Complex I is essential for embryonic stem cell proliferation through NADH-mediated redox signaling

Starkova, T. Y.; Kuzmin, A. A.; Nazarov, I. B.; Skvortsova, E. V.; Ponomartse, S. V.; Drianitchkina, V.; Aksenov, N. D.; Tomilin, A. N.; Sinenko, S. A.

doi:10.64898/2026.10.26.761997

bioRxiv · 10.64898/2026.10.26.761997

Complex I is essential for embryonic stem cell proliferation through NADH-mediated redox signaling

Starkova, T. Y.·Kuzmin, A. A.·Nazarov, I. B.·Skvortsova, E. V.·Ponomartse, S. V.·Drianitchkina, V.·Aksenov, N. D.·Tomilin, A. N.·Sinenko, S. A.

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Abstract

Background: Pluripotent embryonic stem cells (ESCs) possess unlimited self-renewal potential and the capacity to generate all cell types of an organism. Mitochondrial metabolism participates in the regulation of ESC self-renewal and differentiation, yet the specific roles of respiratory complex components in these processes remain largely unknown. Methods: Ndufs1-knockout mouse ESCs were generated using the CRISPR/Cas9 method. Proliferation and cell cycle dynamics were assessed via flow cytometry with propidium iodide and Hoechst 33342/Pyronin Y staining. Mitochondrial physiology, including membrane potential, ROS levels, and mass was evaluated using JC-1, MitoSOX Red, DHE, and MitoTracker Red measurements. Cellular ATP and NAD/NADH levels were determined by luminescent and colorimetric assays. Pluripotency was confirmed by immunocytochemistry, Western blotting, teratoma formation, and directed differentiation into EpiSCs and cardiomyocytes. Downstream molecular and transcriptomic shifts were analyzed using RNA-seq and Western blotting. Results: We demonstrate that while Complex I function is dispensable for the maintenance of pluripotent identity, its loss leads to a profound reduction of ESC proliferation rates. Deficiency of the core CI subunit Ndufs1 induces mitochondrial reductive stress, characterized by a decreased cellular NAD/NADH ratio and elevated ROS levels. We find that cytosolic, but not mitochondrial, NADH accumulation acts as the primary signal limiting ESC proliferation, independent of energy depletion or excessive ROS. This process is associated with the activation of the integrated stress response and suppression of the NRF2/PGC-1/SOD2 antioxidant axis. Conclusions: Our findings identify NADH-driven reductive stress as a key checkpoint controlling ESC proliferation and reveal metabolic flexibility that preserves ESC developmental potential despite severe mitochondrial dysfunction. We propose that Complex I functions as a metabolic rheostat, tuning ESC mitotic activity through mitochondrial-to-nuclear redox signaling.

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Starkova, T. Y., Kuzmin, A. A., Nazarov, I. B., Skvortsova, E. V., Ponomartse, S. V., Drianitchkina, V., Aksenov, N. D., Tomilin, A. N., Sinenko, S. A.. 2026-10-28. Complex I is essential for embryonic stem cell proliferation through NADH-mediated redox signaling. https://doi.org/10.64898/2026.10.26.761997

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