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There is now a CONTENT FREEZE for Mercury while we switch to a new platform. It began on Friday, March 10 at 6pm and will end on Wednesday, March 15 at noon. No new content can be created during this time, but all material in the system as of the beginning of the freeze will be migrated to the new platform, including users and groups. Functionally the new site is identical to the old one. webteam@gatech.edu
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Bone Marrow Niche-Inspired, Multi-Phase Expansion of Megakaryocytic Progenitors with High Polyploidization Potential
Abstract:
Producing platelets from CD41+ megakaryocytic (Mk) cells derived from cultured blood stem and progenitor CD34+ cells would increase the supply of rare blood types and decrease the risk from blood-borne pathogens. However, producing one transfusion dose of 500 billion platelets using current technology would require 5-10 umbilical cord blood harvests or more than a full CD34+ cell harvest from the peripheral blood of donors pretreated with cytokines, so the ex vivo platelet yield must be increased by several orders of magnitude. Because there are profound changes in cell physiology, regulation, and gene expression during Mk commitment and maturation, optimal conditions will differ greatly for the processes of Mk progenitor expansion, Mk cell production, and terminal maturation (polyploidization and proplatelet formation). Inspired by gradients of increasing pH and pO2 with increasing Mk maturation in the bone marrow, we showed that low pH and low pO2 enhance Mk progenitor production, while supervascular pH and pO2 increase Mk ploidy (and platelet-producing potential). We also showed that the vitamin nicotinamide greatly increases Mk ploidy and proplatelet formation in culture. We are developing a 3-step process for Mk cell expansion and maturation with conditions tailored for each step. We identified cytokines in cultures at pH 7.2 and 5% O2 that expand Mk progenitors from 0.01 to 3.5 CD34+CD41+ cells per input CD34+ cell at day 11, while increasing purity from 1% to 17%. However, Mk clonogenic capacity decreased after day 5. Shifting cells to pH 7.4 and 20% O2 at day 5 yielded up to 14 mature Mks per input cell. Simultaneous addition of nicotinamide decreased Mk production, but more than doubled the fraction of high-ploidy (≥ 8N) Mk cells to 40%. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed nicotinamide addition will greatly increase high-ploidy Mk cell production. We hypothesize that nicotinamide increases Mk cell ploidy by inhibiting the SIRT1/2 histone/protein deacetylases. Understanding the underlying mechanisms would facilitate regulatory approval for using nicotinamide to produce platelets for transfusions and may lead to the discovery of even more effective conditions for Mk polyploidization.
Faculty Host: Michelle Dawson, 404.894.5192
