Platelets are transfused for a wide range of thrombotic deficiencies, but there are problems. Platelet collection typically requires pooling harvests from multiple donors. Platelet transfusion risks from bacterial contamination, blood-borne pathogens, and alloimmunization are compounded because patients receive platelets from many donors. Production of autologous or compatible platelets by megakaryocytic cells (Mks) derived from cultured hematopoietic stem and progenitor cells (HSPCs or CD34+ cells) would greatly decrease these risks. However, generating 500 billion platelets for a single transfusion using culture conditions that yield relatively pure (e 75%) Mk populations would require 250 million CD34+ cells. This is equivalent to more than 50 umbilical cord blood harvests or 1-2 harvests of HSPCs from the peripheral blood of donors treated (or mobilized) with growth factors. In order for culture-derived platelet production to be economically feasible, it will be necessary to produce more Mk progenitors per CD34+ cell, obtain a greater number of Mks per Mk progenitor, and increase Mk ploidy (platelet- producing potential). Our objective is to increase the ploidy of culture-derived Mks to levels similar to those found in human bone marrow. We have shown that the vitamin nicotinamide (NIC) greatly increases Mk ploidy in culture. Since Mks in vivo produce several thousand platelets, we anticipate that Mks produced in culture with NIC could generate 1000 platelets. Understanding the mechanisms responsible for NIC-mediated increases in Mk ploidy will facilitate regulatory approval for using NIC to produce platelets for transplantation and is likely to lead to the discovery of even more effective conditions for Mk polyploidization. We propose to use RNA-interference-mediated knockdown to test the hypothesis, based on our preliminary results, that NIC increases Mk ploidy via inhibition of the SIRT1 and SIRT2 Class III histone/protein deacetylases. We will then examine changes in the acetylation of SIRT target proteins involved in the regulation of the cell cycle and/or apoptosis. Finally, we will investigate the roles in megakaryopoiesis of the most promising SIRT targets.
Platelet transfusions are used for a wide range of thrombotic deficiencies and several million units are transfused each year in the USA and Europe. Production of patient-specific or compatible platelets by megakaryocytic cells derived from blood stem cells in culture using Good Manufacturing Practices would greatly decrease adverse immune responses from platelet transfusions, as well as bacterial and viral contamination, but many challenges remain to be addressed. A better understanding of the mechanisms that underlie megakaryocytic differentiation and the development of efficient processes for megakaryocytic progenitor cell proliferation and for the generation and maturation of megakaryocytic cells would bring the large-scale production of culture-derived platelets closer to fruition.
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