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J Exp Med. Apr 1, 1996; 183(4): 1797–1806.
PMCID: PMC2192511

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ross EA, Anderson N, Micklem HS. Serial depletion and regeneration of the murine hematopoietic system. Implications for hematopoietic organization and the study of cellular aging. J Exp Med. 1982 Feb 1;155(2):432–444. [PMC free article] [PubMed]
  • Jordan CT, Lemischka IR. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev. 1990 Feb;4(2):220–232. [PubMed]
  • SIMINOVITCH L, TILL JE, MCCULLOCH EA. DECLINE IN COLONY-FORMING ABILITY OF MARROW CELLS SUBJECTED TO SERIAL TRANSPLANTATION INTO IRRADIATED MICE. J Cell Physiol. 1964 Aug;64:23–31. [PubMed]
  • CUDKOWICZ G, UPTON AC, SHEARER GM, HUGHES WL. LYMPHOCYTE CONTENT AND PROLIFERATIVE CAPACITY OF SERIALLY TRANSPLANTED MOUSE BONE MARROW. Nature. 1964 Jan 11;201:165–167. [PubMed]
  • Mauch P, Hellman S. Loss of hematopoietic stem cell self-renewal after bone marrow transplantation. Blood. 1989 Aug 1;74(2):872–875. [PubMed]
  • Harrison DE, Stone M, Astle CM. Effects of transplantation on the primitive immunohematopoietic stem cell. J Exp Med. 1990 Aug 1;172(2):431–437. [PMC free article] [PubMed]
  • Spangrude GJ, Brooks DM, Tumas DB. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood. 1995 Feb 15;85(4):1006–1016. [PubMed]
  • Van Zant G, Holland BP, Eldridge PW, Chen JJ. Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice. J Exp Med. 1990 May 1;171(5):1547–1565. [PMC free article] [PubMed]
  • Watson JV, Nakeff A, Chambers SH, Smith PJ. Flow cytometric fluorescence emission spectrum analysis of Hoechst-33342-stained DNA in chicken thymocytes. Cytometry. 1985 Jul;6(4):310–315. [PubMed]
  • Pallavicini MG, Summers LJ, Dean PN, Gray JW. Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting. Exp Hematol. 1985 Dec;13(11):1173–1181. [PubMed]
  • Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. 1988 Jul 1;241(4861):58–62. [PubMed]
  • Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med. 1992 Jan 1;175(1):175–184. [PMC free article] [PubMed]
  • Li CL, Johnson GR. Long-term hemopoietic repopulation by Thy-1lo, Lin-, Ly6A/E+ cells. Exp Hematol. 1992 Dec;20(11):1309–1315. [PubMed]
  • Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood. 1980 Jan;55(1):77–81. [PubMed]
  • Harrison DE, Astle CM, Lerner C. Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells. Proc Natl Acad Sci U S A. 1988 Feb;85(3):822–826. [PMC free article] [PubMed]
  • Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood. 1993 Dec 1;82(11):3327–3332. [PubMed]
  • Fleming WH, Alpern EJ, Uchida N, Ikuta K, Spangrude GJ, Weissman IL. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol. 1993 Aug;122(4):897–902. [PMC free article] [PubMed]
  • Spangrude GJ. Hematopoietic stem-cell differentiation. Curr Opin Immunol. 1991 Apr;3(2):171–178. [PubMed]
  • Visser JW, Bauman JG, Mulder AH, Eliason JF, de Leeuw AM. Isolation of murine pluripotent hemopoietic stem cells. J Exp Med. 1984 Jun 1;159(6):1576–1590. [PMC free article] [PubMed]
  • Szilvassy SJ, Lansdorp PM, Humphries RK, Eaves AC, Eaves CJ. Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability. Blood. 1989 Aug 15;74(3):930–939. [PubMed]
  • Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature. 1990 Sep 13;347(6289):188–189. [PubMed]
  • Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence. Exp Hematol. 1985 Nov;13(10):999–1006. [PubMed]
  • Fleming WH, Alpern EJ, Uchida N, Ikuta K, Spangrude GJ, Weissman IL. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol. 1993 Aug;122(4):897–902. [PMC free article] [PubMed]
  • Spangrude GJ, Scollay R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp Hematol. 1990 Sep;18(8):920–926. [PubMed]
  • Neben S, Redfearn WJ, Parra M, Brecher G, Pallavicini MG. Short- and long-term repopulation of lethally irradiated mice by bone marrow stem cells enriched on the basis of light scatter and Hoechst 33342 fluorescence. Exp Hematol. 1991 Oct;19(9):958–967. [PubMed]
  • McAlister I, Wolf NS, Pietrzyk ME, Rabinovitch PS, Priestley G, Jaeger B. Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow. Blood. 1990 Mar 15;75(6):1240–1246. [PubMed]
  • Wolf NS, Koné A, Priestley GV, Bartelmez SH. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol. 1993 May;21(5):614–622. [PubMed]
  • Ellwart JW, Dörmer P. Vitality measurement using spectrum shift in Hoechst 33342 stained cells. Cytometry. 1990;11(2):239–243. [PubMed]
  • Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell. 1991 Jul 12;66(1):85–94. [PubMed]
  • Drach D, Zhao S, Drach J, Mahadevia R, Gattringer C, Huber H, Andreeff M. Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood. 1992 Dec 1;80(11):2729–2734. [PubMed]
  • Spangrude GJ, Johnson GR. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7433–7437. [PMC free article] [PubMed]
  • Li CL, Johnson GR. Rhodamine123 reveals heterogeneity within murine Lin-, Sca-1+ hemopoietic stem cells. J Exp Med. 1992 Jun 1;175(6):1443–1447. [PMC free article] [PubMed]
  • Ploemacher RE, Brons RH. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: evidence for a pre-CFU-S cell. Exp Hematol. 1989 Mar;17(3):263–266. [PubMed]
  • Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol. 1984 Jul;133(1):157–165. [PubMed]
  • Andrews RG, Singer JW, Bernstein ID. Human hematopoietic precursors in long-term culture: single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells. J Exp Med. 1990 Jul 1;172(1):355–358. [PMC free article] [PubMed]
  • Tindle RW, Nichols RA, Chan L, Campana D, Catovsky D, Birnie GD. A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow. Leuk Res. 1985;9(1):1–9. [PubMed]
  • Watt SM, Karhi K, Gatter K, Furley AJ, Katz FE, Healy LE, Altass LJ, Bradley NJ, Sutherland DR, Levinsky R, et al. Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells. Leukemia. 1987 May;1(5):417–426. [PubMed]

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