Live-animal imaging of native haematopoietic stem and progenitor cells

Abstract

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions 1 , 2 . Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche 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 long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM) 3 – 5 . We find that this subset of LT-HSCs resides in close proximity to both 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. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous 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 BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

Calcium regulation of stem cells

“…Trabecular bone may therefore absorb calcium in steady state in young individuals. Intriguingly in this context, a recent report indicates that HSCs localize near areas of bone turnover, which may affect local calcium concentrations . While a recent report showed expansion of HSCs in vitro , among other measures by replacing albumin with polyvinyl alcohol l , low‐calcium cultures can open avenues for HSC manipulation based on their specific physiological features.…”

Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors

“…In the liver of the fetus, or in the bone-marrow of adult, hematopoietic stem and progenitor cells (HSPCs) sense and respond to numerous biochemical stimuli (Pinho and Frenette, 2019). Within the bone-marrow, the vascular network and the bone matrix constitute local niches that impart distinct and specific signals regulating the quiescence, proliferation and differentiation of HSPCs (Morrison and Scadden, 2014) (Christodoulou et al, 2020) (Guezguez et al, 2013). Perturbed interactions between HSPCs and their niches have been associated with blood malignancies and ageing (Verovskaya et al, 2019), underscoring the importance of better understanding how HSPCs sense and respond to stromal and endothelial cells in the bone-marrow (Ceafalan et al, 2018).…”

“…Furthermore, the similarities and differences between the polarities of migrating and anchored HSPCs are still unclear. Such investigation appear all the more necessary that it has recently been revealed that quiescent longterm hematopoietic stem cells are actually non-motile in vivo (Christodoulou et al, 2020). Although a lot has been learned from co-culture experiments, it has remained technically challenging to study the specific role of cell adhesion independently of cell migration.…”

Life Through a Lens: Technological Development and Applications in Intravital Microscopy

“…This technology offers a new way of viewing the metabolic processes of all cells, independent of their origin, phenotype, or state of differentiation, as respiration is fundamental to every cell type. The technology presented by Okkelman et al may complement two other readouts of nutrition and respiration already employed in intravital imaging: the ubiquitous coenzyme‐dependent (NAD(P)H and FAD) metabolism and oxygen availability . However, the performance of the current technology has not been yet applied intravitally, the results presented by the authors convincingly demonstrate its power and feasibility for use with intravital imaging.…”

“…However, even with this success, advances that expand the utility of the technique and enable IVM to provide more complete information about in vivo pathophysiological phenomena at the single‐cell level were still necessary and continue to be made . These recent developments include (1) expanding the spectral and spatiotemporal boundaries of the current technology, (2) retrieving more detailed molecular information on cellular and tissue functions within living organisms, and (3) translating basic science knowledge into clinically relevant information . The publications in this special issue, Intravital Microscopy: Innovations and Applications , review these recent achievements, as well as present new advances in the IVM of osteoimmunological cross talk, musculoskeletal development, infection biology, and cancer research.…”