CD38 Inhibition Enhances Human Hematopoietic Stem Cell Maintenance in <i>Ex Vivo</i> Cultures

Hematopoietic stem cells (HSCs) maintain a delicate balance between self-renewal and differentiation within the bone marrow, ensuring lifelong maintenance of the blood and immune systems. However, these properties are compromised under in vitro culture conditions, posing a significant barrier to investigating HSC function ex vivo and hindering clinical applications, such as ex vivo gene therapy. CD38, a transmembrane glycoprotein, acts as a receptor, adhesion molecule, and NADase ectoenzyme, catalyzing the conversion of NAD+ into nicotinamide (NAM) and other active molecules. It is commonly used as a negative selection marker to enrich human HSCs from the broader CD34+ hematopoietic stem/progenitor cell (HSPC) population, suggesting CD38 may negatively regulate HSC function. To explore the potential impact of CD38 on HSC properties, human mobilized peripheral blood CD34+ cells were cultured for 7 days with and without 78c, a small molecule known to suppress CD38 activity. Treatment with 78c resulted in a 2-fold increase in the percentage of phenotypic HSCs compared to the DMSO control. To address whether HSCs with engraftment potential are maintained with 78c, we quantified human cell engraftment 4 months after transplanting NBSGW mice with 50,000 uncultured CD34+ cells or their 7-day progeny. While limited engraftment was observed in the DMSO control group, cells treated with 78c exhibited engraftment levels akin to uncultured CD34+ cells. Notably, a limiting-dilution secondary transplantation assay showed that the frequency and numbers of HSCs with long-term engraftment potential were preserved in the 78c-cultured group, comparable to uncultured cells, and increased 9- and 38-fold, respectively, compared to the DMSO group (p=0.02). To investigate the mechanism by which CD38 inhibition supports HSC maintenance, we examined whether 7-day culture in the presence of NAM, a known NAD booster, could replicate the effects of CD38 inhibition. NAM slightly increased phenotypic HSCs (characterized by CD34+CD45RA-CD90+ markers) but did not significantly affect human cell engraftment in NBSGW mice, indicating that the negative impact of CD38 on HSC function is not mediated primarily through its NADase activity. Proteomics analysis of CD34+ cells cultured for 24 to 72 hours with DMSO or 78c revealed that 78c-treated cells maintain a proteomic profile closer to uncultured cells compared to DMSO-treated cells. At 24 hours, 78c initially suppressed protein synthesis and translation, as shown by Reactome, GO, and String analyses. By 72 hours, this extended to suppression of unfolded protein and stress response mechanisms. Gene set enrichment analysis (GSEA) of bulk RNA sequencing from HSC-enriched CD34+CD38low cells cultured for 24 hours with 78c revealed significant inhibition of genes involved in protein synthesis and the unfolded protein response. This analysis also highlighted downregulation of the PI3K/AKT/mTOR signaling pathway, a key regulator upstream of cell cycle control, and marked suppression of genes involved in cell proliferation. Consistent with these findings, the DMSO group exhibited 2.8-fold higher cell numbers after 7 days of ex vivo culture, reinforcing the suppressive impact of 78c on cell cycle progression and proliferation. Additional BrdU incorporation experiments to assess cell cycle dynamics showed a 2.6-fold increase in the proportion of CD34+ cells in the G0/G1 phase with 78c treatment compared to the control group (p&amp;lt;0.0001). This effect was mirrored by treatments with cell cycle inhibitors Olomoucine and Hydroxyurea. Further analysis of the PI3K/AKT/mTOR signaling pathway showed that 78c treatment reduced mTOR phosphorylation. Rapamycin, an mTOR inhibitor, replicated the effects of 78c by maintaining HSC stemness and reducing cell proliferation, confirmed by xenotransplantation assays. Collectively, this study reveals that CD38 serves not only as a negative selection marker for LT-HSCs from human CD34+ cells, but also as an important target for enhancing HSC maintenance in ex vivo cultures. Inhibition of CD38 suppresses mTOR signaling and cell cycle progression, thereby safeguarding HSCs from exiting dormancy, disrupting proteostasis, and losing their stemness.