Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

The protocol presented here enables the identification and high-dimensional analysis of muscle stem and progenitor cells by single-cell mass cytometry and their purification by FACS for in-depth studies of their function. This approach can be applied to study regeneration dynamics in disease models and test the efficacy of pharmacological interventions.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Skeletal muscle constitutes the largest tissue by mass in the body and regulates multiple functions, from eyesight to respiration, from posture to movement, as well as metabolism¹. Therefore, maintaining skeletal muscle integrity and function is critical to health. Skeletal muscle tissue, which consists of tightly packed bundles of multinucleated myofibers surrounded by a complex network of nerves and blood vessels, exhibits remarkable regenerative potential^1,2.

The main drivers of skeletal muscle regeneration are adult muscle stem cells (MuSCs). Also known a....

Animal procedures were approved by the Danish animal experiments inspectorate (protocol # 2022-15-0201-01293), and experiments were performed in compliance with the institutional guidelines of Aarhus University. Analgesia (buprenorphine) is provided in drinking water 24 h prior to injury for the mice to adapt to the taste. Supplying buprenorphine in drinking water is continued for 24 h post-injury. Together with a subcutaneous (s.c.) injection of buprenorphine at the time of acute muscle injury, buprenorphine in the drin.......

Here we present an overview of the experimental setup for using this combined approach which includes (i) high-dimensional CyTOF analysis of an acute injury time course by notexin injection to study the cellular and molecular dynamics of stem and progenitor cells in skeletal muscle (Figure 1, top scheme); and (ii) FACS of stem and progenitor cells using two cell surface markers, CD9 and CD104, to isolate these populations and perform in-depth studies of their function (F.......

Skeletal muscle regeneration is a dynamic process that relies on the function of adult stem cells. While prior studies have focused on the role of muscle stem cells during regeneration, their progeny in vivo has been understudied, primarily due to a lack of tools to identify and isolate these cell populations^15,16,17,18. Here, we present a method to simultaneously identify and isolate .......

We thank the members of the FACS Core Facility in the Department of Biomedicine at Aarhus University for technical support. We thank Alexander Schmitz, the manager of the Mass Cytometry Unit at the Department of Biomedicine, for discussion and support. Scientific Illustrations were created using Biorender.com. This work was funded by an Aarhus Universitets Forskningsfond (AUFF) Starting Grant and a Start Package grant (0071113) from Novo Nordisk Foundation to E.P.