Figure 4. Co-culture of LPC and HPC.

(a) Scheme for co-cultures: SCs were derived from FDB muscles of both wild-type and GFP+ rats then cloned through limiting dilutions and after 10 days of culture HPC were seeded on LPC (top panel). Increase in myotube formation derived from HPC. Epifluorescence for GFP+ myotubes (indicated with white arrows, central panel left, bar = 100 µm), and diagrams indicating number of myotubes per clone (central panel, center) and percentage of MyoD+ cells (central panel, right) in co-cultures, compared to control HPC (mean values±s.d., ***p<0.001). Immunostaining for MyoD and epifluorescence for GFP, merged with DAPI on co-cultures (bottom panel, bar = 100 µm). (b) Scheme for culture in conditioned medium: SCs were derived from FDB muscles of wild-type rats then cloned through limiting dilutions. After 10 days of culture LPC-conditioned medium was transferred on HPC (top panel). Myotube formation (indicated with white arrows, central panel left, bar = 100 µm), and diagrams indicating number of myotubes per clone (center) and percentage of MyoD+ cells (right) in cultures with LPC-conditioned medium, compared to control HPC (mean values±s.d., ***p<0.001; middle panel). Immunostaining for MyoD on HPC cells, subjected to LPC-conditioned medium, merged with DAPI (bottom panel, bar = 100 µm). (c) Cartoon of the current working model: pools of Satellite Cells can be isolated from the muscle fiber and cultured in vitro preserving their original diversities. HPC spontaneously differentiate into adipocytes whilst LPC into myoblasts. Notably, HPC may undergo myogenic pathway if cultured together with LPC. LPC and HPC in vitro diversities mirror differences in the capacity of regenerating damaged muscles tissues.