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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+CD31CD45Sca1). 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.

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Figure 1: Representative FACS profiles of quiescent and activated MuSCs.
Figure 2: Dissection of hind limb muscles.
Figure 3: Confirmation of the myogenicity of isolated MuSCs.

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Acknowledgements

We thank P. Huang for technical assistance of dissection and L. Rott for technical assistance with FACS. This work was supported by grants from The Glenn Foundation for Medical Research, the US National Institutes of Health (P01 AG036695, R37 AG023806 and R01 AR062185) and the Department of Veterans Affairs to T.A.R.

Author information

Authors and Affiliations

Authors

Contributions

All authors contributed to the design and interpretation of the experiments; L.L., T.H.C. and G.W.C. conducted the experiments; and L.L. and T.A.R. wrote the manuscript.

Corresponding author

Correspondence to Thomas A Rando.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the VCAM1+Sca1-CD31-CD45- population from muscle as MuSCs using a genetic lineage tracer.

Pax7CreER/+; ROSA26eYFP/+ mice were injected with tamoxifen at 8-10 weeks of age. Mononucleated cells were isolated from limb muscles of these mice at different ages (indicated in parentheses) in the absence of injury or at different times following the injury, as indicated. Cells were then stained with APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Sca1 and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. Cells positive for CD31 and CD45 (CD31+/CD45+), cells positive for Sca1 but negative for CD31/CD45 (CD31-/CD45-, Sca1+), and cells positive for VCAM1 but negative for CD31/CD45/Sca1 (CD31-/CD45-/Sca1-, VCAM1+) were analyzed for YFP-expressing cells. The FITC channel (for the detection of YFP) is shown on the Y axis and FSC-S is shown on the X axis of all plots. qMuSCs: quiescent MuSCs; aMuSCs: activated MuSCs and their progeny; mo: months; DPI: days post-injury; MPI: months post-injury.

Supplementary Figure 2 Typical FACS profiles for single channel staining controls.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) APC anti-mouse CD31 and APC anti-mouse CD45, (B) Pacific Blue anti-mouse Ly-6A/E (Sca1), and (C) Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. The population hierarchy is shown under the plots.

Supplementary Figure 3 Typical FACS profiles for Fluorescence Minus One (FMO) staining controls.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, (B) APC anti-mouse CD31, APC anti-mouse CD45 and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, and (C) APC anti-CD31, APC anti-mouse CD45 and Pacific Blue anti-mouse Ly-6A/E (Sca1). The population hierarchy is shown under the plots.

Supplementary Figure 4 Typical FACS profiles for staining using isotype control antibodies.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) APC IgG2a, APC IgG2b, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, (B) APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue IgG2a, and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, and (C) APC anti-CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1), and Biotin IgG2a followed by PE/Cy7 Streptavidin. The population hierarchy is shown under the plots.

Supplementary Figure 5 Representative FACS profiles of quiescent and activated MuSCs without PI staining.

The digested tissue preparations, as described in this protocol, from uninjured or injured limb muscles were stained with 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) VCAM1+/CD31-/CD45-/Sca1- SCs were isolated from a 2-month-old C57BL/6 mouse according to the protocol described. Cells were then incubated with 0.3 μg/ml PI and analyzed by FACS. (D) Profiles of the unstained activated MuSCs and their progeny. (E) Profiles of activated MuSCs and their progeny after antibody staining. The population hierarchy is shown under the plots. (F) VCAM1+/CD31-/CD45-/Sca1- SCs were isolated from a 2-month-old C57BL/6 mouse injured by BaCl2 injection according to the protocol described. Cells were then incubated with 0.3 μg/ml PI and analyzed by FACS for the percentage of PI-positive cells.

Supplementary Figure 6 Identification and isolation of different cell-types in skeletal muscle.

(A) Distinct separation of mononucleated cells from myofiber debris following isolation from uninjured skeletal muscle. 2,000 events are shown in the FSC-A and SSC plot. The region of the plot containing cellular debris is highlighted in red. (B) Representative FACS profiles to isolate highly pure quiescent MuSCs (P6) and muscle-derived mesenchymal stem cells (P5) simultaneously. The suspension of resident mononucleated cells from uninjured limb muscles was stained with 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. 20,000 events were recorded and are shown in the plots. The population hierarchy is shown under the plots. Note the size difference of the P1 gate in comparison to that in Figure 1A and 1B. Mesenchymal stem cells are much larger than MuSCs and therefore a larger gate is needed to acquire these cells. Therefore we recommend using the FSC-W parameter to further select for small cells within the CD31-/CD45-/Sca1-/VCAM1+ population (P4). (C) Typical FACS profiles to identify and isolate highly pure quiescent MuSCs (P8) and muscle-derived mesenchymal stem cells (P5), as well as populations enriched for hematopoietic (P6) and endothelial (P7) cells simultaneously. The suspension of resident mononucleated cells from uninjured limb muscles was stained with APC anti-mouse CD31, FITC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. 20,000 events were recorded and are shown in the plots. The population hierarchy is shown under the plots. The FSC-W parameter further selected for small cells within the CD31-/CD45-/Sca1-/VCAM1+ population (P4) in order to maximize the purity of MuSCs.

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Liu, L., Cheung, T., Charville, G. et al. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc 10, 1612–1624 (2015). https://doi.org/10.1038/nprot.2015.110

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