# US 8,785,185 B2 · Differentiation of human embryonic stem cells

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- Number: US 8,785,185 B2
- Document ID: US8785185B2
- Status: Granted
- Kind: B2
- Application number: 12/839,551
- Filed: 2010-07-20
- Published: 2011-01-20
- Granted: 2014-07-22
- Earliest claimed: 2009-07-20
- Assignees: Janssen Biotech, Inc.; The Cleveland Clinic Foundation
- Applicants: Jean Xu; Jan Jensen
- Inventors: Jean Xu; Jan Jensen
- Web page: https://patentgrep.com/patent/US8785185B2

## CPC

- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- 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 2501/415: Wnt; Frizzeled
- C12N 2506/02: from embryonic cells
- 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)
- 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 cells expressing markers characteristic of the pancreatic endocrine lineage that co-express PDX1, NKX6.1, but do not express CDX2 and NGN3.

## Claims

### Claim 1

A method to differentiate a population of human pluripotent stem cells into a population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3 comprising the steps of:
(a) culturing human pluripotent stem cells,
(b) differentiating the human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent stem cells with a medium supplemented with a TGF-β receptor agonist,
(c) differentiating the definitive endoderm cells into a population of pancreatic endoderm cells that co-express PDX-1 and NKX6.1, but do not express CDX2 and NGN3 by;
i) culturing the definitive endoderm cells in a first medium supplemented with FGF7, followed by
ii) culturing the cells from step i) in a second medium supplemented with FGF7, a factor capable of inhibiting BMP, activin A, retinoic acid, and a hedgehog signaling pathway inhibitor, and
(d) selecting cells which co-express PDX-1 and NKX6.1 and do not express CDX2 and NGN3 to obtain a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3.

### Claim 2 (depends on 1)

The method of claim 1, wherein the factor capable of inhibiting BMP is noggin.

### Claim 3 (depends on 1)

The method of claim 1, wherein the hedgehog signaling pathway inhibitor is cyclopamine-KAAD.

### Claim 4

A method of differentiating definitive endoderm cells into a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX6.1, but do not express CDX2 and NGN3 by:
(a) treating definitive endoderm cells with a first medium supplemented with FGF7; and
(b) culturing the treated definitive endoderm cells in a second medium supplemented with FGF7, a factor capable of inhibiting BMP, activin A, retinoic acid, and a hedgehog signaling pathway inhibitor to obtain a population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3, and
(c) selecting cells which co-express PDX-1 and NKX6.1 and do not express CDX2 and NGN3 to obtain a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3.