Differentiation of pluripotent stem cells to definitive endoderm lineage

• US 8,741,643 B2
• Filed: 04/18/2007
• Issued: 06/03/2014
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Rezania, Alireza, Xu, Jean
• Primary Examiner(s): Ford, Allison
• Application Number:
  US11/736,908
• Publication Number:
  US 20070254359A1
• Time in Patent Office: 2,603 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• 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 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Claims

1. A method for differentiating pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage, comprising the steps of:

a) Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and lacking a feeder cell layer,

b) Culturing the pluripotent stem cells with activin A, a Wnt ligand, and a GSK-3B inhibitor in a first culture medium, and

c) Culturing the pluripotent stem cells with activin A in a second culture medium wherein the second culture medium does not contain a Wnt ligand, for a period of time sufficient to differentiate the pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage.