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Isolation of satellite cells and transplantation into mice for lineage tracing in muscle


Limited methods exist to assay the direct effects of therapeutic intervention on muscle stem cell fate, proliferation or differentiation in an in vivo context. Here we provide an optimized protocol for muscle stem cell isolation and transplantation into mice to deconvolute heterogeneity within isolated stem cell populations. Viable and pure cell populations are isolated within 2 h and can then be used for therapeutic intervention or transplantation to uncover the repopulating and differentiation potential in mice, a physiologically relevant in vivo context. Effects can be assessed 9 d after transplantation. This methodology analyzes cell and sort purity prior to transplantation to improve reproducibility and outlines novel blocking steps to improve tissue staining and analysis. Experience with surgical procedures in mice is recommended before attempting this protocol. Our system is widely applicable for exploring stem cell dynamics within muscle and has already been used to study heterogeneity within muscle stem cell populations and efficacy of therapeutic intervention on isolated stem cell populations.

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Fig. 1: Overview of the protocol for satellite cell isolation and transplantation.
Fig. 2: Muscle stem cell isolation from hindlimb muscle.
Fig. 3: Muscle stem cell lineage gating and quality control strategy.
Fig. 4: Microdeposition of muscle stem cells into recipient muscle.
Fig. 5: Graft processing and immunohistochemistry strategy.

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Data availability

Data supporting Fig. 3 and Supplementary Fig. 1 are available from the corresponding author upon request.


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P.F. is supported by a fellowship from Canadian Institutes for Health Research. M.A.R. holds the Canada Research Chair in Molecular Genetics. These studies were carried out with grant support to M.A.R. from the NIH (R01AR044031), the Canadian Institutes for Health Research (FDN-148387), E-Rare-2: Canadian Institutes of Health Research/Muscular Dystrophy Canada (ERA-132935), the Muscular Dystrophy Association, the Ontario Institute for Regenerative Medicine and the Stem Cell Network.

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Authors and Affiliations



Conceptualization, P.F. and M.A.R.; methodology, P.F. and M.A.R.; writing, P.F. and M.A.R.; funding acquisition, M.A.R.; resources, M.A.R; supervision, M.A.R.

Corresponding author

Correspondence to Michael A. Rudnicki.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Protocols thanks Robert W. Arpke, Dawn Cornelison and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references that demonstrate the development/use of the Protocol

Wang, Y. X. et al. Cell Stem Cell. 24, 419–432.e6 (2019):

Bentzinger, C. F. et al. Cell Stem Cell. 12, 75–87 (2013):

Kuang, S., Kuroda, K., Le Grand, F., & Rudnicki, M. A. Cell. 129, 999–1010 (2007):

Integrated supplementary information

Supplementary Fig. 1 Cell purity quality control prior to engraftment.

Representative immunofluorescence images with single channels for committed satellite cell transplants (a) and satellite stem cell transplants (b). Blue, DAPI; green, nGFP; red, nTdTomato; white, Pax7. Scale bar, 50 μm. n = 3, mean ± s.d. Experimental protocols for mice used in this study were performed in accordance with the guidelines established by the University of Ottawa Animal Care Committee, which is based on the guidelines of the Canadian Council on Animal Care.

Supplementary Fig. 2 Lipofuscin-quenching validation for transplanted tissue.

a, Representative immunofluorescence images of non-injured, non-transplanted muscle show the high levels of autofluorescence present within skeletal muscle. A trace of the 12 Bit histogram from Zen 2.5 for 488, 546 and 647 nm is shown. b, Lipofuscin-quenched sections from serial sections have significantly decreased background in 488 and 546 nm autofluorescence with a slight increase in 647 nm autofluorescence. c and d, Representative immunofluorescence images from satellite stem cell (nTdTomato) transplants treated without TrueBlack (c) show the significant presence of non-cellular (DAPI-negative) autofluorescence artifact within injured skeletal muscle, which impedes the ability to detect real signal from transplanted cells as shown following blocking with TrueBlack (d). Blue, DAPI; green, nGFP; red, nTdTomato; white, Pax7. Scale bar, 100 μm. Experimental protocols for mice used in this study were performed in accordance with the guidelines established by the University of Ottawa Animal Care Committee, which is based on the guidelines of the Canadian Council on Animal Care.

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Feige, P., Rudnicki, M.A. Isolation of satellite cells and transplantation into mice for lineage tracing in muscle. Nat Protoc 15, 1082–1097 (2020).

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