High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche.