Fig 1.
The schematic presentation of experiments and methods used in this study.
The interactions were analysed on myoblasts and bone marrow mesenchymal stem cells derived from the same donor.
Fig 2.
MDC and MSC 24 h in a direct co-culture.
Images in rows represent the same fields of view. A. Bright field microscopy. A' Fluorescent microscopy where MDC are red (PKH26) and MSC are green (PKH67), nuclei stained with DAPI. It can be observed that cells tend to contact directly. B,C,D—Fluorescent microscopy, where MSC are green (GFP), desmin is stained with red fluorochrom (AlexaFluor® 594) and nuclei are stained with DAPI (blue). Different types of direct contact can be observed—extending filopodia (B), parallel arrangement of different cell types (C) or surrounding one cell by another (D). Scale bars: 20μm.
Fig 3.
Mutual MDC-MSC interaction on myogenic differentiation.
(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).
Fig 4.
The effect of the in vitro oxidative stress on MDC, MSC and MDC-MSC co-culture.
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.
Fig 5.
MDC and MSC migration capacity.
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.
Fig 6.
MDC and MSC proliferation rate.
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.