Serum free cultivation of primate embryonic stem cells

• US 20030190748A1
• Filed: 05/06/2003
• Published: 10/09/2003
• Est. Priority Date: 03/09/2000
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A.
• Granted Patent: US 7,217,569 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Disclosed herein are methods for culturing primate embryonic stem cells. These cells are cultured on a prolonged and stable basis in the presence of exogenously supplied fibroblast growth factor and in the absence of animal serum. Preferably there is also a fibroblast feeder layer. Also disclosed is a culture media containing fibroblast feeder layer and the fibroblast growth factor.

Claims

1. A method of culturing primate embryonic stem cells, comprising:

culturing the stem cells in a culture essentially free of mammalian fetal serum and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer.

2. The method of claim 1, wherein the culture is essentially free of any animal serum.

3. The method of claim 2, wherein the culture also comprises a fibroblast feeder layer.

4. The method of claim 2, wherein the fibroblast growth factor is basic fibroblast growth factor.

5. The method of claim 4, wherein the fibroblast growth factor is human basic fibroblast growth factor which has been produced from a recombinant gene.

6. The method of claim 2, wherein the primate embryonic stem cells are human embryonic stem cells.

7. The method of claim 2, wherein said culturing step includes the embryonic stem cells proliferating in culture for over one month while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.

8. The method of claim 2, wherein the human basic fibroblast growth factor is present in the culture in a concentration of at least 0.1 ng/ml for at least a portion of the method.

15. A cell line derived using the method of claim 1.

9. A method of culturing primate embryonic stem cells, comprising:

culturing the stem cells in a culture essentially free of mammalian fetal serum and in the presence of a growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer.

10. The method of claim 9, wherein the culture is essentially free of any animal serum.

11. The method of claim 10, wherein the culture also comprises a fibroblast feeder layer.

12. The method of claim 10, wherein the primate embryonic stem cells are human embryonic stem cells.

13. The method of claim 10, wherein said culturing step includes the embryonic stem cells proliferating in culture for over one month while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the katyotype of the stem cells.

16. A cell line derived using the method of claim 9.

14. A culture system for culturing primate embryonic stem cells, comprising:

a fibroblast feeder layer; and

fibroblast growth factor supplied by other than just the fibroblast layer;

wherein the culture system is essentially free of animal serum.