Detection of functional haematopoietic stem cell niche using real-time imaging

Abstract

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Osteopontin attenuates aging‐associated phenotypes of hematopoietic stem cells

“…Elucidation of the mechanisms by which aging of the niche affects HSC aging will support the design of rationale therapeutic interventions to attenuate aging in hematopoiesis (Khong et al , 2015; Khatri et al , 2016). While mechanisms through which the niche regulates young HSCs have started to be elucidated (Visnjic et al , 2004; Xie et al , 2009; Méndez‐Ferrer et al , 2010; Nombela‐Arrieta et al , 2013; Bruns et al , 2014; Acar et al , 2015; Gur‐Cohen et al , 2015), data on the extent of aging of the niche and the contribution to aging of HSCs are rare. Changes in the niche composition or function upon aging involve decreased bone formation, enhanced adipogenesis and changes in extracellular matrix (ECM) components (Calvi et al , 2003; Zhang et al , 2003; Naveiras et al , 2009; Geiger et al , 2013).…”

Adipocytic Cells Augment the Support of Primitive Hematopoietic Cells In Vitro But Have No Effect in the Bone Marrow Niche Under Homeostatic Conditions

“…However, to formally rule out adipocytes as being active in the hematopoietic niche under homesostatic conditions, direct visualization of the HSCs in real time would have to be performed. Unfortunately, this has only been achieved with moderate success recently [41,42]. Our functional data thus argue against a role of adipocytes in the HSC niche under homeostatic conditions.…”

Hypoxia mediates low cell-cycle activity and increases the proportion of long-term–reconstituting hematopoietic stem cells during in vitro culture

“…Recent data imply that HSCs reside in areas that are hypoxic, i.e., niches low in oxygen content. Furthermore, it has been demonstrated that vascular cells can secrete signals that regulate HSC maintenance [6][7][8]. Despite that the BM is extensively vascularized, it appears that the nature of the vasculature where HSCs reside is mainly of a sinusoidal type, characterized by slow blood flow that contains venous and poorly oxygenated blood.…”