Valproic acid-expanded cord blood CD34+CD90+ cells are functionally distinct based on their origin

Hematopoietic stem cell (HSC) transplantation is a potentially curative option for patients with hematologic malignancies, but donor shortages impact graft availability. Umbilical cord blood (UCB) is a viable alternative source of HSC; however, the limited numbers present in a single unit have spurred efforts to expand HSC ex vivo. We previously demonstrated that the addition of valproic acid (VPA), an anti-convulsive drug, to CB cell cultures promotes maintenance of functional HSC, but not expansion. However, it has been proposed that VPA primarily induces mitochondrial reprogramming of mature CD34+CD90- cells to more primitive CD34+CD90+ cells, rather than the replication of CD34+CD90+ cells in culture. To determine which fraction of the CD34+CD90+ cells present after culture in VPA were derived from CD34+CD90- vs. CD34+CD90+ cells, we examined the functionality of CD34+CD90+ cells derived from each flow cytometry-sorted population. During culture in VPA there was a significant increase in CD34+CD90+ cell number; the majority arising from pre-existing CD34+CD90+ cells, with minimal contribution from CD34+CD90- cells. Colony-forming unit (CFU) assays revealed reduced plating efficiency and xeno-transplantation studies demonstrated diminished in vivo hematopoietic reconstitution potential of CD34+CD90+ cells derived from relatively committed CD34+CD90- cells. Our findings indicate that while VPA supports CD34+CD90+ cell expansion, the CD34+CD90+ cells derived from CD34+CD90- cells are functionally more differentiated than those derived directly from CD34+CD90+ cells, with increased mitochondrial mass and membrane potential, but reduced regenerative potential. These results emphasize the need for functional assessments of culture-expanded HSCs to accurately determine their therapeutic potential.