Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting

Abstract

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Figure 1. Representative FACS profiles of quiescent and activated MuSCs

The digested tissue preparations, as described in this protocol, from uninjured or injured limb muscles were stained with PI, APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. Cells in the P4 gate are MuSCs. (A) Profiles of the unstained quiescent MuSCs. (B) Profiles of quiescent MuSCs after antibody staining. The population hierarchy is shown under the plots. (C) Profiles of the unstained activated MuSCs and their progeny. (D) Profiles of activated MuSCs and their progeny after antibody staining. The population hierarchy is shown under the plots.

Figure 2. Confirmation of the myogenicity of isolated MuSCs

(A) Immediately following FACS isolation, 5,000 quiescent MuSCs were plated in a well of an 8-well chamber slide that had been coated with Poly-D lysine and ECM. Cells were fixed with 4% paraformaldehyde 6 hours (6 hr), 3 days (3 d) and 5 days (5 d) following plating, and stained with antibodies against Pax7, MyoD1 and Myogenin, respectively. DAPI stains all nuclei. Note the changes in cell size and morphology. Scale bars represent 70 μm in the images and 10 μm in the inserts. (B) Immediately following FACS isolation, 5,000 activated MuSCs or their progeny from limb muscles that had been injured three days earlier were plated in a well of an 8-well chamber slide that had been coated with Poly-D lysine and ECM. Cells were fixed with 4% paraformaldehyde 6 hours later and stained with an anti-MyoD1 antibody. DAPI stains all nuclei. Scale bars represent 70 μm in the image and 10 μm in the insert.