The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model

<div><p>Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC <i>in vitro</i> in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H₂O₂ on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders.</p></div

The schematic presentation of experiments and methods used in this study.

<p>The interactions were analysed on myoblasts and bone marrow mesenchymal stem cells derived from the same donor.</p

The effect of the <i>in vitro</i> oxidative stress on MDC, MSC and MDC-MSC co-culture.

<p>Metabolic activity was measured by MTT assay. Data presented as median, interquartile range and min, max. Upper graphs present absorbance values measured in MDC, MSC or MDC-MSC (A,A',A'', respectively) treated with increasing concentrations of H₂O₂ analysed by Wilcoxon signed-rank test in comparison to the control group. *, p<0.05; Lower graphs present relative values, which is the ratio of sample absorbance to the control absorbance from the same donor (internal control) obtained after treatment with 500, 600 or 700 μM of H₂O₂ (B,B',B'', respectively) analysed with ANOVA Kruskal-Wallis test. Values that differed significantly (p<0.05) are indicated with different lowercase letters. Experiments were done on cells from 6 different donors (same donors for MDC and MSC) and performed in triplicates.</p

MDC and MSC proliferation rate.

<p>Proliferation of MDC (A) and MSC (B) derived from the same donors (n = 6) was measured with BrdU assay. Data presented as means, SEM, SD. The graphs present proliferation of cells cultured in standard GM (CTRL), GM supplemented with bFGF and cells cultured in 100% conditioned medium (cond) derived either from MDC or MSC. Data analysed with Wilcoxon test for related groups. Values that differed significantly (p<0.05) were indicated as following: * in comparison to CTRL group; # in comparison to bFGF group; ^ in comparison to the second conditioned group. Tests were performed in triplicates.</p

MDC and MSC migration capacity.

<p>The migration of MDC and MSC analysed by inserts assay (Ø 8μm). Data presented as mean and SEM. A) The migration rate of unstimulated MDC and MSC (no cells in the lower compartment marked as xxx) analysed with U-Mann Whitney test, ***, p<0.01. B) The graph presents the migration stimulated by cells seeded in the lower compartment. The rate of MDC or MSC migration in relation to the stimulating cell number was calculated. Descriptions on X axis reflect the location of cell types—i.e. MDC-MSC means the MDC cells migrated under stimulation of MSC cells. Data analysed with ANOVA Kruskal-Wallis test. Values that differed significantly (p<0.05) are indicated with different uppercase letters.</p

Mutual MDC-MSC interaction on myogenic differentiation.

<p>(A, B) Fluorescent microscopy where MDC (unlabeled) and MSC (GFP+, labeling efficiency less than 50%) are in a direct co-culture under myogenic differentiation medium (mDM) for 48 h. Images in rows represent the same fields of view. GFP+ myotubes can be observed what proves the ability of MSC to contribute in the myotube formation. Staining against desmin (B) confirms myogenic character of formed structure. scale bars: (A) 50μm, (B) 20 μm. C. Representative Western blot for desmin expression in MDC, MSC and MSC cultured in MDC-conditioned (COND) medium (50%) (all from one donor). D. Graph presents fusion index of MDC after 3 days of culture in mDM under the influence of soluble factors derived from either MDC or MSC (1 μm inserts).</p