Small molecule-mediated human HSC expansion and development of HSC expansion medium

Hematopoietic stem cells (HSC), known by their ability of multipotency and self-renewal are mostly utilized in HSC transplantation for treatment of hematological disorders and malignancies. Umbilical cord blood (UCB) and mobilized peripheral blood (mPB) which are alternatives to bone marrow (BM) for HSC transplantation, are limited by lack of efficient HSC number. To overcome this limitation, ex-vivo expansion of HSCs is a highly promising therapeutic applications rather than usage of double cord blood unit, which causes delay of hematopoietic recovery. We have previously shown that knockout of HSC quiescence genes could increase HSC pool in vivo. Thus, we hypothesized that targeting the HSC quiescence regulators using small molecules may be utilized for ex vivo mPB and UCB-HSC expansion. We aimed to identify the most effective hematopoietic small molecules (HSMs)/HSM combinations and develop good manufacturing practises (GMP)-grade human HSC expansion medium including the identified HSMs for successful HSC transplantation. We used 35 HSMs targeting HSC quiescence genes for HSC expansion. We found that BML-260 and TAME molecules increased the mPB and UCB-HSC content and triggered the re-entry to cell cycle. Colony forming-unit (CFU) assay confirmed their enhanced multilineage potential effects. BML-260 was safer rather than TAME on viability of the expanded cells. Gene expression analysis showed that BML-260 and TAME molecules contribute to HSC expansion through modulation of cell cycle kinetics including p27, Skp2 and Cdh1. On the other hand, the preliminary data about identification of the effectice chemically-defined HSC expansion medium showed that RPMI 1640 basal medium suplemented with 10 percent FBS, cytokines (SCF, TPO, FLT-3), insulin-transferrin-selenium (ITS) and BML-260 molecule could be promising for development of GMP-grade human HSC expansion medium. These findings reveal that these small molecules and this medium composition could be improved for human HSC expansion.