt inhibitor, a bFGF inhibitor, and Noggin, wherein the bFGF inhibitor is PD0325901, PD98059, PD161570, or PD166285, to form differentiating RPE cells that express PAX6 and MITF;
(d) culturing the differentiating RPE cells from step (c) in a third medium comprising retinal media comprising Activin A and Wnt3a to produce cells that have increased expression of MITF and PAX6 and increased RPE differentiation efficiency; and
(e) culturing the cells that have increased expression of MITF and PAX6 and increased RPE differentiation efficiency from step (d) in a fourth medium comprising a RPE cell medium comprising a non-canonical Wnt 5a inducer, DKK1, SU5402, and cyclopamine to produce human RPE cells that express TYR, TYRP1, MYRIP, Cadherin 1 or Cadherin 1 and TRPMI 1 or TRPMI 3, thereby producing human RPE cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the Wnt inhibitor is N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK1-7), 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), Secreted frizzled-related protein (SFRP) 1, SFRP-2, SFRP-3, SFRP-4 or SFRP-5.

### Claim 3 (depends on 1)

The method of claim 1, wherein the Nodal pathway inhibitor is SB-431542.

### Claim 4 (depends on 3)

The method of claim 3, wherein the first medium comprises 3.5 to 9 mM of SB-431542.

### Claim 5 (depends on 1)

The method of claim 1, wherein the retinal induction medium does not comprise serum.

### Claim 6 (depends on 1)

The method of claim 1, wherein the inhibitor of bFGF is PD032590.

### Claim 7 (depends on 6)

The method of claim 6, wherein the second medium comprises 0.5 to 2 mM of PD0325901.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first medium comprises 50 ng/ml of Dickkopf-related protein 1 (DKK1).

### Claim 9 (depends on 1)

The method of claim 1, wherein the third medium comprises 100 to 200 ng/ml of Activin A.

### Claim 10 (depends on 1)

The method of claim 1, wherein the third medium comprises 75 to 150 ng/ml of Wnt3a.

### Claim 11 (depends on 1)

The method of claim 1, wherein the second medium comprises 75 to 150 ng/ml of DKK1.

### Claim 12 (depends on 1)

The method of claim 1, wherein the fourth medium comprises 75 to 150 ng/ml of WNT5a, 75 to 150 ng/ml of DKK1, 5 μM Cycolopamine and 10 μM of SU5402.

### Claim 13 (depends on 1)

The method of claim 1, wherein step (a) comprises culturing the cells in the presence of 1.5% v/v knock out serum replacement.

### Claim 14 (depends on 1)

The method of claim 1, wherein the tissue culture substrate is a transwell plate.

### Claim 15 (depends on 1)

The method of claim 1, further comprising expressing OCT4, SOX2, LIN28 and Nanog in a human fetal RPE cell to produce the human induced pluripotent stem cells.

### Claim 16 (depends on 1)

The method of claim 1, wherein the human induced pluripotent stem cells comprise a nucleic acid encoding a marker operably linked to a RPE cell specific promoter.

### Claim 17 (depends on 16)

The method of claim 16, wherein the retinal specific promoter is a tyrosine kinase.

### Claim 18 (depends on 1)

The method of claim 1, wherein the embryoid bodies are cultured in the first medium for 48 hours.

### Claim 19 (depends on 1)

The method of claim 1, wherein the embryoid bodies are cultured in the second medium for 18 to 24 days.

### Claim 20 (depends on 19)

The method of claim 19, wherein the embryoid bodies are cultured in the second medium for three weeks.

### Claim 21 (depends on 1)

The method of claim 1, wherein the differentiating RPE cells are cultured in the third medium for 18 to 24 days.

### Claim 22 (depends on 21)

The method of claim 21, wherein the differentiating RPE cells are cultured in the third medium for three weeks.

### Claim 23 (depends on 1)

The method of claim 1, wherein the cells are cultured in the fourth medium for 12 to 16 days.

### Claim 24 (depends on 23)

The method of claim 23, wherein the cells are cultured in the fourth medium for two weeks.

### Claim 25 (depends on 1)

The method of claim 1, further comprising maintaining the RPE cells in the fourth medium and 5% v/v fetal serum.

### Claim 26 (depends on 25)

The method of claim 25, comprising maintaining the RPE cells in fourth medium and 5% v/v fetal serum for six to eight weeks.