Gene expression profiling of human HSCs treated with Eltrombopag

Eltrombopag, a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy to stimulate sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. While the functional and molecular effects of Eltrombopag on megakaryopoiesis have been carefully studied in the recent past, insights into its mechanistic impact on the earliest stages of hematopoiesis have been limited. Additionally, previous studies also revealed molecular pathway of Eltrombopag apart from stimulation of TPO signaling, detail characterization remains to be addressed. In this study, we investigated the effects of Eltrombopag, in comparison with TPO, on highly-purified primary hematopoietic stem cells (HSCs) from healthy human donors. The binding of Eltrombopag to TPO-R is species-specific to human and primate cells. we also utilized HSCs isolated mouse as separation-of-function models to directly assess the molecular effect of Eltrombopag on HSCs independent of TPOR stimulation. Bone marrow was enriched for mononuclear cells by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Pittsburgh, PA). Mononuclear cells were further enriched for CD34+ cells by immunomagnetic cell separation techniques (Miltenyi Biotec, Auburn, CA). Then hematopoietic stem cells (Lineage−CD34+CD38−CD49f+) were purified from CD34+ enriched cells by multiparameter-high speed fluorescence-activated cell sorting (FACS) on either FACS Aria II (Becton Dickinson, Franklin Lakes, NJ) or MoFlo Astrios (Beckman Coulter, Indianapolis, IN)