Effective Expansion and Engraftment Of Nonhuman Primate CD34⁺Hematopoietic Stem Cells After Co-Culture With The Small Molecule UM171

Chemical factors that simultaneously maintain and expand definitive hematopoietic stem cells (HSCs) have the potential to greatly improve the clinical outcome in patients transplanted with allogeneic umbilical cord blood or gene corrected autologous CD34⁺ cells, in which the number of cells available for transplantation is limited. The recently discovered small molecules UM729 and UM171 effectively expand HSCs and synergize with StemRegenin1 (SR1) to enhance human HSC expansion, leading to successful long-term engraftment of SCID repopulating cells (SRCs) in immunodeficient mice. In order to facilitate translation of these small molecules for clinical use, we sought to determine the effect of UM729 and UM171 on expansion and transduction of nonhuman primate CD34⁺ cells from various sources. We hypothesized that UM171 would expand definitive CD34⁺ HSCs from macaque umbilical cord blood (Mn UCB), thereby increasing the definitive HSC cell dose available for engraftment. Here, we compared engraftment of the progeny of 250,000 Mn UCB CD34⁺ cells after a 10-day expansion with cytokines with or without SR1, UM729, or UM171 in immunodeficient mice. Although all expansion conditions led to >44-fold increase in the number of CD34⁺ cells across all cohorts, mice transplanted with UM171 treated Mn UCB CD34⁺ cells had a significantly higher level of engraftment compared to all other groups (41% nonhuman primate CD45⁺ cells in peripheral blood at 10 weeks post transplantation, P<0.05). We next wanted to determine whether UM171 expands bone marrow derived CD34⁺ cells. The combination of SR1 and UM171 expanded CD34⁺CD38^- cells from mobilized and steady state bone marrow 9- and 3-fold, respectively. Mobilized CD34⁺ cells with an LT-HSC phenotype (CD34⁺CD38^-Thy1⁺CD45RA^-CD49f⁺) increased 13-fold. This robust expansion in phenotypic LT-HSCs was accompanied by a significant decrease in short-term hematopoietic colony forming cell (CFC) potential, compared to unexpanded CD34⁺ cells (p<0.005). Before translation to a large animal transplantation model, we next wanted to assess the effect of UM171 on transduction and expansion of gene modified HSCs. Macaque bone marrow derived CD34⁺ cells were transduced with GFP expressing lentivirus and alphavirus vectors. UM171 co-culture enhanced viability after lentivirus transduction and GFP⁺ cells with an LT-HSC phenotype expanded 5-fold compared to 1.7-fold expansion of these cells in the absence of UM171. UM171 had no effect on expansion of alphavirus transduced CD34⁺ cells. Studies are currently underway to compare the synergistic effect of SR1 and UM171 on the transduction, expansion, and engraftment of UCB and adult CD34⁺ cells in immunodeficient mice and nonhuman primate recipients, and to clarify the molecular target of UM171 in nonhuman primate HSCs. These findings mark a critical step toward the clinical translation of small molecule based HSC expansion therapeutics for use in UCB and gene modified autologous HSC transplantation.