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

INTRODUCTION: Mesenchymal stromal cells (MSC) have been identified as important cell-based therapy candidates for cartilage, bone and intervertebral disc diseases [1]. During prolonged in vitro expansion prior to administration, MSC become senescent which impairs their therapeutic potential [2]. Here, we aimed to investigate whether extracellular nicotinamide riboside (NR) is beneficial for MSC expansion with respect to delaying the senescence, improving cellular activity and growth kinetics. METHODS: MSC were isolated from human bone marrow aspirates by gradient centrifugation of the bone marrow and subsequent expansion in α-MEM + 10% fetal bovine serum + 2.5 ng/ml bFGF2. The cytotoxicity of NR was measured at day 4 for 29 concentrations in a range from 5 nM to 4 mM. The long-term effects of NR were tested at concentrations of 10, 100 and 1,000 μM by measuring the population doubling level (PDL), relative confluency (real-time live-cell imaging with IncuCyte S3 ® ), mitochondrial activity by resazurin reduction, senescence-associated β-galactosidase assay (SA-β-gal) and NAD/NADH ratio. RESULTS & DISCUSSION: MSC treated with 3 and 4 mM NR had a significantly higher mitochondrial activity at day 4 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. 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 mitochondrial activity was significantly higher with 10 μM NR at P4, P8 and P10 (p<0.01, N=4). 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). CONCLUSIONS: Extracellular NR elevated the intracellular NAD/NADH ratio. NR is not cytotoxic within 4 days of culture at concentrations up to 4 mM. Long-term culture with 10 μM NR improved the growth kinetics markedly in two donors. ACKNOWLEDGEMENTS: Financial support was received by the Competence Center for Applied Biotechnology and Molecular Medicine (CABMM) start-up grant to BG. REFERENCES [1] Vedicherla S et al. J Orthop Res. 2017; 35(1):8-22 [2] Turinetto V et al. Int J Mol Sci. 2016;17(7):1164

FACS reveals more Pluripotent Intervertebral Disc Progenitor Cells compared to MACS and pluriSelect

INTRODUCTION: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue [1,2,3]. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. METHODS: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. RESULTS & DISCUSSION: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS tended to have more colonies than FACS and pluriSelect. Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted Tie2+ NPPCs tended to produce more proteoglycan versus Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. CONCLUSIONS: Based on the parameters tested, isolation of NPPC is possible with all three methods. However, cell yields differed widely. FACS although most invasive, appears to be the most specific sorting method for these Tie2 + cells among the tested methods as Tie2 + cells do not demonstrate osteogenic and adipogenic differentiation. As for cell yield MACS seems to reveal the most, possibly this is due to inclusion of cells expressing Tie2 less strongly