4 research outputs found
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New Approaches to Study Thymic Seeding & Regeneration
Hematopoietic Cell Transplantation (HCT) is a common treatment for patients suffering from a variety of malignant or benign diseases, reconstituting the hematopoietic system after preconditioning treatment. In patients undergoing HCT, the capacity of the thymus to produce functional T cells is inhibited due to damage from cytotoxic preconditioning. Endogenous thymus regeneration depends on the complex relationship between thymus stromal cells (including vascular endothelial cells (EC)) and the recruitment of de novo “seeding” early thymic progenitors (ETPs) from the regenerated bone marrow (BM). However; functional damage to the vascular network (ECs compartment) may alter the hemodynamics and negatively impact ETP homing and thymus regeneration. Traditionally, flow cytometry, immunohistochemistry, ex vivo imaging, and other molecular biology techniques have been applied to study the thymus in preclinical mouse models since direct visualization of the native thymus in live mice was deemed impossible. In my project, we developed a new method for intravital two-photon microscopy of the native thymus to study functional changes to the vascular system after cytotoxic preconditioning. We hypothesize that cytotoxic preconditioning causes functional and anatomical changes in blood vessel architecture, especially cortical vasculature, that negatively impacts ETP homing and leads to long-term changes in the thymus microenvironment. Using our methodology, we were able to quantify the changes to the blood vessel network after sublethal irradiation (4.5 Gy). We were able to quantify blood flow velocity and shear rate in cortical blood vessels and identified a subtle but significant increase in vessel diameter and barrier function ~24 hrs post-sublethal irradiation. We validated this result using tissue clearing and ex vivo imaging. In addition, most cortical blood velocity is <500 μm/s in both control and one day after sublethal irradiation, although no significant changes were observed in blood velocity and shear rate between the groups at this time point. Taken together, our study suggests that native intravital thymus imaging is a powerful technique enabling functional and anatomical characterization of the thymus vascular network. We believe further work will help clarify the changes to the vascular system at later time points and in the context of higher irradiation doses. This method enables a whole new paradigm for studying thymus biology not previously possible. In the second project, we developed whole organ imaging based on a modified tissue clearing method and investigated the performance based on clearing capability, fluorescence preservation, imaging depth, and size deformation. An optical clearing technique is a powerful tool to reduce light scattering for deep-tissue imaging and enable 3-D imaging of thick tissue samples. We hypothesize that by modifying the temperature and pH of the ultimate 3D imaging of solvent-cleared organs (uDISCO) clearing method, we can improve the retention of GFP fluorescence over time without sacrificing the clearing capability. We developed a modified uDISCO clearing method named a-ucDISCO (alkaline-ultimate chilled DISCO) by adjusting the PH and temperature and performed ex vivo imaging of vascular networks of the murine thymus using two-photon microscopy. Our results revealed a significant increase in GFP fluorescence retention over time compared to the standard uDISCO method. This modified clearing method, therefore, represents an alternative approach for three-dimensional whole-organ imaging of samples with endogenous GFP fluorescence
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Live-animal imaging of native haematopoietic stem and progenitor cells.
The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches