abstracts of oral presentations

A4 © 2008 Japan Human Cell Society

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

are aberrantly regulated in cancer and are also central toembryonic pattering. The Wnt signaling pathway has providedan outstanding example of this. Wnt proteins are a large familyof cysteine-rich secreted molecules. Wnt family members activatemultiple intracellular signaling systems. Wnt-1 was identified asan oncogene product that induces mouse mammary tumor.Wnt-1 stabilizes β-catenin, resulting in the expression of mRNAof c-myc and cyclin D1. The β-catenin-dependent pathway isknown to regulate cellular proliferation and differentiation.Genetic alterations of proteins that constitute this pathway areoften observed in human cancers. So far, we have found that Axinforms a complex with β-catenin, APC, and GSK-3β and thatβ-catenin is phosphorylated and ubiquitinated, resulting in thedegradation of β-catenin. This β-catenin destruction complexmodel revealed the molecular mechanism by which geneticalterations of β-catenin, APC, and Axin lead to tumorigenesis.Some of Wnt proteins, including Wnt-5a, activate the β-catenin-independent pathway that primary modulates cell movement, asfirst observed during embryogenesis. Several groups including usfound that this pathway is also involved in tumorigenesis.Abnormal expression of Wnt-5a was observed in 71 of 237gastric cancer cases by means of immunohistochemistry. Thepositivity of Wnt-5a expression was correlated with advancedstages and poor prognosis of gastric cancer. Wnt-5a had theabilities to stimulate cell migration and invasion in gastric cancercells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which play a role in cell migration.Cell migration, membrane ruffling, and turnover of paxillin weresuppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. Wealso found similar relationship between expression of Wnt-5aand aggressiveness of prostate cancer. These results suggest thatWnt-5a stimulates cell migration by regulating focal adhesioncomplexes and that Wnt-5a is not only a prognostic factor butalso a good therapeutic target for gastric and prostate cancers.

In this symposium, the molecular mechanism by whichabnormal activation of the Wnt signaling pathway causes cancerwill be discussed.

S3-4MAKING OF GLIOBLASTOMA-INITIATING CELLS

Toru KONDORIKEN Center for Developmental Biology, Laboratory for Cell Lineage Modulation

Recent progress has demonstrated that malignant tumors containcancer stem cells (CSCs), which self-renew indefinitely and aretumorigenic. However, it is still controversial whether CSCs insolid tumors arise from tissue-specific stem cells, committedprecursor cells, or differentiated cells, although recent studiesshow that leukaemic stem cells are generated from bothhematopoietic stem cells and committed progenitor cells. Weaddressed this question for gliomas by simultaneously activatingoncogene and repressing tumor suppressor gene in neural stemcells (NSCs), oligodendrocyte precursor cells (OPCs), anddifferentiated astrocytes (ASTs). We demonstrate that both OPCsand NSCs, but not ASTs, transform into glioma stem cells underthese conditions, with a widespread reprogramming of gene

expression and formed glioblastomas even when 10 transformedcells were transplanted into the brain of nude mice. Thesefindings suggest that both OPCs and NSCs are cells of origin forglioblastoma stem cells and support the dedifferentiationhypothesis of tumorigenesis.

Abstract of Luncheon Seminar

ROLE OF CELL–ECM INTERACTION IN CANCER CELL MIGRATION AND METASTASIS

Hideyuki SAYADivision of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine

There are multiple steps in the metastasis of cancer cells. Tumor-cells must first be dissociated from the tumor mass and invadeinto the surrounding tissues. In these processes, cell surfaceadhesion molecules play an important role in the interactionbetween the cells and their microenvironments. CD44 is anadhesion molecule that interacts with hyaluronic acid (HA), acomponent of extracellular matrix (ECM), and implicated in awide variety of physiological and pathological processes.Recently, the proteolytic cleavage of CD44 has been emerging askey regulatory events for the CD44 dependent cell-matrixinteraction and signaling pathway. CD44 undergoes sequentialproteolytic cleavage in the ectodomain and intramembranousdomain, resulting in the release of a CD44 intracellular domain(ICD) fragment. The ectodomain cleavage of CD44 is triggered bymultiple stimulations, and identification of molecules and signalscontributing this event is essential for understanding cancerinvasion and metastasis. In the presentation, I would like todemonstrate the underlying mechanism of CD44-mediated cellinvasion and our recent approaches to develop new drugs forcancer invasion and metastasis.

Abstracts of Oral Presentations

O-01THE ABILITY OF THE SPERMATOGENIC CELLS FOR FERTILIZATION AND EMBRYONIC DEVELOPMENT IN MICE

Yorino SATO, Kahei SATODepartment of Applied Life Science, Nihon University Graduate School of Bioresource Sciences

Recently, the immature spermatid has been focused in the area ofthe male sterility in the reproductive assistance medicine. Thereare some reports that showed fertilization and embryonicdevelopmental ability of the spermatid including the immature

Luncheon Seminar

© 2008 Japan Human Cell Society A5


spermatid. The fertilization deficiency or development arrest,however, appear frequently because of the ability of the spermatid.Furthermore, the functional immaturity is observed in the abilityof the spermatid, despite the same karyotype as spermatozoa.However, a functional difference between spermatid andspermatozoa is not clarified. In this study, fertilization anddevelopmental ability of an immature spermatid such as roundspermatid, elongating spermatid, elongated spermatid, and intra-testicular spermatozoa were examined using micromanipulator.

Mature female mice were superovulated with hMG and hCG.Cumulus-oocytes complex were obtained from oviducts, andcumulus cells were removed by hyaluronidase and pippeting.Spermatids were gathered from mature male testis. They wereinjected into MII oocytes using micromanipulator. Following theembryos were cultured in CZB medium and examined about thedevelopmental ability. The control was used epididymal sperma-tozoon. In addition, we examined the changes of Ca levels inoocytes after spermatid injection.

The fertilization rate of the zygote injected the spermatid wasdepending on the degree of spermatid metaboly. In embryonicdevelopmental rate, however, there were no significant differencebetween control and experimental embryo, but the data showedthat elongating, elongated and round spermatid deteriorate theembryonic development slightly.

From the results of the fertility, it is suggested that the ability ofthe oocyte activation is intensified as the spermatid metamorphose.Further, we will study about the ability of embryonic develop-ment and clarify oocyte activation factor in the spermatid.

O-02ENHANCEMENT OF MESENCHYMAL CELL PROLIFERATION BY USING SOLUBILIZED AMNIOTIC MEMBRANE AS SCAFFOLD

Mamoru KOBAYASHI,1 Yasunobu YOKOYAMA,2 Aiko KIKUCHI,1 Isao KAMO,1 Kahei SATO,3 Norio SAKURAGAWA1

1School of Allied Health Sciences, Kitasato University; 2Stem Cell Processing Inc.; 3Graduate School of Bioresource Sciences, Nihon University

Objective: In this study, we used the completely solubilizedamniotic membrane (SAM) as a scaffold by coating culture plates,and evaluated the effect of the SAM to the in vitro proliferation ofseveral types of mesenchymal cells.Materials and Methods: The amniotic membrane removedfrom the placenta and the fetal membrane was washed with PBS(–), and the amniotic epithelial and mesenchymal cells wereremoved by the EDTA treatment. Then, the membrane waswashed with NaOH, and solubilized by boiling in acetic acid.Total protein concentration in the SAM was measured usingabsorbance at 280 nm. Culture dishes were coated with theSAM. The human amniotic mesenchymal cells (AMC), fibroblast,and mesenchymal stem cells (MSC) were cultured on the disheswith or without (control) the SAM coating. To determine theoptimal concentration of SAM to the proliferation of these cells,we prepared dishes coated with several concentrations of SAM. InMSCs, surface markers, such as CD13, CD29, CD44, CD49a-f,

CD90, CD105, and CD166 were detected by the flow cytometryat day 17 of culture with or without the SAM coating. In addition,to estimate the differentiation ability of MSCs, we cultured thesecells in the osteogenic, chondrogenic, and adipogenic media, anddetected specific markers.Results and Discussion: When AMCs were cultured for14 days with the SUM coated by the protein concentration at0.1 µg/cm2, a total cell number was 10 times as high as that of thecontrol. The proliferation of MSCs also was enhanced with theSAM coating. By the flow cytometrical analysis, the high expressionof CD105 was observed in the MSCs cultured with the SAMcoated dishes. These results revealed that the SAM coating notonly enhance the proliferation but maintain the multipotency ofMSCs during the in vitro culture. Our results demonstrate that theSAM is available as scaffold for the mesenchymal cell proliferation,and is containing some factor(s) for the enhancement of that.

O-03SOMATIC NUCLEAR TRANSFER EFFICIENCY OF HANDMADE CLONING IN BOVINE

Toshimi KAGEYAMA,1 Shinji MAKINO,1 Fumihito AONO,2 Keiichi FUKUDA1

1Regenerative Medicine, School of Medicine, Keio University; 2Department of Research and Development, Kato Ladies Clinic

To examine the developmental rate to blastocyst on handmadecloning (HMC). We used bovine ovaries from a slaughterhouse.Bovine cumulus oocyte complexes (COGs) were matured for22 hr in IVMD fluid. The HMC technique includes manualbisection of zona-free oocytes, and the simultaneous fusion of thesomatic cell with two cytoplasts to produce a cloned embryo.

HMC is a rapid and efficient technique that suits large-scaleNT programs. It requires less expertise and time than traditionalNT methods and the cost of equipment is significantly less. Weused fibroblasts from bone marrow and cumulus for somatic NTdonor in HMC.

In conclusion, the development to blastocyst rates of SC NT is24% (fibroblast cell), 41.7% (cumulus cell). These results indicatethat HMC may be a useful tool for experimental cloning research.

O-04AN EFFICIENT BLASTOMERE FIXATION METHOD OF PREIMPLANTATION GENETIC DIAGNOSIS FOR CHROMOSOMAL DISORDER WITH INTERPHASE FISH

Naoki AOYAMA, Tomoko KURODA, Rie YAMADERA, Satoshi KAWACHIYA, Tomoya SEGAWA, Yuji TAKEHARA, Keiichi KATO, Osamu KATOKato Ladies Clinic, Tokyo, Japan

Objective: Good correlation between FISH errors and therange of nuclear diameter as a FISH target area on the glass slideis observed. The blastomere fixation is one of the most criticalsteps because it is responsible for FISH error and cell loss. In this



study, we modified the traditional fixation method with a 1 mLsyringe as fixative dispenser to reduce the cells lost, and to obtainthe wide nuclear diameter.Materials and Methods: The source of embryos were un-transferable due to abnormal morphological (multi PNs orarrested) obtained from patients with informed consentundergoing IVF at our clinic. Embryos were randomly assignedto one of three fixation methods. Method 1: The blastomere wasexposed to hypotonic solution then it was placed on the glassslide. Next, one drop of methanol : acetic acid (3 : 1) was addedon blastomere by 1 mL syringe for fixative dispenser, the nuclearwas washed with fixative and removed protein and phospholipidcompletely for 5 min. FISH analysis was performed with amixture of probes for chromosome 13,16,18,21,22,X and Y(Vysis).Method 2: The traditional method was described by Munne (1998).It was performed under the same equipment, however severaldrops of fixative was added by a 100 µL pipette (Eppendorf).Method 3: Another traditional method was described byDozortsev (2001). The blastomere was placed hypotonic solutionand transferred onto a glass slide containing Tween 20-HClsolution which caused cell membrane breakdown. Three fixationtechniques were evaluated according to the following parameters:(i) cell loss during fixation and (ii) nuclear diameter.Results: (i) The rates of lost cells in Methods 1, 2, and 3 were1.6% (1/64), 6.5% (4/62), and 0% (0/63), respectively. There wasno significant difference in three methods. (ii) This resultsindicate significant differences (P < 0.01) in nuclear diameterafter fixation with an average of (ave. µm ± SD) 71.1 ± 15.6(n = 63) for Method 1, 53.4 ± 21.2 (n = 58) for Method 2, and31.3 ± 7.7 (n = 63) for Method 3, respectively.Conclusions: As shown in the results, the new and simpleblastomere fixation methods in the present study could reducenot only cell loss rate at fixation but also ratio of FISH errorsresulted in minimizing the ET cancel cases of PGD derivedembryos.

O-06FUNCTIONAL KIDNEY ORGANOGENESIS FROM HUMAN MESENCHYMAL STEM CELLS (hMSCs) USING A GROWING RODENT EMBRYO

Takashi YOKOO, Akira FUKUI, Kei MATSUMOTO, Tatsuo HOSOYADepartment of Internal Medicine, The Jikei University School of Medicine

Given the limits of allogenic organ transplantation, an ultimatetherapeutic solution is to establish self-organs from autologousstem cells and transplant them as syngrafts back into donorpatients. To this end, we aimed to establish an “organ factory” tobuild a functional kidney for transplantation. We first confirmedthat cultivation of human mesenchymal stem cells (hMSCs) inrodent embryos enables their differentiation within a spatiallyand temporally appropriate developmental milieu, facilitating thefirst step of nephrogenesis. As a next step, we modified oursystem to achieve complete functional organogenesis. hMSCstransfected with GDNF and LacZ were cultured in the

nephrogenic site of growing rat embryos (E11.5) for 48 husing a whole-embryo culture system, followed by in vitro organculturing for 24 h. Kidney primordia were then transplantedinto the omentum of uninephrectomized rats and grown foranother 2 weeks. The kidneys grew to 64 ± 21 mg (about 1/10th

the volume of native kidney) and showed normal renal structurewith normal vasculature. LacZ-positive cells were scatteredthroughout the neo-kidney and were morphologically identicalto the native glomerular cells, tubular epithelial cells, andinterstitial cells. Transplantation of these primordia into LacZ-transgenic rats revealed that their vasculature was derived fromthe host. Finally, the levels (mg/dL) of urea nitrogen andcreatinine in fluid collected from the expanded ureter weremarkedly higher than the serum levels of the transplanted rats(840.3 ± 184.6 vs. 30.4 ± 10.8 and 10.1 ± 3.1 vs. 0.3 ± 0.2,respectively; P < 0.01), indicating that the neo-kidney canproduce host urine. Taken together, these findings suggest thathMSCs can differentiate into mature neo-kidney, which has thepotential to replace lost kidney function.

O-07HEPATIC CELLS DERIVED FROM HUMAN UNDIFFERENTIATED AMNIO-STEM CELLS AND THE FUNCTIONAL EVALUATION WHEN TRANSPLANT INTO LIVER FAILURE RAT

Isao TABEI,1 Taka NAKAHARA,4 Akihiro OHYAMA,5 Hisashi HASHIMOTO,2 Toshiaki TACHIBANA,2 Isamu ISHIWATA,3 Yuichi ISHIDA,1 Katsuhiko YANAGA,1 Hiroshi ISHIKAWA4

1Jikei University School of Medicine Department of Surgery and 2Department of Anatomy II; 3Ishiwata Obstetrics and Gynecology Hospital; 4Nippon Dental University School of Life Dentistry at Tokyo, Section of Developmental and Regenerative Dentistry, Laboratory of Regenerative Medical Science; 5Aloka Co. LTD, Tokyo, Japan

Objective: To create a functional liver tissue/cells fortransplantation from ethically available human-amnion.Material and Methods: Human early embryo amnion wasdissected in 0.1% trypsin−0.02% EDTA/PBS (–) and cultured ingrowth medium (GM) containing DMEMF 12 + 20% FBS + 0.1%NEA. Small circular cells developed within the single-layeredcells were cloned colonially. SKG-II-SF cell were used asfeeder cells with addition of LIF for cells to proliferate. Next,such cells were cultured in supernatant conditioned mediumof SKG-II-SF cells with LIF. The cells were named HEAC cellsand were used in the following experiments. HEAC cellswere maintained in GM with addition of 1 ng/mL of LIF.Mesenchymal hepatocytes were cultured from GH defect SD ratsusing the filter paper method of Okumura. Embryotrophicfactors (ETFs) are cytokines contained in the dialysis of theserum free conditioned medium from human cervical squamouscancer cell line (SKG-II-SK). The dialysis molecular weight wasbetween 90–120 kD and was freeze dried. Liver failure rats werecreated chemically using carbon tetrachloride with the survivalrate of 33%.



Results: HEAC cell forms various shapes with poor organella,which were mainly small sphere cells or angular adhesive cells.The cells were alkaline phosphatase-positive with long telomere.HEAC cells co-cultured with mesenchymal hepatocytes incollagen sponge (Stem company) in GM with ETFs for over1 month produced a tri-dimension liver like structure. Thisstructural body produced albumin, and was dose dependentagainst GH to produce IGF-1. When transplanted intochemically induced liver failure rats, it prolonged survival rate to85% as compared to the control value of 33%.Discussion: So far no liver-like-structure can be obtainedwithout the presence of mesenchymal hepatocytes or ETFs. Rathepatic mesenchymal cells were used to prove that theparenchymal hepatocytes were of amnion origin.

O-09Hex IS ESSENTIAL FOR CARDIAC MYOGENESIS IN DIFFERENTIATING EMBRYONIC STEM CELLS

Ruri KANEDA,1,2 Yu LIU,1 Michael D SCHNEIDER1

1Center for Cardiovascular Development, Baylor College of Medicine; 2Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine

Background and Hypothesis: We previously demonstratedthat Sox17, an Sry-box-containing transcription factor thatinteracts with the Wnt/β-catenin pathway, is essential for cardiacmyogenesis in differentiating embryonic stem (ES) cells. Sox17shRNA blocks cardiac myogenesis and selectively impairsthe induction of Hex, a member of the homeobox familyof transcription factors, many of which are involved indevelopmental processes. However, the function of Hex in mouseES cells on differentiation to cardiomyocytes has not beendefined. We hypothesized that Hex is essential for cardiacmyogenesis in ES cells.Methods: Lentiviral vectors encoding shRNAs that knockdown Hex were transduced into AB2.2 cells. Transduced cells,distinguished by expression of EGFP, were flow-sorted andsubjected to embryonic body (EB) culture. Total RNA wasextracted from EBs at 10 time points. Real-time QRT–PCR wascarried out for representative genes related to mesodermformation, mesoderm patterning, and cardiac myogenesis.Spontaneously beating EBs were scored.Results: The prevalence of beating EBs and the expression ofcardiogenic transcription factors (Nkx2.5, Tbx5, Mef2c, Gata4and myocardin) and cardiac structural genes (Ryr2 and α-MHC)both were suppressed by Hex shRNA. Hex shRNA did not impairthe progressive down-regulation of Sox2 and Oct4 (masterregulators of pluripotency) or the induction of Brachyury/T andMesp1/2 (markers of primitive and precardiac mesoderm,respectively).Conclusion: Hex is essential for cardiac myogenesis indifferentiating ES cells acting at the stage of cardiac specification.

O-10DIFFERENTIATION OF AMNION CELLS (HAM) INTO THE CELL WITH NEURAL PHENOTYPE

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: Amnion membrane cells has been shown to giverise to cells of different tissues, including neural cells, hepatocyte,cardiomyocyte, chondrocyte, osteoblast and so on, expandingtheir differentiation potential. In this study, we investigated thatcharacterized of differentiated and HAM cells into neural cells 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 pellets were filtered using stainlessmeshes or 100 µm cell strainer and centrifuged two times. Thesediments 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.

To induced differentiation to neural cells, we used the modifi-cation method described by Blondheim et al. (2006). Growthmedium was replaced with differentiation medium I consisting ofαMEM supplemented with 10% FBS, 2 mM glutamine, 10 ng/mLhEGF, 10 ng/mL human fibroblast growth factor-2 (hFGF-2,Sigma), and 1× N2 supplement (Invitrogen-GIBCO) for 48 h.Differentiation medium I was then removed and the HAM cellswashed with PBS and transferred to differentiation medium II,composed of αMEM supplement with 2 mM glutamine, 1× N2supplement, 1 mM dibutyryl cyclic AMP (dbcAMP, Sigma),0.5 mM 3-isobutyl-1 methyl-xanthine (IBMX, Sigma) and 1 µM

all-trans retinoic acid (Sigma) for up to 96 h.Results: The change in morphology of HAM cells could beobserved 24 h after differentiation medium I. At the end ofdifferentiation medium II, HAM cells were changed to typicalneural like cells and the cells with retracted bodies, longprocesses and neuron network. We analyzed the expressionof various neural-related gene, including nestin, neuron-specific enolase (NSE), gilal fibrillary acidic protein (GFAP),neurofilament medium (NF-M). The differentiated HAMcells expressed nestin, NSE, GFAP and NF-M. Immuno-chemical analysis of neural-associated markers revealedHAM cells to differentiate into neural cells were positiveNSE, myelin basic protein (MBP), and β-tubulin isotype III(Tuj-1).Conclusion: These studies demonstrate that HAM cells can bedifferentiated into neural cells by optical differentiation protocol.HAM cells might be an ideal source for neuroregeneration.



O-11ANGIOPOIETIN-1 SUPPORTS INDUCTION OF HEMATOPOIETIC ACTIVITY IN HUMAN CD34– BONE MARROW CELLS

Yoshihiko NAKAMURA,1,4 Takashi YAHATA,1,2 Yukari MUGURUMA,1 Tomoko UNO,1 Tadayuki SATO,4 Hideyuki MATSUZAWA,4 Shunichi KATO,3 Yukari SHIRASUGI,1,2 Tomomitsu HOTTA,1,2 Kiyoshi ANDO1,2

1Division of Hematopoiesis, Research Center for Regenerative Medicine; 2Department of Hematology; 3Department of Cell Transplantation & Regenerative Medicine; 4Teaching and Research Support Center; Tokai University School of Medicine

Objective: Hematopoietic stem cells (HSCs) consist ofheterogenous subpopulations, one of which is CD34– HSCs.The recent development of successful engraftment by intra-bonemarrow transplantation (iBMT) revealed severe combinedimmunodeficiency (scid) mouse-repopulating cell (SRC) activityin human CD34– cord blood (CB) cells. On the other hand, CD34–

cells from bone marrow (BM) cells remain relatively undefined.Here, we investigated pre-SRC populations in human BM CD34– cellsand the effect of the niche-related factor, angiopoietin-1, on them.Methods: Two populations in BM CD34– cells (namely M cellsand S cells) were purified by flow cytometry. Then, they werecocultured with 6 growth factors on the hematopoietic-supportive mouse BM stromal cell line, HESS-5 or AHESS-5 thatwere engineered to produce human angiopoietin-1, since wedetected Tie2 expression on M cells and S cells. Cultured cellswere assessed for their in vitro and in vivo hematopietic activities.Results: After 7 days in coculture, AHESS-5 was stronger moreeffective than HESS-5 in converting M and S cells to CD34+ cells (Mcells: 67.4% versus 17.5%; n = 6, P < 0.001) (S cells: 42.3% versus2.3%; n = 6, P < 0.001). Furthermore, both M and S cells were able toengraft in immunodeficient mice after they were cocultured onAHESS-5.Conclusions: The results suggest that angiopoietin-1 supportsSRC activities in human CD34– BM cells, as murine studiesdemonstrated. Furthermore, the identification of previouslyundetected subpopulations of BM CD34– HSCs unveilsheterogenous components in the stem cell pool.

O-13TELMISARTAN ENHANCES CHOLESTEROL EFFLUX FROM THP-1 MACROPHAGES BY ACTIVATING PPARγγγγ

Kazuhiro NAKAYA, Makoto AYAORI, Tetsuya HISADA, Masatsune OGURA, Emi YAKUSHIJI, Shun-ichi TAKIGUCHI, Harumi UTO-KONDO, Masatoshi KUSUHARA, Fumitaka OHSUZUDepartment of Internal Medicine, National Defense Medical College

The ATP binding cassette transporters A1 and G1 (ABCA1/G1)and scavenger receptor class B type I (SR-BI) are key moleculesin cholesterol efflux and atherogenesis. These genes are regulatedby peroxisome proliferator-activated receptor γ (PPARγ) andliver X receptor (LXR). Telmisartan is an angiotensin type 1receptor blocker which has been reported to act as a ligand

for PPARγ. We investigated whether PPARγ-activating ARBsaffect the expression of these genes and cholesterol efflux frommacrophages.

Telmisartan increased ABCA1, ABCG1 and SR-BI mRNA levelsin THP-1 macrophages in a dose- and time-dependent fashion. Italso increased their protein levels and enhanced apoA-I- and HDL-mediated cholesterol efflux from macrophages. The knockdownof PPARγ by siRNA abolished telmisartan-induced expression ofthese genes. The knockdown of LXRα resulted in the completeand partial abolishment of telmisartan-induced ABCA1 and ABCG1expression, respectively. We also demonstrated that telmisartan-induced SR-BI expression was dependent on the PPARγ pathwaybut not the LXRα pathway. A luciferase assay using an ABCA1promoter construct showed that telmisartan activated ABCA1transcription, which was abolished if the LXR binding elementwas mutated, indicating that increased ABCA1 transcription bytelmisartan is LXR dependent.

Our results showed that telmisartan enhanced bothapoA-I- and HDL-mediated cholesterol efflux from macrophagesby increasing ABCA1, ABCG1 and SR-BI expression viaPPARγ-dependent and LXR-dependent/independent pathways.

O-14MECHANISM OF VASODILATATION WITH N-ACETYLCYSTEINE AND NaHS

Michiko YAMAMOTO, Takeshi ADACHI, Makoto SUEMATSUDepartment of Biochemistry, Keio University School of Medicine

Objective: Hydrogen sulfide (H2S) is known as a toxic gas,however it is identified as the third gaseous mediator forvasodilatation following nitric oxide (NO) and Carbon monoxide.N-acetylcysteine is widely used as an anti-oxidant agents,however, its pharmacological effects is an increase in intracellularcysteine (Cys) levels, which may increase the production of H2Sby cystathionine-γ-lyase (CSE). We focused that the productionsof vascular H2S were mediated via Cys/CSE pathway. In thisstudy, we aimed to test the mechanism of vasodilatation with aCys donor, NAC, and a H2S donor, NaHS.Methods: Vascular function was assessed with isometrictension measurement of aortic rings equipped in the organ bath.After aortic constriction with phenylephrine (PE), a dose-dependentrelaxation to NAC/NaHS (0.3–10 mM) was measured. Somerings were pre-treated with L-NAME (100 µM), a NO synthaseblocker, ODQ (10 µM), a guanylyl cyclase blocker, high potassiumconcentration, and ATP-sensitive potassium channels (IKatp)blocker, glybenclamide (10 µM).Results: Neither L-NAME nor ODQ affected the maximumaortic relaxations to NAC and NaHS. High potassium reducedmaximum relaxations to NAC and NaHS. Glybenclamide reducedthe maximum relaxation to NaHS. Notably, glybenclamidedecreased the speed of relaxation to NAC and NaSH.Conclusion: NO has a little effects of aortic relaxation to NAC/NaSH. The mechanisms of aortic relaxation to NAC/NaSH wereinvolved with hyperpolarization via the activation of potassiumchannels. H2S may activate IKatp to relax aorta especially in theearly phase. From these results, H2S can be a novel vasodilatationmediator with hyperpolarization.



O-15MODULATION OF PROTEIN-METHYLATION IN AORTA FROM ZUCKER RAT

Takeshi ADACHI, Michiko YAMAMOTO, Kyoko ISHIWATA, Makoto SUEMATSUDepartment of Biochemistry, Keio University School of Medicine

Zucker rat is widely employed as a typical experimental model ofmetabolic syndrome, which is characterized with obesity,hyperlipidemia and insulin resistance. Although atherosclerosisin large arteries is the final disease-targets of metabolic syndrome,changes in post-translational protein modifications in aorta ofthis pathological state had not been well studied. Recently we areinvestigating about the post-translational protein modifications,which are induced by gas molecules such as NO, CO, and H2Sand they are important factors for the progression of vasculardiseases. In this study, we aimed to test the involvement ofgaseous molecules and protein-methylation in aorta from Zuckerrat.Methods: Vascular function was assessed with isometrictension measurement of aortic rings equipped in the organ bathand aortic constriction was induced with phenyrephrin (PE).Some rings were pre-treated with an iNOS selective blocker, L-NIL (10 µM). Proteome analysis of aortic protein was performedwith the differential display of two-dimensional electrophoresisusing Ettan Dige method.Results: Although Endothelium-dependent relaxation waspreserved, PE-induced vasoconstriction was significantlydecreased in aorta from Zucker rat (2.6 ± 0.1 g vs. 3.2 ± 0.2 g[Lean Control] with PE 0.1 µM, P < 0.05). The reduced PE-induced constriction was improved with a pre-treatment ofL-NIL, suggesting the involvement of NO from iNOS in thismodel. In proteome analysis, there are few differences of theexpressions of protein spots. The analysis of protein-methylationwith anti-dimethyl arginine antibody revealed the decreasedADMA modifications of many spots in aortic proteins fromZucker rat.Conclusion: In aorta from Zucker rat, the expression offunctional iNOS was indicated. In spite of few differences of theprotein expression, there are many differences of the spots ofprotein-methylation. Further studies are required for theidentifications of pathological relationship among the aortic function,augmentation of NO generation and protein-methylation.

O-16IMMUNOMODULATORY EFFECTS OF INTERLEUKIN-18 ON SPLENIC CELLS

Seitaro FUJISHIMA, Kazuhiko SEKINE, Naoki AIKAWADepartment of Emergency and Critical Care Medicine, Keio University School of Medicine

Rationale: Patients under critical conditions becomesusceptible to secondary insults, such as infection, and often

develop septic shock and acute lung injury (ALI). Previously,we have found that a prior burn insult induces overproductionof proinflammatory cytokines and lethal ALI afterlipopolysaccharide (LPS) challenge in mice (AJP 284: L270-8,2003), and in vivo interleukin-18 (IL-18) supplementationameliorates ALI and improved survival.Purpose: In the present study, to elucidate the mechanisms ofIL-18-derived protection for ALI, we examined the in vitroimmunomodulatory effects of IL-18 at physiologicalconcentrations on splenic cells, by analyzing cytokinesproduction and gene expression profiles.Methods: Spleens were resected aseptically from mice andcells were suspended in RPMI-1640/5% FCS at a concentrationof 1.0 × 106 cells/mL. For the analysis of cytokines production,cells were divided into three groups, preincubated with 30,30/100, or 100 pg/mL of IL-18 for 9 days, and stimulated with10 µg/mL LPS. The 30/100 group was incubated with 30 pg/mLIL-18 for the initial 7 days and then with 100 pg/mL IL-18 for thenext 2 days. After culture, MIP-2 and IL-10 in the supernatantswere analyzed by ELISA. For the analysis of gene expressionprofiles, splenic cells were incubated with RPMI-1640/10%FCS containing 0–400 pg/mL IL-18 for 2 days. Afterculture, total RNA was extracted and gene expression profileswere analyzed using Illumina Sentrix Mouse-6 ExpressionBeadChip. Data analysis was performed by GeneSpringsoftware.Results: MIP-2 production in the 30 pg/mL IL-18 group wassignificantly higher than that in the 30/100 pg/mL and 100 pg/mL groups. MIP-2 production in the 30/100 pg/mL group wassignificantly higher than that in the 100 pg/mL group, suggestinga parallel decrease of MIP-2 production with IL-18concentration. In contrast, there was no significant difference inIL-10 production among the three groups, although there was atendency for production to be decreased in the 100 pg/mL groupas compared with the other groups. With regard to geneexpression profile analysis, we found about 500 genes whoseexpressions were changed more than 1.4 times. Among them,several genes were identified whose expressions were changedmore than 4 times.Conclusions: It is speculated that IL-18, depending on itsconcentrations, could play anti-inflammatory roles throughmodulating gene expressions in immune cells.

O-17VITAMIN E HOMOLOGUES INHIBITED THE ADIPOGENIC DIFFERENTIATION VIA THE Akt PATHWAY

Harumi UTO-KONDO,1 Makoto AYAORI,1 Masatoshi KUSUHARA,1 Fumitaka OHSUZU,1 Kazuo KONDO2

1Department of Internal Medicine, National Defense Medical College; 2Institute of Environmental Science for Human Life, Ochanomizu University

Obesity is a serious health problem, and its prevention ispromoted through diet and exercise. In vivo studies have shownthat the oral administration of γ-tocotrienol derived from



tocotrienol-rich fraction in palm oil (TRF) decreases body fatin rats. However, the mechanism of the inhibitory effect of TRFfor adipocytes has been unclear. In this study, we investigatedthe mechanism of TRF suppression of adipocyte differentiationin 3T3-L1 cells. 3T3-L1 preadipocytes differentiated intoadipocytes in the presence of 10 µg/mL insulin. After 14 days,the mRNA expression levels of peroxisome proliferator-activatedreceptor gamma (PPARγ) and other adipocyte-specific genes weremeasured by real-time PCR, and the expression levels of insulinsignaling molecules, Akt, ERK1/2 and PPARγ were measured bya western blot analysis. When TRF and insulin were added to theculture, the mRNA expression of PPARγ decreased by 73%in comparison to the expression levels of insulin alone. Wesubsequently studied the amount of PPAR γmRNA in thepresence of the major components of TRF (such as γ-tocotrienol,γ-tocotrienol and γ-tocopherol) in order to examine theinhibitory effect of TRF. The PPARγ mRNA expression in thepresence of insulin and γ-tocotrienol or γ-tocotrienol wasdecreased by 55% and 90%, respectively, in comparison to theexpression in the presence of insulin alone. However, whenγ-tocopherol and insulin were added to the culture, theexpression of adipocyte-specific genes increased. Furthermore,two tocotrienols inhibited the insulin-induced accumulation oftriglycerides and the phosphorylation of Akt, but did notinhibit ERK1/2. Therefore, the inhibitory effect of TRF dependson both γ-tocotrienol and γ-tocotrienol, which suppressedPPARγ expression via the Akt pathway in 3T3-L1preadipocytes.

O-18REPRESSION OF GLUCOCORTICOID-RESPONSIVE GENE EXPRESSION BY NUCLEAR PROTEIN HEXIM1

Noriaki SHIMIZU,1 Noritada YOSHIKAWA,1,2 Motoaki SANO,3 Satori TOKUDOME,3 Keiichi FUKUDA,3 Chikao MORIMOTO,1,2 Hirotoshi TANAKA1,2

1Division of Clinical Immunology, Institute of Medical Science, University of Tokyo; 2Department of Rheumatology and Allergy, Institute of Medical Science, University of Tokyo; 3Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine

The nuclear protein HEXIM1 is known to squelch positive-transcription elongation factor b (P-TEFb) using 7SK snRNA as ascaffold, thereby negatively regulating RNA polymerase II-dependent transcriptional elongation. It is rational to expect thatthe liberation of P-TEFb from HEXIM1 occurs in the context ofindividual promoter. On the other hand, we previously reportedthat HEXIM1 directly binds to glucocorticoid receptor (GR) andrepresses GR-mediated transcriptional activation. In HepG2cells, most of such genes that are induced by treatment withsynthetic glucocorticoid, dexamethasone (DEX) were down-regulated when HEXIM1 was overexpressed. The efficiency of therepression showed great diversity dependent on each promotercontext, indicating the variety of the functional relevance ofHEXIM1 and GR on each promoter.

In this report, we analyzed effects of HEXIM1 protein levels onendogenous mRNA expression of glucocorticoid-responsive

Na-K-ATPaseα1, ENacα, and xenobiotic-responsive CYP1A1gene, using quantitative real-time PCR. When a recombinantadenovirus expressing either HEXIM1 or siRNA against HEXIM1was infected in HeLa cells, HEXIM1 protein expression wasincreased to 10-fold or decreased to less than 10% of that of con-trol cells, respectively.

In control HeLa cells, Na-K-ATPaseα1 and ENacα mRNAwere induced 1.1-fold and 8.6-fold, respectively, by 6 h treat-ment with 1 mM DEX. CYP1A1 mRNA was induced 8.9-fold by6 h treatment with 1 mM 3-methylcholanthrene. Overexpressionof HEXIM1 scarcely affected basal mRNA expression of thesegenes. In HEXIM1-overexpressed cells, these genes were induced1.1-fold, 8.5-fold, and 3.7-fold, respectively, after the treatmentwith the cognate ligands. Knockdown of HEXIM1 induced basalmRNA expression of Na-K-ATPaseα1 and ENacα 1.3-fold and11-fold, respectively. In HEXIM1-knocked-down cells, thesegenes were induced 1.6-fold and 1.8-fold by the cognate ligands,respectively.

These results clearly demonstrate that HEXIM1 protein dosagedifferentially modulates basal and ligand-inducible mRNAexpression levels of these genes in a promoter context-dependentmanner. This regulation might contribute to the gene-specificand fine-tuned actions of glucocorticoid such as tissue- or stage-specific and signal-dependent coordination of gene expression.

O-19THE ROLES OF CORTICOSTEROID RECEPTORS IN THE MYOCARDIUM

Noritada YOSHIKAWA,1 Satori TOKUDOME,2 Noriaki SHIMIZU,1 Motoaki SANO,2 Keiichi FUKUDA,2 Chikao MORIMOTO,1 Hirotoshi TANAKA1

1Division of the Clinical Immunology, ACRC, Institute of Medical Science, University of Tokyo; 2Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan

At pharmacological doses, glucocorticoid could, directly orindirectly, induce metabolic and cardiovascular diseases.Moreover, recent reports show that mineralocorticoid has distincteffects on the heart including ventricular remodeling. However,precise mechanism of these unwanted corticosteroids’ actions oncardiovascular systems remains to be elucidated, becauseredundant relationship between the corticosteroids and theirreceptors (GR and MR) exists in the myocardium. Availability ofsuperselective agonist for each receptor would contribute tounveiling these issues. In this report, we studied the effects ofseveral corticosteroids on the nuclear translocation andtransactivation ability of rat GR and MR, and revealed that asynthetic glucocorticoid cortivazol (CVZ) is exclusively selectivefor the GR, and endogenous corticosteroids corticosterone (B)and aldosterone (ALD) act on both receptors. Taking advantageof using CVZ for analyzing GR-specific signal transduction, weanalyzed the effects of these steroids on mRNA expression profilein primary culture of neonatal rat cardiomyocytes using DNAmicroarray techniques. mRNA levels of 30 000 known rat geneswere measured at 3 h after treatments vs. vehicle, and werevealed that 2% of them were increased > 2-fold or decreased



< 50% by either steroids. CVZ induced 77% of total up-regulatedgenes, and 45% and 15% of CVZ-induced genes were alsoinduced with B and ALD, respectively. CVZ repressed 58% oftotal downregulated genes, and 14% and 7% of CVZ-repressedgenes were also repressed with B and ALD, respectively. Althoughfurther analysis must be needed, these results suggest that manycorticosteroid-dependent upregulated genes are controlled viathe GR, and that ALD also regulates GR-dependent signaltransduction in the myocardium. Our approach would define aset of corticosteroid-regulated genes and demonstrate theselectivity of GR- vs. MR-dependent signal transduction in themyocardium.

O-21A STUDY ON EPITHELIAL–MESENCHYMAL TRANSITION (EMT) IN DIFFERENT RENAL DISEASE MODELS

Hirokazu OKADA, Tsutomu INOUE, Hiromichi SUZUKIDepartment of Nephrology, Saitama Medical University

Renal interstitial fibroblasts consist of a mixed cell populationderived from multiple origins. We have conducted a number ofstudies and proposed candidate origins including (i) bonemarrow cells invading into impaired organs as circulatingleukocytes, (ii) matured renal tubular epithelial cells via EMT and(iii) proliferated resident fibroblasts in a normal tissue. In thepresent study, we specifically manipulated genes of proximaltubular epithelial cell in vivo using Cre-LoxP system and studiedthe behavior of proximal tubular epithelium-derived cells in therenal interstitial lesions.

All the mice were with SJL background and consisted of γ-GT.Cre transgenic mice, ROSA (floxed-LacZ) transgenic mice,and Floxed-p35 transgenic mice. The renal disease models usedwere the unilateral urinary obstruction (UUO) model and anti-glomerular basement membrane (anti-GBM) nephritis model.The LacZ+, tubular epithelium-derived cell was identified by LacZstaining using X-gal. The role of apoptosis in the EMT process wasexamined by unfloxed p35 in the proximal tubular cells in F1

mice (γ-GT.Cre × floxed p35). The TUNEL method was used todetect the apoptosis. The qPCR was used for mRNA analyses.

Although the LacZ+ interstitial cells were observed in the inter-stitium of the UUO model after 7 days of urinary obstruction,anti-GBM models were without significant increases in thenumber of LacZ+ interstitial cells. When unfloxed p35 mice withUUO were compared to the wild-type mice with UUO, thenumber of TUNEL-positive cells was decreased significantly.However, there was no significant difference in the levels of inter-stitial fibrosis. Moreover, unfloxed p35/LacZ mice with anti-GBMnephritis revealed that a few LacZ+ interstitial cells appeared inthe fibrous kidney.

In conclusion, the proximal tubular epithelium-derivedinterstitial cells via EMT were detected only in the UUO model.Suppression of the renal tubular apoptosis increased the numberof cells derived from tubular cells. There is a possibility thatapoptosis in the renal tubular epithelial cells decreased thenumber of interstitial cells derived from tubular epithelium viaEMT in the fibrous kidneys.

O-22SUPPRESSION OF Oct4 BY GERM CELL NUCLEAR FACTOR PROMOTES THE DEVELOPMENT OF THE EARLY NEURAL STEM CELL LINEAGE

Wado AKAMATSU,1,2 Austin J COONEY,3 Derek VAN DER KOOY2

1Department of Physiology, Keio University School of Medicine; 2Department of Medical Genetics and Microbiology, University of Toronto; 3Department of Cell Biology, Baylor College of Medicine

The earliest murine LIF-dependent, primitive neural stem cellstransition into FGF 2-dependent, definitive neural stem cellsbetween E7.5 and E8.5 in vivo. In mice lacking GCNF, atranscriptional repressor of Oct4, generation of definitive neuralstem cells was dramatically suppressed, accompanied by asustained expression of Oct4 in the early neuroectoderm.Knockdown of Oct4 in GCNF–/– neural stem cells rescued theGCNF–/– phenotype. Overexpression of Oct4 blocked thedifferentiation of primitive to definitive neural stem cells, but didnot induce the de-differentiation of definitive to primitive neuralstem cells. The Oct4 promoter was methylated during thistransition dependently of GCNF. These data suggest thatprimitive neural stem cell develop into definitive neural stem cellby means of GCNF induced suppression of Oct4. Themethylation of the Oct4 promoter by GCNF explains theasymmetry in this transition between primitive and definitiveneural stem cells by restricting the plasticity of mature neuralstem cells even after GCNF is no longer expressed.

O-23ANALYSIS OF AGGREGATION OF HUMAN NEURAL STEM/PROGENITOR CELLS

Hideki MORI, Tomoko SHOFUDA, Mami YAMASAKI, Yonehiro KANEMURAInstitute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan

Objective: Human neural stem/progenitor cells (NSPCs)proliferate as aggregates in vitro, but the mechanism of theiraggregation is not fully known. Here, to understand theneurosphere-forming process, the ability of human NSPCs toproliferate was examined in terms of the fluctuations in the sizeand number of human neurospheres along with culture time. Wepresent a finding that aggregation promotes the proliferation ofNSPCs.Methods: Approval to use human fetal neural tissues wasobtained from the ethical committees of Osaka National Hospital.Human NSPCs were propagated using DMEM/F-12 baseddefined medium supplemented with EGF, FGF-2, LIF, and B27supplement. Bright-field images of neurospheres were randomlyand automatically captured from each well using a cell-morphology screening system. The projected area of eachneurosphere and the number of neurospheres were analyzed



using an algorithm developed with software for image analysis,and then the size of the neurospheres was defined as anequivalent circle diameter. To evaluate the regional cell densitiesin individual human neurospheres of different sizes, the imagesof the nuclei-stained neurosphere sections were analyzed usingan algorithm. To detect proliferating cells, the sections wereimmunostained with anti-Ki-67 monoclonal antibody.Results: Human neurospheres were formed and enlarged, notonly by clonal proliferation from a single cell, but also by acomplicated combination of cell division and neurosphereaggregation. In addition, we found the increase of proliferationrate depended on the size of the aggregate; that is, the populationdoubling time of the NSPCs gradually decreased as the diameterapproached 250 µm. NSPC’s proliferation was independent oftheir location in neurosphere.Conclusion: Human neurosphere grows larger both throughcell division and agglomeration with other cells or neurospheres.The proliferation rate was linear and depended on the size of theaggregate. This result suggests that the three-dimensionalarchitecture of NSPC aggregates creates a microenvironment thatpromotes the proliferation of human NSPCs.

O-24TUMOR SUPPRESSOR GENE Fbxw7 ACTS AS A CRITICAL FAIL SAFE AGAINST THE PREMATURE LOSS OF HEMATOPOIETIC STEM CELLS AND LEUKEMOGENESIS

Sahoko MATSUOKADepartment of Cell Differentiation, School of Medicine, Keio University

Ubiquitin ligase component Fbxw7 is mutated and functions asa tumor suppressor gene in several human cancer cell lines andprimary cancer cells. However, little has been known about thefunction of Fbxw7 in hematopoiesis. Previously we have reportedthat Fbxw7-deficient mice died at mid-embryonic day withdeficiencies in hematopoietic and vascular development,indicating that Fbxw7 has a pivotal role in hematopoiesis. To assessthe requirement of Fbxw7 in adult hematopoietic cells, we generatedFbxw7-deficient mice by conditional gene targeting. Fbxw7 wasconditionally deleted from Mx-1-Cre (+); Fbxw7fl/– adult mice byinjection of pIpC over 1 week to induce Cre expression.

The number of bone marrow mononuclear cells (BM MNCs)and hematopoietic stem cells (HSCs) was significantly decreasedby 3 months after pIpC treatment. To examine the function ofFbxw7-deficient HSCs, we transplanted BM MNCs from Fbxw7-dificient mice into lethally irradiated recipient mice. In the result,Fbxw7-deficient HSCs are impaired in long-term repopulatingactivity and multipotency. The portion of Fbxw7-deficient HSCsin the G0 phase was markedly decreased. These data suggest thatFbxw7 deletion leads to premature loss of HSCs due to the activecell cycling.

Furthermore, more than half of the Fbxw7-deficient micedeveloped T-cell acute lymphoblastic leukemia within 6 monthsafter pIpC treatment. The leukemic cells of Fbxw7-deficient micedisplayed significant accumulation of c-Myc and Notch1. It is

suggested that accumulated Notch1 and c-Myc proteins inFbxw7-deficient bone marrow cells caused extrathymic develop-ment of T-lineage cells and induced T-ALL.

O-25ACTIVATION OF GCN2-eIF2-ATF4 PATHWAY INDUCES THE SENESCENCE-LIKE PHENOTYPE IN ALDH2*2 TRANSGENIC MICE

Takaharu KATAYAMA,1,2 Motoaki SANO,1 Jin ENDO,1,2 Satori TOKUDOME,1 Shinsuke YUASA,1,2 Shinji MAKINO,1 Fumiyuki HATTORI,1 Toshimi KAGEYAMA,1,2 Kiyomi NISHIMAKI,3 Ikuroh OHSAWA,3 Takeshi ADACHI,4 Shigeo OHTA,3 Makoto SUEMATSU,4 Satoshi OGAWA,2 Keiichi FUKUDA1

1Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine; 2Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine; 3Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School of Tokyo; 4Department of Biochemistry and Integrative Medical Biology, Keio University School of Medicine

Introduction: Aging process is a deep modification of theenergetic metabolism, which results from a cellular stressresponse to the accumulation of macromolecule aggression. Toelucidate myocardial senescence, we developed a novel mousemodel of senescence-like phenotypes, by generating transgenic(Tg) mice that overexpress the inactive variant of aldehydedehydrogenase (ALDH) 2*2 which produce mitochondrialoxidative stress. This study investigated the molecularmechanism of the senescence-like phenotype induction causedby aldehyde-induced macromolecule aggression in ALDH2*2 Tgmice.Methods and Results: (i) The ALDH2*2-Tg mice showeddecreased body weight by virtue of decreased skeletal and cardiacmuscle mass (30%) despite comparable daily food intake. Thefractional synthesis rates of protein in heart and gastrocnemiuswere decreased by 34% and 25%, respectively. There was noincrease in plasma and muscle concentration of 3-methylhistidine (a marker of muscle degradation), indicating thatthe decreased muscle mass was not caused by the augmenteddegradation but rather by the reduction of protein synthesis. (ii)Gene chip analysis revealed that ATF4 (activating transcriptionfactor 4) and its downstream target genes related to amino acidanabolism, glutathione biosynthesis were markedly increased.Phosphorylation of eIF2a (eukaryotic translation initiation factor2 alpha), which represses global protein synthesis but selectivelyenhances the ATF4 translation, was increased in ALDH2*2 Tgmuscles. (iii) Metabolome analysis revealed an increasedproduction of NADPH via enhanced flux towards the pentosephosphate pathway and activation of amino acid anabolismleading to glutathione biosynthesis in accordance with theupregulation of ATF4-dependent genes, while histidine wasselectively decreased by 50%. (iv) GCN2 (amino acid biosensorgeneral control nonderepressible 2, one of the upstream eIF2α



kinases) was activated in the muscles. (v) Furthermore, a highhistidine diet rescued the decreased body weight of transgenicmice.Conclusions: These findings indicated that the induction ofsenescence-like phenotypes was caused by activation of theGCN2-eIF2α-ATF4 pathway triggered by intracellular aminoacid imbalance.

O-26ROLES OF IGFBP-2 IN INVASIVE GROWTH OF GLIOBLASTOMA CELLS

Tsuyoshi FUKUSHIMA, Nobuyasu TAKAHASHI, Shoichiro MUKAI, Makiko KAWAGUCHI, Hiroyuki TANAKA, Hiroaki KATAOKASection of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

Insulin-like growth factor binding protein-2 (IGFBP-2) isexpressed in fetal tissues and is associated with braindevelopment. The expression of IGFBP-2 decreases after birth innormal condition. The elevated expression of IGFBP2 has beenreported in various malignant tumors including glioblastoma(GBM) that is the most common malignant brain tumor with thepoorest prognosis. However, it remains to be determined howIGFBP-2 is involved in the growth and invasive behavior of GBMcells.

We used short hairpin RNA (shRNA) expression retrovirusvector to inactivate the IGFBP-2 gene of human GBM cell lines,U251 and YKG-1 permanently. The knockdown of IGFBP-2resulted in morphological changes of culture cells, decreased sat-uration density in culture, and decreased invasiveness in matrigelinvasion assays. Reversion of IGFBP-2 in IGFBP-2 knockdowncells restored invasion activity. Those effects were independent ofinsulin-like growth factor signaling. The intracranial implanta-tion of these cells in nude mice revealed that the tumorigenicityof the IGFBP-2 knockdown clones was impaired as comparedwith that of the control clones in vivo. Using cDNA microarraysfor transcriptional profiling, we have identified genes whoseexpressions were altered by IGFBP-2 inactivation. One of themwas CD24, a small glycoprotein linked to the plasma membraneby a glycosylphosphatidylinositol lipid anchor, which has beenimplicated in the invasiveness and metastasizing activity of avarious cancers. It has been also reported to be a prognosticablefactor of malignant tumors such as ovarian cancer and prostatecancer. Actually, IGFBP-2 knockdown decreased both expressionlevel of CD24 and CD24 promoter activity of GBM cells. Next, weperformed stable knockdown of CD24 by shRNA expressingretrovirus. Knockdown of CD24 also resulted in decreased inva-siveness of glioblastoma cells, and conversely, overexpression ofCD24 enhanced the invasion. Reversion of CD24 in IGFBP-2knockdown cells restored the invasive activity of GBM cells.

In conclusion, IGFBP-2 and CD24 is involved in invasivegrowth of GBM cells, and CD24 is one of the downstream targetsof IGFBP-2. IGFBP-2, CD24, and the signaling pathways linkingthem may be potential targets of novel therapeutic strategies forthe control of invasive growth of GBM cell.

O-27IDENTIFICATION OF NOVEL DELETION COPY NUMBER VARIATIONS IN BREAST CANCER

Akira KOMATSU,1,3 Koichi NAGASAKI,2 Minoru FUJIMORI,3 Yoshio MIKI1,2

1Department of Molecular Diagnosis, Cancer Institute; 2Genome Center, Japanese Foundation for Cancer Research; 3Department of Surgery, Shinshu University School of Medicine

Breast cancer is the most frequent cancer in female world wideand it has long been known that multiple genetic rearrangementscorrelate with complex biology and clinical behavior. In addition,copy number variations (CNVs) of DNA sequences account for asignificant proportion of normal phenotypic variation and mayhave an important role in human pathological variation. In thisstudy, we performed high-density oligonucleotide array CGHanalyses in 25 breast cancer cell lines to identify a novelhomozygous deletion site. Selected homozygous deletion sites onCGH analyses were confirmed by quantitative PCR (Q-PCR) and4 novel homozygous deletion sites were selected. Thesehomozygous deletion sites on 30 microdissected human breasttumors and paired normal mammary tissues were analyzed by Q-PCR and confirmed as deletion CNVs. Furthermore, we analyzedthese CNVs on blood DNA in 50 breast cancer patients and 50healthy female volunteers. These 4 deletion CNVs had tendenciesthat were observed in more frequency breast cancer patients thanhealthy female. Our results suggested that these CNVs may playimportant role in breast cancer with biological process andclinical behavior.

O-30GERMLINE AND SOMATIC MUTATIONS OF THE BRAF GENE IN OVARIAN CARCINOMA

Masatsugu UEDA,1 Eisaku TOJI,2 Osamu NUNOBIKI,3 Shinji IZUMA,1 Kiyo TORII,1 Sadamu NODA1

1Cytopathology and Gynecology and 2Medical Technology, Osaka Cancer Prevention and Detection Center; 3Medical Technology, Kobe Tokiwa College

Ovarian carcinoma is one of the most lethal neoplasms inwomen, and serous carcinoma is the most common type. Adualistic model of ovarian serous carcinogenesis has beenrecently proposed. High-grade conventional serous carcinomaarises de novo from the surface epithelium or from the epitheliumof inclusion cysts. In contrast, it has been shown that ovarianlow-grade serous carcinoma evolves out of a stepwise progressionfrom benign serous cystadenoma to serous borderline tumor(SBT) to micropapillary serous carcinoma (MPSC) and that BRAFactivation is a very early somatic event in the tumorigenesis. Inthis study, we postulated that BRAF germline mutations could beresponsible for low-grade serous tumor and investigated bothgermline and somatic mutations of the BRAF gene in a series ofovarian cancer patients and cell lines.

Representative surgical blocks were obtained from 104 caseswith ovarian tumors including 21 SBTs, 3 MPSCs, 42 conven-tional high-grade serous carcinomas, 12 mucinous borderline



tumors (MBTs), 10 mucinous carcinomas, 6 endometrioidcarcinomas, and 10 clear cell carcinomas. Tumor tissues weremicrodissected from the sections and blood samples wereobtained from these 104 patients and 101 normal healthy women.10 human ovarian cancer cell lines were also included in themutation analysis. DNA samples extracted from tissue samples,peripheral blood lymphocytes, and cell lines were screened formutations of exon 15 of the BRAF gene by PCR-SSCP and sequenced.

BRAF V599E mutation in histologic samples was found in 5(24%) of 21 SBTs, 1 (33%) of 3 MPSCs, and 1 (17%) of 6endometrioid carcinomas but not detected in 42 conventionalserous carcinomas, 12 mucinous borderline tumors, 10 muci-nous and 10 clear cell carcinomas. No V599E mutation could bedetected in blood samples from these 104 patients. We also foundno BRAF V599E mutation in 101 normal healthy women and 10well-established ovarian cancer cell lines.

These data suggest that BRAF V599E mutation is a very earlysomatic event in tumorigenesis of low-grade serous neoplasmsand that this mutation is not germline-derived.

O-31PROSTATE-SPECIFIC ANTIGEN STIMULATES OSTEOPROTEGERIN PRODUCTION AND INHIBITS RECEPTOR ACTIVATOR OF NUCLEAR FACTOR-κκκκB LIGAND EXPRESSION BY HUMAN OSTEOBLASTS

Hiroyuki Yonou, Yoshihiro NAKAGAMI, Choichiro OZU, Yoshio OHNO, Yutaka HORIGUCHI, Kunihiko YOSHIOKA, Makoto OHORI, Tadashi HATANO, Masaaki TACHIBANADepartment of Urology, Tokyo Medical University

Purpose: Prostate cancer cells produce a large amount ofprostate-specific antigen (PSA), which is widely used as a markerfor this cancer. Even though it is widely used in the diagnosis ofprostate cancer, many aspects of the pathophysiologic role of PSAin bone metastasis remain obscure. The receptor activator ofnuclear factor-κB ligand (RANKL) is essential for the activationof osteoclasts, while osteoprotegerin (OPG) neutralizes the actionof RANKL. Various substances that act on bone have been shownto modulate the production of RANKL and OPG by osteoblasts.Methods: In this study, we investigated the effect of PSA on theexpression of OPG and RANKL mRNA and on proteinproduction in human osteoblast-like cells.Results: After addition of PSA and culture for 72 h, OPGmRNA expression and protein secretion by MG-63 and SaOS-2cells showed a concentration-dependent increase. Whenosteoblasts were incubated with PSA (100 ng/mL), OPG mRNAexpression and protein secretion increased with the passage oftime. α1-antichymotrypsin, which inactivates the serine proteaseactivity of PSA, inhibited the increase of OPG mRNA expressionand protein production in response to PSA, and this effect of PSAwas also inhibited by anti-transforming growth factor-β antibody.Conclusions: Based on our findings, PSA acts on humanosteoblast-like cells via its own serine protease activity andpromotes osteoblast differentiation. In addition, PSA stimulatesOPG production and inhibits RANKL expression of osteoblasts,and inhibits bone resorption by osteoclasts, suggesting that itcontributes to the characteristic osteoblastic features of bonemetastases of prostate cancer.

O-32PACLITAXEL-INDUCED OVARIAN TOXICITY IN RAT

Wataru TARUMI,1,2 Nao SUZUKI,2 Noriyuki TAKAHASHI,2 Youichi KOBAYASHI,2 Kahei SATO,1 Kazushige KIGUCHI,2 Bunpei ISHIZUKA2

1Department of Applied life Science, Graduate School of Bioresources Science, Nihon University; 2Department of Obstetrics and Gynecology, St. Marianna University School of Medicine

In recent years, the prevalence of malignancies such as uterinecancer or breast cancer has shown an upward trend in youngerwomen. Not only may preservation of fertility be impossible, butalso the quality of life can be impaired by premature menopausalsymptoms and other symptoms related to treatment(oophorectomy, anticancer agents, and radiation) in these youngwomen. On the other hand, the result of ovary functionnormalization was found a confirmed fact. Blumenfeld andassociates gave GnRHa therapy to their patients with malignantlymphoma, and reported that 93.7% of the patients showednormalization of ovarian function. Paclitaxel is an anticanceragent frequently used for young women that causes ovariantoxicity, so we investigated protection against such toxicity byGnRHa therapy.

First, we administered GnRHa (3.75 mg/kg) to 8-week-oldfemale Wistar rats subcutaneously and administered paclitaxel(5.0 mg/kg) at the same time. Second, we administered paclitaxel(5.0 mg/kg) for 3 days. After five cycles of treatment, we obtainedserum samples from the GnRHa group and GnRHa + paclitaxelgroup, as well as harvesting the ovaries. We measured E2 and P4levels, the number of 250 µm follicles, the number of climactericfollicles, and the number of corpora lutea. Next, we obtainedserum and ovaries from the control and paclitaxel groups inwhich condition to have been it in D. As was done for the GnRHagroup and the GnRHa + paclitaxel group, we measured the E2level, the P4 levels, the number of 250 µm follicles, the numberof climacteric follicles, and the number of corpora lutea.

In conclusion, we confirmed the occurrence of follicular toxicitydue to paclitaxel for follicles measuring 250 µm. However, ovarianprotection by GnRHa therapy was not confirmed, possibly becauselarge follicles with active cell division are more easily affected byanticancer agents than small or medium follicles. On the otherhand, we could not recognize any protection against toxicity forsmall or medium follicles, so it may be necessary to examinefollicular toxicity and ovarian protection by further studies.

O-33ESTABLISHMENT AND CHARACTERIZATION OF A CELL LINE (OMC-9) ORIGINATING FROM A HUMAN ENDOMETRIAL STROMAL SARCOMA

Takashi YAMADA,1 Yoshiteru KAKUNO,2 Hiroshi MORI1

1Second Department of Pathology and 2Department of Radiology, Osaka Medical College

A new human endometrial stromal sarcoma cell line, designatedOMC-9, was established from the uterine tumor of a 55-year-old



woman. OMC-9 was successively subcultured in 60 months. Themonolayer cultured cells appeared mostly to be spindle-shapedor multipolar, though sometimes polygonal with a tendency topile up without contact inhibition. They had oval nuclei withmultiple nucleoli. Multinucleated giant cells were occasionallyobserved. The chromosomal number shows aneuploidy and themodal chromosomal number is in the diploid range. Thepopulation doubling time was 88 h, the saturation densitywas 1.2 × 105 cells/cm2, the plating efficiency was 15.7% andthe mitotic index was 7.6%. OMC-9 cells were transplantedsubcutaneously to nude mice and produced tumors thatresembled the original tumor. 3 × 106 OMC-9 cells producedTPA 233 U/L during 5 days in culture media. Antitumor effectsof 13 anticancer drugs were compared using the MTT assy. OMC-9 cells were sensitive to ACD, ADM, CBDCA, CDDP, VP-16 in ourstudy. OMC-9 may be useful in investigating endometrial stromalsarcoma of the uterus.

O-34DEVELOPMENT OF MOUSE FETAL GERM CELLS IN VITRO AND IN VIVO: ORGAN CULTURE OF FEMALE GENITAL RIDGES, GRAFT UNDER THE KIDNEY CAPSULE OF SCID MOUSE AND MATURATION IN VITRO OF THE GROWN OOCYTES

Hideyuki 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

Mouse primordial germ cells (PGCs) are first recognizable in theposterior region of the extraembryonic mesoderm at 7.5 dayspost coitum (dpc). They migrate into the genital ridges by 11.5dpc with proliferation. In the female embryo, the germ cellsenter the prophase of the first meiotic division at about 13.5 dpc.The aim of this study is to develop female fetal germ cells in vitroand in vivo. F1 mice (C57BL/6J × CBA/JNCrj) were used in thisstudy. The female mice were mated with the males overnight. Thepregnant females were sacrificed at 12.5 dpc and fetuses wereextracted. The stage of fetal development was defined accordingto the shape of the hind limb bud (McLaren and Buehr, 1990). Thegenital ridges with the adjacent mesonephric region were torninto half fragments and cultured on Millicell-PC membrane inalpha-MEM supplemented with 15% FBS in six-well cluster dish.

The genital ridges cultured for 0, 7, 14 days were extracted atday 28, 21, 14 after grafted under the kidney capsule of SCIDfemale mouse, respectively. The extracted tissues were used forhistology or oocyte maturation in vitro. The oocytes isolated fromthe tissues were cultured for 17 h in alpha-MEM supplementedwith 15% FBS and 10 ng/mL EGF.

The cultured genital ridges developed in the kidney capsule.However, the extension of the culture period prevented thedifferentiation into antral follicle and the full growth of oocyte inthe kidney capsule. Nevertheless, the oocytes had grown to size

of 60–70 µm. These oocytes resumed meiosis, underwent GVBD(37.3%, 22.1%, 10.2%), and progressed to the metaphase II stage(25.3%, 13.0%, 6.8%) in vitro. The matured oocytes were 67.0–71.1 µm in mean diameter.

We have developed a system that allows the successful matu-ration of PGCs using the combination of in vitro and in vivo. Inaddition, this study showed that the long-term culture of the genitalridges affected follicle differentiation after the transplantation.

O-35SUCCESSFUL OVARIAN TISSUE VITRIFICATION IN MAMMAL

N KAGAWA, M KUWAYAMA, Y TAKEHARA, O KATOKato Ladies Clinic, Tokyo, Japan

Recent drastic advances have enabled the cryopreservation ofmammalian gametes. Improved “vitrification” methods, havemade it possible to preserve pre-fertilized oocytes with little lossof viability. Using a highly efficient vitrification method with ultrarapid cooling (Cryotop), we have established very successful humanoocyte banks world wide and have obtained more than 300babies so far to preserve the fertility of cancer patients after BMT.

To preserve fertility in female cancer patients, oocyte vitrifica-tion is one solution. However, it does not work for the childrenof the urgent patients who does not have enough time for one ormore IVF cycles. Ovarian tissue cryopreservation has the poten-tial to solve these problems, and to preserve their natural fertilityafter chemo- and radiotherapy. Similarly to oocyte freezing,ovarian tissue cryopreservation has utilized a conventional slowfreezing method, resulting in limited success. However, encour-aging good results with ovarian tissue freezing are recently beingobtained by using vitrification. Based on the high efficiencyvitrification Cryotop method, we have endeavored to develop apractical vitrification method for mouse, cattle and humanovarian tissues.

In mouse, whole, half and 0.2 mm cubes of BDF1 mouse ovarywere vitrified according to the Cryotop method (Kuwayama2005). Ovarian tissues were first equilibrated in m-199 containing7.5% EG and 7.5% DMSO and vitrified with m-199 containing15% EG, 15% DMSO and 0.5 M sucrose using Cryotop (KitazatoBioPharma, Japan). After thawing samples by plunging in m-199containing 1 M sucrose for 1 min, cryoprotectants were dilutedout with 2 steps using m-199 containing 0.5 and 0 M sucrose.Growing oocytes in preantral follicles of vitrified-thawed ovariantissue were stained by Hoechst and PI to assess their survival.Bovine ovarian tissues were vitrified according to the Cryotopmethod as an animal model for human clinical use because of thesimilar size and structure of the ovary. The size of the ovariantissue was 1 cm × 1 cm according to the successful human ovariantissue transplantation between twins (Silber 2005).

One hundred percent post-thaw survival was obtained for theoocytes of ovarian tissue. Vitrified ovarian tissue was successfullytransplanted into the kidney of SCID mouse with normal growthof follicles. Full grown oocytes were obtained from the allotrans-plant and then matured, fertilized, and cultured in vitro, resultingin normal young after embryo transfer to the recipient. Eighty-eight percent survival was obtained for bovine ovarian tissue



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.

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