Live-Animal Imaging of Native Hematopoietic Stem and Progenitor Cells

Abstract

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. Particularly challenging has been the study of dynamic HSC behaviors in the native niche given that live animal HSC tracking under steady state conditions still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent longterm HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial marrow. We find that this subset of LT-HSCs resides in an endostealsinusoidal niche where they are simultaneously in close proximity to sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. Additionally, our results demonstrate that LTHSCs do not occupy the marrow niches with the deepest hypoxia and that they are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging experiments revealed that LT-HSCs display limited motility compared to the more motile MPPs. However, following activation, LT-HSCs become more motile and expand clonally within spatially restricted domains. These spatial domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities exhibiting bone-remodeling activities (resorption and new bone deposition). In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover, which has not been recognized previously. Overall, our work describes a model that enables live imaging of LT-HSCs in the native niche and provides insight into the dynamic behaviors of hematopoietic stem and progenitor cells, and the heterogeneity of HSC niches.