Hematopoietic stem cell egress and mobilization from the stem cell niche

Abstract

Hematopoietic stem cells (HSCs) produce all mature blood cells throughout the entire life of an organism. HSCs reside in a complex microenvironment, or ‘niche’, where they receive diverse signals that regulate their function. Dysregulation of HSC-intrinsic or extrinsic signals from the niche can lead to hematologic malignancies. To treat hematological disorders, patients undergo conditioning to release or mobilize malignant HSCs from their niche, so that newly transplanted healthy HSCs can colonize the emptied niche. However, traditional myeloablative conditioning strategies for HSCs are often toxic and result in long-term complications. A thorough understanding of HSC egress and mobilization will help develop a safer mobilization-based conditioning regimen for patients. Here, I leveraged the advantages of zebrafish to visualize the release of HSCs, understand mechanisms of HSC egress from the embryonic niche, and target these mechanisms to mobilize HSCs from the adult niche. I created a transgenic line with inducible expression of zebrafish granulocyte colony stimulating factor (G-CSF), the most widely used mobilizing agent, and performed high-resolution confocal microscopy to capture mobilized HSCs in circulation. G-CSF successfully mobilized HSCs from the embryonic niche into circulation, and neutrophils and macrophages were not required for the G-CSF induced mobilization in embryos. Adopting this live-imaging technique, I performed an in vivo live imaging-based chemical screen and identified three putative mobilizing agents in zebrafish embryos, from which one compound, doxepin, successfully mobilized HSCs from the adult hematopoietic niche. Single cell RNA-sequencing of the embryonic niche sinusoidal endothelial cells revealed a downregulation of cathepsin La during development. Morpholino-induced knockdown and chemical inhibition revealed cathepsin La as a potential regulator of HSC maintenance and development of sinusoids in the embryonic niche. Single cell transcriptomics analysis also revealed scavenging receptor-mediated endocytosis as a putative regulator of HSC retention, and disrupting it through administration of dextran sulfate, an inhibitor of scavenger-receptor mediated endocytosis, mobilized HSCs from the adult hematopoietic niche. Lastly, the transcriptomics data also suggested the VCAM1/VLA-4 axis as a regulator of HSC mobilization and chemically or genetically disrupting this interaction mobilized HSCs from the adult hematopoietic niche. Together, this work identifies multiple mechanisms that regulate HSC egress from the hematopoietic niche. These mechanisms can be therapeutically targeted to develop a safe mobilization-based conditioning regimen for HSC transplantation.