Abstract
Degenerative muscle diseases affect muscle tissue integrity and function. Human embryonic stem cells (hESC) are an attractive source of cells to use in regenerative therapies due to their unlimited capacity to divide and ability to specialize into a wide variety of cell types. A practical way to derive therapeutic myogenic stem cells from hESC is lacking. In this study, we demonstrate the development of two serum-free conditions to direct the differentiation of hESC towards a myogenic precursor state. Using TGFß and PI3Kinase inhibitors in combination with bFGF we showed that one week of differentiation is sufficient for hESC to specialize into PAX3+/PAX7+ myogenic precursor cells. These cells also possess the capacity to further differentiate in vitro into more specialized myogenic cells that express MYOD, Myogenin, Desmin and MYHC, and showed engraftment in vivo upon transplantation in immunodeficient mice. Ex vivo myomechanical studies of dystrophic mouse hindlimb muscle showed functional improvement one month post-transplantation. In summary, this study describes a promising system to derive engrafting muscle precursor cells solely using chemical substances in serum-free conditions and without genetic manipulation.
Highlights
Muscular dystrophies are debilitating degenerative muscle diseases caused by defective muscle proteins that disrupt normal homeostasis [1,2]
Differentiation was initiated by embryoid body (EB) formation in pre-differentiation medium KFLy(B)S: KSR medium freshly supplemented with 20 ng/ml basic fibroblast growth factor (bFGF), 10 μM Ly294,002 hydrochloride (Ly; Sigma-Aldrich), 10 μM SB431542 hydrate (SB; Sigma-Aldrich), and for the first 36 hours supplementing with recombinant human bone morphogenetic protein-4 (BMP-4) (10 ng/ml; HumanZyme, Chicago, IL), or pre-differentiation medium KFS: KSR medium freshly supplemented with 40 ng/ml bFGF and 10 μM SB
Inhibition of TGFβ pathway directs myogenic commitment To compare previously described, serum-containing methods for their capacity to induce the formation of myogenic precursors, we differentiated Human embryonic stem cells (hESC) by a selective culture approach using serumcontaining (10% fetal bovine serum (FBS)) medium to enrich for CD73+ mesodermal stem cells followed by a second cell sort for expression of neural cell adhesion molecule (NCAM), a molecule expressed by myogenic cells [16]
Summary
Muscular dystrophies are debilitating degenerative muscle diseases caused by defective muscle proteins that disrupt normal homeostasis [1,2]. These cells are termed satellite cells –stem cells that remain quiescent beneath the basal lamina of muscle fibers under normal conditions Tissue damage triggers these cells to proliferate and differentiate into myoblasts, leading to the formation of multinucleated myotubes and fibers [9,10,11,12]. Dystrophic tissue induces repeated activation of satellite cells, leading to stem cell exhaustion, limiting the ability for further restorative myogenesis. Given their role in muscle formation, growth and repair, satellite cells, or other myogenic precursors with similar characteristics, would be attractive candidates for cell-based therapies for muscular dystrophies. An important therapeutic benefit would be repopulation of the muscle stem cell niche to allow for ongoing homeostasis
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