Identification and Function of Hematopoietic Stem and Progenitor Cell Specific Micrornas.

Abstract 2631

Little is known about microRNA function in hematopoietic stem and progenitor cells (HSPC). Using a lentivector genetic reporter strategy to functionally detect miRNA activity in hematopoietic cells at single cell resolution, we identified several miRNAs which were specifically expressed in mouse and human HSC and early progenitors, defined according to cell surface phenotype and functional repopulation assays. One of these HSPC-specific miRNAs, miR-126, was further studied. We generated a stable miR-126 knockdown (kd) or forced its expression (“knock-in”, ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for >1 year in both primary and secondary recipients. miR-126 kd HSPC displayed enhanced myeloid and/or lymphoid contribution during the early phases of reconstitution, while they subsequently contributed similarly as the control cells. When this steady state bone marrow (BM) was transplanted into secondary recipients, we noted an even more pronounced over-contribution of miR-126 kd cells to hematopoiesis. In the long run, however, some secondary mice showed signs of exhaustion of miR-126 kd cells. These data suggest that miR-126 kd enhances hematopoiesis, likely at the stem/early progenitor level and in particular under stress conditions. On the other hand, forced expression of miR-126 (ki) resulted in an early competitive disadvantage in vivo, with progressively decreasing contribution to all hematopoietic lineages, paralleled by a nearly complete depletion of Kit+Sca+Lin- (KSL) miR-126 ki cells in the BM at 6 weeks after transplant. At 3 weeks post-transplant, when miR-126 ki KSL cells could still be detected, we found an increased proliferative index in these cells as judged by EdU incorporation in vivo, paralleled by a higher hematopoietic output respect to control cells at week 2–4 after transplant. These data suggest that miR-126 ki might favor HSC commitment at the cost of self-renewal. This phenotype was specific for miR-126 and not due to vector toxicity, as we demonstrate stable, long term overexpression of several control miRNAs in vivo. Moreover, miR-126 ki cells showed normal clonogenic activity in vitro. We then optimized a protocol to stably knock down miR-126 in human cord blood (huCB) HSPC, and validated this approach by demonstrating upregulation of previously described miR-126 targets including the beta subunit of phosphoinositide-3-kinase. Manipulation of miR-126 activity changed cell growth and differentiation of huCB, and we show altered activation of key signal transduction pathways upon miR-126 kd. Identification of additional miR-126 targets is ongoing using unbiased proteomic and transcriptomic approaches. In summary, these data suggest that a narrow range of miR-126 activity is required for robust and sustained HSC function, and that its manipulation may provide novel insights into stem cell biology.