Claims
- 1. A method for culturing primate pluripotent stem (pPS) cells, comprising culturing pPS cells in a growth environment essentially free of feeder cells but containing conditioned medium produced by a method comprising:
a) culturing cells in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture; wherein the cells used to condition the medium are from a cell line from a non-malignant source that can proliferate in culture for at least 60 days.
- 2. The method of claim 1, wherein the pPS cells are human embryonic stem (hES) cells.
- 3. The method of claim 1, wherein the cell line used to condition the medium is an immortalized mouse cell line.
- 4. The method of claim 1, wherein the cell line used to condition the medium is a human cell line.
- 5. The method of claim 1, wherein the cell line used to condition the medium is a mesenchymal cell line.
- 6. The method of claim 1, wherein the cell line used to condition the medium is a fibroblast cell line.
- 7. The method of claim 1, wherein the cell line used to condition the medium has been obtained by differentiating human embryonic stem cells ex vivo.
- 8. The method of claim 1, wherein the cell line used to condition the medium is euploid.
- 9. The method of claim 1, wherein the cell line has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.
- 10. A method for preparing a conditioned medium suitable for culturing primate pluripotent stem (pPS) cells in a growth environment essentially free of feeder cells, comprising:
a) culturing cells in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture; wherein the cells used to condition the medium are from a human cell line from a non-malignant source that can proliferate in culture for at least 60 days; and wherein pPS cells can be caused to proliferate without differentiation in an environment essentially free of feeder cells but comprising the medium and a growth factor.
- 11. The method of claim 10, further comprising adding a fibroblast growth factor to the medium before or after harvesting.
- 12. A composition of proliferating pPS cells, comprising undifferentiated pPS cells in a growth environment that includes conditioned medium produced by a method comprising:
a) culturing cells in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture; wherein the cells used to condition the medium are from a cell line from a non-malignant source that can proliferate in culture for at least 60 days.
- 13. A human cell line obtained by differentiating a culture of human embryonic stem (hES) cells, and then selecting mesenchymal or fibroblast-like cells from the culture; wherein medium conditioned by culturing with the selected cells supports growth of pPS cells without differentiation in a culture environment essentially free of feeder cells.
- 14. The human cell line of claim 13, wherein the cell line is a fibroblast cell line.
- 15. The human cell line of claim 13, which has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.
- 16. The human cell line of claim 13, which can be used to produce conditioned medium by a method comprising:
a) culturing the cell line in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture; wherein pPS cells can be caused to proliferate without differentiation in an environment essentially free of feeder cells but comprising the medium and a growth factor.
- 17. A method for preparing a conditioned medium suitable for culturing primate pluripotent stem (pPS) cells in feeder-free culture, comprising:
a) culturing the cell line of claim 13 in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture.
- 18. A conditioned medium to support culturing primate pluripotent stem (pPS) cells without differentiation in feeder-free culture, produced by a method comprising:
a) culturing the cell line of claim 13 in a tissue culture medium, thereby conditioning the medium; and then b) harvesting the conditioned medium from the culture.
- 19. A method for screening cells suitable for producing conditioned medium that supports growth of primate pluripotent stem (pPS) cells without differentiation in feeder-free culture, comprising:
a) providing a growth environment essentially free of feeder cells, wherein growth of pPS cells in the environment in a substantially undifferentiated state is promoted by medium conditioned by primary mouse embryonic fibroblasts (mEF), but wherein a test medium conditioned by cells being screened according to the method is used in the growth environment instead of the mEF conditioned medium; and b) determining whether pPS cells cultured in the growth environment containing the test medium grow without differentiating.
- 20. A device for preparing conditioned medium according to claim 18, comprising:
a) a culture chamber containing a plurality of cells from a cell line that can proliferate in culture for at least 60 days; whereby the cells condition medium in the device in such a manner that renders the medium able to support growth of pPS cells in feeder-free culture without differentiation; and b) a port for withdrawing medium from the culture chamber after conditioning by the cells.
RELATED APPLICATIONS
[0001] This application claims priority to pending U.S. provisional patent applications 60/216,387, filed Jul. 7, 2000; and 60/220,064, filed Jul. 21, 2000.
[0002] The aforelisted priority applications are hereby incorporated herein by reference in their entirety, as are the following: U.S. Ser. No. 60/175,581, filed Jan. 11, 2000; U.S. Ser. No. 09/688,031, filed Oct. 10, 2000; U.S. Ser. No. 09/718,308, filed Nov. 20, 2000; and International Patent Application PCT/US01/01030, filed Jan. 10, 2001.
Provisional Applications (2)
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Number |
Date |
Country |
|
60216387 |
Jul 2000 |
US |
|
60220064 |
Jul 2000 |
US |