ology 134 (2009) 75–81www.elsevier.com/locate/ijcard

International Journal of Cardi

Cardiac differentiation of P19CL6 cells by oxytocin

Fardin Fathi a,⁎, Satoshi Murasawa b, Satoshi Hasegawa b, Takayuki Asahara b,Abbas Jafari Kermani c, Seyed Javad Mowla c

a KDRC, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iranb Group of Vascular Regeneration Research, Kobe Institute of Biomedical Research and Innovation/Riken Center for Developmental Biology,

2-2 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japanc Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran

Received 9 May 2007; received in revised form 31 August 2007; accepted 14 January 2008Available online 5 June 2008

Abstract

Background: It has been reported that P19 embryonal carcinoma (EC) cells differentiate into beating cardiomyocytes under the action ofoxytocin (OT). It has been suggested that dimethylsulfoxide (DMSO) acts via the oxytocin/oxytocin receptor pathway because an oxytocinreceptor antagonist not only blocks oxytocin-induced cardiomyocyte differentiation, but also blocks DMSO-induced differentiation. In thisstudy, the differentiation ability of OT was tested using P19CL6 cells.Methods: P19CL6 cells were cultured as a confluent monolayer and aggregated cells. OT was then added to culture media as an inducingagent. The cells treated with 1% DMSO were used as a positive control group. Differentiated cells were evaluated morphologically andimmunocytochemically, as well as by RT-PCR. In addition, a stable line of green fluorescent protein (GFP)-expressing P19CL6 cells weredifferentiated into beating cardiomyocytes by OT.Results: Aggregated P19CL6 cells could be differentiated into cardiomyocytes, whereas monolayer cells could not differentiate and expressspecific cardiac muscle marker genes. In the control group, both aggregates and monolayer cells could be differentiated into cardiomyocytesby DMSO. In addition, GFP-expressing P19CL6 cells differentiated efficiently into beating cardiomyocytes when treated with OT. Theresults of all evaluations confirmed that the differentiated cells were cardiomyocytes.Conclusions: We concluded that embryoid body formation (cell aggregation) is necessary for the differentiation of P19CL6 cells intocardiomyocytes when using OT as an inducer agent. Furthermore, because of the high rate of differentiation efficiency, GFP-expressingcardiomyocytes derived from P19CL6 cells have the potential to be used for regenerative therapies in experimental models.© 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cardiomyocytes; Cell differentiation; Embryoid body formation; Oxytocin; P19CL6 cells

1. Introduction

Murine P19 embryonal carcinoma (EC) cells are a line ofpluripotent stem cells that can be maintained in culture in anundifferentiated state without the need to supplement themedium with leukemia inhibitory factor (LIF). This cell linecan be induced to differentiate in vitro into the cell types of

⁎ Corresponding author. Tel.: +98 871 6661830; fax: +98 871 6664674.E-mail address: farfath@yahoo.com (F. Fathi).

0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2008.01.046

the three germ layers. P19 EC cells were derived from anexperimental embryo-derived teratocarcinoma in mice andare one of the most intensively studied cells of all pluripotentembryonal carcinoma cell lines [1,2]. The P19 EC cell line isan excellent model to study cell differentiation because thecells differentiate into cardiomyocytes in vitro, therebymimicking the events of early cardioembryogenesis [3]. P19EC cells that have undergone differentiation into beatingcardiomyocytes exhibit the same biochemical and physiolo-gical properties as their embryonic equivalents [4]. P19CL6

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cells are a clonal derivative of P19 EC cells and can beobtained following long-term culture under conditions thatpromote mesodermal differentiation [5]. Unlike P19 CE cellswhose utility is limited because of their multipotentialproperties, P19CL6 cells differentiate efficiently intocardiomyocytes following treatment with DMSO in theabsence of prior embryoid body (EB) formation. Because thedifferentiation of almost all cells into cardiomyocytesrequires the expression of cardiac-specific genes, P19CL6cells are thought to be a useful in vitro model to studycardiomyocyte differentiation [5–7].

Recently, OT was demonstrated to induce cardiac differ-entiation in P19 EC cells [3]. OT, which is expressed primarilyin the hypothalamus, is a female reproductive hormone and isnecessary for uterine contractions during parturition, thetiming and amplification of labor, the facilitation of milkejection during lactation and the regulation of ovulation.Recently, a role for OTas a growth and cellular differentiationfactor has been suggested [8]. In addition, the existence of anoxytocin/oxytocin receptor (OT/OTR) pathway has beendiscovered recently in the heart [9,10]. Moreover, the levelsof expression of OTRs are higher in developing hearts than inadult hearts. Collectively, these findings suggest that OT maybe involved in many processes that include cell growth andcardiomyocyte differentiation [6]. Paquin et al. [3] haveexamined the role of OT in cardiomyocyte differentiationusing P19 EC cells. They reported that OT could cause thedifferentiation of P19 EC cells into beating myocardiocytes. Inaddition, they demonstrated that the cardiogenic action of OTwas specific and mediated by OTR because it could beabolished by an OTR antagonist. Furthermore, they demon-strated that an OTR antagonist not only blocked OT-inducedcardiomyocyte differentiation, but also DMSO-induced differ-entiation. This result indicates that DMSO acts via the OT/OTR pathway and suggests a new role for the OT/OTRpathway in cardiac development and in particular, OT primingof cardiogenesis [3,6]. Although the precise mechanism ofaction of OT in the developing heart is not clear, OT may playan important role in the differentiation of primordial cells,including adult somatic stem cells, into cardiomyocytes fromprimitive cells including adult somatic stem cells. Matsuura etal. were the first to demonstrate that OT can induce Sca-1+cells in adult hearts to differentiate into beating cardiomyo-cytes in vitro [11]. More recently, Hatami et al. has reportedthat OTR are present in undifferentiated embryonic stem cells(ESCs) and that their treatment by OT leads to an increase intheir differentiation into cardiomyocytes [12].

In this study using P19CL6 cells, we report our findingson the mechanism of action of OT-induced differentiationand the question of whether EB formation is necessary forthis transformation. We also report our results on thepreparation of a P19CL6 reporter cell line expressing greenfluorescent protein (GFP) and the results of experiments inwhich these cells efficiently differentiated into beatingcardiomyocytes after their treatment with OT that wasadministered prior to EB formation.

2. Materials and methods

2.1. Cell culture and differentiation

P19CL6 cells were cultured using a previously-describedmethod [5]. In brief, the cells were grown in a 100-mm tissueculture culture-grade dish under adherent conditions with α-minimal essential medium (α-MEM) (Gibco, USA) supple-mented with 10% fetal bovine serum (Gibco, USA), penicillin(100 U/ml), and streptomycin (100 µg/ml) (growth medium)and were maintained in a 5% CO2 atmosphere at 37 °C.

To induce differentiation under adherent conditions,P19CL6 cells were plated at a density of 8×10 4 cells/wellin 6-well tissue culture-grade dishes containing the growthmedium in the presence of 1% DMSO (Sigma, USA)(control group) or 1×10 −7 M OT (Sigma, USA) (experi-mental group). To induce differentiation under EB forma-tion, P19CL6 cells were plated at a density of 1×10 5

10 5 cells/ml in 100 mm bacteriological-grade Petri dishescontaining the growth medium in the presence of 1% DMSO(control group) and 1×10 −7 M OT (experimental group).After four days, the EBs were transferred into 6-well tissueculture-grade dishes. The growth medium was changedevery two days in both culture systems. The days ofexperiment were numbered consecutively after the first dayof DMSO or OT treatment (day 0). The cell populations wereanalyzed at days 6–14 of the entire differentiation protocol,at a time cardiac cells normally beat synchronously.

2.2. Stable transfection

The pEGFP-C1 plasmid was transfected into P19CL6cells. This vector was linearized using the ApaL1 restrictionenzyme and was used for their introduction into 1×10 7 cellsby electroporation (350 V/500 µF). Approximately 24 h aftertransfection, the culture medium was changed with fresh α-MEM containing 400 µg of G418 antibiotic. The mediumcontaining the G418 antibiotic was replaced every two dayswith fresh medium containing the G418 antibiotic and thecells were grown until individual colonies were visible,usually after 8–10 days. We could observe two types ofcolonies that were resistant to the G418 antibiotic (Gibco,USA). Most of the colonies expressed GFP but there weresome that did not express GFP. In order to separate the GFP-expressing cells, we isolated several cell lines using singlecell cloning. Briefly, stable-transfected cells were resus-pended at a density of 5 cells/ml in growth medium. Analiquot (100 µl) of this cell suspension was added to eachwell of a 96-well dish. Proliferated cells were visualized byan inverted fluorescent microscope(TE 300, Nikon, Japan)and five subclones expressing GFP were propagated.

2.3. Cell morphology and immunofluorescence

For morphological examination, P19CL6 cells weregrown directly onto the plastic surface of tissue culture

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vessels. For the immunocytofluorescence studies, beatingcardiomyocytes were transferred onto 4-well culture slidescoated with 0.1% gelatin on day 12. After 4 h, they werefixed by a 20 min incubation in phosphate buffered saline(PBS) containing 4% paraformaldehyde, then rinsed in PBS,and then finally stored in PBS at 4 °C until required. All stepsof fixation, permeabilization, washing, and incubation withantibodies were performed at room temperature. The fixedcells were permeabilized for 15 min in PBS containing0.02% Triton X-100, then blocked for 60 min in PBS TritonX-100 containing 10% serum and 0.02% Triton, and thenincubated with an antibody against troponin I (1:100)(Chemicon, USA) for 60 min and with an antibody againstthe sarcomeric myosin heavy chain (MHC) (1:50) (SantaCruz, USA) for 120 min. The samples were then incubatedwith secondary antibodies conjugated with biotin (1:500)and cyanine 3 dye (Cy3) (1:100) (Pharmingen, USA) forbinding to the antibodies against troponin I and MHC,respectively, for 60 min. A tertiary antibody conjugated withavidin–FITC (1:500) (Pharmingen, USA) was used for

Fig. 1. The effect of DMSO and OT treatment on the cardiac differentiation of P19Cthe presence of 1% DMSO (B, ×200) and 1×10 −7 M OT (D, ×100) in bacteriologi6-well tissue culture plate and cultured in medium without inducer. Each plate wasbeating cells. By days 8 and 9, colonies of beating cells were found in all wells treatat a density of 8×10 4 cells/well onto 6-well plates and cultured for 16 days in the pdifferentiated cell types were found among the cells treated with DMSO (E), wherethat were grown under adherent conditions and treated with OT (G, ×100). Timeagents and conditions has been represented (L). The results are representative of f

binding to the secondary antibody by incubating thespecimens for 30 min. PBS Triton X-100 blocking with2% serum was used for washing the specimens betweenincubations and diluting the antibodies. Coverslips weremounted in VECTASHIELD® Hard+Set™ mounting med-ium containing DAPI and immediately examined under themicroscope(TE 300, Nikon, Japan).

2.4. RT-PCR analysis

Total RNAwas isolated from the cells using an Isogen RNApurification kit (Nippon Gene, Tokyo). One microgram of totalRNA was reverse transcribed into DNA using AvianMyeloblastosis Virus (AMV) reverse transcriptase and oligo(dT) primer. RT-PCRwas performed in a 20µl reactionmixtureusing a thermal cycler. Aliquots of cDNA, which were derivedfrom RNA, were then subjected to PCR amplification withAmpliTaq®Gold kit and primer sets that are specific to the geneof interest. The PCR conditions were: initial denaturation for9min at 95 C followed by 35–40 cycles of denaturation for 30 s

L6 cells. P19CL6 cells were cultivated as aggregates from day 0 to day 4 incal dishes. Aggregates of 1 Petri dish were evenly distributed in the wells of aexamined at 2-day intervals for the number of wells containing colonies ofed with OTand DMSO, respectively (F, H, ×200). P19CL6 cells were seededresence of 1% DMSO (A, ×200) and 1×10 −7 M OT (C, ×200). By day 12,as and no beating cells and many dead cells were observed among the cellscourse of appearance of beating cell colonies upon treatment with differentour independent differentiation experiments.

Fig. 2. The expression of cardiomyocyte-specific genes in P19CL6aggregate-derived cardiomyocytes following DMSO and OT treatment. Toconduct reverse transcriptase (RT)-PCR, total RNA was isolated fromcardiac differentiated cells on days 6 and 14 of the experiment. After DNasetreatment, RT-PCR was performed for analysis of GATA-4, NKX2.5, Tbx5and Tbx20 genes on day 6 of the experiment, and ß-MHC, and α-cardiacactin gene expression on day 14 of the experiment. Murine heart mRNAwasused as the positive control. Cardiac differentiated cells treated by OT andDMSO expressed GATA-4, Nkx2.5, Tbx5 and Tbx20 transcription factorsand ß-MHC, and α-cardiac actin gene expression whereas monolayercultured cells that were treated by OT did not expressed these genes.Undifferentiated cells were used as the negative control and GAPDH wasused as the internal standard. The results are representative of threeindependent differentiation experiments.

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at 94 C, annealing for 60 s at 56–64 C and extension for 30 s at72 C. PCR amplificationwas followed by a final extension stepof 5 min at 72 C.

The amplification of GAPDH mRNA, a constitutivelyand ubiquitously expressed gene, served as an internalstandard for RT-PCR analysis. The following primers wereused: Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) (411 bp); sense 5′-TGTTCCAGTATGACTC-CACTCACGG-3′; antisense 5′-CAGTGATGGCATGGACTGTGGTCAT-3′; GATA binding protein 4 (GATA-4) [11] (186 bp); sense 5′-ACTCTGGAGGCGAGATGGG-3′; antisense 5′-CTCGGCATTACGACGCCACAG-3′; NK2transcription factor related, locus 5 (Nkx2.5) [12] (225 bp),sense 5′-CCTCTAGAGCAGAGCTGCGCGCGGAGATG-3′; antisense 5′-GGTGGCTTCCGTCGCCGCCGTGC-3′;ß-myosin heavy chain (ß-MHC) [13] (205 bp); sense 5′-GCCAACACCAACCTGTCCAAGTTC-3′; antisense 5′-TGCAAAGGCTCCAGGTCTGAGGGC-3′; α-cardiac actin[14] (99 bp); sense 5′-CTGAGATGTCTCTCTCT-CTCTTAG-3′; antisense 5′-ACAATGACTGATGAGA-GATG; T-box transcription factor 5 (Tbx5) (494 bp); sense5′- CACAGCCCCTTCAGCAGCGAGAC-3′; antisense 5′-AGGGGCCCCGAGGTGAAATGAG-3′; T-box transcriptionfactor 20 (Tbx20) (211 bp); sense 5′-ATGGTGATGGCT-GAAGCTC-3′; antisense 5′-GCAAATCCCTGACAA-GGATG-3′.

3. Results

Our observations showed that DMSO (the controlexperiments) stimulated the production of colonies of beatingcells in 36 independently growing cultures, in 18 wells inwhich EB formation had occurred by the day nine and in 18wells in which P19CL6 cells were grown under adherentconditions by the day twelve (Fig. 1). Beating cells werepresent only in the aggregated cells in the presence of OT.Wefound that OT stimulated the production of colonies ofbeating cells in all 18 independently growing cultures by theeighth day, whereas beating cells were not observed in theP19CL6 confluent monolayer cultures (Fig. 1). After sevendays, OT-treated adherent cells became gradually detachedfrom the bottom of the culture dishes and the culture mediumwas filled with dead cells by day 10 of the experiment.Identical results were obtained after four replicates were runusing 6-well tissue culture culture-grade dishes (Fig. 1).

The cells were harvested on differentiation day 14 and thenanalyzed by RT-PCR for the presence of the specific cardiacmuscle markers, such as GATA-4, Nkx2.5, α-cardiac actin, ß-MHC and the expression of the mesodermal cardiogenictranscription factors, Tbx5 and Tbx20. Based upon the resultsof RT-PCR, significant induction of these markers wasdetected in the control experiments and the aggregates whichtreated with OT (Fig. 2). This result demonstrates that OT caninduce cardiomyocyte differentiation in EBs from P19CL6cells, whereas no cardiac gene expression and differentiation(beating cells) were observed in the adherent P19CL6 cells

treated with OT. We then examined whether the treatment ofcell aggregates with OT induced the expression of theintracellular cardiomyocyte markers, sarcomeric β-MHC andtroponin I. Sarcomeric β-MHC is expressed in contractilemuscle cells as is troponin I. The undifferentiated cells werenegative for β-MHC and troponin I (Fig. 3). DMSO and OTinduced the appearance of immunoreactive foci in cellpopulations. The cell aggregates that were not exposed toOTorDMSOwere not positive forMHC or troponin I (Fig. 3).

To evaluate the effect of OTon the differentiation of GFP-expressing P19CL6 cells, the cell aggregations were exposedto OT. GFP-expressing beating cells appeared by day 8 of theexperiment in all 18 wells of the transferred cell aggregates(Fig. 4).

4. Discussion

Our results have demonstrated that OT does not inducecardiomyogenic differentiation of P19CL6 cells that are

Fig. 3. Immunostaining of P19CL6 cell-derived cardiomyocytes after DMSO and OT treatment (×100). P19CL6 cell aggregates were treated from day 0 to day 4with either DMSO or OT and were stained with an antibody against MHC or troponin I on day 12. Immunoreactivity signals were obtained for cell aggregatestreated with DMSO or OT which confirmed expression of sarcomeric myosin heavy chain MHC (M&P) and troponin I (A&D) antigens. Immunoreactive cellswere absent in the negative control group (G). The results are representative of three independent experiments.

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cultured as a confluent monolayer. Many types of stem cellshave been shown to differentiate into cardiomyocytes in vitro[11–16]. Of these stem cells, P19 EC cells were the first stemcell line in which this process was described and this is the cellline that has beenmost extensively characterized [6]. Recently,P19CL6 cells were cloned following long-term culture underconditions that promote mesoderm differentiation. This cellline differentiates easily into beating cardiomyocytes whencultured under adherent conditions or under conditions for EBformation following their treatment with 1% DMSO. Almostall cells derived from this cell line are thought to differentiateinto cardiomyocytes [5]. Because of their ability to differ-entiate into cardiomyocytes, these cells have been used toidentify cardiac-specific transcription factors and to examinetheir role in cardiac cell differentiation. They have also beenused to examine the unique aspects of cardiac cell physiologyduring their differentiation [3]. It has also been found thatP19CL6 cells do not spontaneously differentiate to cardio-myocytes following their aggregation in the presence of fetalcalf serum, as had been observed in the original P19 cell line[17]. For cardiac differentiation of this clone, exposure toDMSO or other cardiogenic factors is essential. Thesecharacteristics give P19CL6 cells a distinct advantage overeither P19 EC cells or pluripotent mouse ES cells fordeveloping a bioassay [18].

Paquin et al. [3] recently reported that OT OT-inducedcardiomyocyte differentiation in P19 EC cells. In this study,

we found that OT did not induce the differentiation of P19CL6cells into cardiomyocytes when they were maintained as aconfluent monolayer culture and without prior EB formation.To investigate whether OT can promote the differentiation ofP19CL6 cells into cardiomyocytes without prior EB forma-tion, we seeded them at a high density of 8×10 4 cells/wellonto 6-well dishes in order to allow them to differentiate intocardiac cells. Paquin et al. [3] reported that OT-induced cardiacdifferentiation is mediated by OTR and the signal transductionpathway(s) has yet to be defined. They reported also that OTOT-induced cardiomyogenic differentiation when added to theculture medium of P19 EC cell aggregates, and that beatingcells appeared earlier than those cells treated with DMSO [3].This effect of OTwas specific andmediated byOTR because itwas abolished by an OT receptor antagonist. Furthermore, anOTR antagonist blocked bothOT- andDMSO-induced cardiacmyocyte differentiation and this result suggested that DMSOacts via the OT/OTR pathway. However, our results suggestedthat the molecular mechanisms for controlling of cardiacdifferentiation by OTand DMSO are different. The expressionof cardiacmusclemarkers, such as GATA-4, NK2.5, Tbx5 andTbx20was evident in the cells that were treated by DMSO andOTafter EB formation, while the expression for these markerswas not observed in the cells grown under adherent cultureconditions following their exposure to OT. When treated withOT, the Sca-1+ cells expressed cardiac genes that includedCsx/Nkx2.5, GATA-4, MEF-2C and ß-MHC. Furthermore,

Fig. 4. Beating cardiomyocytes expressing GFPwere obtained by OT treatment. P19CL6 cells expressingGFP aggregated in the presence of a differentiationmediumcontaining 1×10 −7MOT (A,×100).Onday 4, aggregates (EBs)were transferred to 6-well plates and grown in the absence of theOT (C, day 6, ×100). By day 8 of theexperiment,many colonies of GFP-expressing cells appeared in the treated aggregated cells in all 18wells. Beating cells were observed inmore than 60%of the cells inidentical areas of a culture dish (E, ×200). The previous photomicrographs were taken under a phase contrast microscope (B&D ×100, F ×200). Dotted lines encirclethe colonies of contracting cells (F). The results are representative of three independent experiments.

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some Sca-1+ cells had well-organized sarcomeres and showedspontaneous beating [11]. In this regard, the cardiacdifferentiation of Sca-1+ cells does not require cell aggregationfor the process to proceed. Although OT may play animportant role in the differentiation of primordial cells thatinclude adult somatic stem cells, embryonic and embryonalcarcinoma stem cells into cardiomyocytes, its precisemechanism of the action in this process is not clear [11].

It is known that the efficient differentiation of P19 ECcells depends on the prior formation of non-adhering cellaggregates [6]. However, the molecular events that occurduring cell aggregation and that are necessary for cardiacdifferentiation are not fully understood [6]. Skerjanc et al.[19] have reported that overexpression of Nkx2.5 can inducecardiomyogenesis in aggregated P19 EC cells, but not whenthey are maintained in monolayer cultures. On the otherhand, the addition of soluble bone morphogenic protein 4(BMP4) into the culture media resulted in the requirementfor cell aggregation and induced cardiac differentiation beingbypassed in monolayer cultures of P19 EC cells over-expressing Nkx2.5 [20]. This result demonstrated that cellaggregation is crucial for the generation of the BMP signal in

P19 EC cells. Our study suggested that OT-induced cardiacdifferentiation is not mediated by the expression of BMPsignaling molecules because the induction of BMP signalingcascade can bypass the requirement for prior EB formation.

The low efficiency of P19 EC cells to differentiate is oneof their major limitations [16], compared to the high percentof the beating cells obtained in the last days of experimentwhen using P19CL6 cells [5]. The clinical application ofstem cell therapies will be increased when it becomespossible to produce a well-defined and well-differentiatedcell population in large numbers in culture. Consequently,P19CL6 cells represent a promising stem cell line for thispurpose. On the other hand, the results of previousinvestigations point to a particular involvement of OT inthe priming of cardiogenesis and the involvement of thematernal and embryonal OT/OTR pathways during devel-opment of the embryo. As a result, OT probably assists in thematuration of newly differentiated cardiomyocytes. In thiscontext, we were able to obtain a stable clone of transfectedP19CL6 cells expressing GFP that was capable of differ-entiating into cardiac cells by using OT and inducing theformation of EBs. Their efficiency of differentiation was

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high because more than 60% of the cells becamedifferentiated when compared to the 10–15% differentiationof P19 EC cells, which has already been reported [21], inidentical areas of a culture dish.

In conclusion, EB formation is necessary for thedifferentiation of P19CL6 cells into cardiomyocytes whenthe inducer is OT. Because of the high rate of differentiationefficiency, GFP-expressing cardiomyocytes derived fromP19CL6 cells have the potential to be used for regenerativetherapies in experimental models of heart failure. Our datajustify the continued study on the role of aggregation and OTin process of cardiac differentiation.

Acknowledgement

This study was supported by a grant from Kobe TranslationalResearch Cluster.

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