Megakaryopoiesis in the Mammalian Embryo Is Distinguished From the Adult by Rapid Maturation At Low Ploidy and Generates Platelets with Altered Morphology and Function.

Abstract 2305

In the adult, all platelets are derived from hematopoietic stem cells (HSCs). However, we previously determined that the megakaryocyte (meg) lineage is specified several days before HSC emergence in the murine embryo and that circulating platelets exist in myb-null embryos, which lack HSCs. Pre-HSC meg progenitors arise in the yolk sac beginning at embryonic day 7.5 (E7.5) and have lower proliferative potential than adult meg progenitors. The fetal liver is colonized by over 1,000 meg progenitors by E10.5, before HSCs are found there. By E12.5, there are over one million circulating embryonic platelets that are larger than adullt platelets with smaller α-granules. There are also indications in humans of intrinsic differences between embryonic/fetal and adult thrombopoiesis, including the natural history of several congenital platelet disorders, as well as the small size, rapid maturation, and reduced platelet output of fetal/neonatal meg progenitors. We compared embryonic versus adult megakaryopoiesis and platelet function in the mouse. E12.5 fetal livers contain predominantly small megs with low ploidy (8% >4N versus 33% >4N in the adult marrow). Fetal megs have higher cell surface levels of CD41 and GP1bß and are larger than similar ploidy adult megs. Further evidence of rapid maturation of fetal megs was seen in the punctate localization pattern of endostatin and the presence of α-granules and a forming demarcation membrane system in small E12.5 liver megs. Like their primary counterparts, in vitro-generated embryonic megs from E9.5 yolk sac progenitors have lower ploidy than megs differentiated from bone marrow progenitors and show similar evidence of rapid cytoplasmic maturation. Initial analysis of megs generated from ES cells demonstrated low ploidy and rapid maturation similar to yolk sac-derived megs. These data support the concept that embryonic megakaryopoiesis is characterized by a rapid maturation and low ploidy phenotype that is cell intrinsic and is similar to that observed during ES cell megakaryopoiesis. An analysis of primary fetal versus adult platelets reveals similar patterns of VEGF and endostatin distribution in α-granules. However, there are differences in the expression of several other factors associated with platelet activation and function, including higher expression of the thrombin receptor PAR1 and lower expression of the ADP receptor P2Y₁₂ and P-selectin in embryonic platelets. While primary adult and embryonic platelets have altered side scatter characteristics and binding of the activation-specific Jon/A antibody after thrombin treatment, embryonic platelets fail to express P-selectin on their surface. Taken together, our findings indicate that embryonic/fetal megakaryopoiesis is characterized by low ploidy and rapid maturation, and leads to the generation of platelets with marked differences in size, structure and function compared with adult platelets. A better understanding of hematopoietic ontogeny is particularly relevant to the generation of blood cells from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, whose differentiation recapitulate early embryonic development.