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. This protocol enables isolation of viable and pure stem cell populations from muscle, and describes how to transplant the cells and follow repopulation and differentiation potential in vivo.