Page 71 - 2019_09-HaematologicaMondo-web

miR-127 controls HSC maintenance
AB
CD
EF
Figure 3. Inhibition of miR-127-3p function leads to hematopoietic stem cell (HSC) depletion. (A) Experimental workflow. (B) Level of engraftment of transduced cells over time, measured as percentage of cells expressing the donor marker CD45.2 within total peripheral blood (PB) CD45+ cells. Graphs show engraftment in individual mice transplanted with Lin- cells transduced with 127DR (top, n=11) or empty vector (EV) (bottom, n=9). Tx: transplant. (C) dGFP expression in PB donor monocytes measured by FACS analysis 18 weeks after transplant. (Left) Average of eight mice; (right) representative FACS histogram. (D) Kinetics of multi-lineage differentiation of transduced cells during PB sampling (6-12-18 weeks). Histograms show the absolute cell number, determined by hemocytometer analysis, and stacked columns represent the relative abundance of each population (CD11b+ myeloid cells, CD19+ B cells, CD3+ T cells; CD3R: T cells from recipient), determined by FACS analysis (n=6-8). (E) FACS analysis of 127DR and EV stem and progenitor cells in the bone marrow (BM) of transplanted mice. (Left) Representative FACS analysis of HSC and multi-potent progenitors (MPP) gated on LKS (dot plots) and of their dGFP expression (bottom histograms) compared to that of a representative non-transplanted mouse. (Right) Average of HSC and MPP absolute numbers (n=6-7). (F) Central and peripheral multi-lineage differentiation by transplanted 127DR- and EV-transduced cells in primary recipients. Histograms show absolute BM cell numbers (left) and percentage of donor cells in the spleen (right). Stacked columns represent relative abundance of each lineage (n=6-8). All graphs summarize results from two independent experiments, and BM cells counts are relative to one femur and one tibia.
(Figure 3F). None of the transplanted animals displayed extramedullary hematopoiesis (data not shown), indicating that the observed HSC reduction was not due to abnormal egress from the BM.
To evaluate whether there was a decrease in functional HSC independently of the immunophenotype used to define them, secondary transplants were performed by injecting a high dose of total BM cells from two individual primary recipients in lethally irradiated CD45.1+ second- ary recipient mice. The number of transplanted donor BM cells was sufficient for allowing recovery after irradiation;
however, most of the mice transplanted with cells derived from 127DR BM succumbed 4-6 weeks after transplant, soon after the challenge of the first blood withdrawal, sug- gesting a severe HSC defect (Figure 4A). Four weeks after transplant, donor myeloid chimerism was very high in all mice, without significant differences among the groups of mice transplanted with 127DR or EV transduced BM (Figure 4B), indicating that the early mortality of 127DR BM-transplanted mice was likely not due to homing or engraftment impairment. However, sponging miR127-3p severely compromised myeloid cell and platelet produc-
haematologica | 2019; 104(9)
1749