Objective

Skeletal muscle is maintained by tissue-resident stem cells. Upon injury, muscle stem cells (MuSCs) activate, proliferate, differentiate to repair damaged myofibres and self-renew, through a balance of symmetric (SCD) and asymmetric (ACD) cell divisions. However, most of our current knowledge arises from ex vivo studies, leaving the dynamic behaviour of MuSCs in vivo largely unexplored. We aim to understand how dynamic interactions between MuSCs and niche cells impact stem cell functions in vivo, and how they are affected in disease models.

Methods

We have developed an in vivo intravital imaging setup to track MuSC fate decisions and interactions with cells during muscle regeneration. We set up conditions (anaesthesia, surgery, imaging, new reporters) to record MuSC dynamics directly in vivo for more than 18h. For functional validations, we have defined an in vitro system to assess MuSC fate decisions using live-imaging and single-cell tracking.

Results

Our intravital imaging setup allowed us to capture consecutive MuSC (Pax7 ^CreERT2; Rosa ^mGFP or Rosa ^tdTomato reporters) divisions, migration and differentiation decisions (Myogenin ^ntdTom reporter), as well as dynamic interactions between MuSCs and niche cells (LysM ^EGFP reporter of myeloid cells). To extend our live-imaging modalities, we have generated a new far-red lineage-tracing mouse line, allowing 3-colours intravital imaging. In parallel, we optimised in vitro culture conditions to recapitulate all MuSC division modes (ACD = 11%, proliferative SCD = 49%, differentiative SCD = 40%), which will allow perturbations studies and functional validations of the impact of MuSC-niche cells interactions on MuSC fate decisions.

Conclusions

Altogether, our in vivo and in vitro pipelines will allow us to measure dynamic MuSC fate decisions and niche cell interactions, and their alterations in disease conditions (e.g., Duchenne Muscular Dystrophy). By investigating the cues governing self-renewal and differentiation of adult stem cells, we expect that these studies will provide the community with key knowledge, resources and methodologies for investigating diverse stem cell functions ex vivo and in vivo.