Megakaryocytes (MKs) and the platelets they produce are essential for clot formation, but both are also involved in critical processes such as development, inflammation, homeostasis and regeneration. Platelet biogenesis is canonically thought to occur primarily in the adult bone marrow and the developing fetal liver. As such, our current understanding of MK development stems primarily from the investigation of these tissues. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs, with several groups demonstrating that lung-MKs significantly contribute to circulating platelet mass. While a diversity of cells specific to the bone marrow are known to promote the maturation and trafficking of MKs, investigation into how cells of the lung niche influence the development and function of MKs has not been done. This knowledge gap is highlighted by the fact that platelets have demonstrated lung-specific roles in processes such as development, epithelial protection against bacterial exotoxins, and pulmonary fibrosis. Whether lung-MKs are involved in these processes and/or provide a localized source of platelets that carry out lung-specific functions is unknown. This question led us to hypothesize that symbiotic interactions between lung-MKs and the lung microenvironment aid in the maturation and functionalization of both MKs and the lung itself. Here we propose the use of both in-vivo and in-vitro approaches to investigate this adaptive symbiosis.
In Aim 1, we will perform single cell RNA sequencing and functional assays on MKs isolated from fetal and adult mouse tissues to define a lung-specific MK phenotype. Investigating MKs at different stages of development will bolster our understanding of the maturational trajectory and functionalization of lung-MKs. We will also perform the histological assessment of primary mouse lung tissue to determine the cellular makeup of the lung-MK microenvironment.
In Aim 2, we will develop an in-vitro human induced pluripotent stem cell (iPSC) based coculture system to model the interaction of MKs in the lung microenvironment. Previous work from the Murphy (Sponsor) and Kotton (Co-Sponsor) labs have established methodologies for directing the patterning of iPSCs towards hematopoietic and lung lineages to robustly produce iPSC derived MKs and lung organoids. Harnessing these tools, we will coculture MKs with lung organoids to investigate how reciprocal interactions between each system influences the development and patterning of these tissues. Resultant cells from coculture will then be subjected to functional and transcriptomic studies to investigate how the phenotypic patterning of MKs and lung organoids is influenced by coculture. These studies will improve our understanding of the biology of MK specification and platelet production, and also shed light on the symbiotic relationship between the lung and resident MKs, thus introducing the previously undescribed role of MKs as an integral component of the lung microenvironment.
Megakaryocytes are platelet producing cells canonically thought to be primarily resident in the bone marrow but now hypothesized to be an integral part of the lung microenvironment during development and disease. Little investigation into the role of lung megakaryocytes beyond platelet production has been done, thus we propose to apply next generation sequencing, functional assessments and high-resolution microscopy as a means of characterizing lung megakaryocytes. We will also utilize induced pluripotent stem cell-based models of megakaryocyte and lung development as a means of investigating how symbiotic interactions between megakaryocytes and the lung niche influence the development of both systems.
