Culturing mammalian embryonic stem cells in human foreskin fibroblast conditioned medium
View Patent ↗A cell culture comprising human foreskin cells, the human foreskin cells being capable of maintaining stem cells in an undifferentiated state when co-cultured therewith.
1. A method of maintaining mammalian pluripotent stem cells in an undifferentiated state comprising culturing the mammalian pluripotent stem cells with a human foreskin fibroblast conditioned medium, wherein said foreskin fibroblast conditioned medium is of non-genetically modified human foreskin fibroblasts, and wherein said foreskin fibroblast conditioned medium comprises at least 4 ng/ml of added basic fibroblast growth factor (bFGF), and being capable of maintaining said mammalian pluripotent stem cells in an undifferentiated, proliferative state through at least 87 passages, thereby maintaining the mammalian pluripotent stem cells in the undifferentiated state.
2. The method of claim 1 , wherein said mammalian pluripotent stem cells are human pluripotent stem cells.
3. The method of claim 1 , wherein said mammalian pluripotent stem cells are of embryonic origin.
4. The method of claim 1 , wherein foreskin cells for said human foreskin fibroblast conditioned medium are obtained from an 8-14 day old male individual.
5. The method of claim 1 , wherein said culturing is effected under culturing conditions including a growth medium which comprises serum and/or serum replacement.
6. The method of claim 5 , wherein said serum and/or said serum replacement are provided at a concentration of at least 10%.
7. The method of claim 5 , wherein said serum and/or said serum replacement are provided at a concentration of 15%.
8. The method of claim 5 , wherein said serum is human serum.
9. The method of claim 1 , wherein said culturing is effected under culturing conditions including a growth medium which comprises a factor selected from the group consisting of an anti oxidant and an amino acid.
10. A cell culture comprising:
(i) mammalian pluripotent stem cells, wherein said mammalian pluripotent stem cells are capable of forming teratomas containing cells of the endoderm, mesoderm and ectoderm germ layers; and
(ii) human foreskin fibroblast conditioned medium, wherein said foreskin fibroblast conditioned medium is of non-genetically modified human foreskin fibroblasts, and wherein said foreskin fibroblast conditioned medium comprises at least 4 ng/ml of added basic fibroblast growth factor (bFGF) and being capable of maintaining said mammalian pluripotent stem cells in an undifferentiated, proliferative state least 87 passages.
11. The cell culture of claim 10 , wherein said mammalian pluripotent stem cells are of embryonic origin.
12. The cell culture of claim 10 , wherein said mammalian pluripotent stem cells are human pluripotent stem cells.
13. The cell culture of claim 10 , wherein human foreskin cells for said human foreskin fibroblast conditioned medium are obtained from an 8-14 day old male individual.
14. The cell culture of claim 10 , wherein said cell culture includes a culture medium including serum replacement and/or human serum.
15. The cell culture of claim 14 , wherein said serum replacement and/or said human serum are provided at a concentration of at least 10%.
16. The cell culture of claim 14 , wherein said serum replacement and/or said human serum are provided at a concentration of 15%.
17. A method of producing a conditioned medium suitable for maintaining mammalian pluripotent stem cells in an undifferentiated state, the method comprising:
(a) culturing non-genetically modified human foreskin fibroblast cells in a growth medium which comprises added basic fibroblast growth factor (bFGF) under conditions suitable for producing the conditioned medium; and
(b) collecting said growth medium to thereby produce the conditioned medium, and
(c) validating the ability of the conditioned medium to maintain said mammalian pluripotent stem cells in an undifferentiated state following step (b),
thereby producing the conditioned medium suitable for maintaining the mammalian pluripotent stem cells in an undifferentiated state.
18. The method of claim 17 , wherein said validating is effected by a differentiation assay selected from the group consisting of morphology analysis, karyotype analysis and surface marker analysis.
19. The method of claim 17 , wherein said bFGF is provided at a concentration of 4 ng/ml bFGF.
20. The method of claim 17 , wherein said growth medium further comprises serum replacement.
21. The method of claim 17 , wherein said collecting is performed after 4 hours of said culturing.
22. The method of claim 17 , wherein foreskin cells for said human foreskin fibroblast conditioned medium are obtained from an 8-14 day old male individual.
23. The method of claim 17 , wherein said foreskin fibroblast conditioned medium is suitable for maintaining mammalian pluripotent stem cells in an undifferentiated state through at least 87 passages.
24. A conditioned medium generated according to the method of claim 17 , wherein said conditioned medium comprises at least 4 ng/ml of said bFGF.
25. The conditioned medium of claim 24 , wherein said medium further comprises serum replacement.
26. A foreskin fibroblast conditioned medium which comprises at least 4 ng/ml added basic fibroblast growth factor (bFGF), wherein said foreskin fibroblast conditioned medium is of non-genetically modified human foreskin fibroblasts, wherein said foreskin fibroblast conditioned medium is suitable for maintaining mammalian stem cells in an undifferentiated state through at least 87 passages.