Mitophagy and LPS-induced Tolerance in Mesenchymal Stem Cells (ASC52telo)

Mesenchymal stem cells (MSC) are considered a promising tool for cell therapy due to their regenerative and immunomodulatory properties. However, results from clinical trials remain inconclusive: clinical improvements have been shown in some studies, while other trials did not reveal statistically significant differences from placebo-treated patients. Moreover, some participants experienced adverse effects. Targeted preconditioning of the cells aimed for modifying of their secretome seems to be a strategy to improve the safety and efficacy of MSC-based therapies. Another promising approach is modulation of mitophagy, a key mitochondrial quality-control mechanism that determines stress resilience and immunoregulatory capacity of MSCs. Active mitophagy reduces senescence and preserves immunomodulatory functions of MSCs, thus promoting resolution of inflammation. When mitophagy is impaired, the mitochondrial components and reactive oxygen species may accumulate, thus exacerbating 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 MSCs under different stimulation regimens. 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 schedules. To probe mitophagy process, the cells were exposed to FCCP to depolarize mitochondria and then subjected to confocal microscopy after dual staining of mitochondria and lysosomes. To assess their tolerance, the 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 establishment of endotoxin (LPS) tolerance in mesenchymal stem cells may be regarded as complementary adaptive mechanisms. These processes may be exploited as targets to increase the predictability and clinical efficacy of MSC-based therapies.

1. Chen X., Chen M., Yang Y., Xu C., Lu H., Xu Y., Li X., Wei Y., Zhu Z., Ding Y., Yu W. Lipopolysaccharidepreconditioned mesenchymal stem cell transplantation attenuates critical persistent inflammation immune suppression and catabolism syndrome in mice. Shock, 2022, Vol. 58, no. 5, pp. 417-425.

2. Feng X., Yin W., Wang J., Feng L., Kang Y.J., Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells. Exp. Biol. Med. (Maywood), 2021, Vol. 246, no. 1, pp. 97-105.

3. Kahrizi M.S., Mousavi E., Khosravi A., Rahnama S., Salehi A., Nasrabadi N., Ebrahimzadeh F., Jamali S. Recent advances in pre-conditioned mesenchymal stem/stromal cell (MSCs) therapy in organ failure; a comprehensive review of preclinical studies. Stem Cell Res. Ther., 2023, Vol. 14, no. 1, 155. doi: 10.1186/s13287-023-03374-9.

4. Ko J.H., Lee H.J., Jeong H.J., Kim M.K., Wee W.R., Yoon S.-O., Choi H., Prockop D.J., Oh J.Y. Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye. Proc. Natl. Acad. Sci. U.S.A., 2016, Vol. 113, no. 1, pp. 158-163.

5. Kouchakian M.R., Baghban N., Moniri S.F., Baghban M., Bakhshalizadeh S., Najafzadeh V., Safaei Z., Izanlou S., Khoradmehr A., Nabipour I., Shirazi R., Tamadon A. The Clinical trials of mesenchymal stromal cells therapy. Stem. Cells Int., 2021, 2001, 1634782. doi: 10.1155/2021/1634782.

6. Liu F., Yuan Y., Bai L., Yuan L., Li L., Liu J., Chen Y., Lu Y., Cheng J., Zhang J. LRRc17 Controls BMSC senescence via mitophagy and inhibits the therapeutic effect of BMSCs on ovariectomy-induced bone loss. Redox Biol., 2021, Vol. 43, 101963. doi: 10.1016/j.redox.2021.101963.

7. Mukkala A.N., Jerkic M., Khan Z., Szaszi K., Kapus A., Rotstein O. Therapeutic effects of mesenchymal stromal cells require mitochondrial transfer and quality control. Int. J. Mol. Sci. 2023, Vol. 24, no. 21, 15788. doi: 10.3390/ijms242115788.

8. Phinney D.G., di Giuseppe M., Njah J., Sala E., Shiva S., St Croix C.M., Stolz D.B., Watkins S.C., Di Y.P., Leikauf G.D., Kolls J., Riches D.W.H., Deiuliis G., Kaminski N., Boregowda S.V., McKenna D.H., Ortiz L.A. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat. Commun., 2015, Vol. 6, 8472. doi: 10.1038/ncomms9472.

9. Rayat Pisheh H., Sani M. Mesenchymal stem cells derived exosomes: a new era in cardiac regeneration. Stem. Cell Res. Ther., 2025, Vol. 16, no. 1, 16. doi: 10.1186/s13287-024-04123-2.

10. Tan Y.L., Eng S.P., Hafez P., Abdul Karim N., Law J.X., Ng M.H. Mesenchymal stromal cell mitochondrial transfer as a cell rescue strategy in regenerative medicine: a review of evidence in preclinical models. Stem. Cells Transl. Med., 2022, Vol. 11, no. 8 pp. 814-827.

11. Zhang F., Peng W., Zhang J., Dong W., Wu J., Wang T., Xie Z. P53 and parkin co-regulate mitophagy in bone marrow mesenchymal stem cells to promote the repair of early steroid-induced osteonecrosis of the femoral head. Cell Death Dis., 2020, Vol. 11, no. 1, 42. doi: 10.1038/s41419-020-2238-1.