Conversion of Committed Mouse Myoblasts into Muscle Stem Cells Using Small Molecules [scRNA-Seq]

Quiescent muscle stem cells, commonly known as satellite cells, are crucial for muscle repair and can convert into committed myoblasts capable of proliferation and differentiation upon in vitro culture. However, following prolonged propagation, myoblasts frequently lose myogenic differentiation capacity, limiting their utility in research and clinical applications. Here, we demonstrate that exposing committed mouse myoblasts to a small-molecule cocktail elicits their conversion into expandable and heterogeneous myogenic progenitor cells (MPCs), comprised of muscle stem, progenitor and differentiated cells. Utilizing a new dual-fluorescent reporter for Pax7 and MyoD, we demonstrate that the small molecules de-differentiate Pax7+/MyoD+ myoblasts into Pax7+/MyoD- satellite-like cells within days. This conversion is characterized by upregulation of signaling pathways associated with satellite cells including Notch, Calcitonin and EGFR. Accordingly, genetic ablation of Notch1-expressing cells abrogated MPC cultures but not committed myoblasts. Furthermore, a comparison with in vivo-derived freshly isolated and activated satellite cells through single-cell transcriptomics revealed that the stem cell subset in MPCs shares common features with both cell types, particularly with a sub-population of activated satellite cells. Collectively, our study presents a method to de-differentiate myoblasts into MPCs harboring satellite cell attributes in vitro, offering a new avenue for studying myogenesis and advancing muscle disease therapeutics.