Erythrocytes differentiated in vitro from nanofiber expanded CD133+ cells
View Patent ↗The invention provides, among other things, methods and systems for expanding CD133+ cells. The invention further provides methods and systems for increasing the blood flow to an ischemic tissue in a subject in need thereof, such as to ischemic myocardium. The invention further provides methods and systems for directing differentiation of expanded CD133+ cells. The invention further provides methods and systems for treating a subject with differentiated cells in a subject in need thereof.
1. A method for producing erythrocytes comprising:
(a) providing a purified or selected population of CD133+ cells,
(b) culturing said CD133+ cells on polymeric nanofibers to expand the number of CD133+ cells, and
(c) directing differentiation of the expanded CD133+ cells into erythrocytes, wherein said CD133+ cells are cultured without stromal cells, and wherein directing differentiation comprises sequentially exposing cells to media comprising:
a) Stem Cell Growth Factor 1 (SCF), Erythropoietin (EPO) and Interleukin 3,
b) SCF and EPO, and
c) EPO.
2. The method of claim 1 , wherein said expansion comprises increasing the number of CD133+ cells by at least 300-fold.
3. The method of claim 1 , wherein the CD133+ cells are CD133+CD34− cells, CD133+CD34+ cells, or combinations thereof.
4. The method of claim 1 , wherein the CD133+ cells are CD133+CD34+KDR-CXCR4-cells.
5. The method of claim 1 , wherein at least 10% of the total cells are CD133+.
6. The method of claim 1 , wherein said expansion occurs in about 10 days.
7. The method of claim 1 , wherein said polymeric nanofibers are random nanofiber meshes or films.
8. The method of claim 1 , wherein said polymeric nanofibers are aligned nanofiber meshes or films.
9. The method of claim 1 , wherein said polymeric nanofibers are polyethersulfone (PES) meshes or films.
10. The method of claim 1 , wherein said polymeric nanofibers are surface-conjugated with functional groups.
11. The method of claim 10 , wherein said functional groups are selected from the group consisting of: hydroxyl, carboxyl, and amino groups.
12. The method of claim 1 , wherein said polymeric nanofibers are surface-conjugated with fibronectin.
13. The method of claim 1 , wherein said CD133+ cells are mammalian cells.
14. The method of claim 1 , wherein said CD133+ cells are human cells.
15. The method of claim 1 , wherein the CD133+ cells are isolated from umbilical cord blood, bone marrow or peripheral blood or combinations thereof.
16. The method of claim 1 , wherein the CD133+ cells are enriched from umbilical cord blood, bone marrow or peripheral blood or combinations thereof.
17. The method of claim 16 , wherein the composition comprises CD133+ enriched at least 2-fold over bone marrow mononuclear cells.
18. The method of claim 1 , wherein the expanded CD133+ cells are recultured for further expansion.
19. The method of claim 1 , wherein the erythrocytes are O Rh negative cells.
20. The method of claim 1 , wherein the total number of differentiated cells is increased by at least 4500-fold over the starting number of CD133+ cells.
21. The method of claim 1 , wherein said differentiation occurs in about 6-28 days.
22. The method of claim 1 , wherein the CD133+ cells express a recombinant transgene.
23. The method of claim 22 , wherein the CD133+ cells express a recombinant proangiogenic growth factor.
24. The method of claim 23 , wherein the proangiogenic growth factor is VEGF164, PDGF-BB, or both.
25. The method of claim 24 , wherein VEGF164 and PDGF-BB are expressed in a bicistronic co-delivery vector.
26. A method for producing erythrocytes comprising:
(a) providing a purified or selected population of CD133+ cells,
(b) culturing said CD133+ cells in a bioreactor containing polymeric nanofibers to expand the number of CD133+ cells, and
(c) directing differentiation of the expanded CD133+ cells into erythrocytes, wherein directing differentiation comprises sequentially exposing cells to media comprising:
a) Stem Cell Growth Factor 1 (SCF), Erythropoietin (EPO) and Interleukin 3,
b) SCF and EPO, and
c) EPO.
27. The method of claim 26 , wherein said CD133+ cells are expanded about 300-fold.
28. The method of claim 26 , wherein the total number of differentiated cells is increased by at least 4500-fold over the starting number of CD133+ cells.
29. The method of claim 26 , wherein said differentiation occurs in about 6-28 days.
30. The method of claim 26 , wherein said expansion and differentiation occur in about 28 days.
31. The method of claim 26 , wherein cellular waste production is optimized during culturing.
32. The method of claim 31 , wherein cellular waste is selected from the group consisting of ammonia and lactic acid.
33. The method of claim 26 , wherein CO2 levels, O2 levels and temperature are optimized during culturing.
34. The method of claim 33 , wherein CO2 levels, O2 levels and temperature are re-optimized during different phases of culturing.