Ren Yan, Cui Yan-Ni, Wang Hong-Wei
The Second Clinical Medical College, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China.
Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China.
World J Stem Cells. 2024 Feb 26;16(2):163-175. doi: 10.4252/wjsc.v16.i2.163.
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 , but its effect on hematopoietic stem and progenitor cells (HSPCs, CD34CD38) and functional subtypes of HSCs - short-term repopulating HSCs (ST-HSCs, CD34CD38CD45RACD49f) and long-term repopulating HSCs (LT-HSCs, CD34CD38CD45RACD49fCD90) 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.
To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation.
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.
Compared with the control group, the proportion and expansion folds of HSPCs (CD34CD38) incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased (all < 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 < 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 < 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).
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.
扩大脐血中造血干细胞（HSC）的数量可提高这种重要资源的临床疗效。烟酰胺（NAM）可促进造血干细胞扩增，但其对造血干祖细胞（HSPC，CD34⁺CD38⁻）以及造血干细胞的功能亚型——短期重建造血干细胞（ST-HSC，CD34⁺CD38⁻CD45RA⁻CD49f⁺）和长期重建造血干细胞（LT-HSC，CD34⁺CD38⁻CD45RA⁻CD49f⁺CD90⁺）的影响尚不清楚。作为一种沉默调节蛋白1（SIRT1）抑制剂，NAM参与调节细胞黏附、极性、迁移、增殖和分化。然而，SIRT1在不同组织或细胞中通过促进或抑制分化表现出双重作用。我们推测NAM的浓度可能影响造血干细胞的增殖、分化和SIRT1信号传导。
评估不同浓度NAM对造血干细胞增殖和分化的影响及其潜在作用机制。
使用Macs CD34磁珠从脐血中纯化CD34⁺细胞，并在补充细胞因子的无血清培养基中培养10 - 12 d，根据实验要求添加不同浓度的NAM。采用流式细胞术检测培养细胞的表型、细胞周期分布和凋亡情况。使用实时聚合酶链反应检测编码干性相关因子、趋化因子、缺氧途径成分和抗氧化酶的靶基因的转录水平。使用二氯二氢荧光素二乙酸酯探针评估细胞内活性氧（ROS）的产生。通过细胞计数珠阵列测定不同培养条件对细胞因子平衡的影响。
与对照组相比，用5 mmol/L或10 mmol/L NAM孵育的HSPC（CD34⁺CD38⁻）比例和扩增倍数显著增加（均P<0.05）。5 mmol/L NAM组的ST-HSC比例和扩增倍数显著高于对照组和10 mmol/L NAM组（均P<0.001），而10 mmol/L NAM组的LT-HSC比例和扩增倍数显著高于其他两组（均P<0.05）。当NAM浓度>10 mmol/L时，细胞活力显著下降。此外，与5 mmol/L NAM组相比，10 mmol/L NAM组凋亡细胞比例增加，S期和G2期细胞比例下降。与5 mmol/L NAM组相比，用10 mmol/L NAM孵育的造血干细胞SIRT1表达显著受抑制，细胞内ROS含量增加，编码抗氧化酶（超氧化物歧化酶1、过氧化物还原酶1）的基因表达下调。
低浓度（5 mmol/L）的NAM能更好地调节增殖与分化之间的平衡，从而促进造血干细胞的扩增。这些发现有助于根据扩增需求调整NAM浓度。
