Differentiation of human embryonic stem cells

• US 8,785,185 B2
• Filed: 07/20/2010
• Issued: 07/22/2014
• Est. Priority Date: 07/20/2009
• Status: Active Grant

• Current Assignee: Cleveland Clinic Foundation, Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Cleveland Clinic Foundation, Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Xu, Jean, Jensen, Jan
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/839,551
• Publication Number:
  US 20110014703A1
• Time in Patent Office: 1,463 Days
• Field of Search: 435/325, 435/366, 435/377
• US Class Current: 435/325
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

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

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.

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

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

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.