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The most studied sources of mesenchymal stem/stromal cells (MSCs) are bone marrow and adipose tissue. However skeletal muscle represents an interesting source of diverse subpopulations of MSCs, such as paired box 7 (Pax-7)-positive satellite cells, fibro-/adipogenic progenitors, PW1-positive interstitial cells and others. The specific properties of some of these muscle-derived cells have encouraged the development of cell therapies for muscle regeneration. However, the identity and multilineage potential of the diverse muscle-resident cells should first be evaluated in vitro, followed by in vivo clinical trials to predict their regenerative capacity. Here, we present protocols for the isolation of MSCs from skeletal muscle using enzymatic digestion and mechanical trituration. We also provide a method to determine their specific growth rate, a feature that is of particular interest when designing cell therapies.

Mesenchymal stem/stromal cells (MSCs) can be isolated from several connective tissues in the adult organism by harnessing their propensity for plastic adherence in vitro. Upon culture expansion, the resulting cell cultures are composed of many different cell types at different stages of differentiation. Hence, their identity must be confirmed. Flow cytometry is an indispensable method for accurate quantification of MSC surface antigens. Here, we present a protocol that uses flow cytometry for the identification of MSCs based on the set of surface antigens required by the International Society for Cellular Therapy.

Synovium-derived mesenchymal stem/stromal cells (MSCs) have been shown to have superior features in comparison with MSCs from other tissue sources. As they are far less recognised compared to bone marrow- or adipose tissue-derived MSCs, I provide here a detailed procedure on how to isolate MSCs from human synovium. This includes determination of the proportions of viable cells in ex vivo isolated fractions before the seeding of the cells and a description of how to carry out colony-forming fibroblast assays to quantify the clonogenicity of these cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.