Nature Medicine
3, 1337 - 1345 (1997)
doi:10.1038/nm1297-1337
Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple speciesMargaret A. Goodell1, 6, *, Michael Rosenzweig2, *, Hyung Kim1, Douglas F. Marks2, MaryAnn DeMaria2, Glenn Paradis3, Stephen A. Grupp4, Colin A. Sieff4, Richard C. Mulligan1
& R. Paul Johnson2, 5, 7
1Howard Hughes Medical Institute, Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
2New England Regional Primate Research Center, Harvard Medical School, P.O. Box 9102, One Pine Hill Drive, Southborough, Massachusetts 01772, USA
3Flow Cytometry Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA
4Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA
5Infectious Disease Unit, Massachusetts General Hospital, 13th Street, and Partners AIDS Research Center, Boston, Massachusetts 02115, USA
6Departments of Pediatrics and Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, 77030, USA
*These authors contributed equally to this work.
7Correspondence should be addressed to R.C.M. We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.
REFERENCES
- Metcalf, D. Haemopoietic Cells p. 550 (North-Holland, Amsterdam, 1971).
- 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. 175, 175−184 (1992).
- Spangrude, G.J., Heimfeld, S. & Weissman, I.L. Purification and characterization of mouse hematopoietic stem Cells. Science 241, 58−62 (1988).
- Mulder, A., Bauman, J., Visser, J., Boersma, W. & van, den Engh, G.I. Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant. Cell. Immunol. 88, 401−410 (1984).
- Okada, S. et al. In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic Cells. Blood 80, 3044−3050 (1992).
- Katayama, N. et al. Stage-specific expression of c-kit protein by murine hematopoietic progenitors. Blood 82, 2353−2360 (1993).
- Sutherland, H.J., Eaves, C.J., Eaves, A.C., Dragowska, W. & Lansdorp, P.M. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74, 1563−1570 (1989).
- Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C. & Eaves, C.J. Functional characterization of individual human hematopoietic stem Cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. USA 87, 3584−3588 (1990).
- Baum, C., Weissman, I., Tsukamoto, A., Buckle, A. & Peault, B. Isolation of a candidate human hematopoietic stem-cell population. Proc. Natl. Acad. Sci. USA 89, 2804−2808 (1992).
- Huang, S. & Terstappen, L. Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38- hematopoietic stem Cells. Blood 83, 1515−1526 (1994).
- Berardi, A., Wang, A., Levine, J., Lopez, P. & Scadden, D. Functional isolation and characterization of human hematopoietic stem Cells. Science 267, 104−108 (1995).
- Petzer, A.L., Hogge, D.E., Landsdorp, P.M., Reid, D.S. & Eaves, C.J. Self-renewal of primitive human hematopoietic Cells (long-term-culture-initiating Cells) in vitro and their expansion in defined medium. Proc. Natl. Acad. Sci. USA 93, 1470−1474 (1996).
- Larochelle, A. et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy. Nature Med. 2, 1329−1337 (1996).
- Hao, Q., Thiemann, F., Petersen, D., Smogorzewska, E. & Crooks, G. Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population. Blood 88, 3306−3313 (1996).
- Goodell, M., Brose, K., Paradis, G., Conner, A. & Mulligan, R. Isolation and functional properties of murine hematopoietic stem Cells that are replicating in vivo. J. Exp. Med. 183, 1797−1806 (1996).
- Krause, D. et al. Characterization of murine CD34, a marker for hematopoietic progenitor and stem Cells. Blood 84, 691−701 (1994).
- Morel, F., Szilvassy, S., Travis, M., Chen, B. & Galy, A. Primitive hematopoietic Cells in murine bone marrow express the CD34 antigen. Blood 88, 3774−3784 (1996).
- Osawa, M., Hanada, K., Hamada, H. & Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem Cell. Science 273, 242−245 (1996).
- Jones, R. et al. Characterization of mouse lymphohematopoietic stem Cells lacking spleen colony-forming activity. Blood 88, 487−491 (1996).
- Broxmeyer, H. et al. Human umbilical cord blood: A clinically useful source of transplantable hematopoietic stem/progenitor Cells. Int. J. Cell Cloning 8 (Suppl. 1), 76−89; discussion 89−91 (1990).
- Olweus, J., Lund-Johansen, F., Terstappen, L.W.M.M. Expression of Cell surface markers during differentiation of CD34+, CD38-/lo fetal and adult bone marrow Cells. Immunomethods 5, 179−188 (1994).
- Barcena, A. et al. Phenotypic and functional analysis of T-Cell precursors in the human fetal liver and thymus: CD7 expression in the early stages of T-and myeloid-Cell development. Blood 82, 3401−3414 (1993).
- Rosenzweig, M. et al. In vitro T lymphopoiesis of human and rhesus CD34+ progenitor Cells. Blood 87, 4040−4048 (1996).
- Zanjani, E.D., Flake, A.W., Rice, H., Hedrick, M., Tavassoli, M. Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem Cells in sheep. J. Clin. Invest. 93, 1051−1055 (1994).
- Dunbar, C. Gene transfer to hematopoietic stem Cells: Implications for gene therapy of human disease. Annu. Rev. Med. 47, 11−20 (1996).
- Berenson, R. et al. Engraftment after infusion of CD34+ marrow Cells in patients with breast cancer or neuroblastoma. Blood 77, 1717−1722 (1991).
- Civin, C. et al. Highly purified CD34-positive Cells reconstitute hematopoiesis. J Clin Oncol. 14, 2224−2233 (1996).
- Kohn, D. et al. Engraftment of gene-modified umbilical cord blood Cells in neonates withadenosine deaminase deficiency. Nature Med. 1, 1017−1023 (1995).
- Guide for the Care and Use of Laboratory Animals. DHHS Publication No. [NIH] 85−23 (US Dept. of Health and Human Services, Bethesda, MD, 1985).
- Kawai, T., Wong, J., MacLean, J., Cosimi, A.B., Wee, S. Characterization of a monoclonal antibody (6G12) recognizing the cynomolgus monkey CD3 antigen. Transplant. Proc. 26, 1845−1846 (1994).
- Sutherland, D.R. & Eaves, C.J. Culture of Hematopoietic Cells. pp. 139−162 (Wiley-Liss, 1994).
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