DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20110151561A1
• Filed: 12/16/2010
• Published: 06/23/2011
• Est. Priority Date: 12/23/2009
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: DITOLVO, KEVIN, DAVIS, JANET, PARMENTER, CHRISTINE
• Granted Patent: US 9,133,439 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0613 Cells from endocrine organs pancreas C12N5/0676, gonads C12N5/0681
• CPC Class Codes: C12N 5/0676 Pancreatic 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 to increase the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage.

Claims

1. A method to increase the expression of NGN3 and NKX6.1 in a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a) culturing pluripotent stem cells,

b) differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,

c) differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, supplementing the medium used to differentiate the cells expressing markers characteristic of the definitive endoderm lineage with a compound selected from the group consisting of H-9, H-89, GF 109203×

, HA-1004, PP2, PP1, LY 294002, Wortmannin, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7, and

d) differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.

2. The method of claim 1, wherein the medium used to differentiate the cells expressing markers characteristic of the pancreatic endoderm lineage is supplemented with a compound selected from the group consisting of H-9, H-89, GF 109203×

, HA-1004, PP2, PP1, LY 294002, Wortmannin, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7.