1. Physioxia rewires mitochondrial complex composition to protect stem cell viability.
- Author
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Raabe J, Wittig I, Laurette P, Stathopoulou K, Brand T, Schulze T, Klampe B, Orthey E, Cabrera-Orefice A, Meisterknecht J, Thiemann E, Laufer SD, Shibamiya A, Reinsch M, Fuchs S, Kaiser J, Yang J, Zehr S, Wrona KM, Lorenz K, Lukowski R, Hansen A, Gilsbach R, Brandes RP, Ulmer BM, Eschenhagen T, and Cuello F
- Subjects
- Humans, Oxygen metabolism, Fibroblasts metabolism, Fibroblasts cytology, Oxidative Phosphorylation, Proteomics methods, Cellular Senescence, Cells, Cultured, Cellular Reprogramming genetics, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology, Mitochondria metabolism, Mitochondria genetics, Cell Survival
- Abstract
Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O
2 ) or physioxic (5 % O2 ) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TE is a shareholder and a member of the scientific advisory board of DiNAQOR, which is not relating to this manuscript. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2024
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