# US 9,752,125 B2 · Differentiation of human embryonic stem cells

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- Number: US 9,752,125 B2
- Document ID: US9752125B2
- Status: Granted
- Kind: B2
- Application number: 13/105,285
- Filed: 2011-05-11
- Published: 2011-11-17
- Granted: 2017-09-05
- Earliest claimed: 2010-05-12
- Assignees: Janssen Biotech, Inc.
- Applicants: Jean Xu
- Inventors: Jean Xu
- Web page: https://patentgrep.com/patent/US9752125B2

## CPC

- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- 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 2501/15: 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 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/727: Kinases (EC 2.7.)
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 50% of the cells in the population co-express PDX1 and NKX6.1.

## Claims

### Claim 1

A cell culture comprising:
a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;
human definitive endoderm cells; and
a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,
wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,
wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and
wherein cells in the isolated PDX1 and NKX6.1 co-expressing population express CDX2.

### Claim 2 (depends on 1)

The cell culture of claim 1 wherein greater than 70% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 3 (depends on 1)

The cell culture of claim 1 wherein greater than 80% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 4 (depends on 1)

The cell culture of claim 1 wherein greater than 90% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 5 (depends on 1)

The cell culture of claim 1, wherein cells in the cell population express ISL1 and/or NEUROD1.

### Claim 6 (depends on 1)

The cell culture of claim 1, wherein the protein kinase C activator is selected from the group consisting of (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB), phorbol-12-myristate-13-acetate (PMA), and phorbol-12,13-dibutyrate (PDBu).

### Claim 7

A cell culture comprising:
a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;
human definitive endoderm cells; and
a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,
wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,
wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and
wherein the PDX1 and NKX6.1 co-expressing cells in the isolated population differentiate into insulin secreting cells in vivo.

### Claim 8 (depends on 7)

The cell culture of claim 7, wherein cells in the cell population express ISL1 and/or NEUROD1.

### Claim 9 (depends on 7)

The cell culture of claim 7, wherein greater than 70% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 10 (depends on 7)

The cell culture of claim 7, wherein greater than 80% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 11 (depends on 7)

The cell culture of claim 7, wherein greater than 90% of the cells in the population co-express PDX1 and NKX6.1.

### Claim 12 (depends on 7)

The cell culture of claim 7, wherein the protein kinase C activator is selected from the group consisting of (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB), phorbol-12-myristate-13-acetate (PMA), and phorbol-12,13-dibutyrate (PDBu).

### Claim 13 (depends on 1)

The cell culture of claim 1, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

### Claim 14 (depends on 7)

The cell culture of claim 7, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

### Claim 15

A cell culture comprising:
a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;
human definitive endoderm cells; and
a population comprising pancreatic endoderm cells,
wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 90% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1, and
wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells.

### Claim 16 (depends on 1)

The cell culture of claim 1, wherein cells in the cell population express NEUROD1.

### Claim 17 (depends on 1)

The cell culture of claim 1, wherein the cells are obtained by culturing a population of cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a protein kinase C activator and lacking FGF10.

### Claim 18 (depends on 7)

The cell culture of claim 7, wherein cells in the cell population express NEUROD1.

### Claim 19 (depends on 1)

The cell culture of claim 1, wherein the cells are obtainable by culturing a population of cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a protein kinase C activator and lacking FGF10.

### Claim 20

A cell culture comprising:
a medium supplemented with (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB);
human definitive endoderm cells; and
a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,
wherein the presence of TPB in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,
wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and
wherein cells in the isolated population express CDX2.

### Claim 21 (depends on 1)

The cell culture of claim 1, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

### Claim 22 (depends on 7)

The cell culture of claim 7, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

### Claim 23 (depends on 15)

The cell culture of claim 15, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

### Claim 24 (depends on 20)

The cell culture of claim 20, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.