Gene expression profiling of mouse HSCs treated with Eltrombopag. Gene expression profiling of mouse 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. Overall design: For isolation of mouse HSCs, bone marrow cells were isolated from tibiae, femurs, pelvic bones and vertebrae of 6 to 10-week-old mice. Low-density mononuclear cells (LDMNCs) were enriched by density gradient centrifugation using Histopague 1083 (Sigma-Aldrich, MO). Prospective HSCs (Lin−Sca-1+c-Kit+CD150+CD48−) were sorted on either FACS Aria II (Becton Dickinson, Franklin Lakes, NJ) or MoFlo Astrios (Beckman Coulter, Indianapolis, IN)