Stress Fiber Formation, Mitochondrial Morphology and Membrane Properties of Human Mesenchymal Stem Cells Cultured in Plastic Adherence or in Spherical Aggregates

Relevance: Mesenchymal stem cells (MSCs), when cultivated in adherence to plastic surfaces, develop large bundles of actin filaments with mechanosensitive properties. These stress fibers induce tension forces via focal adhesions bound to the extracellular matrix, thereby influencing elastic properties of the cell membrane relevant for MSC function. Methods: Actin was quantified in plastic adherent MSCs or in aggregate cultures using flow cytometry andWestern blotting. Stress fiber formation, connection to focal adhesions and mitochondrial dynamics were investigated by laser scanning microscopy. Surface morphology and membrane properties of MSCs were performed by atomic force microscopy and migration was measured in a wound closure assay. Results: Total actin protein remained largely unchanged during cultivation of MSCs from passage (P)1 to P3, but distribution of bold stress fibers changed. Ventral stress fibers, anchored at each end to focal adhesions, increased in fiber length during cultivation, while dorsal stress fibers anchored at one end to focal adhesions, increased in number. Mitochondrial branching during cultivation from P1 to P3 gave further evidence for deficits in fusion/fission kinetics. Deflection scanning by AFM confirmed these observations by showing intense bundles of stress fibers in P1 andP3 MSCs, while MSC emerging from aggregates showed discrete stress fiber morphology. Force vs indentation profiles of native MSCs with exclusion of nuclear regions showed the highest Young’s moduli of 8.5 ± 6.4 kPa in P1, followed by 6.5 ± 6.1 kPa in P3 MSCs and 3.6 ± 3.2 kPa for MSCs of spherical aggregates. Conclusion: When MSCs are expanded for potential therapeutic use, stress fiber morphology with increased length of ventral stress fibers and higher number of dorsal stress fibers can increase the inner force of MSCs resulting in changes of their physical properties. Mitochondrial branching in passaged MSCs gave further evidence for a decline in cell function.