Information de reference pour ce titreAccession Number: | 00007529-199607120-00014.
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Author: | Osawa, Masatake *; Hanada, Ken-ichi; Hamada, Hirofumi; Nakauchi, Hiromitsu **
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Institution: | M. Osawa and H. Nakauchi, Department of Immunology, Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science-City, Ibaraki 305, Japan. K.-i. Hanada and H. Hamada, Department of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170, Japan. *Present address: KIRIN Pharmaceutical Research Laboratory, Gunma 371, Japan. **To whom correspondence should be addressed.
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Title: | Long-Term Lymphohematopoietic Reconstitution by a Single CD34-Low/Negative Hematopoietic Stem Cell.[Report]
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Source: | Science. 273(5272):242-245, July 12, 1996.
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Abstract: | Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the mCD34 low to negative fraction. Injection of a single mCD34 (lo/minus), c-Kitplus, Sca-1plus, lineage markers negative (Linminus) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21 percent of recipients. Thus, the purified HSC population should enable analysis of the self-renewal and multilineage differentiation of individual HSCs.
Copyright (C) 1996 by the American Association for the Advancement of Science
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References: | 1. C. I. Civin et al., J. Immunol. 133, 157 (1984); C. M. Baum, I. L. Weissman, A. S. Tsukamoto, A. M. Buckle, B. Peault, Proc. Natl. Acad. Sci. U.S.A. 89, 2804 (1992).
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3. J. Brown, M. F. Greaves, H. V. Molgaard, Int. Immunol. 3, 175 (1991); J. Suda et al., Blood 79, 2288 (1992).
4. M. Osawa, K.-i. Hanada, H. Hamada, H. Nakauchi, unpublished results.
5. ``The BM cell suspension was prepared from C57BL/6 mice (8 to 10 weeks old) and stained with biotinylated antibodies to lineage (anti-lineage) markers (Mac-1, Gr-1, B220, CD4, CD8, and TER119), fluorescein isothiocyanate (FITC)-mCD34 (49E8), phycoerythrin (PE)-Sca-1 (E13-161.7; Pharmingen), and allophycocyanin (APC)-c-Kit (ACK-2). Biotinylated antibodies were detected with Texas red-streptavidin (Gibco-BRL). Stained cells were suspended in staining medium [phosphate-buffered saline, 0. 05% NaN3, and 3% fetal bovine serum (FBS)] containing propidium iodide (PI) (1 micro gram/ml) and analyzed on a FACS Vantage (Becton Dickinson). Residual erythrocytes, debris, doublets, and dead cells were excluded by forward scatter, side scatter, and PI gatings as described [6].''
6. S. Okada et al., Blood 80, 3044 (1992); M. Osawa et al., J. Immunol. 156, 3207 (1996).
7. ``Bone marrow cells were obtained from the tibias and femurs of C57BL/6 mice. The suspension was overlaid with sodium metrizoate (Nycomed; Oslo, Norway) solution (1.086 g/ml) and centrifuged at 400g for 15 min. The low-density cells were harvested and incubated with biotinylated anti-lineage markers (Mac-1, Gr-1, B220, CD4, CD8, and TER119).Linplus cells were depleted with streptavidin-conjugated magnetic beads (PerSeptive Diagnostics, Cambridge, MA). The lineage-depleted cell population was then collected and incubated with FITC-anti-mCD34, PE-Sca-1, Texas red-streptavidin, and APC-anti-c-Kit. Stained cells were sorted with a Clone Cyt (Becton Dickinson) apparatus to deposit the required number of cells into 96-well microtiter plates. Two hundred mCD34minus or mCD34plus cells within the c-Kitplus Sca-1plus Linminus population were sorted and subjected to in vitro colony assay. The sorted cells were plated in 1.0% methylcellulose in alpha-medium (Flow Laboratories, North Ryde, Australia) supplemented with 30% FBS, 1% bovine serum albumin (BSA), 2-mercaptoethanol (10minus 4 mol/liter), and recombinant mouse IL-3 (20 ng/ml) or IL-3 plus stem cell factor (SCF, 20 ng/ml). The colonies that were formed by the stimulation of IL-3 or IL-3 plus SCF were counted after 14 days of incubation at 37 degrees C in a humidified atmosphere of 5% CO2.''
8. ``Bone marrow cells were stained and sorted as described [7]. Two hundred mCD34minus c-Kitplus Sca-1plus Linminus cells or mCD34plus c-Kitplus Sca-1plus Linminus cells were injected intravenously into lethally irradiated mice [9. 5 gray (Gy) total body irradiation]. The spleens were removed on day 12 after injection and fixed in Bouin's solution, and macroscopically visible spleen colonies were counted.''
9. M. Osawa and H. Nakauchi, data not shown.
10. R. J. Jones, J. E. Wagner, P. Celano, M. S. Zicha, S. J. Sharkis, Nature 347, 188 (1990).
11. ``Bone marrow c-Kitplus Sca-1plus Linminus cells were fractionated according to the extent of mCD34 expression. One hundred Fr. 1 or Fr. 2 cells or 500 Fr. 3 cells Figure 2A from Ly5.1 mice were sorted into a well containing 2 times 104 lineage-depleted BM cells from C57BL/6-Ly5.2 mice. In a preliminary experiment, we determined that 2 times 104 lineage-depleted BM cells were the minimum number of cells required for the survival of lethally irradiated recipients and reconstitution of their hematopoiesis. The cells in each well were collected and transferred into lethally irradiated C57BL/6-Ly5.2 mice. Several weeks to months after the transplantation, peripheral blood mononuclear cells were collected from the retro-orbital sinus and stained with FITC-anti-Ly5.1, PE-anti-myeloid (Mac-1 and Gr-1), and APC-anti-lymphoid (Thy1.2 and B220). The proportion of myeloid and lymphoid cells that originated from Ly5.1 cells was estimated by flow cytometry as described [S. Okada et al., Blood 81, 1720 (1993)].''
12. D. S. Krause et al., Blood 84, 691 (1994).
13. M. Onishi et al., ibid. 81, 3217 (1993); J. P. Wineman et al., ibid. 80, 1717 (1992).
14. ``One thousand Fr. 1, Fr. 2, or Fr. 3 cells Figure 2A were sorted and lysed in 50 micro liter of Isogen-LS (Nippon Gene, Tokyo). Total RNA was isolated according to the manufacturer's protocol. The first-strand cDNA reaction was carried out with 100 U of Molony murine leukemia virus reverse transcriptase (Gibco-BRL) in 20 mM tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.5 mM deoxynucleoside triphosphate (dNTP), 10 mM DTT, and 0.25 micro gram of oligo(dT)12-18. For the analysis of mCD34 and hypoxanthine phosphoribosyl transferase (HPRT) gene expression, the following specific primers were used to amplify fragments from one-half of the cDNAs obtained from each of the fractions: mCD34 [5 prime-ATGCAGGTCCACAGGGACACG-3 prime and 5 prime-CTGTCCTGATAGATCAAGTAG-3 prime, generating a 221-base pair (bp) fragment]; and HPRT (5 prime-GCTGGTGAAAAGGACCTCTCG-3 prime and 5 prime-GCAGATGGCCACAGGACTAGA-3 prime, generating a 258-bp fragment). PCR conditions were as follows: 20 mM tris-HCl (pH 8. 4), 50 mM KCl, 1. 5 mM MgCl2, 0. 2 mM dNTPs, 2. 5 U of Ex Taq (Takara Ohtsu, Japan), and 50 pmol of each primer for 30 cycles (94 degrees C, 30 s; 55 degrees C, 30 s; 72 degrees C, 45 s) followed by 5 min at 72 degrees C in a Perkin-Elmer thermocycler. Each PCR product was electrophoresed through 2% agarose, transferred to a nylon membrane, and hybridized with a biotinylated internal oligonucleotide. Hybridized probes were detected with an ECL system (Amersham Life Sciences) according to the manufacturer's protocol.''
15. L. G. Smith, I. L. Weissman, S. Heimfeld, Proc. Natl. Acad. Sci. U.S.A. 88, 2788 (1991).
16. J. Cheng et al., Blood 87, 479 (1996).
17. E. F. Srour et al., ibid. 82, 3333 (1993).
18. ``We thank H. Kodama for discussion, T. Toyoshima for FACS operation, M. Ito for secretarial assistance, and C. Tarlinton for reading the manuscript. Supported by grants from Fujisawa Pharmaceutical, Uehara Memorial Foundation, The Ministry of Education, Science, and Culture, and the Agency for Science and Technology, Japan.''
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Language: | English.
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Document Type: | Reports.
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Journal Subset: | Life Sciences. Physical Science & Engineering.
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ISSN: | 0036-8075
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NLM Journal Code: | 0404511, uj7
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