1. Generation of Rejuvenated Antigen-Specific TCells by Reprogramming to Pluripotency andRedifferentiation

Toshinobu Nishimura1, Shin Kaneko1,9, Ai Kawa-na-Tachikawa2, Yoko Tajima1, Haruo Goto1,Dayong Zhu2, Kaori Nakayama-Hosoya2, ShoichiIriguchi1, Yasushi Uemura6, Takafumi Shimizu1,Naoya Takayama3,10, Daisuke Yamada7, KenNishimura8, Manami Ohtaka8, Nobukazu Watana-be4 , Satoshi Takahashi5 , Aikichi Iwamoto2 ,Haruhiko Koseki7, Mahito Nakanishi8, Koji Eto3,10

and Hiromitsu Nakauchi1: 1Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, 2Division of Infectious Diseases,Advanced Clinical Research Center, 3Stem CellBank, Center for Stem Cell Biology and Regenera-tive Medicine, 4Laboratory of Diagnostic Medicine,Center for Stem Cell Biology and RegenerativeMedicine, 5Division of Molecular Therapy, Ad-vanced Clinical Research Center, The Institute ofMedical Science, The University of Tokyo, Tokyo,Japan, 6Division of Immunology, Aichi CancerCenter Research Institute, Nagoya, Aichi, Japan,7Laboratory for Lymphocyte Development,RIKEN Center for Allergy and Immunology,Yokohama, Japan, 8Research Center for Stem Cell

Engineering, National Institute of Advanced In-dustrial Science and Technology, Ibaraki, Japan,9Present address: Department of Fundamental CellTechnology, Center for iPS Cell Research and Ap-plication (CiRA), Kyoto University, Kyoto, Japan,10Present address: Department of Clinical Applica-tion, CiRA, Kyoto University, Kyoto, Japan

Adoptive immunotherapy with functional T cellsis potentially an effective therapeutic strategy forcombating many types of cancer and viral infection.However, exhaustion of antigen-specific T cells rep-resents a major challenge to this type of approach.In an effort to overcome this problem, we repro-grammed clonally expanded antigen-specific CD8＋T cells from an HIV-1-infected patient to pluripo-tency. The T cell-derived induced pluripotent stemcells were then redifferentiated into CD8＋ T cellsthat had a high proliferative capacity and elongatedtelomeres. These "rejuvenated" cells possessed anti-gen-specific killing activity and exhibited T cell re-ceptor gene-rearrangement patterns identical tothose of the original T cell clone from the patient.We also found that this method can be effective forgenerating specific T cells for other pathology-asso-ciated antigens. Thus, this type of approach mayhave broad applications in the field of adoptive im-

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 Satoshi Yamazaki, Ph.D.Assistant Professor Tomoyuki Yamaguchi, Ph.D.

教 授 医学博士 中 内 啓 光助 教 理学博士 山 﨑 聡助 教 医学博士 山 口 智 之

Recent great progress in stem cell biology has brought about an increase in theprospects for application of stem cell-based therapy. Especially the discovery ofiPSCs, a great step forward in stem-cell research, holds out the promise for devel-opment of novel therapeutic strategies by generating iPSCs from patients. Thegoal of this laboratory is to provide new insights into stem cell biology as well asapproaches to novel therapeutic intervention for various intractable diseases.

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munotherapy.

2. Earliest lineage commitment of hematopoieticstem cell revealed by in vivo five lineage trac-ing system

Ryo Yamamoto1, Yohei Morita2, Jun Ooehara1,Sanae Hamanaka1,3, Masafumi Onodera4, HideoEma5, Hiromitsu Nakauchi1,3: 1Division of StemCell Therapy, Center for Stem Cell Biology andRegenerative Medicine The Institute of MedicalScience, The University of Tokyo, Tokyo, Japan,2Institute of Molecular Medicine and Max-Planck-Research Department of Stem Cell Aging, Univer-sity of Ulm, Ulm, Germany, 3Japan Science Tech-nology Agency, ERATO, Nakauchi Stem Cell andOrgan Regeneration Project, Tokyo, Japan, 4De-partment of Genetics, National Research Institutefor Child Health and Development, Tokyo, Japan,5Department of Cell Differentiation, SakaguchiLaboratory of Developmental Biology, Keio Uni-versity School of Medicine, Tokyo, Japan

While hematopoiesis, one of the best-character-ized cell differentiation systems, has provided im-portant conceptual models for basic processes ofcell differentiation, early differentiation pathways ofhematopoietic stem cells (HSCs) remain obscure.Consensus holds that HSCs give rise to multipotentprogenitors (MPPs) of reduced self-renewal poten-tial and that MPPs eventually produce lineage-com-mitted progenitor cells in a stepwise manner. How-ever, without an assay system that permits clonalassay of potentials for differentiation into all typesof hematopoietic progenitor and lineage cells, de-finitive experimental evidence for hierarchical rela-tionships among HSCs, MPPs, and lineage-commit-ted progenitor cells is lacking.The Ly5 antigen, which is only expressed by leu-

kocytes, is commonly used to distinguish betweendonor, recipient and competitor cells. However it isnot expressed by platelets and erythrocytes hencethe contribution of these populations are not ac-counted for when assessing multi-lineage reconsti-tution.To address this issue, we generated a transgenic

mouse line in which platelets and erythrocytes inaddition to neutrophils/monocytes and T and Bcells express Kusabira-Orange fluorescent protein.Highly enriched HSCs, CD150＋CD41－CD34－/lowc-Kit＋Sca-1＋Lin－ (CD150＋CD41－CD34－KSL) cells andCD150－CD41－CD34－KSL cells were individuallyisolated from KuO transgenic mice. These weretransplanted into lethally irradiated mice (B6-Ly5.2)along with 2 × 105 competitor bone marrow cells(B6-Ly5.1/Ly5.2). Secondary transplantations werecarried out by transplanting 5 × 106 bone marrowcells from the primary recipient mice into anotherlethally irradiated recipient. The peripheral blood

cells in both primary and secondary recipient micewere periodically analyzed.Unexpectedly, in the phenotypically-defined HSC

compartment we could detect not only HSCs butalso progenitors of megakaryocytes, of commonmyeloid cells, and of megakaryocyte-erythroid cellsand these progenitors exhibited long-term repopu-lation activity (rMkPs, rCMPs, and rMEPs respec-tively). This indicates that loss of self-renewal activ-ity in HSCs is not an essential step for lineage com-mitment.These results also suggested that these myeloid

committed progenitors (rCMPs, rMEPs, and rMkPs)were close to HSCs in the developmental pathway.To determine whether HSCs directly give rise to

these progenitors, we next performed paireddaughter cell assays. After a single CD150＋CD41－CD34－KSL cells divided once in culture, daughtercells were separated by micromanipulation tech-niques and individually transplanted into lethallyirradiated mice along with 2 × 105 competitor cells.Interestingly, we detected HSC-HSC, HSC-rMkP,and HSC-rCMP pairs in the paired daughter cells,suggesting that MkP and CMP commitment mayoccur at the level of HSCs through a single divisionof HSCs.In conclusion, our results demonstrated that the

CD 150＋CD 41－CD 34－KSL population containsrMkPs and rCMPs, which HSCs can give rise to viaone division. Here, we propose a revision of the hi-erarchical overview of normal hematopoiesis,namely myeloid bypass pathway from HSCs intomyeloid lineage committed progenitors (rMkPs andrCMPs) without passing through conventional MPPstatus.

3. Mesenchymal progenitor cells in mouse fetalliver regulate differentiation and proliferationof hepatoblasts.

Keiichi Ito1, Ayaka Yanagida1, Ken Okada1, YujiYamazaki2, Hiromitsu Nakauchi1,2, and AkihideKamiya3: 1Division of Stem Cell Therapy, Centerfor Stem Cell and Regenerative Medicine, The In-stitute of Medical Science, The University of To-kyo, Tokyo, Japan, 2NAKAUCHI Stem Cell andOrgan Regeneration Project, Japan Science andTechnology Agency, Tokyo, Japan, 3Laboratoy ofStem Cell Therapy, Institute of Innovative Scienceand Technology, Tokai University, Kanagawa, Ja-pan

Hepatoblasts are somatic progenitor cells of thefetal liver that possess high proliferative capacityand bi-potency for differentiation into both hepato-cytes and cholangiocytes. Although mesenchymalcells are known to be important for liver ontogeny,current understanding of their physiological role inthe fetal liver remains obscure. Here, mesenchymal

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cell populations in the developing liver were puri-fied and their potential to support proliferation anddifferentiation of hepatoblasts was examined. Fetalliver cells were fractionated with a flow cytometerusing antibodies against cell surface markers. Geneexpression of mesenchymal-specific transcripts andmorphological characteristics were analyzed. Theability of the mesenchymal cells to support hepa-toblast function was analyzed using a co-culturesystem. CD45－Ter119－CD71－Dlk1midPDGFRalpha＋cells from the mid-fetal stage liver expressed themesenchymal cell-specific transcription factors H2.0-like homeobox 1 and LIM homeobox 2 at highlevels. Fetal mesenchymal cells make contact withhepatoblasts in vivo and possess the potential todifferentiate into chondrocytes, osteocytes, and adi-pocytes under appropriate cell culture conditions,indicating that these cells are possible candidatesfor mesenchymal stem/progenitor cells. Fetal mes-enchymal cells expressed pleiotrophin, hepatocytegrowth factor, and midkine 1, which are involvedin growth of hepatoblasts. Using the co-culture sys-tem with hepatoblasts and fetal mesenchymal cells,these cells were shown to support proliferation andmaturation of hepatoblasts through indirect and di-rect interactions, respectively. These data suggestedthat Dlk1midPDGFRalpha＋ cells in non-hematopoeticfraction derived from the fetal liver exhibit mesen-chymal stem/progenitor cell characteristics andform a niche to support the development of hepa-toblasts during liver ontogeny.

4. An in vitro expansion system for generationof human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiationpotential

Ayaka Yanagida1, Keiichi Ito1, Hiromi Chikada3,Hiromitsu Nakauchi1,2, and Akihide Kamiya1,3: 1Di-vision of Stem Cell Therapy, Center for Stem Celland Regenerative Medicine, The Institute of Medi-cal Science, The University of Tokyo, Tokyo, Ja-pan, 2Japan Science and Technology Agency,NAKAUCHI Stem Cell and Organ RegenerationProject, Tokyo, Japan, 3Laboratoy of Stem CellTherapy, Institute of Innovative Science and Tech-nology, Tokai University, Kanagawa, Japan.

Hepatoblasts, hepatic stem/progenitor cells inliver development, have a high proliferative poten-tial and the ability to differentiate into both hepato-cytes and cholangiocytes. In regenerative medicineand drug screening for the treatment of severe liverdiseases, human induced pluripotent stem (iPS)cell-derived mature functional hepatocytes are con-sidered to be a potentially good cell source. How-ever, induction of proliferation of these cells is diffi-cult ex vivo. To circumvent this problem, we gener-ated hepatic progenitor-like cells from human iPS

cells using serial cytokine treatments in vitro.Highly proliferative hepatic progenitor-like cellswere purified by fluorescence-activated cell sortingusing antibodies against CD13 and CD133 that areknown cell surface markers of hepatic stem/pro-genitor cells in fetal and adult mouse livers. Whenthe purified CD13highCD133＋ cells were cultured at alow density with feeder cells in the presence ofsuitable growth factors and signaling inhibitors(ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively largecolonies. These colonies consisted of two types ofcells expressing hepatocytic marker genes (hepato-cyte nuclear factor 4alpha and alpha-fetoprotein)and a cholangiocytic marker gene (cytokeratin 7),and continued to proliferate over long periods oftime. In a spheroid formation assay, these cellswere found to express genes required for matureliver function, such as cytochrome P450 3A4 and 7A1. When these cells were cultured in a suitable ex-tracellular matrix gel, they eventually formed acholangiocytic cyst-like structure with epithelial po-larity, suggesting that human iPS cell-derived he-patic progenitor-like cells have a bipotent differen-tiation ability. Collectively these data indicate thatthis novel procedure using an in vitro expansionsystem is useful for not only liver regeneration butalso for the determination of molecular mechanismsthat regulate liver development.

5. Development of an all-in-one inducible lentivi-ral vector for gene specific analysis of repro-gramming.

Tomoyuki Yamaguchi1,2, Sanae Hamanaka1,2, Aki-hide Kamiya2, Motohito Okabe2, Mami Kawarai2,Yukiko Wakiyama1, Ayumi Umino1, TomonariHayama2, Hideyuki Sato1, Yung Sue Lee1, MegumiKato-Itoh1, Hideki Masaki1,2, Toshihiro Koba-yashi1,2, Satoshi Yamazaki1,2, Hiromitsu Nakauchi1,2:1Japan Science and Technology Agency,NAKAUCHI Stem Cell and Organ RegenerationProject, Tokyo, Japan, 2Division of Stem CellTherapy, Center for Stem Cell and RegenerativeMedicine, The Institute of Medical Science, TheUniversity of Tokyo, Tokyo, Japan.

Fair comparison of reprogramming efficienciesand in vitro differentiation capabilities among in-duced pluripotent stem cell (iPSC) lines has beenhampered by the cellular and genetic heterogeneityof de novo infected somatic cells. In order to ad-dress this problem, we constructed a single cassetteall-in-one inducible lentiviral vector (Ai-LV) for theexpression of three reprogramming factors (Oct3/4,Klf4 and Sox2). To obtain multiple types of somaticcells having the same genetic background, we gen-erated reprogrammable chimeric mice using iPSCsderived from Ai-LV infected somatic cells. Then,

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hepatic cells, hematopoietic cells and fibroblastswere isolated at different developmental stagesfrom the chimeric mice, and reprogrammed againto generate 2nd iPSCs. The results revealed that so-matic cells, especially fetal hepatoblasts were repro-grammed 1200 times more efficiently than adult he-patocytes with maximum reprogramming efficiencyreaching 12.5％. However, we found that forced ex-pression of c-Myc compensated for the reduced re-programming efficiency in aged somatic cells with-out affecting cell proliferation. All these findingssuggest that the Ai-LV system enables us to gener-ate a panel of iPSC clones derived from various tis-sues with the same genetic background, and thusprovides an invaluable tool for iPSC research.

6. Generation of Rat Gonad-like Structure fromFetal Primordial Germ Cells by Ectopic XenoTransplantation in Immune deficient Mice

Tomonari Hayama1, Tomoyuki Yamaguchi1,2 ,Ayumi Umino2, Hideyuki Sato2 Megumi Kato-Itoh2, Hiromitsu Nakauchi1,2: 1Division of Stem Cell

Therapy, Center for Stem Cell and RegenerativeMedicine, The Institute of Medical Science, TheUniversity of Tokyo, Tokyo, Japan, 2Japan Scienceand Technology Agency, NAKAUCHI Stem Celland Organ Regeneration Project, Tokyo, Japan

Primordial germ cells (PGCs) are stem cells ofgerm cell lineage in genital ridge of fetus. As PGCsgive rise to definitive oocytes and spermatozoa thatcontribute to new life in the next generation, theyhave been actively studied. However because oftheir rare population, detailed mechanisms of im-portant events (epigenetic modification, mobiliza-tion, proliferation and meiosis) are not clear yet. Onthe other hand, induction of artificial PGC frompluripotent stem cell or generation of gonad-liketissue by co-transplantation of PGCs with fetal go-nadal somatic cells has been reported recently. Toapply these new technologies to zootechnics andhuman treatment, it is necessary to establish ectopicxeno transplantation of PGCs or artificial PGCs togenerate definitive oocytes or spermatozoa in termsof ethics and safety issue.

Publications

1. Toshinobu Nishimura, Shin Kaneko, AiKawana-Tachikawa, Yoko Tajima, Haruo Go-toh, Dayong Zhu, Kaori Nakayama, ShoichiIriguchi, Yasushi Uemura, Takafumi Shimizu,Naoya Takayama, Daisuke Yamada, KenNishimura, Manami Ohtaka, Nobukazu Watan-abe, Satoshi Takahashi, Aikichi Iwamoto,Haruhiko Koseki, Mahito Nakanishi, Koji Etoand Hiromitsu Nakauchi. Generation of rejuve-nated antigen-specific T cells by pluripotencyreprogramming and dedifferentiation. Cell StemCell. (accepted for publication)

2. Oikawa T, Kamiya A, Zeniya M, Chikada H,Hyuck AD, Yamazaki Y, Wauthier E, Tajiri H,Miller LD, Wang XW, Reid LM, Nakauchi H.SALL4, a stem cell biomarker in liver cancers.Hepatology. 2012 Nov 23. doi: 10.1002/hep.26159.

3. Nakajima-Takagi Y, Osawa M, Oshima M,Takagi H, Miyagi S, Endoh M, Endo TA,Takayama N, Eto K, Toyoda T, Koseki H,Nakauchi H, Iwama A. Role of SOX17 in hema-topoietic development from human embryonicstem cells. Blood. 2012 Nov 20.

4. Lin HT, Otsu M, Nakauchi H. Stem cell ther-apy: an exercise in patience and prudence. Phi-los Trans R Soc Lond B Biol Sci. 2013 Jan 5; 368(1609): 20110334. doi: 10.1098/rstb.2011.0334.

5. Matsuno N, Yamamoto H, Watanabe N, UchidaN, Ota H, Nishida A, Ikebe T, Ishiwata K,Nakano N, Tsuji M, Asano-Mori Y, Izutsu K,Masuoka K, Wake A, Yoneyama A, NakauchiH, Taniguchi S. Rapid T-cell chimerism switch

and memory T-cell expansion are associatedwith pre-engraftment immune reaction early af-ter cord blood transplantation. Br J Haematol.2012 Nov 1. doi: 10.1111/bjh.12097.

6. Kamiya A, Nakauchi H. Enrichment and clonalculture of hepatic stem/progenitor cells duringmouse liver development. Methods Mol Biol.2013; 945: 273-86. doi: 10.1007/978-1-62703-125-7_16.

7. Hirabayashi M, Tamura C, Sanbo M, Kato-ItohM, Kobayashi T, Nakauchi H, Hochi S. A retro-spective analysis of germline competence in ratembryonic stem cell lines. Transgenic Res. 2012Aug 9. [Epub ahead of print]

8. Yamaguchi T, Hamanaka S, Kamiya A, OkabeM, Kawarai M, Wakiyama Y, Umino A,Hayama T, Sato H, Lee YS, Kato-Itoh M,Masaki H, Kobayashi T, Yamazaki S, NakauchiH. Development of an All-in-One InducibleLentiviral Vector for Gene Specific Analysis ofReprogramming. PLoS One. 2012; 7(7): e41007.Epub 2012 Jul 18.

9. Nakamura S, Oshima M, Yuan J, Saraya A, Mi-yagi S, Konuma T, Yamazaki S, Osawa M,Nakauchi H, Koseki H, Iwama A. Bmi1 confersresistance to oxidative stress on hematopoieticstem cells. PLoS One . 2012; 7(5): e36209. Epub2012 May 11.

10. Kumano K, Arai S, Hosoi M, Taoka K,Takayama N, Otsu M, Nagae G, Ueda K,Nakazaki K, Kamikubo Y, Eto K, Aburatani H,Nakauchi H, Kurokawa M. Generation of in-

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duced pluripotent stem cells from primarychronic myelogenous leukemia patient samples.Blood . 2012 Jun 28; 119(26): 6234-42. Epub 2012May 16.

11. Tashiro Y, Nishida C, Sato-Kusubata K, Ohki-Koizumi M, Ishihara M, Sato A, Gritli I, Komi-yama H, Sato Y, Dan T, Miyata T, Okumura K,Tomiki Y, Sakamoto K, Nakauchi H, Heissig B,Hattori K. Inhibition of PAI-1 induces neutro-phil-driven neoangiogenesis and promotes tis-sue regeneration via production of angiocrinefactors in mice. Blood. 2012 Jun 28; 119(26):6382-93. Epub 2012 May 9.

12. Sekine K, Takebe T, Suzuki Y, Kamiya A,Nakauchi H, Taniguchi H.Highly efficient gen-eration of definitive endoderm lineage from hu-man induced pluripotent stem cells. TransplantProc . 2012 May; 44(4): 1127-9.

13. Kobayashi T, Kato-Itoh M, Yamaguchi T, Ta-mura C, Sanbo M, Hirabayashi M, Nakauchi H.Identification of Rat Rosa26 Locus Enables Gen-eration of Knock-in Rat Lines Ubiquitously Ex-pressing tdTomato. Stem Cells Dev . 2012 Jun 11.[Epub ahead of print]

14. Nishida C, Kusubata K, Tashiro Y, Gritli I, SatoA, Ohki-Koizumi M, Morita Y, Nagano M,Sakamoto T, Koshikawa N, Kuchimaru T, Ki-zaka-Kondoh S, Seiki M, Nakauchi H, HeissigB, Hattori K. MT1-MMP plays a critical role inhematopoiesis by regulating HIF-mediatedchemokine/cytokine gene transcription withinniche cells. Blood. 2012 Jun 7; 119(23): 5405-16.Epub 2012 Apr 27.

15. Usui J, Kobayashi T, Yamaguchi T, Knisely AS,Nishinakamura R, Nakauchi H. Generation ofKidney from Pluripotent Stem Cells via Blasto-cyst Complementation. Am J Pathol. 2012 Jun;180(6): 2417-26. Epub 2012 Apr 14.

16. Hirabayashi M, Tamura C, Sanbo M, Goto T,

Kato-Itoh M, Kobayashi T, Nakauchi H, HochiS. Ability of tetraploid rat blastocysts to sup-port fetal development after complementationwith embryonic stem cells. Mol Reprod Dev .2012 Jun; 79(6): 402-12. doi: 10.1002/mrd.22043.Epub 2012 May 4.

17. Ghosn EE, Yamamoto R, Hamanaka S, Yang Y,Herzenberg LA, Nakauchi H, Herzenberg LA.Distinct B-cell lineage commitment distin-guishes adult bone marrow hematopoietic stemcells. Proc Natl Acad Sci U S A . 2012 Apr 3; 109(14): 5394-8. Epub 2012 Mar 19.

18. Wang J, Sun Q, Morita Y, Jiang H, Gros A,Lechel A, Hildner K, Guachalla LM, Gompf A,Hartmann D, Schambach A, Wuestefeld T,Dauch D, Schrezenmeier H, Hofmann WK,Nakauchi H, Ju Z, Kestler HA, Zender L, Ru-dolph KL. A Differentiation Checkpoint LimitsHematopoietic Stem Cell Self-Renewal in Re-sponse to DNA Damage. Cell . 2012. 148(5):1001-1014.

19. Oguro H, Yuan J, Tanaka S, Miyagi S, Mochi-zuki-Kashio M, Ichikawa H, Yamazaki S,Koseki H, Nakauchi H, Iwama A. Lethalmyelofibrosis induced by Bmi1-deficient hema-topoietic cells unveils a tumor suppressor func-tion of the polycomb group genes. J Exp Med .209: 445-454, 2012.

20. Ito H, Kamiya A, Ito K, Yanagida A, Okada K,Nakauchi H. In vitro expansion and functionalrecovery of mature hepatocytes from mouseadult liver. Liver Int . 2012 Jan 4. doi: 10.1111/j.1478-3231.2011.02741.x. [Epub ahead of print]

21. Umeyama K, Saito H, Kurome M, Matsunari H,Watanabe M, Nakauchi H, Nagashima H. Char-acterization of the ICSI-mediated gene transfermethod in the production of transgenic pigs.Mol Reprod Dev . 2012 Mar; 79(3): 218-28.

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1. Phenotypic analysis of ATL cells and predic-tion of the onset of ATL from human T-lym-photropic virus type 1 (HTLV-1) asymptomaticcarriers

Tomohiro Ishigaki, Seiichiro Kobayashi1,Nobuhiro Ohno2, Yuji Zaike3, Natsuko Sato, EriWatanabe, Kaoru Uchimaru2 and NobukazuWatanabe: 1Department of Molecular Therapy,2Research Hospital, 3Department of LaboratoryMedicine, IMSUT

Among the one million HTLV-1 carriers in Japan,approximately one thousand progress to ATL everyyear. Through collaborations with the ResearchHospital and two laboratories at IMSUT, we areanalyzing ATL cells using a flow cytometry-basedmethod of phenotypic analysis [HTLV-1 analyzingsystem (HAS)-Flow method] to monitor diseasecondition. In addition, we are analyzing peripheralblood from HTLV-1 carriers to find a predictablephenotypic change of immune cells just before ATLonset in order to begin more effective treatment.

2. Analysis of ATL cells and immune cells afterhematopoietic cell transplantation, DC ther-apy and anti-CCR4 antibody therapy in pa-tients with ATL.

Eri Watanabe, Natsuko Sato, Ilseung Choi4, YokoSuehiro4, Nobuaki Nakano5, Yoshitaka Inoue6, Sei-ichiro Kobayashi, Kaoru Uchimaru, AtaeUtsunomiya5, Takahiro Fukuda6, Naokuni Uike4

and Nobukazu Watanabe: 4Department of Hema-tology, National Kyushu Cancer Center; 5Depart-ment of Hematology, Imamura-bunin Hospital;6Stem Cell Transplantation Division, NationalCancer Center Hospital

In a Japanese study group of cell therapy forATL, hematopoietic cell transplantation, DC ther-apy and anti-CCR4 antibody therapy are plannedfor patients with acute ATL. We are joining thisstudy group and analyzing engraftment and ATLcells using HLA-Flow method and HAS-Flowmethod. In addition, we are analyzing ATL cellsand normal regulatory T cells with their expressionlevels of CCR4 using 12-color flow cytometer.

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

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 celland organ transplantations. Through collaborations with hospitals in Japan, wehave performed several problem-based clinical studies to tackle the issues ofadult T cell leukemia (ATL) and pathogenic conditions after transplantation, e.g.cytomegalovirus infection, graft failure, acute graft versus host disease (GVHD),relapse of leukemia, and recurrence of hepatitis.

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3. Studies for the mechanisms underlying per-sistent chimerism and late rejection after cordblood transplantation in patients with severecombined immunodeficiencies (SCID).

Eri Watanabe, Nobukazu Watanabe, KosukeImai7, Tomohiro Morio7: 7Department of Pediat-rics, Tokyo Medical Dental University

Although T cells and NK cells are lacked in pa-tients with SCID, persistent chimerism and late re-jection sometimes occur after cord blood transplan-tation. We analyze subpopulation-specific chimer-ism using HLA-Flow method and investigate theunderlying mechanisms of these pathogenic condi-tions.

4. Studies for the mechanisms underlying recur-rence of type C hepatitis and rejection afterliving-donor liver transplantation

Nobukazu Watanabe, Akinobu Takaki8, Kazuko

Koike6, Takahito Yagi9: 8Department of Gastroen-terology and Hepatology, 9Department of Gastro-enterological Surgery, Transplant and SurgicalOncology, Okayama University Graduate Schoolof Medicine and Dentistry

Since the 2004 approval of insurance coverage forliving-donor liver transplantations (LDLT), morethan 6,000 LDLTs have been performed in Japan.Although most recipients have a good prognosis,patients with hepatitis C virus (HCV) infection stillface the recurrence of hepatitis after transplantation.In addition, rejection is an important issue becauseimmunosuppressive agents are needed to suppressanti-graft immune reactions. Long-term use of im-munosuppressants, however, can worsen HCV in-fections and future malignancies. To understand themechanism underlying these pathologic conditions,we are investigating the following: chimerismanalysis/HLA-Flow method, detection of regulatoryT cells and allospecific T cells, and identification ofHCV-specific CD8＋ T cells using tetramers.

Publications

Mikami Y, Suzuki S, Ishii Y, Watanabe N, Taka-hashi T, Isokawa K, Honda MJ. The p75 neu-rotrophin receptor regulates MC3T3-E1 osteoblas-

tic differentiation. Differentiation. 84(5): 392-399,2012.

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1. Pleiotropic nature of hematopoietic stem cellresponses to an inflammatory niche environ-ment

Makoto Otsu, Huang-Ting Lin, Jun Ooehara,Chen-Yi Lai, Mozhgan Kharaj, Takashi Ishida,Naoaki Mizuno, Hiromitsu Nakauchi

Hematopoietic stem cells (HSCs) have been util-ized in transplantation settings to treat a range ofintractable disorders. These include hematopoieticmalignancies and primary immunodeficiencies,whereby successful outcomes are defined as sus-tained reconstitution of functional hematopoiesis bytransplanted HSCs. Central to this success is thebone marrow "niche" and the microenvironmentregulated by its constituent cells. It is an anatomicallocation that transplanted HSCs ultimately reside inhence their behavior can be highly susceptible toenvironmental changes. Therefore, niche influencesmay strongly impact upon HSC functions and thesuccess of clinical outcomes. As such, worth seriousconsideration are incidences of negative responsesin the form of "inflammation" following precondi-tioning treatment and/or allogeneic reactions in re-lation to transplantation. In such cases, inflamma-tory cytokines are secreted into the microenviron-ment, which could exert detrimental effects onHSCs. From initial screening experiments, it was

found that interleukin (IL)-1 and tumor necrosisfactor (TNF)- were rapidly produced withinmurine BM in response to total body irradiation(TBI). The detrimental effects of these cytokines ondonor HSCs were substantiated by demonstratingloss of function in both in vivo and ex vivo studies.These results highlighted the pleiotropic nature ofHSC functional responses to these cytokines. Forexample, the pleiotropism became greater with fac-tors such as age (donor and recipient) and the oxy-gen tension of culture.In this presentation, two factors "age" and "in-

flammation" shall be discussed in depth particu-larly in the context of transplantation. These are im-portant considerations that may be critical in devel-oping new strategies to optimize clinical outcomesfor transplanted patients.

2. Stage-specific roles for Cxcr4 signaling inmurine hematopoietic stem/progenitor cells inthe process of bone marrow repopulation

Chen-Yi Lai, Satoshi Yamazaki, Sachie Suzuki,Shigeru Kakuta, Yoichiro Iwakura, Makoto Otsu,Hiromitsu Nakauchi

Hematopoietic cell transplantation has provenbeneficial for various intractable diseases, but howhematopoietic stem/progenitor cells (HSPCs) home

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. 特任准教授 医学博士 大 津 真

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.

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to the bone marrow (BM) microenvironment andinitiate hematopoietic reconstitution remains un-clear. The use of newly elucidated molecular deter-minants for HSPC engraftment should benefit pa-tients. Here we report that modification of Cxcr4signaling in murine HSPCs does not significantlyaffect initial homing/lodging events, but leads to al-teration in subsequent BM repopulation kinetics. Byusing C-terminal truncated Cxcr4 as a gain-of-func-tion effector, we demonstrated that signal augmen-tation likely led to favorable in vivo retention/ex-pansion of primitive cell populations, possibly inpart through alterations in integrin expression pro-file. Unexpectedly however, sustained signal en-hancement even with wild-type Cxcr4 overexpres-sion resulted in impaired peripheral blood (PB) re-constitution, most likely by preventing release ofmature leukocytes from the marrow environment.We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing HSPCs withenhanced repopulation potential following trans-plantation, whilst preserving the ability to releaseHSPC progeny into PB for improved transplanta-tion outcomes.

3. Protection of hematopoietic stem cells fromstress-induced functional impairment by verylow-dose interleukin-1 stimulation

Jun Ooehara, Chen-Yi Lai, Huang-Ting Lin,Satoshi Yamazaki, Makoto Otsu, HiromitsuNakauchi

Background: Hematopoietic stem cell (HSC) genetherapy is a treatment option that potentially pro-vides life-long immune reconstitution for patientswith primary immunodeficiency diseases. Virus-mediated gene transfer is the measure currentlyused to confer functionality on patients' bloodCD34＋ cells (HSPCs). Because this procedure typi-cally requires ex vivo stimulation of HSPCs for 4-5days, it is essential to optimize the cocktail of cy-tokines so that the transduced cells retain the bestreconstitution capability. Here we sought to revisitthe use of interleukin-1 (IL-1), which is the well-known cytokine, but its positive effects on highlypurified HSCs remain doubtful.Results: By analyzing IL-1 receptor knockout

mice, we found impaired reconstitution ability intheir HSCs, suggesting the positive effect of IL-1 atthe stem cell level. We then tested in vitro effects ofIL-1 in a 7-day serum-free culture and observed itsdose-dependent biphasic effects on HSCs: while itforced HSC differentiation at the concentrationshigher than ～10 ng/ml, it clearly enhanced prolif-erative response of the primitive cells at low con-centrations around 0.05 ng/ml. Consistent with this,addition of IL-1 at a limited dose improved in vivoreconstitution capability of cultured HSCs in both

primary and secondary recipients, and the positiveeffects became more evident with certain stress con-ditions including hypoxia and aging.Conclusions: This study provides the possible re-

vival of one of the oldest cytokine as a positive am-plifier/protector of HSCs, potentially culminating inthe improvement in long-term hematopoiesis that isrelevant to clinical gene therapy trials.In vitro studies: HSCs overexpressing wt- or C-

CXCR4 exhibited enhanced chemotaxis and prolif-eration in response to SDF1, confirming the gain-of-function effects of these modifications. CA formingability was greater in HSCs overexpressing C-CXCR4 than control counterparts and absent inCXCR4-KO HSCs, suggesting the critical role ofCXCR4-signaling in HSC proliferation in the pres-ence of stromal support.In vivo studies: 1) the homing/lodging phase. Un-

expectedly, we did not find significant alteration inthe numbers of early progenies detectable in recipi-ent BM 3 days after transplantation of HSCs receiv-ing either loss- or gain-of-function modification toCXCR4, indicating that this signaling is indispensa-ble in HSC homing. 2) the early repopulationphase. Impairment of hematopoietic repopulationin BM became evident for CXCR4-KO HSCsthrough 2-3 wks. On the other hand, HSCs overex-pressing CXCR4, more remarkably of C-mutation,showed enhanced BM repopulation kinetics at ～3wks post transplantation, suggesting the impor-tance of CXCR4 signaling in HSC amplification atthis post-transplantation phase. 3) long-term hema-topoiesis. CXCR4-KO-HSCs showed poor hema-topoietic reconstitution potentials, consistent withprevious observations. Interestingly, impaired pe-ripheral repopulation was also observed with HSCsoverexpressing wt- or C-CXCR4. Further charac-terization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies,which showed multilineage differentiation, but ex-hibited impaired release of mature leukocytes fromthe BM to the peripheral blood. Most importantly,however, test-cell chimerism in the long-term HSCfraction was significantly higher in the mice receiv-ing HSCs overexpressing CXCR4, especially of C-type, than those transplanted with control HSCs,indicating that the augmentation of CXCR4 signal-ing enhanced competitive repopulation ability ofHSCs. These modified HSCs demonstrated repopu-lation abilities also in secondary recipients.We demonstrated that CXCR4 signaling is indis-

pensible for HSC homing and that continuous over-expression of CXCR4 cannot benefit the peripheralreconstitution in contrary to the expectation. Moreimportantly, our studies showed that augmentationof CXCR4 signaling leads to HSC expansion invivo. We thus conclude that CXCR4 signaling has arole in HSC self-renewal and that its regulationmay find the approach that will improve HSC

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transplantation outcomes.

Publications

1. Nishimura S, Manabe I, Nagasaki M, Kakuta S,Iwakura Y, Takayama N, Ooehara J, Otsu M,Kamiya A, Petrich BG, Urano T, Kadono T, SatoS, Aiba A, Yamashita H, Sugiura S, Kadowaki T,Nakauchi H, Eto K, Nagai R. In vivo imagingvisualizes discoid platelet aggregations withoutendothelium disruption and implicates contribu-tion of inflammatory cytokine and integrin sig-naling. Blood. 2012; 119(8): e45-56.

2. Nakanishi M, Otsu M. Development of Sendai

Virus Vectors and Their Potential Applications inGene Therapy and Regenerative Medicine. Cur-rent gene therapy. 2012. Epub 2012/08/28.

3. Kumano K, Arai S, Hosoi M, Taoka K,Takayama N, Otsu M, Nagae G, Ueda K,Nakazaki K, Kamikubo Y, Eto K, Aburatani H,Nakauchi H, Kurokawa M. Generation of in-duced pluripotent stem cells from primarychronic myelogenous leukemia patient samples.Blood. 2012; 119(26): 6234-42.

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1. Inhibition of PAI-1 induces neutrophil-drivenneoangiogenesis and promotes tissue regen-eration via production of angiocrine factors inmice

Yoshihiko Tashiro1, Chiemi Nishida2, Kaori Sato-Kusubata2, Makiko Ohki-Koizumi2, Makoto Ishi-hara2, Aki Sato2, Ismael Gritli2, Hiromitsu Komi-yama1,Yayoi Sato2, Takashi Dan3, Toshio Miyata3,Ko Okumura4, Yuichi Tomiki1, Kazuhiro Saka-moto1, Hiromitsu Nakauchi2, Beate Heissig,2,4 andKoichi Hattori2,4: 1Department of ColoproctologicalSurgery, Juntendo University faculty of Medicine,2Center for Stem Cell Biology and RegenerativeMedicine, Institute of Medical Science at the Uni-versity of Tokyo, 3United Centers for AdvancedResearch and Translational Medicine (ART), To-hoku University Graduate School of Medicine,4Atopy (Allergy) Center, Juntendo UniversitySchool of Medicine

Plasminogen activator inhibitor-1 (PAI-1), an en-dogenous inhibitor of a major fibrinolytic factor, tis-sue-type plasminogen activator, can both promoteand inhibit angiogenesis. However, the physiologicrole and the precise mechanisms underlying the an-giogenic effects of PAI-1 remain unclear. In the pre-sent study, we report that pharmacologic inhibition

of PAI-1 promoted angiogenesis and prevented tis-sue necrosis in a mouse model of hind-limb ische-mia. Improved tissue regeneration was due to anexpansion of circulating and tissue-resident granu-locyte-1 marker (Gr-1(＋)) neutrophils and to in-creased release of the angiogenic factor VEGF-A,the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated in-creased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization andgenetic knockout studies indicated that both the im-proved tissue regeneration and the increase in cir-culating and ischemic tissue-resident Gr-1(＋) neu-trophils depended on the activation of tissue-typeplasminogen activator and matrix metalloprotein-ase-9 and on VEGF-A and FGF-2. These results sug-gest that pharmacologic PAI-1 inhibition activatesthe proangiogenic FGF-2 and VEGF-A pathways,which orchestrates neutrophil-driven angiogenesisand induces cell-driven revascularization and istherefore a potential therapy for ischemic diseases.

2. New functions of the fibrinolytic system inbone marrow cell-derived angiogenesis.

Beate Heissig1,2,3, Makiko Ohki-Koizumi1, Yoshi-hiko Tashiro1, Ismael Gritli1, Kaori Sato-

Center for Stem Cell Biology and Resenerative 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

The major goal of our laboratory is to identify novel therapeutic targets for dis-eases like cancer or inflammatory diseases. We study the role of inflammatorycells and adult stem cells in regenerative and cancer biology. Stem cells fromadult tissues have the unique capacity to repair damaged tissue, a process con-trolled in part by the microenvironment. We currently focused our scientific effortson understanding the mechanism how inflammatory cells control blood vessel for-mation thereby promoting cancer growth or tissue regeneration (1-3), and whichfactors can alter the generation of blood cells (4,5).

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Kusubata2, Koichi Hattori1,3: 1Center for Stem CellBiology and Regenerative Medicine, Institute ofMedical Science at the University of Tokyo, 2Fron-tier Research Initiative, Institute of Medical Sci-ence at the University of Tokyo, 3Atopy (Allergy)Research Center, Juntendo University School ofMedicine

Angiogenesis is a process by which new bloodvessels form from preexisting vasculature. Thisprocess includes differentiation of angioblasts intoendothelial cells with the help of secreted angio-genic factors released from cells such as bone mar-row (BM)-derived cells. The fibrinolytic factor plas-min, which is a serine protease, has been shown topromote endothelial cell migration either directly,by degrading matrix proteins such as fibrin, or in-directly, by converting matrix-bound angiogenicgrowth factors into a soluble form. Plasmin can alsoactivate other pericellular proteases such as matrixmetalloproteinases (MMPs). Recent studies indicatethat plasmin can additionally alter cellular adhesionand migration. We showed that factors of the fibri-nolytic pathway can recruit BM-derived hema-topoietic cells into ischemic/hypoxic tissues by al-tering the activation status of MMPs. These BM-de-rived cells can function as accessory cells that pro-mote angiogenesis by releasing angiogenic signals.This review will discuss recent data regarding therole of the fibrinolytic system in controllingmyeloid cell-driven angiogenesis. We propose thatplasmin/plasminogen may be a potential target notonly for development of effective angiogenic thera-peutic strategies for the treatment of cancer, butalso for development of strategies to promoteischemic tissue regeneration.

3. Plasmin inhibitor reduces lymphoid tumorgrowth by suppressing matrixmetallprotein-ase-9 dependent CD11b＋/F4/80＋ myeloidcell recruitment.

Makoto Ishihara, Chiemi Nishida, YoshihikoTashiro, Ismael Gritli, Jeanette Rosenkvist,Makiko Koizumi, Ryo Yamamoto, Hideo Yagita1,Ko Okumura2 , Momoko Nishikori3 , KeikoWanaka4, Yuko Tsuda5, Yoshio Okada5, HiromitsuNakauchi, Beate Heissig2,6, Koichi Hattori: 1De-partment of Immunology, Juntendo UniversitySchool of Medicine, 2Atopy (Allergy) ResearchCenter, Juntendo University School of Medicine,3Department of Hematology and Oncology, KyotoUniversity, 4Kobe Research Projects on Thrombo-sis and Haemostasis, 5Faculty of PharmaceuticalSciences, Kobe Gakuin University, 6Frontier Re-search Initiative, Institute of Medical Science atthe University of Tokyo

Activation of the fibrinolytic system during lym-

phoma progression is a well-documented clinicalphenomenon. But the mechanism by which the fi-brinolytic system can modulate lymphoma progres-sion has been elusive. The main fibrinolytic en-zyme, plasminogen (Plg)/plasmin (Plm), can acti-vate matrix metalloproteinases (MMPs), like MMP-9, which has been linked to various malignancies.Here we provide the evidence that blockade of Plgreduces lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b＋F4/80＋ myeloidcells locally within the lymphoma tissue. Geneticplasminogen deficiency and drug-mediated Plmblockade delayed lymphoma growth and dimin-ished MMP-9 dependent CD11b＋F4/80＋ myeloidcell infiltration into lymphoma tissues. A neutraliz-ing antibody against CD11b inhibited lymphomagrowth in vivo, which indicates that CD11b＋myeloid cells play a role in lymphoma growth. Plgdeficiency in lymphoma-bearing mice resulted inreduced plasma levels of the growth factors vascu-lar endothelial growth-A and Kit ligand, both ofwhich are known to enhance myeloid cell prolifera-tion. Collectively, the data presented in this studydemonstrate a previously undescribed role of Plmin lymphoproliferative disorders and providestrong evidence that specific blockade of Plg repre-sents a promising approach for the regulation oflymphoma growth.

4. MT1-MMP plays a critical role in hematopoi-esis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells.

Chiemi Nishida1, Kaori Kusubata2, YoshihikoTashiro1, Ismael Gritli1, Aki Sato1, Makiko Ohki-Koizumi1, Yohei Morita1, Makoto Nagano3, Take-haru Sakamoto3, Naohiko Koshikawa3, Shinae Ki-zaka -Kondoh4 , Motoharu Seiki3 , HiromitsuNakauchi1, Beate Heissig1,2,5, Koichi Hattori1,5:1Center for Stem Cell Biology and RegenerativeMedicine, Institute of Medical Science, The Uni-versity of Tokyo, 2Frontier Research Initiative, In-stitute of Medical Science at the University of To-kyo, 3Division of Cancer Cell Research, Institute ofMedical Science, The University of Tokyo, 4De-partment of Biomolecular Engineering, Tokyo In-stitute of Technology, 5Atopy (Allergy) ResearchCenter, Juntendo University School of Medicine

HSC fate decisions are regulated by cell-intrinsicand cell-extrinsic cues. The latter cues are derivedfrom the BM niche. Membrane-type 1 matrix metal-loproteinase (MT1-MMP), which is best known forits proteolytic role in pericellular matrix remodel-ing, is highly expressed in HSCs and stromal/nichecells. We found that, in MT1-MMP(－/－) mice, inaddition to a stem cell defect, the transcription andrelease of kit ligand (KitL), stromal cell-derived fac-tor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-

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7 was impaired, resulting in a trilineage hema-topoietic differentiation block, while addition of ex-ogenous KitL and SDF-1 restored hematopoiesis.Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1)within niche cells, thereby inducing the transcrip-tion of HIF-responsive genes, which induce termi-nal hematopoietic differentiation. Thus, MT1-MMPin niche cells regulates postnatal hematopoiesis, bymodulating hematopoietic HIF-dependent nichefactors that are critical for terminal differentiationand migration.

5. Plasminogen deficiency attenuates post-natalerythropoiesis in male C57BL/6 mice throughdecreased activity of the LH-testosterone axis

Yurai Okaji1, Yoshihiko Tashiro, Ismael Gritli,Chiemi Nishida, Aki Sato, Yoko Ueno1, Sandra DelCanto Gonzalez1, Makiko Ohki-Koizumi, HaruyoAkiyama, Hiromitsu Nakauchi2, Koichi Hattori3,Beate Heissig1,3: 1Frontier Research Initiative, 2Di-vision of Stem cell Therapy, Center for Stem CellBiology and Regenerative Medicine, Institute ofMedical Sciences, University of Tokyo, 3AtopyCenter, Juntendo University

Novel roles for the serine protease plasmin havebeen recently implicated in physiological andpathological processes. However, whether plasminis involved in erythropoiesis, is not known. In the

present study, we studied the consequences of plas-minogen deficiency on erythropoiesis in plasmino-gen deficient (Plg KO) mice. Erythroid differentia-tion was attenuated in male Plg KO mice and re-sulted in erythroblastic accumulation within thespleen and bone marrow, with increased apoptosisin the former, erythrocytosis and splenomegaly,whereas similar erythropoietic defect was lessprominent in female Plg KO mice. In addition,erythrocyte lifespan was shorter in both male andfemale Plg KO mice. Erythropoietin levels werecompensatory increased in both male and femalePlg KO mice, and resulted in a higher frequency ofBFU-E within the spleen and bone marrow. Sur-prisingly, we found that male Plg KO mice but nottheir female counterparts exhibited normochromicnormocytic anemia. The observed gender-linkederythropoietic defect was attributed to decreasedserum testosterone levels in Plg KO mice, as a con-sequence of impaired secretion of the pituitary lu-teinizing hormone (LH) under steady state condi-tion. Surgical castration, causing testosterone defi-ciency and stimulating LH release, attenuatederythroid differentiation and induced anemia in WTanimals, but did not further decrease the hematocritlevels in Plg KO mice. In addition, complementa-tion of LH using human choriogonadotropin, whichincreases testosterone production, improved theerythropoietic defect and anemia in Plg KO mice.The present results identify a novel role for plasminin the hormonal regulation of post-natal erythropoi-esis by the LH-testosterone axis.

Publications

〈Koichi Hattori Group〉1. Yoshihiko Tashiro, Chiemi Nishida, Kaori Sato-

Kusubata, Makiko Ohki-Koizumi, Makoto Ishi-hara, Aki Sato, Ismael Gritli, Hiromitsu Komi-yama, Yayoi Sato, Yuichi Tomiki, HirokazuSakamoto, Takashi Dan, Toshio Miyata, KoOkumura, Hiromitsu Nakauchi, Beate Heissig,and Koichi Hattori: Inhibition of PAI-1 inducesneutrophil-driven neoangiogenesis and pro-motes tissue regeneration via production of an-giocrine factors in mice. Blood. l19(26) 6382-6393. 2012

2. Beate Heissig, Makiko Ohki-Koizumi, YoshihikoTashiro, Ismael Gritli, Kaori Sato-Kusubata andKoichi Hattori. New functions of the fibrinolyticsystem in bone marrow cell-derived angiogene-sis. International Journal of Hematology 95(2).131-137. 2012

3. Ishihara M, Nishida C, Tashiro Y, Gritli I, Ro-senkvist J, Koizumi M, Yamamoto R, Yagita H,Okumura K, Nishikori M, Wanaka K, Tsuda Y,Okada Y, Nakauchi H, Heissig B, Hattori K:,Plasmin inhibitor reduces T-cell lymphoid tu-

mor growth by suppressing matrix metallopro-teinase-9-dependent CD11b＋/F4/80＋ myeloidcell recruitment. Leukemia, 26. 332-339. 2012

4. Chiemi Nishida, Kaori Kusubata, YoshihikoTashiro, Ismael Gritli, Aki Sato, Makiko Ohki-Koizumi, Yohei Morita, Makoto Nagano, Take-haru Sakamoto, Naohiko Koshikawa, TakahiroKuchimaru, Shinae Kizaka-Kondoh, MotoharuSeiki, Hiromitsu Nakauchi, Beate Heissig, andKoichi Hattori. MT1-MMP plays a critical rolein hematopoiesis by regulating HIF-mediatedchemo-/cytokine gene transcription within nichecells. Blood. 119(23) 5405-5416. 2012

5. Okaji Y, Tashiro Y, Gritli I, Nishida C, Sato A,Ueno Y, Del Canto Gonzalez S, Ohki-KoizumiM, Akiyama H, Nakauchi H, Heissig B, HattoriK: Plasminogen deficiency attenuates post-natalerythropoiesis in male C57BL/6 mice throughdecreased activity of the LH-testosterone axis.Exp Hematol, Vol 40, 143-154, 2012

6. 服部浩一，島津浩：骨髄における巨核球系細胞の成熟分化機構，医薬ジャーナル社，２０１３ 印刷中

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7. 服部浩一，宗像慎也：造血系細胞による血管新生制御機構，血管新生研究の最先端，医薬ジャーナル社，５６―６９，２０１３

8. 服部浩一，島津浩：がん微小環境形成に関与する骨髄由来細胞と治療戦略，細胞，ニューサイエンス社，Vol４４．（１１），２０１２

9. 服部浩一，佐藤亜紀：血液線維素溶解系因子による造血系細胞の動態制御機構，臨床血液，日本血液学会，Vol５３．６８０―６８５，２０１２

10. 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

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1. Novel method for efficient production of mul-tipotential hematopoetic progenitors from hu-man pluripotent stem cells

Feng Ma, Yasuhiro Ebihara1, Shinji Mochizuki,Shohei Yamamoto1, Sachiyo Hanada, SahokoMatsuzaka, Yuji Zaike2, Hiromitsu Nakauchi3,Kohichiro Tsuji; 1Department of Pediatric Hema-tology-Oncology, and 2Department of LaboratoryMedicine, Research Hospital, 3Division of StemCell Therapy, Center for Stem Cell Therapy andRegenerative Medicine

ESC are pluripotent cells derived from the innercell mass of preimplantation embryos, and iPSC areinduced from somatic cells by nuclear reprogram-ming. Since both have the ability to be maintainedin culture indefinitely as undifferentiated cells, yetthey are capable of forming more differentiated celltypes, they are expected as a novel source of hu-man transplantable cells for the regenerative medi-cine. We then planed to produce hematopoieticstem cells (HSC) for therapeutic HSC transplanta-tion and functional blood cells for transfusionmedicine from these human pluripotent stem cells.In result, we developed a novel method for the effi-cient production of hematopoietic progenitor cells(HPC) from hESC and hiPSC by co-culture with

AGMS-3 stromal cells which originates from mirineaorta-gonad-mesoneephros (AGM) region at 11 to12 dpc. In the co-culture, various hematopoieticprogenitors were generated, and this hematopoieticactivity was concentrated in cobblestone-like (CS)cells within differentiated human ES or iPS cellcolonies. The CS cells expressed CD34 and retaineda potential for endothelial cells. They also containedHPC, especially erythroid and multipotential HPCat high frequency. The multipotential HPC abun-dant among the CS cells produced all types of ma-ture blood cells, including adult type globin-ex-pressing erythrocytes and tryptase and chymase-double positive mast cells (MC). They showed nei-ther immature properties of PSC nor potentials todifferentiate into endoderm and ectoderm at a clo-nal level. The developed co-culture system of hPSCcan provide a novel source for hematopoietic andblood cells applicable to cellular therapies and drugscreenings.

2. Generation of functional erythrocytes fromhuman ES or iPS cell-derived definitive hema-topoiesis

Feng Ma, Yasuhiro Ebihara1, Shinji Mochizuki,Shohei Yamamoto1, Sachiyo Hanada, SahokoMatsuzaka, Yuji Zaike2, Hiromitsu Nakauchi3,

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 辻 浩一郎特任助教 医学博士 望 月 慎 史

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.

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Kohichiro Tsuji

A critical issue for utilization of hESC or hiPSCin possible clinical use is whether they can deriveterminally mature progenies with the normal func-tion. To solve this, we examined hESC or hiPSC-de-rived erythroid cells in coculture with mFLSC orAGM cells. By the coculture, large quantity of hESCor hiPSC-derived erythroid progenitors allowed usto analyze the development of erythropoiesis at aclone level and to investigate their function as oxy-gen carrier. The results showed that the globin ex-pression in the erythroid cells in individual cloneschanged in a time-dependent manner. In particular,embryonic globin positive erythrocytes decreased,while adult-type globin positive cells increased toalmost 100％ in all single clones we examined, indi-cating they had already been fated to definitive he-matopoiesis. Enucleated erythrocytes also appearedin the clonal erythroid progenies. A comparisonanalysis showed that hESC-derived erythroid cellstook a similar pathway in differentiation to humancord blood CD34＋ progenitor-derived erythrocyteswhen traced by glycophorin A, CD71 and CD81.Furthermore, these hESC-derived erythroid cellscould function as oxygen carrier, and had a suffi-cient glucose-6-phosphate dehydrogenase activity.The present study provided an experimental modelto explore early development of human erythropoi-esis, hemoglobin switching, erythroid pathogenesis,and to discover drugs for hereditary diseases inerythrocyte development. The quantitative produc-tion and their functional maturation indicate thathPSC-derived erythrocytes can be a novel potentialsource for therapeutic transfusion.

3. Derivation of blood cells from human pluripo-tent stem cells in culture without animal se-rum or cells

Yasuhiro Ebihara1, Feng Ma, Shinji Mochizuki,Shohei Yamamoto1, Sachiyo Hanada, SahokoMatsuzaka, Yuji Zaike2, Hiromitsu Nakauchi3,Kohichiro Tsuji

It is inevitable to establish an in vitro culturemethod for the induction of hPSC, such as hESC orhiPSC, to differentiate into mature blood cells with-out animal serum and cells. To achieve this, wefirst induced hPSC to differentiate into mesenchy-mal stem cells (MSC). When human ES or iPS cellscultured on murine embryonic fibroblast (MEF)feeder cells were recultured on gelatin-coated cul-ture dishes with platelet lysate (PL)-containing me-dia in the absence of MEF feeder cells. Cells werepassaged several times with PL containing media,and then MSC were induced after 6 to 8 weeks. TheMSC were spindle-like shaped, revealed a pheno-type of CD45－, CD34－, CD14－, CD105＋, CD

166＋, CD31－, and SEA-4－, and had the ability todifferentiate into mesenchymal tissues such asbone, cartilage and fat in vitro. Murine MEF andundifferentiated hPSC were undetectable in thehPSC-derived MSC by reverse transcription polym-erase chain reaction analysis. We then coculturedhPSC with MSC derived from hPSC themselves un-der serum-free condition. Two weeks later, a num-ber of HPC appeared in the coculture. These HPCwere cultured in hematopoietic colony assay usinghuman serum. In result, hPSC-derived HPC pro-duced various hematopoietic colonies, such asmyeloid, erythroid and multilineage colonies, in-cluding all types of blood cells. The novel culturemethod must be useful for the clinical applicationof hPSC-derived blood cells.

4. Differential production of connective tissue-type and mucosal mast cells from hESC foranti-allergy drug screening

Feng Ma, Yang Wenyu, Yanzheng Gu, YasuhiroEbihara1, Shinji Mochizuki, Shohei Yamamoto1,Sachiyo Hanada, Sahoko Matsuzaka, HiromitsuNakauchi3, Kohichiro Tsuji

MC function as effector cells in allergy and atopicdisease. Therefore, anti-allergy drugs have been es-tablished to diminish MC function. However, sincethe acquisition of an abundance of human MC(hMC) is difficult because of no culture methodproducing massive hMC, most anti-allergy drugstargeted animal MC. Thus, efficient discovery of ef-fective anti-allergy drugs needs to establish the cul-ture system of massive hMC. Then, hESC are con-sidered as a potential cell source for hMC. In hu-man, two types of MC have been characterized;connective tissue-type and mucosal MC (CTMCand MMC, respectively). CTMC contain tryptase,chymase, MC carboxypeptidase and cathepsin G intheir secretory granules, are predominantly locatedin normal skin and in intestinal submucosa, and in-volve in atopic dermatitis. MMC contain tryptase intheir secretory granules, but lack the other pro-teases, are the main type of MC in normal alveolarwall and in small intestinal mucosa, and involve inallergic rhinitis or bronchial asthma. Although MCcan be generated from human adult CD34＋ HPC invitro, these MC are mainly MMC. So far, there lacksan evidence for the direct derivation of CTMC fromadult HPC. We achieved successful production ofhESC-derived CD34＋ HPC, using coculture withAGMS-3 cells for 1-2 weeks. In suspension culturefavoring MC differentiation within 3weeks, hESC-derived progenitors generated mature MC thatshared a chymase/tryptase double positive pheno-type and strongly expressed c-Kit, similar to humanskin derived CTMC. On the other hand, hESC-de-rived multipotential hematopoietic progenitors ob-

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tained in clonal culture developed into MC for alonger time (over 5 weeks) and only expressedtryptase, with no or few chymase, similar to humanCD34＋ cell-derived MMC. Since the current culturesystem of hESC can produce differentially a largenumber 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, YasuhiroEbihara1, Shinji Mochizuki, Shohei Yamamoto1,Sachiyo Hanada, Sahoko Matsuzaka, HiromitsuNakauchi3, Kohichiro Tsuji

Eosinophils are multifunctional leukocytes impli-cated in the pathogenesis of numerous inflamma-tory processes. As the major effectors, eosinophilsfunction in a variety of biological responses, allergicdiseases and helminth infections. It is generally ac-cepted human eosinophils develop through a path-way initially sharing common feature with baso-phils. However, there lacks a clear chart for earlydevelopment of human eosinophils, such as duringembryonic or fetal stages. We established an effi-cient method for producing eosinophils from hESCand hiPSC. By a two-step induction, we first gener-ated multipotential HPC by co-culturing hPSC withAGMS-3 cells for 2 weeks. Then, total co-culturecells were transferred into suspension culture favor-ing eosinophil development with addition of IL-3and other factors (stem cell factor, interleukin-6,thrombopoietin, Flt-3 ligand) . The maturation ofhPSC-derived eosinophils was shown in a time-de-pendent manner, first co-expressing eosinophil-andbasophil-specific markers [eosinophil peroxidase(EPO), and 2D7, respectively], then the portion ofeosinophil markers gradually increased while thatof basophil markers decreased, typically mimickingthe development 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-dependent manner,paralleling to those with EPO. The expression ofeosinophil-specific granule cationic proteins (EPO,MBP, ECP, EDN) and IL-5 receptor mRNA wasalso detected by RT-PCR. Furthermore, transmis-sion electron microscopy (TEM) observation con-firmed the eosinophil property. Eosinophils derivedfrom hiPSCs hold similar characteristics as thosefrom hESCs. Our study provides an experimentalmodel for exploring early genesis of eosinophils, es-pecially in uncovering the mechanisms controllingthe development of the initial innate immune sys-tem of human being in normal and diseased indi-

viduals.

6. Hematopoiesis of human induced pluripotentstem cells derived from patients with Downsyndrome

Natsumi Nishihama, Yasuhiro Ebihara1, ShinjiMochizuki, Shohei Yamamoto1, Feng Ma, WenyuYang, Kiyoshi Yamaguchi4, Masaharu Hiratsuka5,Yoichi Furukawa4, Mitsuo Oshimura5, HiromitsuNakauchi3, Kohichiro Tsuji; 4Division of ClinicalGenome Research, 5Division of Molecular and CellGenetics, Department of Molecular and CellularBiology, Faculty of Medicine, Tottori University

Trisomy 21, genetic hallmark of Down syndrome,is the most frequent human chromosomal abnor-mality. Infants and children with Down Syndrome(DS) are known to have some hematological disor-ders with an increased risk of developing leukemia.Ten to 20％ of newborn with DS are diagnosed asneonatal preleukemic status, transient myeloprolif-erative disorder (TMD), and approximately 30％ ofTMD patients are predisposed to acutemegakaryoblastic leukemia (AMKL). Recently, ac-quired mutations in the N-terminal activation do-main of the GATA1 gene, leading to expression of ashorter GATA1 isoform (GATA1s), have been re-ported in AMKL and TMD, but neither patients normice with germline mutations leading to expressionof GATA1s developed AMKL and TMD in the ab-sent of trisomy 21. These findings suggested thattrisomy 21 itself directly contributes to the develop-ment of AMKL and TMD. However, the role of tri-somy 21 in hematopoiesis, particularly in the hu-man fetus remains poorly understood. To better un-derstand the effects of trisomy 21 on hematopoiesisin embryonic stage and leukemogenesis, we em-ployed hiPSC derived from patients with DS (DS-hiPSC). Six DS-hiPSC and 5 hiPSC lines (control)from healthy donors were all created from skin fi-broblasts and reprogrammed by the defined 3 or 4reprogramming factors (OCT3/4, KLF4, and SOX2,or c-MYC in addition to the 3 factors, respectively).We generated blood cells from DS-hiPSC and con-trols with coculture system using AGMS-3 cells.The cells from hiPSC were harvested at day 11 or12 of coculture and analyzed the presence of hema-topoietic markers and the potentials of hematopoie-tic colony formation. In the experiments usinghiPSC reprogrammed by 3 factors, human CD34 ex-pression in harvested cells from DS-hiPSC or con-trols were detected 10.06 ± 4.35％ and 3.04％, re-spectively. We next examined the hematopoieticcolony formation. Both myeloid and erythroid colo-nies were detected. Number of colonies formedfrom DS-hiPSC was 43.7±11.1 to 74.3±11.2 per aniPS cell colony, which was approximately 2 to 3.5folds the number of control. Similar results were

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obtained in the experiments using hiPSC repro-grammed by 4 factors. These results indicated thathiPSC derived from patients with DS could differ-entiate into multiple hematopoietic cell lineages andthe differentiation into hematopoietic lineage waspromoted in DS patients.

7. Wnt3a stimulates maturation of impaired neu-trophils developed from severe congenitalneutropenia patient-derived pluripotent stemcells

Takafumi Hiramoto, Yasuhiro Ebihara1, Mochi-zuki, Shohei Yamamoto1, Masao Nagasaki6, YoichiFurukawa4, Hiromitsu Nakauchi3, Masao Ko-bayashi7, Kohichiro Tsuji: 6Functional Genomics,Human Genome Center, 7Department of Pediat-rics, Hiroshima University Graduate School ofBiomedical Sciences

The derivation of induced pluripotent stem cells(iPS cells) from individuals of genetic disorders of-fers new opportunities for basic research into thesediseases and the development of therapeutic com-pounds. Severe congenital neutropenia (SCN) is aserious disorder characterized by severe neutro-penia at birth. SCN is associated with heterozygousmutations in the neutrophil elastase (ELANE) gene,but the mechanisms that disrupt neutrophil devel-opment have not yet been clarified because of thecurrent lack of an appropriate disease model. Here,we generated iPS cells from an individual withSCN (SCN-iPS cells). Granulopoiesis from SCN-iPScells revealed neutrophil maturation arrest and lit-tle sensitivity to granulocyte-colony stimulating fac-tor, reflecting a disease status of SCN. Molecularanalysis of the granulopoiesis from the SCN-iPScells versus control iPS cells showed reduced ex-pression of genes related to the Wnt3a/-cateninpathway, (e.g., lymphoid enhancer-binding factor(LEF)-1), whereas Wnt3a administration inducedelevation LEF-1 expression and the maturation ofSCN-iPS cell-derived neutrophils. These results in-dicate that SCN-iPS cells provide a useful diseasemodel for SCN, and the activation of the Wnt3a/-catenin pathway may offer a novel therapy for SCNwith ELANE mutation.

8. Generation of disease-specific human iPScells

Shohei Yamamoto1, Mai Nanya, Yasuhiro Ebi-hara1, Shinji Mochizuki, Sachiyo Hanada, SahokoMatsuzaka, Hiromitsu Nakauchi3, Kohichiro Tsuji

Using developmental technics regarding to hu-man pluripotent stem (iPS) cells, disease-specificiPS cells are generating from patients with a varietyof disease. We have generated some disease-specific

iPS cells. Apart from iPS cells derived from patientswith DS or SCN mentioned above, one is from thepatient with juvenile myelomonocytic leukemia(JMML). We employed hiPS cells derived from pa-tients with JMML (JMML-hiPS cells). 3 JMML-hiPScell lines were all created from bone marrow cellsand reprogrammed by the defined 4 reprogram-ming factors (OCT3/4, KLF4, SOX2, and c-MYC).We generated blood cells from JMML-hiPS cellswith coculture system using AGMS-3 cells. Hema-topoiesis especially myelopoiesis was quite facili-tated in cells derived from JMML-iPS cells, and theresponse to granulocyte macrophage-colony stimu-lating factor (GM-CSF) for hematopoietic colonyformation was highly promoted like the recipientBM cells. These results indicated that JMML-iPScells might reflect the pathophysiology of JMML.The other is from a patient with acute myeloid leu-kemia (AML) developed from 8p11 myeloprolifera-tive syndrome (EMS). EMS is an aggressive chronicmyeloproliferative disorder frequently accompanieswith T or B lymphoblastic lymphoma, and rapidlytransforms into AML. Fibroblast growth factor re-ceptor 1 (FGFR1) has critical role in the pathogene-sis of EMS. We produced hiPS cells derived fromthis patients (EMS-hiPS cells). One EMS-hiPS celllines was created from bone marrow cells fibro-blasts and reprogrammed by the defined 4 repro-gramming factors (OCT3/4, KLF4, SOX2, and c-MYC). We generated blood cells from EMS-hiPScells with coculture system using AGMS-3 cells.EMS-iPS cells produced five-fold more hematopoie-tic colonies (especially monocyte and erythroid line-age) than control iPS cells. When some of FGFR1signal inhibitor was added to the hematopoieticculture, colony formation was suppressed withdose increase at 1/7 level. These results indicatedthat EMS-iPS cells might reflect the pathophysiol-ogy of EMS, and EMS-iPS cells might be useful fordrug sensitivity test for treatment of EMS.

9. The effect of SR1 on hematopoietic cells de-rived from human iPS cells

Mai Nanya, Yasuhiro Ebihara1, Shohei Yama-moto1, Shinji Mochizuki, Sachiyo Hanada, SahokoMatsuzaka, Hiromitsu Nakauchi3, Kohichiro Tsuji

Recently StemRegenin 1 (SR1), an aryl hydrocar-bon receptor (AhR) antagonist was found to ex-pand human CD34＋ cells, which indicated thatSR1 acted with hematopoietic stem cells (HSCs). Toexamine the effect of SR1 on hematopoietic cells de-rived from human iPS cells, we added SR1 whenhuman iPS cells were cocultured with AGM-3 cells.CD34＋ cells generated in coculture system at day14 were expanded two-fold more than control. He-matopoietic cell in suspension culture induced fromCD34＋ cells expanded more than control at five-

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fold increase. The cultured cells from iPS cells incondition with SR1 formed three-fold more hema-topoietic colonies than control. These results indi-

cated that SR1 has capability to increase CD34＋cells including hematopoietic progenitor cells fromiPS cells.

Publications

Ebihara, Y., Takahashi, S., Mochizuki, S., Kato, S.,Kawakita, T., Ooi, J., Yokoyam, K., Nagamura, F.,Tojo A, Asano, S. and Tsuji, K. Unrelated cordblood transplantation after myeloablative condi-tioning regimen in adolescent and young adultpatients with hematologic malignancies: a singleinstitute analysis. Leuk. Res., 36: 128-131, 2012

Ebihara Y, Ma F, Tsuji K. Generation of red bloodcells from human embryonic/induced pluripotentstem cells for blood transfusion. Int J Hematol.95: 610-616, 2012

Yamamoto S, Akiyama K, Oyama N, Hayashi M,Fujimoto Y, Ikeda H, Isoyama K. Fatal hepatic si-nusoidal obstruction syndrome in a child withprimary CNS lymphoma during induction ther-apy. Int J Hematol. 96: 284-6, 2012

Yamamoto S, Ebihara Y, Mochizuki S, Tsuda M,Yuji K, Uchimaru K, Tojo A, Tsuji K. Acute lym-phoblastic leukemia with t(1;19)(q23;p13)/TCF3-PBX1 fusion in an adult male with Down syn-drome. Acta Haematol. 128: 242-243, 2012

Yamamoto S, Yagawa A, Toyama D, Akiyama K,Hayashi M, Mabuchi M, Shimizu T, Ikeda H,Isoyama K. Successful treatment of hepatic sinu-soidal obstructive syndrome after hematopoieticstem cell transplantation in a child using recom-binant thrombomodulin. Acta Haematol. 129: 62-64, 2012

Ebihara Y, Takedani H, Ishige I, Nagamura-InoueT, Wakitani S, Tojo A, Tsuji K. Feasibility of au-tologous bone marrow mesenchymal stem cellscultured with autologous serum for treatment ofhemophilic arthropathy. Haemophilia in press.

Mae H, Ooi J, Takahashi S, Kato S, Kawakita T,Ebihara Y, Tsuji K, Nagamura F, Echizen H, TojoA. Acute kidney injury after myeloablative cordblood transplantation in adults: the efficacy ofstrict monitoring of vancomycin serum troughconcentrations. Transpl Infect Dis. In press.

Yamamoto S, Ebihara Y, Mochizuki S, Kawakita T,Kato S, Ooi J, Takahashi S, Tojo A, Yusa N,Furukawa Y, Oyaizu N, Watanabe J, Sato K,Kimura F, Tsuji K. Quantitative PCR detection ofCEP110-FGFR1 fusion gene in a patien with 8p11syndrome. Leuk Lymhoma in press.

Hiramoto T, Ebihara Y, Mizoguchi Y, Nakamura K,Yamaguchi K, Ueno K, Nariai N, Mochizuki S,Yamamoto S, Nagasaki M, Furukawa Y, Tani K,Nakauchi H, Kobayashi M, Kohichiro Tsuji.Wnt3a stimulates maturation of impaired neutro-phils developed from severe congenital neutro-penia patient-derived pluripotent stem cells. ProcNatl Acad Sci U S A. in press.

Singh VK, Tsuji K, Sharma PB, Chandra R. Multidi-mensional role of CD34 protein in hematopoieticstem cell biology. Int J Sci Tech Man. in press.

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1. Matched HLA haplotype contributes to reducesevere acute GVHD with conserving GVL ef-fect in HLA-mismatched cord blood trans-plantation.

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

We studied the clinical outcomes of 170 consecu-tive adult patients who received unrelated CBT be-tween August 1998 and January 2011 in the insti-tute of medical Science, University of Tokyo. Pa-tients received previous allogeneic transplants wereexcluded from this study. All patients receivedmyeloablative regimens including 12 Gy of total

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.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 聡特任助教 河 北 敏 郎

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 now. Recentyears unrelated cord blood has turned to be our major stem cell source inHSCT. Since 1998 we have performed more than 260 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 suggestedthe possible application of reprogrammed somatic cells to individualized ther-apy for intractable disorders. We are trying to generate iPS cells using lentivi-ral vector and tetracycline-inducible gene expression system for introducingand expressing 3 or 4 factors required for generation of iPS cells with rela-tively homogeneous genetic background. We are also challenging to repro-gram mature blood cells into HSC according to the similar strategy used foriPS cells.

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body irradiation, cyclosporine plus short termmethotraxate for GVHD prophylaxis and almostsame supportive care by the institutional protocol.By low-resolution typing method for HLA-A, -Band -DR loci, 6 patients received matched grafts, 57received 1 antigen-mismatched and 107 received 2antigens-mismatched grafts in the graft-versus-host(GvH) direction. We have determined the HLAhaplotype based on common haplotypes in Japa-nese population referred from the 11th Interna-tional Histocompatibility Workshop and other pre-vious reports. We evaluated the impact of haplo-type matching on cumulative incidences of hema-topoietic recovery, of GVHD, of relapse and of non-relapse mortality (NRM) using the Pepe and Mori'stest. Estimates of overall and disease-free survivalswere calculated using the Kaplan-Meier methodand analyzed by the log-rank test. Thirty-threeamong all 170 pairs were defined as the haplotype-matched pairs sharing same haplotypes in bothgrafts and recipients. The age, sex, cytomegalovirusserological status, diagnosis, risk of the disease atthe transplant, numbers of total nucleated cells andCD34＋ cells at the cryopreserved were not signifi-cantly different between both groups with andwithout matched haplotypes. Engraftment of plate-let after CBT tended to be earlier in haplotype-matched group compared with control groupamong the 1 antigen-mismatched pairs in the host-versus-graft direction (median: 38 days versus 44days) and among the 2 antigens-mismatched pairs(median: 38 days versus 42 days), but those werenot significant. The cumulative incidences of gradesIII and IV acute GVHD in patients with haplotype-matched (7％) were significantly lower than non-matched group (9％) among 2 antigens-mismatchedpairs in the GvH direction (P=0.033). Notably, cu-mulative incidences of relapse tended to be lowerin haplotype-matched patients among this group (3years cumulative incidences were 7％ in haplotype-matched patients versus 21％ in non-matched pa-tients, P=0.086). The haplotype matching effectswere not observed in survival rates, cumulative in-cidences of NRM among any HLA-mismatchedpairs. Those data suggest that untyped variationcarried on the HLA haplotytpe might be better tobe matched. The haplotype matching seemed to ef-fect on lower risk of sever acute GVHD, on theother hand, graft-versus-leukemia effect was con-served in the setting of HLA-mismatched CBT.

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

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

There are many reports of second allogeneic stem

cell transplantation (allo-SCT) using cord blood(CB) for graft failure after initial allo-SCT. How-ever, the efficacy of second allo-SCT using CB forpatients with leukemia relapsed after initial allo-SCT is unknown. We report the results of secondallo-SCT using CB in seven adult patients with leu-kemia relapsed after initial allo-SCT. All patientsreceived a myeloablative conditioning regimen in-cluding oral busulfan 16 mg/kg, intravenouslyfludarabine 100mg/m(2) and cyclophosphamide 120mg/kg. All but one patient had myeloid reconstitu-tion and four patients remain alive at between 4and 40 months after second SCT. We conclude thatsecond myeloablative allo-SCT using CB may befeasible in selected patients with the relativelyyounger age, less organ damage and longer timeinterval between first and second allo-SCT.

3. Establishment of murine iPSC-derived hema-topoietic progenitor cell lines which can yieldmature blood cells.

Izawa K, Yamaguchi K, Furukawa Y, YamamotoM, Tojo A

Hematopoietic stem/progenitor cells (HS/PCs)constitute a quite minor part of bone marrow (BM)nucleated cells and cannot be expanded in vitrowith sustained hematopoietic ability for a longtime. It is also difficult to efficiently direct pluripo-tent stem cells including ESCs and iPSCs towardthe stage of HSCs. GATA2 is an essential transcrip-tion factor for hematopoietic development and isexpressed in HS/PCs. We generated iPSCs (GGKI-iPSCs) from GFP＋ BM cells from heterozygousGATA2-GFP knock-in mice in which GFP cDNAwas inserted into exon 2 of the GATA2 gene. Col-ony forming assays confirmed that HPCs exist in aGFP＋ fraction of BM cells in those mice, suggest-ing that HS/PCs derived from GGKI-iPSCs may beidentified according to GFP expression. First,GGKI-iPSCs were induced toward hematopoieticdifferentiation over an OP9 cell monolayer with acocktail of cytokines. Interestingly, after twomonths of culture, we found that GFP＋ cells werecontinuously proliferating. They increased in num-ber by 100-fold per week and were sustained for atleast 3 months without loss of their properties.Next, GFP＋ cells were FACS-sorted according toits expression level. In a methylcellulose assay, thenumber of colonies from GFPlow cells is 100 timeshigher than that from GFPhigh cells. The microarrayand RT-PCR analysis suggested that GFPlow fractionincludes myeloid signatures, while GFPhigh fractionhas early erythroid signatures. Furthermore, trans-plantation experiments revealed that CD11b＋ ma-ture myeloid cells in PB could be detected in micetransplanted with GFPlow, but not GFPhigh, cells. As aresult, we succeeded in establishment of murine

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iPSC-derived hematopoietic progenitor cell lines which can yield mature blood cells.

Publications

Yamamoto S, Ebihara Y, Mochizuki S, Kawakita T,Kato S, Ooi J, Takahashi S, Tojo A, Yusa N, Fu-rukawa Y, Oyaizu N, Watanabe J, Sato K,Kimura F, Tsuji K. Quantitative PCR detection ofCEP110-FGFR1 fusion gene in a patient with 8p11 syndrome (letter to the editor). Leuk Lym-phoma. In press, 2013 Jan 18. [Epub ahead ofprint]

Mae H, Ooi J, Takahashi S, Kato S, Kawakita T,Ebihara Y, Tsuji K, Nagamura F, Echizen H, TojoA. Acute kidney injury after myeloablative cordblood transplantation in adults: the efficacy ofstrict monitoring of vancomycin serum troughconcentrations. Transplant Infectious Disease. Inpress, 2012 Dec 20. doi: 10.1111/tid.12038. [Epubahead of print]

Ebihara Y, Takedani H, Ishige I, Nagamura-Inoue TWakitani S, Tojo A, Tsuji K. Feasibility of autolo-gous bone marrow mesenchymal stem cells cul-tured with autologous serum for treatment of he-mophilic arthropathy. Hemophilia. 2012 Dec 4.doi: 10.1111/hae.12056. [Epub ahead of print]

Kurosawa S, Yakushijin K, Yamaguchi T, Atsuta Y,Nagamura-Inoue T, Akiyama H, Taniguchi S, Mi-yamura K, Takahashi S, Eto T, Ogawa H,Kurokawa M, Tanaka J, Kawa K, Kato K, SuzukiR, Morishima Y, Sakamaki H, Fukuda T. Changesin incidence and causes of non-relapse mortality

after allogeneic hematopoietic cell transplantationin patients with acute leukemia/myelodysplasticsyndrome: an analysis of the Japan TransplantOutcome Registry. Bone Marrow Transplant. Inpress, 2012 Sep 10

Kanda J, Ichinohe T, Kato S, Uchida N, Terakura S,Fukuda T, Hidaka M, Ueda Y, Kondo T,Taniguchi S, Takahashi S, Nagamura-Inoue T,Tanaka J, Atsuta Y, Miyamura K, Kanda Y. Unre-lated cord blood transplantation vs related trans-plantation with HLA 1-antigen mismatch in thegraft-versus-host direction. Leukemia. 27(2): 286-94, 2013

Kanda J, Atsuta Y, Wake A, Ichinohe T, TakanashiM, Morishima Y, Taniguchi S, Takahashi S,Ogawa H, Ohashi K, Ohno Y, Aotsuka N, OnishiY, Kato K, Nagamura-Inoue T, Kanda Y. Impactof the direction of HLA mismatch on transplantoutcome in single unrelated cord blood trans-plantation. Biol Blood Marrow Transplant., S1083-8791, 2012.

Ebihara Y, Takahashi S, Mochizuki S, Kato S,Kawakita T, Ooi J, Yokoyama K, Nagamura F,Tojo A, Asano S, Tsuji K. Unrelated cord bloodtransplantation after myeloablative conditioningregimen in adolescent patients with hematologicmalignancies: a single institute analysis. LeukRes. 6: 128-31, 2012

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1. Screening of surface antigens of iPS cells us-ing a retrovirus-mediated signal transductionmethod SST-REX.

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

We previously developed a retrovirus-mediatedsignal sequence trap method SST-REX as a screen-ing method for surface and secreted proteins. Wesearched surface antigens of cancer cells or immunecells. Here we used SST-REX to iPS to identify iPS-specific surface antigens, surface antigen "catalog"of iPS cells, and attempted to develop iPS-specificantibodies. So far, we have identified 40 iPS cell an-tigens, found that at least 3 of them were expressedrather specifically in iPS cells and ES cells, and de-veloped specific antibodies to these 3 antigens andinvestigated expressions of these antigens in iPScells. We also investigated the effects of transduc-tion of these antigens on iPS induction, and trans-duction of one of the antigen enhanced reprogram-ming process, though the precise mechanisms re-main to be investigated.

2. RasGRP family proteins and Leukemia

Toshihiko Oki, Jiro Kitaura, Koutarou Nishimura,Akie Maehara, Tomoyuki Uchida, Fumio Naka-hara, and Toshio Kitamura

The Ras guanyl nucleotide-releasing proteins(RasGRPs) are a family of guanine nucleotide-ex-change factors, with four members (RasGRP1-4),which positively regulate Ras and related smallGTPases. In the previous study, we identifiedRasGRP4 using expression cloning as a gene thatfully transformed IL-3-dependent HF6 cells, anddemonstrated that in a mouse bone marrow trans-plantation (BMT) model, RasGRP4 induced acutemyeloid leukemia (AML) and/or T-ALL. On theother hand, it has been reported that RasGRP1transgenic mice developed thymic lymphoma orskin tumors.However, the roles of RasGRP family proteins in

leukemogenesis have not been investigated in de-tail. We have recently characterized leukemogenic-ity of RasGRP1 and 4 in details using a BMT model(Oki et al. Leukemia 2012).RasGRP1 exclusively induced T-cell acute lym-

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 Research Associate 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

Our major interest is to elucidate the mechanisms of pluripotency, self-renewaland the control of cell division and differentiation of stem cells such as ES cells,iPS cells, and hematopoietic stem cells. We have developed the retrovirus-medi-ated efficient gene transfer and several functional expression cloning systems,and utilized these system to our experiment. We are now conducting several pro-jects related to stem cells to characterize stem cells, clarify underling mechanismsof reprogramming, maintenance of pluripotency, and differentiation, and eventuallyto develop new strategies for regenerative medicine.

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phoblastic leukemia/lymphoma (T-ALL) after ashorter latency when compared with RasGRP4. Ac-cordingly, Ba/F3 cells transduced with RasGRP1survived longer under growth factor withdrawal orphorbol ester stimulation than those transducedwith RasGRP4, presumably due to the efficient acti-vation of Ras. Intriguingly, NOTCH1 mutations re-sulting in a gain of function were found in 77% ofthe RasGRP1-mediated mouse T-ALL samples. Inaddition, gain-of-function NOTCH1 mutation wasfound in human T-cell malignancy with elevatedexpression of RasGRP1. Importantly, RasGRP1 andNOTCH1 signaling cooperated in the progressionof T-ALL in the murine model. The leukemogenicadvantage of RasGRP1 over RasGRP4 was attenu-ated by the disruption of a PKC phosphorylationsite (RasGRP1(Thr184)) not present on RasGRP4. Inconclusion, cooperation between aberrant expres-sion of RasGRP1, a strong activator of Ras, and sec-ondary gain-of-function mutations of NOTCH1plays an important role in T-cell leukemogenesis.

3. Development of new retroviral vectors.

Toshikhiko Oki, Jiro Kitaura, Tomoyuki Uchida,Fumi Shibata-Minoshima, and Toshio Kitamura:

We developed an effective retroviral transductionsystem consisted of vectors named as pMXs, pMYs,pMZs and pMCs and packaging cells named asPLAT-E, PLAT-A, and PLAT-F. We developed newvectors like, vectors with luciferase maker (pMX-IL), vectors for GFP or RFP fusion proteins, vectorswith lox sequences for deletion of inserted geneswith Cre-loxP, Tet-On and Tet-Off systems, vectorsfor expression, inhibition, and monitoring the ex-pression of miroRNA (pMXe series). We utilizedthese vectors in studying stem cell biology and alsoin developing the innovative tools for regenerativemedicine

4. Co-ordinate control of cell division and cellfate of by the Rho family small GTPases.

Toshihiko Oki, Kohtaro Nishimura, ToshiyukiKawashima, and Toshio Kitamura:

We previously identified MgcRacGAP throughfunctional cloning as a protein that enhances or in-duces macrophage differentiation of leukemic celllines M1 and HL60. Interestingly, MgcRacGAPplays distinct roles depending on the cell cycle. Inthe interphase, it plays critical roles in activationand nuclear translocation of STAT3 and STAT5 as aRac-GAP. In the metaphase, MgcRacGAP playssome roles in the segregation of chromosomesprobably as Cdc42-GAP. In the mitotic phase,MgcRacGAP plays essential roles in completion ofcytokinesis as a Rho-GAP. Interestingly, Aurora B-

mediated phosphorylation of S387 convertsMgcRacGAP from Rac-GAP to Rho-GAP.We have recently shown that expression of

MgcRacGAP is regulated by cell-cycle dependentmechanism: increase in S/G2/M phase and decreasein early G1 phase, suggesting that MgcRacGAPmay play some roles in G1 check point. Theubiquitin-dependent degradation of MgcRacGAP isone of the mechanisms that account for its decreasein G1 phase. Using the proteome analysis andretroviral transduction, we identified the E3 ligaseinvolved in regulation of MgcRacGAP and the de-gron in MgcRacGAP. Now we are investigating thephysiological roles of this regulation. In summary,our results implicate MgcRacGAP in coordinationof cell cycle progression and cell fate determination.

5. Molecular therapy targeting signal transduc-tion pathways using small molecule com-pounds

Toshiyuki Kawashima, Akiho Tsuchiya, ToshihikoOki, Jiro Kitaura, and Toshio Kitamura:

STAT3 is frequently activated in many cancersand leukemias, and is required for transformationof NIH3T3 cells. Therefore, we have started search-ing for STAT3 inhibitors. We already established anefficient screening protocol for identification ofSTAT3 inhibitors, and identified several compoundsthat inhibit STAT3 activation. Through the screen-ing of a library of small molecule compounds, wefound the compounds RJSI-1 and RJSI-2 that inhib-ited STAT3 activation. RJSI-2 also inhibited activa-tion of STAT1, STAT5, JAK1 and JAK2, howeverRJSI-2 is not a kinase inhibitor. On the other hand,RJSI-1 inhibited nuclear transport of phospho-rylated STAT proteins, implicating a novel mecha-nism in inhibiting STAT proteins. We have alsoshown that these compounds are effective in a tu-mor-burden mouse model. In addition, we collabo-rate with a US biotech venture company in modifi-cation of RSJI-1 for optimization to develop anti-cancer drugs, and have developed JP1156 whichkill the tumor cells with much lower IC50.

6. Development of G0 indicator

Toshihiko Oki, Kotarou Nishimura, Jiro Kitarura,Fumio Nakahara, Asako Sakaue-Sawano2, AtsushiMiyawaki2, Toshio Kitamura: 2Laboratory for CellFunction Dynamics, RIKEN, Wako, Saitama andERATO Miyawaki Life Function Dynamics Pro-ject, JST.

One of the common features of the stem cells isthat they are in quiescent (G0) phase of cell cycle.Several reports indicate that tissue specific stemcells such as hematopietic stem cells and cancer

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stem cells with tumor initiating potentials are in G0phase.Recently we have developed the system to indi-

cate the cells in G0 phase. It is a system to monitorthe amount of the protein X, which is destructedduring G0 to G1 phase and is not expressed in S/G2/M phase, using the cells retrovirally trasducedwith the fusion protein between a fluorescent pro-tein like mVenus and protein X (mVenus-X), as asimilar cell cycle indicator system, fluorescent,ubiquitination-based cell cycle indicator, (Fucci).

mVenus-X positive cells are Ki67 negative quiescentcells and mVenus-X signals are enhanced when thecycling cell enter G0 phase in response to serumstarvation or contact inhibition.Using this system, we identified genetic signa-

tures of G0 cells. Several genes specifically ex-pressed in G0 cells are now being investigated interms of their functions and biological significancein G0 phase. The mVenus-X trasgenic mice havealso been generated to track several kinds of tissuespecific stem cells in vivo.

Publications

Nishimura, K., Oki, T., Kitaura, J., Kuninaka, S.,Saya, H., Sakaue-Sawano, A., Miyawaki, A. andKitamura, T. APCCDH1 targets MgcRac GAP fordestruction in the late M phase. PLOS One inpress.

Takahashi, M., Izawa, K., Kashiwakura, J., Yamani-shi, Y., Enomoto, Y., Kaitani, A., Maehara, A.,Isobe, M., Ito, S., Matsukawa, T., Nakahara, F.,Oki, T., Kajikawa, M., Ra, C., Okayama, Y., Kita-mura, T. and Kitaura, J. Human CD300C deliversan Fc receptor-g-dependent activating signal inmast cells and monocytes and differs from CD300A in ligand recognition. J. Biol. Chem. in press.

Sakane, A., Almair, A., Abdallah, M., Nakano, K.,Honda, K., Ikeda, W., Nishikawa, Y., Matsumoto,M., Matsushita, N., Kitamura, T. and Sasaki, T.(2012) Rab13 small G protein and Junctional Rab13-binding protein (JRAB) orchestrate actin cy-toskeletal organization during epithelial junc-tional development. J. Biol. Chem. 287, 42455-42968.

Enomoto, Y., Kitaura, J., Shimanuki, M., Kato, N.,Nishimura, K., Takahashi, M., Nakamura, H., Ki-tamura, T., and Sonoki T. (2012) MicroRNA-125b-1 accelerates a C-terminal mutant of C/EBP (C/EBP-C(m))-induced myeloid leukemia. Int. J.Hematol. 96, 334-341.

Shibata, T., Takamura, N., Motoi, Y., Goto, Y.,Karuppuchamy, T., Izawa, K., Akashi-Takamura,S., Tanimura, N., Kunisawa, J., Kiyono, H., Akira,S., Kitamura, T., Kitaura, J., and Miyake K. (2012)PRAT4A-dependent expression of cell surfaceTLR5 on neutrophils, classical monocytes anddendritic cells. Int Immunol. 24; 613-623.

Izawa, K., Yamanishi, Y., Maehara, A., Takahashi,M., Isobe, M., Ito, S., Kaitani, A., Matsukawa, T.,Matsuoka, T., Nakahara, F., Oki, T., Kiyonari, H.,Abe, T., Okumura, K., Kitamura, T., and Kitaura,J. (2012) LMIR3 negatively regulates mast cell ac-tivation and allergic responses by binding to ex-tracellular ceramide. Immunity 37: 827-839.

Yamanishi, Y., Takahashi, M., Izawa, K., Isobe, M.,Ito, S., Tsuchiya, A., Maehara, A., Uchida, T., To-gami, K., Enomoto, Y., Nakahara, F., Oki, T., Ka-

jikawa, M., Kurihara, H., Kitamura, T., and Ki-taura, J. (2012) A soluble form of LMIR5/CD300bamplifies lipppolisaccharide-induced lethal in-flammation in sepsis. J. Immunol. 189, 1773-1779.

Kitamura, T., and Inoue, D. (2012) HDAC inhibitor-induced thrombocytepenia is caused by its unex-pected target. Exp. Hematol. 40, 695-697.

Komori, T., Doi, A., Nosaka, T., Furuta, H., Aka-mizu, T., Kitamura, T., Senba, E. and Morikawa,Y. (2012) Regulation of AMP-activated proteinkinase signaling by AFF4 protein, member of AF4 (ALL-fused gene from chromosome 4) family oftranscription factors, in hypothalamic neurons. J.Biol. Chem. 287, 19985-19996.

Doki, N., Kitaura, J., Inoue, D., Kato, N., Kagiyama,Y., Uchida, T., Togami, K., Isobe, M., Ito, S., Mae-hara, A., Izawa, K., Oki, T., Harada, Y., Naka-hara, F., Harada, H., and Kitamura, T. (2012) Fynis not essential for Bcr-Abl-induced leukemogene-sis in mouse bone marrow transplantation mod-els. Int. J. Hematol. 95: 167-175.

Oki, T., Kitaura, J., Watanabe-Okochi, N., Nishi-mura, K., Maehara, A., Uchida, T., Komeno, Y.,Nakahara, F., Harada, Y., Sonoki, T., Harada, H.,and Kitamura, T. (2012) Aberrant expression ofRasGRP1 cooperates with gain -of - functionNOTCH1 mutations in T-cell leukemogenesis.Leukemia 26: 1038-1045.

Nakamura, M., Kitaura, J., Enomoto, Y., Lu, Y.,Nishimura, K., Isobe, M., Ozaki, K., Komeno, Y.,Nakahara, F., Oki, T., Kume, H., Homma, Y., andKitamura, T. (2012) TSC-22 is a negative-feedbackregulator of Ras/Raf signaling: Implications fortumorigenesis. Cancer Science 103: 26-33.

Suzuki, K., Ono, R., Ohishi, K., Masuya, M., Kata-oka, I., Liu, B., Nakamori, Y., Ino, K., Monma, F.,Hamada, H., Kitamura, T., Katayama, N., andNosaka, T. (2012) IKAROS isoform 6 enhancesBCR-ABL-mediated proliferation of humanCD34＋ hematopoietic cells on stromal cells. Int JOncology 40: 53-62.

Shibata-Minoshima, F., Oki, T., Doki, N., Nakahara,F., Kageyama, S., Kitaura, J., Fukuoka, J. and Ki-tamura, T. (2012) Identification of RHOXF2

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(PEPP2) as a cancer-promoting gene by expres-sion cloning. Int J Oncology 40: 93-98.

Kawamura, S., Sato, I., Wada, T., Yamaguchi, K., Li,Y., Li, D., Zhao, X., Ueno, S., Aoki, H., Tochigi,T., Kuwahara, M., Kitamura, T., Takahashi, K.,Moriya, S., and Miyagi, T. (2012) Plasma mem-

brane-associated sialidase (NEU3) regulates pro-gression of prostate cancer to androgen-inde-pendent growth through modulation of androgenreceptor signaling. Cell Death and Differentiation19: 170-179.

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1. Plasminogen deficiency attenuates post-natalerythropoiesis in male C57BL/6 mice throughdecreased activity of the LH-testosterone axis

Yurai Okaji, Yoshihiko Tashiro1, Ismael Gritli1,Chiemi Nishida1, Aki Sato1, Yoko Ueno, SandraDel Canto Gonzalez, Makiko Ohki-Koizumi1,Haruyo Akiyama1, Hiromitsu Nakauchi2, KoichiHattori1,3, Beate Heissig3: 1Laboratory of Stem CellRegulation and 2Division of Stem Cell Therapy,Center for Stem Cell Biology and RegenerativeMedicine, Institute of Medical Sciences, Universityof Tokyo, 3Atopy Center, Juntendo University

Novel roles for the serine protease plasmin havebeen recently implicated in physiological andpathological processes. However, whether plasminis involved in erythropoiesis, is not known. In thepresent study, we studied the consequences of plas-minogen deficiency on erythropoiesis in plasmino-gen deficient (Plg KO) mice. Erythroid differentia-tion was attenuated in male Plg KO mice and re-sulted in erythroblastic accumulation within thespleen and bone marrow, with increased apoptosisin the former, erythrocytosis and splenomegaly,whereas similar erythropoietic defect was lessprominent in female Plg KO mice. In addition,erythrocyte lifespan was shorter in both male andfemale Plg KO mice. Erythropoietin levels were

compensatory increased in both male and femalePlg KO mice, and resulted in a higher frequency ofBFU-E within the spleen and bone marrow. Sur-prisingly, we found that male Plg KO mice but nottheir female counterparts exhibited normochromicnormocytic anemia. The observed gender-linkederythropoietic defect was attributed to decreasedserum testosterone levels in Plg KO mice, as a con-sequence of impaired secretion of the pituitary lu-teinizing hormone (LH) under steady state condi-tion. Surgical castration, causing testosterone defi-ciency and stimulating LH release, attenuatederythroid differentiation and induced anemia in WTanimals, but did not further decrease the hematocritlevels in Plg KO mice. In addition, complementa-tion of LH using human choriogonadotropin, whichincreases testosterone production, improved theerythropoietic defect and anemia in Plg KO mice.The present results identify a novel role for plasminin the hormonal regulation of post-natal erythropoi-esis by the LH-testosterone axis.

2. MT1-MMP plays a critical role in hematopoi-esis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells

Chiemi Nishida1, Kaori Kusubata, YoshihikoTashiro1, Ismael Gritli1, Aki Sato1, Makiko Ohki-Koizumi1, Yohei Morita2, Makoto Nagano3, Take-

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Dynamics幹細胞ダイナミクス解析分野

Associate Professor Beate Heissig, M.D. 准教授 医学博士 ハイジッヒ ベアーテ

Proteases can perform highly selective and limited cleavage of specific substratesincluding growth factors and their receptors, cell adhesion molecules, cytokines,apoptotic ligand and angiogenic factors. To understand the molecular mechanismunderlying hematopoietic stem cell differentiation, we investigated the role of pro-teases in the regulation of blood cell formation and blood vessel formation usinggene deficient mice.

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haru Sakamoto3, Naohiko Koshikawa3, TakahiroKuchimaru4, Shinae Kizaka-Kondoh4, MotoharuSeiki3, Hiromitsu Nakauchi2, Koichi Hattori1,5,Beate Heissig5: 1Department of Stem Cell Regula-tion and 2Division of Stem Cell Therapy, Centerfor Stem Cell Biology and Regenerative Medicine,Institute of Medical Science, The University of To-kyo, 3Division of Cancer Cell Research, Institute ofMedical Science, The University of Tokyo, 4De-partment of Biomolecular Engineering, Tokyo In-stitute of Technology, 5Atopy (Allergy) ResearchCenter, Juntendo University School of Medicine

HSC fate decisions are regulated by cell-intrinsicand cell-extrinsic cues. The latter cues are derivedfrom the BM niche. Membrane-type 1 matrix metal-loproteinase (MT1-MMP), which is best known forits proteolytic role in pericellular matrix remodel-ing, is highly expressed in HSCs and stromal/nichecells. We found that, in MT1-MMP(－/－) mice, inaddition to a stem cell defect, the transcription andrelease of kit ligand (KitL), stromal cell-derived fac-tor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hema-topoietic differentiation block, while addition of ex-ogenous KitL and SDF-1 restored hematopoiesis.Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1)within niche cells, thereby inducing the transcrip-tion of HIF-responsive genes, which induce termi-nal hematopoietic differentiation. Thus, MT1-MMPin niche cells regulates postnatal hematopoiesis, bymodulating hematopoietic HIF-dependent nichefactors that are critical for terminal differentiationand migration.

3. Inhibition of PAI-1 induces neutrophil-drivenneoangiogenesis and promotes tissue regen-eration via production of angiocrine factors inmice

Yoshihiko Tashiro1,2, Chiemi Nishida1, Kaori Sato-Kusubata, Makiko Ohki-Koizumi1, Makoto Ishi-hara1, Aki Sato1, Ismael Gritli1, Hiromitsu Komi-yama1,2, Yayoi Sato1, Takashi Dan3, Toshio Mi-yata3, Ko Okumura4, Yuichi Tomiki2, KazuhiroSakamoto2, Hiromitsu Nakauchi5, Koichi Hattori1,4,Beate Heissig4: 1Department of Stem Cell Regula-tion Center for Stem Cell Biology and Regenera-tive Medicine, Institute of Medical Science at theUniversity of Tokyo, 2Department of Coloproc-tological Surgery, Juntendo University faculty ofMedicine, 3United Centers for Advanced Researchand Translational Medicine (ART), Tohoku Uni-versity Graduate School of Medicine, 4Atopy (Al-lergy) Center, Juntendo University School ofMedicine, 5Division of Stem Cell Therapy, Centerfor Stem Cell Biology and Regenerative Medicine,

Institute of Medical Science, The University of To-kyo

Plasminogen activator inhibitor-1 (PAI-1), an en-dogenous inhibitor of a major fibrinolytic factor, tis-sue-type plasminogen activator, can both promoteand inhibit angiogenesis. However, the physiologicrole and the precise mechanisms underlying the an-giogenic effects of PAI-1 remain unclear. In the pre-sent study, we report that pharmacologic inhibitionof PAI-1 promoted angiogenesis and prevented tis-sue necrosis in a mouse model of hind-limb ische-mia. Improved tissue regeneration was due to anexpansion of circulating and tissue-resident granu-locyte-1 marker (Gr-1(＋)) neutrophils and to in-creased release of the angiogenic factor VEGF-A,the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated in-creased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization andgenetic knockout studies indicated that both the im-proved tissue regeneration and the increase in cir-culating and ischemic tissue-resident Gr-1(＋) neu-trophils depended on the activation of tissue-typeplasminogen activator and matrix metalloprotein-ase-9 and on VEGF-A and FGF-2. These results sug-gest that pharmacologic PAI-1 inhibition activatesthe proangiogenic FGF-2 and VEGF-A pathways,which orchestrates neutrophil-driven angiogenesisand induces cell-driven revascularization and istherefore a potential therapy for ischemic diseases.

4. New functions of the fibrinolytic system inbone marrow cell-derived angiogenesis

Beate Heissig1, Makiko Ohki-Koizumi2, YoshihikoTashiro2, Ismael Gritli2, Kaori Sato-Kusubata, Koi-chi Hattori1,2 : 1Atopy (Allergy) Research Center,Juntendo University School of Medicine, 2Depart-ment of Stem Cell Regulation Center for StemCell Biology and Regenerative Medicine, Instituteof Medical Science at the University of Tokyo

Angiogenesis is a process by which new bloodvessels form from preexisting vasculature. Thisprocess includes differentiation of angioblasts intoendothelial cells with the help of secreted angio-genic factors released from cells such as bone mar-row (BM)-derived cells. The fibrinolytic factor plas-min, which is a serine protease, has been shown topromote endothelial cell migration either directly,by degrading matrix proteins such as fibrin, or in-directly, by converting matrix-bound angiogenicgrowth factors into a soluble form. Plasmin can alsoactivate other pericellular proteases such as matrixmetalloproteinases (MMPs). Recent studies indicatethat plasmin can additionally alter cellular adhesionand migration. We showed that factors of the fibri-

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nolytic pathway can recruit BM-derived hema-topoietic cells into ischemic/hypoxic tissues by al-tering the activation status of MMPs. These BM-de-rived cells can function as accessory cells that pro-mote angiogenesis by releasing angiogenic signals.This review will discuss recent data regarding therole of the fibrinolytic system in controllingmyeloid cell-driven angiogenesis. We propose thatplasmin/plasminogen may be a potential target notonly for development of effective angiogenic thera-peutic strategies for the treatment of cancer, butalso for development of strategies to promoteischemic tissue regeneration.

5. Plasmin inhibitor reduces lymphoid tumorgrowth by suppressing matrixmetallprotein-ase-9 dependent CD11b＋/F4/80＋ myeloidcell recruitment

Makoto Ishihara1, Chiemi Nishida1, YoshihikoTashiro1, Ismael Gritli1, Jeanette Rosenkvist1,Makiko Koizumi1, Yurai Okaji, Ryo Yamamoto2,Hideo Yagita3, Ko Okumura4, Momoko Nishikori5,Keiko Wanaka6, Yuko Tsuda7, Yoshio Okada7, Hi-romitsu Nakauchi2, Koichi Hattori1,4, Beate Heis-sig4: 1Department of Stem Cell Regulation Centerfor Stem Cell Biology and Regenerative Medicine,Institute of Medical Science at the University ofTokyo, 2Laboratory of Stem Cell Therapy, Centerfor Experimental Medicine, Institute of MedicalScience, The University of Tokyo, 3Department ofImmunology, Juntendo University School of Medi-cine, 4Atopy (Allergy) Research Center, JuntendoUniversity School of Medicine, 5Department of He-

matology and Oncology, Kyoto University, 6KobeResearch Projects on Thrombosis and Haemosta-sis, 7Faculty of Pharmaceutical Sciences, KobeGakuin University

Activation of the fibrinolytic system during lym-phoma progression is a well-documented clinicalphenomenon. But the mechanism by which the fi-brinolytic system can modulate lymphoma progres-sion has been elusive. The main fibrinolytic en-zyme, plasminogen (Plg)/plasmin (Plm), can acti-vate matrix metalloproteinases (MMPs), like MMP-9, which has been linked to various malignancies.Here we provide the evidence that blockade of Plgreduces lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b＋F4/80＋ myeloidcells locally within the lymphoma tissue. Geneticplasminogen deficiency and drug-mediated Plmblockade delayed lymphoma growth and dimin-ished MMP-9 dependent CD11b＋F4/80＋ myeloidcell infiltration into lymphoma tissues. A neutraliz-ing antibody against CD11b inhibited lymphomagrowth in vivo, which indicates that CD11b＋myeloid cells play a role in lymphoma growth. Plgdeficiency in lymphoma-bearing mice resulted inreduced plasma levels of the growth factors vascu-lar endothelial growth-A and Kit ligand, both ofwhich are known to enhance myeloid cell prolifera-tion. Collectively, the data presented in this studydemonstrate a previously undescribed role of Plmin lymphoproliferative disorders and providestrong evidence that specific blockade of Plg repre-sents a promising approach for the regulation oflymphoma growth.

Publications

1. Okaji Y, Tashiro Y, Gritli I, Nishida C, Sato A,Ueno Y, Del Canto Gonzalez S, Ohki-KoizumiM, Akiyama H, Nakauchi H, Hattori K, HeissigB. Plasminogen deficiency attenuates post-natalerythropoiesis in male C57BL/6 mice through de-creased activity of the LH-testosterone axis. ExpHematol. 40: 143-154, 2012.

2. Nishida C, Kusubata K, Tashiro Y, Gritli I, SatoA, Ohki-Koizumi M, Morita Y, Nagano M,Sakamoto T, Koishikawa N, Kuchimaru T, Ki-zaka-Kondoh S, Seiki M, Nakauchi H, Heissig B,Hattori K. MT1-MMP plays a critical role in he-matopoiesis by regulating HIF - mediatedchemokine/cytokine gene transcription withinniche cells. Blood. 119: 5405-16. 2012.

3. Tashiro Y, Nishida C, Sato-Kusubata K, Ohki-Koizumi M, Ishihara M, Sato A, Gritli I, Komi-yama H, Sato Y, Dan T, Miyata T, Okumura K,

Tomiki Y, Sakamoto K, Nakauchi H, Heissig B,Hattori K. Inhibition of PAI-1 induces neutro-phil-driven neoangiogenesis and promotes tissueregeneration via production of angiocrine factorsin mice. Blood. 119: 6382-93, 2012.

4. Heissig B, Ohki-Koizumi M, Tashiro Y, Gritli I,Sato-Kusubata K, Hattori K. New functions ofthe fibrinolytic system in bone marrow cell-de-rived angiogenesis. Int J Hematol. 95: 131-7,2012.

5. Ishihara M, Nishida C, Tashiro Y, Gritli I, Ro-senkvist J, Koizumi M, Okaji Y, Yamamoto R,Yagita H, Okumura K, Nishikori M, Wanaka K,Yuko Tsuda Y, Okada Y, Nakauchi H, Hattori K,Heissig B. Plasmin inhibitor reduces lymphoidtumor growth by suppressing matrix metallpro-teinase-9 dependent CD11b＋/F4/80＋ myeloidcell recruitment. Leukemia. 26: 332-9, 2012.

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