# US 10,307,444 B2 · Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells

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- Number: US 10,307,444 B2
- Document ID: US10307444B2
- Status: Granted
- Kind: B2
- Application number: 14/214,598
- Filed: 2014-03-14
- Published: 2014-10-02
- Granted: 2019-06-04
- Earliest claimed: 2013-03-15
- Assignees: Astellas Institute for Regenerative Medicine
- Applicants: Astellas Institute for Regenerative Medicine
- Inventors: Robert P. Lanza; Shi-Jiang Lu; Wei Wang
- Web page: https://patentgrep.com/patent/US10307444B2

## CPC

- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- 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
- 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 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain}
- C12N 5/0623: {Stem cells}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 2500/33: other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/33: Insulin
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2506/02: from embryonic cells
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods are provided for the production of photoreceptor cells and photoreceptor progenitor cells from pluripotent stem cells. Additionally provided are compositions of photoreceptor cells and photoreceptor cells, as well as methods for the therapeutic use thereof. Exemplary methods may produce substantially pure cultures of photoreceptor cells and/or photoreceptor cells.

## Claims

### Claim 1

A method of producing photoreceptor progenitor cells, comprising
(a) culturing pluripotent stem cells in the presence of a BMP signaling inhibitor to produce eye field progenitor cells;
(b) culturing the eye field progenitor cells under low adherence or non-adherent conditions in a neural differentiation medium in the absence of the BMP signaling inhibitor to form cell spheres;
(c) culturing the cell spheres under adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form attached cells;
(d) dissociating the attached cells and culturing the cells under low adherence or non-adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form further cell spheres;
(e) culturing the further cell spheres under adherent conditions in the neural differentiation medium in the absence of the BMP signaling inhibitor to form further attached cells;
(f) repeating steps (d) and (e) at least one additional time; and
thereby obtaining a preparation of photoreceptor progenitor cells, wherein 75%-100% of the photoreceptor progenitor cells in the preparation are PAX6(+) and CHX10(−) at the mRNA level.

### Claim 2 (depends on 1)

The method of claim 1, wherein said neural differentiation medium comprises Neurobasal medium, D-glucose, N2 supplement, B27 supplement, non-essential amino acids, and (i) glutamine, (ii) a stabilized form dipeptide from L-glutamine or L-alanyl-L-glutamine, or (iii) both glutamine and a stabilized form dipeptide from L-glutamine or L-alanyl-L-glutamine.

### Claim 3 (depends on 2)

The method of claim 2, wherein said neural differentiation medium comprises between about 400 and about 500 mg/ml D-glucose, one or both of streptomycin and penicillin, N2 supplement in a concentration of about 0.1 to 5%, B27 supplement in a concentration of about 0.05-5.0%, and non-essential amino acids in a concentration of about 0.1 mM.

### Claim 4 (depends on 3)

The method of claim 3, wherein said neural differentiation medium comprises about 2% N2 supplement.

### Claim 5 (depends on 3)

The method of claim 3, wherein said neural differentiation medium comprises about 0.05-2.0% B27 supplement.

### Claim 6 (depends on 3)

The method of claim 3, wherein said neural differentiation medium comprises about 2% B27 supplement.

### Claim 7 (depends on 1)

The method of claim 1, wherein step (a) comprises culturing the pluripotent stem cells under adherent conditions.

### Claim 8 (depends on 1)

The method of claim 1, further comprising differentiating said photoreceptor progenitor cells into photoreceptors.

### Claim 9 (depends on 1)

The method of claim 1, wherein the BMP signaling inhibitor is selected from the group consisting of Noggin polypeptide, dorsomorphin, LDN-193189, and any combination thereof.

### Claim 10 (depends on 9)

The method of claim 9, wherein the BMP signaling inhibitor is a Noggin polypeptide.

### Claim 11 (depends on 1)

The method of claim 1, wherein the photoreceptor progenitor cells express one or more of the Nr2e3, Trβ2, RORβ and NRL markers.

### Claim 12 (depends on 1)

The method of claim 1, wherein the photoreceptor progenitor cells are further characterized as mRNA transcript positive for Nr2e3, Trβ2, RORβ and NRL as detected by qPCR.

### Claim 13 (depends on 1)

The method of claim 1, wherein the photoreceptor progenitor cells are characterized by differentiation to photoreceptor cells upon treatment with retinoic acid.

### Claim 14 (depends on 1)

The method of claim 1, wherein the photoreceptor progenitor cells formed in step (c) retain plasticity to differentiate into either rods and cones.

### Claim 15 (depends on 1)

The method of claim 1, wherein the eye field progenitor cells are characterized as immunocytochemically PAX6+ and RX1+ and OCT4− and NANOG−.

### Claim 16 (depends on 15)

The method of claim 15, wherein the eye field progenitor cells are further characterized as Six3+, Six6+, Lhx2+, Tbx3+, SOX2+ and Nestin+ by immunostaining or flow cytometry.

### Claim 17 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

### Claim 18 (depends on 1)

The method of claim 1, wherein the photoreceptor progenitor cells are capable of differentiating into photoreceptor cells in vitro.

### Claim 19 (depends on 18)

The method of claim 18, wherein the photoreceptor progenitor cells differentiate into photoreceptor cells in vitro in the presence of retinoic acid and taurine.

### Claim 20 (depends on 18)

The method of claim 18, wherein the photoreceptor cells express rhodopsin, opsin, recoverin, and PDE6a.

### Claim 21 (depends on 1)

The method of claim 1, wherein the neural differentiation medium further comprises transferrin and insulin.