MITOPHAGY AND LPS-INDUCED TOLERANCE IN MESENCHYMAL STEM CELLS (ASC52TELO)

Mesenchymal stem cells are considered a promising tool for cell therapy because of their regenerative and immunomodulatory properties. However, results from clinical trials remain inconclusive: in some studies participants showed improvements, while in others there were no statistically significant differences from placebo; moreover, some participants experienced adverse effects. One strategy to improve the safety and efficacy of MSC-based therapies is targeted preconditioning to modify their secretome. Another promising approach is modulation of mitophagy – a key mitochondrial quality-control mechanism that determines mesenchymal stem cells stress resilience and immunoregulatory capacity. Active mitophagy reduces senescence and preserves the immunomodulatory functions of mesenchymal stem cells, through which they can promote resolution of inflammation. When mitophagy is impaired, mitochondrial components and reactive oxygen species can accumulate, which may exacerbate local inflammation. As a result, the therapeutic potential of mesenchymal stem cells may be diminished. Thus, the aim of this study was to compare mitophagy and inflammatory tolerance of mesenchymal stem cells under different stimulations. In the present study we compared both strategies by examining the mitophagic response of mesenchymal stem cells to mitochondrial stress and the development of LPS-induced tolerance. We used the hTERT-immortalized adipose-derived line ASC52telo in two complementary experimental schemes. To probe mitophagy, cells were exposed to FCCP to depolarize mitochondria and then imaged by confocal microscopy after dual staining of mitochondria and lysosomes. To assess tolerance, cells were stimulated twice with LPS and cytokine secretion was measured. We found that FCCP induced pronounced mitochondrial fragmentation and activation of mitophagy. In a separate set of experiments, repeated LPS stimulation led to a marked reduction in TNF and CCL2 secretion. Thus, activation of mitophagy in response to acute mitochondrial stress and the establishment of endotoxin (LPS) tolerance in mesenchymal stem cells operate as complementary adaptive mechanisms. These processes may be exploited as targets to increase the predictability and clinical efficacy of MSC-based therapies.

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