Stem Cells, Vol. 14, No. 5, 584-591,
September 1996
© 1996 AlphaMed Press
Changes in Markers, Receptors and Adhesion Molecules Expressed on Murine Hemopoietic Stem Cells After a Single Injection of 5-Fluorouracil
Nobuhiro Nishioa,
Hiroko Hishaa,
Hajime Ogatab,
Muneo Inabaa,
Yoshihisa Yamamotoa,
Yasuo Amoha,
Ryoji Yasumizua,
Ken-ichi Hanadac,
Hirofumi Hamadac,
Susumu Ikeharaa
a First Department of Pathology,
b Pediatrics, Kansai Medical University, Osaka, Japan;
c Department of Molecular Biology, Cancer Institute, Tokyo, Japan
Key Words. Hemopoietic stem cells • c-kit • CD34 • IL-6R • Sca-1
Correspondence: Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570, Japan.
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Abstract
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Cytokines play a crucial role in the differentiation and proliferation of hemopoietic cells, and it has recently been found that adhesion molecules play crucial roles not only in differentiation and proliferation, but also in the homing and other functions of hemopoietic cells. We have very recently established a new method for purifying pluripotent hemopoietic stem cells (P-HSC) in mice by injecting 5-fluorouracil (5-FU). The P-HSC were found to be low-density, lineage marker-negative (Lin–), CD71– and major histocompatibility complex class Ihigh. In the present study, we analyze changes in the expression of various HSC markers (Sca-1 and CD34), receptors (c-kit and interleukin-6 receptor [IL-6R]) and adhesion molecules (very late activation antigen-4 [VLA-4], lymphocyte function-associated antigen-1 [LFA-1], and CD44) after 5-FU injection. The percentage of Sca-1+ cells increases after 5-FU treatment, reaching a maximum on day 3, whereas the percentage of IL-6R+ cells decreases, reaching a minimum on day 3. The percentage of CD34+ cells does not change after 5-FU treatment. The percentages of both c-kitlow and c-kithigh cells decrease, reaching a minimum on day 3 after 5-FU treatment, whereas the percentage of c-kit– cells reciprocally increases, reaching a maximum on day 3. However, there is no change in the expression of adhesion molecules (VLA-4, LFA-1 and CD44) on the P-HSC.
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Introduction
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An effective method of purifying pluripotent hemopoietic stem cells (P-HSC) would contribute not only to clarifying the mechanism behind hemopoietic differentiation and self-renewal of P-HSC, but also to the use of P-HSC in bone marrow transplantation (BMT). Until now, a number of laboratories have reported different purification procedures, including those based on physical characteristics (size and cell density), negative selection to remove lineage-committed cells and positive selection using various markers [1-9]. Visser et al. purified stem cells using density centrifugation followed by positive selection, utilizing both binding ability to wheat germ agglutinin (WGA) and anti-H-2KAb with a cell sorter [2]. It is also known that the administration of high doses of 5-fluorouracil (5-FU) preferentially kills cells in the cycling phase while sparing more primitive dormant cells [10-17]. We have purified dormant stem cells (WGA-binding cells: WGA+ cells) after 5-FU treatment (both in vivo and in vitro) [3]. Spangrude et al. purified Lin–, Thy-1low and Sca-1+ cells as HSC [5]. Smith et al. have reported that 7% of Lin–, Thy-1low and Sca-1+ cells show pluripotent hemopoietic ability in vivo [18]. Jones et al. separated P-HSC using counterflow centrifugal elutriation based on size and density [9].
We have previously reported that interleukin 3 receptor-negative (IL-3R–) progenitor cells are more primitive than IL-3R+ cells [4]. It has also been reported that the transferrin receptor (CD71) is expressed on proliferating committed progenitors but not on noncycling immature progenitors [19-21], and that P-HSC express a high level of major histocompatibility complex (MHC) class I but not class II molecules [22-24]. Based on these findings, we have very recently succeeded in purifying P-HSC. P-HSC were purified from bone marrow cells on day 4 after a single i.v. injection of 5-FU, followed by sorting CD71– class Ihigh cells. This method achieved a 2000-fold enrichment of P-HSC [22]. When only four male P-HSC, thus purified, were transplanted into syngeneic female recipients together with female compromised cells, the production of donor-derived lymphoid and myeloid cells was observed for at least eight months after the transplantation [22]. Accordingly, it seems that long-term hematopoietic repopulation may result from even these few P-HSC.
In the present study, using this purification method, we investigate changes in the expression of various markers (Sca-1 and CD34), receptors (c-kit and IL-6R) and adhesion molecules (very late activation antigen-4 [VLA-4], lymphocyte function-associated antigen-1 [LFA-1] and CD44) on P-HSC.
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Materials and Methods
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Purification of P-HSC
Female C3H/HeJ mice (8 to 12 weeks old) were purchased from Japan Clea Inc. (Osaka, Japan) and maintained in our animal facility. 5-FU (150 mg/kg) was injected i.v. into the mice one to six days before killing. Bone marrow cell (BMC) suspensions were obtained from the femurs, tibias and humeri through needles. The BMC suspensions were fractionated on Percoll (Pharmacia; Uppsala, Sweden) discontinuous density gradient. The low-density fraction (LD) cells, ranging between 1.063 and 1.075 (40% and 60% Percoll solutions), were collected, and lineage marker-positive cells then removed using immunomagnetic beads. Briefly, the LD cells were incubated with a cocktail of anti-CD4 (RM4-5, rat IgG2b class), anti-CD8 (53-6,7, IgG2a), anti-B220 (RA3-6B2, IgG2a), anti-Mac-1 (M1/70, IgG2b), anti-Gr-1 (RB6-8C5, IgG2b) and TER119 monoclonal antibodies (mAb). Except TER119 mAb, these mAb were purchased from Pharmingen (San Diego, CA). The TER119 mAb was kindly donated by Dr. Kina, Department of Molecular Pathology, Chest Disease Research Institute, Kyoto University. After washing, the labeled cells were removed using immunomagnetic beads coated with sheep antirat IgG (Dynabeads M-450; Dynal & Co.; Oslo, Norway). LD/Lin– cells were used for subsequent three-color staining.
Antibodies
mAb against H-2Kk (clone: AF3-12.1), CD71 (C2), LFA-1 (M17/4), VLA-4 (R1-2), CD44 (1M7) and Sca-1 (E13 161-7) antigens (Ag) were purchased from Pharmingen. A mAb against IL-6R (RS11) was a kind gift from Prof. Tadamitsu Kishimoto, 3rd Department of Internal Medicine, Osaka University. A mAb against c-kit (ACK4) was a kind gift from Prof. Shin-ichi Nishikawa, Department of Molecular Genetics, Kyoto University [25]. A mAb against CD34 was a kind gift from Drs. Ken-ichi Hanada and Hirofumi Hamada, Department of Molecular Biology, Cancer Institute, Tokyo. Fluorescein isothiocyanate-conjugated-avidin (FITC-avidin) and phycoerythrin-coupled avidin (PE-avidin) were purchased from Becton Dickinson (Mountain View, CA). Cychrome-avidin was purchased from Pharmingen.
Labeling of Cells and Immunofluorescence Analyses
LD/Lin– cells were divided into aliquots; stained with anti-IL-6R (RS11), anti-LFA-1, anti-VLA-4 or anti-CD44 mAb, respectively; then stained with FITC-conjugated mouse antirat Ig. The cells were further stained with normal rat Ig, followed by anti-CD71-PE, biotinated-H-2K and cychrome-avidin. The LD/Lin– cells were also stained with biotinated-anti-Sca-1 or biotinated-anti-CD34 mAb, followed by cychrome-avidin, anti-H-2K-FITC and anti-CD71-PE. The cells were analyzed on a FACScan (Becton Dickinson; San Jose, CA). Forward-angle and perpendicular-light scatter were used to eliminate any aggregated cells or debris. Data analyses were carried out using the Lysis II software.
All experiments were carried out three times or more. Reproducible results were obtained, and only representative data are shown here.
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Results
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Effects of 5-FU Treatment on Cell Numbers at Various Purification Steps
BMC were collected from days 1 to 6 after a single i.v. injection of 5-FU. LD cells were obtained by Percoll discontinuous density gradient. The LD cells were further purified by negative selection using immunomagnetic beads to obtain LD/Lin– cells. Finally, CD71–H-2high cells (P-HSC) were sorted using a FACStar. The number of cells obtained per mouse at each purification step is shown in Figure 1
. The number of LD/Lin– cells decreased one day after 5-FU treatment and reached a minimum level on day 3, whereas an increase in cell number was observed from day 4 after 5-FU treatment. The numbers of LD cells and whole BMC began to increase on days 5 and 6 after 5-FU injection, respectively. These time lags in the recovery of BMC, LD cells and LD/Lin– cells after 5-FU treatment seem to reflect the recovery of hemopoiesis from P-HSC. Since P-HSC are 5-FU-resistant and contained in the LD/Lin– population, it seems that an approximately 1000-fold enrichment of P-HSC is obtained in the LD/Lin– cells on day 3 or 4 after 5-FU treatment (approximately 108 original whole BMC —> 105 LD/Lin– cells (on day 3 or 4)), as we previously reported [22].
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Figure 1. Changes in cell counts of BMC, LD cells and LD/Lin– cells per mouse after single injection of 5-FU. Bone marrow cells from femurs, tibias and humeruses after single 5-FU injection (150 mg/kg i.v.) were collected each day for six days. The numbers of whole BMC, LD cells and LD/Lin– cells are shown.
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Changes in P-HSC Markers after 5-FU Treatment
LD/Lin– cells obtained from 5-FU-treated or nontreated (control) mice were three-color-stained using anti-H-2, anti-CD71, anti-IL-6R, anti-c-kit, anti-CD44, anti-VLA-4, anti-LFA-1 and anti-CD34 mAb. Figure 2A shows the typical forward- and side-scatter pattern of stained cells from control mice, while Figure 2B shows the staining pattern of CD71 and H-2. The square gate in Figure 2B shows the H-2high and CD71– population. The solid lines in Figure 2C show the fluorescence-intensity pattern of cells in the blast window of Figure 2A (control mice), whereas the dotted lines show a representative pattern of 5-FU-treated mice. The cells were separated into two (negative and positive) or three (negative, low-positive and high-positive) fractions according to the indicated parameters. The criteria for positively stained cells in the H-2high and CD71– population are shown in Figure 2C. Figure 2D shows the daily change in the percentage of positive cells by various mAb after 5-FU treatment.
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Figure 2. Changes in markers (c-kit, IL-6R, Sca-1 and CD34) expressed on Lin–CD71– H-2high cells after single 5-FU treatment. A) Typical forward- and side-scatter patterns of blast cells from control mice. The square gate shows the blast window. B) Coexpression of CD71 and H-2K on blast cells from control mice. The square gate shows a CD71– and H-2high fraction. C) Fluorescence-intensity patterns of cells in the blast window of (A) are shown; the cells were separated into two (negative and positive) fractions, or three (negative, low-positive and high-positive) fractions according to the indicated parameters. D) Daily changes in the percentage of positive fractions of Lin–CD71– H-2high cells stained with various mAb. The c-kit– fraction is shown in the same figure.
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c-kit Expression
In non-5-FU-treated control mice, 70% of the CD71– class Ihigh cells were c-kit–, 20% were c-kitlow, and 10% were c-kithigh (Fig. 2D
). After 5-FU treatment, the percentages of c-kitlow and c-kithigh cells decreased; both fractions reached a minimum level on day 3 after 5-FU treatment. In contrast, the percentage of c-kit– cells increased; most P-HSC were c-kit– on day 3 after 5-FU injection.
IL-6R Expression
A significant decrease in the percentage of IL-6R+ cells was observed after 5-FU treatment; the percentage declined from 40% to 8% on day 3 after 5-FU treatment (Fig. 2D). The expression, however, increased thereafter and reached the control level on day 6.
Sca-1 Expression
It has been shown that most primitive stem cells are Sca-1+. We have confirmed that an increase in Sca-1+ cells is seen on day 3 after 5-FU treatment (Fig. 2D).
CD34 and CD44 Expression
No apparent changes in the percentages of CD34+ and CD44+ cells on P-HSC were observed after 5-FU treatment (Figs. 2D and 3B).
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Figure 3. Changes in adhesion molecules (CD44, VLA-4 and LFA-1) expressed on Lin–CD71– H-2high cells after single 5-FU treatment. A) Fluorescence-intensity patterns of cells in the blast window are shown; the cells were separated into two (negative and positive) fractions, according to the indicated parameters. B) Daily changes in the percentage of positive fractions of Lin–CD71– H-2high cells stained with various mAb. Lin–CD71– H-2high fractions are shown in the square gates of Figures 2A and 2B.
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VLA-4 and LFA-1 Expression
The changes in the VLA-4 and LFA-1 adhesion molecules on the P-HSC were opposite. VLA-4 expression decreased on day 1 after 5-FU treatment, while LFA-1 expression increased the same day (Fig. 3B).
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Discussion
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In the present study, we used a new method of purifying P-HSC to examine changes in markers of P-HSC after 5-FU injection. The number of LD/Lin– cells decreased one day after 5-FU treatment and reached a minimum on day 3, whereas an increase in cell numbers was observed from day 4 after 5-FU treatment. The numbers of LD cells and whole BMC began to increase on days 5 and 6 after 5-FU injection, respectively (Fig. 1)[22].
We have previously shown that IL-3R– progenitor cells are more primitive than IL-3R+ cells [4]. In the present study, we examined the expression of IL-6R on CD71– H-2high cells. On day 3 after 5-FU treatment, the expression of IL-6R on P-HSC was lowest, whereas that of Sca-1 reached its maximum (Fig. 2). Therefore, it seems likely that IL-6R– and Sca-1+ are P-HSC.
The proto-oncogene, c-kit, is known to encode a transmembrane receptor of tyrosine kinase gene families, which are implicated in the control of hemopoietic cell and melanocyte development [26]. It has been reported that c-kit ligand (stem cell factor) can support the development of both early pluripotent progenitors and later committed progenitors into many different lineages in the presence of other cytokines. It is also commonly accepted that c-kit ligand alone has little ability to stimulate the growth of hemopoietic progenitors or to induce them into the cycling phase, which requires the presence of other growth factors. The expression of c-kit has been documented in a variety of murine progenitors, including stem cells with marrow-repopulating ability [27-29]. Okada et al. reported that HSCs exclusively expressed the c-kit molecule [28], whereas Katayama et al. reported that dormant primitive progenitors express low levels of c-kit [30].
We have very recently shown that CD71– class Ihigh cells have long-term reconstituting ability, and that they are c-kit– or c-kitlow [22]. Using Ly5 congeneic mice, we have just obtained direct evidence that P-HSC are c-kit–, based on FACS analysis; c-kit–/class Ihigh/CD71–/Lin– cells have long-term reconstituting activity while c-kitlow cells have no such activity [manuscript in preparation]. In the present study, both c-kitlow and c-kithigh fractions decreased, reaching a minimum three days after 5-FU treatment. In contrast, there was an apparent increase in the percentage of c-kit– cells at the same time. Therefore, it seems likely that c-kit– are P-HSC.
It is well known that CD34+ and CD33– cells are the most primitive stem cells in humans and mice [31, 32]. In the present study, there was no apparent change after 5-FU treatment in the CD34+ fraction among P-HSC (Fig. 2). CD34+ P-HSC were not enriched after 5-FU treatment. Such disparity in CD34 expression on P-HSC in humans and mice may be ascribed to the difference in species.
LFA-1, VLA-4 and CD44 are well-known adhesion molecules. CD44 is a hyaluronic acid receptor. It has been reported that CD44 affects the differentiation of HSC through interaction among HSC and stromal cells. The ligands of LFA-1 (CD11a/CD18) are ICAM-1 and ICAM-2. The ligands of VLA-4 are VCAM, fibronectin, etc. It has also been reported that the administration of anti-VLA-4Ab or anti-CD44Ab primarily inhibited lymphopoiesis [33, 34]. Nakauchi et al. have reported that LFA-1– hemopoietic progenitors are more primitive than LFA-1+ hemopoietic progenitors [35]. In the present study, there was no apparent change in CD44 expression (Fig. 3). However, VLA-4 expression decreased on day 1 after 5-FU treatment, while LFA-1 expression increased the same day.
In conclusion, these findings suggest that most P-HSC are c-kit–, IL-6R– and Sca-1+.
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Acknowledgements
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We thank Ms. Y. Shinno and Ms. Y. Matsui for their expert technical assistance, and Ms. Y. Ando for manuscript preparation.
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Footnotes
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Provisionally accepted April 8, 1996.
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References
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-
Harrison DE, Astle CM, Lerner C. Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells. Proc Natl Acad Sci USA 1988;85:822-826.[Abstract/Free Full Text]
-
Visser JWM, Bauman JGJ, Mulder AH et al. Isolation of murine pluripotent hemopoietic stem cells. Exp Med 1984;169:1576-1590.
-
Inaba M, Ogata H, Toki J et al. Isolation of murine pluripotent hemopoietic stem cells in the G0 phase. Biochem Biophys Res Commun 1987;147:687-694.[Medline]
-
Ogata H, Taniguchi S, Inaba M et al. Separation of hemopoietic stem cells into two populations and their characterization. Blood 1992;80:91-95.[Abstract/Free Full Text]
-
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58-62.[Abstract/Free Full Text]
-
Bertoncello I, Bradley TR, Watt SM. Multiparameter analysis of transplantable hematopoietic stem cells. The separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence. Exp Hematol 1991;19:95-100.[Medline]
-
Spangrude GJ, Smith L, Uchida N et al. Mouse hematopoietic stem cells. Blood 1991;78:1395-1402.[Free Full Text]
-
Iscove N. Searching for stem cells. Nature 1990;347:126-127.[Medline]
-
Jones RJ, Wagner JE, Celano P et al. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990;347:188-189.[Medline]
-
Hodgson GS, Bradley TR. Properties of haematopoietic stem cells surviving 5-fluorouracil treatment. Nature 1979;281:381-383.[Medline]
-
Hodgson GS, Bradley TR, Radley JM. The organ-ization of hemopoietic tissue as inferred from the effects of 5-fluorouracil. Exp Hematol 1982;10:26-35.[Medline]
-
Van Zant G. Studies of hematopoietic stem cells spared by 5-fluorouracil. J Exp Med 1984;159:679-690.[Abstract/Free Full Text]
-
Lerner C, Harrison DE. 5-fluorouracil spares hemopoietic stem cells responsible for long-term repopulation. Exp Hematol 1990;18:114-118.[Medline]
-
Hoyer M, Nielsen OS. Influence of dose on regeneration of murine hematopoietic stem cells after total body irradiation and 5-fluorouracil. Oncology 1992;49:166-172.[Medline]
-
Hirayama F, Shih JP, Awgulewitsch A et al. Clonal proliferation of murine lymphohemopoietic progenitors in culture. Proc Natl Acad Sci USA 1992;89:5907-5912.[Abstract/Free Full Text]
-
Baker JE, Wolfe JH, Rowe LB et al. Advantages of gradient versus 5-fluorouracil enrichment of stem cells for retroviral-mediated gene transfer. Exp Hematol 1993;21:47-54.[Medline]
-
Harrison DE, Lerner CP. Most primitive hemopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 1991;78:1237-1240.[Abstract/Free Full Text]
-
Smith LG, Weissman IL, Heimfeld S. Clonal analysis of hemopoietic stem-cell differentiation in vivo. Proc Natl Acad Sci USA 1991;88:2788-2792.[Abstract/Free Full Text]
-
Lesley J, Domingo DL, Schulte R et al. Effect of an anti-murine transferrin receptor-ricin A conjugation on bone marrow stem and progenitor cells treated in vitro. Exp Cell Res 1984;150:400-407.[Medline]
-
Jefferies WA, Brandon MR, Williams AF et al. Analysis of lymphopoietic stem cells with a monoclonal antibody to the rat transferrin receptor. Immunology 1985;54:333-341.[Medline]
-
Trowbridge IS, Shackelford DA. Structure and function of transferrin receptors and their relationship to cell growth. Biochem Soc Symp 1986;51:117-129.[Medline]
-
Ogata H, Bradley WG, Inaba M et al. Long-term haematopoietic repopulation with only a few stem cells. Proc Natl Acad Sci USA 1995;92:5945-5949.[Abstract/Free Full Text]
-
Szilvassy SJ, Lansdorp PM, Humphries RK et al. Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability. Blood 1989;74:930-939.[Abstract/Free Full Text]
-
Szilvassy SJ, Cory S. Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow. Blood 1993;81:2310-2320.[Abstract/Free Full Text]
-
Nishikawa S, Kusakabe M, Yoshinaga K et al. In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody. EMBO J 1991;10:2111-2118.[Medline]
-
Tsuji K, Zsebo KM, Ogawa M. Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit. Blood 1991;78:1223-1229.[Abstract/Free Full Text]
-
Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci USA 1992;89:1502-1506.[Abstract/Free Full Text]
-
Okada S, Nakauchi H, Nagayoshi K et al. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 1991;78:1706-1712.[Abstract/Free Full Text]
-
Ploemacher RE, Brons RHC. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hematopoietic stem cell compartment following irradiation. Exp Hematol 1989;17:263-266.[Medline]
-
Katayama N, Clark SC, Ogawa M. Stage-specific expression of c-kit protein by murine hematopoietic progenitors. Blood 1993;81:610-616.[Abstract/Free Full Text]
-
Lansdorp PM, Dragowska W. Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. J Exp Med 1992;175:1501-1509.[Abstract/Free Full Text]
-
Krause DS, Ito T, Fackler MJ et al. Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells. Blood 1994;84:691-701.[Abstract/Free Full Text]
-
Miyake K, Weissman IL, Greenberger JS et al. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med 1991;173:599-607.[Abstract/Free Full Text]
-
Miyake K, Medina KL, Hayashi SI et al. Monoclonal antibodies to Pgp1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures. J Exp Med 1990;171:477-488.[Abstract/Free Full Text]
-
Gunji Y, Nakauchi H, Miura Y et al. Expression and function of adhesion molecules on human hematopoietic stem cells. Blood 1992;80:429-436.[Abstract/Free Full Text]
Received March 11, 1996;
accepted for publication April 19, 1996.
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Abstract
Introduction
