Megakaryopoiesis, Thrombopoiesis and the Microenvironment

Abstract

Bone marrow microenvironment has been suggested to be critical for platelet generation. Understanding the environmental cues that regulate megakaryocytes development and platelet production remains a major goal of developmental and clinical biology. My thesis projects focus on understanding interactions between bone marrow microenvironment and thrombopoiesis, so as to develop a novel method to produce platelets in vitro. I first investigated soluble factors affecting megakaryopoiesis and thrombopoiesis by performing a genome-wide search to identify plasma membrane receptors whose ligands may play important functional roles in this developmental process. Thirty-night transmembrane receptor genes were identified. The robustness of this dataset was tested by selecting 7 receptor-associated genes and examining the ability of their matched-ligands to modulate megakaryocytopoiesis. Overall, 6 of 7 of the plasma membrane receptors have functional roles in megakaryocyte (MK) and platelet biology. These data also indicate for the first time that adiponectin plays a regulatory role in MK development. Most importantly, these results support the notion that there is a strong likelihood that the 40 transmembrane genes constitute a MK receptome that will be an important resource to the research community for deciphering the complex repertoire of environmental cues that regulate megakaryocytopoiesis and/or modulate platelet function. I also reconstituted an in vitro 3D microvascular niche, which allowed me to study the final maturation of MKs and the close interactions between MKs and endothelium. MKs migrate toward microvessels via CXCR4-mediated pathways, transmigrate across endothelium and release functional platelets into vessel lumen. I observed two transmigration modes: paracellular transmigration through cell-cell junction and pericellular transmigration directly through endothelial cells. Transmigration of MKs leaves pores in the endothelium and makes the microvessel leaky. This study suggests that big MK fragments may leave bone marrow and enter the blood steam. A stage of MKs with platelet territories was discovered in our study, indicating a transit stage between demarcation membrane system and proplatelet. Collectively, by developing 3D MK-microvessel coculture system, we successfully study the crosstalk between MKs and microvessel and produce and collect platelets from microvessel. This system would serve as an alternative human thrombopoiesis model, complementing animal models and clinical studies, and potentially serve as a platform for large-scale productions of platelets for transfusion.