1 Department of Medicine, Columbia University, New York 10032; and 2 Department of Physiology, Cornell University, New York, New York 10021
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ABSTRACT |
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Renal
epithelial cells derive from either cells of the metanephric mesenchyme
or ureteric bud cells, but the origin of other renal cells is unclear.
To test whether metanephric mesenchymal cells generate cells other than
epithelial, we examined the developmental potential of a metanephric
mesenchymal cell line (7.1.1 cells) and of primary cultures of
metanephric mesenchymal cells. 7.1.1 Cells express both
mesenchymal and epithelial markers and, on confluence, form
well-defined monolayers expressing epithelial junctional proteins.
However, 7.1.1 cells as well as primary cultures of metanephric
mesenchymal cells also generate spindle-shaped cells that are positive
for -smooth muscle actin, indicating that they are myofibroblasts
and/or smooth muscle; this differentiation pathway is inhibited by
collagen IV and enhanced by fetal calf serum or transforming growth
factor-
1. Transforming growth factor-
1 also induces expression of smooth muscle proteins, indicating that the
cells differentiate into smooth muscle. 7.1.1 Cells as well as primary
cultures of metanephric mesenchymal cells also express vascular
endothelial growth factor receptor 2 and Tie-2, suggesting that the
metanephric mesenchymal cells that generate epithelia may also
differentiate into endothelial cells. The pluripotency of the 7.1.1 cells is self-renewing. The data suggest that the metanephric
mesenchyme contains embryonic renal stem cells.
nephrogenesis; cell differentiation; myofibroblast; transforming
growth factor-; collagen IV
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INTRODUCTION |
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CELL DIVISION IS INFREQUENT in the adult kidney, but this organ has the capacity to regenerate and there is abundant cellular proliferation during recovery from diseases, such as acute tubular necrosis (35). The origin of newly generated renal cells is largely undefined, but by analogy to other organs, organ-specific pluripotent cells (i.e., renal stem cells) are likely precursors of new cells. Although several strategies may be attempted to identify adult renal stem cells (1), we reasoned that such cells would share molecular and functional characteristics with embryonic renal stem cells. However, unlike blood cells, all of which derive from a single bone marrow stem cell, the origin of the different lineages in solid organs, such as the kidney, is obscure, and it is unknown whether the embryonic kidney contains organ-specific pluripotent cells that generate the many cell types present in the adult kidney.
Renal embryogenesis starts when a branch of the Wolffian duct called the ureteric bud invades an aggregate of mesenchymal cells called the metanephric mesenchyme. Reciprocal interactions between the metanephric mesenchyme and the ureteric bud result in growth and branching of the duct while the mesenchyme grows and converts to epithelia. The ureteric bud cells give rise to the epithelia of the collecting duct, and the metanephric mesenchymal cells give rise to the epithelia of the rest of the nephron.
Epithelia are the most prominent cells in the adult kidney, but this organ contains many other cell types, including mesangial, vascular smooth muscle, endothelial, and renal medullary interstitial cells, as well as myofibroblasts, fibroblasts, macrophages, and neurons. Of all these nonepithelial cells, and likely because nephron capillaries have such a critical role in nephron function, the potential origin of renal endothelial cells has long attracted attention. Embryonic kidneys grown in vitro do not develop capillaries, and when the developing kidneys of mice were grown on quail allantoic membranes, glomeruli were vascularized by quail endothelia, suggesting that renal endothelial cells may be exogenous to the kidney (33). However, recent studies by Robert et al. (30, 31) demonstrated that when embryonic kidneys are first grown in vitro and then grafted into the anterior eye chamber of a normal animal, the kidney develops endothelial cells (including glomerular capillaries) that are of graft origin. These studies suggest that embryonic kidneys contain endothelial cell precursors (angioblasts). However, it is not clear whether these angioblasts invade the embryonic kidney from outside, are a mesenchymal population of cells distinct from the metanephrogenic mesenchymal cells that will became renal epithelia, or derive from some distinct progenitor cell. Even less is known about the origin of the renal vascular smooth muscle and mesangial cells, and during kidney development, cells with smooth muscle characteristics are generated much later than nephrons and capillaries (5).
Except for epithelia and endothelia, the developmental origin of other renal cells remains little explored, and as an initial approach, three possibilities can be postulated: 1) there is migration of fully differentiated cell types into the developing kidney; 2) the embryonic kidney contains progenitor cells, each giving rise to an adult organ-specific cell type; and 3) the embryonic kidney contains progenitor cells with the ability to generate many cell types (i.e., multipotent progenitors or embryonic renal stem cells).
To test whether the embryonic kidney possesses organ-specific progenitor cells capable of differentiating into several cell types, we examined the differentiation potential of cells from rat metanephric mesenchymes. The mesenchymes were isolated at day 13 of embryonic age (E13), the day when, in the rat, the ureteric bud invades the metanephric mesenchyme, inducing its conversion to epithelia and starting renal development. We used an immortalized cell line of metanephric mesenchymal cells and primary cultures of these cells. The results indicate that metanephric mesenchyme contains cells that are pluripotent and can differentiate, in addition to epithelia, into myofibroblasts and smooth muscle cells and likely also into endothelial cells.
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METHODS |
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Cell lines. The metanephric mesenchymal cell line (7.1.1 cells) has been previously described (17, 18). Cells were grown in MEM containing 10% fetal calf serum (FCS), 2 mM glutamine, and penicillin plus streptomycin and incubated at 37°C in a 5% CO2 atmosphere. When cultures were passed, cells were dissociated with 0.5% trypsin in calcium- and magnesium-free HBSS with 0.02% EDTA. The cells were also cultured in the absence of FCS (serum-free media), in DMEM-Ham's F-12 supplemented with 5 µg/ml insulin and transferrin, 5 ng/ml sodium selenite, and 20 ng/ml of dexamethasone and L-thyroxine (all from Sigma) plus glutamine and antibiotics as described above. For passage, cells cultured in serum-free media were dissociated with calcium- and magnesium-free HBSS containing 0.02% EDTA without trypsin.
Primary cultures of metanephric mesenchymal cells were obtained from rat E13 mesenchymes dissociated as described (3) and grown in either of the two culture media described for the 7.1.1 cells but always supplemented with 50 ng/ml basic FGF and 10 ng/ml transforming growth factor (TGF)-RT-PCR.
RNA from cells was isolated and transcribed as previously described
(26), and PCR was performed with the primers shown in Table 1.
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Antibodies and reagents.
The following antibodies were used: monoclonal to -smooth muscle
actin (
-SMA), clone 1A4 (Sigma); monoclonal to calponin, clone M3556
(DAKO); monoclonal to E-cadherin, clone 36 (Transduction Laboratories);
polyclonal to cytokeratin (Zymed Laboratories); polyclonal to neural
cell adhesion molecule (NCAM; Chemicon); monoclonal to myosin light
chain kinase (MLCK), clone K36 (Sigma); monoclonal to rat endothelial
cell antibody 1, clone HIS52 (Serotec); Tie-2 polyclonals (RDI and
Alpha Diagnostic, no. 1), and monoclonal Ab33 (the gift of Dr. C. Kontos); monoclonal to smooth muscle myosin heavy chain, clone M3558
(DAKO); VEGF receptor 2 [VEGFR-2; R&D Systems and Cruz (SC505 and
SC315)]; polyclonal to vimentin (Chemicon); polyclonal to WT-1 (Santa
Cruz); and polyclonal to ZO-1 (Zymed Laboratories).
Immunodetection. Immunofluorescence was done in methanol-fixed cells or tissue sections as described (26, 27). Immunohistochemistry was done in methanol-fixed cells, serially blocked with 3% H2O2, 10% FCS/2% donkey serum (both dialyzed), and avidin/biotin (Vector Laboratories). After blocking, sections were incubated with the corresponding primary antibodies, and after washing, sections were incubated with a biotin-linked donkey secondary antibody. Avidin-linked horseradish peroxidase was next added with the ABC kit (Vector Laboratories), and peroxidase activity was localized with diaminobenzidine (DAB Substrate Pack, Biogenex) and enhanced by diaminobenzidine-enhancing solution (Vector Laboratories). Slides were finally counterstained with hematoxylin.
Immunobloting was done in protein homogenates as previously described (26).Cloning experiments.
7.1.1 Cells were dissociated with trypsin, diluted, and plated at a
density of ~30 cells/35-mm tissue culture dish. The cells were grown
in MEM containing 10% FCS and supplemented with 2 mM glutamine and
antibiotics. Five days later, individual clones were stained with
-SMA and ZO-1 antibodies. In addition, individual clones were
isolated with clonal rings and again cloned. These secondary clones
were expanded and analyzed for their ability to differentiate into
epithelia and into myofibroblast/smooth muscle.
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RESULTS |
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Metanephric mesenchymal cells. To examine the differentiation potential of metanephric mesenchymal cells, two preparations of cells were used. Most experiments were performed with a clonal cell line (7.1.1 cells) of metanephric mesenchymal cells. This cell line was generated as previously described (17, 18) and cloned by serial dilution. Briefly, metanephric mesenchymes from embryos at E13 were dissected from the ureteric bud and trypsin dissociated, and the cells were immortalized by the introduction of SV40 large T antigen using ts58 retrovirus. Transfected cells were selected with G4198. A clone was established and named 7.1.1 cells. Southern blots for the large T antigen in three subclones showed identical insertion sites, confirming that the line derives from a single clone (not shown).
Experiments were also performed with primary cultures of rat metanephric mesenchymal cells isolated from E13 embryos, the same age in which the 7.1.1 cells were isolated.Expression of mesenchymal and epithelial markers in 7.1.1 cells.
RT-PCR of 7.1.1 cells revealed expression of the genes for the
mesenchymal proteins vimentin, NCAM, and WT-1 (Fig.
1A); the latter two are
essential for kidney development (7, 21). In
addition, the cells also expressed the genes for E-cadherin, Pax-2, and
Wnt-4, all epithelial proteins with important roles during
nephrogenesis (21, 29). Surprisingly, despite the fact that the cell line derives from metanephric mesenchymes obtained the
day that rat renal development starts, RT-PCR was also positive for
mRNA of several genes characteristic of fully differentiated renal
epithelia, such as megalin, aminopeptidase A, the ClC-5 chloride
channel, and the thiazide-sensitive Na-Cl cotransporter.
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Expression of smooth muscle and endothelial genes in 7.1.1 cells.
To examine the possibility that 7.1.1 cells may generate progeny other
than epithelial cells, we examined them for expression of genes
characteristic of vascular cells (Fig.
2A). mRNA for several proteins
characteristic of smooth muscle, including -SMA, calponin, and
tropoelastin, were detected. The figure further shows that mRNA for
several proteins characteristic of endothelial or endothelial cell
precursors [VEGFR-2, Tie-2, CD31, and von Willebrand factor (vWF)]
was also detected. Because the expression of endothelial genes was
somewhat unexpected, to rule out the possibility that it was due to the
transformation of the cells, two other immortalized renal cell lines
were examined for the presence of VEGFR-2 and Tie-2. Although RT-PCR in
7.1.1 cells easily detected the mRNA for these two receptors, a mouse
ureteric bud cell line (2) and a rabbit collecting duct
cell line (9) transformed with the same virus used for
7.1.1 cells were both negative (Fig. 2B).
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Differentiation of metanephric mesenchymal cells into
myofibroblasts and/or smooth muscle.
The presence of mRNA for several smooth muscle genes in 7.1.1 cells
raised the possibility that these cells, in addition to giving rise to
epithelia, may also be capable of differentiating into myofibroblast
and/or smooth muscle. To explore this, we first examined for the
presence of -SMA by immunofluorescent microscopy. We found that when
the cells were grown at low density, individual cells expressed
-SMA
as well as the epithelial marker ZO-1 (Fig. 3A, a) . However, in confluent
cultures, the
-SMA-positive cells became spindle shaped, no longer
expressed ZO-1 (Fig. 3A, b), and, by changing the depth of
field, could be seen to grow on top of the monolayer of cells
expressing ZO-1. In contrast to the 7.1.1 cells, cells from the
ureteric bud cell line did not express
-SMA. These results indicate
that although most 7.1.1 cells differentiate into epithelia, they can
also differentiate into myofibroblasts and/or smooth muscle.
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Regulation of metanephric mesenchymal cell differentiation into
myofibroblasts and/or smooth muscle.
Because E13 kidneys do not contain myofibroblasts and/or
smooth muscle cells (Fig. 3B, a), the expression of -SMA
by 7.1.1 cells and primary cultures of metanephric mesenchymal cells in vitro could be due to one or the combination of two factors. First, there may be factors in the embryonic kidney that inhibit
differentiation of metanephric mesenchymal cells into a myofibroblast
and/or smooth muscle fate (i.e.,
-SMA expression). Second, there may
be factors in vitro that facilitate differentiation of mesenchymal
cells into this fate. As the metanephric mesenchyme initiates
differentiation into epithelia, there is loss of collagens I and III
and appearance of collagen IV (10) so that
mesenchymal-to-epithelial conversion and collagen IV expression are
closely linked (10, 22). Hence, we postulated that
collagen IV may facilitate mesenchymal conversion to epithelia, thereby
preventing differentiation of induced metanephric mesenchymal cells
into myofibroblasts and/or smooth muscle cells. Indeed, immunoblots
demonstrated that when 7.1.1 cells were grown on collagen IV for
7-10 days,
-SMA became undetectable (Fig. 4A). Similarly,
immunofluorescence of 7.1.1 cells (Fig. 4A, a) and of
primary cultures of metanephric mesenchymal cells (Fig. 4A,
b) grown on collagen IV revealed that all cells expressed ZO-1 and that
cells expressing
-SMA disappeared from the cultures. These results
indicate that collagen IV prevents differentiation of induced
metanephric mesenchymal cells into myofibroblasts and/or smooth muscle
cells.
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Endothelial phenotype of metanephric mesenchymal cells.
The presence of mRNA for VEGFR-2 and for Tie-2 in 7.1.1 cells suggested
that metanephric mesenchymal cells could, in addition to generating
epithelia, myofibroblasts, and smooth muscle cells, also be precursors
for endothelial cells. Accordingly, we examined the endothelial
phenotype of the 7.1.1 cells. Immunoblots with three antibodies to
distinct protein domains of VEGFR-2 demonstrated that similar to the
endothelial cell line MS-1, the 7.1.1 cells synthesize VEGFR-2 (Fig.
5A). The blot with antibody
SC-315 shows that the peptide used to raise the antibody fully blocked
the signal.
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DISCUSSION |
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When the ureteric bud invades the metanephric mesenchyme and development of the kidney starts, the renal anlage contains ureteric bud cells (progenitors of the collecting duct epithelia) and metanephric mesenchymal cells. Of the latter, two distinct populations of cells have been identified: cells that are the progenitors of the nephron epithelia (3), excluding the collecting duct, and metanephric mesenchymal stroma cells (15), which presumably are the progenitors of some interstitial cells. Cells expressing VEGFR-2 have also been identified in the metanephric mesenchyme (30, 31) and are believed to be endothelial precursors (i.e., angioblasts). It is unclear, however, whether these angioblasts are distinct from the mesenchymal epithelial precursors or indeed whether they are exogenous to the embryonic kidney.
The origin of the nonepithelial cell types in adult kidney is unknown
and, except for endothelial cells, little explored. To determine
whether a renal embryonic cell could generate several of the cell types
present in adult kidney, we examined the differentiation potential of
metanephric mesenchymal cells isolated on the first day of kidney
development (E13 in the rat). We used two types of
metanephric mesenchymal cells. With an immortalized clonal cell line
(7.1.1 cells), we found that the cells express mesenchymal as well as
epithelial proteins, suggesting that the mesenchyme from which the
cells were derived had initiated, after ureteric bud induction, its
transformation into epithelia (3, 34). When cultured in
vitro, the cells develop monolayers, express epithelial junctional
proteins, and have well-defined apical and basolateral domains,
indicating that the cells differentiate into epithelia. In addition,
RT-PCR showed that the cells express a variety of renal genes
characteristic of fully differentiated nephron epithelia, suggesting
that these cells are kidney-specific mesenchymal cells. We also found
that the cells possess the capacity to differentiate into cell types
other than epithelia and, with the appropriate culture conditions,
differentiate into cells that are spindle shaped, do not express ZO-1,
do not make a monolayer, and express -SMA, indicating that they are
myofibroblasts and/or smooth muscle cells. Although expression of
-SMA is not indicative of a smooth muscle phenotype, the appearance
of cells with several specific smooth muscle proteins (Figs.
2A and 4C) suggests that 7.1.1 cells can indeed
fully differentiate into smooth muscle cells.
We identified two exogenous cues that regulate differentiation of the
7.1.1 cells toward myofibroblast and/or smooth muscle. Seeding the
cells on collagen IV decreased, and FCS and TGF-1 enhanced, the number of spindle-shaped cells expressing
-SMA. Whether these effects result from selection by collagen IV and TGF-
1 of cells with an already determined phenotype
(resulting from asymmetrical cell division) or these factors act by
inducing expression of a genetic program that causes a specific cell
fate is now unclear. Because TGF-
1 was found to
transdifferentiate mammary epithelia into mesenchyme (24),
it would appear that at least in the case of this cytokine, the second
possibility is more likely. Regardless, it is worth noting that the
appearance of myofibroblasts in the renal parenchyma is a prominent
feature of many renal diseases, and these cells are believed to
participate in the mechanism of disease (reviewed in Ref.
13). Although it has long been known that
TGF-
1 increases production of renal extracellular matrix
(4) and may increase the number of renal myofibroblasts
(11), it may be worth exploring the possibility that
instability of collagen IV may also be a mechanism contributing to
myofibroblast appearance and fibrosis in renal diseases. Of note,
humans with genetic mutations of different collagen IV chains (36) or animals with deletion of these genes
(6) develop interstitial fibrosis with renal failure.
At the embryonic age at which the primary cultures of metanephric
mesenchymal cells were isolated (E13), -SMA is not
detectable in the cells of the metanephric mesenchyme in vivo, but
isolation and overnight culture causes most of these cells to become
spindle shaped and express
-SMA (Fig. 3B). Similar to the
7.1.1 cells, this differentiation was markedly inhibited by collagen IV
because this matrix protein restricted the cells toward an epithelial fate. The similar behavior of the 7.1.1 cell line and the primary cultures of metanephric mesenchymal cells indicate that the plasticity of the line is not due to its transformation but rather reflects the
embryonic origin of the cells. In agreement with our results, previous
studies have shown that isolation of neuronal stem cells with
propagating genes does not alter the ability of the cells to enter into
multiple differentiation pathways (12, 14).
In a previous study using a lineage marker (19), it was found that metanephric mesenchymal cells could generate all the different types of nephron epithelia (except collecting duct), indicating that these cells are renal epithelial stem cells. Although in that study no vascular cells were detected, the present results indicate that metanephric mesenchymal cells have a broad differentiation potential and, in addition to epithelia, can generate myofibroblasts and smooth muscle cells. In addition, because clonal experiments demonstrated that the ability of the cells to differentiate into either epithelia or myofibroblast/smooth muscle is self-renewing, it is apparent that some metanephric mesenchymal epithelial precursors are embryonic renal stem cells.
Because fetal and adult organ-specific stem cells are likely to have similar molecular characteristics, characterization of renal embryonic renal stem cells may provide clues to identify adult renal stem cells.
We also found that the 7.1.1 cells express two receptor tyrosine kinases, VEGFR-2 and Tie-2, that are the hallmark of an endothelial or endothelial precursor phenotype. The two receptors were also detected in primary cultures of metanephric mesenchymal cells and in vivo metanephric mesenchymes. Although somewhat surprising, these results are consistent with previous reports detecting VEGFR-2 (31) or its mRNA (25, 30) in metanephric mesenchyme and the mitogenic effect of VEGF in many cells, including epithelia, of isolated renal mesenchymes (37). Although the presence of Tie-2 in the embryonic kidney has been suspected to be restricted to well-differentiated endothelial cells (40), there has been no previous analysis of early metanephric mesenchyme. Furthermore, in E13 rat embryos, Tie-2 has been found in tongue and intestinal mesenchymes as well as heart epicardium (23). Of note is that as renal development advances and shortly after E13, Tie-2 quickly becomes restricted to endothelial cells (not shown), which is in agreement with findings from in situ hybridization (40).
The finding of VEGFR-2 and of Tie-2 in 7.1.1 and primary cultures of metanephric mesenchymal cells suggests that metanephric mesenchymal cells that are epithelial precursors and the angioblasts previously detected in the embryonic kidney (30, 31) are the same cells; i.e., metanephric mesenchymal cells that generate epithelia are also progenitors of endothelial cells. Recent studies have shown that most early mesoderms contain cells with characteristics of endothelial precursors (reviewed in Ref. 16), but our studies suggest that at least in the kidney, the endothelial precursors may be the same cells that under appropriate developmental cues became nephron epithelia, myofibroblasts, or smooth muscle. Interestingly, a vascular progenitor capable of giving rise to both smooth muscle and endothelial cells was recently derived from embryonic stem cells (38); the fate of this vascular progenitor is determined by the specific growth factors to which it is exposed.
Confirmation that our cell line and metanephrogenic mesenchymal epithelial precursors are also endothelial progenitors is presently not available. Despite synthesis of VEGFR-2 and Tie-2 and detection by RT-PCR of genes that are characteristic of fully differentiated endothelial cells (e.g., vWF, CD 31), proteins that characterize mature endothelia, including VE-cadherin, vWF, and CD31, could not be detected in 7.1.1 cell immunoblots (not shown). In addition, multiple attempts to induce the cells to increase the synthesis of these proteins and become fully differentiated endothelial cells were unsuccessful. Needless to say, it is possible that the appropriate differentiation signals were not provided or that our conditions contained inhibitory cues for endothelial differentiation, much like collagen IV is for smooth muscle fate. Alternatively, the presence of Tie-2 and VEGFR-2 in metanephric mesenchymal cells may have an as yet undefined function.
Regardless of whether the metanephric mesenchymal cells that generate epithelia also are endothelial progenitors, the finding that they are embryonic renal stem cells makes their phenotype (i.e., simultaneous expression of mesenchymal and epithelial proteins, ability to differentiate into epithelia or myofibroblast/smooth muscle and VEGFR-2+, and Tie-2+) potentially useful in the identification of adult renal stem cells. Work in the central nervous system suggests that embryonic and adult stem cells share molecular characteristics and developmental potential (28).
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ACKNOWLEDGEMENTS |
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We thank David Williams for help with electron microscopy and Irene Diaz for secretarial assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46934.
Address for reprint requests and other correspondence: J. Oliver, Dept. of Medicine, Columbia Univ., 630 West 168 St., New York, NY 10032 (E-mail: jao7{at}columbia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajprenal.00375.2001
Received 26 December 2001; accepted in final form 20 May 2002.
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