Stem cell based regenerative medicine has been a focus of attention worldwide.In particular, recent development of the iPS cell technology has enabled genera-tion of patient’s pluripotent stem cells (PSCs), opening up the way to regenerativemedicine using patient’s own PSC-derived cells. The goal of this laboratory is toprovide new insights into stem cell biology as well as approaches to novel thera-peutic intervention for various intractable diseases.

1. Nonmyelinating Schwann cells maintainhematopoietic stem cell hibernation in thebone marrow niche.

Satoshi Yamazaki1 , Hideo Ema2, GöranKarlsson3, Tomoyuki Yamaguchi1,2, HiroyukiMiyoshi4,8, Seiji Shioda5, Makoto M. Taketo4,Stefan Karlsson3, Atsushi Iwama6,7, HiromitsuNakauchi1,2: 1Japan Science and TechnologyAgency, ERATO, Chiyoda-ku, Tokyo 102-0075,Japan, 2Division of Stem Cell Therapy, Centerfor Stem Cell Biology and Medicine, Instituteof Medical Science, University of Tokyo, Tokyo108-8639, Japan, 3Department of MolecularMedicine and Gene Therapy, Lund Stem CellCenter, Lund University Hospital, BMC A12,221 84 Lund, Sweden, 4Department of Pharma-cology, Graduate School of Medicine, KyotoUniversity, Kyoto 606-8501, Japan, 5Depart-ment of Anatomy, Showa University School ofMedicine, 1-5-8 Hatanodai, Shinagawa-ku, To-kyo 142-8555, Japan, 6CREST, Sanbancho,Chiyoda-ku, Tokyo 102-0075, Japan, 7Depart-ment of Cellular and Molecular Medicine,Graduate School of Medicine, Chiba Univer-sity, Chiba 260-8670, Japan

Hematopoietic stem cells (HSCs) reside andself-renew in the bone marrow (BM) niche.Overall, the signaling that regulates stem celldormancy in the HSC niche remains controver-sial. Here, we demonstrate that TGF-β type IIreceptor-deficient HSCs show low-level Smadactivation and impaired long-term repopulatingactivity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β isproduced as a latent form by a variety of cells,so we searched for those that express activatormolecules for latent TGF-β. NonmyelinatingSchwann cells in BM proved responsible for ac-tivation. These glial cells ensheathed autonomicnerves, expressed HSC niche factor genes, andwere in contact with a substantial proportion ofHSCs. Autonomic nerve denervation reducedthe number of these active TGF-β-producingcells and led to rapid loss of HSCs from BM. Wepropose that glial cells are components of a BMniche and maintain HSC hibernation by regulat-ing activation of latent TGF-β.

2. Generation of germline-competent rat in-duced pluripotent stem cells.

Sanae Hamanaka1,2, Tomoyuki Yamaguchi1,2＊,

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Therapy幹細胞治療分野

Professor Hiromitsu Nakauchi, M.D., Ph.DAssistant Professor Akihide Kamiya, Ph.D.Assistant Professor Shin Kaneko, M.D., Ph.D.

教 授 医学博士 中 内 啓 光助 教 理学博士 紙 谷 聡 英助 教 医学博士 金 子 新

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Toshihiro Kobayashi1,2, Megumi Kato-Itoh2,Satoshi Yamazaki2, Hideyuki Sato2, AyumiUmino2, Yukiko Wakiyama2, Mami Arai2,Makoto Sanbo3, Masumi Hirabayashi3,4, Hiro-mitsu Nakauchi1,2＊: 1Japan Science TechnologyAgency, Exploratory Research for AdvancedTechnology (ERATO), Nakauchi Stem Cell andOrgan Regeneration Project, Tokyo, Japan,2Division of Stem Cell Therapy, Center forStem Cell Biology and Regenerative Medicine,Institute of Medical Science, University of To-kyo, Tokyo, Japan, 3Center for Genetic Analy-sis of Behavior, National Institute for Physi-ological Sciences, Okazaki, Japan, 4School ofLife Science, The Graduate University for Ad-vanced Studies, Okazaki, Japan

BACKGROUND: Recent progress in rat pluri-potent stem cell technology has been remark-able. Particularly salient is the demonstrationthat embryonic stem cells (ESCs) in the rat(rESCs) can contribute to germline transmission,permitting generation of gene-modified rats asis now done using mouse ESCs (mESCs) ormouse induced pluripotent stem cells (iPSCs;miPSCs). However, determinations of whetherrat iPSCs (riPSCs) can contribute to germ cellsare not published. Here we report the germlinecompetency of riPSCs.

METHODOLOGY/PRINCIPAL FINDINGS:We generated riPSCs by transducing threemouse reprogramming factors (Oct3/4, Klf4,and Sox2) into rat somatic cells, followed by cul-ture in the presence of exogenous rat leukemiainhibitory factor (rLIF) and small molecules thatspecifically inhibit GSK3, MEK, and FGF recep-tor tyrosine kinases. We found that, like rESCs,our riPSCs can contribute to germline transmis-sion. Furthermore we found, by immunostainingof testis from mouse-rat interspecific chimeraswith antibody against mouse vasa homolog, thatriPSCs can contribute to embryonic develop-ment with chimera formation in mice (rat-mouseinterspecific chimeras) and to interspecific germ-lines.

CONCLUSIONS/SIGNIFICANCE: Our dataclearly demonstrate that using only three repro-gramming factors (Oct3/4, Klf4, and Sox2) ratsomatic cells can be reprogrammed into aground state. Our generated riPSCs exhibitedgermline transmission in either rat-rat intras-pecific or mouse-rat interspecific chimeras.

3. Prospective isolation and characterizationof bi-potent progenitor cells in earlymouse liver development.

Ken Okada, Akihide Kamiya, Keiichi Ito, Aya-ka Yanagida, Hidenori Ito, Hiroki Kondou,

Hiroshi Nishina and Hiromitsu Nakauchi

Outgrowth of the foregut endoderm to formthe liver bud is considered the initial event ofliver development. Hepatic stem/progenitorcells (HSPCs) in the liver bud are postulated tomigrate into septum transversum mesenchymeat around embryonic day (E) 9 in mice. Thestudies of liver development focused on themid-fetal stage (E11.5-14.5) have identifiedHSPCs at this stage. However, the in vitro char-acteristics of HSPCs before E11.5 have not beenelucidated. This is probably partly because puri-fication and characterization of HSPCs in early-fetal livers have not been fully established. Topermit detailed phenotypic analyses of early-fetal HSPC candidates, we developed a new co-culture system, using mouse embryonic fibro-blast cells. In this co-culture system, CD13＋Dlk＋

cells purified from mouse early-fetal livers (E9.5and E10.5) formed colonies composed of bothalbumin-positive hepatocytic cells and cytokera-tin (CK) 19-positive cholangiocytic cells, indicat-ing that early-fetal CD13＋Dlk＋ cells have prop-erties of bi-potent progenitor cells. Inhibition ofsignaling by Rho-associated coiled-coil contain-ing protein kinase (Rock) or by non-musclemyosin II (downstream from Rock) was neces-sary for effective expansion of early-fetalCD13＋Dlk＋ cells in vitro. In sorted CD13＋Dlk＋

cells, expression of the hepatocyte marker genesalbumin and α-fetoprotein increased with fetalliver age, while expression of CK19 and Sox17,endodermal progenitor cell markers, was high-est at E9.5 but decreased dramatically thereafter.These first prospective studies of early-fetalHSPC candidates demonstrate that bi-potentstem/progenitor cells exist before E11.5 and im-plicate Rock-myosin II signaling in their devel-opment.

4. In vitro expansion and functional recoveryof mature hepatocytes from mouse adultliver.

Hidenori Ito, Akihide Kamiya, Keiichi Ito,Ayaka Yanagida, Ken Okada, and HiromitsuNakauchi

Mature hepatocytes retain the ability to regen-erate the liver lobule fully in vivo following in-jury. Several cytokines and soluble factors (he-patocyte growth factors, epidermal growth fac-tors, insulin, and nicotinamide) are known to beimportant for proliferation of mature hepato-cytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradu-ally lost their specific functions, particularlythose in drug metabolism. We have explored

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and established a new culture system for expan-sion of functional hepatocytes. We evaluatedtwo approaches to efficient expansion of maturehepatocytes: 1. Co-culture with mouse embry-onic fibroblasts (MEF); 2. Addition to culture ofinhibitors of cell signals involved in liver regen-eration. After expansion steps, 3-dimensionalspheroid-forming culture was used to re-inducemature hepatocellular function. The addition ofinhibitors for tumor growth factor (TGF) β andglycogen synthase kinase (GSK) 3β efficiently in-duced in vitro expansion of mature hepatocytes.Although expression of hepatocellular functionalgenes decreased after expansion in monolayerculture, their expression and the activity of cyto-chrome P450 enzymes significantly increasedwith spheroid formation. Furthermore, when he-patocytes were co-cultured with MEF, additionof a MAPK/ERK kinase (MEK) inhibitor at thespheroid formation step enhanced drug-metabolism-related gene expression. Combina-tion of the MEF co-culture system with the ad-dition of inhibitors of TGFβ and GSK3β inducedin vitro expansion of hepatocytes. Moreover, ex-pression of mature hepatocellular genes and theactivity of drug-metabolism enzymes in ex-panded hepatocytes were re-induced after sphe-roid culture.

5. Generation of antigen specific functional Tcells from human induced pluripotentstem cells of a single T cell origin.

Toshinobu Nishimura1, Shin Kaneko1, AiKawana-Tachikawa2, and Hiromitsu Naka-uchi1: 1Division of Stem Cell Therapy, Centerfor Stem Cell Biology and Regenerative Medi-cine, 2Division of Infectious Disease, The Ad-vanced Clinical Research Center, Institute ofMedical Science, The University of Tokyo, To-kyo, Japan

Adoptive immunotherapy with antigen-specific T cells expressing a limited TCRs reper-toire is a promising approach to fight againstvarious types of cancers or chronic viral infec-tions. However, its effectiveness declines due tothe exhaustion of antigen-specific T cells. Toovercome this problem, we have explored thepotential of induced pluripotent stem (iPS) celltechnology and generated T cell-derived iPS (T-iPS) cells. Furthermore, we have succeeded inredifferentiation of T-iPS cells into functional Tcells specific to the original antigen. As was ex-pected, invariance of the antigen-specificity wasevidenced by the fact that TCR gene rearrange-ment patterns in the re-differentiated T cellswere identical to those found in their original Tcell. Taken together, regeneration of antigen-

specific immune systems using iPS cell technol-ogy will shed new light on the field of adoptiveimmunotherapy.

6. A pluripotent stem cell specific cell-suicide system for prophylaxis and treat-ment of teratocarcinoma in autologousiPSC-based cell and gene therapy

Yumiko Okimoto, Shin Kaneko, Yutaka Yasui,Satoshi Akune, and Hiromitsu Nakauchi: Divi-sion of Stem Cell Therapy, Center for StemCell Biology and Regenerative Medicine, Insti-tute of Medical Science, The University of To-kyo, Tokyo, Japan

Induced pluripotent stem cells (iPSCs) havebeen expected for their potential use as cellsources of regenerative medicine. iPSC-basedtherapy, however, would have a latent risk ofteratomagenesis by contamination of undifferen-tiated iPSCs in the graft. Aiming at realizingsafer iPSC-based therapies by reducing the risk,we’ve been trying to develop an inducible “cell-suicide” system which enables undifferentiatedcells to be eliminated in vitro and in vivo.

We have developed a lentiviral vector NpTkto express herpes simplex virus thymidinekinase (HSV-TK) gene under the control ofNanog promoter. The vector was designed towork with prodrug ganciclovir (GCV) to inducecell-suicide only in undifferentiated iPSCs.C57BL/6 mouse tail-tip fibroblast derivedmurine iPSCs were transduced by the vectorand tested for GCV sensitivity. Under a leuke-mia inhibitory factor (LIF) containing culturecondition, NpTk/iPSCs showed clear cell deathwithin 48 hours with GCV. On the contrary, dif-ferentiating NpTk/iPSCs in embryoid body (EB)showed GCV resistance as they decrease Nanogexpression. In addition, when NpTk/iPSCs havedifferentiated into pre-hematopoietic stem cellsin the presence of GCV, functional hematopoie-tic cells were successfully obtained without con-tamination of significant number of undifferenti-ated cells. These data suggested that iPSC-differentiation with the cell-suicide systemworks well for reduceing the risk of undifferen-tiated cell contamination during cell processing.

Next, we inoculated 1×106 NpTk/iPSCs to B6mice with or without GCV treatment to observewhether the undifferentiated cell specific suicidesystem can prevent teratomagenesis in vivo. Thegroup starting 2-week GCV treatment simulta-neously with iPSC-innoculation showed effectiveprevention of teratomagenesis and the groupstarting 4-week GCV after teratoma formationshowed effective elimination of pathologicallyimmature cells in teratoma.

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In conclusion, the results indicate the feasibil-ity of “pluripotent cell specific promoter-drivencell-suicide system” in vitro and in vivo, as asafety switch for prevention and treatment ofteratocarcinoma in iPSC-based therapies.

7. The entities and molecular pathogenesisof Primary Myelofibrosis

Takafumi Shimizu, Shin Kaneko, Shoichi Iri-guchi, Keiichi Ito, Satoshi Yamazaki, and Hiro-mitsu Nakauchi: Division of Stem Cell Ther-apy, Center for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science,The University of Tokyo, Tokyo, Japan

The gain-of-function JAK 2 mutation,JAK2V617F, is the most common molecular ab-normality in Myeloproliferative neoplasms(MPNs) and appears in most of the patientswith polycythemia vera (PV) and in half of thepatients with essential thrombocythemia (ET)and primary myelofibrosis (PMF). A number ofJAK2V617F mediated mouse models with fea-tures of PV or ET have been reported, however,typical PMF model with the JAK2V617F had notbeen reported. We have succeeded to generatePMF mouse model by transplantation of hema-topoietic stem cells (HSCs) with STAT5a(1*6),the constitutive active STAT5a, a direct down-stream molecule of JAK2. Clinical conditions ofthe model were closely resemble to that of thehuman and fulfilled the WHO criteria.

Through the detail analysis of the model, werevealed that intensive STAT5a activity inducingc-Myc expression leads to increment of atypicalMegakaryocytes (at-Mks). The at-Mks producingTGF-β, Oncostatin M and VEGF evoke mesen-chymal stem cells (MSCs) abnormalities, whichleads to aberrant fibrogenesis and angiogenesisin bone marrow (BM). These microenvironmentfailure, especially loss of nestin＋ MSCs, re-sulted in reduction of HSC niches and followedHSC-exhaustion even to genetically unmodifiedHSC.

Taken together, the specific entity of PMF isHSCs exhaustion associated to MSCs abnormali-ties.

8. Elucidation of the mechanism of the devel-opment of pulmonary alvelor proteinosisaccompanied with aberrant productions ofTh1 cytokines.

Shoichi Iriguchi1, Shin Kaneko1, Yukio Ishii2,and Hiromitsu Nakauchi1: 1Division of StemCell Therapy, Center for Stem Cell Biologyand Regenerative Medicine, Institute of Medi-cal Science, The University of Tokyo, Tokyo,Japan, 2Department of Respiratory Medicine,Institute of Clinical Medicine, University ofTsukuba, Tsukuba, Japan

Mononuclear phagocyte system plays an im-portant role in maintaining homeostasis and,under the inflammations and infections, triggersimmune response to combat against invadingpathogens. To date, it has been shown that al-tered regulations of the mononuclear phagocytesystem involved in the pathogenesis of certaindiseases. Among them, the pathogenesis ofnearly 90％ of congenital and acquired pulmo-nary alveolar proteinosis (PAP) patients hasbeen identified to the disrupted granulocyte/macrophage-colony stimulating factor (GM-CSF)signaling in alveolar macrophages. However, lit-tle is known about the pathogensis of secondaryPAP. In this study, we found that CD2-T-bettg/tg

mice developed PAP independent of the disrup-tion of GM-CSF signaling under the influence ofsevere infiltration of T lymphocytes producingaberrant amount of type 1 helper T lymphocyte(Th1) cytokines in lung. Further study on thehematopoietic system of the mice together withthe results of splenocyte transplantation experi-ment indicated that the over-expression of T-betin T lymphocytes induces inflammatory condi-tion and increases influx of inflammatory mono-cytes from bone marrow into the peripheral tis-sues of T-bettg/tg mice. Moreover, in vitro BM col-ony assay of T-bettg/tg raised the possibility thatthere could have been defects in monocyte/macrophage lineage commitment and the defectwas partially corrected with the addition of M-CSF. Combined together, these results suggestthat the imbalance in the activations of IFN-γ, M-CSF, and GM-CSF signalings in lung triggeredby the Th1/Th2 cytokine production imbalanceresults in the development of pulmonary alveo-lar proteinosis. Our findings propose a newpathway in the pathogenesis of pulmonary al-veolar proteinosis.

Publications

1. Ito H, Kamiya A, Ito K, Yanagida A, OkadaK, Nakauchi H. In vitro expansion and func-

tional recovery of mature hepatocytes frommouse adult liver. Liver Int. 2012 Jan 4. doi:

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10.1111/j.1478-3231.2011.02741. x. [Epubahead of print]

2. Umeyama K, Saito H, Kurome M, MatsunariH, Watanabe M, Nakauchi H, Nagashima H.Characterization of the ICSI-mediated genetransfer method in the production of trans-genic pigs. Mol Reprod Dev . 2011 Dec 15.doi:10.1002/mrd.22015. [Epub ahead ofprint]

3. Yamazaki S, Ema H, Karlsson G, YamaguchiT, Miyoshi H, Shioda S, Taketo MM,Karlsson S, Iwama A, Nakauchi H. Non-myelinating schwann cells maintain hema-topoietic stem cell hibernation in the bonemarrow niche. Cell . 2011 Nov 23; 147(5):1146-58.

4. Suzuki N, Yamazaki S, Ema H, YamaguchiT, Nakauchi H, Takaki S. Homeostasis of he-matopoietic stem cells regulated by themyeloproliferative disease associated-geneproduct Lnk/Sh2b3 via Bcl-xL. Exp Hematol .2011 Nov 17. [Epub ahead of print]

5. Umemoto T, Yamato M, Ishihara J, Shira-tsuchi Y, Utsumi M, Morita Y, Tsukui H,Terasawa M, Shibata T, Nishida K, Ko-bayashi Y, Petrich BG, Nakauchi H, Eto K,Okano T . Integrin αvβ 3 regulatesthrombopoietin-mediated maintenance ofhematopoietic stem cells. Blood . 2011 Nov16. [Epub ahead of print]

6. Nishimura S, Manabe I, Nagasaki M, KakutaS, Iwakura Y, Takayama N, Ooehara J, OtsuM, Kamiya A, Petrich B, Urano T, KadonoT, Sato S, Aiba A, Yamashita H, Sugiura S,Kadowaki T, Nakauchi H, Eto K, Nagai R.In vivo imaging visualizes discoid plateletaggregations without endothelium disrup-tion and implicates contribution of inflam-matory cytokine and integrin signaling.Blood . 2011 Nov 16. [Epub ahead of print]

7. Okaji Y, Tashiro Y, Gritli I, Nishida C, SatoA, Ueno Y, Del Canto Gonzalez S, Ohki-Koizumi M, Akiyama H, Nakauchi H,Hattori K, Heissig B. Plasminogen deficiencyattenuates postnatal erythropoiesis in maleC57BL/6 mice through decreased activity ofthe LH-testosterone axis. Exp Hematol . 2011Nov 3. [Epub ahead of print]

8. Watanabe M, Kurome M, Matsunari H,Nakano K, Umeyema K, Shiota A, NakauchiH, Nagashima H. The creation of transgenicpigs expressing human proteins using BAC-derived, full-length genes and intracytoplas-mic sperm injection-mediated gene transfer.Transgenic Res . 2011 Oct 25. [Epub ahead ofprint]

9. Watabe Y, Baba Y, Nakauchi H, Mizota A,Watanabe S. The role of Zic family zinc fin-ger transcription factors in the proliferation

and differentiation of retinal progenitorcells. Biochem Biophys Res Commun . 2011 Nov11; 415(1): 42-7. Epub 2011 Oct 15.

10. Nishino T, Wang C, Mochizuki-Kashio M,Osawa M, Nakauchi H, Iwama A. Ex vivoexpansion of human hematopoietic stemcells by garcinol, a potent inhibitor of his-tone acetyltransferase. PLoS One . 2011; 6(9):e24298. Epub 2011 Sep 12.

11. Ishihara M, Nishida C, Tashiro Y, Gritli I,Rosenkvist J, Koizumi M, Okaji Y, Yama-moto R, Yagita H, Okumura K, Nishikori M,Wanaka K, Tsuda Y, Okada Y, Nakauchi H,Heissig B, Hattori K. Plasmin inhibitor re-duces T-cell lymphoid tumor growth bysuppressing matrix metalloproteinase-9-dependent CD11b(＋)/F4/80(＋) myeloidcell recruitment. Leukemia. 2011 Sep 20.[Epub ahead of print]

12. Saito R, Nakauchi H, Watanabe S. Serine/threonine kinase, Melk, regulates prolifera-tion and glial differentiation of retinal pro-genitor cells. Cancer Sci . 2011 Sep 16. [Epubahead of print]

13. Yoshida K, Sanada M, Shiraishi Y, NowakD, Nagata Y, Yamamoto R, Sato Y, Sato-Otsubo A, Kon A, Nagasaki M, Chalkidis G,Suzuki Y, Shiosaka M, Kawahata R, Yama-guchi T, Otsu M, Obara N, Sakata-Yanagi-moto M, Ishiyama K, Mori H, Nolte F, Hof-mann WK, Miyawaki S, Sugano S, HaferlachC, Koeffler HP, Shih LY, Haferlach T, ChibaS, Nakauchi H, Miyano S, Ogawa S. Fre-quent pathway mutations of splicing ma-chinery in myelodysplasia. Nature . 2011 Sep11; 478(7367): 64-9

14. Okada K, Kamiya A, Ito K, Yanagida A, ItoH, Kondou H, Nishina H, Nakauchi H. Pro-spective Isolation and Characterization of Bi-potent Progenitor Cells in Early Mouse LiverDevelopment. Stem Cells Dev . 2011 Oct 18.[Epub ahead of print]

15. Hamanaka S, Yamaguchi T, Kobayashi T,Kato-Itoh M, Yamazaki S, Sato H, Umino A,Wakiyama Y, Arai M, Sanbo M, HirabayashiM, Nakauchi H. Generation of germline-competent rat induced pluripotent stemcells. PLoS One . 2011; 6(7): e22008. Epub2011 Jul 15.

16. Kawahara M, Chen J, Sogo T, Teng J, OtsuM, Onodera M, Nakauchi H, Ueda H, Naga-mune T. Growth promotion of geneticallymodified hematopoietic progenitors usingan antibody/c-Mpl chimera. Cytokine. 2011Sep; 55(3): 402-8. Epub 2011 Jun 22.

17. Yokoi T, Kobayashi H, Shimada Y, Eto Y,Ishige N, Kitagawa T, Otsu M, Nakauchi H,Ida H, Ohashi T. Minimum requirement ofdonor cells to reduce the glycolipid storage

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following bone marrow transplantation in amurine model of Fabry disease. J Gene Med .2011 May; 13(5): 262-8

18. Onozuka I, Kakinuma S, Kamiya A, MiyoshiM, Sakamoto N, Kiyohashi K, Watanabe T,Funaoka Y, Ueyama M, Nakagawa M,Koshikawa N, Seiki M, Nakauchi H, Wata-nabe M. Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrixmetalloproteinase-2 deficient mice. Biochem

Biophys Res Commun . 2011 Feb 12. [Epubahead of print]

19. Tanimura S, Tadokoro Y, Inomata K, BinhNT, Nishie W, Yamazaki S, Nakauchi H, Ta-naka Y, McMillan JR, Sawamura D, YanceyK, Shimizu H, Nishimura EK. Hair folliclestem cells provide a functional niche formelanocyte stem cells. Cell Stem Cell . 2011Feb 4; 8(2): 177-87.

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The Laboratory of Diagnostic Medicine was established in January 2009 as a divi-sion of the Center for Stem Cell Biology and Regenerative Medicine. Our majorpurpose is to conduct clinical research and develop analytical methods of patho-genic conditions during infectious disease, cancer and hematopoietic stem cell andorgan transplantations. Through collaborations with hospitals in Japan, we haveperformed several problem-based clinical studies to tackle the issues of adult Tcell leukemia (ATL) and pathogenic conditions after transplantation, e.g. cytomega-lovirus infection, graft failure, acute graft versus host disease (GVHD), relapse ofleukemia, and recurrence of hepatitis. In addition, we also started a new project tomake allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies(ASHmAbs) using HLA-transgenic mouse and HLA-tetramers for the purpose of aflow cytometry-based method of chimerism analysis (HLA-Flow method).

1. Clinical studies through collaborationswith hospitals

a) Phenotypic analysis of ATL cells and pre-diction of the onset of ATL from human T-lymphotropic virus type 1 (HTLV-1) asymp-tomatic carriers

Tomohiro Ishigaki , Seiichiro Kobayashi1 ,Nobuhiro Ohno2, Yuji Zaike3, Eri Watanabe,Kaoru Uchimaru2 and Nobukazu Watanabe:1Department of Molecular Therapy, 2ResearchHospital, 3Department of Laboratory Medicine,IMSUT

Among the one million HTLV-1 carriers in Ja-pan, approximately one thousand progress toATL every year. Through collaborations withthe Research Hospital and two laboratories atIMSUT, we are analyzing ATL cells using a flowcytometry-based method of phenotypic analysis[HTLV-1 associated system (HAS)-Flow method]to monitor disease condition. In addition, we areanalyzing peripheral blood from HTLV-1 carri-

ers to find a predictable phenotypic change ofimmune cells just before ATL onset in order tobegin more effective treatment.

b) Analysis of engraftment, ATL cells, and Tcell activation after cord blood transplanta-tion in patients with ATL.

Eri Watanabe, Reiko Ishida, Ilseung Choi4,Yoko Suehiro4, Seiichiro Kobayashi, KaoruUchimaru, Naokuni Uike4 and NobukazuWatanabe: 4Department of Hematology, Na-tional Kyushu Cancer Center

In a Japanese study group of cell therapy forATL, cord blood transplantation is planned forpatients with acute ATL. We are joining thisstudy group and analyzing engraftment andATL cells using HLA-Flow method and HAS-Flow method. In addition, we are analyzing Tcell activation using intracellular cytokine stain-ing for the purpose of regulation of immuno-suppressants, e.g. cyclosporine A.

Center for Stem Cell Biology and Regenerative Medicine

Laboratory of Diagnostic Medicine病態解析領域

Project Associate Professor Nobukazu Watanabe, M.D., Ph.D. 特任准教授 医学博士 渡 辺 信 和

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c) Studies for the mechanisms underlyingpersistent chimerism and late rejection af-ter cord blood transplantation in patientswith severe combined immunodeficiencies(SCID).

Eri Watanabe, Nobukazu Watanabe, TomohiroMorio5: 5Department of Pediatrics, TokyoMedical Dental University

Although T cells and NK cells are lacked inpatients with SCID, persistent chimerism andlate rejection sometimes occur after cord bloodtransplantation. We analyze subpopulation-specific chimerism using HLA-Flow method andinvestigate the underlying mechanisms of thesepathogenic conditions.

d) Studies for the mechanisms underlying re-currence of type C hepatitis and rejectionafter living-donor liver transplantation

Nobukazu Watanabe , Akinobu Takaki6 ,Kazuko Koike6, Takahito Yagi7: 6Departmentof Gastroenterology and Hepatology, 7Depart-ment of Gastroenterological Surgery, Trans-plant and Surgical Oncology, Okayama Uni-versity Graduate School of Medicine and Den-tistry

Since the 2004 approval of insurance coveragefor living-donor liver transplantations (LDLT),more than 6,000 LDLTs have been performed inJapan. Although most recipients have a goodprognosis, patients with hepatitis C virus (HCV)infection still face the recurrence of hepatitis af-

ter transplantation. In addition, rejection is animportant issue because immunosuppressiveagents are needed to suppress anti-graft im-mune reactions. Long-term use of immunosup-pressants, however, can worsen HCV infectionsand future malignancies. To understand themechanism underlying these pathologic condi-tions, we are investigating the following: chi-merism analysis/HLA-Flow method, detectionof regulatory T cells and allospecific T cells, andidentification of HCV-specific CD8＋ T cells us-ing tetramers.

2. Generation of allele-specific anti-HLA mon-oclonal antibodies (ASHmAbs)

Yusuke Nakauchi, Stephanie C. Napier, SatoshiYamazaki8, Nobukazu Watanabe, and Hiro-mitsu Nakauchi8: 8JST-ERATO, IMSUT.

The difficulty in generating ASHmAbs is wellknown. We recently established a novel methodfor generating ASHmAb. Our strategy involvessuppressing the production of non-allele-specificanti-HLA antibodies against xenogeneic deter-minants of HLA molecules by immunizing hu-man HLA-A2, A24, and B51 transgenic miceagainst non-HLA-A2, A24, and B51 HLA te-tramers. ASHmAbs generated in this mannerwill be useful for HLA typing and for clinicaldiagnoses, such as flow cytometry-based chi-merism analysis for early detection of graft fail-ure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplanta-tion.

Publications

Suguro, H., Mikami, Y., Koshi, R., Ogiso, B.,Watanabe, E., Watanabe, N., Honda, M.J.,Komiyama, K. and Asano, M. Novel approachfor transient protein expression in primarycultures of human dental pulp-derived cells.Protein Expr. Purif. 78(2): 143-148, 2011.

Tian, Y., Kobayashi, S., Ohno, N., Isobe, M.,Tsuda, M., Zaike, Y., Watanabe, N., Tani, K.,Tojo, A. and Uchimaru, K. Leukemic T cellsare specifically enriched in a unique CD3(dim) CD7(low) subpopulation of CD4(＋) Tcells in acute-type adult T-cell leukemia. Can-cer Sci. 102(3): 569-577, 2011.

Mikami, Y., Senoo, M., Lee, M., Yamada, K.,

Ochiai, K., Honda, M.J., Watanabe, E., Watan-abe, N. and Somei, M. Takagi, M. InhibitoryEffects of a Tryptamine Derivative on Ultra-violet Radiation-Induced Apoptosis in MC3T3-E1 Mouse Osteoblasts. J. Pharmacol. Sci. 115(2): 214-220, 2011.

Yoshikazu, M., Ishii, I., Watanabe, N., Shirak-awa, T., Suzuki, S., Irie, S., Isokawa, K. andHonda, M.J. CD271/p75NTR inhibites the dif-ferentiation of mesenchymal stem cells intoosteogenic, adipogenic, chondrogenic, andmyogenic lineages. Stem Cells and Develop-ment, 20(5): 901-913, 2011.

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Stem cells represent a precious cell source usable in various types of regenera-tive medicine. Hematopoietic stem cells provide a good example of the potential ofstem cells as seen in successful cases in both the hematopoietic transplantationand gene therapy. Pluripotent stem cells are the emerging cell sources that maybe utilized either for the basic research or to cure the diseases. Our aim is to es-tablish safe and efficacious treatment for the patients suffering from diseases withno curative treatment available.

1. In vitro modeling of neutrophil develop-ment and function using iPSCs: clinicalvalues in understanding the pathophysiol-ogy and applications in the treatment ofchronic granulomatous disease

Lin Huan-Ting, Makoto Otsu, Naoya Takayama,Koji Eto, Hiromitsu Nakauchi

Neutrophil (NEU) differentiation is a dynamicprocess that can be recaptured in vitro using in-duced pluripotent stem cells (iPSCs). This proc-ess can be manipulated by cytokines, allowingfor the study of these cells at specific stages intheir development. Most importantly from aclinical perspective, by generating patient au-tologous iPSCs, it is possible to recapitulate aspecific disease phenotype. Chronic granuloma-tous disease (CGD) is a congenital NEU disor-der characterized by the impaired generation ofreactive oxygen species (ROS). The transplanta-tion of gene modified CD34＋ cells is the possiblecure. In this study, we aim to highlight the clini-cal value of iPSCs as a disease model in eluci-dating the underlying pathophysiology and inassessing the effective recovery of cell functionsfollowing gene modification.

Peripheral blood (PB) CD34＋ cells were iso-

lated from two CGD patients with gp91phox orp47phox deficiency and from a healthy donor.Cells were reprogrammed using a Sendai virusvector expressing Oct4/Sox2/Klf4/c-Myc. Self-inactivating lentiviral or alpharetroviral vectorswere used to insert either gp91 or p47 cDNAinto iPSCs. Neutrophil differentiation was in-duced using VEGF and G-CSF. WG, MPO andALP staining was done to assess cell morphol-ogy. The immunophenotype profile of differenti-ating cells was assessed using macrophage andneutrophil specific antigens. Neutrophils werestimulated with PMA to analyze ROS produc-tion by the DHR flow cytometry assay. Neutro-phil extracellular traps (NETs) were visualizedby staining with SYTO 13 and anti-MPO anti-body. Neutrophils from healthy donors servedas the control.

Mature PB-NEUs and control iPSC-NEUs dis-played the classic multi-lobed appearance of thenucleus. ROS was generated at comparable lev-els and both populations were able to formNETs. However, CGD iPSC-derived NEUs (gp91phox and p47) appeared to show impaired de-velopment. This was suggested by the fact thatat day 7 of the differentiation culture, only aproportion of the population stained positive forALP, which is a protein found in secretory ves-

Center for Stem Cell Biology and Regenerative Medicine

Stem Cell Bankステムセルバンク

Associate Professor Makoto Otsu, M.D., Ph.D.Associate Professor Koji Eto, M.D., Ph.D.

特任准教授 医学博士 大 津 真特任准教授 医学博士 江 藤 浩 之

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icles. At the same time point, nearly all controliPSC-NEUs stained positive for ALP. CGD iPSC-derived NEUs were ROS negative at all timepoints and displayed impaired formation ofNETs. The loss of these functions however, wasrecovered in NEUs differentiated from genemodified CGD iPSCs.

This is the first report to show that in vitrodifferentiated NEUs have the capacity to formNETs. Only mature NEUs with a complete rep-ertoire of cellular components and normal ROSgenerating capacity have this property. Alongwith other matching characteristics to PB NEUs,these results may be taken as validation of theaccuracy with which this model can be utilisedto mimic NEU physiology and development invivo. Indeed, in this instance it has been utilizedto uncover a previously unreported impairmentin NEU development in CGD. Further work isrequired to determine its implications. Further-more, we have shown that vector mediated genetransfer can recover the characteristic loss offunction associated with CGD NEUs. It is possi-ble that this disease model may also be used tostudy insertion site profiling for example. Thisallows therapeutic vectors to be evaluated forclinical safety thus minimizing the potential riskof genotoxicity and possible harm to patients.

2. Evidence for the involvement of CXCR4signaling in in vivo self-renewal of trans-planted hematopoietic stem cells

Chen-Yi Lai, Sachie Suzuki, Motohito Okabe,Satoshi Yamazaki, Makoto Otsu, HiromitsuNakauchi

Hematopoietic stem cells (HSCs) represent theunique cell population capable of self-renewaland multi-lineage differentiation, thereby life-long sustainment of the hematopoiesis. HSCtransplantation has proven beneficial for variousdiseases, it is therefore important to elucidatethe molecular determinants for successful HSCengraftment. Signaling through the chemokinereceptor CXCR4 has been implicated in HSC en-graftment by the observation that transplanta-tion of HSCs lacking this molecule results inpoor hematopoietic reconstitution. Because thisimpairment, however, can be attributed to thedefects in any of the post-transplantation proc-esses that include bone marrow (BM)-homing,-repopulation, or -retention, it is still unclearwhether CXCR4 plays an essential role in HSCself-renewal upon transplantation.

To elucidate the role of CXCR4 signaling inHSC self-renewal in conjunction with transplan-tation, we used a purified CD34neg/low c-Kit＋ Sca-1＋ Lineage-markerneg population as the defined

stem cell source. As a loss-of-function study,CXCR4 was conditionally deleted in HSCs be-fore transplantation. As a gain-of-function study,we generated the HSC populations overexpress-ing either wild-type (wt)- or C-terminal trun-cated (∆C)-CXCR4, the latter of which is knownto exhibit enhancement in the SDF-1 signaling,by gene transfer and subsequent cell sorting. Wecompared these cells with control HSCs in in vi-tro assays with regard to the biological charac-teristics including chemotaxis, proliferation, col-ony formation, and cobblestone-area (CA) form-ing ability . To dissect in vivo post-transplantation processes, we investigated hema-topoietic repopulation kinetics in the recipientBM at the homing/lodging phase (within 1 wk)and the early repopulation phase (2-3 wks) forthe above test HSCs. The self-renewal potentialof each HSC population was estimated by com-petitive repopulation assay.

In vitro studies: HSCs overexpressing wt- or∆C-CXCR4 exhibited enhanced chemotaxis andproliferation in response to SDF1, confirmingthe gain-of-function effects of these modifica-tions. CA forming ability was greater in HSCsoverexpressing ∆C-CXCR4 than control counter-parts and absent in CXCR4-KO HSCs, suggest-ing the critical role of CXCR4-signaling in HSCproliferation in the presence of stromal support.

In vivo studies: 1) the homing/lodging phase.Unexpectedly, we did not find significant altera-tion in the numbers of early progenies detect-able in recipient BM 3 days after transplantationof HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating thatthis signaling is indispensable in HSC homing.2) the early repopulation phase. Impairment ofhematopoietic repopulation in BM became evi-dent for CXCR4-KO HSCs through 2-3 wks. Onthe other hand, HSCs overexpressing CXCR4,more remarkably of ∆C-mutation, showed en-hanced BM repopulation kinetics at ～3 wkspost transplantation, suggesting the importanceof CXCR4 signaling in HSC amplification at thispost-transplantation phase. 3) long-term hema-topoiesis. CXCR4-KO-HSCs showed poor hema-topoietic reconstitution potentials, consistentwith previous observations. Interestingly, im-paired peripheral repopulation was also ob-served with HSCs overexpressing wt- or ∆C-CXCR4. Further characterization revealed thatthe recipients of CXCR4-overexpressing HSCsdid retain their progenies, which showed mul-tilineage differentiation, but exhibited impairedrelease of mature leukocytes from the BM to theperipheral blood. Most importantly, however,test-cell chimerism in the long-term HSC frac-tion was significantly higher in the mice receiv-ing HSCs overexpressing CXCR4, especially of

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∆C-type, than those transplanted with controlHSCs, indicating that the augmentation ofCXCR4 signaling enhanced competitive repopu-lation ability of HSCs. These modified HSCsdemonstrated repopulation abilities also in sec-ondary recipients.

We demonstrated that CXCR4 signaling is in-dispensible for HSC homing and that continu-ous overexpression of CXCR4 cannot benefit theperipheral reconstitution in contrary to the ex-pectation. More importantly, our studiesshowed that augmentation of CXCR4 signalingleads to HSC expansion in vivo. We thus con-clude that CXCR4 signaling has a role in HSCself-renewal and that its regulation may find theapproach that will improve HSC transplantationoutcomes.

3. Platelet production system using an im-mortalized megakaryocyte cell line derivedfrom human pluripotent stem cells

Sou Nakamura, Naoya Takayama, HiromitsuNakauchi, Koji Eto

Human induced pluripotent stem cells(hiPSCs) are a promising source of blood cells,including platelets, for transfusion. However,there remains a need for: 1) a method to obtainlarge numbers of cells, 2) a system to providecells of a predefined quality, and 3) a method toovercome the storage limitations of plateletscaused by their short shelf life.

To address these issues, we attempted to es-tablish an immortalized megakaryocyte progeni-tor cell line derived from hiPSCs. We recentlyshowed that the temporal profile of c-MYC acti-vation during megakaryopoiesis is critical fornormal platelet production from hiPSCs; that is,peak activation of c-MYC in megakaryocyte pro-genitors must be followed by a reduction of c-MYC expression for further maturation(Takayama et al. J Exp Med, 2010). Mechanisticanalysis revealed that overexpression (O/E) ofc-MYC increased megakaryocyte numbers butalso induced apoptosis and senescence. Here wedemonstrate that this phenomenon is primarilyregulated by induction of the INK4A and ARF

genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/Eand BCL-XL (negative regulator of caspase fam-ily) O/E, and c) c-MYC O/E and BMI1 (nega-tive regulator for both INK4A and ARF genes)O/E in hematopoietic progenitors derived fromhuman embryonic stem cells (hESCs), we foundthat only c-MYC and BMI1 O/E (protocol b) in-creased numbers of CD41a＋/CD42b＋ non-polyploid megakaryocytes in an exponentialmanner for over 3 months. Neither c-MYC O/Eand p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficientto maintain an increase in the megakaryocytepopulation , which suggests that down-regulation of INK4A and ARF contributesmainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thusappears that we were able to establish an im-mortalized megakaryocyte cell line (MKCL).

As mentioned, a decline in c-MYC activationduring hiPSC-derived megakaryocyte matura-tion is required for generation of functional CD41a＋/CD42b＋ platelets in vitro. Excessively sus-tained c-MYC expression in megakaryocyteswas accompanied by increased ARF and INK4Aexpression and decreased GATA1 and NF-E2expression, eventually leading to megakaryocytesenescence and apoptosis, and CD41a＋/CD42blow/- platelet generation. By using an inducibleexpression vector system with c-MYC and BMI1in this context, the MKCL was capable of gener-ating polyploid megakaryocytes (＞8N; 40％).The MKCL also subsequently showed proplate-let formation leading to the release of “func-tional” CD41a＋/CD42b＋ platelets. Furthermore,following transfusion of 6×108 platelets origi-nally derived from the our immortalized MKCLinto immunodeficient NOG mice, the plateletsappeared to exhibit normal circulation a highdegree of chimerism (human CD41a/ humanCD41a＋mouse CD41 was ～67％ at 2 hrs and26％ at 24 hrs post transfusion). We thereforepropose that establishment of immortalizedMKPCs through gene manipulation could po-tentially provide a stable supply of platelets at apredefined quality and quantity for transfusiontherapy.

Publications

1. Kawahara, M., Chen, J., Sogo, T., Teng, J.,Otsu, M., Onodera, M., Nakauchi, H., Ueda,H., Nagamune, T. Growth promotion of ge-netically modified hematopoietic progenitorsusing an antibody/c-Mpl chimera. Cytokine55: 402-408, 2011.

2. Kunishima, S., Kashiwagi, H., Otsu, M.,

Takayama, N., Eto, K., Onodera, M., Miya-jima, Y., Takamatsu, Y., Suzumiya, J., Matsu-bara, K., Tomiyama, Y., Saito, H. Heterozy-gous ITGA2B R995W mutation inducing con-stitutive activation of the alphaIIbbeta3 recep-tor affects proplatelet formation and causescongenital macrothrombocytopenia. Blood

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117: 5479-5484, 2011.3. Maeyama, Y., Otsu, M., Kubo, S., Yamano, T.,

Iimura, Y., Onodera, M., Kondo, S., Saki-yama, Y., Ariga, T. Intracellular estrogenreceptor-binding fragment-associated antigen9 exerts in vivo tumor-promoting effects viaits coiled-coil region. Int. J. Oncol. 39: 41-49,2011.

4. Nishimura, S., Manabe, I., Nagasaki, M.,Kakuta, S., Iwakura, Y., Takayama, N., Ooe-hara, J., Otsu, M., Kamiya, A., Petrich, B.,Urano, T., Kadono, T., Sato, S., Aiba, A.,Yamashita, H., Sugiura, S., Kadowaki, T.,Nakauchi, H., Eto, K., Nagai, R. In vivo imag-ing visualizes discoid platelet aggregationswithout endothelium disruption and impli-cates contribution of inflammatory cytokineand integrin signaling. Blood, 2011.

5. Yokoi, T., Kobayashi, H., Shimada, Y., Eto, Y.,

Ishige, N., Kitagawa, T., Otsu, M., Nakauchi,H., Ida, H., Ohashi, T. Minimum requirementof donor cells to reduce the glycolipid storagefollowing bone marrow transplantation in amurine model of Fabry disease. J. Gene. Med.13: 262-268, 2011.

6. Yoshida, K., Sanada, M., Shiraishi, Y.,Nowak, D., Nagata, Y., Yamamoto, R., Sato,Y., Sato-Otsubo, A., Kon, A., Nagasaki, M.,Chalkidis, G., Suzuki, Y., Shiosaka, M., Kawa-hata, R., Yamaguchi, T., Otsu, M., Obara, N.,Sakata-Yanagimoto, M., Ishiyama, K., Mori,H., Nolte, F., Hofmann, W.K., Miyawaki, S.,Sugano, S., Haferlach, C., Koeffler, H.P., Shih,L.Y., Haferlach, T., Chiba, S., Nakauchi, H.,Miyano, S., Ogawa, S. Frequent pathway mu-tations of splicing machinery in myelodyspla-sia. Nature 478: 64-69, 2011.

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The major goal of our laboratory is to understand the role of mesenchymal andadult stem cells in regenerative and cancer biology. Stem cells from adult tissueshave the unique capacity to repair damaged tissue, a process controlled in part bythe microenvironment. Proteases, as part of the microenvironment act as process-ing enzymes that perform highly selective and limited cleavage of specific sub-strates including growth factors and their receptors, cell adhesion molecules, cy-tokines, chemokines, apoptotic ligands and angiogenic factors. We currently fo-cused our scientific efforts on understanding the mechanism how the fibrinolyticpathway controls tumor cell growth (1), a process in part due to its ability alteringblood cell formation (hematopoiesis) (2). The goal of this laboratory is to identifynovel therapeutic targets for diseases like cancer or inflammatory diseases.

1. Plasmin inhibitor reduces lymphoid tumorgrowth by suppressingmatrixmetallproteinase-9 dependent CD11b＋/F4/80＋ myeloid cell recruitment.

Makoto Ishihara, Chiemi Nishida, YoshihikoTashiro, Ismael Gritli, Jeanette Rosenkvist,Makiko Koizumi, Ryo Yamamoto, HideoYagita1, Ko Okumura2, Momoko Nishikori3,Keiko Wanaka4, Yuko Tsuda5, Yoshio Okada5,Hiromitsu Nakauchi, Beate Heissig2,6, KoichiHattori: 1Department of Immunology, Jun-tendo University School of Medicine, 2-1-1Hongo, Bunkyo-ku, Tokyo 113-8421, Japan,2Atopy (Allergy) Research Center, JuntendoUniversity School of Medicine, 2-1-1 Hongo,Bunkyo-ku, Tokyo 113-8421, Japan, 3Depart-ment of Hematology and Oncology, Kyoto Uni-versity, 54 Shogoin Kawahara-cho, Sakyo-ku,Kyoto, 606-8507, 4Kobe Research Projects onThrombosis and Haemostasis, 3-15-18 Asahi-gaoka, Tarumi-ku, Kobe, 655-0033, Japan,5Faculty of Pharmaceutical Sciences, Kobe

Gakuin University, 518 Arise, Ikawadani-cho,Nishi-ku, Kobe 651-2180 Japan, 6Frontier Re-search Initiative, Institute of Medical Science atthe University of Tokyo, 4-6-1 Shirokanedai,Minato-ku, Tokyo 108-8639, Japan

Activation of the fibrinolytic system duringlymphoma progression is a well-documentedclinical phenomenon. But the mechanism bywhich the fibrinolytic system can modulate lym-phoma progression has been elusive. The mainfibrinolytic enzyme, plasminogen (Plg)/plasmin(Plm), can activate matrix metalloproteinases(MMPs), like MMP-9, which has been linked tovarious malignancies. Here we provide the evi-dence that blockade of Plg reduces lymphomagrowth by inhibiting MMP-9-dependent recruit-ment of CD11b＋F4/80＋ myeloid cells locallywithin the lymphoma tissue. Genetic plasmino-gen deficiency and drug-mediated Plm blockadedelayed lymphoma growth and diminishedMMP-9 dependent CD11b＋F4/80＋ myeloid cellinfiltration into lymphoma tissues. A neutraliz-

Center for Stem Cell and Regenerative Medicine

Laboratory of Stem Cell Regulation幹細胞制御領域

Associate Professor (Project) Koichi Hattori, M.D., Ph.D. 特任准教授 医学博士 服 部 浩 一

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ing antibody against CD11b inhibited lym-phoma growth in vivo, which indicates that CD11b＋ myeloid cells play a role in lymphomagrowth. Plg deficiency in lymphoma-bearingmice resulted in reduced plasma levels of thegrowth factors vascular endothelial growth-Aand Kit ligand, both of which are known to en-hance myeloid cell proliferation. Collectively,the data presented in this study demonstrate apreviously undescribed role of Plm in lym-phoproliferative disorders and provide strongevidence that specific blockade of Plg representsa promising approach for the regulation of lym-phoma growth.

2. Tumor necrosis factor receptor-associatedfactor (TRAF) 2 controls homeostasis ofthe colon to prevent spontaneous develop-ment of murine inflammatory bowel dis-ease.

Jiang-Hu Piao1,2, Mizuho Hasegawa3, Beate He-issig, Koichi Hattori, Kazuyoshi Takeda1, Yoi-chiro Iwakura4, Ko Okumura1,2, Naohiro Ino-hara3, Hiroyasu Nakano1.: 1Department of Im-munology, 2Atopy Research Center, JuntendoUniversity School of Medicine, 2-1-1 Hongo,Bunkyo-ku, Tokyo 113-8421, Japan, 3Depart-ment of Pathology, University of MichiganMedical School, MSRB2, C5741500 E. MedicalCenter Dr. Ann Arbor, MI 48109, USA, 4Cen-ter for Experimental Medicine and Systems Bi-ology, Institute of Medical Science, Universityof Tokyo, Tokyo 108-8639, Japan, and CREST,Japan Science and Technology Agency, Sai-

tama 332-0012, Japan.

Fine-tuning of host cell responses to commen-sal bacteria plays a crucial role in maintaininghomeostasis of the gut. Here, we show that tu-mor necrosis factor receptor-associated factor(Traf)2(－/－) mice spontaneously developed se-vere colitis and succumbed within 3 weeks afterbirth. Histological analysis revealed that apopto-sis of colonic epithelial cells was enhanced, andB cells diffusely infiltrated into the submucosallayer of the colon of Traf2(－/－) mice. Expres-sion of proinflammatory cytokines, includingTnfa, Il17a, and Ifng, was up-regulated, whereasexpression of antimicrobial peptides was down-regulated in the colon of Traf2(－/－) mice.Moreover, a number of IL-17-producing helperT cells were increased in the colonic laminapropria of Traf2(－/－) mice. These cellular al-terations resulted in drastic changes in the colo-nic microbiota of Traf2(－/－) mice comparedwith Traf2(＋/＋) mice. Treatment of Traf2(－/－) mice with antibiotics ameliorated colitisalong with down-regulation of proinflammatorycytokines and prolonged survival, suggestingthat the altered colonic microbiota might con-tribute to exacerbation of colitis. Finally, dele-tion of Tnfr1, but not Il17a, dramatically amelio-rated colitis in Traf2(－/－) mice by preventingapoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, andrestoration of wild-type commensal bacteria. To-gether, TRAF2 plays a crucial role in controllinghomeostasis of the colon.

Publications

1. Beate Heissig, Makiko Ohki-Koizumi, Yoshi-hiko Tashiro, Ismael Gritli, Kaori Sato-Kusubata and Koichi Hattori. New functionsof the fibrinolytic system in bone marrowcell-derived angiogenesis International Jour-nal of Hematology Doi 10.1007/s12185-012-1016-y (Published online:07 Feb 2012)

2. Okaji Y, Tashiro Y, Gritli I, Nishida C, SatoA, Ueno Y, Del Canto Gonzalez S, Ohki-Koizumi M, Akiyama H, Nakauchi H, HattoriK, Heissig B. Plasminogen deficiency attenu-ates post-natal erythropoiesis in male C57BL/6 mice through decreased activity of the LH-testosterone axis. Exp. Hematology, Vol 40,143-154, 2012

3. Ishihara M, Nishida C, Tashiro Y, Gritli I, Ro-senkvist J, Koizumi M, Okaji Y, Yamamoto R,Yagita H, Okumura K, Nishikori M, WanakaK, Yuko Tsuda Y, Okada Y, Nakauchi H,Hattori K, Heissig B. Plasmin inhibitor re-

duces lymphoid tumor growth by suppress-ing matrix metallproteinase-9 dependent CD11b＋／F4/80＋ myeloid cell recruitment. Leu-kemia, Sep 20. Doi:10.1038/leu.2001.203.[Epub ahead of print], 2011.

4. Piao JH, Hasegawa M, Heissig B, Hattori K,Takeda K, Iwakura Y, Okumura K, InoharaN, Nakano H. Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeosta-sis of the colon to prevent spontaneous devel-opment of murine inflammatory bowel dis-ease. J Biol Chem 286(20): 17879-88, 2011.

5. 服部浩一，佐藤亜紀：造血幹細胞動態と血管新生制御機構，診断と治療社，血管再生治療，３８―４５．２０１２

6. 服部浩一，西田知恵美：線溶系を起点とした造血制御機構，医薬ジャーナル社，血液フロンティア，Vol２１．５７―６４．２０１１

7. 服部浩一，佐藤亜紀：凝固・線溶系活性調整による血管新生制御機構，メディカルレ

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ビュー社，血管医学Vol１２ ６５―７０ ２０１１．8. 服部浩一，西田知恵美：血液線維素溶解系因

子による骨髄細胞の動態制御，中外医学社Annual Review２０１１血液 １８７―１９５

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Our major goal is to cure patients suffering from life-threatening diseases by thetreatment with processing of various stem cells. Currently our efforts are directedtoward the establishment of novel therapies using human pluripotent stem cells(hPSC), such as embryonic stem cells and induced pluripotent stem cells (ESCand iPSC, respectively), and the analysis of pathogenesis of a variety of disordersbased on disease-specific iPS cells.

1. Novel method for efficient production ofmultipotential hematopoetic progenitorsfrom human pluripotent stem cells

Feng Ma, Yasuhiro Ebihara1, Shinji Mochi-zuki, Sachiyo Hanada, Emiko Matsuzaka, YujiZaike2, Hiromitsu Nakauchi3, Kohichiro Tsuji: 1

Department of Pediatric Hematology-Oncology,and 2Department of Laboratory Medicine, Re-search Hospital, 3Division of Stem Cell Ther-apy, Center for Stem Cell Biology and Regen-erative Medicine.

ESC are pluripotent cells derived from the in-ner cell mass of preimplantation embryos, andiPSC are induced from somatic cells by nuclearreprogramming. Since both have the ability tobe maintained in culture indefinitely as undiffer-entiated cells, yet they are capable of formingmore differentiated cell types, they are expectedas a novel source of human transplantable cellsfor the regenerative medicine. We then planedto produce hematopoietic stem cells (HSC) fortherapeutic HSC transplantation and functionalblood cells for transfusion medicine from thesehuman pluripotent stem cells. In result, we de-veloped a novel method for the efficient produc-tion of hematopoietic progenitor cells (HPC)

from hESC and hiPSC by co-culture withAGMS-3 stromal cells which originates frommirine aorta-gonad-mesoneephros (AGM) regionat 11 to 12 dpc. In the co-culture, various hema-topoietic progenitors were generated, and thishematopoietic activity was concentrated incobblestone-like (CS) cells within differentiatedhuman ES or iPS cell colonies. The CS cells ex-pressed CD34 and retained a potential for endo-thelial cells. They also contained HPC, especiallyerythroid and multipotential HPC at high fre-quency. The multipotential HPC abundantamong the CS cells produced all types of ma-ture blood cells, including adult type β globin-expressing erythrocytes and tryptase andchymase-double positive mast cells (MC). Theyshowed neither immature properties of PSC norpotentials to differentiate into endoderm and ec-toderm at a clonal level. The developed co-culture system of hPSC can provide a novelsource for hematopoietic and blood cells appli-cable to cellular therapies and drug screenings.

2. Generation of functional erythrocytes fromhuman ES or iPS cell-derived definitive he-matopoiesis

Feng Ma, Yasuhiro Ebihara1, Shinji Mochi-

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Processing幹細胞プロセシング分野

Associate Professor Kohichiro TsujiProject Assistant Professor Shinji Mochizuki

准教授 医学博士 � 浩一郎特任助教 医学博士 望 月 慎 史

233

zuki, Sachiyo Hanada, Emiko Matsuzaka, YujiZaike2, Hiromitsu Nakauchi3, Kohichiro Tsuji

A critical issue for utilization of hESC orhiPSC in possible clinical use is whether theycan derive terminally mature progenies with thenormal function. To solve this, we examinedhESC or hiPSC-derived erythroid cells in cocul-ture with mFLSC or AGM cells. By the cocul-ture, large quantity of hESC or hiPSC-derivederythroid progenitors allowed us to analyze thedevelopment of erythropoiesis at a clone leveland to investigate their function as oxygen car-rier. The results showed that the globin expres-sion in the erythroid cells in individual cloneschanged in a time-dependent manner. In par-ticular, embryonic ε globin positive erythrocytesdecreased, while adult-type β globin positivecells increased to almost 100％ in all singleclones we examined, indicating they had al-ready been fated to definitive hematopoiesis.Enucleated erythrocytes also appeared in theclonal erythroid progenies. A comparison analy-sis showed that hESC-derived erythroid cellstook a similar pathway in differentiation to hu-man cord blood CD34＋ progenitor-derivederythrocytes when traced by glycophorin A, CD71 and CD81. Furthermore, these hESC-derivederythroid cells could function as oxygen carrier,and had a sufficient glucose-6-phosphate dehy-drogenase activity. The present study providedan experimental model to explore early develop-ment of human erythropoiesis, hemoglobinswitching, erythroid pathogenesis, and to dis-cover drugs for hereditary diseases in erythro-cyte development. The quantitative productionand their functional maturation indicate thathPSC-derived erythrocytes can be a novel poten-tial source for therapeutic transfusion.

3. Derivation of blood cells from human pluri-potent stem cells in culture without animalserum or cells

Yasuhiro Ebihara1, Feng Ma, Shinji Mochi-zuki, Sachiyo Hanada, Emiko Matsuzaka, YujiZaike2, Hiromitsu Nakauchi3, Kohichiro Tsuji

It is inevitable to establish an in vitro culturemethod for the induction of hPSC, such as hESCor hiPSC, to differentiate into mature blood cellswithout animal serum and cells. To achieve this,we first induced hPSC to differentiate into mes-enchymal stem cells (MSC). When human ES oriPS cells cultured on murine embryonic fibro-blast (MEF) feeder cells were recultured ongelatin-coated culture dishes with platelet lysate(PL)-containing media in the absence of MEFfeeder cells. Cells were passaged several times

with PL containing media, and then MSC wereinduced after 6 to 8 weeks. The MSC werespindle-like shaped, revealed a phenotype of CD45－, CD34－, CD14－, CD105＋, CD166＋, CD31－, and SEA-4－, and had the ability to differ-entiate into mesenchymal tissues such as bone,cartilage and fat in vitro. Murine MEF and un-differentiated hPSC were undetectable in thehPSC-derived MSC by reverse transcription po-lymerase chain reaction analysis. We then cocul-tured hPSC with MSC derived from hPSC them-selves under serum-free condition. Two weekslater, a number of HPC appeared in the cocul-ture. These HPC were cultured in hematopoieticcolony assay using human serum. In result,hPSC-derived HPC produced various hema-topoietic colonies, such as myeloid, erythroidand multilineage colonies, including all types ofblood cells. The novel culture method must beuseful for the clinical application of hPSC-derived blood cells.

4. Differential production of connectivetissue-type and mucosal mast cells fromhESC for anti-allergy drug screening

Feng Ma, Yang Wenyu, Yanzheng Gu,Yasuhiro Ebihara1, Shinji Mochizuki, SachiyoHanada , Emiko Matsuzaka , HiromitsuNakauchi3, Kohichiro Tsuji

MC function as effector cells in allergy andatopic disease. Therefore, anti-allergy drugshave been established to diminish MC function.However, since the acquisition of an abundanceof human MC (hMC) is difficult because of noculture method producing massive hMC, mostanti-allergy drugs targeted animal MC. Thus, ef-ficient discovery of effective anti-allergy drugsneeds to establish the culture system of massivehMC. Then, hESC are considered as a potentialcell source for hMC. In human, two types of MChave been characterized; connective tissue-typeand mucosal MC (CTMC and MMC, respec-tively). CTMC contain tryptase, chymase, MCcarboxypeptidase and cathepsin G in their secre-tory granules, are predominantly located in nor-mal skin and in intestinal submucosa, and in-volve in atopic dermatitis. MMC contain tryp-tase in their secretory granules, but lack theother proteases, are the main type of MC in nor-mal alveolar wall and in small intestinal mu-cosa, and involve in allergic rhinitis or bronchialasthma. Although MC can be generated fromhuman adult CD34＋ HPC in vitro, these MC aremainly MMC. So far, there lacks an evidence forthe direct derivation of CTMC from adult HPC.We achieved successful production of hESC-derived CD34＋ HPC, using coculture with

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AGMS-3 cells for 1-2 weeks. In suspension cul-ture favoring MC differentiation within 3weeks,hESC-derived progenitors generated mature MCthat shared a chymase / tryptase double posi-tive phenotype and strongly expressed c-Kit,similar to human skin derived CTMC. On theother hand, hESC-derived multipotential hema-topoietic progenitors obtained in clonal culturedeveloped into MC for a longer time (over 5weeks) and only expressed tryptase, with no orfew chymase, similar to human CD34＋ cell-derived MMC. Since the current culture systemof hESC can produce differentially a large num-ber of CTMC and MMC, our study may high-light a new understanding for MC developmentand finally benefit the screening for anto-allergydrugs.

5. Generation of mature eosinophils from hu-man pluripotent stem cells

Feng Ma, Yang Wenyu, Yanzheng Gu,Yasuhiro Ebihara1, Shinji Mochizuki, SachiyoHanada , Emiko Matsuzaka , HiromitsuNakauchi3, Kohichiro Tsuji

Eosinophils are multifunctional leukocytes im-plicated in the pathogenesis of numerous in-flammatory processes. As the major effectors,eosinophils function in a variety of biological re-sponses, allergic diseases and helminth infec-tions. It is generally accepted human eosinophilsdevelop through a pathway initially sharingcommon feature with basophils. However, therelacks a clear chart for early development of hu-man eosinophils, such as during embryonic orfetal stages. We established an efficient methodfor producing eosinophils from hESC andhiPSC. By a two-step induction, we first gener-ated multipotential HPC by co-culturing hPSCwith AGMS-3 cells for 2 weeks. Then, total co-culture cells were transferred into suspensionculture favoring eosinophil development withaddition of IL-3 and other factors (stem cell fac-tor, interleukin-6, thrombopoietin, Flt-3 ligand) .The maturation of hPSC-derived eosinophilswas shown in a time-dependent manner, firstco-expressing eosinophil-and basophil-specificmarkers [eosinophil peroxidase (EPO), and 2D7,respectively], then the portion of eosinophilmarkers gradually increased while that of baso-phil markers decreased, typically mimicking thedevelopment of eosinophils from human adulthematopoietic progenitors. By flowcytometricanalysis, an eosinophil-specific surface marker,Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependentmanner, paralleling to those with EPO. The ex-pression of eosinophil-specific granule cationic

proteins (EPO, MBP, ECP, EDN) and IL-5 recep-tor mRNA was also detected by RT-PCR. Fur-thermore, transmission electron microscopy(TEM) observation confirmed the eosinophilproperty. Eosinophils derived from hiPSCs holdsimilar characteristics as those from hESCs. Ourstudy provides an experimental model for ex-ploring early genesis of eosinophils, especiallyin uncovering the mechanisms controlling thedevelopment of the initial innate immune sys-tem of human being in normal and diseased in-dividuals.

6. Hematopoiesis of human induced pluripo-tent stem cells derived from patients withDown syndrome

Natsumi Nishihama, Yasuhiro Ebihara2, ShinjiMochizuki, Feng Ma, Wenyu Yang, KiyoshiYamaguchi4, Masaharu Hiratsuka5, Yoichi Fu-rukawa4 , Mitsuo Oshimura5 , HiromitsuNakauchi3, Kohichiro Tsuji: 4Division of Clini-cal Genome Research, 5Division of Molecularand Cell Genetics, Department of Molecularand Cellular Biology, Faculty of Medicine, Tot-tori University.

Trisomy 21, genetic hallmark of Down syn-drome, is the most frequent human chromoso-mal abnormality. Infants and children withDown Syndrome (DS) are known to have somehematological disorders with an increased riskof developing leukemia. Ten to 20％ of newbornwith DS are diagnosed as neonatal preleukemicstatus, transient myeloproliferative disorder(TMD), and approximately 30％ of TMD pa-tients are predisposed to acute megakaryoblasticleukemia (AMKL). Recently, acquired mutationsin the N-terminal activation domain of theGATA1 gene, leading to expression of a shorterGATA1 isoform (GATA1s), have been reportedin AMKL and TMD, but neither patients normice with germline mutations leading to expres-sion of GATA1s developed AMKL and TMD inthe absent of trisomy 21. These findings sug-gested that trisomy 21 itself directly contributesto the development of AMKL and TMD. How-ever, the role of trisomy 21 in hematopoiesis,particularly in the human fetus remains poorlyunderstood. To better understand the effects oftrisomy 21 on hematopoiesis in embryonic stageand leukemogenesis, we employed hiPSC de-rived from patients with DS (DS-hiPSC). Six DS-hiPSC and 5 hiPSC lines (control) from healthydonors were all created from skin fibroblastsand reprogrammed by the defined 3 or 4 repro-gramming factors (OCT3/4, KLF4, and SOX2, orc-MYC in addition to the 3 factors, respectively).We generated blood cells from DS-hiPSC and

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controls with coculture system using AGMS-3cells. The cells from hiPSC were harvested atday 11 or 12 of coculture and analyzed the pres-ence of hematopoietic markers and the poten-tials of hematopoietic colony formation. In theexperiments using hiPSC reprogrammed by 3factors, human CD34 expression in harvestedcells from DS-hiPSC or controls were detected10.06±4.35％ and 3.04％, respectively. We nextexamined the hematopoietic colony formation.Both myeloid and erythroid colonies were de-tected. Number of colonies formed from DS-hiPSC was 43.7±11.1 to 74.3±11.2 per an iPScell colony, which was approximately 2 to 3.5folds the number of control. Similar results wereobtained in the experiments using hiPSC repro-grammed by 4 factors. These results indicatedthat hiPSC derived from patients with DS coulddifferentiate into multiple hematopoietic celllineages and the differentiation into hematopoie-tic lineage was promoted in DS patients.

7. Suppressed neutrophil development in he-matopoiesis of induced pluripotent stemcells derived from a severe congenitalneutropenia patient with ELA2 mutation

Takafumi Hiramoto, Yasuhiro Ebihara1, ShinjiMochizuki, Shohei Yamamoto1, Masao Na-gasaki6 , Yoichi Furukawa4 , HiromitsuNakauchi3, Masao Kobayashi7, Kohichiro Tsuji:6Functional Genomics, Human Genome Center,7Department of Pediatrics, Hiroshima Univer-sity Graduate School of Biomedical Sciences.

Severe congenital neutropenia (SCN) is a raredisorder characterized by severe neutropeniapresent at birth, an arrest of neutrophilic differ-entiation at the promyelocyte or myelocytestage, and a propensity to develop acutemyeloid leukemia and myelodysplasia. Muta-tions of the ELA2 gene encoding neutrophilelastase have been identified in the majoritycases of SCN, but the mechanisms which dis-rupt neutrophil development in SCN with ELA2mutation have been unclear. To understand themechanisms, we established three hiPSC clonesfrom bone marrow stromal cells of a patienthaving heterozygous mutation in ELA2 gene atexon 5, 707 region by transfection with retrovi-rus vector which expressed human OCT3/4,

SOX2, KLF4, and c-MYC (SCN-iPSC). We thenexamined the hematopoietic differentiation ofSCN-iPSC and control iPSC which were gener-ated from healthy donors by the same methodto SCN-iPS cells, using coculture system withAGMS-3 cells. The cocultured cells were har-vested at day 12, and CD34＋ cells were sepa-rated. Hematopoietic colony assay was per-formed using these CD34＋ cells. Although num-ber and size of erythroid and mixed-lineagecolonies derived from SCN-iPS cells were almostsimilar to control, those of myeloid colonies de-rived from SCN-iPSC were significantly less andsmaller than control. In particular, we could de-tect few number of neutrophil colonies fromSCN-iPSC. Since SCN patients need granulocytecolony-stimulating factor (G-CSF) treatment toincrease peripheral neutrophils, we conductedthe hematopoietic colony assay with G-CSFalone to examine the sensitivity of granulopoi-esis derived from SCN-iPSC and control iPSC toG-CSF. Myeloid colony formation reached a pla-teau at 1 to 10 ng/mL of G-CSF in control iPScells, while the number and size of myeloidcolonies gradually increased at up to 1000 ng/mL in SCN-iPS cells, but did not attain the con-trol level. In suspension culture with myeloiddifferentiation-oriented cytokines including 10ng/mL of G-CSF, CD34＋ cells from control iPScell increased 23.3-fold for 2 weeks, and matureneutrophils predominantly occupied in the cul-tured cells. By contrast, CD34＋ cells from SCN-iPS cells gradually decreased, and few neutro-phils, but mainly monocytic cells were con-tained in the culture. We also carried out mi-croarray analysis using CD34＋ cells stimulatedby myeloid differentiation-oriented cytokines for2 days to identify the genes which led to im-paired granulopoiesis in SCN-iPS cells. As a re-sult, LEF-1, C/EBP alpha, Cyclin D1 and BCL2were downregulated in the cultured cells fromSCN-iPS cells compared with those from controliPS cells. These results demonstrated that thedevelopment of neutrophils was selectively im-paired in the hematopoiesis derived from SCN-iPSC, and that the stimulation of higher concen-tration of G-CSF compensated the impaired de-velopment of neutrophils to some extent, indi-cating SCN-iPSC can provide a useful tool tounderstand pathogenesis of SCN with ELA2mutation.

Publications

Tsuda, M., Ebihara, Y., Mochizuki, S., Uchimaru,K., Tojo, A. and Tsuji, K. Reduced dose che-motherapy for acute promyelocytic leukemiawith adult Down syndrome. Br. J. Haematol.

155: 130-132, 2011.Iwatsuki-Horimoto, K., Horimoto, T., Tamura,

D., Kiso, M., Kawakami, E., Hatakeyama, S.,Ebihara, Y., Koibuchi, T., Fujii, T., Takahashi,

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K., Shimojima, M., Sakai-Tagawa, Y., Ito, M.,Sakabe, S., Iwasa, A., Takahashi, K., Ishii, T.,Gorai, T., Tsuji, K., Iwamoto, A. andKawaoka, Y. Sero-prevalence of pandemic (H1N1) 2009 influenza A virus among schoolchil-dren and their parents in Tokyo, Japan. Clin.Vaccine Immunol. 18: 860-866, 2011.

Ma, F., Yang, W. and Tsuji, K. Regenerativemedicine based on human ES and iPS cells. InStem Cells in Medicine. Edited by Isobe. K.(Transworld Research Network, Kerala). pp51-70, 2011.

Ma, F., Yang, W., Ebihara, Y. and Tsuji, K. Gen-eration of blood cells from human embryonicstem cells and their possible clinical utiliza-tion. In Embryonic Stem Cells. Edited by At-wood, C. (Intech, Vienna), pp 239-250, 2011.

Kato, M., Koh, K., Kikuchi, A., Toyama, D., Mo-chizuki, S., Uchisaka, N., Nagatoshi, Y.,Tanaka, R., Oh-Ishi, T., Nozawa, K., Oguma,E. and Hanada, R. Case series of pediatricacute leukemia without a peripheral blood ab-normality, detected by magnetic resonanceimaging. Int. J. Hematol. 93: 787-790, 2011.

Yamamoto, S., Ikeda, H., Toyama, D., Hayashi,M., Akiyama, K., Suzuki, M., Tanaka, Y.,Watanabe, T., Fujimoto, Y., Hosaki, Y., Nishi-hira H. and Isoyama, K. Quality of long-termcryopreserved umbilical cord blood units forhematopoietic cell transplantation. Int. J. He-

matol. 93: 99-105, 2011.Sugi, T., Kato, K., Kobayashi, K., Kurokawa, H.,

Takemae, H., Gong, H., Recuenco, F. C.,Iwanaga, T., Horimoto, T. and Akashi, H. 1NM-PP1 treatment of mice infected withToxoplasma gondii . J. Vet. Med. Sci. 73: 1377-1379, 2011.

Kurokawa, H., Kato, K., Iwanaga, T., Sugi, T.,Sudo, A., Kobayashi, K., Gong, H., Takemae,H., Recuenco, F. C., Horimoto, T. and Akashi,H. Identification of Toxoplasma gondii cAMPdependent protein kinase and its role in thetachyzoite growth. PloS One 6: e22492, 2011.

Ma, F., Gu, Y., Nishihama, N., Ebihara, Y. andTsuji, K. Differentiation of human embryonicand induced pluripotent stem cells in cocul-ture with murine stromal cells. In Human Em-bryonic and Induced Pluripotent Stem Cells:Lineage-specific Differentiation Protocols ,Springer Protocol Handbooks. Edited by Ye,K., Jin, S. (Springer Science, New York), pp266-335, 2011.

Ebihara, Y., Takahashi, S., Mochizuki, S., Kato,S., Kawakita, T., Ooi, J., Yokoyam, K., Naga-mura, F., Tojo A, Asano, S. and Tsuji, K. Un-related cord blood transplantation aftermyeloablative conditioning regimen in adoles-cent and young adult patients with hema-tologic malignancies: a single institute analy-sis. Leuk. Res., in press.

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We are conducting clinical stem cell transplantation, especially using unrelatedcord blood as a promising alternative donor in IMSUT research hospital. We arealso engaged in the clinical and basic research for promotion of transplantation aswell as regenerative medicine.(1) Hematopoietic Stem Cell Transplantation (HSCT)

Our facility is a main hub of hematopoietic stem cell transplantation (HSCT)centers in Japan. In close association with Department of Hematology/Oncol-ogy in the IMSUT research hospital, as many as 600 cases of allogeneicHSCT have been performed and HSCT-related complications including acute/chronic GVHD and opportunistic infection have been treated until 2010. Re-cent years unrelated cord blood has turned to be our major stem cell sourcein HSCT. Since 1998 we have performed more than 250 cases of cord bloodTransplantation (CBT) in adults and demonstrated outstanding clinical resultsamong domestic and overseas HSCT centers. During such a transition of ourstem cell source, immunological reconstitution from the CB graft, optimal useof immunosuppressive agents as well as viral infection/ reactivation are be-coming our main theme to be elucidated, and we are now approaching theseissues in collaboration with other divisions in the center.

(2) iPS cell and hematopoietic stem cell (HSC) researchRecent development of induced pluripotent stem (iPS) cells has suggested thepossible application of reprogrammed somatic cells to individualized therapyfor intractable disorders. We are trying to generate iPS cells using lentiviralvector and tetracycline-inducible gene expression system for introducing andexpressing 3 or 4 factors required for generation of iPS cells with relativelyhomogeneous genetic background. We are also challenging to reprogram ma-ture blood cells into HSC according to the similar strategy used for iPS cells.

1. Matched HLA Haplotype Contributes toReduce Sever Acute GVHD with Conserv-ing GVL Effect in HLA-Mismatched CordBlood Transplantation.

Takahashi S, Ooi J, Kato S, Kawakita T, Tojo A

We studied the clinical outcomes of 170 con-secutive adult patients who received unrelatedCBT between August 1998 and January 2011 inthe institute of medical Science, University ofTokyo. Patients received previous allogeneictransplants were excluded from this study. All

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Transplantation幹細胞移植分野

Professor Arinobu Tojo, M.D., D.M.Sc.Associate Professor Satoshi Takahashi, M.D., D.M.Sc.Research Associate Seiko Kato, M.D., D.M.Sc.Project Research Associate Toshiro Kawakita, M.D.

教 授 医学博士 東 條 有 伸准教授 医学博士 高 橋 聡助 教 医学博士 加 藤 せい子特任助教 河 北 敏 郎

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patients received myeloablative regimens in-cluding 12 Gy of total body irradiation, cy-closporine plus short term methotraxate forGVHD prophylaxis and almost same supportivecare by the institutional protocol. By low-resolution typing method for HLA-A, -B and -DR loci, 6 patients received matched grafts, 57received 1 antigen-mismatched and 107 received2 antigens-mismatched grafts in the graft-versus-host (GvH) direction. We have deter-mined the HLA haplotype based on commonhaplotypes in Japanese population referred fromthe 11th International Histocompatibility Work-shop and other previous reports. We evaluatedthe impact of haplotype matching on cumulativeincidences of hematopoietic recovery, of GVHD,of relapse and of non-relapse mortality (NRM)using the Pepe and Mori’s test. Estimates ofoverall and disease-free survivals were calcu-lated using the Kaplan-Meier method and ana-lyzed by the log-rank test. Thirty-three amongall 170 pairs were defined as the haplotype-matched pairs sharing same haplotypes in bothgrafts and recipients. The age, sex, cytomega-lovirus serological status, diagnosis, risk of thedisease at the transplant, numbers of total nucle-ated cells and CD34＋ cells at the cryopreservedwere not significantly different between bothgroups with and without matched haplotypes.Engraftment of platelet after CBT tended to beearlier in haplotype-matched group comparedwith control group among the 1 antigen-mismatched pairs in the host-versus-graft direc-tion (median: 38 days versus 44 days) andamong the 2 antigens-mismatched pairs (me-dian: 38 days versus 42 days), but those werenot significant. The cumulative incidences ofgrades III and IV acute GVHD in patients withhaplotype-matched (7％) were significantlylower than non-matched group (9％) among 2antigens-mismatched pairs in the GvH direction(P＝0.033). Notably, cumulative incidences of re-lapse tended to be lower in haplotype-matchedpatients among this group (3 years cumulativeincidences were 7％ in haplotype-matched pa-tients versus 21％ in non-matched patients, P＝0.086). The haplotype matching effects were notobserved in survival rates, cumulative inci-dences of NRM among any HLA-mismatchedpairs. Those data suggest that untyped variationcarried on the HLA haplotytpe might be betterto be matched. The haplotype matching seemedto effect on lower risk of sever acute GVHD, onthe other hand, graft-versus-leukemia effect wasconserved in the setting of HLA-mismatchedCBT. Second myeloablative allogeneic stem celltransplantation (SCT) using cord blood for leu-kemia relapsed after initial allogeneic SCT.

2. Unrelated cord blood transplantation (CBT)after myeloablative conditioning in adultswith advanced myelodysplastic syn-dromes.

Sato A, Ooi J, Takahashi S, Tsukada N, KatoS, Kawakita T, Yagyu T, Tojo A

We analyzed the disease-specific outcomes ofadult patients with advanced myelodysplasticsyndrome (MDS) treated with cord blood trans-plantation (CBT) after myeloablative condition-ing. Between August 1998 and June 2009, 33adult patients with advanced MDS were treatedwith unrelated CBT. The diagnoses at transplan-tation included refractory anemia with excessblasts (n＝7) and MDS-related secondary AML(sAML) (n＝26). All patients received four frac-tionated 12 Gy TBI and chemotherapy asmyeloablative conditioning. The median agewas 42 years, the median weight was 55 kg andthe median number of cryopreserved nucleatedcells was 2.51×10(7) cells per kg. The cumula-tive incidence of neutrophil recovery at day 50was 91％. Neutrophil recovery was significantlyfaster in sAML patients (P＝0.04). The cumula-tive incidence of plt recovery at day 200 was 88％. Plt recovery was significantly faster in CMVseronegative patients (P＜0.001). The cumulativeincidence of grade II-IV acute GVHD (aGVHD)and extensive-type chronic GVHD was 67 and34％, respectively. Degree of HLA mismatchhad a significant impact on the incidence ofgrade II-IV aGVHD (P＝0.021). TRM and relapseat 5-years was 14 and 16％, respectively. Theprobability of EFS at 5 years was 70％. No fac-tor was associated with TRM, relapse and EFS.These results suggest that adult advanced MDSpatients without suitable related or unrelatedBM donors should be considered as candidatesfor CBT.

3. Second myeloablative allogeneic stem celltransplantation (SCT) using cord blood forleukemia relapsed after initial allogeneicSCT.

Ooi J, Takahashi S, Tsukada N, Kato S, SatoA, Uchimaru K, Tojo A

There are many reports of second allogeneicstem cell transplantation (allo-SCT) using cordblood (CB) for graft failure after initial allo-SCT.However, the efficacy of second allo-SCT usingCB for patients with leukemia relapsed after in-itial allo-SCT is unknown. We report the resultsof second allo-SCT using CB in seven adult pa-tients with leukemia relapsed after initial allo-SCT. All patients received a myeloablative con-

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ditioning regimen including oral busulfan 16 mg/kg, intravenously fludarabine 100mg/m(2) andcyclophosphamide 120 mg/kg. All but one pa-tient had myeloid reconstitution and four pa-tients remain alive at between 4 and 40 monthsafter second SCT. We conclude that secondmyeloablative allo-SCT using CB may be feasi-ble in selected patients with the relativelyyounger age, less organ damage and longertime interval between first and second allo-SCT.

4. Bcr-Abl impairs T cell development frommurine induced pluripotent stem cells andhematopoietic stem cells.

Bidisha C, Izawa K, Harnprasopwat R, Taka-hashi K, Kobayashi K, Tojo A

Chronic myeloid leukemia (CML) is a clonalmyeloproliferative disorder generally believed tooriginate from a hematopoietic stem cell carry-ing the BCR-ABL fusion gene, which generallyencodes 210kD and 190kD constitutively activetyrosine kinases termed as p210 and p190, re-spectively. In spite of the putative stem cell ori-gin and the competence for differentiation to-ward mature B cells, there is a longstandingconsensus that CML never involves the T celllineage at least in chronic phase. To gain insightinto this apparent conflict, we used in vitro Tcell differentiation model from murine pluripo-tent stem cells (PSCs) as well as hematopoieticstem cells (HSCs). C57BL/6 MEFs were repro-grammed using a polycistronic lentiviral Tet-Onvector encoding human Oct4, Sox2 and Klf4,which were tandemly linked via porcineteschovirus-1 2A peptides, together with anotherlentiviral vector expressing rtTA driven by theEF-1α promoter. Almost all the vector sequencesincluding the transgenes were deleted byadenovirus-mediated transduction of Cre recom-

binase after derivation of iPSCs, and only rem-nant 291-bp LTRs containing a single loxP siteremained in the genome. A clone of MEF-iPSCswere retrovirally transduced with p190∆ccER, aligand-controllable p190-estrogen receptor fusionprotein, whose tyrosine kinase activity abso-lutely depends on 4-hydroxytamoxyfen (4-HT).For T cell lineage differentiation, p190∆ccER-MEF-iPSCs were recovered from a feeder-freeculture supplemented with LIF and plated ontoa subconfluent OP9-DL1 monolayer in the pres-ence of Flt3 ligand and IL7 with or without 0.5µM 4-HT. After 3 weeks of culture, iPSC-derived blood cells were collected and subjectedto FACS analysis for their lineage confirmation.About 70％ of lymphocyte-like cells from the 4-HT(－) culture expressed CD3, but only 20％ ofcounterparts from the 4-HT(＋) culture ex-pressed CD3, suggesting impaied T cell devel-opment by Bcr-Abl. Next, c-Kit＋Sca1＋Lin－ (KSL)bone marrow cells were prepared by FACS from8-weeks old C57BL/6 mice treated with 5-FU.KSL cells were similarly transduced with p190∆ccER and were subjected to the OP9-DL1 co-culture system with or without 0.5 µM 4-HT.After 2 weeks of culture, 95％ of lymphocytesfrom the 4-HT(－) culture revealed CD3＋TCRβ＋phenotype, but only 30％ of those were doublepositive in the presence of 4-HT. In addition, 90％ of lymphocytes from the 4-HT(－) cultureprogressed to the DN2 stage with c-Kit-CD44＋

CD25＋ phenotype, whereas 50％ of those fromthe 4-HT(－) culture arrested at the DN1 stageshowing c-Kit＋CD44＋CD25－. Since IL7 plays acentral role at the stage from DN1 to DN2 ofprogenitor T cells, Bcr-Abl is suggested to im-pair T cell development possibly through inter-fering with the IL7 signal. The precise mecha-nism underlying impaired T lymphopoiesis byBcr-Abl is under investigation.

Publications

Sato A, Ooi J, Takahashi S, Tsukada N, Kato S,Kawakita T, Yagyu T, Nagamura F, Iseki T,Tojo A, Asano S. Unrelated cord blood trans-plantation after myeloablative conditioning inadults with advanced myelodysplastic syn-dromes. Bone Marrow Transplant, 46: 257-61,2011

Waki F, Masuoka K, Fukuda T, Kanda Y, Naka-mae M, Yakushijin K, Togami K, Nishiwaki K,Ueda Y, Kawano F, Kasai M, Nagafuji K,Hagihara M, Hatanaka K, Taniwaki M, MaedaY, Shirafuji N, Mori T, Utsunomiya A, Eto T,Nakagawa H, Murata M, Uchida T, Iida H,Yakushiji K, Yamashita T, Wake A, Takahashi

S, Takaue Y, Taniguchi S. Feasibility ofReduced-intensity Cord Blood Transplantationas Salvage Therapy for Graft Failure: Resultsof a Nationwide Survey of 80 Adult Patients.Biol Blood Marrow Transplant. 17: 841-51,2011

Kako S, Morita S, Sakamaki H, Ogawa H,Fukuda T, Takahashi S, Kanamori H, OnizukaM, Iwato K, Suzuki R, Atsuta Y, Kyo T,Sakura T, Jinnai I, Takeuchi J, Miyazaki Y, Mi-yawaki S, Ohnishi K, Naoe T, Kanda Y. A de-cision analysis of allogeneic hematopoieticstem cell transplantation in adult patientswith Philadelphia chromosome-negative acute

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lymphoblastic leukemia in first remission whohave an HLA-matched sibling donor. Leuke-

mia. 25: 259-65, 2011

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Our major interest is to elucidate the mechanisms of pluripotency, self-renewaland the control of cell division and differentiation of stem cells like ES cells, iPScells, and hematopoietic stem cells. We have developed the retrovirus-mediatedefficient gene transfer and several functional expression cloning systems, and util-ized these system to our experiment. We are now conducting several projects re-lated to stem cells to characterize stem cells, clarify underling mechanisms of re-programming, maintenance of pluripotency, and differentiation, and eventually todevelop new strategies for regenerative medicine.

1. Screening of surface antigens of iPS cellsusing a retrovirus-mediated signal trans-duction method SST-REX.

Toshikhiko Oki, Jiro Kitaura, Masunori Ka-jikawa1, and Toshio Kitamura: 1ACTGen,Komagane, Nagano.

We previously developed a retrovirus-mediated signal sequence trap method SST-REXas a screening method for surface and secretedproteins. We searched surface antigens of cancercells or immune cells. Here we used SST-REXfor iPS to identify iPS-specific surface antigens, asurface antigen “catalog” of iPS cells, and at-tempted to develop iPS-specific antibodies. Sofar, we have identified 40 iPS cell antigens,found that at least 3 of them were expressedrather specifically in iPS cells and ES cells, anddeveloped specific antibodies to these 3 antigensand investigated expressions of these antigens iniPS cells. We also investigated the effects oftransduction of these antigens on iPS induction,and transduction of one of the antigen enhancedreprogramming process, though the precisemechanisms remain to be investigated.

2. RasGRP family proteins and Leukemia

Toshihiko Oki, Jiro Kitaura, KoutarouNishimura, Akie Maehara, Tomoyuki Uchida,Fumio Nakahara, and Toshio Kitamura

The Ras guanyl nucleotide-releasing proteins(RasGRPs) are a family of guanine nucleotide-exchange factors, with four members (RasGRP1-4), which positively regulate Ras and relatedsmall GTPases. In the previous study, we identi-fied RasGRP4 using expression cloning as agene that fully transformed IL-3-dependent HF6cells, and demonstrated that in a mouse bonemarrow transplantation (BMT) model, RasGRP4induced acute myeloid leukemia (AML) and/orT-ALL. On the other hand, it has been reportedthat RasGRP1 transgenic mice developed thymiclymphoma or skin tumors.

However, the roles of RasGRP family proteinsin leukemogenesis have not been investigated indetail. We have recently characterized leukemo-genicity of RasGRP1 and 4 in details using aBMT model (Oki et al. Leukemia 2012).

RasGRP1 exclusively induced T-cell acutelymphoblastic leukemia/lymphoma (T-ALL) af-ter a shorter latency when compared with

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Signaling幹細胞シグナル制御部門

Professor Toshio Kitamura, M.D., D.M.Sc.Project Assistant Professor Toshihiko Oki, M.D., D.M.Sc.

教 授 医学博士 北 村 俊 雄特任助教 医学博士 沖 俊 彦

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RasGRP4. Accordingly, Ba/F3 cells transducedwith RasGRP1 survived longer under growthfactor withdrawal or phorbol ester stimulationthan those transduced with RasGRP4, presum-ably due to the efficient activation of Ras. In-triguingly, NOTCH1 mutations resulting in again of function were found in 77％ of theRasGRP1-mediated mouse T-ALL samples. Inaddition, gain-of-function NOTCH1 mutationwas found in human T-cell malignancy with ele-vated expression of RasGRP1. Importantly,RasGRP1 and NOTCH1 signaling cooperated inthe progression of T-ALL in the murine model.The leukemogenic advantage of RasGRP1 overRasGRP4 was attenuated by the disruption of aPKC phosphorylation site (RasGRP1(Thr184))not present on RasGRP4. In conclusion, coopera-tion between aberrant expression of RasGRP1, astrong activator of Ras, and secondary gain-of-function mutations of NOTCH1 plays an impor-tant role in T-cell leukemogenesis.

3. Development of new retroviral vectors.

Toshikhiko Oki, Jiro Kitaura, TomoyukiUchida, Fumi Shibata-Minoshima, and ToshioKitamura

We developed an effective retroviral transduc-tion system consisted of vectors named aspMXs, pMYs, pMZs and pMCs and packagingcells named as PLAT-E, PLAT-A, and PLAT-F.We developed new vectors like, vectors with lu-ciferase maker (pMX-IL), vectors for GFP or RFPfusion proteins, vectors with lox sequences fordeletion of inserted genes with Cre-loxP, Tet-Onand Tet-Off systems, vectors for expression, in-hibition, and monitoring the expression of mi-roRNA (pMXe series). We utilized these vectorsin studying stem cell biology and also in devel-oping the innovative tools for regenerativemedicine

4. Co-ordinate control of cell division andcell fate of by the Rho family smallGTPases.

Toshihiko Oki, Kohtaro Nishimura, ToshiyukiKawashima, and Toshio Kitamura

We previously identified MgcRacGAPthrough functional cloning as a protein that en-hances or induces macrophage differentiation ofleukemic cell lines M1 and HL60. Interestingly,MgcRacGAP plays distinct roles depending onthe cell cycle. In the interphase, it plays criticalroles in activation and nuclear translocation ofSTAT3 and STAT5 as a Rac-GAP. In the meta-phase, MgcRacGAP plays some roles in the seg-

regation of chromosomes probably as a Cdc42-GAP. In the mitotic phase, MgcRacGAP playsessential roles in completion of cytokinesis as aRho-GAP. Interestingly, Aurora B-mediatedphosphorylation of S387 converts MgcRacGAPfrom Rac-GAP to Rho-GAP.

We have recently shown that expression ofMgcRacGAP is regulated by cell-cycle depend-ent mechanism: increase in S/G2/M phase anddecrease in early G1 phase, suggesting thatMgcRacGAP may play some roles in G1 checkpoint. The ubiquitin-dependent degradation ofMgcRacGAP is one of the mechanisms that ac-count for its decrease in G1 phase. Using theproteome analysis and retroviral transduction,we identified the E3 ligase involved in regula-tion of MgcRacGAP and the degron inMgcRacGAP. Now we are investigating thephysiological roles of this regulation. In sum-mary, our results implicate MgcRacGAP in coor-dination of cell cycle progression and cell fatedetermination.

5. Pepp2 as a candidate oncogene for gastriccancer and leukemia.

Fumi Shibata-Minoshima, Toshihiko Oki, Fu-mio Nakahara, Jiro Kitaura, Junya Fukuoka,and Toshio Kitamura

We have searched for potential oncogenes byscreening cDNA libraries derived from gastriccancer cell lines, pancreatic cancer cell lines, andglioma cell lines, using retrovirus-mediated ex-pression cloning. Several genes were identifiedincluding PIM-1, PIM-2, PIM-3, GADD45B andReproductive HOmeobox genes on the X chro-mosome gene F2 (RHOXF2) as candidate on-cognes (Fumi Shibata-Minoshima et al. Interna-tional Journal of Oncology 2011). Although nomutation in these genes was found, these mole-cules were highly expressed in cancer cell lines,suggesting that they play important roles in celltransformation. Among them, we focused on atranscriptional repressor RHOXF2. Transductionof RHOXF2 rendered HF6 cells factor-independent, while knockdown of RHOXF2 in-hibited growth of a gastric cancer cell line HGC27 in which RHOXF2 is highly expressed. In ad-dition, RHOXF2-transfuced HF6 cells quickly in-duced leukemia when transplanted to sub-lethally irradiated mice. Moreover, RHOXF2 ishighly expressed in some of leukemia cell linesand a variety of human cancer samples includ-ing colon cancers and lung cancers. Thus, theseresults indicated that RHOXF2 is involved incarcinogenesis.

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6. Molecular therapy targeting signal trans-duction pathways using small moleculecompounds

Toshiyuki Kawashima, Akiho Tsuchiya, Toshi-hiko Oki, Jiro Kitaura, and Toshio Kitamura

STAT3 is frequently activated in many cancersand leukemias, and is required for transforma-tion of NIH3T3 cells. Therefore, we searched forSTAT3 inhibitors. We already established an ef-ficient screening protocol for identification ofSTAT3 inhibitors, and identified several com-pounds that inhibit STAT3 activation. Throughthe screening of a library of small molecule

compounds, we found the compounds RJSI-1and RJSI-2 that inhibited STAT3 activation. RJSI-2 also inhibited activation of STAT1, STAT5,JAK1 and JAK2, however RJSI-2 is not a kinaseinhibitor. On the other hand, RJSI-1 inhibitednuclear transport of phosphorylated STAT pro-teins, implicating a novel mechanism in inhibit-ing STAT proteins. We have also shown thatthese compounds are effective in a tumor-burden mouse model. In addition, we collabo-rate with a US biotech venture company inmodification of RSJI-1 for optimization to de-velop anti-cancer drugs, and have developed JP1156 which kill the tumor cells with much lowerIC50.

Publications

Doki, N., Kitaura, J., Inoue, D., Kato, N., Kagi-yama, Y., Uchida, T., Togami, K., Isobe, M.,Ito, S., Maehara, A., Izawa, K., Oki, T., Ha-rada, Y., Nakahara, F., Harada, H., and Kita-mura, T. Fyn is not essential for Bcr-Abl-induced leukemogenesis in mouse bone mar-row transplantation models. Int. J. Hematol.95: 167-175.

Oki, T., Kitaura, J., Watanabe-Okochi, N.,Nishimura, K., Maehara, A., Uchida, T.,Komeno, Y., Nakahara, F., Harada, Y., Sonoki,T., Harada, H., and Kitamura, T. Aberrant ex-pression of RasGRP1 cooperates with gain-of-function NOTCH1 mutations in T-cell leu-kemogenesis. Leukemia in press.

Tran, P.T., Bendapaudi, P.K., Lin, H.J., Choi, P.,Koh, S., Chen, J., Horng, G., Hughs, N.P.,Schwartz, L.H., Miller, J.H., Kawashima, T.,Kitamura, T., Paik, D., and Felsher, D.W. Sur-vival and death signals can predict tumor re-sponse to therapy after oncogene inactivation.Science Translational Medicine, in press.

Nakamura, M., Kitaura, J., Enomoto, Y., Lu, Y.,Nishimura, K., Isobe, M., Ozaki, K., Komeno,Y., Nakahara, F., Oki, T., Kume, H., Homma,Y., and Kitamura, T. TSC-22 is a negative-feedback regulator of Ras/Raf signaling: Im-plications for tumorigenesis. Cancer Science103: 26-33.

Shibata-Minoshima, F., Oki, T., Doki, N., Naka-hara, F., Kageyama, S., Kitaura, J., Fukuoka, J.and Kitamura, T. Identification of RHOXF2(PEPP2) as a cancer-promoting gene by ex-pression cloning. Int J Oncology 40: 93-98,

2012.Kawamura, S., Sato, I., Wada, T., Yamaguchi, K.,

Li, Y., Li, D., Zhao, X., Ueno, S., Aoki, H., To-chigi, T., Kuwahara, M., Kitamura, T., Taka-hashi, K., Moriya, S., and Miyagi, T. Plasmamembrane-associated sialidase (NEU3) regu-lates progression of prostate cancer toandrogen-independent growth through modu-lation of androgen receptor signaling. CellDeath and Differentiation 19: 170-179, 2012.

Enomoto, Y., Kitaura, J., Hatakeyama, K., Wata-nuki, J., Akasaka, T., Kato, N., Shimanuki, M.,Nishimura, K., Takahashi, M., Taniwaki, M.,Haferlach, C., Siebert, S., Dyer, M.J.S., Asou,N., Hideki Nakakuma, ＊Kitamura, T., and＊Sonoki, T. Eµ/miR-125b transgenic mice de-velop lethal B-cell malignancies. Leukemia 25:1849-1856, 2011.

Kato, N., Kitaura, J., Doki, N., Komeno, Y.,Watanabe-Okochi N., Togami, K., Nakahara,F., Oki, T., Enomoto, Y., Fukuchi, Y., Naka-jima, H., Harada, Y., Harada, H., and Kita-mura. T. Two types of C/EBPa mutationsplay distinct roles in leukemogenesis: Lessonsfrom clinical data and BMT models. Blood117: 221-233, 2011.

Yoshimi, Y., Goyama, S., Watanabe-Okochi, N.,Yoshiki, Y., Nannya, Y., Nitta, E., Arai, S.,Sato, T., Shimabe, M., Nakagawa, M., Imai, Y.,Kitamura, T. and Kurokawa, M. Evi1 re-presses PTEN expression by interacting withpolycomb complexes and activates PI3K/AKT/mTOR signaling. Blood 117: 3617-3628,2011.

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