title: Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
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.
authors.affiliation: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Goodell
authors.forename: K
authors.initials: K
authors.lastname: Brose
authors.forename: G
authors.initials: G
authors.lastname: Paradis
authors.forename: A S
authors.initials: AS
authors.lastname: Conner
authors.forename: R C
authors.initials: RC
authors.lastname: Mulligan
mesh_terms: D000818:Animals; D000950:Antigens, Ly; D001562:Benzimidazoles; D001853:Bone Marrow / Q000254:growth & development; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D002455:Cell Division; D002469:Cell Separation; D005260:Female; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology*; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D011835:Radiation Protection; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000950:Antigens, Ly; D001562:Benzimidazoles; D005456:Fluorescent Dyes; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D014700:Verapamil; C017807:bisbenzimide ethoxide trihydrochloride