State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.

The microenvironment is critical for stem cell maintenance and can be of cellular and non-cellular composition, including secreted growth factors and extracellular matrix (ECM)1–3. Although Notch and other signalling pathways have been reported to regulate quiescence4–9, the composition and source of niche molecules remain largely unknown. Here, we show that adult muscle satellite (stem) cells produce ECM collagens to maintain quiescence cell-autonomously. By ChIP-sequencing we identified NOTCH/RBPJ-bound regulatory elements adjacent to specific collagen genes, whose expression is deregulated in Notch mutant mice. Moreover, we show that satellite cell produced collagen V (COLV) is a critical component of the quiescent niche, as conditional deletion of Col5a1 leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by CALCR, for which COLV acts as a surrogate local ligand. Strikingly, systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects scored in COLV null satellite cells. This study unveils a Notch/COLV/CALCR signalling cascade that cell-autonomously maintains the satellite cell quiescent state and raises the possibility of a similar reciprocal mechanism acting in diverse stem cell populations.

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