Single-cell characterization of skeletal muscle mononuclear populations in physiological and pathological conditions

The adult skeletal muscle has the ability to regenerate in response to mechanical or chemical damage, stress caused by genetic mutations or increased workload. This regenerative property is mainly due to muscle resident mononuclear progenitors with the cooperation of additional cell types recruited from non-muscle compartments in response to specific stimuli. The complex cross talk between these heterogeneous cell types regulates muscle regeneration and homeostasis. However, contrasting evidence is reported in literature about the existence and abundance of these populations and their role and contribution to regeneration in the adult healthy or diseased muscle. In this context, my PhD project aimed at providing a quantitative characterization of the mononuclear populations that regulate muscle homeostasis in physiological and pathological conditions. Biological processes that are mediated by cell-cell interactions in heterogeneous populations are best approached by methods that have single cell resolution. For this reason, I decided to take advantages of the recently developed, mass cytometry platform (CyTOF2, DVS Sciences), a multiparametric single-cell approach, in order to study the complexity of the muscle system. The technology relies on the preparation, from solid tissues, of cell suspensions by enzymatic digestion followed by analysis of single cell reactivity to an antibody panel that allows the discrimination of cell populations and characterization of their activation state. For this purpose, I have assembled and characterized an antibody panel for the discrimination of muscle progenitor-cell populations by mass cytometry. The panel is formed by antibody probes, directed against antigens of relevance in myology, that bind either surface markers, to define cell populations, or intracellular epitopes reflecting protein expression and activation. This panel has been used to assess different mononuclear cell extraction methods for studying the complexity of the skeletal muscle tissue. Here, we show that the assembled collection of antibodies, albeit limited and incomplete, is suitable for the identification of most of the main skeletal muscle populations by mass cytometry. Additionally, by comparing three different enzymatic extraction methods, we conclude that according to the protocol utilized, the yields of the distinct cell types and their heterogeneity vary significantly. In addition, we adopted the assembled antibody panel and the optimized extraction protocol I to analyze by mass cytometry the muscle regeneration process. By this approach, I have described the dynamics at a single-cell level of muscle regeneration after CTX-induced injury by mass cytometry, and we have estimated their abundance. Furthermore, I studied the distribution at single-cell level and the abundance of the main muscle mononuclear populations of a 30 days-old male mdx mouse, in a state of degeneration/regeneration process, highlighting the activation state of each cell type. Although the details of the cellular and molecular mechanisms remain to be elucidated and additional efforts are required to expand the antibody panel described in the present thesis, I conclude that the approach developed here can provide a complete overview of the complexity of the muscle tissue and that represents a powerful tool to identify yet uncharacterized populations. In addition, in order to develop rational strategies to modify the differentiation trajectories of skeletal muscle populations and control their differentiation fate, I started a screening project aimed at identifying small molecules that perturb differentiation decisions. I used a strategy that takes advantage of the selective optimization of side activities (SOSA) approach. I performed a high-throughput and high-content screening of small molecules (Prestwick library) approved by the U.S. Food and Drug Administration (FDA). In this context, I screened 560 small molecules and developed fluorescence microscopy readouts to monitor cell differentiation into skeletal muscle, adipocytes or osteoblasts in a heterogeneous mixture of diverse muscle cell populations. Five chemicals are selected as putative differentiation modulators, able to perturb muscle mononuclear cells. Among these perturbagens, we have identified azathioprine, an immunosuppressive drug, as an inhibitor of the adipogenic differentiation program and atracurium besylate, a neuromuscular-blocking drug, as an inducer of skeletal muscle differentiation