SummaryHematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic hneages 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 ceils that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to conrain 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 efl]ux 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-G~ and S-G2M subsets of fresh purified HSC. Transplantation of these subsets of HSC revealed that 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.

A subset of stem cells, termed the ''side population'' (SP), has been identified in several tissues in mammalian species. These cells maintain a high efflux capability for antimitotic drugs. We have investigated whether functionally equivalent stem cells also may be detected in human cancers. We initially examined primary tumor cells from 23 patients with neuroblastoma and cell lines derived from a range of other tumors. A distinct SP was found in neuroblastoma cells from 15 of 23 patients (65%). The SP was capable of sustained expansion ex vivo and showed evidence for asymmetric division, generating both SP and non-SP progeny. These cells also expressed high levels of ABCG2 and ABCA3 transporter genes and had a greater capacity to expel cytotoxic drugs, such as mitoxantrone, resulting in better survival. A SP also was detected in breast cancer, lung cancer, and glioblastoma cell lines, suggesting that this phenotype defines a class of cancer stem cells with inherently high resistance to chemotherapeutic agents that should be targeted during the treatment of malignant disease.