Int J Stem Cells.  2018 Jun;11(1):13-25. 10.15283/ijsc18033.

Enhancement of Replication and Differentiation Potential of Human Bone Marrow Stem Cells by Nicotinamide Treatment

• Ok JS
• Song SB
• Hwang ES

• KMID: 2413497
• DOI: http://doi.org/10.15283/ijsc18033

Abstract

BACKGROUND AND OBJECTIVES
Therapies using mesenchymal stem cells (MSCs) generally require substantial expansion of cell populations. However, the replicative life span of MSCs is limited and their multipotency declines over continued passages, imposing a limitation on their application especially in aged individuals. In an effort to increase MSC life span, we tested the effects of nicotinamide (NAM), a precursor of NAD+ that has been shown to reduce reactive oxygen species generation and delay the onset of replicative senescence in fibroblasts.
METHODS
Bone marrow stem cells (BMSCs) from healthy donors were cultivated in the presence of 5 mM NAM until the end of their life span. The levels of proliferation and differentiation to osteogenic, adipogenic, and chondrogenic lineages of BMSCs were compared between populations incubated in the absence or presence of NAM.
RESULTS
The replicative life span was substantially increased with a significant delay in the onset of senescence, and differentiation to all tested lineages was increased. Furthermore, differentiation was sustained and the adipogenic switch from osteogenesis to adipogenesis was attenuated in late-passage BMSCs.
CONCLUSIONS
NAM could be considered as an important biological agent to expand and sustain the multipotency of BMSCs and thus broaden the application of stem cells in cell therapies.

Keyword

Bone marrow stem cell; Replicative life span; Differentiation; Nicotinamide; ROS; Mitochondria

MeSH Terms

Adipogenesis
Aging
Bone Marrow*
Cell Aging
Fibroblasts
Humans*
Mesenchymal Stromal Cells
Mitochondria
Niacinamide*
Osteogenesis
Reactive Oxygen Species
Stem Cells*
Tissue Donors
Niacinamide
Reactive Oxygen Species

Figure

• 

  Fig. 1 Effect of NAM treatment on replicative life span and senescence onset of BMSCs. (A) BMSC lines from individuals of different ages (31, 35, and 51 years) were cultivated in MSCGM supplemented with (○) or without (●) 5 mM NAM. Cells were passaged in a 1:4 ratio until they stopped dividing. (B) The number of population doublings between day 10 and day 40 of the cultivation in (A) was counted, and normalized to doublings in the absence of NAM. Relative numbers are plotted. (C) Cryo-preserved cells (from the 31-year-old donor) at different passage numbers were thawed, sub-cultured in 6-well plates for 2 days, and stained for SA β-Gal.

• 

  Fig. 2 Effect of NAM treatment on ROS levels, SIRT1 activity, and mitochondria content. (A) BMSCs (from the 31-year-old donor) at PD 8 were incubated without (−) and with (+) 5 mM NAM for 10 days and then stained with MitoSox, DHR123, and DHE to determine the levels of mitochondrial superoxide, hydroxyl radical, and cytosolic superoxide. Values are plotted relative to the cells in day 0 plates. The significance of the difference between (−) and (+) NAM was determined by ANOVA test (Dunnett’s test) (*p<0.1; **p<0.01) (B) BMSCs were cultured in the presence of NAM and the NAD+/NADH ratio was determined at the indicated time points. The significance of the difference compared to the day 0 sample was determined by ANOVA test (Dunnett’s test) (*p<0.1; **p<0.01). (C) BMSCs incubated with 5 mM NAM or 0.16 nM SRT1720 for 0, 1, or 2 days were collected and lysed. The extracts were subjected to western blotting for acetylated or total p53 or Erk protein (upper panel), or LC3 proteins or β-actin (lower panel). (D) The cells were stained with nonyl acridine orange (NAO) to determine mitochondria content.

• 

  Fig. 3 Effect of NAM treatment on osteogenesis. (A) BMSCs cultured in the absence or presence of NAM for PD 4, 6, 8, or 10 (or PD13 in NAM (+) culture) were plated in 24-well plates and induced to osteogenesis in OIM containing NAM for 12 days. Cells were stained with Alizarin Red S and photographed under a microscope. (B, C) BMSCs cultured in MSCGM in the absence or presence of 5 mM NAM for PD 6 or 8 were plated in 24-well plates and induced to osteogenesis in OIM with or without 5 mM NAM for 12 days. Cells in each wells were stained with Alizarin Red S and photographed (B) or extracted with acid and neutralized, and applied for quantitation through observance at 405 nm in spectrometry. The significance of the difference between (−) and (+) NAM in MSGCM was determined by ANOVA test (Dunnett’s test) (*p<0.1; **p<0.01).

• 

  Fig. 4 Effects of NAM treatment on adipogenesis and chondrogenesis. (A) BMSCs cultured in the absence or presence of 5 mM NAM for PD 8 were plated in 24-well plates and induced to adipogenesis for 9 days in AIM without or with NAM. Cells were stained with 0.2% Oil red O and photographed under a microscope. (B) For quantification of lipid accumulation, BMSCs were treated as in (A) (except for incubation in 6-well plates), induced to adipogenesis, and stained with Oil Red O. Oil droplets were dissolved in isopropanol and absorbance at 518 nm was determined using a spectrophotometer. Two biological repeats from two independent experiment were quantitated and mean values were plotted. The significance of the difference between (−) and (+) NAM in MSGCM was determined by ANOVA test (Dunnett’s test) (*p<0.1; **p<0.01). (C) BMSCs cultured in MSCGM in the absence or presence of 5 mM NAM for PD 8 were plated in 24-well plates and induced to chondrogenesis for 15 days in CIM without NAM or containing 5 mM NAM. Cells were stained with Alician Blue solution and the spheroids were visualized under light microscopy and photographed.

• 

  Fig. 5 Effect of NAM treatment on mRNA expression during osteogenesis and chondrogenesis. BMSCs at PD 6 or PD 10 (cultured in the absence of presence of NAM) were induced to either osteogenesis (A, B) or chondrogenesis (C, D) in the absence or presence of NAM. Cells were collected at day 7 (for osteogensis) or day 3 (for chondrogenesis) of differentiation and the mRNA levels of Runx2 (A), COL1a1 (B), Sox9 (C), and COL2a1 (D) were quantitated by real-time PCR. Mean values of three biological repeats were normalized against those of untreated and uninduced cells and the relative values were plotted. The significance of the difference between (−) and (+) NAM was determined by ANOVA test (Dunnett’s test) (*p<0.1; **p<0.01).

• 

  Fig. 6 Effect of NAM treatment on adipogenic switch of BMSCs. BMSCs cultured to PD 8, 10, or 12 in the absence or presence of NAM were induced to osteogenesis for 12 days, stained with Oil red O, and photographed. Images of PD 8 and PD 12 cells are shown in (A). The lipid stain was dissolved and quantitated. Two biological repeats were assayed and the mean values were plotted. The significance of the difference was determined by ANOVA test (Dunnett’s test) (*p<0.1).

Reference

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