Methods of deriving differentiated cells from stem cells

• US 8,986,995 B2
• Filed: 09/14/2012
• Issued: 03/24/2015
• 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:
  US13/620,082
• Publication Number:
  US 20130045534A1
• Time in Patent Office: 921 Days
• Field of Search: 435/477, 435/325
• 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 stem cells in the presence of trichostatin A (TSA), with the proviso that activin A is not present; and

b) thereafter, culturing the pluripotent stem cells in the absence of TSA, and in a media comprising activin A

thereby producing definitive endoderm cells.

2. The method of claim 1, wherein the definitive endoderm cells express one or more markers selected from the group consisting of SOX17, CXCR4, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 FoxA2, Brachyury, and CER1.

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

4. The method of claim 3, wherein the cells are further treated to form liver or pancreas cells.

5. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells, parthenogenetic stem cells, or induced pluripotent stem (iPS) cells.

6. The method of claim 1, wherein the culturing of the cells in the presence of TSA occurs for about 12-48 hours.

7. The method of claim 1, wherein the culturing in the absence of TSA occurs for about 6-96 hours, or about 6-72 hours, preferably about 24-72 hours.

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

M.

9. The method of claim 1, wherein the cells are mammalian cells.