Differentiation of Pluripotent Stem Cells

• US 20120052576A1
• Filed: 08/17/2011
• Published: 03/01/2012
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Granted Patent: US 9,181,528 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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 insulin producing cells. In particular, the present invention provides a method utilizing an agent that degrades retinoic acid to produce a population of pancreatic endocrine precursor cells.

Claims

1. A method to derive a population of pancreatic endocrine precursor cells from pluripotent stem cells comprising the steps of:

a. Culturing a population of pluripotent stem cells;

b. Differentiating the pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage;

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into a population of primitive gut tube cells;

d. Differentiating the population of primitive gut tube cells into a population of posterior foregut cells; and

e. Treating the population of posterior foregut cells with a medium supplemented with an agent that degrades retinoic acid.

2. The method of claim 1, wherein the agent that degrades retinoic acid is a CYP26A inhibitor.

3. The method of claim 2, wherein the CYP26A inhibitor is used at a concentration from about 1 nM to about 1000 nM.

4. The method of claim 2, wherein the CYP26A inhibitor is used at a concentration from about 10 nM to about 100 nM.

5. The method of claim 2, wherein the CYP26A inhibitor is N-{4-[2-Ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl}-1,3-benzothiazol-2-amine.