Methods of deriving definitive endoderm cells from pluripotent parthenogenetic stem cells

• US 8,268,621 B2
• Filed: 12/02/2009
• Issued: 09/18/2012
• Est. Priority Date: 12/03/2008
• Status: Active Grant

• Current Assignee: International Stem Cell Corporation
• Original Assignee: International Stem Cell Corporation
• Inventors: Turovets, Nikolay, Agapova, Larisa, Semechkin, Andrey, Janus, Jeffrey
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/629,813
• Publication Number:
  US 20100173414A1
• Time in Patent Office: 1,021 Days
• Field of Search: 435/377, 435/384
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

The present invention provides a method of generating definitive endoderm, mesoderm, or ectoderm cells. The method includes culturing embryonic stem cells, parthenogenetic cells, or induced pluripotent stem cells in the presence of a demethylation agent, a histone deacetylase inhibitor, or a combination thereof, and thereafter, culturing the stem cells in the absence of the agent or combination of agents, to produce definitive endoderm cells, mesoderm, or ectoderm cells.

Claims

1. A method of producing definitive endoderm cells comprising:

a) culturing pluripotent parthenogenetic stem cells lacking paternal imprinting in the presence of trichostatin A (TSA) with the proviso that Activin A is not present, wherein the TSA alters the epigenetic status of the cell; and

b) thereafter, culturing the parthenogenetic stem cells in the absence of the TSA and in the presence of Activin A, thereby producing definitive endoderm cells.

2. The method of claim 1, wherein the step of culturing the parthenogenetic stem cells in the absence of the TSA further occurs in the presence of Wnt3a.

3. The method of claim 1, wherein the TSA is present in a concentration of about 1 nM to about 1 μ

M.

4. The method of claim 1, wherein the culturing in the presence of the TSA occurs for about 24 hours.

5. The method of claim 1, wherein the culturing in the absence of the TSA occurs for about 6-72 hours.

6. The method of claim 1, wherein the parthenogenetic stem cells are LLC-6p, LLC-12ph, LLC-2p, or LLC-15ph cells.

7. The method of claim 1, wherein the definitive endoderm cells express one or more markers selected from the group consisting of FoxA2, Sox17, CXCR4, brachyury, and CER1.

8. The method of claim 1, wherein the definitive endoderm cells do not express or express decreased levels as compared to untreated stem cells of E-cadherin, Oct4, or both.

9. The method of claim 1, wherein the culturing steps are carried out under defined media conditions.

10. The method of claim 1, wherein the definitive endoderm cells are further treated to form cells of the gastrointestinal tract, respiratory tract, or endocrine system.

11. The method of claim 1, wherein the definitive endoderm cells are further treated to form liver cells or pancreatic cells.