NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro

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.

In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells

“…Generally, extensive OXPHOS leads to the exhaustion of mitochondria and accumulation of ROS, which can account for the replicative senescence (66,67). BM-hMSCs have been shown to acquire culture-induced senescence with decreased proliferation rate, altered metabolism, and impaired mitochondrial functions (9,11,12). However, hASCs only exhibit certain characteristics of cellular senescence under the similar time-frame and culture conditions to BM-MSCs, as shown in this study.…”

“…Proteomics analysis was performed for hASCs at P4, P8, and P12 following the workflow established in our lab ( Supplementary Figure S15 ) ( 11 , 31 , 59 ). A total of 1,467, 1,505, and 1,310 different proteins were identified for P4, P8, and P12 groups, respectively.…”

“…Though as the most widely acknowledged source, human bone marrow contains low amount of hMSCs (~0.001-0.01%) (8), and it is impractical to directly apply hMSCs from bone marrow without in vitro culture expansion. Moreover, the extensive passaging of BM-hMSCs results in significant alterations on cellular behavior termed as replicative senescence, which compromises hMSCs' therapeutic potentials (5,(9)(10)(11)(12). For example, 1-year survival rates in graft vs host disease were found to drop from 75% (in patients who received BM-hMSCs from passages 1-2) to 21% (in patients who received passages 3-4 cells) (13).…”

“…Previous studies have revealed that the increase of heterogeneity, metabolic reconfiguration, and loss of cellular homeostasis could be potential reasons for BM-hMSC senescence during culture expansion (6,11,(28)(29)(30)(31). BM-hMSCs exhibit metabolic plasticity that can adapt themselves to artificial environments in order to maintain stem cell functions, specifically by reconfiguring energy production from glycolysis towards oxidative phosphorylation (OXPHOS), which contributes to the loss of mitochondrial fitness and replicative senescence (5,11,29,30). Moreover, metabolism and stem cell fate are inherently intertwined to regulate immunomodulation in BM-hMSCs (5,30,32).…”

“…Recently, the NAD+/NADH redox cycle and corresponding enzyme Sirtuins have been proposed to connect aging/senescence to cellular homeostasis (35)(36)(37)(38)(39). Our previous study revealed that the culture-induced alterations of NAD + /NADH redox balance contribute to metabolic alteration, mitochondrial dysfunction, loss of autophagy, and impaired stem cell functions in BM-hMSCs with replicative senescence (11). Other studies have documented similar findings for the role of NAD+ biosynthesis and metabolisms in age-related functional decline of stem cells (40,41).…”