ENGRAFTMENT POTENTIAL OF CD34+ CELLS FROM SEVERE APLASTIC ANEMIA PATIENTS EXPANDED WITH UM171

Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34+ cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34+ cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34+ cells from patients with AA expanded with UM171 in a murine xenotransplantation model. CD34+ cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×104UCB CD34+ cells or 8.250 AA patient CD34+ cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. We found that both progenies of UCB and AA BM CD34+ cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45+ cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded