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

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.

by weight; Sigma Aldrich, St. Louis, MO) by vortex, followed by centrifugation at 8,000xg for 5 minutes. An equal volume of Ehrlich reagent (2% p-dimethylaminobenzaldehyde in glacial acetic acid) was added to the clarified supernatant, and optical density at 490 nm was measured.

Supplementary Method M3. Proteomics Analysis.
Cells were harvested when reached 80% confluency and hMSC pellets were resuspended in protein extracting buffer containing protease inhibitor. The samples were ultra-sonicated for 2 min on ice and the extracted protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA). The proteins were then digested by modified Filter Aided Sample Prep (FASP) method 2 . Briefly, 100 µg protein was vacuum-dried and resuspended in 8 M urea solution to a final volume of 200 µL, then 10 mM dithiothreitol (DTT) and 50 mM iodoacetamide (IAA) were added for reduction and alkylation respectively. Samples were transferred to a 10 kDa filter and centrifuged with 14000 g for 30 min. After washing with 200 µL of 8 M urea and 200 µL of ammonium bicarbonate, the extracts were centrifuged at 14000 g for 30 min. Then 2 µg trypsin was added for digestion at 37°C overnight. After that, peptides were collected and vacuum-dried.
An externally calibrated Thermo Q Exactive HF (high-resolution electrospray tandem mass spectrometer, MS, Thermo Scientific) was used in conjunction with Dionex UltiMate3000 RSLCnano System. The solution of 1 µg peptides in 0.1% formic acid was injected into a 50 μL loop and loaded onto the trap column (Thermo µ-Precolumn 5 mm, with nanoViper tubing 30 µm i.d. × 10 cm). The flow rate was set to 300 nL/min for separation on the analytical column (Acclaim pepmap RSLC 75 μM× 15 cm nanoviper). Mobile phase A was composed of 99.9% H2O (EMD Omni Solvent) with 0.1% formic acid and mobile phase B was composed of 99.9% acetonitrile with 0.1% formic acid. A 120 min-stepped gradient from 3% to 45% of phase B was performed. The LC eluent was directly nano-sprayed into Q Exactive HF MS. During the chromatographic separation, the Q Exactive HF was operated in a data-dependent mode and under direct control of the Thermo Excalibur 3.1.66 (Thermo Scientific). The MS data were acquired at 20 data-dependent collisional-induced-dissociation (CID) MS/MS scans per full scan (350 to 1700 m/z). The spray voltage for Thermo Scientific™ LTQ was 2.0 kV and the capillary temperature was set at 200°C. A survey full scan (m/z = 350-1700) and the five most intense ions were selected for a zoom scan to determine the charge state, after which MS/MS was triggered in Pulsed-Q Dissociation mode (PQD) with minimum signal required (1000), isolation width 2.0, normalized collision energy 27.0. All measurements were performed at room temperature. Raw files were analyzed by Maxquant 1.6 followed protein identification and relative comparison in Scaffold 4.4. Gene ontology (GO) annotation was carried out by WebGestalt while canonical pathway, diseases and functions analysis was performed by Ingenuity Pathway Analysis (IPA) (Qiagen).
Proteins were separated by 15% BIS-Tris-SDS gels and transferred onto a nitrocellulose membrane (Bio-rad, Hercules, CA). For the detection of non-phosphorylated proteins, the membranes were blocked for 30 min in 3% skim milk (w/v) in Tris-buffered saline (10mM Tris-HCl, pH 7.5, and 150mM NaCl) with 0.1% Tween 20 (v/v) (TBST), or in 3% bovine serum albumin in TBST. Membranes were incubated overnight in the presence of the primary antibody diluted (1:1000) in the corresponding blocking buffer at 4°C. Afterward, the membranes were washed four times for 10 min each with TBST and then incubated with an IR secondary (LI-COR, Lincoln, NE) at 1:10,000 for 180 min at room temperature. Blots were then washed another four times for 10 min each with TBST and processed using the LI-COR OdysseyCLx (LI-COR, Lincoln, NE). Images were analyzed using ImageJ software for band density, and the band density of proteins of interest was normalized to the band density of endogenous control β-actin.

Supplementary Figures
Supplementary Figure S1. Immuno-response of culture-expanded human mesenchymal stem cells ( . The x-axis corresponds to the -log of the P-value (Fisher's exact test) and the orange points on each pathway bar represent the ratio of the number of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes that map to that pathway. (B) DEPs between P12 and P4 hMSCs enriched in the disease and disorder category from IPAs (top 5). Figure S10. Proteomics analysis reveals the potential pathways related to NAD + /NADH redox cycle: Fatty acid β-oxidation. (A) Differential expression of proteins that are encoded with genes related to fatty acid β-oxidation pathway (P12 over P4). (B) Differential protein expression is denoted as pink (upregulated) and green (downregulated) in the Fatty acid βoxidation canonical pathway. (C) Simplified illustration of involvement of NAD + /NADH in fatty acid β-oxidation pathway.  Figure S14. Intracellular NAD + level and NAD + /NADH ratio measured in alternative donors and passages of hMSCs. (A) and (B) NAD + and NADH level and NAD + /NADH ratio of donor 2 hMSCs at P4, P7, and P9. (C) and (D) NAD + and NADH level and NAD + /NADH ratio of donor 3 hMSCs at P4, P5, P6, P9, P10, P11, and P12.

Supplementary
Supplementary Figure S15. Proposed mechanism of regulatory role of NAD + /NADH redox cycle in hMSCs cellular homeostasis during in vitro culture expansion. Culture expansion of hMSCs results in accumulation of DNA damage, which further activates PARP signal and causes the intracellular NAD + decrease. Imbalanced NAD + /NADH level causes NAD + dependent Sirtuin inactivation, which down-regulates several pathways, including mitochondrial biogenesis, antioxydant protection, immunomodulation. Dysfunction of mitochondria further accumulates NADH and consumes NAD + to maintain cellular function, leading to energy metabolism shift from glycolysis towards OXPHOS. Maintaining intracellular NAD + pool size via supplement of NAD + precursors could enhance hMSCs resistance to senescence during replicative expansion.
Supplementary Figure S16. Nuclear magnetic resonance (NMR) spectrum of human mesenchymal stem cell (hMSC) culture medium, NAD + , NADH, and NAD + precursor NAM. NMR spectra of different samples were obtained on a Bruker 500M spectrometer at 200 MHz. The spectrum was taken in deuterated chloroform at 20 o C. The peaks for fresh culture medium (green), NAD + (yellow), NADH (red), and NAM (black) were labeled in the spectra.  Tables   Supplementary Table S1. KEGG identified key pathways that are altered during culture expansion of hMSCs.