Direct Intravital Imaging of the Bone Marrow and Splenic Hematopoietic Niches in Individual Mice to Define the Early Engraftment Kinetics Following HSC-Transplant

Hematopoietic stem cells (HSCs) give rise to and maintain the entire hematopoietic system for the life of an organism. This remarkable feat has established HSC transplant as an indispensable tool for treating a variety of hematological disorders. Yet the initial steps of homing, engraftment, and clonal expansion, which lead to eventual long-term hematopoietic recovery after HSC transplant, remain incompletely characterized. Given the determinative effect that early HSC activity has on transplant outcomes, a more complete understanding of initial engraftment dynamics is imperative for bettering HSC therapies.

Preliminary studies aimed at functional characterization of classic HSC and hematopoietic stem and progenitor cell (HSPC) populations-namely the CD150 ⁺CD48 ^-Sca-1 ⁺c-Kit ⁺Lin ^- (SLAM SKL) and Sca-1 ⁺c-Kit ⁺Lin ^- (SKL) populations, respectively-revealed that these populations exhibit disparate early engraftment dynamics. Using previously developed intravital imaging techniques, we were able to partially characterize early HSC and HSPC engraftment dynamics in mice competitively transplanted with GFP ⁺ SLAM SKL and DsRed ⁺ SKL cells. The SKL population was found to primarily engraft in the bone marrow, completely recapitulating engraftment behavior of transplanted whole bone marrow. In contrast, the more purified SLAM SKL population engrafted poorly in the marrow space and instead preferentially engrafted in the spleen, where it produced the majority of donor-derived blood at early stages (7 days) after competitive transplant into lethally irradiated mice. However, by 14 days post-transplant, SLAM SKL-derived cells migrated from the spleen to repopulate the majority of bone marrow space. These results reflect the dynamic nature of hematopoietic recovery in a myeloablative model and highlight the need for in vivo imaging techniques to fully understand hematopoietic reconstitution by the SLAM SKL population.

In order to further dissect the interactive processes of bone marrow hematopoiesis and splenic extramedullary hematopoiesis, we have developed a novel, multi-organ intravital imaging technique that allows for simultaneous analysis of defined hematopoietic compartments in a single animal. Our multimodal imaging approach combines direct visualization of fluorescently labeled hematopoietic cells in the spleen via our recently developed spleen window, with concomitant observation of hematopoietic cells in tibia marrow environment. Our spleen window is a specially engineered biocompatible ring with an affixed coverslip to allow for direct, non-invasive microscopic visualization of labeled hematopoietic cells in the spleen. The spleen window can be installed with the tibia window in an individual mouse. Multimodal mice can be visualized repeatedly over a minimum of 7 days post-HSC transplant to follow individual cell behaviors within the living recipient. Preliminary results from competitive repopulation assays utilizing our multimodal imaging approach suggest that the SLAM SKL population is an active one that confers rapid hematopoietic recovery in lethally irradiated recipients primarily from extramedullary hematopoiesis in the spleen (CFU-S). The results of ongoing work characterizing the active use of the splenic and marrow niches will be presented.