ISOLATION AND FUNCTIONAL-PROPERTIES OF MURINE HEMATOPOIETIC STEM-CELLS THAT ARE REPLICATING IN-VIVO

Hematopoietic stem cells (HSC) are multipotent cells that reside in th e bone marrow and replenish all adult hematopoietic lineages throughou t the lifetime of the animal. While experimenting with staining of mur ine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission w avelengths revealed a small and distinct subset of whole bone marrow c ells that had phenotypic markers of multipotential HSC. These cells we re shown in competitive repopulation experiments to contain the vast m ajority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these H oechst-stained side population (SP) cells were shown to protect recipi ents from lethal irradiation at low cell doses, and to contribute to b oth lymphoid and myeloid lineages. The formation of the Hoechst SP pro file was blocked when staining was performed in the presence of verapa mil, indicating that thr distinctly low staining pattern of the SP cel ls 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 a ctivity 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-G(2)M . This also enabled the purification of the G(0)G(1) and S-G(2)M subse ts of fresh purified HSC. Transplantation of these subsets of HSC reve aled that S-G(2)M HSC had a reconstitution capacity equivalent to quie scent stem cells. These findings have implications for models of hemat opoietic cell development and or the development of genetic therapies for diseases involving hematopoietic cells.