Abstract 25


Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) microenvironments called stem cell niches (HSCNs). Two types of HSCNs have been reported, involving osteoblasts (osteoblastic niche) and blood sinusoids (vascular niche). Various hematopoietic and non-hematopoietic cell types are contributing to the regulation of HSCs and HSCNs, however, the bone marrow microenvironment has not yet been precisely characterized and the fine localization, composition and regulation of the niches remain highly controversial.

Intravital microscopy of the calvarium (IVMC) is the only non-invasive option for high resolution imaging of bone marrow HSCNs. However it is unclear if IVMC provides data representative of all BM compartments. Indeed it has been shown that endochondral ossification, the process used for long bones growth, is required for the formation of HSCNs. By contrast, the calvarium is a flat bone which develops through intra-membranous ossifications, therefore questioning the presence of HSCNs in this bone (Chan CK et al, Nature 2009).


Combined confocal and multiphoton excitation intravital microscopy was used for multiparametric observation of live bone marrow in intact bones after in-vivo contrasting procedures. Bioluminescence imaging was used to quantify the systemic trafficking and proliferation of murine HSCs and human leukemia. Flow cytometry and histology were used to sort specific populations of cells, analyze the frequency of mouse and human stem cells or leukemic cells at steady state or after transplantation, quantify perfusion efficiency and hypoxia and cross-validate in-vivo imaging procedures.


Thanks to advanced imaging modalities we realized a thorough study of potential niche markers and HSCs distribution at homeostasis and during hematopoietic reconstitution in calavia, epiphyses and diaphyses. We report important heterogeneity between these compartments in terms of bone remodelling activity (BRA) and blood vessel fraction (BVF). Although BVF was surprisingly high in any compartment, including in very close proximity to any endosteal surface, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted following mouse and human HSC transplantation. Unexpectedly, the macro-anatomical distribution of mouse and human HSCs at steady state is homogeneous across the skeleton and independent of these 2 markers. These data suggest the existence of “reconstituting niches” which would be distinct from “homeostatic niches”. Importantly, this study provides the first evidence that both types of niches are fully functional in the calvarium, including for mouse and human HSCs, indicating that endochondral ossification is dispensable for adult HSCNs.

The model is currently being exploited to analyze leukemia/microenvironment interactions in live bone marrow.


This study confirms and extends our recent statement considering the critical need for multimodal imaging (Lassailly F et al, Blood 2010). Further more, it demonstrates that combination of different imaging modalities for in-vivo and ex-vivo analysis is a powerful strategy allowing to shed a new light on the structure of the bone marrow microenvironment and improve our understanding of stem cells/niches interactions in health and disease.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.