Defined culture conditions of human embryonic stem cells

• US 9,101,590 B2
• Filed: 07/28/2006
• Issued: 08/11/2015
• Est. Priority Date: 07/29/2005
• Status: Active Grant

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Snyder, Michael P., Lu, Joyce J.
• Primary Examiner(s): Ware, Debbie K
• Application Number:
  US11/989,363
• Publication Number:
  US 20110076253A1
• Time in Patent Office: 3,301 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/40 Transferrins, e.g. lactoferrins, ovotransferrins

Abstract

The present invention relates to compositions and methods for culturing stem cells, particularly embryonic stem cells. Specifically, the invention relates to a culture medium that supports proliferation of substantially undifferentiated stem cells, while maintaining potency of the cells. An an embodiment, the culture medium is defined and supports proliferation of substantially undifferentiated embryonic stem cells in essentially serum free and feeder cell free conditions. Compositions for making the medium and methods using the culture medium are also provided.

Claims

1. A culture medium for the maintenance and proliferation of substantially undifferentiated stem cells, wherein said culture medium comprises a basal medium, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising fibroblast growth factor (FGF), wherein a population of substantially undifferentiated stem cells cultured in said culture medium proliferates while maintaining a substantially undifferentiated state for at least about 4 passages.

2. The culture medium of claim 1, wherein said protein comprising a TNF member is selected from the group consisting of APRIL, an APRIL substitute, BAFF, a BAFF substitute and combinations thereof.

3. The culture medium of claim 1, wherein said protein comprising a member of the Wnt family is Wnt3a or a Wnt3a substitute.

4. The culture medium of claim 1, wherein said basal medium is selected from the group consisting of DMEM and DMEM/F12, further wherein said insulin is present at about 160 ug/ml, wherein said transferrin is present at about 88 ug/ml, wherein said member of the Wnt family is present at about 100 ng/ml, and wherein said member of the TNF family is present at about 100 ng/ml.

5. The culture medium of claim 1, further comprising a protein comprising albumin.

6. The culture medium of claim 1, further comprising cholesterol.

7. The culture medium of claim 6, wherein said cholesterol is chemically-defined cholesterol.

8. The culture medium of claim 1, wherein said protein comprising FGF is basic fibroblast growth factor (bFGF).

9. The culture medium of claim 1, wherein any of said proteins are recombinantly synthesized, chemically synthesized or isolated from a human biological sample.

10. The culture medium of claim 1, wherein any of said proteins are recombinantly synthesized and/or chemically synthesized.

11. The culture medium of claim 1, wherein said medium is essentially free of animal serum.