The existence of a hematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that hematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow (BM) microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in BM cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where the majority closely interacts with sinusoidal and non-sinusoidal BM microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF-1α, regardless of localization throughout the BM, adjacency to vascular structures or cell cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.

In the version of this Technical Report originally published, reference to a study (L. Wang et al. Identification of a clonally expanding haematopoietic compartment in bone marrow. EMBO J. 32, 219-230; 2013) that used confocal microscopy to analyse the presence of phenotypically defined HSPCs in previously unreported and localized structures termed hemospheres, published while the current manuscript was under consideration, was inadvertently omitted. This has now been added as reference 42, and the following paragraph has been added to the Discussion section.

3417 The identification of specific microenvironments, in which Hematopoietic Stem and Progenitor Cells (HSPCs), reside within the BM is a major challenge in stem cell biology. Yet the extreme rarity of HSCs, their dynamic nature, and the lack of unique specific markers to identify them, have precluded an accurate definition of HSC niches to date. Using Laser Scanning Cytometry, a powerful emerging quantitative imaging technology that enables analysis of whole femoral sections at the single cell level, we have mapped the global distribution of hematopoietic stem and progenitor cells within femoral bone marrow cavities, and analyzed their inmediate surrounding microenvironment. Systematic mapping of the global distribution of endogenous HSPC-enriched populations in the BM, revealed an accumulation of these cells inside endosteal regions (ER <100μm from inner bone surface), but not necessarily in contact with endosteal surfaces. Interestingly, the vast majority of HSPCs were found in direct association with BM micrrovessels, further supporting previous work, which suggested bone marrow endothelium as a major component of HSPC niches. By employing a novel imaging approach, we provide a three-dimensional (3D) microscopic overview of the unique BM vascular network found in endosteal zones, which contain the transition of bone-lining arterioles and capillaries to the sinusoidal network. Of note, HSPC association to vascular structures is not restricted to sinusoids. A significant fraction of HSPCs lied adjacent to non-sinusoidal endothelium. Using five-color imaging cytometry and pimonidazole incorporation, we have assessed the hypoxic state of HSPCs in different BM microenvironments. Our in situ analysis reveals that intracellular hypoxia is a hallmark of HSPCs, independent of their distance to bone surfaces, and more importantly, regardless of their perivascular localization. These studies provide unequivocal anatomical evidence for the intrinsic rather than environmental regulation of intracellular hypoxia in HSPCs and challenge the hypothesis of a “super hypoxic” HSPC niche. Disclosures: No relevant conflicts of interest to declare.