Feeder independent extended culture of embryonic stem cells

• US 20060014279A1
• Filed: 05/20/2005
• Published: 01/19/2006
• Est. Priority Date: 05/21/2004
• Status: Active Grant

• Current Assignee: WiCell Research Institute, Inc.
• Original Assignee: WiCell Research Institute, Inc.
• Inventors: Thomson, James A., Xu, Ren-He
• Granted Patent: US 7,514,260 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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

Previous methods for culturing human embryonic stem cells have required either fibroblast feeder cells or a medium which has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. It has now been found that if an antagonist of bone morphogenic protein is added to the medium in which the stem cells are cultured, together with fibroblast growth factor, the stem cells will remain undifferentiated indefinitely, even without feeder cells or conditioned medium.

Claims

1. A method for culturing human embryonic stem cells in a feeder independent culture, the method comprising the step of

culturing the human embryonic stem cells in a medium including salts, vitamins, amino acids, glucose, a fibroblast growth factor and a bone morphogenic protein antagonist in sufficient amount to maintain the stem cells in an undifferentiated state.

2. The method of claim 1 wherein the medium includes the fibroblast growth factor in a concentration of at least 4 ng/ml.

3. The method of claim 1 wherein the bone morphogenic protein antagonist is noggin.

4. The method of claim 1 wherein the fibroblast growth factor is bFGF at a concentration of 40 ng/ml.

5. In a method of culturing human embryonic stem cells in a medium including salts, vitamins, amino acids, and a fibroblast growth factor, the improvement comprising adding to the medium an amount of an antagonist of bone morphogenic protein signaling in an amount sufficient to maintain the cells in an undifferentiated state without exposure of the stem cells or the medium to feeder cells.

6. In a method of culturing human embryonic stem cells in a medium including salts, vitamins, amino acids, and a fibroblast growth factor, the improvement comprising inhibiting the action of bone morphogenic protein signaling such that the stem cells can be maintained in an undifferentiated state without exposure to feeder cells or to medium exposed to feeder cells.

7. An in vitro cell culture comprising in a culture vessel:

human embryonic stem cells and

a culture medium, the culture medium comprising salts, vitamins, amino acids, glucose, a fibroblast growth factor and a bone morphogenic protein signaling antagonist in sufficient amount to maintain the stem cells in an undifferentiated feeder independent state, the medium being free of feeder cells and never having been exposed to feeder cells.

8. A cell culture as claimed in claim 7 wherein the bone morphogenic protein antagonist is selected from the group consisting of factors inhibiting BMP signaling extracellularly by physical interaction including noggin, gremlin, chordin, inhibin, follistatin, twisted gastrulation, and members of the DAN family.

9. A cell culture as claimed in claim 8 wherein the bone morphogenic protein signaling antagonist is noggin.

10. A cell culture as claimed in claim 7 wherein the bone morphogenic protein antagonist is selected from the group consisting of factors inhibiting BMP signaling factors inhibiting BMP signaling intracellularly, including FGF, TGFβ

, Nodal, and Activin.

11. A cell culture as claimed in claim 7 wherein the medium includes the fibroblast growth factor in a concentration of at least 4 ng/ml of bFGF.

12. A cell culture as claimed in claim 7 wherein the medium further comprises proteins selected from the group consisting of albumin, insulin and transferrin.

13. A cell culture as claimed in claim 12 wherein the proteins are human.

14. A cell culture as claimed in claim 12 wherein the proteins are recombinant proteins.

15. A medium for culturing embryonic stem cells in a feeder independent culture, the medium comprising salts, vitamins, amino acids, glucose, a fibroblast growth factor and a bone morphogenic protein antagonist in sufficient amount to maintain stem cells grown in the medium in an undifferentiated state without exposure to feeder cells or medium exposed to feeder cells.

16. The medium of claim 15 wherein the bone morphogenic protein antagonist is selected from the group consisting of factors inhibiting BMP signaling extracellularly by physical interaction including noggin, gremlin, chordin, inhibin, follistatin, twisted gastrulation, and members of the DAN family.

17. The medium of claim 15 wherein the bone morphogenic protein antagonist is selected from the group consisting of factors inhibiting BMP signaling factors inhibiting BMP signaling intracellularly, including FGF, TGFβ

, Nodal, and Activin

18. The medium of claim 15 wherein the bone protein antagonist is noggin.

19. The medium of claim 15, wherein the medium further comprises proteins selected from the group consisting of albumin, insulin and transferrin.

20. The medium of claim 15 wherein the fibroblast growth factor is bFGF present in the medium at a concentration of at least 4 ng/ml.