Methods and compositions for treatment of muscle disease with iPSC-induced human skeletal muscle stem cells
Human skeletal muscle stem cells were generated from facioscapulohumeral muscular dystrophy (FSHD) and healthy control iPSC using a transgene-free skeletal muscle differentiation protocol and production of stable iMyoblasts. Analyses revealed that FSHD and healthy control iMyoblasts are embryonic-like myogenic cells that undergo myotube differentiation ex vivo by growth factor depletion and are efficiently transplantable into the tibialis anterior (TA) muscles of NSG mice, where human muscle under-goes embryonic-to-adult myosin isoform switching. The DUX4 FSHD disease gene maintains its hypomethylated disease state inFSHD iPSC and iMyoblast, and its expression is upregulated during myotube differentiation and in muscle xenografts. Consequently, these iMyoblasts accurately exhibit the molecular pathology of human muscular dystrophies and are useful for the development of drug, gene editing and stem cell therapeutics.
1 . A method, comprising:
a) providing induced pluripotent stem cells (iPSCs) derived from reprogrammed primary myoblasts or primary fibroblasts;
b) stimulating said induced pluripotent stem cells in an S3 differentiation media to create induced primary myocytes and a first plurality of undifferentiated myogenic stem cells;
c) propagating said first plurality of undifferentiated myogenic stem cells in a transgene-free growth factor media to create induced secondary myocytes, wherein said induced secondary myocytes retain a genetic identity identical to that of said induced pluripotent stem cells; and
d) expanding said induced secondary myocytes to create a second plurality of undifferentiated stem cells, wherein said second plurality of undifferentiated stem cells retain a genetic identity to that of said induced pluripotent stem cells.
2 . The method of claim 1 , wherein said expanding said induced secondary myocytes further creates a myotube tissue.
3 . The method of claim 2 , wherein said myotube tissue further differentiates into a striated muscle tissue.
4 . The method of claim 1 , wherein said iPSCs are derived from a healthy subject.
5 . The method of claim 1 , wherein said iPSCs are derived from a subject diagnosed with facioscapulohumeral muscular dystrophy 1.
6 . The method of claim 1 , wherein said iPSCs are derived from a subject diagnosed with fukutin-related protein dystroglycanopathy.
7 . The method of claim 1 , wherein said iPSCs are derived from a subject diagnosed with limb-girdle muscular dystrophy R7.
8 . The method of claim 1 , wherein said method further comprises propagating said second plurality of undifferentiated stem cells in the presence of said myotube tissue.
9 . The method of claim 1 , wherein said second plurality of undifferentiated stem cells retain an autonomous expression of PAX3 and MYOD1 muscle master regulatory genes.
10 . The method of claim 9 , wherein said muscle master regulatory genes are upregulated.
11 . The method of claim 1 , wherein second plurality of undifferentiated stem cells retain a commitment to myotube differentiation.
12 . The method of claim 1 , wherein said genetic identity of said expanded second plurality of undifferentiated stem cells is stable.
13 . The method of claim 1 , wherein said method further comprises inducing said expanded second plurality of undifferentiated stem cells into induced tertiary myoblasts with a growth medium.