# US 10,870,832 B2 · Use of small molecules to enhance MAFA expression in pancreatic endocrine cells

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- Number: US 10,870,832 B2
- Document ID: US10870832B2
- Status: Granted
- Kind: B2
- Application number: 16/007,600
- Filed: 2018-06-13
- Published: 2018-10-11
- Granted: 2020-12-22
- Earliest claimed: 2014-05-16
- Assignees: Janssen Biotech, Inc.
- Applicants: Janssen Biotech, Inc.
- Inventors: Alireza Rezania
- Web page: https://patentgrep.com/patent/US10870832B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- C12N 2500/38: Vitamins
- 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 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/375: Thyroid stimulating hormone [TSH]
- C12N 2501/40: Regulators of development
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/72: Transferases [EC 2.]
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2506/22: from pancreatic 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

## Abstract

The present invention provides methods, cell cultures and differentiation media to promote differentiation of pluripotent stem cells to pancreatic endocrine cells of a mature phenotype. The resulting pancreatic endocrine cells express single hormonal insulin, PDX1, NKX6.1, and MAFA. In one or more differentiation stages, culturing may be carried out in a culture vessel at the air-liquid interface.

## Claims

### Claim 1

A method for producing pancreatic endocrine cells expressing MAFA comprising:
(a) obtaining a population of cells comprising pancreatic endoderm and endocrine precursor cells;
(b) differentiating the population of cells comprising pancreatic endoderm and endocrine precursor cells into a pancreatic endocrine cell population in a medium comprising (i) a SHH signaling pathway antagonist, (ii) a BMP inhibitor, (iii) T3, a T3 analogue, T4, a T4 analogue or a mixture thereof, and (iv) an ALK5 inhibitor; and
(c) culturing the pancreatic endocrine cell population in a further medium comprising an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT 1L, or a combination thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the aurora kinase inhibitor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the aurora kinase inhibitor is aurora kinase inhibitor II, SNS 314 mesylate, GSK1070916 or TAK-901.

### Claim 4 (depends on 3)

The method of claim 3, wherein the aurora kinase inhibitor is aurora kinase inhibitor II.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the RSK inhibitor.

### Claim 6 (depends on 5)

The method of claim 5, wherein the RSK inhibitor is RSK inhibitor II.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the inhibitor of protein methyltransferase DOT1L.

### Claim 8 (depends on 7)

The method of claim 7, wherein the inhibitor of protein methyltransferase DOT1L is EPZ-5676.

### Claim 9 (depends on 1)

The method of claim 1, wherein the a) the population of pancreatic endoderm and endocrine precursor cells and/or b) the pancreatic endocrine cell population, are cultured in suspension cultures.

### Claim 10

A method for producing a pancreatic endocrine cell population expressing MAFA, comprising:
(a) obtaining a pancreatic endoderm and endocrine precursor cell population; and
(b) culturing the pancreatic endoderm and endocrine precursor cell population in a medium, wherein the medium comprises (i) a SMO inhibitor or a SHH signaling pathway antagonist, (ii) a BMP inhibitor, (iii) T3, an analogue of T3, T4, an analogue of T4 or a mixture thereof, (iv) an ALK5 inhibitor, and (v) an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, or a combination thereof,
thereby producing the pancreatic endocrine cell population, wherein cells in the pancreatic cell endocrine population express MAFA.

### Claim 11 (depends on 10)

The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising with the aurora kinase inhibitor.

### Claim 12 (depends on 11)

The method of claim 11, wherein the aurora kinase inhibitor is aurora kinase inhibitor II, SNS 314 mesylate, GSK1070916 or TAK-901.

### Claim 13 (depends on 10)

The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising the RSK inhibitor.

### Claim 14 (depends on 13)

The method of claim 13, wherein the RSK inhibitor is RSK inhibitor II.

### Claim 15 (depends on 10)

The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising the inhibitor of protein methyltransferase DOT1L.

### Claim 16 (depends on 15)

The method of claim 15, wherein the inhibitor of protein methyltransferase DOTIL is EPZ-5676.