PARENT SESSION

Experimental and Clinical Therapeutics

(PP152) Primitive hematopoietic stem cells are sequestered in a hypoxic microenvironment within the bone marrow: implications for oxygen-dependent cytotoxic injury.

Parmar, Kalindi^*,1, Vergilio, Jo-Anne², Clyne, James¹, Sackstein, Robert³, Mauch, Peter^{1, 4}, Down, Julian⁵, ¹ Department of Radiation Oncology, Boston, MA, USA² Department of Pathology, Boston, MA, USA³ Department of Dermatology and Medicine, Boston, MA⁴ Department of Radiation Oncology, Boston, MA⁵ Research, Cambridge, MA

ABSTRACT- The spatial organization of hematopoietic cell (HC) subsets of differing proliferative potential and its association with stromal elements within the bone marrow microenvironment has come under renewed interest. In the present study we have investigated how different HC subsets are distributed along a Hoechst 33342 (Ho) 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 Ho at 10 and 5 min before marrow cell harvesting. Flow cytometric (FC) analysis revealed a wide distribution of Ho 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 repopulating cells as determined in vitro in the so-called cobblestone area-forming cell (CAFC) assay and in vivo by long-term engraftment of congenically marked CD45.1 bone marrow transplanted in irradiated recipients. The primitive CAFC subset appearing at day 28 to 35 in culture was shown to be progressively enriched with decreasing Ho fluorescence. The higher concentration of hematopoietic stem cells (HSCs) at the end of the Ho gradient was confirmed from their greater ability to reconstitute recipients at 3 months post-transplant. We further investigated whether HSCs exist in a very low oxygen tension by administering pimonidazole (PIM) in vivo and performing FC analysis on sorted HSCs residing in high Ho 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-PIM antibody that recognizes PIM adducts formed only under hypoxic conditions. Finally, in vivo administration of the hypoxic cytotoxin tirapazamine was shown to be selectively toxic to the primitive stem cell subset with about 90% depletion of day 35 CAFCs. These results represent the first direct evidence that the hematopoietic cell hierarchy is spatially organized in relation to blood vessels and that the stem cell niche exists at the lowest end of an oxygen gradient. These findings have important implications in the general regulation of hematopoiesis in situ as well as the sensitivity of hypoxic stem cells and their better-oxygenated descendents towards ionizing radiation.

Key words: hypoxia, hematopoietic stem cells, spatial organization, oxygen gradient