Mol. Hum. Reprod. Advance Access originally published online on October 15, 2008
Molecular Human Reproduction 2008 14(11):619-625; doi:10.1093/molehr/gan058
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Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells
1Reproductive Medicine Center, Peking University Third Hospital, Beijing 100083, People's Republic of China 2Stem Cell Research Center, Peking University Third Hospital, Beijing 100083, People's Republic of China 3Neuroscience Research Institute, Peking University, Beijing 100083, People's Republic of China
4 Correspondence address. E-mail: chenguian2008{at}bjmu.edu.cn
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Abstract |
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During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the ‘right’ therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and β-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT–PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K+. Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH+ cells which may be used in cell therapy for PD in future.
Key words: human embryonic stem cell/in vitro differentiation/TH-positive cells/transplantation
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Introduction |
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We all know pharmacological treatment for Parkinson's disease (PD) with L-3, 4-dithydroxyphenylalanine works initially, but over time there is reduced efficiency along with motor complications (Arenas, 2002; Perrier et al., 2004; Takagi et al., 2005). A potential therapeutic approach to PD which has been assayed for the last decades is the implantation of dopamine (DA)-producing cells in the striatum. Among the various classes of DA cells used, the best results have been obtained with fetal midbrain neurons (Dunnett et al., 2001; Vitalis et al., 2005). Indeed, over 15 years of clinical experience, the transplantation of human fetal mesencephalic brain tissues into the caudate-putamen of patients with PD could promote significant and sustained clinical improvement (Taylor and Minger, 2005). However, the technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissues have limited the application of this therapy (Sonntag et al., 2005). Human embryonic stem cells (hESCs), derived from the inner cell mass of preimplantation blastocysts, can proliferate indefinitely and are able to differentiate into cell types of all three germ layers in vivo and in vitro (Thomson et al., 1998). These properties of hESCs make them an excellent candidate for cell-replacement therapy in PD, and this requires the development of simple and reliable cell differentiation protocols.
Several protocols could direct hESCs towards differentiation into DA neurons, by using complex media with undefined components or coculture with various cells of animal origin (Buytaert-Hoefen et al., 2004; Perrier et al., 2004; Park et al., 2004; Yan et al., 2005; Lou et al., 2006). After transplantation, a few of these hES-derived DA neurons could survive (Schulz et al., 2004; Zeng et al., 2004; Park et al., 2005; Brederlau et al., 2006; Iacovitti et al., 2007), but only two reports indicated the functional benefit from these grafts (Sonntag et al., 2007; Geeta et al., 2008) in rat PD models.
were cultured on a feeder layer of mitotic-inactivated (3000 rads gamma irradiation) mouse embryonic fibroblasts (mEFs; Abbondanzo et al., 1993) with a daily change of medium consisted ofIn this study, we tried to enrich the functional tyrosine hydroxylase (TH)-positive cells differentiated from our hESCs line PKU1.1 (Peng and Chen, 2006) using a chemically defined culture system and transplanted those cells into rat models of PD to detect whether the derived TH+ cells could survive and lead to functional improvements in the PD rats.
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Materials and Methods |
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hESCs culture
hES cells, PKU1.1 (passages 63–102, a monoclonal cell line; Peng and Chen, 2006), derived from preimplantation human blastocyst in Reproductive Medical Center of Peking University Third Hospital, were cultured on a feeder layer of mitotic-inactivated (3000 rads gamma irradiation) mouse embryonic fibroblasts (mEFs; Abbondanzo et al., 1993) with a daily change of medium consisted of 80% knock-outTM DMEM (Gibco/BRL, Carlsbad, CA, USA), 20% knock-outTM Serum Replacer (Gibco/BRL), 4 ng/ml basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ, USA), 0.1 mmol/l β-mercaptoethanol (Gibco/BRL), 2 mmol/l glutamine (Gibco/BRL), 1% non-essential amino acid stock (Gibco/BRL), 50 IU/ml penicillin (Sigma, St Louis, MO, USA) and streptomycin (Sigma). Cells were cultured at 37°C in 6% CO2 in air and passed every 6–7 days by dissociation with 1 mg/ml collagenase IV (Gibco/BRL) treatment (Stage 1).
Differentiation and enrichment of NPCs
After incubation with collagenase IV for about 5 min, hES cells were then detached mechanically, pelleted, resuspended and cultured for 5–6 days in bacteriological Petri dishes (Greiner Bio-one) with daily medium change to form aggregated embryoid body (EB) (Stage 2). The EB medium consisted of 80% DMEM/F12 (Gibco/BRL) with knock-outTM Serum Replacer, β-mercaptoethanol, glutamine, non-essential amino acid stock, penicillin and streptomycin same as stem cell culture medium, but without bFGF. After EB formation to a certain size (diameter 0.2–0.45 cm) with cystic cavities, tissue culture dishes coated with poly-L-lysine were used for further culture, and N2 medium consisting of DMEM/F12, insulin (25 µg/ml), transferrin (100 µg/ml), progesterone (20 nmol/l), putrescine (60 µmol/l), sodium selenite (30 nmol/l), heparin (2 µg/ml) (all from Sigma) and bFGF (20 ng/ml) was used for cell differentiation into neural progenitor cells (NPCs). Two weeks later, cells were isolated mechanically and replated. Cells grew in adherence dishes and were passed every 6 days by mere mechanical detachment and dissociation (Stage 3).
TH+ cell differentiation
The detached, enriched NPCs were replated again onto 10-cm tissue culture dishes precoated with poly-L-ornithine (15 µg/ml, Sigma)/laminin (1 µg/ml, Sigma) at the density of 5–5.5 x 104 cells cm–2. Medium was changed every 2 days, and growth factors [100 ng/ml fibroblast growth factor 8 (FGF8), 200 ng/ml sonic hedgehog (SHH), 1 ng/ml transforming growth factor type β3 (TGF-β3), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF) (all from R&D Systems, Minneapolis, MN, USA) and 0.5 mM dibutyryl cAMP (db-cAMP) (Sigma)] were added in different combinations at various time points as follows: SHH and FGF8 were supplemented in N2 medium for the culture during the first 14 days at this stage, then GDNF, TGF-β3 and db-cAMP were added instead of the two previous factors for six more days (Stage 4).
Reverse transcription-polymerase chain reaction
Total RNA was extracted from cultured cells by using Trizol (Invitrogen, Carlsbad, CA, USA). First-stand cDNA was generated with a Superscript first-stand synthesis kit (Invitrogen). A total of 3 µg RNA treated by DNase was used in each cDNA synthesis, in a total volume of 25 µl. PCR amplification was performed by using a standard procedure with Taq Polymerase (Promega, Madison, WI, USA). The number of cycles was 35 with denaturation at 95°C for 30 s and elongation at 72°C for 60 s. The PCR cycle was preceded by an initial denaturation of 5 min at 95°C and followed by a final extension of 10 min at 72°C. The primers, product lengths and annealing temperatures were shown in Table I.
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Negative control was achieved by omitting transcriptase during RT or cDNA samples during PCR
DA measurement
During the Stage 4, cultured media for each 48 h at Days 1, 8, 14 and 20 were collected. And activity-dependent DA release from the cultured cells at Day 14 and 20 was first measured by culture cells in DMEM/F12 medium for 30 min at 37°C, and then replacing it with DMEM/F12 containing 56 mM KCl for 30 more minutes. DA in the collected media were stabilized by 7.5% orthophosphoric acid and 0.22 mg/ml metabisulfite, then stored at –80°C until examination (Studer et al., 1996).
DA was extracted by aluminum adsorption and eluted in 0.4 M perchloric acid (Anton and Sayre 1962). Then the aliquots (50 µl) were separated by a reverse phase C18 column (25 x 0.46 cm, Waters, Cotland, NY, USA) maintained at 30°C with a column heater. The mobile-phase consisted of 63.5 mmol citric acid, 61.0 mmol sodium citrate, 0.1 mmol EDTA, 3% methyl cyanide was pumped at a flow rate of 1.0 ml/min using Water's solvent delivery system. Electroactive compounds were analysed at 0.7 V using an analytical cell and an amperometric detector (Waters). DA levels were calculated using external DA standard injected before and after each detected sample immediately. Every assay was duplicated, and data were pooled from three different collected culture media.
Transplantation
Animals were housed and treated following National Institutes of Health guidelines. Adult Sprague–Dawley female rats (200–220 g) were anaesthetized with chloral hydrate (0.35 g/kg, i.p.) and injected with 6-hydroxydopamine (6-OHDA, 8 µg/4 µl in saline, Sigma) into the right medial forebrain bundle [–4.4 mm anterior/posterior (AP), 1.2 mm medial/lateral (ML) relative to bregma and 7.8 mm below the dura] over 4 min. Four weeks later, the unilaterally 6-OHDA-lesioned rats were tested for rotational behaviour in response to subcutaneous apomorphine (0.5 mg/kg) injections in an automated rotometer. Animals showing significant ipsilateral rotations (>4 rpm) were used for transplantation studies.
The cultured cells at Stage 3 and Stage 4 were isolated and resuspended in new media at a concentration about 105 cells/µl. Total 5 µl of the cell suspensions were used for an intrastriatal (1.1 mm AP and 2.7 mm ML relative to bregma and 5 mm, 4.5 mm below the dura) injection over 5 min through a 10 µl syringe with a pulled-glass micropipette under pump. After each injection, the needle was left for an additional 5 min and then slowly withdrawn. To inhibit immunological rejection, injections of cyclosporine A (10 mg/kg, i.p.) were given to the animals daily starting 48 h prior to grafting and continuing for 5 days, then followed by addition of cyclosporine (0.1 mg/ml) to the drinking water until death. In sham animals, 5 µl of N2 medium was injected and the cyclosporine A (0.1 mg/ml) solution was given daily after operation.
The rats were tested for rotational behaviour again 4 weeks after transplantation. Then they were anaesthetized and perfused transcardially with 4% paraformaldehyde in PBS. The whole brains were removed and subjected for immunostaining assay as described later.
Immunostaining on cultured cells and brain slices
Cultured cells were fixed by 4% paraformaldehyde in PBS for 30 min at room temperature, while rat brain was perfused overnight. The rat brain tissues were then dehydrated in a series of alcohol gradients (70–100%), embedded in paraffin and sectioned at 6 µm routinely. The specific primary antibodies were used as follows: rabbit anti-nestin (1:100, Boster Biotechnology Company, Wuhan), mouse anti-β-tubulin isotype III (TuJ1, 1:1000, Sigma), mouse anti-TH (1:1000, Sigma), mouse anti-human nuclei (HN, 1:100, Chemicon, Temecula, CA, USA). For visualization, appropriate fluorescence-tagged second antibodies or peroxydase-conjugated antibodies (1:100, Zhongsan Biotechnology Company, Beijing) against the first ones were used. The immunostaining procedures were performed according to the manufacturer's instruction. Cell nuclei were stained with 5 ng/ml 4', 6'-diamidino-2-phenylindole (Sigma) in immunofluorescence assay. Negative controls were set by using PBS instead of the primary or secondary antibodies.
Statistical analysis
Statistical analysis was performed using the SPSS statistical package (SYSTAT Software Inc.). Differences were evaluated by Student's t-test, Fisher's exact test and one-way ANOVA. P-value <0.05 was considered statistically significant.
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Morphology and cell markers of neural progenitors derived from hESCS
The schematic diagram of the protocol was shown in Fig. 1A. Five days later, the formed EBs (Fig. 1C) were transferred to tissue dishes and sequentially cultured with defined neural culture medium. The touched cells migrated out from the colony centre and formed a monolayer rosette formation after 2 weeks. Then they were detached mechanically into small clumps and transferred to new dishes with fresh medium same as above. Cells were passed in this way every 6 days. After 5 weeks of culture, the Stage 3 differentiated cells (Fig. 1D) showed a meshy morphology as epithelial cell appearance. Immunofluorescence staining revealed the majority of these cells (>90% of total cells) were nestin-positive (Fig. 1E), but negative for TuJ1 and TH, which is special for neural precursor cells.
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Characteristics of TH+ cells differentiated from hES cells
After being treated with SHH and FGF8 for 2 weeks at Stage 4, culture cells in the colony centre developed neural-like structures and displayed a bipolar or multipolar characteristics with extensive development of fine processes, similar to the neurons (Stage 4; Fig. 2A). Immunofluorescence of these cultured cells showed nestin (Fig. 2B) and TuJ1-positive (Fig. 2C), more like large networks neurons. Some of these TuJ1-positive cells also expressed TH. But for an additional 6 days culture, TH+ cells increased expeditiously (Fig. 2D) and the vast majority of cells (>90%) co-expressed TH and TuJ1 (Fig. 2E–G). But at this time, nestin still existed in cytoplasm of most cells.
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In addition to the immunofluorescence staining, RT–PCR analysis was performed to further detect three dopaminergic cell markers, including TH, dopamine transporter (DAT) and transcription factor Nurr1, as well as Nestin. During the differentiation process, Nestin expression became lower and lower, while the Nurr1 expression was much higher at the end of Stage 4 compared with the beginning of cell culture. On the agarose gel, TH band could be seen at Day 14 after the beginning of Stage 4, but DAT band appeared only at Day 20 of Stage 4 (Fig. 2H).
DA release from hES-derived TH+ cells in vitro
Reverse-phase HPLC was used to examine the ability of hES-derived cells to release DA. The results were shown in the Fig. 3.
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DA release was not detected at Day 1 and 8, but later at Day 14 and 20, DA was detected in the media under 56 mM KCl-induced depolarization stimulus, but the amounts were very low without K+ stimulation. More DA was detected at Day 20 than those at Day 14. These observations suggested that the hES-derived TH+ cells had dopaminergic neuron function when they were cultured for about 2 weeks at Stage 4.
Transplantation of hES-derived cells into PD rats
We transplanted the hES-derived cells into the striatum of rats that had received unilateral lesions with 6-OHDA 4 weeks before and had been tested for apomorphine-induced rotation to verify lesion completeness. Two differentiated stage cells were used for transplantation, early neural precursors (cells at the end of Stage 3; Group 1) and TH+ cells after 20 more days of differentiation culture (cells at the end of Stage 4; Group 2). Eighty rats were used to make hemi-Parkinsonian models, but only 27 rats were suited for this study after 4 weeks and were divided into three groups according to function. At 2 and 4 weeks after transplantation, the survival rates of animals were shown in Fig. 4A. There were no statistically significant differences between the three groups.
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The graft function was assessed by reversal of apomorphine-induced rotation behaviour at 4 weeks after cell injection. The majority of animals in Group 1 (4/5) and some in Group 2 (2/6) showed a significant (>30%) recovery in rotation behaviour, but no one in control group. The average percentage decrease in rotation scores as compared with pre-transplantation values was shown in Fig. 4B. We observed that in both Group 1 and Group 2 there were considerable decreases of the mean number of turns, and the changes of rotation behaviour were also great compared with the control group, but the significant differences were only found in Group 1. These observations suggest that transplantation of early neural precursors as well as TH+ cells derived from hESCs can lead to functional improvements in the PD rats.
According to immunostaining analysis, we found hES-derived TH+ cells were identified around the graft sites in both Group 1 and Group 2, as shown in Fig. 4C–F, but the number of TH+ cells was limited. And those cells showed small cell bodies with short processes in rat brains, unlike in culture dishes.
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Discussion |
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The differentiation of DA neurons from hESCs has been reported since 2004. Generally there are two different methods, as in the mouse. One is coculturing ES cells with a stromal cell line, like PA6 (a clonal pre-adipocyte stromal cell line isolated from newborn mouse calvarias), and this method was termed stromal cell-derived inducing activity (SDIA) (Kawasaki et al., 2000, 2002). The SDIA method provides efficient derivation of DA neurons from hES cells, by using the MS5, S2 cells for coculture (Perrier et al., 2004). Up to 79% of all cells expressed specific cell marker TH, which is the rate-limiting enzyme in the synthesis of DA. Later, in addition, Buytaert-Hoefen and other investigators applied PA6 stromal cells for coculture to yield high percentage of DA neurons at the end of the differentiation process (up to 90% of all cells). However, the mechanism of SDIA is unclear, and considering clinical cell-replacement therapy as a goal of hESCs research, cell lines in contact with animal products had contamination risks as well as undefined origins (Yan et al., 2005). The other protocol is a multiple-step method involving EB formation followed by selection for nestin-positive cells and addition of neurotrophic factors, as reported by Park et al. (2004), Yan et al. (2005), Iacovitti et al., (2007) and Geeta et al. (2008), but the efficiency was much lower than that of coculture methods when used to generate functional DA neurons from hES cells, only 20–60% TH+ cells were obtained.
In this study, we tried to adopt and modify multiple-step culture method to improve the differentiation efficiency. According to the experimental results, we proved that our human ES cell line, PKU1.1, which has maintained in an undifferentiated state for 102 passages could directly differentiate into TH+ neurons in a chemically defined system by several steps: the generation of EBs (Stage 2), selection and proliferation for NPCs (Stage 3) and differentiation of the progenitor cells to TH+ cells (Stage 4). At the beginning of Stage 4, we cultured cells by adding FGF8 and SHH which dramatically increased the percentage of TH+ cells (>90%), in comparison with previous results by using bFGF and TGF-
(Park et al., 2004). Although the intrinsic control of dopaminergic fate specification remains to be clarified, FGF8 and SHH could efficiently promote differentiation of DA neurons from ESC-derived neuroepithelium as described previously (Ye et al., 1998; Hynes and Rosenthal, 1999; Lee et al., 2000). The generation of DA neurons requires the ventralis signal SHH, in conjunction with factors that define AP patterning, such as FGF8, FGF4 and retinoic acid. Then the expression of transcription factors involved in midbrain DA neuron development, Lmx1b, En1, nuclear orphan receptor 1 and Pitx3 increases sequentially, at last TH appears in the cells (Perrier et al., 2004). In this experiment, we also observed treatment of these two factors for a period more than 10 days at Stage 4 and found that a high percentage of the NPCs could differentiate into TH+ cells.
The phenotypes of hES-derived TH+ cells were also confirmed by RT–PCR. We observed Nurr1 mRNA expressed at the Stage 3 before DA neuron differentiation, but not TuJ1 and TH. After treated with FGF8 and SHH for 14 days, the levels of TH and Nurr1 mRNA were up-regulated, but DAT mRNA was still not detected. Since absence of DAT could lead to either excess, unregulated dopaminergic transmission or to premature loss of synaptic stores of the transmitter (Park et al., 2005), we think these cells are immature at this time. But when we added three survival-promoting factors (GDNF, db-cAMP and TGF-β3) in the final differentiation stage for six more days, we observed most of cells expressed both TuJ1 and TH in cytoplasm, and DAT mRNA could also be detected. Furthermore, the HPLC detection also suggested TH+ cells were able to synthesis and release more DA after treatment with survival-promoting factors. These may indicate that TH+ cells became more mature. The effects of these three factors have been demonstrated previously. GDNF is considered as a stimulus for the differentiation of mesencephalic neurons, and a potent survival factor for maintaining DA neurons via inhibition of apoptosis (Lin et al., 1993; Clarkson et al., 1995; Widmer et al., 2000; Brundin., 2002). While the db-cAMP is implicated in promoting DA neuron yield from CNS cultures and maintaining genes phosphorylation or reactivating of protein kinase B to avoid apoptosis (Branton et al., 1998). TGF-β3 at terminal stage of ES cell differentiation could significantly enhance mRNA expressions of Nurr1, TH and bcl-2, known to be an anti-apoptotic gene (Flanders et al., 1998; Krieglstein et al., 1998; Rolletschek et al., 2001).
So far, there are several reports on the transplantation of hES-derived cells into Parkinsonian models, but the cell survival rate was low (Schulz et al., 2004; Zeng et al., 2004; Park et al., 2005; Brederlau et al., 2006; Iacovitti et al., 2007); and only Sonntag's and Geeta’s experiments showed the functional benefit from the grafts, which adopted the SDIA method by coculture with MS5-Wnt1 cells (Sonntag et al., 2007) or just transplanted NPCs (Geeta et al., 2008).
We applied intrastriatal transplantation of hemi-Parkinsonian rats to test the function of our hES-derived TH+ cells in vivo. Four weeks after implantation, we detected hES-derived cells in both Groups 1 and 2, but the number of TH+ cells presenting in the brain was limited. The reason maybe caused by the fact: DA neurons are highly sensitive to enzymatic treatment and physical dissociation (Yoshizaki et al., 2004; Yamazoe and Iwata, 2006). For transplantation, DA neurons should be collected from the culture dish and it is difficult to collect them without damaging the cells. And also the rat striatum environment may be not ideal for the survival of transplanted hES-derived TH+ cells. We found some Parkinsonian rats were behaviourally improved in both experimental groups, and it seemed more improvements took place in Group 1. But as the number of animal models was so limited, we could not conclude that the cells in Group 1 were more effective. More work should be done to achieve survival, maintain and function of hES-derived neurons in vivo. The PD rat behaviour was improved in Group 1 in this experiment, and it implied: the progenitor cells were able to further differentiate into TH+ cells in vivo or the progenitor cells may support the endogenous progenitor cells and lead to the behavioural improvment (Geeta et al., 2008).
In conclusion, we used a chemically defined culture system to efficiently generate TH+ cells from our hESCs line, PKU1.1. We proved that in order to produce functional DA cells in vitro, it was necessary to treat cells with the survival-promoting factors, like GDNF, TGF-β3 and db-cAMP, at the final differentiation stage. And these cells could survive and reduce the apomorphine-induced rotation behaviour of Parkinson's rat models. The differentiation protocol presented here will be useful in further studies of human midbrain DA neurons development as well as potential cell-replacement therapy of PD in future.
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National Natural Sciences Foundation of China (30672239).
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Acknowledgments |
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The authors acknowledge Dr Hongmei Peng for providing hESCs. We are grateful to Mr. Qiuming Geng for excellent technical assistance in HPLC. We would like to thank Professor Xiaoming Wang for providing an opportunity to work on animal experiment and Haiyan Yu for assistance in immunofluorescence experiment.
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Submitted on August 6, 2008; resubmitted on September 11, 2008; accepted on October 1, 2008.
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