Cui Yanni, Ren Yan, Ren Fanggang, Zhang Yaofang, Wang Hongwei
The Second Clinical Medical College, Shanxi Medical University, Taiyuan, China.
Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan, China.
Regen Ther. 2024 Mar 27;27:191-199. doi: 10.1016/j.reth.2024.03.011. eCollection 2024 Dec.
Several approaches to expand human hematopoietic stem cells (HSCs) have been reported, but the ability of these methods to expand long-term hematopoietic stem cells (LT-HSCs) remains to be improved, which limits the application of HSCs-based therapies.
CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and then cultured for 12 d in a serum-free medium. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Colony-forming cell (CFC) assays can evaluate multi-lineage differentiation potential of HSCs. Real-time polymerase chain reaction was employed to detect the expression of genes related to self-renewal programs and antioxidant activity. DCFH-DA 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.
Here, we show a combination, Nicotinamide (NAM) combined with pyrimidoindole derivative UM171, can massively expand LT-HSCs , and the expanded cells maintained the capability of self-renewal and multilineage differentiation. Additionally, our data indicated that UM171 promoted self-renewal of HSCs by inducing HSCs entry into the cell cycle and activating Notch and Wnt pathways, but the infinite occurrence of this process may lead to mitochondrial metabolism disorder and differentiation of HSCs. NAM kept HSCs in their primitive and dormant states by reducing intracellular ROS levels and upregulating the expression of stemness related genes, so we believed that NAM can act as a brake to control the above process.
The discovery of the synergistic effect of NAM and UM171 for expanding LT-HSCs provides a new strategy in solving the clinical issue of limited numbers of HSCs.
已有多种方法用于扩增人类造血干细胞（HSCs），但这些方法扩增长期造血干细胞（LT-HSCs）的能力仍有待提高，这限制了基于HSCs的疗法的应用。
使用MacsCD34磁珠从脐带血中纯化CD34+细胞，然后在无血清培养基中培养12天。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。集落形成细胞（CFC）分析可评估HSCs的多谱系分化潜能。采用实时聚合酶链反应检测与自我更新程序和抗氧化活性相关基因的表达。使用DCFH-DA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。
在此，我们发现烟酰胺（NAM）与嘧啶吲哚衍生物UM171联合使用可大量扩增LT-HSCs，且扩增后的细胞保持了自我更新和多谱系分化的能力。此外，我们的数据表明，UM171通过诱导HSCs进入细胞周期并激活Notch和Wnt信号通路来促进HSCs的自我更新，但这一过程的无限发生可能导致线粒体代谢紊乱和HSCs分化。NAM通过降低细胞内ROS水平和上调干性相关基因的表达，使HSCs保持原始和休眠状态，因此我们认为NAM可作为控制上述过程的制动器。
NAM和UM171联合扩增LT-HSCs的协同作用的发现，为解决HSCs数量有限的临床问题提供了新策略。
