Abstract
Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.
Highlights
Hematopoietic stem cells (HSC) 1 are multipotent cells that reside in the bone marrow and give rise to all adult hematopoietic lineages of mice
We attempted to use the fluorescent vital dye Hoechst 33342, which binds D N A in live cells, and is an indicator of cell cycle as it relates to D N A content [9], to isolate replicating and quiescent cell populations
Med. 9 The Rockefeller University Press 9 0022-1007/96/04/1797/10 $2.00 Volume 183 April 1996 1797-1806 whole bone marrow when Hoechst fluorescence was displayed simultaneously at two emission wavelengths. This led us to identify a small subset of Hoechst-staining cells that were well separated from the rest of the bone marrow, contained a majority of cells that had cell surface markers of HSC, and were enriched for HSC activity at least 1,000fold. We show that this distinct staining pattern by the HSC is due to a high level of dye efl]ux activity that may be mediated by p-glycoprotein or a related molecule
Summary
The cells were resuspended in media containing goat anti-rat antibody conjugated to PE (mouse serum adsorbed; Caltag Laboratories), and incubated for 10 rain on ice. After washing, the cells were resuspended in 1/3 volume rat serum (Cappel Laboratories, Cochranville, PA) and 2/3 HBSS+. After 10 rain on ice, biotinylated Sca-1 antibody (E13 161-7) was added for 10 rain on ice. After washing, the cells were stained with avidin-FITC (Becton Dickinson & Co.) for 10 rain on ice. After the final wash, the bone marrow cells were filtered through a 70-/.LM nylon filter (Falcon Plastics, Cockeysville, MD) and resuspended in HBSS + containing 2 ~g/ml propidium iodide (PI). The bone marrow was sometimes magnetically preenriched for Sca-1 + cells using the MACS | (Miltenyi Biotec, Sunnyvale, CA) and streptavidin microbeads. This resulted in a 5-10-fold enrichment for Sca-1 + cells
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