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Research Article |
Prenatal Diagnosis Center,3 Cathay General Hospital, Taipei 106, Taiwan
School of Medicine,4 Fu Jen Catholic University, Hsinchung 242, Taiwan
College of Medicine,5 Taipei Medical University, Taipei 110, Taiwan
Bioresource Collection and Research Center,6 Food Industry Research and Development Institute, Hsinchu 300, Taiwan
Department of Medical Research,7 Taichung Veterans General Hospital, Taichung 407, Taiwan
ABSTRACT
Recent evidence has shown that amniotic fluid may be a novel source of fetal stem cells for therapeutic transplantation. We previously developed a two-stage culture protocol to isolate a population of amniotic fluid-derived mesenchymal stem cells (AFMSCs) from second-trimester amniocentesis. AFMSCs maintain the capacity to differentiate into multiple mesenchymal lineages and neuron-like cells. It is unclear whether amniotic fluid contains heterogeneous populations of stem cells or a subpopulation of primitive stem cells that are similar to marrow stromal cells showing the behavior of neural progenitors. In this study, we showed a subpopulation of amniotic fluid-derived stem cells (AF-SCs) at the single-cell level by limiting dilution. We found that NANOG- and POU5F1 (also known as OCT4)-expressing cells still existed in the expanded single cell-derived AF-SCs. Aside from the common mesenchymal characteristics, these clonal AF-SCs also exhibit multiple phenotypes of neural-derived cells such as NES, TUBB3, NEFH, NEUNA60, GALC, and GFAP expressions both before and after neural induction. Most importantly, HPLC analysis showed the evidence of dopamine release in the extract of dopaminergic-induced clonal AF-SCs. The results of this study suggest that besides being an easily accessible and expandable source of fetal stem cells, amniotic fluid will provide a promising source of neural progenitor cells that may be used in future cellular therapies for neurodegenerative diseases and nervous system injuries.
amniotic fluid cells, amniotic fluid-derived stem cells, developmental biology, dopamine, dopamine release, mesenchymal stem cells, neural stem cells, pregnancy
INTRODUCTION
Despite the well-established culturing of amniotic fluid cells (AFCs) in routine prenatal genetic diagnosis, many questions concerning the nature and in vivo origin of these cells have not been entirely resolved [1–3]. Amniotic fluid contains a heterogeneous population of cells, which are contributed mainly from the fetal skin; the fetal digestive, respiratory, and urinary tract; and the placental membranes [4–7]. Recent discoveries of stem cell populations in amniotic fluid have postulated that the amniotic fluid is a promising alternative source of fetal stem cells for cellular therapy [8–13].
We previously developed a novel two-stage culture protocol to isolate amniotic fluid-derived mesenchymal stem cells (AFMSCs) from second-trimester amniocentesis without interfering with the process of fetal karyotyping [14]. Aside from the common mesenchymal lineages (adipocytes and osteocytes), our cultured AFMSCs have also been successfully differentiated into neuron-like cells. Recent reports have shown that adult bone marrow and umbilical cord blood contain a subpopulation of pluripotent-like mesenchymal stem cells that can be isolated at the single-cell level and have the capacity to differentiate into cells of all three germ layers [15–18]. Moreover, several animal model studies have also demonstrated that marrow stromal cells could mimic the behavior of neural stem cells by participating in many aspects of normal brain development, and could promote recovery of spinal cord injury [19, 20]. These indicated that bone marrow might constitute a potential source of neural progenitors for treating a variety of central nervous system disorders. Do amniotic fluid-derived stem cells (AF-SCs) have a similar capability to marrow stromal cells? It is still unknown whether they contain a heterogeneous population of stem cells from different germ layers or a subpopulation of primitive precursors that are capable to overcome germ layer commitment. In this study, we attempted to isolate AF-SCs at the single-cell level and evaluate their phenotypic characteristics and differentiation potentials in vitro.
MATERIALS AND METHODS
Cell Culture
Amniotic fluid was obtained by amniocentesis performed between 16 and 20 wk of gestation for fetal karyotyping. Initial AF-SCs were cultured from nonadhering cells of the primary AFC culture (NA-AFCs) as previously described [5, 6]. Single-cell derived AF-SC clones were established by limiting dilution. Briefly, the primary plating-adhering AF-SCs were trypsinized, serially diluted to 1 cell/ml, and seeded 200 µl onto 96-well plates in alpha-modified Minimum Essential Medium (
-MEM; Invitrogen) supplemented with 20% fetal bovine serum (FBS; Hyclone) and 4 ng/ml basic fibroblast growth factor (FGF2; R&D Systems) and incubated at 37°C with 5% humidified CO2. The clonal AF-SCs were expanded serially with a split ratio of 1:3 and tested for their genetic markers, cellular surface antigens, and differentiation potentials. The Institutional Review Board of Cathay General Hospital, Taipei, Taiwan, approved this protocol, and each patient signed a written informed consent.
Flow Cytometry Analysis
The specific cell surface antigens of the clonal AF-SCs at passage 7–8 were characterized by flow cytometry analyses. Cells in culture were trypsinzed and stained with fluorescein isothiocyanate (FITC)- or phycoerythrin-conjugated antibodies against ITGAM, ANPEP, CD34, THY1, HLA-A,B,C, and HLA-DR,DP,DQ (BD PharMingen); CD14, ITGB1, CD44, and ENG (Eurolone); and SH2 and SH3 (American Type Culture Collection). Thereafter, the cells were analyzed using a flow cytometry (Becton Dickinson).
RT-PCR Procedure
Total RNA was extracted from the clonal AF-SCs by using TRI Reagent (MRC Inc.) according to the manufacturer's instructions. RT-PCR was performed using the One Step RT-PCR kit (Qiagen Inc.) with specific DNA primers as follows: NANOG (426 bp), sense, 5'-GCGCGGTCTTGGCTCACTGC-3', antisense, 5'-GCCTCCCAATCCCAAACAATACGA-3'; POU5F1 (also known as OCT4) (247 bp) sense, 5'CGTGAAGCTGGAGAAGGAGAAGCTG-3', antisense, 5'-CAAGGGCCGCAGCTTACACATGTTC-3'; NES (200 bp) sense, 5'-ATCGCGCCAGCCCTCATCAGC-3', antisense, 5'-TTTTCCGTGTAGCCAGCCTTGTCG-3'; TUBB3 (175 bp) sense, 5'-CATGGACAGTGTCCGCTCAG-3', antisense, 5'-CAGGCAGTCGCAGTTTTCAC-3'; NEFH (400 bp) sense, 5'-TGAACACAGACGCTATGCGCTCAG-3', antisense, 5'-CACCTTTATGTGAGTGGACACAGAG-3'; GFAP (317 bp) sense, 5'-CTGGAGGTTGAGAGGGACAATCT-3', antisense, 5'-TACTGCGTGCGGATCTCTTTC-3'; and ACTB (396 bp) sense, 5'-TGGCACCACACCTTCTACAATGAGC-3', antisense, 5'-GCACAGCTTCTCCTTAATGTCACGC-3'. The conditions for all RT-PCR in this study were initially at 50°C for 30 min and 95°C for 15 min for reverse transcription, followed by 35 cycles, with each cycle consisting of denaturing at 94°C for 1 min, annealing at 57°C for 1 min, elongation at 72°C for 1 min, and the final extension at 72°C for 10 min. The amplified DNA fragments were visualized through 2% agarose gel electrophoreses, stained, and photographed under UV light. NTERA-2 cl.D1 cells (ATCC CRL-1973, a pluripotent human testicular embryonic carcinoma cell line) and HeLa cells (ATCC CCL-2, a human cervix adencarcinoma cell line) were used as a positive control and a negative control respectively for RT-PCR expression analysis.
Differentiation Assay of Single Cell-Derived Clonal AF-SCs
The clonal AF-SCs at the 12th passage were grown to 70%–90% confluence and shifted to osteogenic medium (-MEM supplemented with 10% FBS, 0.1 µM dexamethason, 10 mM ß-glycerol phosphate, 50 µM ascorbate) or adipogenic medium (-MEM supplemented with 10% FBS, 1 µM dexamethasone, 5 µg/ml insulin, 0.5 mM isobutylmethylxanthine, and 60 µM indomethacin) for 3 wk. The differentiation potential for osteogenesis was assessed by the mineralization of calcium accumulation by Alizarin Red S (Sigma) staining and alkaline phosphatase activity (Sigma) staining. For adipogenic differentiation, intracellular lipid droplets could be observed under microscope and confirmed by Oil Red O (Sigma) staining. For differentiation of neural cells, the clonal AF-SCs were incubated with -MEM supplemented with 20% FBS, 1 mM ß-mercaptoethanol, and 5 ng/ml FGF2 for 24 h, and then treated with neuronal induction media composed of Dulbecco modified Eagle medium (DMEM) and 10 mM ß-mercaptoethanol for 5 h. Immunocytochemical stain and RT-PCR were used to assess the capacity of neuronal differentiation.
Immunocytochemical Analyses
For cellular NANOG and POU5F1 expression analyses, the clonal AF-SCs, HeLa cells (negative control), and NTERA-2 cl.D1 cells (positive control) were washed, fixed, and incubated with human anti-NANOG polyclonal antibody (R&D Systems) and mouse anti-POU5F1 monoclonal antibody (Santa Cruz Biotechnology), respectively, overnight at 4°C. For neural gene expression analyses, the noninduced and induced clonal AF-SCs were washed, fixed, and incubated with murine specific monoclonal antibodies directed against the following markers of neural progenitor cells or mature neural cells overnight at 4°C: NES (nestin, monoclonal mouse IgG1, 1:200; Chemicon), TUBB3 (tubulin ß-III, monoclonal mouse IgG1, 1:400; Chemicon), NEFH (neurofilament, polyclonal rabbit IgG, 1:400; Chemicon), NEUNA60 (neuron-specific nuclear protein, also know as NeuN, monoclonal mouse IgG1, 1:100; Chemicon), GALC (galactocerebroside, monoclonal mouse IgG3, 1:200; Chemicon), and GFAP (glial fibrillary acid protein, polyclonal rabbit IgG, 1:1000; Chemicon). Thereafter, the cells were washed and incubated with a secondary antibody of FITC-conjugated goat anti-mouse IgG (Chemicon) or rhodamine-conjugated goat anti-rabbit IgG (Chemicon) at room temperature for 1 h. Cell nuclei were counterstained with 1 µg/ml 4,6-diamino-2-phenylindole (DAPI; Molecular Probes) in PBS for 5 min and mounted in Vectashield mounting medium (Vector Laboratories).
Dopamine Release Assay
The clonal AF-SCs were plated onto 24-well plates at a density of 104 cells per well. After 24 h, the medium was replaced by dopaminergic differentiation medium composed of 90% DMEM/F-12 (1:1), 10% FBS, 10 µM forskolin (Sigma), and 50 ng/ml FGF8 (PeproTech) for 7 days. For dopamine release assay, cells were washed twice and incubated for 2 min with a low-K+ solution (20 mM HEPES, pH 7.4, 140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4. and 11 mM glucose, all from Sigma). After aspiration, 1 ml per well of high-K+ solution (20 mM HEPES, pH 7.4, 85 mM NaCl, 60 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose) was added for 10 min, and then the solution was collected for dopamine analysis [21]. Concentrations of norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine (DA) in the solutions were determined by a microbore HPLC system (Beckman Coulter, Fullerton, CA) using a microbore column packed with Inertsil-2 C18 particles (GL Science) and a dual potentiostat amperometric detector (AS-4C with MF-1020 electrode; Bioanalytical Systems) as described in our previous report [22]. The identity of each peak of the chromatogram was confirmed by its retention time, redox ratio, spiking, and superimposition-aligment techniques that were provided by Beckman Gold Data Analysis Software (Beckman Coulter).
RESULTS
Single Cell-Derived AF-SCs Clones in Culture
Initially more than 100 AF-SCs clones were obtained from 8 different amniotic fluid samples after plating the primary AF-SCs (passage 0) onto 96-well plates by limiting dilution with a cell concentration of 0.2 cells per well theoretically. To facilitate proliferation, these primary AF-SCs clones were passed and grown first onto 24-well plates and then transferred each time the culture reached 80%–90% confluence to 12-well plates, 6-well plates, 25-T culture flasks, and finally to 10-cm culture dishes. Only about two to five single-cell-derived AF-SCs clones were maintained in every amniotic fluid sample, and the subsequent passages were carried on in 10-cm culture dishes under the same culture condition.
These AF-SCs clones showed a homogenous fibroblastlike morphology (Fig. 1, a1 and a2) and grew to 90% confluence of the subsequent passage culture in 3 days (Fig. 1a3). Two AF-SCs clones (93507 and 93502) were further tested on their cellular phenotypic characteristics and differentiation potentials in this report. At the time of writing, we have completed the 26th passages of these two clones, and their chromosome analyses remained as a normal karyotype at the 25th passage.
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POU5F1 and NANOG Expressions by Clonal AF-SCs
RT-PCR and immunocytochemical staining were performed for the analysis of pluripotency markers POU5F1 and NANOG expression by the clonal AF-SCs. Both POU5F1 and NANOG gene expressions were positive for mRNA and protein levels of the clonal AF-SCs (Fig. 1, b and c).
Flow Cytometry Analysis of the Clonal AF-SCs
The characteristics of cell surface antigens of the clonal AF-SCs at passage 7–8 were analyzed by flow cytometry. The single cell-derived AF-SCs had an immunophenotype similar to that of common MSCs. SH2, SH3, ANPEP, ITGB1, CD44, THY1, and HLA-A,B,C (MHC class I) were strongly positive; CD14 and ENG were low positive; and ITGAM, CD34, and HLA-DR,DP,DQ (MHC class II) were negative, as shown in Figure 2.
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Adipogenic and Osteogenic Differentiation of the Clonal AF-SCs
To evaluate the mesenchymal differentiation potential of the single-cell derived AF-SCs, cells at the 12th passage were cultured under conditions that favored adipogenic and osteogenic differentiation respectively.
Adipogenic tendency of the clonal AF-SCs was apparent after 1 wk of incubation with adipogenic induction medium. Between the culture periods of 2–3 wk, almost all cells contained numerous Oil Red O positive lipid droplets (Fig. 3a). Similarly, after culturing the clonal AF-SCs with osteogenic induction medium for 3 wk, most of the cells showed the nodules of calcium mineralization in the culture by Alizarin Red S staining (Fig. 3b) and became alkaline phosphatase-positive (Fig. 3c). Nontreated control cultures of clonal AF-SCs did not show any of the above differentiated phenomena (Fig. 3, d–f).
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Neural Marker Expression by the Clonal AF-SCs
To evaluate neural differentiation capacity of the clonal AF-SCs, cells at the 13th passage were used for neural induction (Fig. 4a1). The clonal AF-SCs were incubated in a serum-containing preinduction medium for 24 h (Fig. 4a2), and then treated with serum depletion for 5 h. Initially, cytoplasm in some of the AF-SCs retracted toward the nucleus and began to form an elongated cell body within several hours of pretreatment. After incubation under serum-free conditions, a morphologic change to a rounded cell body increased progressively, and then axonal outgrowth was observed in 1–3 h (Fig. 4a3). At the fifth hour, three-fourths of the cell bodies became spherical with multiple cell processes, a neuron-like morphology (Fig. 4a4).
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RT-PCR was performed on those cells of the NA-AFCs and the clonal AF-SCs before and after neural induction. All three types of cells were positive on neural gene expression of NES, TUBB3, NEFH, and GFAP, respectively (Fig. 4b). However, the expression of NEFH and GFAP on the NA-AFCs seemed to be low. It may need to be verified by quantative RT-PCR in future study.
Further immunocytochemical analyses were performed on the noninduced and induced single cell-derived AF-SCs. Both cells showed positive neural expressions of NES (an intermediate filament protein expressed by neural stem or progenitor cells), TUBB3 (a neuronal cytoskeletal dimer), NEFH (a marker of neurofilament located primarily in the cytoplasm of mature neurons), GFAP (a structure element of fibrillary astrocytes), NEUNA60 (a marker for early neurons), and GALC (a marker for oligodenrocytes) as shown in Fig. 4, d–i. The percentage of positive cells of these markers before and after neural induction were 96 ± 4% versus 88 ± 5% for NES; 91 ± 8% versus 97 ± 3% for TUBB3; 80 ± 6% versus 97 ± 6% for NEFH; 43 ± 13% versus 97 ± 5% for GFAP; 82 ± 2% versus 84 ± 12% for NEUNA60; and 55 ± 7% versus 92 ± 14% for GALC. Nontreated control cultures did not show any of the above stains.
Dopamine Release by the Clonal AF-SCs after Dopaminergic Induction
In addition to neuronal-like morphology and specific neural gene expressions, production of dopamine is an important hallmark of functional neurons. After 7 days of dopaminergic differentiation, the induced AF-SCs produced a significant basal level of DOPAC and DA when depolarized by high-K+ stimulation for 10 min (Fig. 4c). The DOPAC and DA concentrations in the culture medium were ca. 0.057 nM and 0.849 nM, respectively. The undifferentiated AF-SCs controls did not show any production of dopamnine.
DISCUSSION
In this study, we successfully obtained single-cell derived AF-SCs clones from culturing NA-AFCs of primary amniocytes culture by limiting dilution in -MEM supplemented with 20% FBS and 4 ng/ml FGF2. The clonal AF-SCs can be expanded rapidly and express common characteristics of mesenchymal stem cells as well as neural stem cells in vitro. Under appropriate culture conditions, these clonal cells still maintained the capacity to differentiate into multiple cell types such as mesodemal lineages, adiopocytes and osteocytes, and ectodermal lineages, neuronal cells, and glial cells. These observations indicate that human amniotic fluid contains a subpopulation of multipotent stem cells, which might be the precursors of both mesenchymal and neural stem cells. During cultivation, these primitive precursors may receive a specific commitment into directions of either mesenchymal or neural lineages, depending on culture conditions. This phenomenon illustrated that the epithelial-mesenchymal transition may exist in fetal stem cells derived from second-trimester amniotic fluid, which is a variant of transdifferentiation and a well-recognized mechanism for dispersing cells in vertebrate embryos [23–26]. Most importantly, HPLC analysis showed evidence of dopamine release in the extract of dopaminogenic-induced AF-SCs when they were depolarized by high K+. This finding suggests that these clonal AF-SCs are able to synthesize and release dopamine and may be a favorable candidate for cellular therapy of neurodegenerative diseases and central nervous system injuries.
Under our culture protocol, a small portion of the clonal AF-SCs spontaneously differentiated into neural cells, as identified by both morphological transformation into neuronal-like phenotype and immunochemical expression of markers (NES, TUBB3, NEFH, NEUNA60, GALC, and GFAP) for neural progenitor cells and mature neural cells during the passage cultures. However, when these clonal AF-SCs were treated with neural differentiation medium for 5 h, more than three fourths of them showed a neuronal-like appearance, and the expression of mature neural markers (NEFH, GALC, and GFAP) was also increased. Our data confirm a recent report by Prusa et al. [11], which described evidence that native human amniotic fluid contains neurogenic cells, and that differentiation occurs sporadically in standard culture conditions but is strongly increased in neurogenic induction medium. In the past, the unexpected presence of neural cells in AFC cultures would indicate that the fetus had a neural tube defect [3, 6]. However, both the study of Prusa et al. [11] and our study argue for the existence of neural progenitor cells in second-trimester amniotic fluids from normal pregnancies. There are three possibilities that could describe this discrepancy. One explanation is that stem cells normally adhere later than other cells during AFC cultivation. Another possibility is that routine AFC culture conditions do not favor neurogenic cell growth and that thus the neural progenitor cells are contained in NA-AFCs instead. A third explanation is that, in fact, neural progenitor cells do not exist in native amniotic fluid, but rather, pluripotent precursor cells exist in NA-AFCs and require culturing under specific conditions to make the commitment to becoming neural progenitor cells. These explanations may need to be verified in future studies.
Amniotic fluid contains a heterogeneous population of cells from fetal origin. In't Anker et al. recently reported that amnion is a novel source of fetal MSCs and the potential site contributing to MSCs in amniotic fluid [12, 13]. Sakuragawa et al. [27] demonstrated that amnion mesenchyme cells (AMCs) expressed markers of neuroglial progenitor cells both before and after neural induction, and that they could be differentiated into neuroglial phenotypes by optimal differentiation protocol. These AMCs were vimentin (VIM)-positive up to 97% and KRT19-positive 3.6% in cultivation [27]. The clonal AF-SCs we cultured in this study also had the same neuroglial phenotypes as AMCs, but showed no VIM or KRT19 expression (data not shown). Thus, we suggest that amnion is unlikely to be the main contributing source for fetal MSCs in amniotic fluid.
Cellular therapy using neural stem cells (NSCs) is a potential strategy for the treatment of neurodegenerative disorders and central nervous system injuries [28–30]. Usually, NSCs can be derived from embryonic stem cells or from two principal adult neurogenic regions of the brain, the hippocampus and the subventricular zone [31–33]. Each of these sources, though successful, has its limitations, whether they involve inadequate tissue supply or are immersed in ethical controversy. Therefore, looking for alternative sources of NSCs has become meaningful research [20, 34–39]. As an alternative source of NSCs, amniotic fluid provides several advantages compared to other sources such as adult bone marrow, human amniotic epithelial cells, umbilical cord blood, Wharton's jelly, and adipose tissue. First, amniotic fluid contains a subpopulation of primitive stem cells in early fetal life. In a preliminary study, we analyzed the telomere length [40] of our single-cell derived AF-SCs (at passage 6), umbilical cord blood-derived MSCs, and bone marrow-derived MSCs (both at passage 4, not single-cell clones); they were ca. 14.3 kb, 12.0 kb, and 10.0 kb, respectively (data not shown). This finding indicated that AF-SCs had a higher proliferation capacity than that of the postnatal adult stem cells mentioned above. Second, amniotic fluid contains a pool of various stem cells, which may coordinate their capacities and improve the efficiency of tissue repair during cellular transplantation. Third, amniotic fluid is easily obtained and has a higher success rate with regard to isolating stem cells. Finally, amniotic fluid-derived stem cells open an unprecedented approach for autologous intrauterine fetal gene and cellular therapy.
Under routine culture conditions for fetal karyotyping, AFCs can be divided into two major categories: adhering and nonadhering cells [4–6]. However, many questions concerning the nature and in vivo origin of the NA-AFCs have not been entirely defined [7]. In our preliminary study, the total number of NA-AFCs was between 5 and 8 x 105 cells/ml. However, only 5% of these cells, morphologically small and rounded, were alive and could adhere to a culture flask under specific conditions. In our two-stage culture protocol, the NA-AFCs were collected from supernatant of primary cultures using serum-free Chang's medium [41]. This was followed by 7–10 days of incubation with no added nutrition, a condition similar to serum deprivation as reported by Pochampally et al. [42], who described a resulting subpopulation of human marrow stromal cells with enhanced expression of POU5F1 and other embryonic gene expressions. Therefore, besides containing populations of stem cells from different germ layers, NA-AFCs may also have a subpopulation of pluripotent-like stem cells with enhanced POU5F1 expression [10, 14]. We have previously reported that NA-AFCs contained a population of multipotent MSCs with fibroblastic-like phenotype, which retained the capacity to differentiate into various mesenchymal lineages and neuronal cells [14]. In the present study, we have further demonstrated that clonal AF-SCs could differentiate into both mesenchymal and neural lineages, indicating that they are multipotent precursors. More interesting issues of NA-AFCs could still be addressed in the near future, such as how many populations of stem cells are in NA-AFCs, strategies to isolate them, the number of stem cells in each trimester and their lifespan throughout pregnancy, and the role of stem cells inside the amniotic cavity during fetal development and any association with fetal compromise.
In conclusion, we have demonstrated that human amniotic fluid contains a subpopulation of clonally multipotent fetal stem cells. Besides the common mesenchymal lineage, these cells also have the capacity to differentiate into multiple neural lineages and release dopamine in vitro. These findings support that human amniotic fluid provides a great alternative source of neural stem cells for the treatment of neurodegenerative disorders and central nervous system injuries.
FOOTNOTES
2 Correspondence: Ming-Song Tsai, Prenatal Diagnosis Center, Department of OBS/GYN, Cathay General Hospital, 280 Jen-Ai Road, Section 4, Taipei, 106, Taiwan. FAX: 886 2 23259530; mstsai{at}cgh.org.tw 
1 Supported by grants from the National Science Council, Taiwan (NSC 92–2314-B-281–007 and NSC 93–2314-B-281–004) and the Ministry of Economic Affairs, Taiwan (93-EC-17-A-17-R7–0525).
Received: 2 August 2005.
First decision: 29 August 2005.
Accepted: 23 November 2005.
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