Abstract
Early hemopoietic precursors have been extensively studied using short- term assays based on colony formation or in vivo reconstitution that do not run beyond a few weeks. However, little information is available on the phenotype of the stem cells that are detectable in 6-12-mo transplantation assays, and their relationship to cells detected in short-term assays is not known. In this study, we investigated the phenotype and separability by cell sorting of a spectrum of hemopoietic precursor cells in normal adult mouse marrow, including cells quantitated in a 1 yr competitive transplantation assay in vivo as well as in short-term colony assays in vitro and in vivo. Two principal findings emerged. The first was that cells detected in a variety of short-term assays--CFU-S12 (spleen colony-forming cells), CFCmulti (multilineage colony-forming cells), pre-CFCmulti (precursors of CFCmulti), CFC-E/Mg (erythroid/megakaryocyte CFC) and CFC-G/M (granulocyte/macrophage CFC)--were phenotypically similar and could not be separated from one another using a panel of markers useful in segregating them from more differentiated cells, including buoyant density, sedimentation velocity, adhesiveness to plastic, light scatter, high rhodamine-123 retention, and expression of surface wheat- germ agglutinin (WGA)-binding carbohydrate, H-2K, CD45, AA4.1, heat stable antigen (HSA), CD71, and Ly6A/Sca-1 antigens. Long-term reconstituting (LTR) cells quantitated in vivo differed little from the other precursors in expression of many of the above markers. However, they differed somewhat in lower sedimentation velocity and lower expression of WGA-binding surface carbohydrate, and most strikingly in their conditional adhesiveness to plastic, very low retention of Rh123 and high level expression of Ly6A/Sca-1, to a degree that would permit the quantitative separation of the two precursor classes from each other. The results provide a comprehensive characterization of LTR cells measured to 12 mo in vivo and a direct and quantitative analysis of their separation from cells detected in colony assays.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aihara Y., Bühring H. J., Aihara M., Klein J. An attempt to produce "pre-T" cell hybridomas and to identify their antigens. Eur J Immunol. 1986 Nov;16(11):1391–1399. doi: 10.1002/eji.1830161113. [DOI] [PubMed] [Google Scholar]
- Basch R. S., Oh Y. D., Saha C. S., Fredrickson G. G., Hirst J. A. Separation of self-renewing hematopoietic progenitors on the basis of CD45 (T-200) antigen expression. Exp Hematol. 1992 Jan;20(1):11–16. [PubMed] [Google Scholar]
- 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]
- Boggs D. R., Boggs S. S., Saxe D. F., Gress L. A., Canfield D. R. Hematopoietic stem cells with high proliferative potential. Assay of their concentration in marrow by the frequency and duration of cure of W/Wv mice. J Clin Invest. 1982 Aug;70(2):242–253. doi: 10.1172/JCI110611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boggs D. R., Saxe D. F., Boggs S. S. Aging and hematopoiesis. II. The ability of bone marrow cells from young and aged mice to cure and maintain cure in W/Wv. Transplantation. 1984 Mar;37(3):300–306. [PubMed] [Google Scholar]
- Bruce J., Symington F. W., McKearn T. J., Sprent J. A monoclonal antibody discriminating between subsets of T and B cells. J Immunol. 1981 Dec;127(6):2496–2501. [PubMed] [Google Scholar]
- Capel B., Hawley R. G., Mintz B. Long- and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers. Blood. 1990 Jun 15;75(12):2267–2270. [PubMed] [Google Scholar]
- Eppig J. J., Kozak L. P., Eicher E. M., Stevens L. C. Ovarian teratomas in mice are derived from oocytes that have completed the first meiotic division. Nature. 1977 Oct 6;269(5628):517–518. doi: 10.1038/269517a0. [DOI] [PubMed] [Google Scholar]
- Fogh J. Cultivation, characterization, and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl Cancer Inst Monogr. 1978 Dec;(49):5–9. [PubMed] [Google Scholar]
- 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]
- 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]
- Harrison D. E., Jordan C. T., Zhong R. K., Astle C. M. Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations. Exp Hematol. 1993 Feb;21(2):206–219. [PubMed] [Google Scholar]
- Harrison D. E., Lerner C. P. Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood. 1991 Sep 1;78(5):1237–1240. [PubMed] [Google Scholar]
- Harrison D. E. Lifesparing ability (in lethally irradiated mice) of W-W mouse marrow with no macroscopic colonies. Radiat Res. 1972 Dec;52(3):553–563. [PubMed] [Google Scholar]
- Harrison D. E., Zhong R. K. The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10134–10138. doi: 10.1073/pnas.89.21.10134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hodgson G. S., Bradley T. R. Properties of haematopoietic stem cells surviving 5-fluorouracil treatment: evidence for a pre-CFU-S cell? Nature. 1979 Oct 4;281(5730):381–382. doi: 10.1038/281381a0. [DOI] [PubMed] [Google Scholar]
- Iscove N. N., Shaw A. R., Keller G. Net increase of pluripotential hematopoietic precursors in suspension culture in response to IL-1 and IL-3. J Immunol. 1989 Apr 1;142(7):2332–2337. [PubMed] [Google Scholar]
- Iscove N. N., Yan X. Q. Precursors (pre-CFCmulti) of multilineage hemopoietic colony-forming cells quantitated in vitro. Uniqueness of IL-1 requirement, partial separation from pluripotential colony-forming cells, and correlation with long term reconstituting cells in vivo. J Immunol. 1990 Jul 1;145(1):190–195. [PubMed] [Google Scholar]
- 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]
- 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]
- Jordan C. T., McKearn J. P., Lemischka I. R. Cellular and developmental properties of fetal hematopoietic stem cells. Cell. 1990 Jun 15;61(6):953–963. doi: 10.1016/0092-8674(90)90061-i. [DOI] [PubMed] [Google Scholar]
- Karasuyama H., Melchers F. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur J Immunol. 1988 Jan;18(1):97–104. doi: 10.1002/eji.1830180115. [DOI] [PubMed] [Google Scholar]
- Keller G., Snodgrass R. Life span of multipotential hematopoietic stem cells in vivo. J Exp Med. 1990 May 1;171(5):1407–1418. doi: 10.1084/jem.171.5.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerk D. K., Henry E. A., Eaves A. C., Eaves C. J. Two classes of primitive pluripotent hemopoietic progenitor cells: separation by adherence. J Cell Physiol. 1985 Oct;125(1):127–134. doi: 10.1002/jcp.1041250117. [DOI] [PubMed] [Google Scholar]
- Kiefer F., Wagner E. F., Keller G. Fractionation of mouse bone marrow by adherence separates primitive hematopoietic stem cells from in vitro colony-forming cells and spleen colony-forming cells. Blood. 1991 Nov 15;78(10):2577–2582. [PubMed] [Google Scholar]
- Kincade P. W., Lee G., Watanabe T., Sun L., Scheid M. P. Antigens displayed on murine B lymphocyte precursors. J Immunol. 1981 Dec;127(6):2262–2268. [PubMed] [Google Scholar]
- Kitamura Y., Yokoyama M., Matsuda H., Ohno T., Mori K. J. Spleen colony-forming cell as common precursor for tissue mast cells and granulocytes. Nature. 1981 May 14;291(5811):159–160. doi: 10.1038/291159a0. [DOI] [PubMed] [Google Scholar]
- Ledbetter J. A., Herzenberg L. A. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979;47:63–90. doi: 10.1111/j.1600-065x.1979.tb00289.x. [DOI] [PubMed] [Google Scholar]
- Lerner C., Harrison D. E. 5-Fluorouracil spares hemopoietic stem cells responsible for long-term repopulation. Exp Hematol. 1990 Feb;18(2):114–118. [PubMed] [Google Scholar]
- Lesley J., Hyman R., Schulte R., Trotter J. Expression of transferrin receptor on murine hematopoietic progenitors. Cell Immunol. 1984 Jan;83(1):14–25. doi: 10.1016/0008-8749(84)90220-x. [DOI] [PubMed] [Google Scholar]
- 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]
- Lord B. I., Spooncer E. Isolation of haemopoietic spleen colony forming cells. Lymphokine Res. 1986 Winter;5(1):59–72. [PubMed] [Google Scholar]
- Mauch P., Greenberger J. S., Botnick L., Hannon E., Hellman S. Evidence for structured variation in self-renewal capacity within long-term bone marrow cultures. Proc Natl Acad Sci U S A. 1980 May;77(5):2927–2930. doi: 10.1073/pnas.77.5.2927. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKearn J. P., Baum C., Davie J. M. Cell surface antigens expressed by subsets of pre-B cells and B cells. J Immunol. 1984 Jan;132(1):332–339. [PubMed] [Google Scholar]
- Miller B. A., Antognetti G., Springer T. A. Identification of cell surface antigens present on murine hematopoietic stem cells. J Immunol. 1985 May;134(5):3286–3290. [PubMed] [Google Scholar]
- Mulder A. H., Visser J. W. Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence. Exp Hematol. 1987 Jan;15(1):99–104. [PubMed] [Google Scholar]
- Muller-Sieburg C. E., Whitlock C. A., Weissman I. L. Isolation of two early B lymphocyte progenitors from mouse marrow: a committed pre-pre-B cell and a clonogenic Thy-1-lo hematopoietic stem cell. Cell. 1986 Feb 28;44(4):653–662. doi: 10.1016/0092-8674(86)90274-6. [DOI] [PubMed] [Google Scholar]
- Okada S., Nakauchi H., Nagayoshi K., Nishikawa S., Miura Y., Suda T. In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. 1992 Dec 15;80(12):3044–3050. [PubMed] [Google Scholar]
- Orlic D., Bodine D. M. Pluripotent hematopoietic stem cells of low and high density can repopulate W/Wv mice. Exp Hematol. 1992 Dec;20(11):1291–1295. [PubMed] [Google Scholar]
- Ploemacher R. E., Brons N. H. Isolation of hemopoietic stem cell subsets from murine bone marrow: II. Evidence for an early precursor of day-12 CFU-S and cells associated with radioprotective ability. Exp Hematol. 1988 Jan;16(1):27–32. [PubMed] [Google Scholar]
- 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]
- Ploemacher R. E., van der Loo J. C., van Beurden C. A., Baert M. R. Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo. Leukemia. 1993 Jan;7(1):120–130. [PubMed] [Google Scholar]
- Quesniaux V. F., Graham G. J., Pragnell I., Donaldson D., Wolpe S. D., Iscove N. N., Fagg B. Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo. Blood. 1993 Mar 15;81(6):1497–1504. [PubMed] [Google Scholar]
- SIMINOVITCH L., MCCULLOCH E. A., TILL J. E. THE DISTRIBUTION OF COLONY-FORMING CELLS AMONG SPLEEN COLONIES. J Cell Physiol. 1963 Dec;62:327–336. doi: 10.1002/jcp.1030620313. [DOI] [PubMed] [Google Scholar]
- 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]
- Snodgrass R., Keller G. Clonal fluctuation within the haematopoietic system of mice reconstituted with retrovirus-infected stem cells. EMBO J. 1987 Dec 20;6(13):3955–3960. doi: 10.1002/j.1460-2075.1987.tb02737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Springer T., Galfrè G., Secher D. S., Milstein C. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur J Immunol. 1978 Aug;8(8):539–551. doi: 10.1002/eji.1830080802. [DOI] [PubMed] [Google Scholar]
- Suda J., Suda T., Ogawa M. Analysis of differentiation of mouse hemopoietic stem cells in culture by sequential replating of paired progenitors. Blood. 1984 Aug;64(2):393–399. [PubMed] [Google Scholar]
- Szilvassy S. J., Cory S. Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow. Blood. 1993 May 1;81(9):2310–2320. [PubMed] [Google Scholar]
- Szilvassy S. J., Fraser C. C., Eaves C. J., Lansdorp P. M., Eaves A. C., Humphries R. K. Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8798–8802. doi: 10.1073/pnas.86.22.8798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- TILL J. E., MCCULLOCH E. A., SIMINOVITCH L. A STOCHASTIC MODEL OF STEM CELL PROLIFERATION, BASED ON THE GROWTH OF SPLEEN COLONY-FORMING CELLS. Proc Natl Acad Sci U S A. 1964 Jan;51:29–36. doi: 10.1073/pnas.51.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TILL J. E., McCULLOCH E. A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res. 1961 Feb;14:213–222. [PubMed] [Google Scholar]
- Till J. E., McCulloch E. A. The 'f-factor' of the spleen-colony assay for hemopoietic stem cells. Ser Haematol. 1972;5(2):15–21. [PubMed] [Google Scholar]
- Trowbridge I. S., Lesley J., Schulte R. Murine cell surface transferrin receptor: studies with an anti-receptor monoclonal antibody. J Cell Physiol. 1982 Sep;112(3):403–410. doi: 10.1002/jcp.1041120314. [DOI] [PubMed] [Google Scholar]
- 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]
- Visser J. W., de Vries P. Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling. Blood Cells. 1988;14(2-3):369–384. [PubMed] [Google Scholar]
- Wingfield P., Payton M., Tavernier J., Barnes M., Shaw A., Rose K., Simona M. G., Demczuk S., Williamson K., Dayer J. M. Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli. Eur J Biochem. 1986 Nov 3;160(3):491–497. doi: 10.1111/j.1432-1033.1986.tb10066.x. [DOI] [PubMed] [Google Scholar]
- Worton R. G., McCulloch E. A., Till J. E. Physical separation of hemopoietic stem cells differing in their capacity for self-renewal. J Exp Med. 1969 Jul 1;130(1):91–103. doi: 10.1084/jem.130.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van de Rijn M., Heimfeld S., Spangrude G. J., Weissman I. L. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4634–4638. doi: 10.1073/pnas.86.12.4634. [DOI] [PMC free article] [PubMed] [Google Scholar]