Abstract
Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.
Highlights
Human mesenchymal stem cells promote endogenous tissue regeneration and have become a promising candidate for cell therapy
Increased DNA damage was observed using the Comet assay during culture expansion, indicated by increased tail/body length in Human mesenchymal stem cells (hMSCs) at P12 compared to cells at P5 (Fig. 1d). hMSCs at late passage exhibited increased population doubling (PD) time (~6 days of P12 vs. 2.8 days of P5) (Fig. 1e), reduced capacity for self-renewal (~10 colony-forming unitfibroblasts (CFU-F) colonies at P12 vs. 96 colonies at P5; Fig. 1f), downregulation of stem cell genes Oct[4] and Sox[2] compared to early passage cells (P5) (Fig. 1g)
Loss of autophagy indicated the breakdown of cellular homeostasis of hMSCs during in vitro culture expansion
Summary
Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. In vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Recent studies have demonstrated that hMSCs exhibit metabolic plasticity both in vivo and in vitro, which contributes to cellular properties and aging. Upon isolation of hMSCs from in vivo niche for expansion, in vitro nutrient-enriched environment supports rapid cell proliferation, which requires energy and anabolic macromolecules for daughter cell replication. In this process, catabolic and anabolic pathways are interconnected and together play essential roles in providing energetic sources as well as metabolites to maintain cellular homeostasis[17]
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