Differentiation of human pluripotent stem cells

• US 9,234,178 B2
• Filed: 10/23/2009
• Issued: 01/12/2016
• Est. Priority Date: 10/31/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza, Fryer, Benjamin
• Primary Examiner(s): Hibbert, Catherine S
• Application Number:
  US12/604,457
• Publication Number:
  US 20100112693A1
• Time in Patent Office: 2,272 Days
• Field of Search: None
• US Class Current: 1/1
• 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 2501/155 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.

Claims

1. A method for increasing the expression of insulin and MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage, the method comprising the steps of:

a. sequentially differentiating human pluripotent stem cells to obtain cells expressing markers characteristic of the pancreatic endocrine lineage;

b. culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising an added amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of insulin and MAFA as compared to cells expressing markers characteristic of the pancreatic endocrine lineage that are not cultured in medium comprising the added cyclin-dependent kinase inhibitor;

wherein the cyclin-dependent kinase inhibitor is Ethyl-(6-hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate.

2. The method of claim 1, wherein the ethyl-(6-hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 μ

M to about 10 μ

M for about one to seven days.