Niche-specific endothelial mechanisms regulating the extravasation of hematopoietic stem cells

PROJECT SUMMARY Hematopoietic stem cell (HSC) transplantation is a potentially curative treatment for numerous blood and immune disorders. The process itself, however, is inefficient and can leave patients immunocompromised and vulnerable to life-threatening infections for up to a year post-transplantation. Only once donor HSCs engraft in the recipient’s bone marrow niches can the stem cells regenerate a new immune system. In this proposal, we aim to elucidate the key endothelial mechanisms that direct circulating HSCs across the blood vessel wall and into their niche. Our studies will pave the way for the development of novel therapeutics to accelerate post- transplant immune recovery by enhancing the niche migration of HSCs. In mammals, the trans-endothelial migration of HSCs occurs deep inside opaque bones, which are largely inaccessible to imaging and experimentation in live animals. The transparent zebrafish embryo, by contrast, which has a blood and vascular system highly similar to that of humans, enables direct visualization of HSC migration with unprecedented sub-cellular resolution. We previously identified a conserved gene expression signature for HSC niche endothelial cells, which includes molecules with cell adhesion and endocytosis/vesicle trafficking functions. Consistent with this, intravenous injection of fluorescent endocytosis reporters revealed a high level of endocytosis activity, with each niche endothelial cell harboring a discrete cluster of endocytic activity. Using high-resolution time-lapse microscopy, we observed adherent HSCs initiating trans-endothelial migration into the niche specifically through these endocytic clusters. In preliminary studies, we disrupted candidate factors from our niche endothelial signature, which blocked endocytosis in the niche endothelium and impaired HSC migration. Together, our data suggest that a combination of specific cell adhesion and endocytosis/vesicle trafficking machinery within the endothelium facilitate the extravasation of HSCs into their niche, a hypothesis we will test in our proposed aims. In Aim 1, we employ high-resolution live cell imaging in combination with structure-function studies and in vivo proximity-dependent biotinylation in zebrafish, to define the endothelial receptor domains and binding partners that mediate the adhesion and trans-endothelial migration of HSCs. In Aim 2, we use loss-of-function chemical and genetic tools in combination with overexpression studies, both in the zebrafish and a biomimetic human blood vessel model, to determine the conserved vascular endocytosis and membrane trafficking machinery that is necessary and sufficient to trigger HSC extravasation. Together, our studies will provide novel molecular targets for the design of new endothelial-focused therapeutics aimed at more efficiently guiding HSCs into the bone marrow, which could accelerate immune system recovery following HSC transplantation.