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

Abstract

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 Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

“…Regardless of whether FACS is used, optimization of enzymatic digestion conditions for cell viability and yield is critical, as in our experience this represents the most common point of experimental failure in scRNA‐seq studies. In keeping with this, comparison of in vivo transcriptional profiles on fixed cells to cells undergoing a typical isolation protocol in muscle suggests that enzymatic digestion is the major step that can disrupt the in vivo transcriptional profile . This emphasizes the importance of minimizing the duration and harshness of enzymatic digestion, and we note that brief digestion protocols can provide robust yields of mesenchymal cells, particularly in younger mice .…”

“…This emphasizes the importance of minimizing the duration and harshness of enzymatic digestion, and we note that brief digestion protocols can provide robust yields of mesenchymal cells, particularly in younger mice . Alternatively, there are several approaches designed to circumvent isolation‐associated artifacts, including in vivo fixation before cell isolation, though fixation can negatively impact cell isolation efficiency and RNA quality . In another approach, transgenic expression of Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) only in cell types of interest allows for selective labeling, capture, and bulk sequencing of transcripts only from this cell type after whole‐tissue RNA extraction .…”

“…However, this method requires a suitably specific promoter that allows targeting UPRT expression only to the cell type of interest, and experience to date with cre lines suggests that such a promoter may be elusive in the skeletal system. Recent work on muscle comparing post‐isolation to “in vivo” transcriptional profiles provides insight into the likely scope and degree of the impact of tissue digestion and isolation; these procedures induce an immediate early stress response and loss of quiescence that may occur predominantly in subpopulations of cells . Particular care is warranted in assessing whether such isolation‐associated activating transcriptional changes may either drive cell clustering or confound efforts to assess the transcriptional response to environmental or genetic perturbations.…”

“…These biases will largely be determined by the enzymatic digestion protocol employed. For these cell types with challenging isolation requirements, epigenetic readouts such as single‐cell ATAC‐seq may offer a more stable method to assess cell state in the face of harsh isolation procedures and can be multiplexed with scRNA‐seq using recent methods, though it is noted that epigenetic features can also be impacted by cell isolation protocols . The field of neuroscience in particular has had to deal with challenges in disassociating their tissue of interest into a single‐cell suspension and has found success in instead performing sequencing of single nuclei through a family of microfluidics or flow cytometry–based approaches .…”

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells

“…On FACS plots, we found that the freshly isolated p110α ‐null MuSCs were even smaller in size than the control MuSCs based on the mean forward scatter (FSC) values (Fig EV4C). This was most likely due to the fact that the control “MuSCs” was already partially activated during the isolation process (van den Brink et al , ; Machado et al , ; van Velthoven et al , ). Consistently, on freshly isolated myofibers, we found that the size of the mutant MuSCs was indeed smaller than that of the control MuSCs (Fig EV4D and E).…”