# US 12,110,503 B2 · Method for differentiation of ocular cells and use thereof

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- Number: US 12,110,503 B2
- Document ID: US12110503B2
- Status: Granted
- Kind: B2
- Application number: 17/049,018
- Filed: 2019-04-22
- Published: 2021-08-05
- Granted: 2024-10-08
- Earliest claimed: 2018-04-20
- Assignees: FUJIFILM Cellular Dynamics, Inc.
- Applicants: FUJIFILM Cellular Dynamics, Inc.
- Inventors: Lucas Chase; Kyle Wallace; Benjamin Meline; Andrew Dias; Brandon Shelley; Marisa Fenn; Debjani Phillips; Matthew Sternfeld; Nathan Meyer
- Web page: https://patentgrep.com/patent/US12110503B2

## 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
- C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- C12N 2500/38: Vitamins
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/734: Proteases (EC 3.4.)
- C12N 2501/999: Small molecules not provided for elsewhere
- 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)
- 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

Provided herein are methods of producing a photoreceptor precursor (PRP) cell population derived from stem cells. Further provided herein are methods of using the PRP cell populations, such as for therapeutics.

## Claims

### Claim 1

A method for producing a population of neural retinal progenitors (NRPs) comprising:
(a) obtaining a starting population of human induced pluripotent stem cells (iPSCs);
(b) culturing the cells in a first retinal differentiation medium (RD1) comprising a BMP inhibitor to further differentiate the cells to anterior neuroectoderm cells;
(c) inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in a second retinal differentiation medium (RD2) essentially free of BMP inhibitors to form retinal progenitor cells (RPCs); and
(d) culturing the RPCs in a retinal maturation (RM) medium to produce NRPs.

### Claim 2 (depends on 1)

The method of claim 1, further comprising culturing the iPSCs in retinal induction medium (RIM) to initiate differentiation of the cells into anterior neuroectoderm cells prior to culturing the cells in RD1.

### Claim 3 (depends on 2)

The method of claim 2, wherein the RIM is essentially free of activin A.

### Claim 4 (depends on 2)

The method of claim 2, wherein the RIM comprises a BMP inhibitor, a TGFβ inhibitor, and/or IGF-1.

### Claim 5 (depends on 4)

The method of claim 4, wherein the RIM further comprises a WNT inhibitor.

### Claim 6 (depends on 4)

The method of claim 4, wherein RIM is essentially free of or free of CKI-7.

### Claim 7 (depends on 1)

The method of claim 1, further comprising culturing the population of NRPs as suspension aggregates in medium comprising a γ-secretase inhibitor.

### Claim 8 (depends on 7)

The method of claim 7, wherein the medium further comprises a ROCK inhibitor or blebbistatin.

### Claim 9 (depends on 1)

The method of claim 1, wherein the culturing of steps (a)-(d) is further defined as adherent 2-dimensional culture.

### Claim 10 (depends on 1)

The method of claim 1, wherein the RD1 medium comprises IGF-1, a TGFβ inhibitor, a Wnt inhibitor, and a MEK inhibitor.

### Claim 11 (depends on 1)

The method of claim 1, wherein the RD2 medium comprises IGF-1, a TGFβ inhibitor, a Wnt inhibitor, and a MEK inhibitor.

### Claim 12 (depends on 11)

The method of claim 11, wherein the RD2 medium is essentially free of LDN193189.

### Claim 13 (depends on 1)

The method of claim 1, wherein the RM medium comprises nicotinamide and ascorbic acid and is essentially free of Activin A.

### Claim 14 (depends on 13)

The method of claim 13, wherein the RM medium further comprises a γ-secretase inhibitor.

### Claim 15 (depends on 1)

The method of claim 1, further comprising detecting an increase in VSX2 expression of the anterior neuroectoderm cells to determine differentiation potential.

### Claim 16 (depends on 1)

The method of claim 1, wherein at least 90% of the cells after culturing in RD2 express PMEL17.

### Claim 17 (depends on 1)

The method of claim 1, wherein at least 30% of the cells after culturing in RD2 express VSX2.

### Claim 18 (depends on 1)

The method of claim 1, wherein the RPCs express PAX6, MITF, and/or PMEL.

### Claim 19 (depends on 1)

The method of claim 1, wherein at least 70% of the cells after culture in RM express PAX6 and CHX10 (VSX2).

### Claim 20 (depends on 1)

The method of claim 1, wherein the NRPs express one or more of the markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL.

### Claim 21 (depends on 1)

A pharmaceutical composition comprising the NRPs produced according to claim 1 and a pharmaceutically acceptable carrier.

### Claim 22 (depends on 1)

A method for producing a population of photoreceptor precursor cells (PRPs) comprising:
(a) obtaining a starting population of NRPs according to claim 1;
(b) further culturing the NRPs in PRP maturation medium (PM) comprising a γ-secretase inhibitor for a period of time sufficient to produce a population of PRPs.

### Claim 23 (depends on 22)

A pharmaceutical composition comprising the PRPs produced according to claim 22 and a pharmaceutically acceptable carrier.

### Claim 24 (depends on 1)

A method for producing a population of PRPs comprising:
(a) obtaining a starting population of NRPs according to claim 1;
(b) culturing the NRPs as aggregates in RM medium;
(c) further culturing the NRPs in RM medium comprising a γ secretase inhibitor to produce PRPs; and
(d) culturing the PRPs in PRP maturation medium (PM) further comprising a cyclin-dependent kinase inhibitor for a period of time sufficient to produce a population of mature PRPs.

### Claim 25 (depends on 24)

The method of claim 24, wherein the PM medium further comprises a CDK4/6 inhibitor.

### Claim 26 (depends on 24)

The method of claim 24, wherein culturing of step (d) is further defined as adherent 2-dimensional culture.

### Claim 27 (depends on 1)

A method for producing a population of optic vesicles (OVs) comprising:
(a) obtaining a starting population of RPCs according to claim 1; and
(b) further culturing the RPCs as suspension aggregates in RM medium or PM medium for a period of time sufficient to produce a population of OVs.

### Claim 28 (depends on 27)

The method of claim 27, wherein the population of OVs comprises cells at least 50% positive for VSX2 and/or at least 20% positive for RCVRN.

### Claim 29

A composition comprising an NRP population derived from iPSCs, wherein at least 90% of the cells in the NRP population express PAX6, at least 90% of the cells in the NRP population express PMEL17, and at least 70% of the cells in the NRP population express VSX2.