Differentiation of human embryonic stem cells

• US 8,785,184 B2
• Filed: 07/19/2010
• Issued: 07/22/2014
• Est. Priority Date: 07/20/2009
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Xu, Jean
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/839,041
• Publication Number:
  US 20110014702A1
• Time in Patent Office: 1,464 Days
• Field of Search: 435/325, 435/366, 435/377
• US Class Current: 435/325
• CPC Class Codes: 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
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/19 Growth and differentiation factors [GDF]
• 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 5/0676 Pancreatic cells

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 cells capable of producing insulin following transplantation into an animal.

Claims

1. A method to differentiate a population of human pluripotent stem cells into 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 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 treating the definitive endoderm cells with a first medium supplemented with FGF7, followed by culturing the cells in a second medium supplemented with FGF7, a factor capable of inhibiting BMP, retinoic acid, and a hedgehog signaling pathway inhibitor, and a TGF-β

receptor agonist selected from the group consisting of;

activin B, TGFβ

-I, TGFβ

-II, GDF-8, and GDF-11, 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 hedgehog signaling pathway inhibitor is cyclopamine-KAAD.