# US 12,215,354 B2 · Differentiation of human embryonic stem cells into single hormonal insulin positive cells

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- Number: US 12,215,354 B2
- Document ID: US12215354B2
- Status: Granted
- Kind: B2
- Application number: 17/749,735
- Filed: 2022-05-20
- Published: 2022-09-01
- Granted: 2025-02-04
- Earliest claimed: 2011-12-22
- Assignees: Janssen Biotech, Inc.
- Applicants: Janssen Biotech, Inc.
- Inventors: Alireza Rezania
- Web page: https://patentgrep.com/patent/US12215354B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- C12N 2500/34: Sugars
- C12N 2500/38: Vitamins
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- 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 2501/19: Growth and differentiation factors [GDF]
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/02: from embryonic cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of cells, wherein greater than 10% of the cells in the population express markers characteristic of single hormonal pancreatic beta cells.

## Claims

### Claim 1

An in vitro method for the stepwise differentiation of pluripotent cells into a population of cells comprising pancreatic endocrine cells, comprising:
differentiating the pluripotent cells into foregut endoderm cells; and
differentiating the foregut endoderm cells in a first medium supplemented with retinoic acid, a shh inhibitor and a BMP inhibitor, thereby producing the population of cells comprising the pancreatic endocrine cells.

### Claim 2 (depends on 1)

The in vitro method of claim 1, wherein the shh inhibitor is SANT-1.

### Claim 3 (depends on 1)

The in vitro method of claim 1, wherein the BMP inhibitor is LDN-193189 or Noggin.

### Claim 4 (depends on 1)

The in vitro method of claim 1, wherein differentiating the foregut endoderm cells in the first medium supplemented with retinoic acid, the shh inhibitor and the BMP inhibitor produces foregut precursor cells, and wherein the method further comprises:
differentiating the foregut precursor cells in a second medium supplemented with a shh inhibitor.

### Claim 5 (depends on 4)

The in vitro method of claim 4, wherein the second medium is further supplemented with a BMP inhibitor.

### Claim 6 (depends on 5)

The in vitro method of claim 5, wherein the BMP inhibitor is LDN-193189 or Noggin.

### Claim 7 (depends on 4)

The in vitro method of claim 4, wherein the second medium is further supplemented with an ALK5 inhibitor.

### Claim 8 (depends on 4)

The in vitro method of claim 4, wherein the shh inhibitor in the second medium is SANT-1.

### Claim 9 (depends on 4)

The in vitro method of claim 4, wherein the second medium is further supplemented with retinoic acid.

### Claim 10 (depends on 4)

The in vitro method of claim 4, wherein the second medium is further supplemented with ascorbic acid.

### Claim 11 (depends on 4)

The in vitro method of claim 4, wherein the second medium further comprises a BMP inhibitor, and wherein differentiating the foregut precursor cells in the second medium supplemented with the shh inhibitor and the BMP inhibitor produces pancreatic endoderm cells and pancreatic endocrine precursor cells, and wherein the method further comprises:
differentiating the pancreatic endoderm cells and the pancreatic endocrine precursor cells to form pancreatic endocrine cells in a third medium supplemented with a shh inhibitor.

### Claim 12 (depends on 11)

The in vitro method of claim 11, wherein the shh inhibitor in the third second-medium is SANT-1.

### Claim 13 (depends on 11)

The in vitro method of claim 11, wherein the third medium is further supplemented with retinoic acid.

### Claim 14 (depends on 11)

The in vitro method of claim 11, wherein the third medium is further supplemented with ascorbic acid.

### Claim 15 (depends on 11)

The in vitro method of claim 11, wherein the third medium is further supplemented with an ALK5 inhibitor.

### Claim 16 (depends on 1)

The in vitro method of claim 1, wherein the pancreatic endocrine cells are single hormonal insulin-producing cells which are also NKX6.1+ and PDX-1+.