# US 11,898,168 B2 · Methods of promoting esophageal differentiation of pluripotent stem cells

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- Number: US 11,898,168 B2
- Document ID: US11898168B2
- Status: Granted
- Kind: B2
- Application number: 16/156,401
- Filed: 2018-10-10
- Published: 2019-04-18
- Granted: 2024-02-13
- Earliest claimed: 2017-10-12
- Assignees: The Trustees of Columbia University in the City of New York
- Applicants: The Trustees of Columbia University in the City of New York
- Inventors: Jianwen Que; Yongchun Zhang
- Web page: https://patentgrep.com/patent/US11898168B2

## CPC

- C12N 5/0679: {Cells of the gastro-intestinal tract}
- A61K 35/37: Digestive system
- C12N 2500/38: Vitamins
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/99: Serum-free medium
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2506/23: from cells of the gastro-intestinal tract
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2513/00: 3D culture
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- 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)

## Abstract

The current invention provides for methods of promoting differentiation of human pluripotent stem cells into esophageal progenitor cells as well as the cells obtained from the methods, solutions, compositions, and pharmaceutical compositions comprising such cells. The current invention also provides for methods of using the esophageal progenitor cells for treatment and prevention of disease, and kits.

## Claims

### Claim 1

A method for inducing esophageal differentiation of pluripotent stem cells, comprising the steps of
a. differentiating the pluripotent stem cells into endoderm cells by culturing the pluripotent stem cells in an endoderm differentiation medium for about one day to about four days;
b. differentiating the endoderm cells produced from step a. into anterior foregut cells by culturing the endoderm cells in an anterior foregut differentiation medium and (i) contacting or incubating the cultured endoderm cells with Noggin and SB431542 for about 24 hours, and (ii) further contacting or incubating the cultured endoderm cells with SB431542 and IWP-2 and wherein step b(ii) does not comprise contacting or incubating the cultured endoderm cells with Noggin for about 24 hours;
c. further culturing the anterior foregut cells produced from step b. in a first esophageal differentiation medium for about two days to ten days, wherein the first esophageal differentiation medium comprises Noggin and SB431542; and
d. further culturing the cells produced from step c in a second esophageal progenitor cell differentiation medium under conditions and for a time sufficient to allow the cells produced by step c to differentiate into esophageal progenitor cells, wherein the esophageal progenitor cells are SOX2+p63+EPCAM+ITGβ+NKX2.1-.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pluripotent stem cell is derived from a mouse or a human.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pluripotent stem cell is chosen from the group consisting of embryonic stem cells and induced pluripotent stem cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the Noggin in step b. is used in an amount ranging from about 50 ng/ml to about 200 ng/ml.

### Claim 5 (depends on 1)

The method of claim 1, wherein the SB431542 in step b. is used in an amount ranging from about 1 μM to about 25 μM.

### Claim 6 (depends on 1)

The method of claim 1, wherein step b. is performed starting at about day 4 to about day 5 for about 48 hours to about 72 hours.

### Claim 7 (depends on 1)

The method of claim 1, wherein the IWP-2 is used in an amount ranging from about 0.5 μM to about 2 μM.

### Claim 8 (depends on 1)

The method of claim 1, wherein the Noggin in step c. is used in an amount ranging from about 50 ng/ml to about 200 ng/ml.

### Claim 9 (depends on 1)

The method of claim 1, wherein the SB431542 in step c. is used in an amount ranging from about 1 μM to about 25 μM.

### Claim 10 (depends on 1)

The method of claim 1, wherein step c. is performed starting at about day 6 to about day 8 for about 2 days to about 10 days.

### Claim 11 (depends on 1)

The method of claim 1, wherein step d. is performed for about 2 days to about 10 days.

### Claim 12 (depends on 1)

The method of claim 1, wherein the endoderm differentiation medium is serum free.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first esophageal differentiation medium further comprises EGF and FGF10.

### Claim 14 (depends on 1)

The method of claim 1, further comprising purifying the esophageal progenitor cells obtained in step d. using the cell surface markers EPCAM+ and ITGμ4+.

### Claim 15 (depends on 14)

The method of claim 14, wherein antibodies for EPCAM+ and ITGμ4+ are used to purify the esophageal progenitor cells.

### Claim 16 (depends on 1)

The method of claim 1, wherein step (b)(ii) takes place for about 24 hours subsequent to step b(i).