<em>In Vivo</em> 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Abstract

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood [1][2] .We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments

“…This was gradually reduced to approximately 1% as anaesthesia stabilised. Surgery to attach the headpiece was then performed as described in16. Large 3 dimensional ‘tilescans’ of the entire BM cavity space were acquired by stitching adjacent, high-resolution Z-stack images using a surgically implanted imaging window that ensures steady positioning of mice on the microscope.…”

“…Large 3 dimensional ‘tilescans’ of the entire BM cavity space were acquired by stitching adjacent, high-resolution Z-stack images using a surgically implanted imaging window that ensures steady positioning of mice on the microscope. The calvarium has been demonstrated to be equivalent to the long bones such as the femur with regards to haematopoietic stem cell frequency, function and localisation14,22, and is the only bone marrow compartment that allows longitudinal imaging through minimally invasive surgery16,32. Blood vessels were highlighted by I.V.…”

“…The window was bandaged, and mice were allowed to recover from anaesthesia. Due to the lock-and-key mechanism of the imaging window16, mice could then be re-anaesthetised and accurately repositioned on the microscope stage and the same BM areas re-imaged. After each imaging, analgesia was administered via oral buprenorphine in raspberry jelly at a dose of approximately 0.8mg/kg.…”

“…To address these questions, we monitored leukaemia growth in mouse calvarium bone marrow by intravital microscopy14–16. We used a tile-based imaging approach akin to Google Earth that allows tissue-wide visualisation of heterogeneous BM microenvironments (Fig.…”

Current Developments in Mobilization of Hematopoietic Stem and Progenitor Cells and Their Interaction with Niches in Bone Marrow

“…Homing of HSPCs to the osteoblastic niche was only found after irradiation of mice, but not without irradiation, explaining the need for conditioning in an attempt to 'open' marrow space, i.e., niches for transplanted HSPCs. Improved in vivo imaging techniques were used for these studies which make use of fluorescence markers to tag HSPCs as well as stromal compartments [25]. In these models, transplanted HSPCs were found to primarily locate in the vicinity of vasculature and near N-cadherin+ pre-osteoblastic cells [26].…”