Protocol for the isolation of mouse muscle stem cells using fluorescence-activated cell sorting

Abstract

Muscle stem cells (MuSCs) are the building blocks for regenerating skeletal muscle after trauma. If we intend to maximize the therapeutic potential of MuSCs, we must further study their molecular and functional properties. Here, we present a protocol for the isolation of mouse MuSCs via a two-step enzymatic and mechanical dissociation of skeletal muscle coupled with fluorescence-activated cell sorting (FACS). FACS-isolated MuSCs can be used for various downstream applications including cell culture, cell transduction, immunofluorescence, and gene expression assays. For complete details on the use and execution of this protocol, please refer to Almada et al. (2021).¹.

Keywords: Antibody; Cell Differentiation; Cell isolation; Flow Cytometry; Gene Expression; Stem Cells.

Graphical abstract

Figure 1

Extraction of hindlimb, triceps, and abdominal skeletal muscles from adult mice Mouse skeletal muscle dissection. (A) Create a transverse incision along the waist and peel away skin and underlying fascia to access (B) hindlimb, (C) forelimb, and (D) abdominal skeletal muscles.

Figure 2

Preparing glass Pasteur pipets for trituration Safely cut Pasteur pipets at the dotted line and place a bulb on the cut end of the pipet. We note that the users can fire-polish the uncut end to create different size diameters which may help with the mincing step.

Figure 3

Mechanical dissociation of skeletal muscle to release single fibers Trituration of skeletal muscle releases myofibers. (A) Digested muscle is poured into a dish containing PBS and subsequently triturated. (B) A gentle round of trituration releases myofibers, with (C) a second round of trituration liberating additional myofibers from muscle. (D) If optimally performed, the third round of trituration results in muscles appearing paler and less opaque because of releasing most of the myofibers. In a subsequent digestion step, MuSCs can be released from the basal lamina of myofibers.

Figure 4

Gravitational sedimentation of myofibers Phases of gravitational sedimentation. (A) Interstitial cells (ICs) are dispersed in the supernatant after the first round of sedimentation. (B) By the second round, the supernatant becomes clearer, as IC cells are progressively depleted from the supernatant. (C) By the third round of gravitational sedimentation, the supernatant is visibly clear and myofibers are enriched at the bottom of the tube.

Figure 5

FACS gating plots of β1-integrin and CXCR4+ MuSCs FACS gating strategy for sorting prospective MuSCs. (A) Debris are removed. (B) Single cells are isolated. (C) PI-negative and Calcein blue-positive cells (live) are gated. (D) Muscle-resident FAPs (Sca1⁺), blood cells (CD45⁺, CD11b⁺, TER119⁺) and endothelial cells (CD31⁺) are excluded by negative selection markers. (E) MuSCs are identified as double-positive for β₁-integrin and CXCR4.

Figure 6

Immunofluorescence staining of cultured primary muscle progenitor cells and myotubes (A) 3,000 MuSCs were cultured for 2 days in growth media supplemented with bFGF and stained for PAX7 and Myogenin (MYOG). MuSCs grown in GM are positive for PAX7 but negative for MYOG. (B) 10,000 MuSC-derived myoblasts were cultured for 3 days in differentiation media and stained for PAX7 and MYOG. Myotubes express no PAX7 but high levels of MYOG. Staining was performed with anti-PAX7 (1:20, DSHB) and anti-MYOG (1:100, BD Biosciences).