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FOCUS ON HEMATOLOGY
From the Department of Clinical Oncology and Department
of Molecular Medicine, Institute of Medical Science, University of
Tokyo, Japan; Department of Internal Medicine, Keio University, Tokyo,
Japan; and Department of Pediatrics, Kyoto University, Japan.
There is controversy as to whether murine definitive hematopoiesis
originates from yolk sac (YS) or the intraembryonic region. This study reports the generation of definitive hematopoietic stem cells (HSCs) from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (dpc). YS
and P-Sp cells at 8.5 dpc generated no definitive hematopoiesis-derived colony-forming cells in cocultures with AGM-S3 cells, but spleen colony-forming cells and HSCs capable of reconstituting definitive hematopoiesis in adult mice simultaneously appeared on day 4 of coculture. Precursors for definitive HSCs were present in YS and P-Sp
at 8.0 dpc, a time when YS and embryo were not connected by blood
vessels. It is proposed that precursors with the potential to generate
definitive HSCs appear independently in YS and intraembryonic P-Sp and
that the P-Sp or AGM region affords the microenvironment that
facilitates generation of definitive hematopoiesis from precursors.
(Blood. 2001;98:6-12) The hematopoietic system of vertebrates, derived
from the mesodermal germ layer in early embryogenesis, occurs in 2 waves. At early stages, the first wave, primitive hematopoiesis, takes place in the visceral yolk sac (YS) or its analog. The primitive hematopoiesis is followed by a second wave, known as definitive hematopoiesis and which is observed in intraembryonic areas.
Experiments using chimeric embryos in nonmammalian vertebrates
demonstrated that mesodermally derived ventral compartments (YS or its
analog) and dorsal compartments (intraembryonic region) contribute to hematopoiesis in a different manner. In the avian species, the intraembryonic region containing the dorsal aorta is responsible for
definitive hematopoiesis, while the YS produces only transient embryonic hematopoiesis.1,2 In amphibians, the
intraembryonic region containing the pronephros is the major source of
definitive hematopoiesis.3,4 The ontogenic source of
mammalian definitive hematopoiesis has remained controversial because
the in utero development of mammals excludes embryo grafting
experiments. Results of earlier mouse studies led to the general
acceptance of a model that murine definitive hematopoiesis begins in
YS, shifts to fetal liver (FL), and finally resides in bone marrow
(BM), in contrast to the conclusion derived from nonmammal
vertebrates.5,6 However, recent studies have shown that
early development of murine hematopoiesis is more complex than
heretofore considered.
During mouse embryogenesis, primitive hematopoiesis appears at 7.5 days
post coitum (dpc), a time when primitive nucleated erythrocytes are
visible in YS blood islands.5 Primitive erythrocytes are
distinguishable from definitive ones, which are first detected in FL at
9 dpc, as based on the specific expression of embryonic-type globins.7 Colony-forming cells (CFCs) are also detectable
as early as 7.5 dpc in YS and later at 9 dpc in FL.5,8
However, spleen colony-forming cells (CFU-Ss) and long-term
repopulating hematopoietic stem cells (LTR-HSCs) are absent in YS
before the circulation has been established.9,10 Recently,
it has been shown that intraembryonic paraaortic splanchnopleures
(P-Sp), which goes on to differentiate into the dorsal aorta, genital ridge/gonad, and pro/mesonephros (aorta-gonad-mesonephros, or AGM)
region, harbors lymphohematopoietic progenitors, CFU-Ss, and LTR-HSCs
prior to liver colonization.11,12 Lymphohematopoietic progenitors are simultaneously identifiable in P-Sp and YS at 8.5 dpc.13 CFU-Ss appear at the YS and AGM region at late 9 dpc, but the number and frequency of CFU-Ss in the AGM region greatly
exceed those in the YS.9 HSCs capable of long-term multilineage repopulation in irradiated adult recipients are
first noted in the AGM region at 10 dpc, before such activity can be observed in YS and FL.10 In contrast, other studies have
shown that HSCs in 9 dpc YS as well as P-Sp can contribute to
definitive hematopoiesis when conditioned newborn mice instead of
irradiated adult mice serve as recipients for hematopoietic
transplantation.14,15
While the developmental relationship between primitive and definitive
hematopoiesis in murine embryogenesis remains unanswered, localization
and developmental stage are critical factors for embryonic
hematopoiesis; hence the importance of the microenvironment surrounding
embryonic hematopoietic cells or their precursors. To better understand
the roles of the microenvironment in the development of murine
embryonic hematopoiesis, several stromal cell lines were isolated from
various tissues at different stages of development, such as YS at 9.5, 10, and 10.5 dpc and FL at 14 dpc, and their supportive activities on
hematopoiesis were analyzed.16-19 We and another
group recently established endothelial cell lines, AGM-S3 and DAS
104-4, from the AGM region at 10.5 and 11.0 dpc,
respectively.20,21 These cell lines support the development of murine and human immature hematopoietic cells including LTR-HSCs.
In the present work, we asked if AGM-S3 cells have the potential to
support the generation of LTR-HSCs capable of reconstituting adult
definitive hematopoiesis from early YS and intraembryonic P-Sp, since
it has been shown that LTR-HSCs initiate autonomously within the 10 dpc
AGM region in an in vitro organ culture system.11 When cocultured with AGM-S3, cells isolated from 8.0 and 8.5 dpc P-Sp
generate CFU-Ss and LTR-HSCs. Surprisingly, definitive LTR-HSCs were
also generated from YS even at 8.0 dpc, a time when YS and the embryo
were not connected by blood vessels. We propose that precursors capable
of generating definitive hematopoiesis are already present at early YS
and are independent of simultaneously existing P-Sp and that the P-Sp
or AGM region provides the microenvironment for generation of
definitive hematopoiesis from precursors.
Mice
Cell preparation
Antibodies The antibodies used for immunofluorescence staining included 104 (anti-Ly-5.2), RB6-8C5 (anti-Gr-1), M1/70 (anti-Mac-1), RA3-6B2 (anti-CD45R/B220), and 30-H12 (anti-Thy-1.2), all purchased from PharMingen (San Diego, CA). All antibody incubations were carried out for 30 minutes on ice.Culture with stromal cell lines YS and P-Sp cells were cultured with stromal cell lines in -minimal essential medium (-MEM; Flow Laboratories, Rockville, MD) containing 10% FBS. Cells including stromal ones were harvested using 0.05% trypsin containing 0.53 mM EDTA (Gibco, Grand Island, NY)
on the designated day of culture and passed through a nylon mesh.
AGM-S3 stromal cell line was established from 10.5 dpc AGM region-derived cells as described previously.21 In brief,
AGM tissues were removed from 10.5 dpc embryos of C3H/HeN mice,
dissected to some pieces, and cultured in 24-well plates overnight with a drop of -MEM containing 10% FBS at 37°C in a humidified
atmosphere flushed with 5% CO2 in air, and 1 mL culture
medium was added to the well the next day. When the adherent cells
appeared around the tissues 1 week later, the AGM tissues were removed.
After 1 additional week of incubation, the adherent cells were
harvested from the well using 0.05% trypsin plus 0.53 mM EDTA and were
plated in 6-well plates. After 2 weeks of incubation, the cultured
cells were irradiated with 900-rad  -ray to deplete hematopoietic
cells. The medium was replaced with fresh culture medium once weekly. Adherent cells were harvested 2 weeks later using trypsin, and 50 to
100 cells were seeded into 24-well plates. After 3 weeks of incubation,
cells expanded in a well were harvested and used for cell cloning by
the limiting dilution technique.
Cell sorting BM mononuclear cell suspensions were separated using a density gradient centrifugation method and stained with fluorescein isothiocyanate (FITC)-anti-Ly-5.2. The stained cells were sorted on a FACSVantage (Becton Dickinson, Mountain View, CA) as reported.25Transplantation YS and P-Sp cells from Ly-5.2 mice were transferred together with 1 × 105 unfractionated BM mononuclear cells from Ly-5.1 mice into sublethally irradiated Ly-5.1 mice. Peripheral blood (PB) was collected from the tail vein of the recipient mice for up to 6 months after transplantation. Red blood cells were removed, and the nucleated PB cells were stained with FITC-anti-Ly-5.2 and phycoerythrin-antimyeloid cells (Mac-1 and Gr-1), anti-B lymphocytes (B220), or anti-T lymphocytes (Thy-1) and analyzed on a FACScan (Becton Dickinson). The mice in which donor-derived (Ly-5.2+) cells made up more than 1% of all B220+, Thy-1+, and Mac-1+/Gr-1+ cells in PB were scored as being positive for successful reconstitution.CFC assay Clonal cell culture was done in triplicate as described.26,27 Briefly, 1 mL culture mixture containing cells from YS or P-Sp cells of Ly-5.2 mice or their progenies cultured with AGM-S3 cells,-MEM, 1.2% methylcellulose (Shinetsu Chemical,
Tokyo, Japan), 30% FBS, 1% deionized fraction V bovine serum albumin (Sigma), 10 4 M mercaptoethanol (Eastman Organic
Chemicals, Rochester, NY), 100 ng/mL rat stem cell factor (SCF; Amgen,
Thousand Oaks, CA) and human interleukin (IL)-6 (Tosoh, Kanagawa,
Japan), 20 ng/mL mouse IL-3 (Kirin Brewery, Tokyo, Japan) and human
thrombopoietin (Kirin), 2 U/mL human erythropoietin (Kirin) and 10 ng/mL human granulocyte colony-stimulating factor (G-CSF) (Kirin) was
plated in each of 35-mm suspension culture dishes and incubated at
37°C in a humidified atmosphere flushed with 5% CO2 in
air. Colony types were determined on days 7 to 14 of incubation
by in situ observation using an inverted microscope and according to
the criteria described.26,28
CFU-S assay Eight-week-old C57BL/6 recipient mice were irradiated (9.5 Gy) with a 60Co source and intravenously given donor cells. On day 8 after injection, the number of colonies in the recipient spleen were counted.RT-PCR analysis for hemoglobin Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed using a previously described method with some modification.21,24 Messenger RNA (mRNA) was prepared from individual erythroid bursts or erythrocyte-containing hematopoietic mixed colonies using QuickPrep Micro mRNA Purification kit (Pharmacia, Uppsala, Sweden), and reverse-transcribed using T-primed First-Strand Kit (Pharmacia). Hemoglobin-specific complementary DNA was amplified with Taq DNA polymerase using pairs of oligonucleotide primers as follows29:-globin-5',
CTCTCTGGGGAAGACAAAAGCAAC-3'; -globin-3', GGTGGCTAGCCAAGGTCACCAGAC-3'.  major-globin-5',
CTGACAGATGCTCTCTTGGG-3'; major-globin-3',
CACAACCCCAGAAACAGACA-3'. H1-globin-5', AGTCCCCATGGAGTCAAAGA-3'; H1-globin-3', CTCAAGGAGACCTTTGCTCA-3'.  -globin-5',
GGAGAGTCCATTAAGAACCTAGACAA-3'; -globin-3',
CTGTGAATTCATTGCCGAAGTGAC-3'.  -globin-5', GCTCAGGCCGAGCCCATTGG-3'; -globin-3', TAGCGGTACTTCTCAGTCAG-3'. -actin-5',
GTGGGCCGCTCTAGGCACCAA-3'; -actin-3', CTCTTTGATGTCACGCACGATTTC-3'.
Samples were denatured at 94°C for 5 minutes, followed by
amplification rounds consisting of 94°C for 30 seconds (denaturing),
55°C to 60°C for 1 minute (annealing), and 72°C for 2 minutes
(extension) for 40 cycles. Products were separated on a 2.0% agarose
gel, stained with ethidium bromide, and photographed. Messenger RNA
derived from granulocyte/macrophage colonies, adult BM cells, and 8.0 dpc YS cells were used as negative controls and controls for definitive
and primitive erythropoiesis, respectively.
Generation of blood cells from 8.5 dpc P-Sp and YS cocultured with AGM-S3 cells When the cells isolated from 8.5 dpc P-Sp were cocultured with AGM-S3 cells, small round cells were first evident on day 2 or 3 of culture (Figure 1A) and gradually increased up to day 7 (Figure 1B). Cytospin preparation on day 7 of culture showed that most of the nonadherent cells were macrophages and some immature cells were present. The cells isolated from 8.5 dpc YS also produced blood cells, mainly macrophages, with a similar time course.
Generation of CFCs from 8.5 dpc P-Sp and YS cocultured with AGM-S3 cells To determine if the blood cells generated from early YS and P-Sp contain CFCs, cells isolated from YS and P-Sp at 8.5 dpc (0.5 embryo equivalents) were cultured with SCF, IL-3, IL-6, G-CSF, erythropoietin, and thrombopoietin. As shown in Table 1, YS at 8.5 dpc contained various types of CFCs. When the YS cells were cocultured with AGM-S3 cells, CFCs slightly increased until day 2 of culture and then decreased, and CFCs were not detected after day 6 of culture. P-Sp at 8.5 dpc contained only a small number of CFCs, and these disappeared by day 4.
The hemoglobin types of erythrocytes in erythroid bursts or
erythrocyte-containing mixed colonies were analyzed by RT-PCR, the
objective being to determine if the CFCs derived from primitive or
definitive hematopoiesis. In the culture of cells isolated from 8.5 dpc
YS, all 7 erythroid bursts and 7 mixed colonies examined expressed both
embryonic hemoglobins, such as
Generation of CFU-Ss from 8.5 dpc P-Sp and YS cocultured with AGM-S3 cells We next examined whether cells generated from 8.5 dpc P-Sp and YS contain CFU-Ss. As shown in Table 2, 8.5 dpc P-Sp and YS (1 embryo equivalent) contained no CFU-Ss. Cells isolated from 8.5 dpc P-Sp and YS were cocultured with AGM-S3 cells for 4 days and then assayed daily for CFU-Ss. No CFU-S activity was found in the cells cultured from P-Sp and YS for 1 to 3 days. However, both P-Sp and YS generated a substantial number of CFU-Ss in the coculture with AGM-S3 cells at day 4. Therefore, AGM-S3 cells can induce generation of CFU-Ss from early YS and P-Sp.
Generation of LTR-HSCs from 8.5 dpc P-Sp and YS cocultured with AGM-S3 cells We then examined the generation of HSCs capable of reconstituting hematopoiesis from 8.5 dpc P-Sp and YS in the coculture with AGM-S3 cells using a competitive repopulating assay (Table 3). We first transplanted only AGM-S3 cells cultured for 6 days into 8 Ly-5.1 mice to examine whether AGM-S3 cells have the hematopoietic repopulating ability. Twelve weeks after transplantation, there were no Ly-5.2 cells in PB of all recipients. When cells isolated from 8.5 dpc P-Sp and YS (2 embryo equivalents) of Ly-5.2 mice were respectively transplanted into 9 irradiated Ly-5.1 adult mice, no recipient mouse showed successful engraftment. Four recipients transplanted with 8.5 dpc P-Sp or YS cells cultured without stroma for 6 days also showed no successful engraftment. However, all 4 recipients transplanted with 8.5 dpc P-Sp or YS cells cocultured with AGM-S3 cells for 6 days had Ly-5.2+ donor-type myeloid and lymphoid cells in the PB at 12 weeks after transplantation (Table 3 and Figure 3). Figure 4 shows representative PB profiles of mice transplanted with cells cocultured from 8.5 dpc P-Sp (Figure 4A) and YS (Figure 4B) with AGM-S3 cells for 6 days. Stable chimerism was maintained for 6 months in recipients engrafted with the progeny of 8.5 dpc P-Sp or YS.
To confirm that LTR-HSCs generated in the coculture with AGM-S3 cells
produce definitive erythrocytes, we cultured donor-type Ly-5.2+ cells sorted from BM cells of recipients engrafted
with the cells harvested on day 6 of coculture of early YS and P-Sp 20 weeks after the transplantation, and hemoglobin types of erythrocytes were determined in case of erythroid bursts and erythrocyte-containing mixed colonies derived from the sorted cells. All of 3 and 4 erythroid bursts and 4 and 5 mixed colonies derived from donor-type BM cells of
the mice transplanted with the progeny of 8.5 dpc P-Sp and YS cells,
respectively, contained definitive erythrocytes expressing adult but
not embryonic hemoglobins (Figure 5 and
data not shown). These findings indicate that AGM-S3 cells can support
the generation of definitive LTR-HSCs from early YS and P-Sp.
The time course of the generation of definitive LTR-HSCs was also
examined. Progenies of 8.5 dpc P-Sp or YS (2 embryo equivalents) cocultured with AGM-S3 cells for 2 to 4 days were transplanted (Table
4). None of 4 mice transplanted with the
cells harvested at days 2 and 3 of coculture of P-Sp or YS showed
successful engraftment. However, 9 of 11 and 5 of 10 recipients
transplanted with cells harvested on day 4 of coculture of 8.5 dpc P-Sp
or YS, respectively, were successfully engrafted. Thus, LTR-HSCs as
well as CFU-Ss were simultaneously generated from 8.5 dpc P-Sp and YS
on day 4 of coculture with AGM-S3 cells.
We also determined if generation of LTR-HSCs from early YS or P-Sp could be induced by other stromal cell lines, such as adult BM-derived MS-5 cells,31 OP9 cells established from newborn calvaria of the op/op mice that possess a mutation in the macrophage colony-stimulating factor (M-CSF) gene,32 or fibroblastoid stromal cells 3T3 cells. When 8.5 dpc P-Sp or YS cells (5 embryo equivalents) cocultured with these stromal cells for 6 days were respectively transplanted into 4 recipients, no mice showed successful engraftment. Generation of LTR-HSCs from 8.0 dpc P-Sp and YS cocultured with AGM-S3 cells The above results indicate that both 8.5 dpc P-Sp and YS have the potential to generate definitive LTR-HSCs in coculture with AGM-S3 cells. However, because the blood connection between YS and embryo has already been established at 8.5 dpc, precursors for the definitive LTR-HSCs may pass through the circulation from one site to another. We then examined the generation of LTR-HSCs from P-Sp and YS at 8.0 dpc, when YS and embryo are not yet connected by blood vessels. When 8.0 dpc P-Sp or YS cells (2 embryo equivalents) cocultured with AGM-S3 cells for 4 days were transplanted, 6 of 8 and 5 of 7 recipients had donor-derived myeloid and lymphoid cells in PB 10 weeks after transplantation (Table 5 and Figure 6). Figure 7 shows representative PB profiles of mice transplanted with cells cocultured from 8.0 dpc P-Sp (Figure 7A) and YS (Figure 7B) with AGM-S3 cells 12 weeks after transplantation. Both mice had donor-derived Gr-1/Mac-1+ myeloid cells, B220+ B cells, and Thy-1+ T cells, the chimerism of which was maintained for more than 6 months. Therefore, precursors capable of generating definitive LTR-HSCs appear independently in early YS and P-Sp.
The origin of mouse definitive HSCs Whether mouse definitive HSCs originate from YS or intraembryonic regions has been controversial. In the present study, we found the generation of the definitive hematopoiesis from both early YS and intraembryonic P-Sp on AGM-S3 stromal cells. In the transplantation experiments using adult mice, the progeny of early YS and P-Sp had the potential of mutilineage reconstitution of definitive hematopoiesis in congeneic recipients, and the stable engraftment was evident for 6 months after transplantation. These findings indicate that both early YS and P-Sp can generate definitive HSCs with a similar repopulating potential by coculture with AGM-S3 stromal cells. In addition, the generation of definitive HSCs was noted even at 8.0 dpc, a time before blood vessels link YS and embryo. Hence, the precursors with the potential to generate definitive HSCs appear independently in the 2 sites, even if the precursors may move to another site.However, the present result does not mean that the cells with the potential in both YS and P-Sp contribute to definitive hematopoiesis in vivo. Because the localization and ontogenetic stage are critical factors for development of embryonic hematopoiesis, it is unclear whether early YS and P-Sp can generate definitive HSCs in their circumstances in vivo. Engrafting experiments using a whole embryo culture might clarify the in vivo fate of the precursors with the potential to generate LTR-HSCs in coculture with AGM-S3 cells. Environment inducive for generation of definitive HSCs AGM-S3 stromal cells established from the 10.5 dpc AGM region induced definitive CFU-Ss and LTR-HSCs from 8.0 and 8.5 dpc P-Sp, but other stromal cells such as MS-5, OP9, and 3T3 cells could not do so. This finding indicates that AGM-S3 cells specifically express the molecule(s) capable of supporting the generation of CFU-Ss and LTR-HSCs. Our previous study showed that AGM-S3 cells express SCF, IL-11, and oncostatin M,21 but any combinations of these cytokines did not induce the generation of CFU-Ss or LTR-HSCs from early P-Sp (data not shown). Yet to be identified molecule(s) may be responsible for the generation of CFU-Ss or LTR-HSCs. However, 8.0 or 8.5 dpc P-Sp seems to take a longer time to generate CFU-S and LTR-HSCs in cocultures with AGM-S3 cells than is required in vivo. Molecules expressed on AGM-S3 cells may not completely reconstitute the hematopoietic microenvironment of the AGM region for generation of CFU-Ss and LTR-HSCs.The present observation that definitive CFCs were not evident even on day 10 of culture despite the appearance of CFU-Ss and LTR-HSCs on day 4 indicates that AGM-S3 cells could not support the differentiation of CFCs from LTR-HSCs or CFU-Ss generated from P-Sp. In the developing mouse embryo, CFU-Ss appear in the AGM region and simultaneously in YS at late 9 dpc, and LTR-HSCs are detected in the AGM region at 10 dpc, while no hematopoietic clusters or blood islands indicative of the differentiation or maturation of hematopoietic cells are found in AGM region.11 Thus, the AGM region plays a specific role in the development of embryonic hematopoiesis. The hematopoietic activity of AGM-S3 cells on the generation of CFU-Ss and LTR-HSCs, but not CFCs, from P-Sp is in accord with the specific function of the AGM region as a hematopoietic tissue. In this context, it is of interest that numerous macrophages were generated in the coculture with AGM-S3 cells from early YS and P-Sp, although they do not express M-CSF.21 It may be that these macrophages originate from primitive hematopoiesis and are regulated by mechanisms differing from these related macrophages in definitive hematopoiesis.33 AGM-S3 cells also supported the generation of CFU-Ss and LTR-HSCs from early YS. In contrast to our result, Medvinsky et al11 showed that when 10 dpc YS or P-Sp explants were cultured for 3 days in isolation, only P-Sp generated CFU-Ss and LTR-HSCs. Because the explants are considered to include various cells surrounding the precursors for CFU-Ss and LTR-HSCs, the discrepancy between our result and theirs reflects differences in the function of the microenvironment in YS and P-Sp. The AGM region may provide the specific microenvironment for the generation of CFU-Ss and LTR-HSCs from their precursors. Precursors for definitive LTR-HSCs and P-Sp When cells isolated from early YS and P-Sp were cocultured with AGM-S3 cells, they generated CFU-Ss and LTR-HSCs with a similar time course. The similar hematopoietic activity of early YS and P-Sp suggests that the precursors in both sites resemble each other, albeit being generated independently. Yoder et al14 reported the presence of the cells capable of repopulating in conditioned neonatal but not adult mice in 9.0 dpc YS and P-Sp. They showed that YS contained a larger number of neonate-repopulating cells than P-Sp at 9.0 dpc, thereby suggesting that cells in YS arise autonomously of those in P-Sp. Precursors that generated adult-repopulating cells on AGM-S3 cells may also produce the neonate-repopulating cells in vivo.The order of appearance of hematopoietic activities observed during early mouse embryogenesis is reserved compared with the events that occur during adult hematopoiesis.34 In the developing mouse embryo, CFU-S activity is first detected at late 9 dpc, whereas stem cell activity is not found until late 10 dpc. In the present study, CFU-Ss and LTR-HSCs were generated from early YS and P-Sp on day 4 of coculture with AGM-S3 cells. One possibility is that CFU-Ss are the progeny of LTR-HSCs generated from early YS and P-Sp, although we detected no time lag of their appearance because of the difference of the sensitivity of assay systems used for CFU-Ss and LTR-HSCs. Another possibility is that CFU-Ss and LTR-HSCs are independently generated. In this case, there are 2 other possibilities: Firstly, CFU-Ss and LTR-HSCs are derived from the same progenitors and, secondly, have different ancestors. Use of single cells isolated by various markers such as c-Kit, CD34,23 Sca-1,35 or VE-cadherin36 may answer these questions.
Submitted August 23, 2000; accepted March 14, 2001.
Supported by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan and, in part, by research funding from Kirin Brewery (T.N.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kohichiro Tsuji, Dept of Clinical Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: tsujik{at}ims.u-tokyo.ac.jp.
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Abstract
Introduction
