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. 1996 Apr 1;183(4):1797–1806. doi: 10.1084/jem.183.4.1797

Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo

PMCID: PMC2192511  PMID: 8666936

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

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  1. Andrews R. G., Singer J. W., Bernstein I. D. 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. doi: 10.1084/jem.172.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertoncello I., Hodgson G. S., Bradley T. R. 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] [Google Scholar]
  3. CUDKOWICZ G., UPTON A. C., SHEARER G. M., HUGHES W. L. LYMPHOCYTE CONTENT AND PROLIFERATIVE CAPACITY OF SERIALLY TRANSPLANTED MOUSE BONE MARROW. Nature. 1964 Jan 11;201:165–167. doi: 10.1038/201165a0. [DOI] [PubMed] [Google Scholar]
  4. Chaudhary P. M., Roninson I. B. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell. 1991 Jul 12;66(1):85–94. doi: 10.1016/0092-8674(91)90141-k. [DOI] [PubMed] [Google Scholar]
  5. Civin C. I., Strauss L. C., Brovall C., Fackler M. J., Schwartz J. F., Shaper J. H. 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] [Google Scholar]
  6. 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] [Google Scholar]
  7. Ellwart J. W., Dörmer P. Vitality measurement using spectrum shift in Hoechst 33342 stained cells. Cytometry. 1990;11(2):239–243. doi: 10.1002/cyto.990110204. [DOI] [PubMed] [Google Scholar]
  8. Fleming W. H., Alpern E. J., Uchida N., Ikuta K., Spangrude G. J., Weissman I. L. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol. 1993 Aug;122(4):897–902. doi: 10.1083/jcb.122.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fleming W. H., Alpern E. J., Uchida N., Ikuta K., Spangrude G. J., Weissman I. L. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol. 1993 Aug;122(4):897–902. doi: 10.1083/jcb.122.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harrison D. E., Astle C. M., 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. doi: 10.1073/pnas.85.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harrison D. E. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood. 1980 Jan;55(1):77–81. [PubMed] [Google Scholar]
  12. Harrison D. E., Stone M., Astle C. M. Effects of transplantation on the primitive immunohematopoietic stem cell. J Exp Med. 1990 Aug 1;172(2):431–437. doi: 10.1084/jem.172.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones R. J., Wagner J. E., Celano P., Zicha M. S., Sharkis S. J. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature. 1990 Sep 13;347(6289):188–189. doi: 10.1038/347188a0. [DOI] [PubMed] [Google Scholar]
  14. Jordan C. T., Lemischka I. R. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev. 1990 Feb;4(2):220–232. doi: 10.1101/gad.4.2.220. [DOI] [PubMed] [Google Scholar]
  15. Li C. L., Johnson G. R. Long-term hemopoietic repopulation by Thy-1lo, Lin-, Ly6A/E+ cells. Exp Hematol. 1992 Dec;20(11):1309–1315. [PubMed] [Google Scholar]
  16. Li C. L., Johnson G. R. Rhodamine123 reveals heterogeneity within murine Lin-, Sca-1+ hemopoietic stem cells. J Exp Med. 1992 Jun 1;175(6):1443–1447. doi: 10.1084/jem.175.6.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mauch P., Hellman S. Loss of hematopoietic stem cell self-renewal after bone marrow transplantation. Blood. 1989 Aug 1;74(2):872–875. [PubMed] [Google Scholar]
  18. McAlister I., Wolf N. S., Pietrzyk M. E., Rabinovitch P. S., 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] [Google Scholar]
  19. Neben S., Redfearn W. J., Parra M., Brecher G., Pallavicini M. G. 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] [Google Scholar]
  20. Pallavicini M. G., Summers L. J., Dean P. N., Gray J. W. Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting. Exp Hematol. 1985 Dec;13(11):1173–1181. [PubMed] [Google Scholar]
  21. Ploemacher R. E., Brons R. H. 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] [Google Scholar]
  22. Ross E. A., Anderson N., Micklem H. S. 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. doi: 10.1084/jem.155.2.432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. SIMINOVITCH L., TILL J. E., MCCULLOCH E. A. DECLINE IN COLONY-FORMING ABILITY OF MARROW CELLS SUBJECTED TO SERIAL TRANSPLANTATION INTO IRRADIATED MICE. J Cell Physiol. 1964 Aug;64:23–31. doi: 10.1002/jcp.1030640104. [DOI] [PubMed] [Google Scholar]
  24. Spangrude G. J., Brooks D. M. 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] [Google Scholar]
  25. Spangrude G. J., Brooks D. M., Tumas D. B. 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] [Google Scholar]
  26. Spangrude G. J., Heimfeld S., Weissman I. L. Purification and characterization of mouse hematopoietic stem cells. Science. 1988 Jul 1;241(4861):58–62. doi: 10.1126/science.2898810. [DOI] [PubMed] [Google Scholar]
  27. Spangrude G. J. Hematopoietic stem-cell differentiation. Curr Opin Immunol. 1991 Apr;3(2):171–178. doi: 10.1016/0952-7915(91)90046-4. [DOI] [PubMed] [Google Scholar]
  28. Spangrude G. J., Johnson G. R. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7433–7437. doi: 10.1073/pnas.87.19.7433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spangrude G. J., Scollay R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp Hematol. 1990 Sep;18(8):920–926. [PubMed] [Google Scholar]
  30. Szilvassy S. J., Lansdorp P. M., Humphries R. K., Eaves A. C., Eaves C. J. 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] [Google Scholar]
  31. Tindle R. W., Nichols R. A., Chan L., Campana D., Catovsky D., Birnie G. D. 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. doi: 10.1016/0145-2126(85)90016-5. [DOI] [PubMed] [Google Scholar]
  32. Uchida N., Weissman I. L. 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. doi: 10.1084/jem.175.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Van Zant G., Holland B. P., Eldridge P. W., Chen J. J. Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice. J Exp Med. 1990 May 1;171(5):1547–1565. doi: 10.1084/jem.171.5.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Visser J. W., Bauman J. G., Mulder A. H., Eliason J. F., de Leeuw A. M. Isolation of murine pluripotent hemopoietic stem cells. J Exp Med. 1984 Jun 1;159(6):1576–1590. doi: 10.1084/jem.159.6.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Watson J. V., Nakeff A., Chambers S. H., Smith P. J. Flow cytometric fluorescence emission spectrum analysis of Hoechst-33342-stained DNA in chicken thymocytes. Cytometry. 1985 Jul;6(4):310–315. doi: 10.1002/cyto.990060406. [DOI] [PubMed] [Google Scholar]
  36. Watt S. M., Karhi K., Gatter K., Furley A. J., Katz F. E., Healy L. E., Altass L. J., Bradley N. J., Sutherland D. R., Levinsky R. 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] [Google Scholar]
  37. Wolf N. S., Koné A., Priestley G. V., Bartelmez S. H. 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] [Google Scholar]

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