Coordinated mitochondrial quality control underlies oxidative stress resilience in MUSE stem cells

Abstract

Reactive oxygen species (ROS) regulate stem-cell function, but sustained exposure causes oxidative damage, mitochondrial dysfunction and exhaustion. Multilineage-differentiating Stress-Enduring (MUSE) cells, a rare SSEA-3⁺ MSC subset, tolerate ROS-rich environments, although the mechanisms remain unclear. We compared MUSE, Non-MUSE (SSEA-3⁻) and MSCs after H₂O₂ exposure, assessing ROS clearance, mtDNA integrity, 8-oxo-dG, antioxidant and BER responses, mitochondrial morphology/function, mitophagy and biogenesis, with focus on the PGC-1α/NRF2 axis. Three independent biological replicates were analyzed by imaging, qPCR, flow cytometry and biochemical assays. MUSE cells showed greater resistance to oxidative stress than MSCs and Non-MUSE cells. After H₂O₂, they displayed lower ROS accumulation (~15–25%), preserved mtDNA integrity, reduced 8-oxo-dG, rapid induction of antioxidant and BER-related genes, and maintenance of mitochondrial morphology, membrane potential and respiratory activity. MUSE cells also showed enhanced mitophagy, with >2-fold increased LAMP1–MitoTracker colocalization and induction of PINK1/phospho-Ub-S65, together with increased biogenesis, including ~45% higher COX-I/SDHA ratio, TFAM induction and PGC-1α activation at 48 h. MUSE cells coordinate antioxidant defense, DNA repair, mitophagy and biogenesis through a remove-and-replace program that preserves mitochondrial function during oxidative stress. Proposed model of mitochondrial quality control in MUSE stem cells for stress endurance. Mitochondrial ROS accumulation activates the PINK1/p-Ub-S65 pathway, initiating mitophagy to remove damaged organelles. Concurrently, oxidative stress promotes nuclear translocation of PGC1α and activation of the NRF1/NRF2 transcriptional axis, leading to upregulation of: (1) antioxidant genes (SOD1, CAT, GPX1); (2) DNA repair genes (MUTYH, OGG1); and (3) mitochondrial proteins. The base excision repair (BER) pathway is activated to repair mtDNA damage, while TFAM mediates mitochondrial biogenesis, ultimately generating new healthy mitochondria as evidenced by the increased COX-I/SDH-A ratio at 48 hours post-stress (generated with BioRender).