Differentiation of human embryonic stem cells

• US 9,593,310 B2
• Filed: 08/31/2015
• Issued: 03/14/2017
• Est. Priority Date: 12/23/2009
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Xu, Jean
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US14/840,297
• Publication Number:
  US 20160032250A1
• Time in Patent Office: 561 Days
• Field of Search: 435/377
• US Class Current: 1/1
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon.

Claims

1. An in vitro culture of a population of cells that co-express NKX6.1 and insulin in a culture medium supplemented with (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam, wherein less than 10% of the cells in the population express glucagon, and wherein the population of cells is obtained by a step-wise differentiation protocol which comprises culturing pancreatic endoderm cells in the medium supplemented with (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.

2. The in vitro culture of claim 1, wherein at least 30% of the cells express NKX6.1.

3. The in vitro culture of claim 1, wherein at least 40% of the cells express NKX.6.1.

4. The in vitro culture of claim 1, wherein at least 50% of the cells express NKX6.1.

5. The in vitro culture of claim 1, wherein at least 60% of the cells express NKX6.1.

6. The in vitro culture of claim 1, wherein at least 5% of the cells express insulin.

7. The in vitro culture of claim 1, wherein the medium is supplemented with one or more of noggin or a TGFβ

receptor signaling inhibitor.

8. The in vitro culture of claim 7, wherein the TGFβ

receptor signaling inhibitor is an inhibitor of ALK5.