Myogenic differentiation of stem cells and uses thereof
Disclosed are methods of differentiating stem cells into muscle cells by growing the cells in a myogenic culture medium. The differentiated cells can be used as a source of cells for transplantation in a patient in need thereof.
1. A method of inducing differentiation of human induced pluripotent stem cells (iPSCs) into myogenic cells comprising i) culturing said iPSCs in a myogenic differentiation medium to induce differentiation into mesenchymal-like cells expressing TBX1 and TBX4; and ii) culturing said mesenchymal-like cells in the presence of at least one myogenic factor to complete differentiation into myogenic cells.
2. The method of claim 1 , wherein said myogenic differentiation medium comprises basic fibroblast growth factor (bFGF) and/or insulin.
3. The method of claim 2 , further comprising first growing said iPSCs in a proliferation medium.
4. The method of claim 3 , wherein said proliferation medium comprises between about 10 and about 30% of serum.
5. The method of claim 3 , wherein said myogenic differentiation medium and said proliferation medium are substantially the same as Myogenic Culture Medium 1 (MCM1).
6. The method of claim 5 , wherein said iPSCs are CD73 positive cells.
7. The method of claim 6 , wherein the presence of said at least one myogenic factor is provided by:
i) Treating said iPSCs with at least one myogenic factor protein;
ii) Inducing said at least one myogenic factor expression in said iPSCs; or
iii) Introducing in said iPSCs at least one nucleic acid capable of expressing said at least one myogenic factor.
8. The method of claim 7 , wherein said at least one myogenic factor is MyoD, Pax3, Pax7, Myf5 or myogenin.
9. The method of claim 8 , wherein said at least one myogenic factor is MyoD.
10. The method of claim 9 , wherein said MyoD is provided by introducing in said iPSCs a nucleic acid sequence capable of expressing MyoD.
11. The method of claim 10 , wherein said MyoD is introduced in said iPSCs with an adeno-associated viral vector; a retroviral vector, a lentiviral vector, a non-integrative lentiviral vector or a non-viral vector.
12. The method of claim 11 , comprising growing said iPSCs as single cells.
13. The method of claim 12 , wherein said myogenic cells express at least one of the following myogenic markers: Pax7, MyoD, myogenin, CD56, desmin and MHC and express low levels of Rex-1.
14. The method of claim 2 , wherein said myogenic differentiation medium comprises between about 0.5 and about 2% of serum.
15. The method of claim 2 , wherein said myogenic differentiation medium comprises between about 0.5% and about 5% of serum.
16. The method of claim 1 , wherein said iPSCs are derived from a fibroblast of a Duchenne muscular dystrophy patient.
17. A method of transplanting myogenic cells in a subject comprising implanting in said subject myogenic cells prepared by i) culturing human induced pluripotent stem cells (iPSCs) in a myogenic differentiation medium to induce differentiation into mesenchymal-like cells expressing TBX1 and TBX4; and ii) culturing said mesenchymal-like cells in the presence of a myogenic factor to complete differentiation into myogenic cells.
18. The method of claim 17 , wherein said transplantation is for increasing muscle mass in a subject suffering from Duchenne muscular dystrophy.
19. The method of claim 17 , wherein said myogenic differentiation medium is substantially the same as Myogenic Culture Medium 1 (MCM1).