Defining the Bone Marrow Stem Cell Niche According to a Hoechst Dye Diffusion Gradient and Hypoxia.

The spatial organization of hematopoietic cell subsets of differing proliferative potential within the bone marrow microenvironment has come under increasing interest. In particular, it has been suggested that hematopoietic stem cells (HSCs) normally reside in a regulatory niche situated at the endosteal bone surface and in close proximity to osteoblasts. In the present study we have investigated how different hematopoietic cell subsets are distributed along a Hoechst dye perfusion gradient that may reflect the distance from marrow blood vessels and the level of oxygenation. C57BL/6J mice were intravenously injected with two doses of Hoechst 33342 at 10 and 5 min before marrow cell harvesting, a period that we determined was insufficient for active dye exclusion in vitro. Flow cytometric analysis revealed a wide distribution of Hoechst staining over 3 logs fluorescence intensity. The cells were then sorted from 6 different regions of the Hoechst gradient and evaluated for short- and long-term in vitro repopulating cells in the cobblestone area-forming cell (CAFC) assay. The primitive CAFC subset appearing at day 28 in culture was shown to be progressively enriched with decreasing Hoechst fluorescence while the short-term repopulating day 7 CAFC subset frequencies was the highest at an intermediate level of Hoechst staining. We further investigated whether primitive HSCs exist in a very low oxygen tension by administering the reductive 2-nitroimidazole compound pimonidazole in vivo and performed flow cytometric analysis on sorted primitive HSCs residing in high Hoechst dye effluxing side population (SP). In comparison to whole bone marrow or non-SP cells, the SP fraction showed increased intracellular staining with an anti-pimonidazole antibody that recognizes pimonidazole adducts formed only under hypoxic conditions (less than pO² of 10 mm Hg). Comparison with thymocytes that are already known to be hypoxic in vivo (