PF450 HIGH PLOIDY LARGE CYTOPLASMIC MEGAKARYOCYTES: KEY NEGATIVE REGULATORS OF HEMATOPOIETIC STEM CELLS AND ESSENTIAL FOR PLATELET PRODUCTION
Authors: S. Heazlewood, T. Ahmad, B. Cao, H. Cao, M. Domingues, B. Williams, C. Heazlewood, M. Fulton, S. Li, J. White, F. Kraus, M. Ryan, B. Kile, M.-L. Anko, S. Nilsson
Journal: HemaSphere (2019)

Background: Megakaryocytes (MK) are the large multiploidal bone marrow (BM) cells responsible for generating platelets. Recently, MK have been demonstrated to be key components of the BM stem cell niche, directly regulating hematopoietic stem cells (HSC) through maintenance of cell quiescence. Traditionally, MK have been fractionated using ploidy and it's generally accepted that high ploidy MK are responsible for platelet generation. However, whether all high ploidy MK exhibit these functional effects or whether there are specific sub-populations within high ploidy MK remains unknown. Aims: To identify specific sub-populations of MK and functionally identify their roles in platelet generation and HSC maintenance. Methods: Flow cytometry was used to assess different populations of high ploidy MK (16N, 32N or 64N), which were sub-fractionated as large cytoplasmic MK (LCM) or small cytoplasmic MK (SCM) based on the expression of surface markers including CD41. Transmission emission microscopy (TEM) was used to characterise ultrastructural differences between LCM and SCM and transplant of these MK populations were used to assess their platelet generation potential. A mouse model with normal MK numbers, but essentially devoid of LCM, was used to assess the effect of LCM on platelet generation and HSC homeostasis in BM. BM devoid of LCM were assessed for changes in key soluble factors known to regulate HSC and flow cytometry was used to assess changes in key adhesion receptors expressed by HSC. Limiting dilution and competitive transplant experiments were used to assess the reconstitution potential of HSC in the presence and absence of LCM. Results: We identified a novel sub-population of MK, large cytoplasmic MK (LCM), that are essential for platelet formation, but also critical as negative regulators of hematopoietic stem and progenitors (HSPC). Mice devoid of LCM, demonstrated pronounced thrombocytopenia, but also a significantly increased BM HSPC pool concurrent with endogenous mobilization evidenced by significant HSPC in the peripheral blood and spleen. Notably, LCM-mediated HSC regulation is attributed to several key factors including thrombopoietin, platelet factor-4 and thrombin-cleaved osteopontin. Furthermore, HSC from mice essentially devoid of LCM had significantly reduced expression of adhesion receptors including CXCR4 and a4 and a9 integrins, which together contributes to the endogenous HSC mobilization and splenomegaly. Thrombocytopenia, the absence of LCM and the increased HSPC pool were recapitulated when HSC isolated from a microenvironment devoid of LCM were used to reconstitute hematopoiesis in lethally irradiated mice. However, in contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment essentially devoid of LCM, sufficient WT generated LCM prevented thrombocytopenia as well as resulting in normal regulation of the HSPC pool. Importantly, we provide evidence that these sub-fractions of MK are conserved in humans where we hypothesize LCM have similar functions. Summary/Conclusion: As the cells responsible for platelet generation and maintenance of HSC quiescence, LCM are a critical component of the BM stem cell niche. The data suggests therapeutic efforts to increase platelet production in vitro or in vivo specifically need to increase LCM generation, with better understanding of this previously unrecognised MK sub-population leading to greater understanding of normal and aberrant blood cell production