Skeletal muscle plasticity mainly relies upon a population of resident muscle stem cells (MuSCs), and homeostatic maintenance and regeneration of the skeletal muscle rely upon the support of a population of muscle resident cells named fibro-adipogenic progenitors (FAPs). Isolating cells from these muscles allows for models to develop more complex studies to understand how these pathological mechanisms work.
We performed a standardized method for the simultaneous isolation of FAPs and MuSCs from muscle of adult human using fluorescence-activated cell sorting (FACS). Pure populations of FAPs and MuSCs were isolated using a FACS-based technique, and their purity was subsequently assessed by immunostaining cells with specific cell surface markers and genetic means. Sorted cells should be cultured immediately after sorting, in an appropriate medium on collagen I coated plates.
The protocol consists of three main sections that highly impact the yield of FAPs and MuSCs: the mechanical and enzymatic muscle digestion, the generation of a mononucleated cell suspension through the 20 G needle, and the final isolation of single cells through FACS. MuSCs were identified as CD31−/CD45−/CD56+, while FAPs were identified as CD31−/CD45-/PDGFRα+. Flow cytometric identification of FAPs and MuSCs was validated by cell culture and immunostaining of FACS-isolated FAPs and MuSCs. After activation, MuSCs, now called myoblasts, start to proliferate and fuse with damaged muscle fibers or with one another forming new myotubes. We validated their structure in light microscopy and MHC, PAX7, myoD amrker. FAPs are a population of skeletal muscle-resident MSCs capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage.
Identification, isolation, and cell culture of human muscle stem/progenitor cells represent powerful tools that will help us to understand the role of these cells in different conditions and facilitate the development of safe and effective new treatments for diseases.