# US 9,752,119 B2 · Substantially pure human retinal progenitor, forebrain progenitor, and retinal pigment epithelium cell cultures and methods of making the same

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- Number: US 9,752,119 B2
- Document ID: US9752119B2
- Status: Granted
- Kind: B2
- Application number: 15/143,141
- Filed: 2016-04-29
- Published: 2016-09-01
- Granted: 2017-09-05
- Earliest claimed: 2009-08-24
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: David Matthew Gamm; Jason Stephen Meyer
- Web page: https://patentgrep.com/patent/US9752119B2

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- A61P 27/02: Ophthalmic agents
- 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 5/0602: {Vertebrate cells}
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/91: Heparin
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

Methods for producing substantially pure cultures of human neural retinal progenitor cells, forebrain progenitor cells, and retinal pigment epithelial cells are disclosed. In addition, the successful differentiation of human embryonic stem cells and human induced pluripotent stem cells through the major developmental stages of human retinogenesis is disclosed.

## Claims

### Claim 1

A method for generating a cell culture wherein at least 90% of the cells are human retinal pigment epithelium (RPE) cells, comprising
culturing a population of floating human vesicular neurospheres in the presence of Activin until pigmented RPE neurospheres form, wherein the human vesicular neurospheres have a hollow center and an outer ring-like laminar layer comprising human retinal progenitor cells oriented radially outwards relative to the neurosphere center, and wherein the outer ring-like laminar layer appears phase-bright and golden in color when observed using bright-field microscopy,
identifying a plurality of pigmented RPE neurospheres within the population, and
isolating the plurality of pigmented RPE neurospheres from the population and introducing the isolated neurospheres into a culture medium to obtain a cell culture comprising pigmented RPE neurospheres wherein at least 90% of the cells in the cell culture comprising pigmented RPE neurospheres are RPE cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the culturing is serum free.

### Claim 3 (depends on 1)

The method of claim 1, wherein the RPE cells comprise the genetic background of a subject having a retinal degenerative disease.

### Claim 4 (depends on 1)

The method of claim 1, wherein the culture medium is RPE propagation medium and the method further comprises the step of maintaining the RPE neurospheres in RPE propagation medium to proliferate RPE cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein the RPE propagation medium comprises FGF2, EGF, and heparin.

### Claim 6

A method for generating a cell culture comprising a population of human photoreceptor cells, comprising
culturing a population of human vesicular neurospheres in retinal differentiation medium until Crx+ photoreceptor cells form, wherein the human vesicular neurospheres have a hollow center and an outer ring-like laminar layer comprising human retinal progenitor cells oriented radially outwards relative to the neurosphere center, and wherein the outer ring-like laminar layer appears phase-bright and golden in color when observed using bright-field microscopy.

### Claim 7 (depends on 6)

The method of claim 6, wherein the human vesicular neurospheres are cultured for at least 50 days to produce Crx+ photoreceptor cells.

### Claim 8 (depends on 7)

The method of claim 7, wherein the human vesicular neurospheres are cultured between 50 days and 3 months to produce Crx+ photoreceptor cells.

### Claim 9 (depends on 8)

The method of claim 8, wherein the human vesicular neurospheres are cultured between 50-80 days to produce Crx+ photoreceptor cells.

### Claim 10 (depends on 6)

The method of claim 6, wherein the photoreceptor cells also express recoverin.

### Claim 11 (depends on 6)

The method of claim 6, wherein the photoreceptor cells also express opsin.