Thermostable FGF-2 mutant having enhanced stability
Thermostable FGF-2 proteins having enhanced ability to support human pluripotent stem cell cultures are provided. Also provided are methods and compositions utilizing thermostable FGF-2 proteins.
1. An isolated fibroblast growth factor-two, FGF-2, polypeptide, as depicted in SEQ ID NO: 2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid position 128, wherein the isolated FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).
2. An isolated nucleic acid encoding the isolated FGF-2 of claim 1 .
3. An isolated genetically modified cell producing the isolated fibroblast growth factor of claim 1 .
4. A method for culturing human pluripotent stem cells, the method comprising the step of culturing a human pluripotent stem cell in a medium comprising a K128-substituted thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO: 2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at position 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).
5. The method of claim 4 , wherein the human pluripotent cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.
6. The method of claim 4 , wherein the medium further comprises heparin.
7. The method of claim 4 , wherein the medium comprises the thermostable FGF-2 at a concentration of less than 40 ng/ml.
8. The method of claim 7 , wherein the medium comprises the thermostable FGF-2 at a concentration less than 10 ng/ml.
9. The method of claim 8 , wherein the medium comprises the thermostable FGF-2 at a concentration less than 3 ng/ml.
10. The method of claim 9 , wherein the medium comprises a thermostable FGF-2 at a concentration less than 1 ng/ml.
11. A fully defined culture medium suitable for culturing human pluripotent cells in an undifferentiated state, the medium comprising thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).
12. The fully-defined medium of claim 11 , wherein the fully defined medium comprises thermostable FGF-2 at less than 40 ng/ml.
13. The fully-defined medium of claim 12 , wherein the fully defined medium comprises thermostable FGF-2 at less than 10 ng/ml.
14. The fully-defined medium of claim 13 , wherein the fully medium comprises thermostable FGF-2 at less than 3 ng/ml.
15. The fully-defined medium of claim 14 , wherein the concentration of the thermostable FGF-2 in the fully defined medium is equal to or less than 1 ng/ml.
16. A composition comprising:
a human pluripotent stem cell;
a medium suitable for culturing the human pluripotent cell in an undifferentiated state; and
a thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).
17. The composition of claim 16 , wherein the medium comprises thermostable FGF-2 at less than 40 ng/ml.
18. The composition of claim 17 , wherein the medium comprises thermostable FGF-2 at less than 10 ng/ml.
19. The composition of claim 18 , wherein the medium comprises thermostable FGF-2 at less than 3 ng/ml.
20. The composition of claim 19 , wherein the medium comprises thermostable FGF-2 at less than 1 ng/ml 1.
21. A method for culturing reprogrammed human somatic cells comprising:
culturing reprogrammed human somatic cells in a medium until they express markers indicative of induced pluripotent stem cells, wherein the medium comprises water, salts, amino acids, vitamins, a carbon source, insulin, selenium and thermostable fibroblast growth factor-two, FGF-2, as depicted in SEQ ID NO:2, that differs from wild type FGF-2, as depicted in SEQ ID NO: 1, at amino acid 128 of the wild type FGF-2, wherein the thermostable FGF-2 contains an asparagine submitted for lysine at position 128 (a K128N substitution).
22. The method of claim 21 , wherein the medium comprises thermostable FGF-2 at less than 40 ng/ml.
23. The method of claim 22 , wherein the medium comprises thermostable FGF-2 at less than 10 ng/ml.
24. The method of claim 23 , wherein the medium comprises thermostable FGF-2 at less than 3 ng/ml.
25. The method of claim 22 , wherein the medium comprises thermostable FGF-2 at less than 1 ng/ml.
26. The method of claim 21 , wherein the medium is serum-free.