Abstracts of Poster Presentations

A16 © 2008 Japan Human Cell Society

Proceedings of the 25th Annual Meeting of the Japan Human Cell Society 2007

after vitrification. In total, 8 ovarian tissues were successfullyautotransplanted to 4 cattle. Human ovarian tissue from a cancerpatient, and from donors for ovarian transplantation (with informedconsent) was vitrified according to the cattle method. After thawing,the same high post-thaw survival (89%) was obtained.

In conclusion, these results indicate that the ultra rapid coolingvitrification method in the present study has a potential forclinical use in human ovarian tissue cryopreservation.

Abstracts of Poster Presentations

P-01DEVELOPMENT OF NEW TREATMENT OF THE OOCYTES FOR THE MITOCHONDRIAL DISEASE PATIENTS: EFFECTS OF OOPLASMIC REPLACEMENT AT PRONUCLEAR STAGE

F AONO, M KUWAYAMA, Y TAKEHARA, O KATOKato Ladies Clinic, Tokyo, Japan

Introduction: We have previously reported to show theevidence of positive effect of improving the developmental abilityof defective oocytes from old cattle by ooplasmic replacement atthe germinal vesicle stage. The oocyte cytoplasm that hadmutated mitochondrial DNA in mitochondrial disease patientsmay be replaced to normal one from donated normal oocytes bymicromanipulation. Namely, the ooplasmic replacement can beconsidered a possible treatment method for the oocytes ofmitochondrial disease. We evaluate that the effect of number oftransferred pronuclear on developmental ability of reconstructedoocytes at the pronuclear stage for mouse, and the effect ofthe stage of oocytes for the cytoplasmic replacement ondevelopmental ability of reconstructed oocytes for cattle.Materials and Methods: In experiment 1, pronuclear (PN)stage oocytes were collected from B6C3F1 female mice 16 hafter hCG injection. The both female and male PNs or only femalePN were removed from the oocytes by the micromanipulation inM2 containing 10 µg/mL cytochalasin B (CB) (Sigma, USA). Thekaryoplasts containing PNs or PN were inserted into theperivitelline space of the enucleated ooplasm, and they weregiven 2 times of direct current (DC) pulses, 1 kv/cm for 70 µsecfor fusion. The reconstructed oocytes were cultured fordevelopment to the blastocyst in M16 under a humid atmosphereof 5% CO2 in air at 37 °C for 4 days. Some of the blastocysts weretransferred to the uterine of recipient females to evaluate thenormal developmental ability to the term. The body and placentaweight at the birth of the youngs was measured, and theirreproductive ability was confirmed by outbreeding after sexualmaturation. In experiment 2, germinal vesicle (GV) stage oocyteswere obtained from bovine ovaries that stored in saline at12 °C for 24 h after collection at a local slaughterhouse. Theooplasmic replacement was performed at the GV or PN stageusing the similar technique in experiment 1. All of the oocyteswere cultured for in vitro maturation in IVMD101 (ResearchInstitute for the Functional Peptides, Japan) under a humidatmosphere of 5% CO2 in air at 38.5 °C for 22 h. The maturedoocytes, to induce parthenogenesis, were cultured in TCM199

containing 5 µg/mL of ionomycin (Sigma, USA) for 5 min, andthen they were cultured in SOF containing 5 µg/mL of DMAP(Sigma, USA) for 6 h. All of the oocytes were cultured in SOFcontaining 1% FBS (Hyclone, USA) under a humid atmosphereof 5% CO2, 5% O2, 90% N2 at 38.5 °C for total of 8 days. Afterculture for 48 h, cleavage of the embryo was observed, and theincidence of blastocyst was observed on days 6, 7 and 8.Results: In experiment 1, Both PNs and PN groups werecomparable with cleavage (71 vs 71), blastocyst (60 vs 63) anddevelopmental rates to the term (36 vs 31%), respectively. Butthose were significant-low compared to control group (89, 80,and 54%). The youngs were normal body (1.17, 1.18 and 1.18 g)and placenta (0.89, 0.9 and 0.9 g) weight and showed normalfertility after sexual maturation. The number of transferred PN(s)has no influence with their developmental ability for reconstructedoocytes at the PN stage in mouse. In experiment 2, although thecleavage rates were similar among the GV, PN and control groups(90, 96 and 95%), blastocyst rates in GV (23) were significant-low compared with PN (42) and control groups (55%).Conclusions: In conclusion, these results indicate that thereconstructed oocytes by ooplasmic replacement at PN stage havenormal ability of development to the term.

P-02IN VITRO DIFFERENTIATION OF MESENCHYMAL CELLS DERIVED FROM HUMAN AMNIOTIC MEMBRANES INTO HEPATOCYTE-LIKE CELLS

Tomoharu TAMAGAWA,1 Isamu ISHIWATA,1 Hiroshi ISHIKAWA,2 Yukio NAKAMURA3

1Ishiwata Obstetrics and Gynecology Hospital; 2Laboratory of Regenerative Medical Science, The Nippon Dental University; 3Cell Engineering Division BioResource Center, RIKEN

Objective: It has been reported that human hepatocytes couldbe obtained following the induction of differentiation fromembryonic stem cells, bone marrow cells and amnion epithelialcell, and so on. In this study, we investigated that characteristic ofdifferentiated and undifferentiated HAM cells into hepatocyte in vitro.Materials and Methods: Amniotic membranes were rinsedthree times with PBS, minced with a sharp pair of scissors andincubated at 37 °C for 45 min with 0.25% trypsin-EDTA. Thepellets were centrifuged at 1500 rpm for 5 min. The sedimentswere incubated with αMEM supplemented with 10% FBS,1.0 mg/mL collagenase, 0.1% (w/v) dispase and 0.1% (w/v)papain for 60 min. The pallets were filtered using stainlessmeshes or 100 µm cell strainer and centrifuged two times.The sediments were suspended in growth medium (αMEMsupplemented with 10% FBS, 10 ng/mL hEGF, and 10 ng/mLhLIF), placed in 60 mm plastic dishes and incubated at 37 °C,5% CO2 in air.

The cells were seeded at a density of 5 × 105 cells in type Icollagen-coated plastic dishes and cultured in growth medium.When the cells were confluent, media were replaced with hepa-tocye differentiation medium (αMEM consisting of 10% FBS,20 ng/mL hHGF, 10 ng/mL hbFGF, 10 ng/mL oncostatin M(hOSM) and 0.1 mM dexamethasone). The hepatocyte differenti-ation medium was changed every three days and the cells werecultured for 3 weeks.

© 2008 Japan Human Cell Society A17


Results: We analyzed the expression of various hepatocyte-related gene, including α-fetoprotein (α-FP), albumin, α1-antitrypsin(α1-AT), cytokeratin 18 (CK18), glucose-6-phosphatase (G6Pase),ornithine transcarbamylase (OTC) and hepatocyte nuclearfactor-4 α (HNF-4α). Undifferentiated HAM cells anddifferentiated HAM cells expressed albumin, α-FP, α1-AT andCK18 but did not express G6Pase and OTC. HNF-4α gene wasdetected in two HAM populations out of four populations.

Immunochemical analysis of hepatocyte-associated markersrevealed HAM cells to differentiate into hepatocyte were positiveα-FP, albumin, CK18 and CK19. We analyzed of stored glycogenby Periodic acid-Schiff (PAS) stain of differentiated HAM cellsand undifferentiated HAM cells. In the cells after induction ofdifferentiation storage of glycogen was clearly detected by PASstaining.Conclusion: These results strongly suggested that HAM cellscould differentiate into hepatocyte or at least hapatocyte-likecells. Therefore, HAM cells can be easily obtained with a low riskof pathogen infection and a good resource. HAM cells mightcontribute to the new therapeutic concepts for liver diseases.

P-03DIFFERENTIATION AND TRANSPLANTATION OF COMMON MARMOSET EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTES

H CHEN, F HATTORI, W LI, M MURATA, K KIMURA, E SASAKI, S OIKAWA, Y TANIOKA, K FUKUDADepartment of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine

Objective: Human embryonic stem (ES) cells are an attractivesource for cardiomyocyte replacement therapy. In preclinicalstudies, the evaluation of this concept with non-human primatesis essential. We have developed a non-genetic based purificationtechnique which is applicable to mouse, monkey and human EScell-derived cardiomyocytes. Here, we report on the detailedcharacterization of Common Marmoset ES cell (CMESC)-derived cardiomyocytes (CMESC-CM) and the development of atransplantable pure-myocardial cell sheet.Methods and Results: In the floating culture system, CMESCefficiently developed embryoid bodies (EBs) and an average of10–20% of EB began spontaneously contracting EBs in about15 days. The immunohistochemical studies showed that thebeating cells expressed sets of cardiac proteins includingsarcomeric actinin, tropomyosin, myosin heavy chain, myosinlight chain, ANP, GATA4 and Nkx2.5. RT-PCR revealed theexpression-time courses of those mentioned above and Tbx5,Tbx20 genes. These data showed that CMESC-derived beatingcells were cardiomyocytes. The ultrastructural study of CMESC-CM showed the existence of less-organized myofibers andless-abundant mitochondria compared to adult cardiomyocytes.The action potential recordings indicated that they hadsinus node-like action potentials irrespective of EB-age.BrdU uptake experiment revealed that CMESC-derivedcardiomyocytes still had a highly proliferative capacity. Wepurified the CMESC-CM using the mitochondrial abundance-based method, and cultured on a fibrin-coated dishes to form

myocardial cell sheets. We were able to detach them from thedishes without obvious cell damage. These cell sheets are readyfor transplantation.Conclusions: Our study revealed that CMESC-CM have featuresthat might be suitable for in vivo expansion and maturation.Common Marmoset ES-derived cardiomyocytes should providea convenient human-analogical allotransplant model.

P-04DNA DECAY OF TETRAPLOID MOUSE H1(ES) CELLS

Kohzaburo FUJIKAWA-YAMAMOTO, Minoru MIYAGOSHI, Hiroko YAMAGISHI, Xianwen LUODivision of Cell Medicine, Institute of Medical Science, Kanazawa Medical University

Introduction: We have studied the polyploidization, theestablishment of polyploid cells and the characterization ofpolyploid mammalian cells, and proposed a hypothesisof genome structure of polyploid cells. The DNA content ofmammalian diploid cells is well preserved during subculturing;however, that of polyploid cells is not. The DNA content ofpolyploid cells sometimes decreases gradually, and occasionally itdecreases abruptly by half. Although a few studies of DNAdegradation in polyploid cells have been reported, clearexplanation has not been done. The DNA degradation process oftetraploid mouse H-(ES) cells will be presented.Experiments: Tetraploid H-(ES) cells were subcultured over180 days. The cellular DNA content was determined throughDNA histograms obtained by flow cytometry.Results and Discussion: In the presence of LIF, the cellularDNA content at time t, f(t), was described as f(t) = Ipexp[-at/{exp(-bt) + ct}], were Ip = 4, a = 0.01, b = 0.03 and c = 0.052.Though, in the absence of LIF, f(t) could be described with thesame parameters, abrupt return to diploid was also observed.These results will be clearly explained with new hypothesis forgenome structure of polyploid cells.

P-05ESTABLISHMENT AND CHARACTERIZATION OF THE HEPFT CELL LINE DERIVED FROM HEPATOID CARCINOMA OF THE FALLOPIAN TUBE – SPECIAL REFERENCE OF AFP LECTIN AFFINITY AND HISTOGENESIS

Iwao ISHIWATA,1 Makoto YASUDA,1 Takashi HIRANO,2 Hiroshi ISHIKAWA3

1Department of Obstetrics and Gynecology, Jikei University School of Medicine; 2Applied Gene Technology Research Group, Institute of Biological Resources and Function, National Institute of Advanced Industrial Science and Technology; 3Laboratory of Regenerative Medical Science, The Nippon Dental University

A cell line designated “HEPFT” was established from a humanFallopian Tubal hepatoid carcinoma of 79-year-old Japanese



woman. This line grew well without interruption for 13 monthsand was subcultivated over 35 times. The cells were polygonaland spindle in shape and showed neoplastic and pleomorphicfeatures such as coarse chromatin granules, irregularly shapednuclei and multiple large nucleoli. The cells grew in multilayerand often formed papillary cell aggregates. The bilepigmentations were stained positively by iodine staining. AFPwas detected in HEPFT cells under fluorescence microscopy. Anumber of cells had numerous small Golgi apparatus; microvilliand desmosomes and junctional complexes were frequentlyobserved on the surface of or between the cells. The narrowbilecanaliculus is observed. The cells proliferated rapidly, and thepopulation doubling time was about 45 h. The chromosomenumber showed a wide distribution of aneuploidy. The modalchromosome number was stable in the hyper triploid range andmany marker chromosomes were observed. The subfraction ofAFP of the patients sera contained L3: 90.6%, L1: 9.4% and L2:0.0%, and that of the conditioned media of HEPFT line wasL3: 75.2%, L1: 24.5%, and L2: 0.0%, respectively. The recentlydevelopment bacterial artificial chromosome (BAC) arraycomparative genomic hybridization (CGH) can facilitate detailanalysis with high resolution and sensitivity. Different profiles ofgenomic copy-number abnormalities are demonstrated on variouschromosomal regions in HEPFT cells. The AFP gene of 4q13-3gained slightly in 0.0322 copy number. Thus, the expression ofthe messenger RNA of AFP was analyzed by RT-PCR. RT-PCRassay indicated the expression of genes specific for AFP. TheHEPFT line will serve as a patient experimental materials forclarifying the dynamics of the AFP production and secretion aswell as histogenesis of hepatoid carcinoma.

P-06INTRAMOLECULAR CONTROL OF PROTEIN STABILITY, SUBNUCLEAR COMPARTMENTALIZATION, AND COACTIVATOR FUNCTION OF PGC-1αααα

Motoaki SANO,1,2 Satori TOKUDOME,1 Noriaki SHIMIZU,3 Noritada YOSHIKAWA,3 Jin ENDO,1 Takaharu KATAYAMA,1 Shinji MAKINO,1 Fumiyuki HATTORI,1 Hirotoshi TANAKA,3 Keiichi FUKUDA1

1Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Department of Internal Medicine, Keio University School of Medicine; 2Japan Science and Technology Agency, SAKIGAKE; 3Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo

Introduction: PPARγ coactivator (PGC)-1α is critical foractivating mitochondrial respiration and metabolism to maintainhomeostasis in response to diverse metabolic stresses. Reducedactivity of PGC-1α has been associated with heart failure. Thesignaling for induction of PGC-1α at the mRNA levels have beenwell studied, however, it remains unknown how PGC-1α isinactivated to maintain adequate protein levels. This study isdesigned to investigate how PGC-1α protein stability are regulatedand are coupled with its potent coactivator function.Methods and Results: Here we found that PGC-1α is ashort-lived and inherently aggregation-prone protein. (i) PGC-1αhas two evolutionally conserved PEST-like motifs. (ii)

Immunofluorescent and biochemical fractionation studiesrevealed that PGC-1α localized throughout the nucleoplasm andwas rapidly destroyed via the ubiquitin-proteasome pathway.(iii) Upon proteasome inhibition, PGC-1α formed insolublepolyubiquitinated aggregates. (iv) Ubiquitination of PGC-1αdepended on the integrity of the C-terminus containing arginine-serine rich domains and an RNA recognition motif. (v)Interestingly, ectopically expressed C-terminal fragment of PGC-1α was autonomously ubiquitinated and aggregated with PML(promyelocytic leukemia proteins). (vi) Cooperation of the N-terminal region containing two PEST-like motifs was required forprevention of aggregation and targeting of the polyubiquitinatedPGC-1α for degradation. This region thereby negativelycontrolled the aggregation properties of the C-terminal region toregulate protein turnover and intranuclear compartmentalizationof PGC-1α. (vii) Exogenous expression of the C-terminalfragment of PGC-1α interfered with degradation of full-lengthPGC-1α and enhanced its coactivation properties.Conclusions: The expression levels of PGC-1α are controlledby protein degradation through the ubiquitin-proteasomepathway. Both proteasome inhibitor and truncation of N-terminalregulatory motifs facilitated intranuclear aggregation of thisprotein. The protein stability, intranuclear localization, andcoactivator function of PGC-1α are finely regulated viaintramolecular interactions among distinct structural domains.

P-07BONE MARROW-DERIVED CELLS ARE INVOLVED IN THE PATHOGENESIS OF CARDIAC HYPERTROPHY IN RESPONSE TO PRESSURE OVER-LOAD

Jin ENDO,1,2 Motoaki SANO,1 Jun FUJITA,1,2 Kentaro HAYASHIDA,1,2 Shinsuke YUASA,1,2 Shinji MAKINO,1 Satoshi OGAWA,2 Keiichi FUKUDA1

1Department of Regenerative Medicine and Advanced Cardiac Therapeutics and 2Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine

Purpose: Bone marrow (BM) cells possess a broad differentiationpotential, and can form various cell lineages in response topathophysiological cues. This study investigated whether BM-derived cells contribute to the pathogenesis of cardiac hypertrophy,and the possible cellular mechanisms involved in such a role.Methods and Results: Lethally irradiated wild-type mice weretransplanted with BM cells from EGFP transgenic mice. Thechimeric mice were subjected to either prolonged hypoxia ortransverse aortic constriction. BM-derived EGFP-expressingcardiomyocytes increased in number over time, emergingpredominantly in the pressure-overloaded ventricular myocardium.To discriminate whether BM-derived cardiomyocytes werederived from cell fusion or transdifferentiation at the single celllevel, lethally-irradiated Cre mice were transplanted with BM cellsfrom the double-conditional Cre reporter mouse line Z/EG. BM-derived cardiomyocytes were shown to arise from both cell fusionand transdifferentiation. Interestingly, BM-derived myofibroblastsexpressing both vimentin and a-smooth muscle actin (a-SMA)were concentrated at the perivascular fibrotic area. These cellsinitially expressed MAC-1/CD14, but lost expression of thesemarkers during the chronic phase, suggesting that they were



derived from monocytes. A similar phenomenon occurred incultured human monocytes, most of which ultimately expressedvimentin and a-SMA.Conclusion: We found that BM-derived cells are involved inthe pathogenesis of cardiac hypertrophy, via dual mechanisms ofcell fusion and transdifferentiation. Moreover, we suggested thatBM-derived monocytes might play an important role in theformation of perivascular fibrosis through directly differentiatinginto myofibroblasts.

P-09FACTORS AFFECTING MORPHOLOGICAL CHANGES AND OOCYTE MATURATION IN PREANTRAL FOLLICLES AND OOCYTE-GRANULOSA CELL COMPLEXES DURING CULTURE IN VITRO

Hideyuki H MOTOHASHI,1,2 Tadashi SANKAI,2 Kahei SATO,3 Hidemi KADA1

1Department of Bioproduction Technology, Junior College of Tokyo University of Agriculture; 2Tsukuba Primate Research Center, National Institute of Biomedical Innovation; 3Department of Applied Biological Science, College of Bioresource Sciences, Nihon University

Preantral follicle (PF) culture and oocyte-granulosa cellcomplexes (OGCs) culture are one of the effective methods forthe growth and development in vitro of mouse immature oocytes.In these culture systems, the follicular tissue supporting growthof the oocyte causes a form change during culture. In normaldevelopment in vitro, the granulosa cells three-dimensionallyproliferate on the culture dish or the insert membrane. However,some of the follicles and the OGCs flatten out during culture.These tissues do not react to induction of maturation andovulation in vitro. Therefore, the flat tissues were clearly a loss foroocyte growth and development in vitro. In this study, factorsinfluencing this phenomenon and maturation competence ofoocytes derived from the flat tissues were investigated.

Three week and 12 days female CD-1 mice were used in thePF culture and the OGCs culture, respectively.

PF culture: Early preantral follicles (100–130 µm) wereisolated from ovaries by mechanical dissection. The follicles wereindividually cultured for 12 days in micro-droplets of mediumsupplemented with 5% FBS, 1% ITS and 10 mIU/mL FSH undera paraffin oil. The oocyte maturation was induced by addition offinal concentration of 1.5 IU/mL hCG and 5 ng/mL EGF.

OGCs culture: The OGCs were collected by collagenase diges-tion and group-cultured for 12 days on Millicell-PC membrane inmedium supplemented with 5% FBS. The oocyte maturation wasinduced in medium with 10 ng/mL EGF

The maturation competence of oocytes was evaluated at17 h after the maturation induction. The flat tissue appearedwithout affecting a culture method and the kind of the culturemedium. The appearance frequency of the flat tissue was signifi-cantly higher in groups of the follicles and the OGCs with inter-stitial cells than that without the cells.

The oocytes isolated from the flat tissue were competent toresume meiosis and progress to metaphase II stage. The meanoocyte size was approximately 70 µm. In conclusion, thisstudy shows that relevance between existence of interstitial cells

and appearance frequency of the flat tissue. In addition, the mat-uration competence in the oocytes isolated from the flat tissueswas demonstrated in this study.

P-10EXPRESSION OF MELATONIN RECEPTOR IN HUMAN ENDOMETRIAL CANCER CELL

Mari WATANABE, Yoichi KOBAYASHI, Wataru TARUMI, Noriyuki TAKAHASHI, Masanori ITO, Tathuru OHARA, Nao SUZUKI, Kazushige KIGUCHI, Bunpei ISHIZUKADepartment of Obstetrics and Gynecology St. Mariannna University School of Medicine

Purpose: In previous study, we found that melatonin inhibitsproliferation of Ishikawa, cells derived from endometrial cancer,and this effect was inhibited by estradiol. Also, it revealed thatthere are melatonin binding sites on membranes in Ishikawa cells.A detailed analysis of the melatonin receptor (MT1, MT2) concerningto inhibition of cell proliferation was performed in this research.Method: (i) Examination of melatonin receptor subtype1 × 105 of Ishikawa cells were seeded in each plate and incubatedunder 5% CO2 atmospheric condition 37 °C for 96 h with1 × 10−9 M melatonin (M group). Other groups were incubatedwith melatonin and luzindole (MT1 + MT2 antagonist) : M + LUZgroup, or melatonin and 4P-PDOT (MT2 selective antagonist) :M + PDOT group. After 96 h of incubation, cells were harvestedfrom plates and count the total number of cells. Moreover,melatonin receptor mRNA expression was analyzed by RT-PCR.Expression and the localization of the receptor protein were testedwith the immunocytochemistry. (ii) Examination of influence ofE2. Cells were incubated in the presence or absence of 1 × 10−10

M estradiol in the condition of described above. Thereaftermelatonin receptor mRNA expression was analyzed by RT-PCR.Result and Consideration: Neither M group nor theM + PDOT group had a significant difference, and significantdifference was observed at M + LUZ group. Moreover, theexpression of MT1 receptor detected with RT-PCR despite ofestradiol existence. MT1 receptor was detected on the membraneof Ishikawa cells with immunocytochemistry. In conclusion, it isclear that melatonin inhibits proliferation of Ishikawa throughMT1 receptor. Moreover, both melatonin and estradiol had noinfluence at the expression of MT1 receptor and it was suggestedthat estradiol effects suppressively on melatonin except decreaseMT1 receptor expression.

P-11DETERMINATION OF CYTOKINES IN ASPIRATION FLUIDS OF CYSTIC BRAIN TUMORS BY CYTOMETRIC BEADS ARRAY

Qiang LI,1 Hideyuki OSHIGE,1 Takahiro YAMAHARA,1 Takashi RYU,1 Tetsuya OISHI,1 Toshitaka SENO,1 Takuya KAWAGUCHI,1 Yunbo ZHEN,1 Tomoyuki MURAKAMI,2 Keiji KAWAMOTO1

1Department of Neurosurgery, Kansai Medical University, Moriguchi, Japan; 2Department of Respiratory Medicine, National Sanyo Hospital, Respiratory Disease Center, Ube, Japan



Purpose: Cystic lesions are frequently found in malignant ornonmalignant brain tumors. A new flow cytometric technique,cytometric beads array (CBA), is a tool to simultaneously measurenot only absolute abundance of various cytokines but also theirrelative abundance in a small amount of liquid sample. Westudied 28 samples of cystic fluids from patients with cystic braintumors by CBA.Materials and Methods: In this study, cystic fluids werecollected from 28 patients with cystic brain tumors concludedmetastatic brain tumors, glioma, craniophcarnyngioma andhemangioblastoma. Cystic fluids concentrations of IL-2, IL-4,IL-5, IL-10, TNF-alpha, and INF-gamma were simultaneouslymeasured by CBA using a Human Th1/Th2 Cytokines CytmetricBead Array KitTM (BD). We also detected the expression of IL-5,IL-10 receptors in specimens from the IL-5 and IL-10 CBA-positive cases by immunohistochemistry.Results: Positive expression rates of 6 Cytokines in 28 caseswere IL-5 (20/28 cases) = IL-10 (20/28 cases) > IL-2 (10/28 cases) >INF-gamma (8/28 cases) > TNF-alpha (4/28 cases) > IL-4 (2/28 cases). IL-10 positive was found in all of metastatic braintumors (7/7 cases) and the positive rates of 6 cytokines were thehighest in metastatic brain tumors than in other tumors. Thepercentages of IL-5 positive and the percentage of IL-10 positivewere high in gliomas, but TNF-alpha and INF-gamma was notdetected in any case of gliomas. In the cases of IL-5 positive, cellsof tumor block specimens expressed IL-5 receptors, and IL-10receptors were detected in the IL-10 positive cases byimmunohistochemistry.Conclusion: We confirmed that cystic fluids contain particularcytokines which may play important roles in pathogenesis of thecyst formation associated with cystic brain tumors.

P-12TECHNICAL METHODS FOR LIGHT AND ELECTRON MICROSCOPIC OBSERVATION OF SMALL AMOUNTS OF SORTED CELLS BY FCM

Yunbo ZHEN, Qiang LI, Tetsuya OISHI, Hideyuki OSHIGE, Takahiro YAMAHARA, Toshitaka SENO, Takuya KAWAGUCHI, Toshika SENBA, Keiji KAWAMOTODepartment of Neurosurgery, Kansai Medical University

Background: The light and electron microscopic (LM, EM)observation are necessary for small amounts of cells sorted byflow cytometry (FCM), but this useful technique is notdeveloped.Methods: Glioma cell lines are detached by Trypsin and fixedwith formaldehyde, ethanol alchohol and glutalaldehyde,following by nuclear staining of propidium iodide. Each singlecell is sorted according to cell cycles by flow cytometer (Epics,BD. Company).

Sorted cells are centrifuged with cytospin and submitted forLM. Beam capsule including glutalaldehide was used for fixationand embedded Epon for EM. 300, 1000, 3000 and 5000 of cellswere submitted for the observation of LM and EM.Results: Minimum number of cells is required more than300 cells for LM and were observed more than 1000 cells for EM.

Conclusion: Small amounts of sorted cells are wellobserved for LM and EM by using our designed method detectionmethod.

P-13Zac1 IS AN ESSENTIAL CARDIAC TRANSCRIPTION FACTOR

Shinsuke YUASA,1,2 Takeshi ONIZUKA,1 Kenichiro SHIMOJI,1 Yohei OHNO,1 Jin ENDO,1 Hideaki KANAZAWA,1 Hirotaka YADA,1 Naritaka KIMURA,2 Takashi KAWAKAMI,1 Tomomi KAGEYAMA,1 Takaharu KATAYAMA,1 Takahide ARAI,1 Mitsushige MURATA,1 Daihiko HAKUNO,1 Kensuke KIMURA,1,2 Shinji MAKINO,2 Motoaki SANO,2 Satoshi OGAWA,1 Keiichi FUKUDA2

1Cardiopulmonary Division, Department of Internal Medicine and 2Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine

Objective: Although there are many essential cardiactranscription factors, the mechanisms of heart development andhomeostasis remain poorly understood. The transcription factorshave the central role of gene expression, pathogenesis and organmorphogenesis. To isolate the novel cardiac transcription factor,we performed gene chip analysis by using cardiac myocytedifferentiating embryonic stem cells and isolated the Zac1, a zincfinger protein which was identified as an anti-proliferativetranscription factor. This study was designed to investigate themolecular and functional characterization of Zac1 as a cardiactranscription factor.Methods and Result: (i) Whole-mount in situ hybridizationand immunostaining of Zac1 was performed at mouse embryostages E7.5, E8.5 and E9.5. Zac1 was strongly expressed in thewhole heart from E8.5 embryo to adult. (ii) Luciferase constructsunder the control of either ANF, BNP, and αMHC promoterwere co-transfected with Zac-1 expression vector, and itstranscriptional activity was compared with other cardiactranscription factors. Zac1 had strong transcription activity forthese promoters compared with Nkx2.5, Gata4, SRF, Tbx5 andMEF2C. (iii) Gel mobility shift assay revealed that Zac1 wasdirectly associated DNA within ANF promoter. (iv) The 18 kindsof Zac1 deletion mutants were constructed. Structure-functionalanalysis revealed that the C-terminal of Zac1 had transcriptionalactivity and N-terminal had DNA binding activity. (v) Zac1 DNAbinding site within the ANF promoter was also determined. Itwas adjacent to the Nkx2.5 binding site. Luciferase analysisrevealed that Zac1 has a synergistic transcriptional activity withNkx2.5. GST pull down analysis showed that Zac1 5th and 6th

zinc finger domains directly bound to Nkx2.5 homeodomain.(vi) Zac1 has a PKC phosphorylation site. PKC activationincreased the wild type Zac1 transcriptional activity. Zac1 T167Nwas mutated at the PKC phosphhorylation site. Zac1 T167Nabolished PKC-dependent transcriptional activity. (vii) Zac1knock-out mouse showed severe cardiac deformity at theembryonic stage.Conclusions: These findings indicated that Zac1 is one of theessential cardiac transcription factors, and it collaborates withother cardiac transcription factors.



P-14FOCAL ADHESION KINASE SIGNALING REGULATES CARDIOGENESIS OF EMBRYONIC STEM CELLS

Daihiko HAKUNO,1 Tomosaburou TAKAHASHI,2 J LAMMERDING,3 Lee RT3

1Division of Cardiology, Department of Internal Medicine, Keio University School of Medicine; 2Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine; 3Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA

The signaling steps that induce cardiac differentiation inembryonic stem (ES) cells are incompletely understood. Weexamined the effect of adhesion signaling including Src and focaladhesion kinase (FAK) on cardiogenesis in mouse ES cells usingα-myosin heavy chain promoter-driven enhanced green fluorescentprotein or luciferase as reporters. Cardiac transcription factorsincluding Nkx2.5 and Tbx5 mRNA were first expressed at day 4in hanging drop embryoid bodies, and adhesion of embryoidbodies to surfaces at or before that day strongly inhibiteddifferentiation of ES cells to cardiomyocytes. Since adhesionsignaling could suppress cardiogenesis through Src kinases,embryoid bodies were exposed to the small molecule PP2,known as a Src family kinase inhibitor. PP2 during embryoidbody adhesion dramatically increased cardiomyocyte differentiationand decreased mRNA expression of neuronal cellular adhesionmolecule and α-fetoprotein, neuroectodermal, and endodermalmarkers, respectively. Surprisingly, although there was aninteraction between Src and FAK in cardiogenesis, the pro-cardiogenic effect of PP2 appeared incompletely explained by Srckinase inhibition, since another Src family kinase inhibitor,SU6656, failed to induce cardiogenesis. Instead, PP2 specificallyinhibited adhesion-induced FAK phosphorylation. In ES cellsstably expressing FAK-related nonkinase, which functions as adominant negative FAK, cell migration from embryoid bodieswas inhibited, whereas α-myosin heavy chain expression andmyosin-stained cardiomyocytes were increased, suggesting thatreducing cell motility may contribute to cardiogenesis. Thesedata indicate that FAK is a key regulator of cardiogenesis inmouse ES cells and that FAK signaling within embryoid bodiescan direct stem cell lineage commitment.

P-15DOMINANT NEGATIVE SUPPRESSION OF RAD LEADS TO QT PROLONGATION AND CAUSES VENTRICULAR ARRHYTHMIAS VIA MODULATION OF L-TYPE CA2++++ CHANNELS IN THE HEART

Hirotaka YADACardiopulmonary Division, Keio University

Disorders of L-type Ca2+ channels can cause severe cardiacarrhythmias. A subclass of small GTP-binding proteins, the RGKfamily, regulates L-type Ca2+ current (I(Ca,L)) in heterologousexpression systems. Among these proteins, Rad (Ras associated

with diabetes) is highly expressed in the heart, although itsrole in the heart remains unknown. Here we show thatoverexpression of dominant negative mutant Rad (S105N) led toan increase in I(Ca,L) and action potential prolongation viaup-regulation of L-type Ca2+ channel expression in the plasmamembrane of guinea pig ventricular cardiomyocytes. To verifythe in vivo physiological role of Rad in the heart, a mousemodel of cardiac-specific Rad suppression was created byoverexpressing S105N Rad, using the alpha-myosin heavy chainpromoter. Microelectrode studies revealed that action potentialduration was significantly prolonged with visible identificationof a small plateau phase in S105N Rad transgenic mice,when compared with wild-type littermate mice. Telemetricelectrocardiograms on unrestrained mice revealed that S105NRad transgenic mice had significant QT prolongation and diversearrhythmias such as sinus node dysfunction, atrioventricularblock, and ventricular extrasystoles, whereas no arrhythmiaswere observed in wild-type mice. Furthermore, administration ofepinephrine induced frequent ventricular extrasystoles andventricular tachycardia in S105N Rad transgenic mice. This studyprovides novel evidence that the suppression of Rad activityin the heart can induce ventricular tachycardia, suggesting thatthe Rad-associated signaling pathway may play a role inarrhythmogenesis in diverse cardiac diseases.

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