# US 9,752,126 B2 · Differentiation of human pluripotent stem cells

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- Number: US 9,752,126 B2
- Document ID: US9752126B2
- Status: Granted
- Kind: B2
- Application number: 14/963,436
- Filed: 2015-12-09
- Published: 2016-06-09
- Granted: 2017-09-05
- Earliest claimed: 2008-10-31
- Assignees: Janssen Biotech, Inc.
- Applicants: Janssen Biotech, Inc.
- Inventors: Alireza Rezania; Benjamin Fryer
- Web page: https://patentgrep.com/patent/US9752126B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/39: Steroid hormones
- C12N 2501/405: Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/70: Enzymes
- C12N 2501/998: Proteins not provided for elsewhere
- 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 a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of MAFA.

## Claims

### Claim 1

A method for increasing the expression of insulin and MAF bZIP transcription factor A (“MAFA”) in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of:
a. sequentially differentiating human pluripotent stem cells to obtain cells expressing markers characteristic of the pancreatic endocrine lineage; and
b. culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of insulin and MAFA as compared to cells expressing markers characteristic of the pancreatic endocrine lineage that are not cultured in medium comprising the added cyclin-dependent kinase inhibitor;
wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cyclin-dependent kinase inhibitor is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 μM to about 10 μM for about one to seven days.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

### Claim 5 (depends on 1)

The method of claim 1, wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

### Claim 6

A method for increasing the expression of insulin and MAF bZIP transcription factor A (“MAFA”) in a population of cells comprising pancreatic endocrine cells comprising culturing the population of cells in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in the expression of insulin and MAFA in the population as compared to a population of cells comprising pancreatic endocrine cells not cultured in medium comprising the added cyclin-dependent kinase inhibitor, wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

### Claim 7 (depends on 6)

The method of claim 6, wherein the method comprises adding the cyclin-dependent kinase inhibitor at a concentration from about 0.1 μM to about 10 μM for about one to seven days.

### Claim 8 (depends on 6)

The method of claim 6, wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

### Claim 9 (depends on 6)

The method of claim 6, wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

### Claim 10 (depends on 6)

The method of claim 6, wherein the pancreatic endocrine cells are obtained by differentiating human pluripotent stem cells.

### Claim 11 (depends on 10)

The method of claim 10, wherein the human pluripotent stem cells are human embryonic cells.