ABSTRACT Replacing donor-based platelet (Plt) transfusions with product prepared from human embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or a hematopoietic progenitor cell (HPC) source has been pursued by a number of groups with several proposing that such a product will soon be available. Plts are important in a number of clinically challenging situations and to have a product with less potential side-affects, and greater and more reliable availability and function would be welcomed. Most published efforts have begun with ESCs or iPSCs differentiated into megakaryocytes (Megs) in culture, then harvesting and charactering the ex vivo-released Plts both in vitro and after infusion into immunodeficient mice, most often NOD-SCID/?- interferon receptor deficient (NSG) mice. Reports of the use of plastic sieve-like bioreactors have been published describing the release of pro-Plts and Plts from Megs using shear forces. Our interpretation of these data from all strategies is that the yield of ex vivo-released Plts has bee insufficient to raise Plt counts in infused NSG mice, unless macrophage depletion is used, in contrast to infusing human donor-Plts that is successful without macrophage depletion (in our hands). Moreover, compared to donor-Plts, these products do not have the same size distribution or infused half-life, and are minimally functional. In our studies beginning with eithr iPSC-derived Megs or adult HPC-derived Megs, we confirm the limited nature of ex vivo-generated Plts and offer direct Meg infusion into recipient mice as an alternative approach, generating in vivo-Plts from Megs entrapped in the pulmonary vasculature. These Plts are physically and functionally more like infused donor-Plts and have a similar prolonged half-life even without macrophage depletion. The strategy is robust. We can raise the Plt count in thrombocytopenic animals and also cure a Plt-based bleeding diathesis. We now wish to extend these findings to bring our efforts closer to clinical application in the following 3 Specific Aims (SAs): SA#1: Enhancing Meg quality and yield from stem cells. IPSCs yield yolk sac-like primitive Megs that have low ploidy and Plt yield/Meg. Our group has identified a definitive (Def) hematopoiesis strategy and will pursue such cells to make Def-Megs and test their Plt yield and functionality. Alternative strategies to optimize yield and functionality will also be pursued. SA#: Characterization/correction of ex vivo-derived Plts. We have characterized some of the injury that Megs and Plts incur during ex vivo preparation and will investigate the temporal profiles of these injuries and whether we can prevent these injuries and improve both the Megs and ex vivo-Plts for infusion, extending Plt half-lives and functionality in recipient mice. SA#3: Characterization and development of the infused Meg technology. We will pursue strategies to genetically modify Megs to increase Plt yield per infused Meg and Plt half-life. We will also test other key aspects of infusing Megs to enhance safety and/or to gain insights related to this strategy that may be applicable to developing a physiologic Plt bioreactor.