OC 75.1 - A 3D microvasculature assay reveals novel sub-cellular dynamics of megakaryocyte endothelial barrier interactions, proplatelet formation and release.

Background: Megakaryocytes (MKs) generate platelets through podosome and proplatelet intermediates. The mechanisms by which MKs interact with and extravasate through the endothelial vessel wall, or how subpopulations of MKs multi-task to also generate niche supporting cytokines or extracellular vesicles (EVs) remain unclear.

Aims: To investigate MK-endothelial interactions, we have developed a 3D microvascular system, with which we aim to (1) identify how MKs penetrate the vascular space, (2) determine what cytoskeletal mechanisms power the transition from podosome to proplatelet formation, and (3) define how individual platelets and EVs are released.

Methods: Human umbilical vascular endothelial cells, human lung fibroblasts, and mouse hematopoietic stem and progenitor cells (HSPCs) were seeded into a fibrin hydrogel. The co-cultures self-assembled into open lumens and were labelled for live-cell super resolution microscopy of proplatelet formation and MK-endothelial interactions. Mature 3D networks were fixed and cross sectioned for scanning electron microscopy.

Results: We successfully created a 3D self-forming co-culture that supports HSPC differentiation into MKs. MK-podosome structures were observed extending at the endothelial barrier by electron microscopy. MKs have been observed extending proplatelets within the vessel lumens and releasing platelet like particles (Figure 1). In our alternate microfluidic system, we observed that individual mouse megakaryocytes can simultaneously form proplatelets and release vesicles (Figure 2).

Conclusion(s):

Conclusion: Our results suggest that our co-culture systems are capable of recapitulating thrombopoiesis and the study of subcellular MK-endothelial cell interactions. Simultaneous formation of MK-EVs and platelets suggests the mechanism and the timing of the release of these structures could be linked. We are going to continue to increase the complexity of our model with the addition of flow, retroviral expression systems for cytoskeletal markers and by including CRISPR-Cas9 edited MKs.