Methods and compositions for stem cell self-renewal
The present invention relates to methods for expanding a stem cell population. More particularly, the invention relates, inter alia, to methods and compositions for expanding a stem cell population, particularly a hematopoietic stem cell population.
1. A method for ex vivo expansion of a population of hematopoietic stem cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, the method comprising inhibiting PTEN and GSK-3β in the population of stem cells to expand the number of stem cells.
2. The method according to claim 1 , wherein:
(a) inhibiting PTEN comprises contacting the hematopoietic stem cell with an inhibitor of PTEN selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof; and
(b) inhibiting GSK-3β comprises contacting the hematopoietic stem cell with an inhibitor of a molecule in the Wnt pathway selected from a group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof.
3. The method according to claim 2 , wherein inhibiting PTEN and inhibiting GSK-3β comprises contacting the stem cells with a small molecule inhibitor of PTEN and a small molecule inhibitor of GSK-3β.
4. The method according to claim 3 , wherein the small molecule inhibitor of PTEN and the small molecule inhibitor of GSK-3β are reversible inhibitors.
5. The method according to claim 4 , wherein the reversible PTEN inhibitor is selected from the group consisting of shikonin, a bisperoxovanadium compound, SF-1751 (Semafore Pharmaceuticals), pharmaceutical salts thereof, and combinations thereof.
6. The method according to claim 5 , wherein the bisperoxovanadium compound is selected from the group consisting of bpV(phen)2, bpV(pic), pharmaceutical salts thereof, and combinations thereof.
7. The method according to claim 4 , wherein the reversible GSK-3β inhibitor is selected from the group consisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, University of Rochester), pharmaceutically acceptable salts thereof, and combinations thereof.
8. The method according to claim 4 , wherein the PTEN inhibitor is bpV(pic) and the GSK-3β inhibitor is CHIR99201.
9. The method according claim 1 , wherein the expansion of the hematopoietic stem cells is by a factor of at least 40-fold.
10. The method according to claim 1 , wherein the expansion of the number of stem cells is by a factor of at least 80-fold.
11. The method according to claim 1 , wherein the expansion of the number of hematopoietic stem cells is by a factor of at least 150-fold.
12. The method according to claim 1 , wherein the expansion of the number of hematopoietic stem cells is by a factor of at least 250-fold.
13. A method for ex vivo expansion of a hematopoietic stem cell population comprising inhibiting PTEN and GSK-3β in the hematopoietic stem cell population to expand the number of hematopoietic stem cells without significant differentiation of the hematopoietic stem cell population.
14. A method for ex vivo expansion of an hematopoietic stem cell (HSC) population obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, the method comprising inhibiting PTEN and GSK-3β in the HSC population to expand the HSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the HSC population, which is sufficient for subsequent transplantation into a patient in need thereof.
15. The method according to any one of claim 13 or 14 , wherein:
(a) inhibiting PTEN comprises contacting the hematopoietic stem cell population with a PTEN inhibitor selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof; and
(b) inhibiting GSK-3β comprises contacting the hematopoietic stem cell population with a GSK-3β inhibitor selected from a group consisting of
a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof.
16. The method according to claim 15 , wherein inhibiting PTEN and GSK-3β comprises contacting the heatopoietic stem cell population with a small molecule inhibitor of PTEN and a small molecule inhibitor of GSK-3β .
17. The method according to claim 16 , wherein the small molecule inhibitor of PTEN and the small molecule inhibitor of GSK-3β are reversible inhibitors.
18. The method according to claim 17 , wherein the reversible PTEN inhibitor is selected from the group consisting of shikonin, a bisperoxovanadium compound, SF-1751 (Semafore Pharmaceuticals), pharmaceutical salts thereof, and combinations thereof.
19. The method according to claim 18 , wherein the bisperoxovanadium compound is selected from the group consisting of bpV(phen)2, bpV(pic), pharmaceutical salts thereof, and combinations thereof.
20. The method according to claim 17 , wherein the reversible GSK-3β inhibitor is selected from the group consisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX), SB216763, SB415286, 15, CGP60474, Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12(Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, University of Rochester), pharmaceutically acceptable salts thereof, and combinations thereof.
21. The method according to claim 17 , wherein the PTEN inhibitor is bpV(pic), and the GSK-3β inhibitor is CHIR99201.
22. The method according to claim 13 , wherein the HSC is obtained from a mammalian tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.
23. The method according to claim 14 , wherein the expansion of the number of hematopoietic stem cells is by a factor selected from the group consisting of at least 40-fold, at least 80-fold, at least 150-fold, and at least 250-fold.
24. A method for ex vivo expansion of hematopoietic stem cells (HSCs) by at least 40-fold, the expanded HSCs being competent to reconstitute an HSC lineage upon transplantation into a mammalian patient in need thereof, the method comprising culturing a population of HSCs in a suitable culture medium comprising a PTEN inhibitor and a GSK-3β inhibitor.
25. The method according to claim 24 , wherein the HSCs are obtained from a mammalian tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.
26. The method according to claim 24 , wherein the mammal is a human.
27. The method according to claim 24 , wherein the PTEN inhibitor is bpV(pic), and the GSK-3β inhibitor is CHIR99201.
28. The method according to claim 24 , wherein the culture media comprises from about 100 to about 1000 nM of the PTEN inhibitor.
29. The method according to claims 24 , wherein the culture media comprises from about 50 nM to about 500 nM of the GSK-3β inhibitor.
30. The method according to claim 24 , wherein the expansion of the number of HSCs is by a factor selected from the group consisting of at least 80-fold, at least 150-fold, and at least 250-fold.