HIGH PLOIDY LARGE CYTOPLASMIC MEGAKARYOCYTES ARE CRITICAL FOR PLATELET PRODUCTION AND ARE KEY NEGATIVE REGULATORS OF HEMATOPOIETIC STEM CELLS

Traditionally, megakaryocytes (MK) are known for platelet generation; with their role as regulators of hematopoietic stem cells (HSC) only recently being appreciated. While it is generally accepted high ploidy MK produce platelets, we have now identified that the vast majority of platelets are generated from a novel sub-population of MK, large cytoplasmic MK (LCM). When prospectively isolated from mouse marrow and transplanted, LCM readily form platelets in vivo. We have further characterized LCM using flow cytometry, TEM and performed mitochondrial network analysis via confocal microscopy. Importantly, we have identified this MK sub-population to be critical negative regulators of HSC. Using a mouse model with normal MK numbers, but essentially devoid of LCM (LCMKO), we demonstrate pronounced thrombocytopenia and significantly increased HSC pool concurrent with endogenous mobilization. Furthermore, we identified LCM negative regulation of HSC is via thrombopoietin, platelet factor-4 and thrombin-cleaved osteopontin. Thrombocytopenia, the absence of LCM and an increased HSC pool were recapitulated when HSC isolated from LCMKO mice were used to reconstitute hematopoiesis in irradiated mice. In contrast, following a competitive transplant using minimal numbers of WT HSC with HSC from LCMKO mice, WT HSC were capable of recapitulating the LCM population, preventing thrombocytopenia and resulting in a normal HSC pool. Importantly, we provide evidence that LCM is conserved in humans, where we hypothesize they have similar functions. LCM are an important component of the niche, regulating HSC as well as generating platelets; therefore, therapeutic efforts to increase platelet production needs to specifically increase LCM. Furthermore, by understanding how different MK sub-populations regulate HSC, we will better understand the mechanisms governing steady-state hematopoiesis.