Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq). APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNAseq confirmed the enrichment of HSC signature genes in 24-h, 7-day, and 14-day APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). Overall design: The setup comprised HSPCs expanded for 7 days in single (A_7d, P_7d, U_7d), minus-one (AP_7d, AU_7d, PU_7d), and complete APU medium (APU_7d) in comparison to the basal medium (SFT3_7d) and uncultivated CD34+CD38-CD45RA- cells (uncultivated) (Fig. 2B). Additionally, we aimed to verify the HSC expansion capability of 0.2 µM Poma in the APU combination (APU_0.2_7d) compared to the standard concentration of 2 µM Poma described by Ebina. We also compared the short-term APU cultivation without IL-3 to detect initial intracellular signaling cascades after 24h (APU_24h, SFT_24h). Later, we included HSPCs that had been expanded for 14 days to assess long-term cultivation effects. The dataset also includes two samples that were not part of the manuscript. Here, the HSPCs were cultivated for 24h with conditioned medium from native mesenchymal stromal cells (MSCs) and TGFß-selected MSCs.