GSE161075
RNA seq dataset of parental and reserve cells (FRCs Cycle 1 and SRCs Cycle 3)
Loss of regenerative capacity during ex vivo expansion of muscle stem cells hampers the development of cell-based therapies for skeletal muscle disorders. Here, we developed a method to isolate regenerative reserve cell fractions from expanded murine primary cultures using differential adhesion properties and the induction of subsequent cycles of proliferation and differentiation. Fast-adhering reserve cells (FRCs) were enriched for cells expressing myogenin and contributed efficiently to muscle regeneration following transplantation in immunodeficient hosts. Slow adhering reserve cells (SRCs) generated higher number of PAX7+ cells under differentiating conditions and predominantly regenerated muscle after a secondary injury, suggesting engraftment as muscle stem cells. Transcriptomic analysis revealed that FRCs were enriched for markers of myogenic differentiation, while SRCs were characterized by a distinctive set of cell-adhesion genes. Overall, reserve cells provide a valuable source for the study and identification of molecular determinants of regeneration using ex vivo-expanded muscle cells. Overall design: Reserve cell populations were passaged twice after isolation in proliferation conditions before harvesting. Samples were harvested using RLT buffer. RNA was extracted using the RNeasy minikit with DNase treatment. Total RNA for triplicates of PAR, FRCs Cycle 1, and SRCs Cycle 3 were checked for quality on an Agilent Technologies 2100 Bioanalyzer using a RNA nano assay. All samples had RIN value of 10. Triplicate RNA-Seq libraries were prepared according to the Illumina TruSeq stranded mRNA protocol. Sequencing-by-synthesis was performed using the HiSeq 2500 with a single read 50-cycle protocol followed by dual index sequencing