Expansion of Platelets for Transfusion

Abstract SCI-47

The clinical need for platelets transfusion continues to grow. Platelet units have all the usual concerns with other donor-derived blood products and in addition have concerns over the need to store the platelets at room temperature and of the resulting increased risk of bacterial contamination. Several groups have been able to develop platelets from embryonic stem cells, but achieving significant numbers of pro-platelets and/or platelets from in vitro grown megakaryocytes (Megs) remains elusive. Three sites of thrombopoiesis in vivo have been described: 1) intramedullary, 2) at the medullary/vascular junction, or 3) intrapulmonary from circulating Megs. We investigated whether infusing murine Megs into mice can release a large number of functional platelets both to better understand thrombopoiesis and also to see if such an approach may be an alternative to generating platelets for clinical usage. We show that infused fetal-liver-derived Megs results in 100–200 platelets/Meg. Levels as high as 12% of the total platelets in the recipient mouse were from the infused Megs. These platelets had a delayed presentation, peaking at 90 mins. In spite of these being new platelets, their half-life was only ∼75% of infused platelets. The shortened half-life was not due to the fact that these platelets were fetal in nature in that infused adult-liver-derived Megs had a similar temporal profile. These derived platelets had normal-size distribution, were not activated, and had not been affected by ADAM17 during in vitro culture as determined by surface glycoprotein 1b alpha. Platelet functionality was shown by their incorporation into clots in arterioles and venules in a cremaster laser injury model and in a ferric chloride carotid artery injury model. Using BrdU-labeled Megs we show that most of the Megs are trapped in the lungs with a few also appearing in the splenic red pulp. None were seen in other tissues. Staining with species-specific CD41 antibodies showed that detectable Meg cytoplasm peaked in the pulmonary vasculature at 30 min, and like the nuclei, was mostly gone by 5 hrs. We estimate that only ∼1% of the available pulmonary capillary bed is affected by the infused Megs, and most studied animals survived the infusion of the Megs. These studies support the lungs as a potential site for thrombopoiesis and that in vitro differentiated Megs can be infused directly avoiding the challenge of developing ex vivo technology to release large number of non-activated, but functional platelets.