Abstract
Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.
Highlights
Satellite cells are the adult stem cells of skeletal muscle and support postnatal muscle growth and repair
Primary satellite cells were isolated from 3-month-old adult C57BL/6-β-actin-Enhanced green fluorescent protein (EGFP) mouse muscle by enzymatic digestion followed by fluorescence-activated cell sorting (FACS) to select cells exhibiting the surface marker phenotype CD45−/TER119−/CD11b−/SCA1−/ CXCR4+/ITGB1+, as described previously [22]
Freshly isolated satellite cells have begun to activate, and most cells are positive for Pax7 and Myoblast determination protein 1 (MyoD) (Fig. S1A-B)
Summary
Satellite cells are the adult stem cells of skeletal muscle and support postnatal muscle growth and repair. Adult satellite cells, freshly isolated from skeletal muscle and treated with CEP-701 ex vivo for 5 days, exhibited enhanced muscle engraftment efficiency, as compared to vehicle-treated controls. Consistent with these results, systemic administration of CEP-701 following muscle injury enhanced myofiber regeneration and was accompanied by an in vivo expansion of satellite cell numbers. Based on the putative targets of CEP-701 [18], we identified the proto-oncogene RET as the predominant RTK present on adult satellite cells and showed that the RET ligand GDNF can inhibit satellite cell proliferation in vitro Together, these results implicate RTK signaling, and signaling via GDNF/RET, in skeletal muscle regeneration. This work’s identification of RET inhibitors as candidate therapeutics demonstrates the utility of performing direct chemical screening on primary stem cell populations to treat muscle-wasting conditions
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