Summary In various human pathologies there are countless alterations in platelet production or function. Yet many of these pathogenesis and the required targeted therapies remain unknown, resulting in palliative treatments. In vivo, megakaryocytes associate with the bone marrow microvasculature where they extend proplatelets that protrude through the vascular endothelium into the lumen and release platelets into the blood stream. The scientific and clinical communities are actively searching for new modes to generate functional platelets ex vivo to address clinical needs as well as for insight into fundamental studies of mechanisms. We hypothesize that engineering a 3D bone marrow mimic, as demonstrated in our current grant, will propel mechanistic understanding of platelet shedding and determine future protocols for therapeutic inquiry. To test our hypothesis, in Aim 1 we will utilize non-thrombogenic silk protein biomaterial in a modified ex vivo three dimensional (3D) tissue model of the bone marrow to study platelet release from megakaryocytes derived from human induced pluripotent stem cells (hiPSCs); to compare with the results in the current grant using megakaryocytes from umbilical cord blood progenitors. In Aim 2 we will focus on the use of the bioreactor systems to study the effects of thrombopoietin mimetics on human megakaryocytes derived from patients affected by inherited thrombocytopenias and healthy controls. In Aim 3 we will conduct studies to assess the functionality of the platelets released in the bioreactor systems in vitro and in vivo. The outcome of these studies is expected to be unprecedented insight into mechanisms that control platelet formation. These insights will build on our ability to generate functional human platelets ex vivo but with significant improvements in cell sources, disease insight and functional assessments in this renewal proposal. The development of a unified solution based on the proposed science and technologies will clarify the impact of thrombopoietin mimetics on human megakaryocyte behaviour in terms of activation of intracellular signaling, differentiation, interaction with the extracellular environment and platelet production, all with major implications for human health. Importantly, the successful outcome of this project will provide researchers with new specialized tools for predicting the efficacy and safety of new drugs to address megakaryocyte- related diseases. In addition, the results will provide an important next step towards clinically relevant sources and supplies of functional human platelets for patient treatments.