Isolation and Characterization of hSkMCs
The cell populations derived from human skeletal muscle biopsies (Fig. 1a), obtained by surgical resection, were amplified in Matrigel® coated plates in order to increase cell adherence and maintain cell phenotype. In fact, the isolated cells, when plated for the first time in a dish (passage 0), attach showing a rounded shape that persists for 2–3 days, with a slow proliferation rate (Fig. 1b). After that period, cells become flatter and show an elongated morphology with 2–4 cytoplasmic extensions (Fig. 1c).
Expanded cells were subsequently characterized by flow cytometry in order to verify their phenotype, analyzing the cluster of differentiation (CD) marker surface proteins (CD44, CD90, CD105, CD56, CD34, CD45) and one of the most reliable markers of the satellite cells (PAX-7).
The phenotype analysis revealed that isolated hSkMCs expressed the surface markers CD44, CD90, CD105, commonly used to identify mesenchymal stem cells, with a very high percentage of positiveness. In contrast, the hematopoietic lineage marker CD45 was negative. Regarding PAX-7, analysis showed the presence of the nuclear transcription factor in 99.12% of total cells (Fig. 2a). On the contrary, CD34 and CD56, commonly used to identify satellite cells derived from mouse skeletal muscle, turned out to be non-specific for satellite cells derived from human skeletal muscle tissues; in fact, their presence on the cell surface is close to 0% and 9%, respectively (Fig. 2).
Immunofluorescent staining of PAX-7, observed in LSCM, allowed the expression of the nuclear marker to be assessed. As expected from cytometry, it is clearly shown that the PAX-7 protein is present in the nuclei in all the expanded hSkMCs, confirming the isolation of human satellite cells (Fig. 2b).
Moreover, the gene expression of PAX-7 was analyzed over time, to verify its presence at cellular passages used for the experiments. As shown by Real-Time qPCR, we confirmed PAX-7 presence, but, as the passages increase, the gene expression of PAX-7 decreases, reducing the differentiating potential of cells (Fig. 2c). For that reason, we have limited the use of hSkMCs to passage 4.
Multipotentiality of hSkMCs
The multipotent evaluation of hSkMCs was assessed by the induction toward the adipogenic, osteogenic and myogenic phenotypes, using appropriate media described in “Materials and Methods” section.
Adipogenic differentiation was performed culturing the hSkMCs in AM for 7 days, and confirmed, using Oil Red O staining, by the multiple intracellular lipid-filled droplets accumulation and microscopic observations in brightfield. In contrast, control cells grown in GM for the same time did not show any formation of lipid droplets (Fig. 3a, b).
Osteogenic induction of hSkMCs was assessed with OM up to 15 days and observed monitoring the production of the mineralized calcium deposits, thanks to the fluorophore calcein added to the medium. Epifluorescence microscopic observations have shown calcein uptake in the calcium nodules after 15 days of osteogenic induction; in contrast, the control cells grown in GM for the same time did not show any deposition of calcium deposits (Fig. 3c, d).
Afterwards, the hSkMCs were differentiated toward the myogenic phenotype using DM for 9 days. During this period, cells started to approach one another, fusing with one another. Observations in phase contrast microscopy have revealed the presence of multinucleated elongated cells (containing from 3 to more than 8 nuclei) referable to myotubes (Fig. 3e, f).
In order to confirm the myogenic induction of hSkMCs, we have performed, using Real-Time qPCR, the analysis of the MRFs (MyoD-1, Myf-5, MRF-4, Myogenin), the main myogenic differentiation genes, Desmin and MHC, after cultivating cells in MM up to 9 days. The results have shown a significant increase in the expressions of these genes during myogenesis, except for Myf-5, with respect to the control in GM (Time 0), indicating that differentiation occurred (Fig. 6). Moreover, differentiation has been supported by the presence of significant increases in the gene expression of Desmin and MHC, which represent essential proteins for proper muscular structure and function (Fig. 4).
In particular, MHC was assessed at different time points (T0, 3, 6, 9 days) in order to follow the effective myogenesis over time (Fig. 4f). Since MHC is one of the most important proteins in skeletal muscle, and it is essential for contraction and muscle movement, it was analyzed by immunofluorescence staining. The microscopic observation of hSkMCs after 9 days in MM has shown the presence of MHC, demonstrating the suitability of our in vitro myogenesis model (Fig. 5).
Gene Expression Analysis of Hormone Receptors in hSkMCs During In Vitro Myogenesis
The hSkMCs were differentiated using the MM and, after 9 days of induction, we analyzed the gene expression of the hormone receptors, in order to characterize the maturation and endocrine properties of the cellular model during myogenesis. In particular, we analyzed the following genes by Real-Time qPCR: VDR, TRα, TRβ, GCR, PTH-1R, IFG-1R, LRP-5, LRP-6 and Irisin.
The results of the hormone receptors analyzed have shown significant increases in gene expressions during cell differentiation with respect to the control group in growth medium, demonstrating the formation of the skeletal muscle as an endocrine apparatus during myogenesis (Fig. 6). Subsequently, the expression of Irisin, a hormone secreted by skeletal muscle, specifically suggests the development and maturation of new myofibers, since it represents a myokine secreted by mature endocrine tissue.