Effects of different concentrations of nicotinamide on hematopoietic stem cells cultured in vitro

Abstract

BACKGROUND

In vitro expansion to increase numbers of hematopoietic stem cells (HSCs) in cord blood could improve clinical efficacy of this vital resource. Nicotinamide (NAM) can promote HSC expansion ex vivo, but its effect on hematopoietic stem and progenitor cells (HSPCs, CD34⁺CD38) and functional subtypes of HSCs – short-term repopulating HSCs (ST-HSCs, CD34⁺CD38CD45RACD49f⁺) and long-term repopulating HSCs (LT-HSCs, CD34⁺CD38CD45RACD49f⁺CD90⁺) is not yet known. As a sirtuin 1 (SIRT1) inhibitor, NAM participates in regulating cell adhesion, polarity, migration, proliferation, and differentiation. However, SIRT1 exhibits dual effects by promoting or inhibiting differentiation in different tissues or cells. We propose that the concentration of NAM may influence proliferation, differentiation, and SIRT1 signaling of HSCs.

AIM

To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation.

METHODS

CD34⁺ cells were purified from umbilical cord blood using MacsCD34 beads, and cultured for 10–12 d in a serum-free medium supplemented with cytokines, with different concentrations of NAM added according to experimental requirements. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Real-time polymerase chain reaction was used to detect the transcription levels of target genes encoding stemness-related factors, chemokines, components of hypoxia pathways, and antioxidant enzymes. Dichloro-dihydro-fluorescein diacetate probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array.

RESULTS

Compared with the control group, the proportion and expansion folds of HSPCs (CD34⁺CD38) incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased (all P < 0.05). The ST-HSCs ratio and fold expansion of the 5 mmol/L NAM group were significantly higher than those of the control and 10 mmol/L NAM groups (all P < 0.001), whereas the LT-HSCs ratio and fold expansion of the 10 mmol/L NAM group were significantly higher than those of the other two groups (all P < 0.05). When the NAM concentration was > 10 mmol/L, cell viability significantly decreased. In addition, compared with the 5 mmol/L NAM group, the proportion of apoptotic cells in the 10 mmol/L NAM group increased and the proportion of cells in S and G2 phase decreased. Compared with the 5 mmol/L NAM group, the HSCs incubated with 10 mmol/L NAM exhibited significantly inhibited SIRT1 expression, increased intracellular ROS content, and downregulated expression of genes encoding antioxidant enzymes (superoxide dismutase 1, peroxiredoxin 1).

CONCLUSION

Low concentrations (5 mmol/L) of NAM can better regulate the balance between proliferation and differentiation, thereby promoting expansion of HSCs. These findings allow adjustment of NAM concentrations according to expansion needs.

Keywords: Hematopoietic stem cells, Nicotinamide, Concentration, Proliferation, Differentiation, Sirtuin 1

Core tip: This study reveals the dominant subgroups of hematopoietic stem cells (HSCs) and molecular mechanisms underlying the effects of different nicotinamide (NAM) concentrations. Activation or inhibition of sirtuin 1 (SIRT1) is determined by the concentration of NAM. High concentrations inhibit SIRT1 but are not conducive to self-renewal of HSCs, whereas low concentrations balance HSC proliferation and differentiation by regulating the SIRT1–HIF1A pathway and reactive oxygen species production, effectively promoting in vitro expansion of the stem cells. These findings could allow adjustment of NAM concentrations according to expansion needs and may help predict small molecules that synergistically promote expansion with NAM.