Investigation of the effect of nicotinamide riboside on primary human bone-marrow derived mesenchymal stromal cells in vitro

Abstract

Introduction: Mesenchymal stromal cells (MSC) have been identified as the most prominent cell-based therapy candidates for cartilage, bone and intervertebral disc diseases due to their intrinsic ability to differentiate into chondrocytes and osteocytes.1–4 One of the major problems involved in MSC-based cell therapy is the necessity to expand the cells in vitro to obtain sufficient cells for administration.5 During prolonged expansion, MSC become senescent which impairs their therapeutic potential.5,6 Here, we aimed to investigate whether extracellular nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD), is beneficial for MSC expansion in respective of growth kinetics, mitochondrial activity and senescence. Methods: MSC were isolated from human bone marrow aspirates by gradient centrifugation and subsequent expansion in α-MEM + 10% FBS + 2.5 ng/ml bFGF-2. Immunophenotyping was performed by flow cytometry. The cytotoxicity of NR was measured at day 4 for 29 concentrations in a range from 0.005 μM to 4’000 μM. The long-term effect of NR was tested at concentrations of 10, 100 and 1’000 μM by measuring the population doubling level (PDL), relative confluency (IncuCyte S3®), mitochondrial activity by resazurin reduction, senescence-associated β-galactosidase assay (SA-β-gal) and NAD/NADH ratio. Results: The isolated cells displayed a typical MSC immunophenotype (CD73+, CD90+, CD105+, CD34-, CD45-, CD14-). NR exhibited no acute cytotoxicity at any tested concentration. MSC treated with 3000 μM and 4’000 μM NR had a significantly higher mitochondrial activity than the negative control (p=0.0027 and p<0.0001 respectively, N=3). However, in the weeks 3 to 8, cells treated with ≥100 μM NR died reaching a maximum PDL of 13.43 (N=4). In two donors, the experimental group with 10 μM NR reached a 2-fold higher PDL than the negative control, on the other hand, two donors exhibited no difference in PDL between the two groups. The relative confluency at passage (P) 2 after 6 days in culture was higher with 10 μM NR compared to the negative control (35.00 ± 9.29% and 26.19 ± 5.41% respectively, mean ± SD, N=2). The absolute mitochondrial activity was significantly higher with 10 μM NR at P4, P8 and P10 (p<0.01, N=4), albeit, when normalized to the cell count, no difference in mitochondrial activity was observed suggesting that the higher absolute mitochondrial activity was caused by the higher cell count, rather than higher cellular activity. At all passages, the percentage of SA-β-gal positive cells was under 5%, except in the negative control medium at P11 (18.17% ± 18.18%, mean ± SD, N=1). All experimental groups treated with NR had a higher NAD/NADH ratio which exhibited a dose-dependent trend (N=1). Conclusion: Extracellular NR elevated the intracellular NAD/NADH ratio presumably by serving as a precursor of NAD. NR is not cytotoxic within 4 days of culture at concentrations up to 4’000 μM, though in long-term culture already 100 μM proved to be cytotoxic. Long-term culture with 10 μM NR improved the growth kinetics and mitochondrial activity markedly in two donors. The mechanism of this has yet to be determined by measuring the telomere lengths and by analyzing the gene expression of sirtuins (which are triggered by the NAD/NADH ratio) and senescence-associated genes. 1. Squillaro, T., Peluso, G. & Galderisi, U. Clinical Trials with Mesenchymal Stem Cells: An Update. Cell Transplant. 25, 1–53 (2015). 2. Trounson, A. & McDonald, C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell Stem Cell 17, 11–22 (2015). 3. Sakai, D. & Schol, J. Cell therapy for intervertebral disc repair: Clinical perspective. Journal of Orthopaedic Translation (2017). doi:10.1016/j.jot.2017.02.002 4. Vedicherla, S. & Buckley, C. T. Cell-based therapies for intervertebral disc and cartilage regeneration— Current concepts, parallels, and perspectives. Journal of Orthopaedic Research (2017). doi:10.1002/jor.23268 5. Turinetto, V., Vitale, E. & Giachino, C. Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy. Int. J. Mol. Sci. 17, 1164 (2016). 6. Sepulveda, J. C. et al. Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal endotoxemia model. Stem Cells 32, 1865–77 (2014).