Isolation of Muscle Stem Cells from Mouse Skeletal Muscle

Abstract

Isolation of muscle stem cells from skeletal muscle is a critical step for the study of skeletal myogenesis and regeneration. Although stem cell isolation has been performed for decades, the emergence of flow cytometry with defined cell surface markers, or transgenic mouse models, has allowed the efficient isolation of highly enriched stem cell populations. Here, we describe the isolation of mouse muscle stem cells using two different combinations of enzyme treatments allowing the release of mononucleated muscle stem cells from their niche. Mouse muscle stem cells can be further isolated as a highly enriched population by flow cytometry using fluorescent reporters or cell surface markers. We will present advantages and drawbacks of these different approaches.

Comparison of multiple transcriptomes exposes unified and divergent features of quiescent and activated skeletal muscle stem cells

“…In mouse skeletal muscle, the transcription factor Pax7 marks satellite cells during quiescence and proliferation, and it has been used to identify and isolate myogenic populations from skeletal muscle [ 2 , 3 ]. Myogenic cells have also been isolated by fluorescence-activated cell sorting (FACS) using a variety of surface markers, including α7-integrin, VCAM, and CD34 [ 4 ]. Although these cells have been extensively studied by transcriptome, and to a more limited extent by proteome profiling, different methods have been used to isolate and profile myogenic cells thereby making comparisons laborious and challenging.…”

Comparison of multiple transcriptomes exposes unified and divergent features of quiescent and activated skeletal muscle stem cells

“…Jun and Fos ), skeletal muscles were fixed immediately in 0.5% for 1 h in paraformaldehyde (PFA) using a protocol based on the notion that transcripts are stabilized by PFA fixation [18] [Machado et al, in press; P. Mourikis and F. Relaix, personal communication]. Briefly, PFA fixed and unfixed skeletal muscles were minced as described [4], fixed samples were incubated with collagenase at double the normal concentration and mRNA was isolated following FACS based on size, granulosity and GFP levels using a FACS Aria II (BD Bioscience). Total RNA was extracted from fixed cells with RecoverAll™ Total Nucleic Acid Isolation Kit Ambion, ThermoFisher), according to manufacturer instructions.…”

“…In mouse skeletal muscle, the transcription factor Pax7 marks MuSCs during quiescence and proliferation, and it has been used to identify and isolate myogenic populations from skeletal muscle [2, 3]. Myogenic cells have also been isolated by Fluorescence Activated Cell Sorting (FACS) using a variety of surface markers, including α7-integrin, VCAM and CD34 [4] Although these cells have been extensively studied by transcriptome, and to a more limited extend by proteome profiling, different methods have been used to isolate and profile myogenic cells thereby making comparisons laborious and challenging. To address this issue, it is necessary to generate comprehensive catalogs of gene expression data of myogenic cells across distinct states and in different conditions.…”

Notchless defines a stage-specific requirement for ribosome biogenesis during lineage progression in adult skeletal myogenesis

“…Quiescent and activated satellite cells were isolated from total limb muscles or injured TA muscles, respectively (Gayraud-Morel et al, 2017). Briefly, dissected muscles were collected into cold Dulbecco's Modified Eagle Medium (DMEM), minced with fine scissors and transferred into a 50 ml Falcon tube containing 20 ml of DMEM, 0.1% collagenase D (1088866, Roche), 0.25% trypsin (15090-046, Gibco), 0.1% DNAse1 10 mg/ml (11284932001, Roche) solution.…”