Continuous NF-κB pathway inhibition promotes expansion of human phenotypical hematopoietic stem/progenitor cells through metabolism regulation

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.