Compositions and methods useful for culturing differentiable cells
The present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.
1. A method of expanding pluripotent stem cells, said method comprising
a) culturing the pluripotent stem cells on a solid surface in an adherent culture in a cell medium, the cell medium comprising a basal salt nutrient solution and an ErbB3 ligand, where the cell medium is essentially free of serum, wherein the pluripotent stem cells can give rise to all three germ layers and wherein the ErbB3 ligand binds to an ErbB3 receptor, which in turn dimerizes to an ErbB2 receptor to form an ErbB2/ErbB3 heterodimer, thereby activating the tyrosine kinase activity of the ErbB2 receptor in the ErbB2/ErbB3 heterodimer; and
b) maintaining the pluripotent stem cells in the adherent culture in the cell medium and in appropriate culture conditions for more than one day, wherein the cell medium is changed periodically, wherein the pluripotent stem cells proliferate and do not differentiate.
2. The method of claim 1 , wherein the cell medium is free of exogenous insulin and insulin substitutes.
3. The method of claim 1 , wherein the cell medium further comprises insulin-like growth factors or a functional fragment thereof.
4. The method of claim 1 , wherein the ErbB3 ligand in the cell medium is selected from the group consisting of Neuregulin-β, Heregulin-β (HRG-β), Heregulin-α (HRG-α), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β (NRG2-β), Epiregulin, Biregulin and functional fragments thereof.
5. The method of claim 4 , wherein the ErbB3 ligand is HRG-β or a functional fragment thereof.
6. The method of claim 5 , wherein the cell medium further comprises transforming growth factor beta (TGF-β), a TGF-β family member or a functional fragment thereof.
7. The method of claim 6 , wherein the TGF-β family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and functional fragments thereof.
8. The method of claim 7 , wherein said TGF-β family member is Activin A.
9. The method of claim 8 , wherein the cell medium further comprises insulin-like growth factors or a functional fragment thereof.
10. The method of claim 9 , wherein the cell medium is free of exogenous fibroblast growth factor.
11. The method of claim 9 , wherein the cell medium further comprises at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and variants functional fragments thereof.
12. The method of claim 11 , wherein the at least one FGF is FGF-7, FGF-10 and FGF-22.
13. The method of claim 12 , wherein the cell medium comprises a serum albumin (SA).
14. The method of claim 13 , wherein the SA is bovine SA (BSA) or human SA (HSA).
15. The method of claim 14 , wherein the concentration of the SA is more than about 0.2%, volume to volume (v/v).
16. The method of claim 14 , wherein the concentration of SA is less than about 5% v/v.
17. The method of claim 1 , wherein the pluripotent stem cells are embryonic stem cells.
18. The method of claim 1 , wherein the pluripotent stem cells are selected from the group consisting of ES cells, EPL cells, ICM/epiblast cells, EG cells, and pluripotent cells derived by dedifferentiation or nuclear transfer.
19. A method of expanding primate embryonic stem cells, the method comprising:
a) culturing the primate embryonic stem cells on a solid surface in an adherent culture in a cell medium, the cell medium comprising a basal salt nutrient solution and an ErbB3 ligand, where the cell medium is essentially free of serum, wherein the primate embryonic stem cells can give rise to all three germ layers and wherein the ErbB3 ligand binds to an ErbB3 receptor, which in turn dimerizes to an ErbB2 receptor to form an ErbB2/ErbB3 heterodimer, thereby activating the tyrosine kinase activity of the ErbB2 receptor in the ErbB2/ErbB3 heterodimer; and
b) maintaining the primate embryonic stem cells in the adherent culture in the cell medium and in appropriate culture conditions for more than one day, wherein the cell medium is changed periodically, wherein the primate embryonic stem cells proliferate and do not differentiate.
20. The method of claim 19 , wherein the cells are maintained in the adherent culture and in appropriate culture conditions for at least two days.
21. The method of claim 19 , wherein neither insulin nor an insulin substitute is provided to said embryonic stem cells.
22. The method of claim 19 , further comprising providing insulin-like growth factor or a functional fragment thereof to said embryonic stem cells.
23. The method of claim 19 , wherein the ErbB3 ligand in the cell medium is selected from the group consisting of Neuregulin-β, Heregulin-β (HRG-β), Heregulin-α (HRG-α), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β (NRG2-β), Epiregulin, Biregulin and functional fragments thereof.
24. The method of claim 23 , wherein said ligand is HRG-β or a functional fragment thereof.
25. The method of claim 24 , further comprising providing to said cells transforming growth factor beta (TGF-β), a TGF-β family member or a functional fragment thereof.
26. The method of claim 25 , wherein said TGF-β family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and functional fragments thereof.
27. The method of claim 26 , further comprising providing insulin-like growth factor or a functional fragment thereof to said embryonic stem cells.
28. The method of claim 27 , further comprising providing at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and functional fragments thereof to said embryonic stem cells.
29. The method of claim 28 , wherein said at least one FGF is FGF-7, FGF-10 and FGF-22.
30. The method of claim 29 , further comprising providing a serum albumin (SA) to said embryonic stem cells.
31. The method of claim 30 , wherein the SA is bovine SA (BSA) or human SA (HSA).
32. The method of claim 31 , wherein the concentration of the SA is more than about 0.2%, volume to volume (v/v).
33. The method of claim 31 , wherein the concentration of SA is less than about 5% v/v.