Methods of Deriving Differentiated Cells from Stem Cells

• US 20100173414A1
• Filed: 12/02/2009
• Published: 07/08/2010
• Est. Priority Date: 12/03/2008
• Status: Active Grant

• Current Assignee: International Stem Cell Corporation
• Original Assignee: International Stem Cell Corporation
• Inventors: Janus, Jeffrey, Semechkin, Andrey, Turovets, Nikolay, Agapova, Larisa
• Granted Patent: US 8,268,621 B2
• Time in Patent Office: Days
• 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, mesoderm, ectoderm, or endoderm cells comprising:

a) culturing stem cells in the presence of an agent, wherein the agent alters the epigenetic status of the cell; and

b) thereafter, culturing the stem cells in the absence of the agent,

thereby producing definitive endoderm, mesoderm, ectoderm, or endoderm cells.

2. The method of claim 1, wherein the agent that alters the epigenetic status of the cell is selected from the group consisting of a demethylation agent, a histone deacetylase inhibitor, and a combination thereof.

3. The method of claim 2, wherein the histone deacetylase (HDAC) inhibitor is selected from the group consisting of trichostatin A, vorinostat (SAHA), belinostat (PXD101), LAQ824/LBH589, trapoxin B, entinostat (MS-275), CI994, mocetinostat (MGCD0103), phenylbutyrate, valproic acid, isovalerate, valerate, valproate, nicotinamide, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes, apicidin, FK228, and sodium butyrate.

4. The method of claim 3, wherein the HDAC inhibitor is trichostatin A (TSA).

5. The method of claim 1, wherein the demethylation agent is 5-azacytidine or 5-aza-2′

deoxycytidine.

6. The method of claim 1, wherein the stem cells are selected from the group consisting of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, and somatic stem cells.

7. The method of claim 6, wherein the stem cells are parthenogenetic stem cells or induced pluripotent stem cells.

8. The method of claim 6, wherein the stem cells are parthenogenetic stem cells.

9. The method of claim 1, wherein culturing the stem cells in the absence of the agent occurs in the presence of Activin A, Wnt3a, or a combination thereof.

10. The method of claim 4, wherein the TSA is present in a concentration of about 1 nM to about 1 μ

M.

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

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

13. The method of claim 8, wherein the parthenogenic cells are LLC-6p, LLC-12ph, LLC-2p, or LLC-15ph cells.

14. The method of claim 1, wherein the definitive endoderm cells express one or more markers selected from the group consisting of FoxA2, Sox 17, CXCR4, brachyury, and CER1.

15. 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.

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

17. The method of claim 1, wherein definitive endoderm cells are produced.

18. The method of claim 17, wherein the definitive endoderm cells are further treated to form cells of the gastrointestinal tract, respiratory tract, or endocrine system.

19. The method of claim 17, wherein the definitive endoderm cells are further treated to form liver cells or pancreatic cells.

20. A culture of definitive endoderm cells produced by a method of claim 1.

21. A culture of liver cells or pancreatic cells produced by a method of claim 19.