In vitro production of muscle stem cells
An in vitro method of producing a population of muscle stem cells comprising co-culturing pluripotent stem cells, embryonic fibroblast cells and endothelial cells in 3D cell culture.
1. A method of producing a population of muscle stem cells in vitro, the method comprising:
admixing pluripotent stem cells, embryonic fibroblasts and endothelial cells and establishing at least one 3D spheroid comprising the pluripotent stem cells, the embryonic stem cells and the endothelial cells;
co-culturing the pluripotent stem cells, the embryonic fibroblast cells and the endothelial cells in a 3D cell culture, wherein
(a) the pluripotent stem cells and the embryonic fibroblast cells are in a ratio of about 0.3-4.1;
(b) the pluripotent stem cells and the endothelial cells are in a ratio of about 0.3-4:1; and
(c) the embryonic fibroblast cells and the endothelial cells are in a ratio of about 1-2:1; and
wherein the muscle stem cells produced are skeletal muscle stem cells.
2. The method of claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
3. The method of claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells.
4. The method of claim 1 , wherein the embryonic fibroblast cells are mammalian embryonic fibroblast cells.
5. The method of claim 1 , wherein the endothelial cells are mammalian endothelial cells.
6. The method of claim 1 , wherein the pluripotent stem cells, the embryonic fibroblast cells and the endothelial cells are in a ratio of about 0.3-4:1:1.
7. The method of claim 1 , wherein the pluripotent stem cells, the embryonic fibroblast cells and the endothelial cells are co-cultured for about 3-30 days.
8. The method of claim 1 , wherein the 3D cell culture further comprises fibroblasts other than embryonic fibroblasts, smooth muscle cells and/or myoblast cells.
9. The method of claim 1 , wherein the muscle stem cells are Pax7 + and/or NCAM + and/or CD82 + .
10. The method of claim 1 , wherein the muscle stem cells are negative for commitment factors.
11. The method of claim 1 , wherein the pluripotent stem cells and the embryonic fibroblast cells are in a ratio of about 1.5-2.5:1.
12. The method of claim 1 , wherein the pluripotent stem cells and endothelial cells are in a ratio of about 1.5-2.5:1.
13. The method of claim 1 , wherein the endothelial cells are mouse or human endothelial cells.
14. The method of claim 1 , wherein
(a) the pluripotent stem cells and the embryonic fibroblast cells are in a ratio of about 2:1;
(b) the pluripotent stem cells and the endothelial cells are in a ratio of about 2:1; and/or
(c) the embryonic fibroblast cells and the endothelial cells are in a ratio of about 1:1.
15. The method of claim 1 , wherein the muscle stem cells are at least two selected from the group consisting of Pax7 + , NCAM + , CD82 + , GATA4 + and FGF5 + .
16. The method of claim 1 , wherein the muscle stem cells are at least two selected from the group consisting of Myf5 − , Myogenin − , MyoD − and MYH2 − .