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HEMATOPOIESIS
From the Institute of Molecular and Cellular
Biosciences, the University of Tokyo, Japan; and CREST, Core Research
for Evolutional Science and Technology of Japan Science and Technology,
Tokyo, Japan.
During mammalian development, definitive hematopoietic stem cells
(HSCs) arise in the aorta-gonad-mesonephros (AGM) region and colonize
the fetal liver (FL) before hematopoiesis occurs in the bone marrow.
The FL is a unique hematopoietic organ where both HSCs and mature blood
cells are actively generated along with functional maturation of
hepatic cells as a metabolic organ. To characterize HSCs and FL
microenvironments during development, this study establishes a
coculture system composed of AGM-originated HSCs and FL
nonhematopoietic cells. The results demonstrate that FL cells support
significant expansion of lineage-committed hematopoietic cells as well
as immature progenitors. More important, long-term repopulating
activity was amplified from AGM-originated HSCs in this coculture
system. Engraftment of HSCs to the bone marrow was strongly enhanced by
coculture. In addition, AGM HSCs produced significantly more
hematopoietic cells than E14.5 and E18.5 FL HSCs in vitro. These
results suggest that the FL microenvironment not only stimulates
expansion of the hematopoietic system, but also possibly modifies the
characteristics of AGM HSCs. Thus, this coculture system recapitulates
the developmental process of HSCs and the FL microenvironment and
provides a novel means to study the development of hematopoiesis.
(Blood. 2002;99:1190-1196) Development of the hematopoietic system is a
complex process that takes place in several distinct hematopoietic
microenvironments.1-3 The initial hematopoietic activity,
known as primitive hematopoiesis, appears in the blood island of the
yolk sac (YS) at embryonic day 7.5 (E7.5) in mice. Adult-type
definitive hematopoiesis begins in the aorta-gonad-mesonephros (AGM)
region at E10.54,5 and thereafter shifts to the fetal
liver (FL) at E12.5, where massive production of various hematopoietic
cells occurs. Then, near birth, hematopoiesis shifts to the bone marrow
and spleen. Therefore, the FL is a main hematopoietic organ during the
embryonic period. The transition of hematopoietic sites accompanies
changes in the characteristics of hematopoietic activity.3
Primitive hematopoiesis in the YS is characterized by production of
nucleated erythrocytes with fetal hemoglobin and by the absence of
lymphoid and myeloid cells except for macrophages.6-8
However, YS cells are unable to reconstitute all types of hematopoietic
cells in lethally irradiated adult mice for more than several months
(long-term repopulating hematopoietic stem cells [LTR HSCs]), which
is the most important characteristic of definitive HSCs.9
HSCs with such activity first appear in the AGM region at E10.5 and
thereafter colonize the FL and bone marrow. Therefore, the AGM region
has been considered the origin of definitive HSCs. Interestingly,
however, YS-derived hematopoietic cells were shown to reconstitute the
entire hematopoietic system when grafted into the liver of
busulfan-treated newborn recipients.10-12 These findings
suggest that YS-derived hematopoietic cells acquire the HSC activity by
the interaction with a specific hematopoietic microenvironment in the
liver of newborn mice in which hematopoiesis is sustained. In addition,
the CD34+/c-Kit+ rather than the
CD34 Although HSCs in bone marrow replicate at a constant rate to maintain
hematopoiesis throughout life,14,15 HSCs in embryonic organs are believed to proliferate actively during
development.3 Accumulating evidence suggests that
definitive HSCs differentiate from their precursors, hemangioblasts, in
the AGM at about E10.5 to E11.516 and colonize to the FL,
where the most dramatic expansion of HSCs occurs from E11.5 through
E14.5.17,18 Thereafter, the expansion of HSCs declines
along with development18 and is essentially terminated
around birth. Thus, the proliferative potential of HSCs can be
different from stage to stage during development. The FL provides a
distinct hematopoietic microenvironment that enables significant
proliferation of HSCs, especially in the early phase of its
development. On the other hand, fetal hepatic cells undergo their own
maturation process to become a center of metabolism, while they
function as a major hematopoietic tissue.19,20 The maturation process that leads to expression of various liver-specific genes is also regulated by extracellular signals such as hormones, cytokines, and extracellular matrices. Thus, in the FL, the 2 different
cellular systems coexist and undergo their own developmental processes.
To clarify the mechanism of hematopoietic development in the AGM
region, we previously developed a primary culture system of AGM-derived
cells and demonstrated that oncostatin M (OSM), an interleukin-6 (IL-6)
family cytokine, stimulates development of multilineage progenitors
from the AGM cells in vitro.21 We also established a
primary culture system of nonhematopoietic cells from E14.5 liver and
found that OSM in the presence of dexamethasone (Dex), a synthetic
glucocorticoid, induces maturation of fetal hepatocytes, as evidenced
by morphologic changes that closely resemble differentiated
hepatocytes, expression of various liver enzymes, accumulation of
intracellular glycogen, lipid synthesis, and clearance of
ammonia.22,23 Moreover, those FL cells also supported
hematopoiesis in vitro and interestingly, such activity declined along
with liver development.24 OSM is expressed in CD45+ hematopoietic cells in FL, whereas the OSM receptor
is expressed predominantly in hepatocytes, suggesting that OSM is a
paracrine factor in FL.22
Because HSCs generated in AGM are expected to proliferate in FL, we
describe here a coculture system in which HSCs derived from E11.5 AGM
(AGM HSCs) are cultivated in the microenvironment created by FL
nonhematopoietic cells. AGM HSCs proliferated more efficiently than FL
HSCs in the in vitro FL microenvironment, and, most important, LTR-HSC
activity was significantly increased in this system. These culture
systems enabled us to analyze the characteristics of HSCs and
microenvironments in AGM and FL separately and provided novel in vitro
models to study the development of hematopoiesis and hepatocytes.
Mice
Antibodies and cytokines
AGM culture Primary culture of the AGM cells was performed as described previously.21 In brief, the AGM region was excised from E11.5 GFP+ C57BL/6 embryos and dissociated into a single-cell suspension by trypsin digestion. The cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 15% fetal calf serum (FCS) in the presence of various cytokines: 100 ng/mL stem cell factor (SCF), 1 ng/mL basic fibroblast growth factor (bFGF; Gibco-BRL, Carlsbad, CA), 10 ng/mL murine leukemia inhibitory factor (mLIF), and 10 ng/mL mOSM. After 10 days of incubation, nonadherent hematopoietic cells spontaneously generated in cultures were harvested and analyzed for expression of cell-surface markers and progenitor activities.AGM/FL coculture FL cells were isolated from the E14.5 embryos as described previously22 and used as hematopoietic stromal cells.24 The cells were suspended in DMEM supplemented with 10% FCS, Insulin-Ferritin-X solution (Gibco-BRL), and 1 × 10 7 M Dex, and inoculated onto 0.1%
gelatin-coated plastic dishes. A few hours later, the cells were washed
extensively with phosphate-buffered saline to eliminate hematopoietic
cells. Two days later, sources of hematopoietic cells were overlaid
onto FL cells and cultured in DMEM supplemented with 15% FCS in the
presence of various cytokines, 100 ng/mL SCF, 10 ng/mL mOSM,
Insulin-Ferritin-X solution, and 1 × 107 M Dex. After
incubation for 10 days, the cells were harvested for further analysis.
Flow cytometry Cells generated in vitro or from the recipient mice were filtered through a nylon mesh to remove cell aggregates and debris. After staining with a monoclonal antibody according to the manufacturer's protocol, cells were analyzed by fluorescence-activated cell sorting (FACS) with the FACS-Calibur system or sorted by the FACS-Vantage system (Becton Dickinson, San Jose, CA) for further analysisCulture colony-forming unit assay For analysis of the progenitor activity of cells generated during cultures, donor-derived GFP+ hematopoietic cells were sorted by FACS Vantage (Becton Dickinson) and applied for assays. Two thousand hematopoietic cells were suspended in 1 mL -minimal
essential medium containing 0.8% methylcellulose, 30% FCS, 1%
deionized bovine serum albumin, 100 µM 2-mercaptoethanol, 10 ng/mL
IL-3, 100 ng/mL IL-6, 2 U/mL EPO, and 100 ng/mL SCF and plated onto
plastic dishes (35 mm in diameter). Colony types were judged from the
morphology under microscopic observation on day 14 after
plating.27
In vivo repopulating assay Adult mice (C57BL/6 males, 9-12 weeks old) were exposed to a single dose of 10 Gy from a 137Cs source. At this dose, all irradiated mice died within 2 weeks. Test cells were filtered through a nylon mesh of 70 µm (Cell Strainer; Becton Dickinson, San Jose, CA) and injected intravenously into the tail vein. Normal bone marrow cells (2 × 105) were coinjected with the test cells for radioprotection. Under this experimental condition, 10% to 20% of mice undergoing transplantation died within 2 weeks. Several months later, peripheral blood cells were collected from the tail vein and analyzed by flow cytometry. To examine the tissue distribution of donor cells, we killed the recipient mice; collected blood cells from the bone marrow, thymus, spleen, and peripheral blood; and analyzed them by flow cytometry.
Establishment of a coculture system composed of AGM HSCs and FL nonhematopoietic cells Definitive HSCs generated in AGM immediately colonize to the FL, in which they replicate themselves and generate numerous mature blood cells. To clarify the interaction between HSCs and the FL microenvironment, we developed the coculture system (Figure 1). AGM cells isolated from transgenic mice expressing GFP were inoculated in subconfluent cultures of nonhematopoietic FL cells from nontransgenic mice and incubated in the presence of cytokines and hormones. After 10 days of incubation, numerous cells floating above the hepatic stromal layer were generated. Flow cytometric analysis of hematopoietic cell-surface markers as well as microscopic observation indicated that these floating cells were hematopoietic cells (data not shown). To compare the differences in the hematopoietic microenvironments between AGM and FL, we counted the number of GFP+ floating cells by FACS and found that the AGM/FL coculture produced more hematopoietic cells than the AGM culture (Figure 2). It is therefore likely that the FL microenvironment is more suitable than that of AGM for AGM HSCs to produce hematopoietic cells.
We next examined the factors required for hematopoiesis in the 2 culture systems. SCF was essential for hematopoiesis in both AGM culture and AGM/FL coculture (data not shown). In contrast to AGM culture, which requires OSM for hematopoiesis, OSM was not absolutely required for the production of hematopoietic cells in AGM/FL coculture (Figure 2, lane b). In agreement with our previous observation24 that hematopoiesis from FL-derived HSCs in the FL microenvironment in vitro was inhibited by OSM and Dex, the number of cells produced in AGM/FL coculture was significantly reduced in the presence of OSM and Dex (Figure 2, lanes b-d). Characteristics of AGM HSCs and FL HSCs Because LTR HSCs in AGM were reported to express CD34 and c-Kit,28 we fractionated AGM-derived cells into 2 populations: the CD34+/c-Kit+ cells and the remainder (ie, CD34+/c-Kit,
CD34/c-Kit+, and
CD34/c-Kit cells), and examined their
potential to generate hematopoietic cells in the AGM/FL coculture
system (Figure 3). We found that hematopoietic cells were produced only from the
CD34+/c-Kit+ population in AGM, but not from
the remaining cell populations (data not shown). Furthermore, the
efficiency of expansion was higher in the culture with
CD34+/ c-Kit+ cells than in the culture with
the whole AGM cells. These findings indicate that the
CD34+/c-Kit+ cells in AGM produce hematopoietic
cells, and the remaining cells neither give rise to hematopoietic cells
nor enhance hematopoiesis in the coculture system.
Because LTR HSCs in FL are present in the
lineage Because the floating hematopoietic cells were likely to be a mixture of
lineage-committed cells and immature cells, we examined hematopoietic
progenitor activity of these cells by in vitro colony-formation assays
(Table 1). The colony-forming activity
was found only in CD34+/c-Kit+ cells in AGM.
Whereas the frequency of colony-forming unit-culture (CFU-C) was not
significantly different among the culture conditions, CFU-GEMM
was markedly expanded in the presence of OSM (Figure 4). In conclusion, OSM enhanced the
production of total hematopoietic cells and immature hematopoietic
progenitors from AGM HSCs. Although the addition of Dex suppressed the
production of total hematopoietic cells, it enhanced the production of
immature progenitors from AGM HSCs in the FL microenvironment. Because
OSM and Dex induce maturation of hepatic cells,22
maturation of nonhematopoietic FL cells may provide a more suitable
microenvironment for expansion of immature progenitors, but not for
production of mature blood cells from AGM HSCs.
Repopulating activity of HSCs generated in vitro Because the most important characteristic of HSCs is the ability to reconstitute the entire hematopoietic system in vivo, we transplanted the GFP+ hematopoietic cells generated in vitro into lethally irradiated adult mice through the tail vein and analyzed the donor contribution in the peripheral blood of recipient mice. We compared the repopulating activity in the following 3 cell populations: total hematopoietic cells generated in the AGM culture originating from 6 embryos, and the cells generated in the AGM/FL coculture originating from either whole AGM cells (1 × 105) or the CD34+/c-Kit+ fraction of AGM cells (2 × 103), both of which were equivalent to one third of the AGM cells from a single embryo. Two months after transplantation, GFP+ cells in the peripheral blood of recipients were analyzed by FACS (Figure 5A). GFP+ cells were found in the myeloid (Mac-1+, Gr-1+ cells), erythroid (Ter119+ cells), and lymphoid (B220+, Thy-1+ cells) compartments, as described previously.16 When the cells derived from the AGM culture were transplanted, approximately 12% of peripheral blood cells were found to be GFP+ (Figure 5A, lane a). However, the cells from the AGM/FL coculture originating from only one third of a single embryo were sufficient to achieve a comparable level of donor contribution (Figure 5A, lanes c, f), suggesting that the number of in vivo repopulating cells was increased during the AGM/FL coculture. As in immature progenitors, the repopulation activity was dependent on the presence of OSM during coculture, and the addition of Dex further elevated the efficiency of repopulation (Figure 5A, lanes d, g). These findings indicate that fetal hepatic cells provide a better hematopoietic microenvironment than AGM for the maintenance and expansion of the HSC activity of AGM HSCs.
To analyze LTR-HSC activity in the cultured cells, we examined the donor contribution in peripheral blood for a longer period after transplantation (Figure 5B). The donor contribution by freshly isolated AGM cells in recipient mice was 15% at 1 month after transplantation and reached 53% by 3 months after transplantation. This level was maintained for at least 5 months. Flow cytometric analysis of hematopoietic lineage markers in the recipient indicated that donor cells were present in myeloid, lymphoid, and erythroid compartments (data not shown). Consistent with previous studies,4,5 these results indicate that E11.5 AGM contained LTR HSCs. On the other hand, donor contribution of cultured AGM cells was approximately 14% during the first 4 months after transplantation and thereafter declined to 2%, indicating that the majority of hematopoietic progenitor cells generated in AGM culture were short-term repopulating cells (STRCs)/committed progenitors and that AGM culture was unable to maintain LTR HSCs effectively. In sharp contrast, when AGM cells were cultivated in the FL microenvironment in the presence of SCF and OSM, a high level of donor contribution (up to 70%) was readily achieved within 1 month and was maintained for at least 5 months. These results indicate that AGM/FL cocultured cells contained a significant number of LTR HSCs and STRCs. It is generally accepted that LTR HSCs contribute to hematopoiesis, but STRCs disappear at 5 months after transplantation.29,30 The level of donor contribution by AGM/FL cocultured cells was higher (82%) than that by freshly isolated AGM cells (52%), even though the number of cells added to the AGM/FL coculture was 9-fold less than that to the AGM culture. It is therefore likely that the FL microenvironment increases LTR HSCs derived from AGM in the presence of SCF and OSM. Engraftment of HSCs to the bone marrow The donor contribution in the peripheral blood as shown above strongly suggested that LTR HSCs are generated in AGM/FL coculture from AGM HSCs, whereas AGM culture produces mainly STRCs. Therefore, there is a clear difference in characteristics of hematopoietic progenitor cells generated from AGM HSCs between the AGM and FL microenvironments. Because hematopoiesis from STRCs is not sustained longer than 4 months after transplantation,29,30 we analyzed the donor contribution in various hematopoietic organs at 3 months after transplantation (Figure 6A). The cells generated in AGM culture were poorly engrafted in every organ tested. The efficiency of engraftment of progenitor cells to hematopoietic organs relative to peripheral blood cells revealed that engraftment to the bone marrow was particularly low in comparison with other organs (Figure 6B, left panel). The level of donor contribution in peripheral blood was similar to that in spleen (Figure 6B, left panel), suggesting that spleen is the dominant hematopoietic site for cultured AGM cells. In contrast, the AGM/FL cocultured cells contributed at high levels to all organs including the bone marrow (Figure 6A), and there was no substantial difference in the efficiency of engraftment to each organ (Figure 6B, right panel). Taken together with the results of the long-term repopulation analysis, the data suggest that cultivation of AGM cells in the AGM microenvironment generates mainly STRCs, which reside in the spleen even 3 months after transplantation. In contrast, AGM/FL coculture in the presence of OSM generates both STRCs/committed progenitor cells and LTR HSCs from AGM HSCs, which effectively colonize in spleen and bone marrow.
HSCs are the best characterized stem cell system and have been used to study various concepts in stem cell biology.31 To clarify the characteristics of HSCs and the microenvironment during embryonic development, we have developed a primary coculture system composed of AGM-derived hematopoietic cells and nonhematopoietic FL cells. This system is capable of supporting AGM HSCs to generate a number of lineage-committed progenitors and LTR HSCs. The cell population in AGM contributing to the production of hematopoietic cells in the FL microenvironment was found exclusively in CD34+/c-Kit+ cells, consistent with the previous findings that HSCs in both AGM and FL were detected only in the CD34+/c-Kit+ fraction.28 HSCs change their proliferation potential during development, and AGM HSCs produced significantly more hematopoietic cells than FL HSCs in the FL microenvironment. Furthermore, E18.5 FL HSCs produced fewer hematopoietic cells than E14.5 FL HSCs in the coculture system (Figure 3, lanes h-m). These findings strongly suggest that the proliferation potential of HSCs to produce hematopoietic cells decreases along with development. Alteration of HSC characteristics is also suggested by their response to OSM in the FL microenvironment: OSM enhanced the generation of hematopoietic cells from the AGM-derived CD34+/ c-Kit+ cells, whereas it suppressed hematopoiesis from the CD34+/ c-Kit+ cells derived from FL at E14.5 and E18.5 in coculture with E14.5 FL cells. The most striking difference between the AGM culture and the AGM/FL coculture was their ability to support expansion of hematopoietic progenitors and LTR HSCs. Consistent with previous observations,5,28 we also found LTR-HSC activity in freshly isolated E11.5 AGM cells (Figure 5B). The donor contribution in peripheral blood in our system was relatively lower than those in previous reports.5,28 In previous studies, donor cells with a genetic marker were detected by polymerase chain reaction (PCR)-based methods, whereas we used the GFP transgenic mouse as a source of donor cells and detected donor cells in the recipients by flow cytometry. Although PCR is very sensitive to detect donor cells, flow cytometry provides a more quantitative means to estimate donor contribution. Thus, the difference between our results and those of previous studies may be due to a difference in the detection systems. Although the AGM culture increased the number of total hematopoietic cells, cultured AGM cells exhibited mainly STRC/committed progenitor activity. In sharp contrast, the AGM cells grown in the FL microenvironment showed remarkable LTR-HSC activity (Figure 5B). Whereas 6 embryos were required for LTR using freshly isolated AGM cells, AGM/FL cocultured cells originated from only two thirds of a single embryo were sufficient to reconstitute hematopoiesis with higher levels of donor contribution as early as 1 month after engraftment, and it lasted at least 5 months. These findings indicate that LTR HSCs and STRC/committed progenitor cells are generated from AGM HSCs in coculture and contribute to high levels of hematopoietic reconstitution immediately after transplantation. The efficiency of the in vivo repopulating activity was highest when coculture was performed in the presence of both OSM and Dex, consistent with the generation of immature progenitors (Figure 4). These findings apparently contradict the results shown in Figures 2 and 3, as well as previous observations24 showing that the number of total hematopoietic cells generated in the FL microenvironment was reduced in the presence of both OSM and Dex regardless of the source of hematopoietic cells. However, as described previously,24 lineage-committed cells were more susceptible to OSM and Dex in the FL microenvironment. Therefore, it is conceivable that OSM and Dex affect the multilineage progenitor (CFU GEMM) and/or lineage-committed cells. Our previous findings showed that the combination of OSM and Dex causes functional maturation of hepatic cells in primary FL culture to express metabolic enzymes.22 By this combination, LTR-HSC activity was strikingly expanded while the production of mature blood cells was suppressed. In conclusion, these findings suggest that our culture system is an excellent in vitro model of FL development, in which expansion of LTR HSCs from AGM HSCs and maturation of hepatic cells progress coordinately. The cells from the AGM/FL coculture were efficiently repopulated not only in the peripheral blood, but also in various hematopoietic organs including the bone marrow at 3 months after transplantation. In contrast, the cells from the AGM culture poorly colonized in recipients, particularly in the bone marrow (Figure 6). These findings are well correlated with the previous observation that colonization to the bone marrow is a necessary step for transplanted HSCs to maintain their LTR activity immediately after transplantation.32 Consistent with this, the donor contribution in the peripheral blood by the cells from AGM culture gradually decreased to a level similar to that in the bone marrow. It was shown previously that the bone marrow homing activity of HSCs derived from adult bone marrow declines by in vitro culture, despite a significant increase in the number of hematopoietic cells.33 Because the spleen contains abundant committed progenitors, our results with transplantation of AGM cultured cells indicate that transplanted STRCs/committed progenitor cells also reside in spleen. The AGM/FL coculture system allowed dramatic expansion of multilineage progenitor cells as well as LTR HSCs with high efficiency of engraftment to the bone marrow. These findings also support that engraftment in bone marrow is necessary for maintenance of LTR-HSC activity in the recipient. Because LTR-HSC activity was amplified in vitro, this system will be useful for gene transfer to LTR HSCs by retroviral vectors. Although the molecular basis of how HSCs repopulate to hematopoietic organs is yet to be investigated, it is possible that engraftment is mediated by soluble as well as membrane-bound proteins such as chemokines and integrins. Therefore, the FL microenvironment stimulated with OSM and Dex may modulate expression of the receptors for such molecules in HSCs. Alternatively, it may selectively expand HSCs expressing these molecules. The coculture system also recapitulates these alterations of HSCs in vitro. Taking these findings together with our previous
observations,22-24 a model for development of the
hematopoietic system during embryonic life can be suggested (Figure
7). Definitive HSCs first arise in the
AGM region from hemangioblasts. Because the AGM microenvironment poorly
supports hematopoiesis, HSCs immediately colonize to the FL, where they
proliferate as authentic LTR HSCs and produce mature hematopoietic
cells with the help of the FL microenvironment. During this process,
highly proliferative AGM HSCs change their characteristics to FL HSCs,
with less proliferative potential. However, as reported
previously,22 fetal hepatic cells are also stimulated with
factors such as OSM, which are produced by hematopoietic cells, and
undergo their own developmental program to become a metabolic organ
rather than a hematopoietic microenvironment. Thus, the 2 different
cellular systems existing in the same organ coordinately regulate their
developmental processes. The present in vitro system provides a novel
means to dissect such complex processes at the cellular and molecular
levels.
We are grateful to Dr Masaru Okabe of Osaka University for
providing us with GFP transgenic mice and to Kirin and Ajinomoto for
cytokines. The whole-body irradiation of mice was done at Cs-137
Submitted June 25, 2001; accepted October 15, 2001.
Supported in part by Grants-in-Aid for Scientific Research and Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology; and the Organization for Pharmaceutical Safety and Research.
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: Atsushi Miyajima, Institute of Molecular and Cellular Biosciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; e-mail: miyajima{at}ims.u-tokyo.ac.jp.
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© 2002 by The American Society of Hematology.
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Abstract
Introduction
-ray Irradiation Facilities for Biological Research, Research Center
for Nuclear Science and Technology, University of Tokyo, with kind
assistance by Mr Hoshio Eguchi.