In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells

Abstract

Summary: 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. : Machado et al. demonstrate that muscle stem cells undergo changes in transcripts and histone modifications during isolation. The authors develop an in situ fixation-based methodology, which allows capture of cells in their native state. In light of these findings, some high-throughput analyses of tissue extracted cells may need to be revisited. Keywords: muscle stem cells, quiescence, early response genes, RNA-seq, ChIP-seq, methylation, satellite cells