Cultured hematopoietic stem cells and method for expansion and analysis thereof
View Patent ↗Hematopoietic stem cells and methods for ex vivo expansion of hematopoietic stem cells are provided. The methods comprise culturing the cells in a media containing an effective amount insulin-like growth factor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF), under conditions sufficient for expansion of said cells. Methods for identifying expanded hematopoeitc stem cells and kits for ex vivo expansion of hematopoietic stem cells are also provided.
1. A method for expanding hematopoietic stem cells ex vivo, comprising culturing a population of cells that includes hematopoietic stem cells in a medium comprising insulin-like growth factor-2 (IGF-2) and at least one factor selected from the group consisting of fibroblast growth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF), under conditions sufficient for expansion of the hematopoietic stem cells, thereby expanding hematopoietic stem cells ex vivo.
2. The method of claim 1 , wherein at least two factors from the group are present in the medium.
3. The method of claim 1 , wherein at least three factors from the group are present in the medium.
4. The method of claim 1 , wherein the FGF is FGF-1.
5. The method of claim 1 , wherein the medium comprises IGF-2, FGF-1, SCF, and TPO.
6. The method of claim 1 , wherein the IGF-2 is present at a concentration of about 20 ng/ml.
7. The method of claim 1 , wherein FGF is present at a concentration of about 10 ng/ml.
8. The method of claim 1 , wherein TPO is present at a concentration of about 20 ng/ml.
9. The method of claim 1 , wherein SCF is present at a concentration of about 10 ng/ml.
10. The method of claim 1 , wherein the cells are cultured for about two weeks.
11. The method of claim 1 , wherein said population of cells comprises total bone marrow.
12. The method of claim 1 , wherein said population of cells comprises a sub-population of total bone marrow cells.
13. The method of claim 1 , wherein the expanded hematopoietic stem cells are at least about 8-fold enriched for long term hematopoietic stem cells.
14. The method of claim 1 , wherein said culture medium comprises serum-free medium.
15. The method of claim 1 , further comprising the step of separating cells from the population of cells.
16. The method of claim 15 , wherein said separating is performed after culturing said population of cells.
17. The method of claim 15 , wherein the step of separating comprises selecting cells that express Kit.
18. The method of claim 17 , where the step of separating comprises selecting cells that express IGF-2 receptor.
19. The method of claim 15 , wherein the step of separating comprises selecting cells that express Kit and IGF-2 receptor.
20. The method of claim 15 , wherein the step of separating comprises selecting cells that do not express at least one negative cell surface marker.
21. The method of claim 20 , wherein the step of separating is performed after culturing said population of cells.
22. The method of claim 20 , wherein the step of separating comprises selecting cells that do not express Lin.
23. The method of claim 1 , further comprising the step of separating cells the population of cells by selecting cells that express at least one positive cell surface marker and that do not express at least one negative cell surface marker.
24. The method of claim 23 , wherein the step of separating is performed after culturing said population of cells.
25. The method of claim 23 , wherein the method comprises selecting cells that express Kit and that do not express Lin.
26. The method of claim 25 , wherein the method comprises selecting cells that express IGF-2 receptor.
27. The method of claim 23 , wherein the method comprises selecting cells that express IGF-2 receptor and that do not express Lin.