Hum. Reprod. Advance Access published online on October 24, 2007
Human Reproduction, doi:10.1093/humrep/dem335
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Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines
1 Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden 2 Department of Biosciences and Nutrition at Novum, Karolinska University Hospital Huddinge, SE 141 86 Stockholm, Sweden 3 Department of Molecular Medicine and Surgery, Cardiothoracic Surgery and Anaesthesiology Unit, Karolinska Institutet, Stockholm, Sweden 4 Department of Molecular Medicine and Surgery, Clinical Genetics Unit, Karolinska Institutet, Stockholm, Sweden
5 Correspondence address. E-mail: outi.hovatta{at}ki.se
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Abstract |
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BACKGROUND: For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement.
METHODS: We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT–PCR) and in vivo (teratoma growth).
RESULTS: Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005–2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003–2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present.
CONCLUSIONS: Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.
Key words: human embryonic stem cells/inner cell mass/mechanical isolation/human skin fibroblasts/derivation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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The first derivations of human embryonic stem cell (hESC) lines were carried out by using immunosurgery for isolation of the inner cell mass (ICM). Fetal calf serum (FCS) was a constituent of the culture medium, and mouse fibroblasts were used as feeder cells. Since then, the potential use of such cell lines for cell transplantation has been widely recognized (Keller and Snodgrass, 1999
; Edwards, 2001).
The use of animal-derived culture components in cell line derivation and culture systems is not desirable (Skottman et al., 2006). These components bear a risk of transmitting infections. They also contain immunogenic non-human sialoproteins (Martin et al., 2005).
In our laboratory, the first step towards creating xeno-free hESC lines was to use post-natal human skin fibroblasts as feeder cells in both derivation and propagation of hESCs (Hovatta et al., 2003), and at present, 28 permanent hESC lines have been derived using such feeders, of which 25 are fully characterized (HS181–HS429) (Table 1). Human placental fibroblasts have also been successfully used in derivation of hESCs (Genbacev et al., 2005; Simon et al., 2005), and propagation of existing lines has been possible with many types of human cells, as reviewed by Skottman and Hovatta (2006).
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Initially, we used FCS in the derivation medium. In its place, we later used serum replacement (SR) medium in the cultures (Koivisto et al., 2004
) as well as in derivation (Inzunza et al., 2005). Basic fibroblast growth factor (bFGF) (Amit et al., 2000) has been added in such culture media. The SR medium used for these lines still contained animal proteins.
Feeder-free derivation on a matrix manufactured from mouse tissues has been successful (Klimanskaya et al., 2005), but the animal protein-containing matrix is not optimal for cells aimed at use in human transplantation. Ludwig et al. (2006b) reported feeder-free derivation of two new hESC lines using a combination of a high concentration in chemically defined culture conditions: one of the lines was chromosomally abnormal (47, XXY) from the beginning, and the other one had gained an extra chromosome by passage 40 (Ludwig et al., 2006b). Hence, the normality of cells derived under such conditions is not yet clear, even though the karyotype 47, XXY is common in human embryos. Both the above-mentioned studies used immunosurgery, which involves use of animal components and enzymes for derivation of the new hESC lines.
To eliminate one source of animal-derived substances from hESC, we have used mechanical isolation of the ICM for the derivation of our eight latest cell lines. Mechanical isolation has been reported earlier (Simon et al., 2005; Ellerstrom et al., 2006) with the difference that the whole blastocyst was plated on the feeder layer and later the throphectoderm cells were removed. Five of our eight cell lines derived by mechanical isolation of the ICM have been fully characterized (HS415, HS420, HS422, HS426 and HS429), and they are described here in detail. Three more lines, HS475, HS480 and HS481 are currently being characterized.
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Materials and Methods |
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Blastocysts
We have approval from the Ethics Committee of the Karolinska Institute for derivation, characterization and early differentiation of hESC lines from donated supernumerary embryos. Blastocysts were obtained as donations from infertile couples undergoing IVF treatment at our fertility unit. Both partners signed an informed consent form after receiving oral and written information. Only embryos that could not be used in infertility treatment were used in stem cell line derivation. No re-imbursement was paid to the donors.
During September 2005–January 2006, 19 blastocysts on Days 6–8 after fertilization were received for hESC derivation from the Fertility Unit of Karolinska University Hospital Huddinge. During February 2006–November 2006, eight more embryos were obtained for derivation in these conditions. The embryos had been donated at the cleavage stage and were further cultured to blastocysts in the Fertility Laboratory. Blastocyst quality was defined according to the criteria presented by Gardner et al. (2000). According to these criteria, quality 1 means an early blastocyst with the blastocoel occupying <50% of the volume, 2 means an early blastocyst with the blastocoel expanded to 50–80% of the volume, 3 means a non-expanded blastocyst with a large blastocoel, 4 means an expanded blastocyst, 5 means that the blastocyst has started to hatch and 6 means that the blastocyst is completely hatched. The first letter describes the ICM: ‘A’ means a high number of tightly packed cells, ‘B’, a few cells, loosely packed, ‘C’, almost no cells. The second letter reflects the cell number in the trophectoderm: ‘A’ means many cells and ‘B’ means few cells. Hence, e.g. 4AA means an expanded blastocyst with a normal-looking cell number in both the ICM and trophectoderm.
Culture medium and feeder cells
The medium used for culture and derivation of the stem cells consisted of Knockout Dulbecco's modified Eagle's medium supplemented (20%) with Knockout SR, 2 mM Glutamax, 0.5% penicillin–streptomycin, 1% non-essential amino acids (all from Gibco Invitrogen Corporation, Paisley, UK), 0.5 mM 2-mercaptoethanol (Sigma-Aldrich Co. St Louis, USA.) and bFGF (R&H Systems, Oxon, UK) at 8 ng/ml.
As feeder cells, human foreskin fibroblasts (CRL-2429; ATCC, Manassas, VA, USA) (Hovatta et al., 2003) were used. They were mitotically inactivated by irradiation (40 Gy), and 100 000 cells were plated onto 2.84-cm2 dishes (Falcon) and left overnight to form a confluent monolayer.
The medium for culture of the feeder cells consisted of Iscove's medium supplemented (10%) with FCS and 0.5% penicillin–streptomycin (all from Gibco Invitrogen Corporation).
Mechanical isolation of the icm
The ICM of the blastocyst was isolated by using a specially made flexible metal needle, made of tungsten, with a diameter of 0.125 mm, made for us at the Helsinki School of Micromechanics (Espoo, Finland) The tip was made thin and sharp using electrolysis. We are working with Hunter Scientific Ltd, Essex, UK, to make these needles commercially available as indicated on page 7. Another blunter needle was used to hold the blastocysts while cutting out the ICM. The needles were fixed to hand-pieces of pencil-thickness for manual operation under a stereo-microscope (Nikon x1500). The blastocyst was first cleaned in three or four drops of SR medium and the last drop was then drawn out with the needle so that the blastocyst became attached to the surface of the well. With the blastocyst attached to the plastic, it was possible to make a hole in the zone pellucida with the needle to open up the blastocyst and by two to three cuts removing the ICM from the trophectoderm and place the ICM onto a fresh feeder plate. The procedure took 
2–3 min. The needles are expected to become available to other teams in the near future because discussions with a company are ongoing. This technique is applied manually, and it does not require any expensive equipment. (Detailed specification of the design and manufacture of the tools will be offered upon request.) The ICM was allowed to grow on the human skin feeder cells for 12–15 days before the first transfer of the growing cells to a new feeder plate.
Propagation of the lines
The hESC lines were propagated as described earlier by Inzunza et al. (2005). Splitting of the hESC colonies was always performed mechanically at 6- to 8-day intervals. Colonies, containing on average 20 000 cells, were cut into smaller pieces (6–8) and then transferred onto fresh feeder cells. For passaging, only non-differentiated cells were chosen, as judged by morphology.
Immunohistochemical characterization
Immunohistochemical characterization of the new hESC lines obtained by mechanical isolation of the ICM was carried out at passage levels 5–18. The primary antibodies were specific for Oct-4 (Chemicon, Temecula, CA, USA), nanog, TRA-1-60, TRA-1-81, SSEA-1 and SSEA-4 (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Human foreskin fibroblasts were used as negative control cells.
The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, washed with PBS and blocked with 5% serum (goat, fetal bovine or rabbit depending on the antibodies to be used). Permeabilization (not for surface markers) was carried out using 5% blocking buffer consisting of 0.02% TritonX-100 in PBS. Primary antibodies were added in 5% blocking buffer overnight at 4°C, and the cells washed three times with PBS to remove any unbound antibodies. The secondary antibodies, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG), Cy3-conjugated goat anti-rabbit IgG or FITC-conjugated bovine anti-goat IgG (all from Chemicon) were diluted 1:200 in 5% blocking buffer and applied to the cells for 60 min at room temperature in the dark. After two washes in PBS, the cells were counterstained with either Hoechst 33 342 (2 µg/ml, Sigma, St Louis, USA) or propidium iodide (PI, 1 µg/ml, Sigma) for 10 min for nuclear staining. Primary antibodies were the following: rabbit anti-Oct-4 monoclonal IgG (1:80), rabbit anti-nanog polyclonal (1:200) (both markers are intracellular) (Chemicon), mouse anti-SSEA-4 monoclonal IgG (1:200), mouse anti- SSEA-1 (1:200), mouse anti-TRA-1-60 (1:200), mouse anti-TRA-1-81 (1:200) and mouse anti-nestin human-specific IgG (1:200), all from Santa Cruz Biotechnology Inc. In addition to negative control cells, we had negative controls without primary antibodies and controls with non-immune serum. Stained cells were viewed with a Zeiss Axiovert 200M inverted microscope (Zeiss) equipped with fluorescence optics and appropriate filters, and images were acquired with Openlab 3.1.3 equipment.
Testing pluripotency in vitro
Embryoid bodies (EBs) were formed by culturing aggregates of hESCs in suspension in hESC culture medium without bFGF for 3 weeks before harvesting. The presence of tissues originating from the three embryonic germ cell layers was demonstrated using immunohistochemistry and RT–PCR.
Immunohistochemistry
The EBs were fixed for 1 h and stained for markers of the three embryonic germ layers; bone morphogenetic protein-4 (BMP-4) for mesoderm (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), nestin for ectoderm (Chemicon) and alpha-fetoprotein (AFP) for endoderm (Sigma). Primary antibodies were added in 5% blocking buffer overnight at 4°C, and the EBs washed three times with PBS to remove any unbound antibodies. The secondary antibodies, FITC-conjugated goat anti-mouse IgG, Cy3-conjugated goat anti-rabbit IgG or FITC-conjugated bovine anti-goat IgG (all from Chemicon) were diluted 1:200 in 5% blocking buffer and applied to the cells for 60 min at room temperature in the dark. After two washes in PBS the cells were counterstained with either Hoechst 33 342 (2 µg/ml, Sigma) or PI (1 µg/ml, Sigma) for 10 min for nuclear staining.
Rt–pcr analysis
In order to test for pluripotency, RT–PCR was also performed in regard to the same three markers, nestin, BMP-4 and AFP. Total RNA was extracted from EBs of the five new hESC lines using the Qiagen RNeasy Mini Kit protocol. Total RNA was calculated using a Nano-Drop Spectrophotometer and ND-1000, v.3.2.1 software. One microgram of total RNA was reverse-transcribed with Moloney Murine Leukemia Virus reverse transcriptase (Applied Biosystems) in a 20 µl reaction volume containing the manufacturer's buffer supplemented with 4 mM dNTPs, 20 U RNase inhibitor and 2.5 µM random hexanucleotides. The RT–PCR mixture was then diluted to 100 µl and PCR was performed with 10 µl of complementary DNA and 1.5 mM MgCl2 and 0.4 mM dNTPs in PCR buffer (reaction volume 50 µl) using 2.5 U Taq DNA polymerase (Promega). PCR cycle parameters were 94°C for 3 min, followed by 30 or 35 cycles at 94°C for 30 s, annealing at primer-specific temperature for 30 s, and 72°C for 30 s. Final extension was at 72°C for 7 min. The sequences of primers used were as follows: bMP-4 (sense, 5'-TTTGTTCAAGATTGGCTGTC-3'; antisense, 5'-AGATCCCGCATGTAGTCC-3') AFP (sense, 5'-CTTTGGGCTGCTCGCTATGA-3'; antisense, 5'-TGGCTTGGAAAGTTCGGGTC-3') and nestin (sense, 5'-GAAACTCAAGCACCAC-3'; antisense, 5'-TTTTAAACTCCAGCCATCC-3').
Teratomas
The pluripotency of these five lines in vivo was tested as previously described (Inzunza et al., 2004; 2005). Cells from passages 5 to 24 from all five cell lines (HS415, HS420, HS422, HS426 and HS429) were mechanically harvested from the culture plates. Five colonies (103–104 hESCs) were washed twice in PBS and subsequently implanted beneath the testicular capsule of young (6-week) male mice with severe combined immunodeficiency (C.B.-17/GbmsTac-scid-bgDF N7, M&B, Ry, Denmark). A total of 11 animals were used. Cell lines HS415, HS420, HS426 and HS429 were injected into two animals each, and three animals were used for the injection of line HS422. Teratoma growth was determined by palpation every week, and cystic teratomas were formed in all mice. Eleven weeks after implantation, the mice were sacrificed by cervical dislocation. The teratomas were fixed, and sections were stained with haematoxylin and eosin. The presence of tissue components of all three embryonic germ cell layers was analysed from the stained sections.
Karyotyping
Karyotyping of cell lines was carried out as described earlier by Inzunza et al. (2005) at passages 8–18. Samples of cells were treated with colcemid KaryoMAX (0.07 µg/ml; GIBCO, Paisley, UK) overnight. After washing, the cells were incubated in 0.4% trypsin solution (GIBCO, Paisley, UK) for 2–3 min. Cells were treated with collagenase (1400 IU/ml; Worthington, Lakewood, NJ, USA) at 37°C for 20 min and harvested using standard procedures. The metaphases were analysed after Q-banding. 14–24 total of metaphases have been analysed for each cell line.
Freezing
Samples of the lines were frozen by means of vitrification in pulled open straws, using ethylene glycol, dimethylsulphoxide (DMSO) (20% each) and 1 M sucrose as cryoprotectant, as described by Reubinoff et al. (2001). For larger quantities of hESC material, we have also used a slow freezing method. Up to 20 colonies were collected in a cryo-tube with as small amount of medium as possible, and 400–500 µl SR medium without bFGF supplemented with 10% DMSO was added. The cryo-tube was then placed in a Nalgene Cryo 1°C Freezing Container (5100–0001) (Nalge Nunc International, Rochester, USA) with isopropanol and placed in –70°C freezer overnight. The cryo-tube was then stored in liquid nitrogen. Thawed samples could be grown from the thawed aggregates.
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Results |
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Of the 19 blastocysts obtained during 2005–2006, 5 permanent hESC lines were obtained (Table II). The isolation of the ICM was successful in all. Eighteen ICMs attached to the feeder layer, and nine started to grow out (Table II). A stem cell colony-like growth continued from five blastocysts (Fig. 1). Mechanical splitting could be carried out 12–15 days later in all five early lines, and thereafter, new colonies could be passaged as cell lines. Samples from each line have been frozen, and they have started to grow after thawing. In addition to these five characterized lines. We have three new lines derived in similar conditions from eight blastocysts, now at passage levels 13–20. The characterization of these lines is ongoing. The five lines have been fully characterized; they express Oct-4, nanog, SSEA-4, TRA-1-60 and Tra-1-81, and they are negative for SSEA-1. All are karyotypically normal. Lines HS415 and HS429 have a normal female 46, XX chromosome constitution and HS420, HS422 and HS426 have a normal male 46, XY karyotype.
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EBs were formed from each line, and tissue components expressing AFP, BMP-4 and nestin were identified by RT–PCR (Fig. 2) and by immunohistochemistry (Fig. 3). All five lines formed teratomas that contained components of the three embryonic germ cell layers (Fig. 4). The lines have for the time being been in culture from 12 to 54 passages.
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We wanted also to analyse the efficiency of derivation after mechanical isolation of the ICM. It was not possible to carry out a prospective comparison using donated human embryos, and we carried out a retrospective analysis between immunosurgery and mechanical isolation of the ICM, knowing the biases of historical controls. During 2003–2005 we had derived 16 hESC lines in otherwise similar conditions but using immunosurgery (Table I) (Inzunza et al., 2005
). During that period, we obtained 16 hESC lines from 100 blastocysts. Five hESC lines from 19 blastocysts (26%) appear to be at least as effective as 16 lines from 100 embryos (16%). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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We managed to establish 5 permanent pluripotent hESC lines from 19 supernumerary blastocysts, which proved the feasibility of this method. Mechanical isolation of the ICM using a simple two-needle system proved successful. The isolation procedure was fast, and it did not require any major financial or time investments.
In our unit, we have wide experience of deriving new hESC lines (Hovatta et al., 2003; Inzunza et al., 2005), and we have produced a total of 25 permanent cell lines. In addition, there were several early cell lines that faded off after some passages. The latest 21 lines were all derived using human foreskin fibroblasts as feeder cells, and SR medium instead of fetal bovine serum (Table I). We are systematically improving the derivation and culture conditions in order to reach clinical quality of the cell lines. Mechanical removal of the zona pellucida, and isolation of the ICM was our logical next step in that direction.
Immunosurgery is not the optimal derivation method when poor quality, discarded embryos with hardly visible ICMs are used. Immunosurgery also involves animal-derived substances, mouse antibodies and guinea pig complement, which are not desirable in the context of cell transplantation. By using our mechanical isolation method, we could avoid the use of mouse antibodies and guinea pig complement, and it was not necessary to use pronase or Tyrode's acid, as the zona pellucida could be cut away at the same time. The procedure was therefore also time-saving.
The efficient, successful derivation of five cell lines from 19 blastocysts appeared to be better than using immunosurgery (16 cell lines from 100 blastocysts) in similar culture medium (Inzunza et al., 2005). However, it was not possible to carry out a comparative study and many other factors may have influenced the improved results in the latest series of derivations. It is possible that we have steadily improved our handling of the cells in recent years, even though the medium, feeder cells and culture plates have been similar since 2003, when we began the derivations using the present SR medium (Inzunza et al., 2005).
Mechanical isolation of the ICM has previously been successfully used in the derivation of two lines, as reported by Genbacev et al. (2005) and by Mummery, (2004) and van de Stolpe et al. (2005). Another method, which has not been successful in our hands (Inzunza et al., 2005), is to plate the whole blastocyst on feeders, and then remove the trophectodermal cells during the early stages of culture (Heins et al., 2004; Simon et al., 2005; Ellerstrom et al., 2006).
Even though mechanical isolation of the ICM was a clear step forward in hESC derivation, our present culture system is still not fully optimized as regards clinical quality (Skottman and Hovatta, 2006), as the SR medium still contains animal proteins. The use of chemically defined culture media has been reported, in which animal components in SR media have been replaced by more defined media and supplements. Li et al., (2005) as well as Genbacev et al., (2005) used X-VIVO 10 with bFGF, Vallier et al., (2005) used chemically defined media with activin A, nodal and bFGF, and Ludwig et al. (2006a and b) workers used defined culture media with bFGF, LiCl, 
-aminobutyric acid, transforming growth factor-
and pipecolic acid. Optimizing such media and using them is definitely the way forward as regards obtaining clinical quality cell lines.
The next step towards entirely defined derivation and maintenance of hESCs is a combination of mechanical isolation of the ICM, the use of human feeder cells derived and cultured in defined medium, and the use of a defined culture medium in hESC culture. In addition, an improved and more defined culture system in feeder-free conditions is desirable. However, deriving and culturing hESCs under such demanding conditions would need continuous controls as regards genetic and epigenetic normality of the cells. The reported feeder-free derivation systems have still contained non-defined components such as immunosurgery (Ludwig et al., 2006b) and a mouse-derived non-defined matrix (Klimanskaya et al., 2005). Nevertheless, by combining all these defined components, it will soon be possible to derive clinical quality hESC lines.
Our lines are available for researchers.
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Funding |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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This project was funded by the Swedish Research Council.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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We thank Dr Björn Rozell for his help in the teratoma evaluation, the personnel of the IVF Unit of the Fertility Unit, Karolinska University Hospital Huddinge for their help regarding the donated blastocysts, and Nicholas Bolton for revising the language. We also thank the Swedish Research Council and the Regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute, and the Linnéa och Josef Carlsson foundation for support.
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Footnotes |
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These authors contributed equally to this work. 
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Submitted on April 14, 2007; resubmitted on September 4, 2007; accepted on September 18, 2007.
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