Hematopoietic stem/progenitor cells (HSPCs) ex vivo expansion is critical in facilitating their widespread clinical application. NF-κB pathway is implicated in the energy homeostasis and metabolic adaptation. To explore the effect of NF-κB pathway on the ex vivo HSPC expansion and metabolism, the 50 nM–1 μM inhibitor of NF-κB pathway TPCA-1 was used to expand cord blood derived CD34⁺ cells in serum-free culture. The expansion folds, function, mitochondrial profile and metabolism of HSPCs were determined. After 10 days of culture with 100 nM TPCA-1, the expansion of total cells， CD34⁺CD38⁻ cells， and CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells were significantly increased compared to the cytokine priming alone. Notably, TPCA-1 treatment generated ~ 2-fold greater percentage of CD34⁺EPCR⁺ and CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells compared to cytokine only conditions. Moreover, TPCA-1 expanded CD34⁺ cells displayed enhanced serial colonies forming potential and secondary expansion capability. NF-κB inhibition increased the expression of self-renewal related genes, while downregulated the expression of mitochondrial biogenesis regulator (Pgc1α) and mitochondrial chaperones and proteases (ClpP, Hsp10, Hsp60). Mitochondrial mass and membrane potential were markedly decreased with TPCA-1 treatment, leading to the reduced mitochondrial reactive oxygen species (ROS) level in HSPCs. NF-κB inhibition displayed augmented glycolysis rate with compromising mitochondrial metabolism. This study demonstrated that NF-κB pathway inhibition improved glycolysis and limited ROS production that promoted the ex vivo expansion and maintenance of functional HSPCs.

造血干/祖细胞（HSPC）离体扩增对于促进其广泛的临床应用至关重要。NF-κB通路与能量稳态和代谢适应有关。为了探讨NF-κB途径对离体HSPC扩增和代谢的影响，使用50 nM–1μMNF-κB途径TPCA-1抑制剂在无血清培养物中扩增脐血来源的CD34 ⁺细胞。测定了HSPC的扩增倍数，功能，线粒体分布和代谢。后用100nM TPCA-1，总细胞的扩增，CD34培养10天后⁺ CD38 ^-细胞，和CD34 ⁺ CD38 ^- CD45RA ^- CD90与单独的细胞因子引发相比，⁺ CD49f ⁺细胞显着增加。值得注意的是，与仅使用细胞因子的情况相比，TPCA-1处理产生的CD34 ⁺ EPCR ⁺和CD34 ⁺ CD38 ^- CD45RA ^- CD90 ⁺ CD49f ⁺细胞的百分比高出约2倍。此外，TPCA-1扩增的CD34 ⁺细胞显示出增强的连续菌落，形成了潜在的和二次扩增能力。NF-κB抑制增加了自我更新相关基因的表达，同时下调了线粒体生物发生调节因子（Pgc1α）和线粒体伴侣和蛋白酶的表达（ClpP，Hsp10，Hsp60）。TPCA-1处理可显着降低线粒体质量和膜电位，从而导致HSPC中线粒体活性氧（ROS）水平降低。NF-κB抑制显示出增加的糖酵解速率，并损害线粒体代谢。这项研究表明，NF-κB途径抑制作用改善了糖酵解作用，并限制了ROS的产生，从而促进了功能性HSPC的离体扩增和维持。