Hematopoietic potential of murine skeletal muscle–derived CD45−Sca-1+c-kit− cells

Objective Somatic stem cells, which are poorly defined in postnatal mammalian tissues, are attractive candidates for examination of stem cell plasticity. Our goal was to determine the identity of neonatal muscle-derived cells that contain hematopoietic potential and to explore the status of CD45 expression on these cells. Materials and Methods Skeletal muscle from thighs of 4- to 7-day-old mice was harvested, enzymatically digested, and flow cytometrically sorted to yield CD45−Sca-1+c-kit− cells. These cells were examined in hematopoietic colony-forming assays and competitive repopulation assays, and were expanded ex vivo. Additionally, CD45, c-kit, PU.1, and β globin major expression was tracked over time in cultured cells to assess the possibility of manipulating stem cell differentiation in vitro. Results Freshly isolated CD45−Sca-1+c-kit− cells were devoid of hematopoietic lineage markers and contained no colony-forming activity but displayed superior long-term competitive repopulating ability when compared to freshly isolated muscle-derived CD45+Sca-1+c-kit+ cells. CD45−Sca-1+c-kit− cells expanded ex vivo in 5 ng/mL murine stem cell factor, mFlt-3L, and megakaryocyte growth and development factor (MGDF) for 9 days increased their in vivo hematopoietic repopulating potential 5.3-fold relative to fresh cells. Although fresh cells did not transcribe mRNA of several hematopoietic genes, a small fraction of cells cultured for 9 days acquired cell surface c-kit, and only these cells expressed c-kit and PU.1 mRNA and maintained competitive repopulating ability, suggesting at least myeloid and perhaps lymphoid developmental potential. Conclusion Neonatal murine muscle-derived cells expressing the phenotype CD45−Sca-1+ c-kit− are putative adult somatic stem cells with in vitro and in vivo hematopoietic differentiation potential.