Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation

The limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34⁺ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34⁺CD90^- cells into CD34⁺CD90⁺ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.