# US 9,752,118 B2 · Method of directed differentiation producing corneal endothelial cells from neural crest stem cells by PDGFB and DKK2, compositions thereof, and uses thereof

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- Number: US 9,752,118 B2
- Document ID: US9752118B2
- Status: Granted
- Kind: B2
- Application number: 14/362,775
- Filed: 2012-12-06
- Published: 2014-12-18
- Granted: 2017-09-05
- Earliest claimed: 2012-12-06
- Assignees: Astellas Institute for Regenerative Medicine
- Applicants: Astellas Institute for Regenerative Medicine
- Inventors: Kathryn L. McCabe; Shi-Jiang Lu; Robert P. Lanza
- Web page: https://patentgrep.com/patent/US9752118B2

## CPC

- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- A61K 9/0048: {Eye, e.g. artificial tears}
- 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
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61L 27/3808: {Endothelial cells}
- A61L 27/3839: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)}
- A61L 27/3895: {using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions}
- C12N 5/0623: {Stem cells}
- A61L 2430/16: for reconstruction of eye parts, e.g. intraocular lens, cornea
- C12N 2500/60: Buffer, e.g. pH regulation, osmotic pressure
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/135: Platelet-derived growth factor [PDGF]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/40: Regulators of development
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2506/08: from cells of the nervous system
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/50: Proteins
- 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)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)

## Abstract

This disclosure generally relates to cell-based therapies for treatment of visual disorders, including disorders of the cornea. Methods are exemplified for directed differentiation of corneal cells from stem cells. Compositions of corneal endothelial cells and uses thereof are also provided. Exemplary compositions exhibit improved cell density and/or more “youthful” gene expression relative to cells obtained from donated tissue.

## Claims

### Claim 1

A method of producing corneal endothelial cells (CECs), comprising
contacting embryonic stem (ES) cells with Noggin and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) to induce differentiation of the ES cells into neural crest stem cells (NCSCs); and
contacting the NCSCs with dickkopf-related protein 2 (DKK2), and platelet-derived growth factor subunit B (PDGFB), to induce differentiation of the NCSCs into CECs that express Na+/K+ ATPase, ZO-1, KLF13, AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, Carbonic anhydrase II (CA2), Anion exchanger 2 (AE2/SCL4A2), Solute Carrier Family 16 A1 (SCL16A1), SCL16A3, Carbonic anhydrase 12 (CA12), Carbonic Anhydrase 4 (CA4), cystic fibrosis transmembrane conductance regulator (CFTR) and transcription factor FoxC1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method comprises contacting NCSCs with DKK2 having a concentration of between 2 ng/ml and 20 ng/ml.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method comprises contacting NCSCs with PDGFB having a concentration of between 2 ng/ml and 20 ng/ml.

### Claim 4 (depends on 1)

The method of claim 1, further comprising culturing the CECs on a matrix.

### Claim 5 (depends on 4)

The method of claim 4, wherein the matrix is selected from: laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, a human basement membrane extract, and any combination thereof.

### Claim 6 (depends on 4)

The method of claim 4, wherein the matrix is a soluble preparation obtained from EHS mouse sarcoma cells (MATRIGEL®).

### Claim 7 (depends on 1)

The method of claim 1, wherein the NCSCs express Sox10, AP2, HNK1, Pax3, PAX7 and p75 (NGFR).

### Claim 8 (depends on 1)

The method of claim 1, wherein the CECs do not express vWF and CD31.

### Claim 9 (depends on 1)

The method of claim 1, wherein the ES cells are human ES cells and the CECs are human CECs.