Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities

Abstract

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy.

Keywords: human serum; mesenchymal stem cells; myogenic differentiation; platelet-rich plasma; skeletal muscle.

Figure 1

Human MSC (hMSC) characterization by flow cytometric analysis. Scheme representing the time points analyzed in the flow cytometric analysis, from skeletal muscle-derived hMSCs isolation up to late doubling time: total mononucleated cells freshly isolated from the tissue (t0), new formed colonies after 15 days (t1), expansion passage 3 (t2), and expansion passage 9 (t3). Flow cytometry analysis for CD56, CD90, and CD45 in relation to different culture media: α-MEM and Cyto-Grow, showing the histograms of the signal for each antibody used and the dot plot displaying the double positivity for CD90 and CD56.

Figure 2

Culture conditions influence cell behavior. Immunofluorescences against MyHC (red) and Ki67 (green) reveal the remarkable differences in terms of differentiation and proliferation employing Cyto-Grow commercial medium compared with α-MEM (nuclei labeled in blue by DAPI). The respective quantifications are reported as fusion index and rate of proliferating nuclei (Ki67 positive). Statistical analysis: one-way ANOVA and Tukey’s test. ^**p < 0.01, ^***p < 0.001 (n = 3). Scale bars: MyHC = 200 μm; Ki67 = 100 μm.

Figure 3

Effect of human serum on hMSC proliferation and differentiation. hMSC proliferation and differentiation analyzed upon different medium supplementations exposure. α-MEM medium added with: Ctrl (20% FBS), Low (5% Human Serum), Medium (10% Human Serum), and High (20% Human Serum). Anti MyHC (red) immunolabeling on hMSCs cultured in different conditions as indicated; nuclei were marked with DAPI (blue). Cell growth is indicated as proliferation curve evaluated up to 9 days. Myogenic evaluation relative to MyHC signal is represented as fusion index at day 9 of culturing in the investigated conditions. Statistical analysis: one-way ANOVA and Tukey’s test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (n = 3). C–F: scale bar 250 μm.

Figure 4

platelet-rich plasma (PrP) affects hMSC proliferation and myogenesis. Immunofluorescences against MyHC (red) and Ki67 (green) revealing differences in proliferation and differentiation in relation to different PrP concentration. α-MEM supplemented with: Ctrl (20% FBS), Low (5 × 10⁵ platelets/ml), Medium (1 × 10⁶ platelets/ml), and High (1.5 × 10⁶ platelets/ml); nuclei were counterstained in blue by DAPI. The relative quantification is reported as fusion index for muscle differentiation, proliferating curve, and rate of Ki67 positive nuclei for cell doubling. Statistical analysis: one-way ANOVA and Tukey’s test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (n = 3). Scale bars: MyHC = 200 μm; Ki67 = 100 μm.