center for stem cell biology and regenerative …1. novel method for efficient production of...

1. Investigation of bi-potent differentiation of he-patoblasts using inducible diphtheria toxin re-ceptor-transgenic mice

Yanagida A1,2, Mizuno N1, Yamazaki Y1, Kato-ItohM1, Umino A1, Sato H1, Ito K1,3, Yamaguchi T1,Nakauchi H1,4, Kamiya A5: 1Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science,University of Tokyo, 2Wellcome Trust-Medical Re-search Council Cambridge Stem Cell Institute,University of Cambridge, 3Laboratory of Biochem-istry and Molecular Biology, The Rockefeller Uni-versity, 4Institute for Stem Cell Biology and Re-generative Medicine, Stanford University School ofMedicine, Stanford, 5Department of Molecular LifeSciences, Tokai University School of Medicine

Hepatic progenitor cells, called hepatoblasts, arehighly proliferative and exhibit bi-potential differ-entiation into hepatocytes and cholangiocytes in thefetal liver. Thus, they are the ideal source for trans-plantation therapy. Although several studies havebeen performed in vitro, the molecular mechanismsregulating hepatoblast differentiation in vivo fol-lowing transplantation remain poorly understood.

The aim of this study was to investigate an in vivomodel to analyze hepatoblast bi-potency and prolif-erative ability.Hepatic transplantation model using Cre-induc-

ible diphtheria toxin receptor transgenic mice(iDTR), and albafpCre mice expressing Cre underthe control of albumin and -fetoprotein regulatoryelements were established. Fresh hepatoblasts weretransplanted into diphtheria toxin (DT)-injectediDTRalbafpCre mice and analyzed their differentia-tion and proliferation abilities by immunostainingand genes expression profiles.Fresh hepatoblasts transplanted into DT-injected

iDTRalbafpCre mice engrafted and differentiatedinto both hepatocytes and cholangiocytes. Addition-ally, the number of engrafted hepatoblast-derivedhepatocytes increased following partial hepatec-tomy and serial DT injections. Expression levels ofhepatic functional genes in transplanted hepa-toblast-derived hepatocytes were similar to that ofnormal hepatocytes.In our iDTRalbafpCre transplantation model,

fresh hepatoblasts could differentiate into hepato-cytes and cholangiocytes. In addition, these donorcells were induced to proliferate by the followingliver injury stimulation. This result suggests that

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.DProject Associate Professor Tomoyuki Yamaguchi, Ph.D.Assistant Professor Satoshi Yamazaki, Ph.D.Assistant Professor Hideki Masaki, Ph.D.

教授医学博士中内啓光特任准教授医学博士山口智之助教生命科学博士山﨑聡助教理学博士正木英樹

Recent great progress in stem cell biology has brought about increase in the pros-pect for application of stem cell-based therapy. Especially the discovery of iPSCs,a great step forward in stem-cell research, holds out the promise of developmentof novel therapeutic strategies by generating iPSCs from patients. The goal of thislaboratory is to provide new insights into stem cell biology as well as approachesto novel therapeutic intervention for various intractable diseases.

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this model is valuable for investigating hepatoblastdifferentiation pathways in vivo. This article is pro-tected by copyright. All rights reserved.

2. A Safeguard System for Induced PluripotentStem Cell-Derived Rejuvenated T Cell Therapy

Ando M1, Nishimura T2, Yamazaki S1, YamaguchiT1, Kawana-Tachikawa A3, Hayama T1, NakauchiY1, Ando J4, Ota Y5, Takahashi S6, Nishimura K7,Ohtaka M8, Nakanishi M8, Miles JJ9, Burrows SR9,Brenner MK10, Nakauchi H11: 1Division of StemCell Therapy, Center for Stem Cell Biology andRegenerative Medicine, Institute of Medical Sci-ence, University of Tokyo, 2Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science,University of Tokyo, Institute for Stem Cell Biol-ogy and Regenerative Medicine, Stanford Univer-sity School of Medicine, 3Division of Infectious Dis-eases, Advanced Clinical Research Center, Insti-tute of Medical Science, University of Tokyo,AIDS Research Center, National Institute of Infec-tious Diseases, 4Department of Hematology, Jun-tendo University School of Medicine, 5Departmentof Pathology, Research Hospital, Institute of Medi-cal Science, University of Tokyo, 6Division of Mo-lecular Therapy, Advanced Clinical Research Cen-ter, Institute of Medical Science, University of To-kyo, 7Laboratory of Gene Regulation, Faculty ofMedicine, University of Tsukuba, 8BiotechnologyResearch Institute for Drug Discovery, NationalInstitute of Advanced Industrial Science and Tech-nology, 9QIMR Berghofer Medical Research Insti-tute, 10Center for Cell and Gene Therapy, BaylorCollege of Medicine, Texas Children's Hospital,and Houston Methodist Hospital, Feigin Center,11Division of Stem Cell Therapy, Center for StemCell Biology and Regenerative Medicine, Instituteof Medical Science, University of Tokyo, Institutefor Stem Cell Biology and Regenerative Medicine,Stanford University School of Medicine.

The discovery of induced pluripotent stem cells(iPSCs) has created promising new avenues fortherapies in regenerative medicine. However, thetumorigenic potential of undifferentiated iPSCs is amajor safety concern for clinical translation. To ad-dress this issue, we demonstrated the efficacy ofsuicide gene therapy by introducing inducible cas-pase-9 (iC9) into iPSCs. Activation of iC9 with aspecific chemical inducer of dimerization (CID) in-itiates a caspase cascade that eliminates iPSCs andtumors originated from iPSCs. We introduced thisiC9/CID safeguard system into a previously re-ported iPSC-derived, rejuvenated cytotoxic T lym-phocyte (rejCTL) therapy model and confirmed thatwe can generate rejCTLs from iPSCs expressinghigh levels of iC9 without disturbing antigen-spe-

cific killing activity. iC9-expressing rejCTLs exertantitumor effects in vivo. The system efficiently andsafely induces apoptosis in these rejCTLs. These re-sults unite to suggest that the iC9/CID safeguardsystem is a promising tool for future iPSC-mediatedapproaches to clinical therapy.

3. Interspecific in vitro assay for the chimera-forming ability of human pluripotent stemcells. Development

Masaki H1, Kato-Itoh M1, Umino A1, Sato H1, Ha-manaka S1, Kobayashi T1, Yamaguchi T1, Ni-shimura K2, Ohtaka M2, Nakanishi M2, NakauchiH3: 1Division of Stem Cell Therapy, Center forStem Cell Biology and Regenerative Medicine, TheInstitute of Medical Science, The University of To-kyo, 2Research Center for Stem Cell Engineering,National Institute of Advanced Industrial Scienceand Technology (AIST), 3Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, The Institute of Medical Science,The University of Tokyo, Institute for Stem CellBiology and Regenerative Medicine, Stanford Uni-versity School of Medicine

Functional assay limitations are an emerging is-sue in characterizing human pluripotent stem cells(PSCs). With rodent PSCs, chimera formation usingpre-implantation embryos is the gold-standard as-say of pluripotency (competence of progeny to dif-ferentiate into all three germ layers). In humanPSCs (hPSCs), however, this can only be monitoredvia teratoma formation or in vitro differentiation, asethical concerns preclude generation of human-hu-man or human-animal chimeras. To circumvent thisissue, we developed a functional assay utilizing in-terspecific blastocyst injection and in vitro culture(interspecies in vitro chimera assay) that enablesthe development and observation of embryos up toheadfold stage. The assay uses mouse pre-implanta-tion embryos and rat, monkey and human PSCs tocreate interspecies chimeras cultured in vitro to theearly egg-cylinder stage. Intra- and interspecific chi-mera assays with rodent PSC lines were performedto confirm the consistency of results in vitro and invivo. The behavior of chimeras developed in vitroappeared to recapitulate that of chimeras developedin vivo; that is, PSC-derived cells survived andwere integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecificin vitro chimera assay is useful in evaluating thechimera-forming ability of rodent PSCs. However,when human induced PSCs (both conventional andnaïve-like types) were injected into mouse embryosand cultured, some human cells survived but weresegregated; unlike epiblast-stage rodent PSCs, theynever integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-

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human interspecies in vitro chimera assay does notaccurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily moreclosely related species as host embryos might benecessary to evaluate the developmental potency ofhPSCs.

4. Successful Reprogramming of Epiblast StemCells by Blocking Nuclear Localization of -Catenin. Stem Cell Reports.

Murayama H1, Masaki H1, Sato H1, Hayama T1,Yamaguchi T1, Nakauchi H2: 1Division of StemCell Therapy, Center for Stem Cell Biology andRegenerative Medicine, Institute of Medical Sci-ence, University of Tokyo, 2Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science,University of Tokyo, Institute for Stem Cell Biol-ogy and Regenerative Medicine, Stanford Univer-sity School of Medicine

Epiblast stem cells (EpiSCs) in mice and rats areprimed pluripotent stem cells (PSCs). They barelycontribute to chimeric embryos when injected intoblastocysts. Reprogramming of EpiSCs to embry-onic stem cell (ESC)-like cells (rESCs) may occur inresponse to LIF-STAT3 signaling; however, low re-programming efficiency hampers potential use ofrESCs in generating chimeras. Here, we describedramatic improvement of conversion efficiencyfrom primed to naive-like PSCs through upregula-tion of E-cadherin in the presence of the cytokineLIF. Analysis revealed that blocking nuclear local-ization of -CATENIN with small-molecule inhibi-tors significantly enhances reprogramming effi-ciency of mouse EpiSCs. Although activation ofWnt/-catenin signals has been thought desirablefor maintenance of naive PSCs, this study providesthe evidence that inhibition of nuclear translocationof -CATENIN enhances conversion of mouseEpiSCs to naive-like PSCs (rESCs). This affords bet-ter understanding of gene regulatory circuits under-lying pluripotency and reprogramming of PSCs.

5. Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanizedmouse model.

Nakauchi Y1, Yamazaki S1, Napier SC2, Usui J3,Ota Y4, Takahashi S5, Watanabe N2, Nakauchi H6:1Division of Stem Cell Therapy, Centre for StemCell Biology and Regenerative Medicine, Instituteof Medical Science, University of Tokyo, 2Labora-tory of Diagnostic Medicine, Division of Stem CellTherapy, Centre for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science,University of Tokyo, 3Department of Nephrology,Graduate School of Comprehensive Human Sci-ences, University of Tsukuba, 4Department of Pa-thology, Research Hospital, Institute of MedicalScience, University of Tokyo, 5Department of He-matology/Oncology, Institute of Medical Science,University of Tokyo, 6Division of Stem Cell Ther-apy, Centre for Stem Cell Biology and Regenera-tive Medicine, Institute of Medical Science, Uni-versity of Tokyo, Institute of Stem Cell Biologyand Regenerative Medicine, Stanford UniversitySchool of Medicine, Stanford

Graft-versus-host disease (GVHD), mediated bydonor-derived alloreactive T cells, is a major causeof nonrelapse mortality in allogeneic hematopoieticstem cell transplantation. Its therapy is not well-defined. We established allele-specific anti-humanleukocyte antigen (HLA) monoclonal antibodies(ASHmAbs) that specifically target HLA molecules,with steady death of target-expressing cells. Onesuch ASHmAb, against HLA-A＊02:01 (A2-kASHmAb), was examined in a xenogeneic GVHDmouse model. To induce fatal GVHD, non-irradi-ated NOD/Shi-scid/IL-2R(null) mice were injectedwith healthy donor human peripheral blood mono-nuclear cells, some expressing HLA-A＊02:01, somenot. Administration of A2-kASHmAb promoted thesurvival of mice injected with HLA-A＊02:01-ex-pressing peripheral blood mononuclear cells (p <0.0001) and, in humanized NOD/Shi-scid/IL-2R(null) mice, immediately cleared HLA-A＊02:01-ex-pressing human blood cells from mouse peripheralblood. Human peripheral blood mononuclear cellswere again detectable in mouse blood 2 to 4 weeksafter A2-kASHmAb administration, suggesting thatkASHmAb may be safely administered to GVHDpatients without permanently ablating the graft.This approach, different from those in existingGVHD pharmacotherapy, may open a new door fortreatment of GVHD in HLA-mismatched allogeneichematopoietic stem cell transplantation.

Publications

1. Yanagida A, Nakauchi H, Kamiya A. Genera-tion and In Vitro Expansion of Hepatic Progeni-tor Cells from Human iPS Cells. Methods MolBiol. 2016; 1357: 295-310. doi: 10.1007/7651_2015_199. PubMed PMID: 25697415.

2. Yokoi K, Akiyama K, Kaneshiro E, Higuchi T,Shimada Y, Kobayashi H, Akiyama M, Otsu M,Nakauchi H, Ohashi T, Ida H. Effect of donorchimerism to reduce the level of glycosamino-glycans following bone marrow transplantation

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in a murine model of mucopolysaccharidosistype II. J Inherit Metab Dis. 2015; 38(2): 333-40.doi: 10.1007/s10545-014-9800-x. PubMed PMID:25503568.

3. Yanagida A, Mizuno N, Yamazaki Y, Kato-ItohM, Umino A, Sato H, Ito K, Yamaguchi T,Nakauchi H, Kamiya A. Investigation of bi-potent differentiation of hepatoblasts using in-ducible diphtheria toxin receptor-transgenicmice. Hepatol Res. 2015. doi: 10.1111/hepr.12622.PubMed PMID: 26584962.

4. Yanagida A, Chikada H, Ito K, Umino A, Kato-Itoh M, Yamazaki Y, Sato H, Kobayashi T, Ya-maguchi T, Nakayama KI, Nakauchi H, KamiyaA. Liver maturation deficiency in p57(Kip2)－/－

mice occurs in a hepatocytic p57(Kip2) expres-sion-independent manner. Dev Biol. 2015; 407(2): 331-43. doi: 10.1016/j.ydbio.2015.07.004.PubMed PMID: 26165599.

5. Yamamoto S, Otsu M, Matsuzaka E, Konishi C,Takagi H, Hanada S, Mochizuki S, Nakauchi H,Imai K, Tsuji K, Ebihara Y. Screening of drugsto treat 8p11 myeloproliferative syndrome us-ing patient-derived induced pluripotent stemcells with fusion gene CEP110-FGFR1. PLoSOne. 2015; 10(3): e0120841. doi: 10.1371/journal.pone. 0120841. PubMed PMID : 25803811 ;PMCID: PMC4372437.

6. Wu JM, Hsueh YC, Ch'ang HJ, Luo CY, WuLW, Nakauchi H, Hsieh PC. Circulating cellscontribute to cardiomyocyte regeneration afterinjury. Circ Res. 2015; 116(4): 633-41. doi:10.1161/CIRCRESAHA. 116.304564. PubMedPMID: 25398235.

7. Toriumi T, Takayama N, Murakami M, Sato M,Yuguchi M, Yamazaki Y, Eto K, Otsu M,Nakauchi H, Shirakawa T, Isokawa K, HondaMJ. Characterization of mesenchymal progeni-tor cells in the crown and root pulp of primaryteeth. Biomed Res. 2015; 36(1): 31-45. doi:10.2220 / biomedres. 36.31. PubMed PMID :25749149.

8. Takeuchi Y, Takeuchi E, Ishida T, Onodera M,Nakauchi H, Otsu M. Curative haploidenticalBMT in a murine model of X-linked chronicgranulomatous disease. Int J Hematol. 2015; 102(1): 111-20. doi: 10.1007/s12185-015-1799-8.PubMed PMID: 25921405.

9. Takebe T, Enomura M, Yoshizawa E, KimuraM, Koike H, Ueno Y, Matsuzaki T, Yamazaki T,Toyohara T, Osafune K, Nakauchi H,Yoshikawa HY, Taniguchi H. Vascularized andComplex Organ Buds from Diverse Tissues viaMesenchymal Cell-Driven Condensation. CellStem Cell. 2015; 16(5): 556-65. doi: 10.1016/j.stem.2015.03.004. PubMed PMID: 25891906.

10. Sharma A, Sebastiano V, Scott CT, Magnus D,Koyano-Nakagawa N, Garry DJ, Witte ON,Nakauchi H, Wu JC, Weissman IL, Wu SM. Lift

NIH restrictions on chimera research. Science.2015 ; 350 ( 6261 ) : 640. doi : 10.1126 / sci-ence.350.6261.640-a. PubMed PMID: 26542560.

11. Sato A, Nishida C, Sato-Kusubata K, IshiharaM, Tashiro Y, Gritli I, Shimazu H, Munakata S,Yagita H, Okumura K, Tsuda Y, Okada Y, TojoA, Nakauchi H, Takahashi S, Heissig B, HattoriK. Inhibition of plasmin attenuates murineacute graft-versus-host disease mortality bysuppressing the matrix metalloproteinase-9-de-pendent inflammatory cytokine storm and ef-fector cell trafficking. Leukemia. 2015; 29(1): 145-56. doi: 10.1038/leu.2014.151. PubMed PMID:24791857.

12. Saito T, Yano F, Mori D, Kawata M, Hoshi K,Takato T, Masaki H, Otsu M, Eto K, NakauchiH, Chung UI, Tanaka S. Hyaline cartilage for-mation and tumorigenesis of implanted tissuesderived from human induced pluripotent stemcells. Biomed Res. 2015; 36(3): 179-86. doi:10.2220 / biomedres. 36.179. PubMed PMID :26106047.

13. Rashid T, Takebe T, Nakauchi H. Novel strate-gies for liver therapy using stem cells. Gut.2015; 64(1): 1-4. doi: 10.1136/gutjnl-2014-307480.PubMed PMID: 25183202.

14. Otani S, Kakinuma S, Kamiya A, Goto F,Kaneko S, Miyoshi M, Tsunoda T, Asano Y,Kawai-Kitahata F, Nitta S, Nakata T, OkamotoR, Itsui Y, Nakagawa M, Azuma S, Asahina Y,Yamaguchi T, Koshikawa N, Seiki M, NakauchiH, Watanabe M. Matrix metalloproteinase-14mediates formation of bile ducts and hepaticmaturation of fetal hepatic progenitor cells. Bio-chem Biophys Res Commun. 2015. doi: 10.1016/j.bbrc.2015.12.105. PubMed PMID: 26724533.

15. Okeyo KO, Kurosawa O, Yamazaki S, Oana H,Kotera H, Nakauchi H, Washizu M. Cell Adhe-sion Minimization by a Novel Mesh CultureMethod Mechanically Directs Trophoblast Dif-ferentiation and Self-Assembly Organization ofHuman Pluripotent Stem Cells. Tissue Eng PartC Methods. 2015; 21(10): 1105-15. doi: 10.1089/ten.TEC.2015.0038. PubMed PMID: 25914965;PMCID: PMC4593890.

16. Nakauchi Y, Yamazaki S, Napier SC, Usui J,Ota Y, Takahashi S, Watanabe N, Nakauchi H.Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA mon-oclonal antibody in a humanized mouse model.Exp Hematol. 2015; 43(2): 79-88 e1-4. doi: 10.1016 / j. exphem.2014.10.008. PubMed PMID :25448490.

17. Naka K, Jomen Y, Ishihara K, Kim J, IshimotoT, Bae EJ, Mohney RP, Stirdivant SM, OshimaH, Oshima M, Kim DW, Nakauchi H, TakiharaY, Kato Y, Ooshima A, Kim SJ. Dipeptide spe-cies regulate p38MAPK-Smad3 signalling tomaintain chronic myelogenous leukaemia stem

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cells. Nat Commun. 2015; 6: 8039. doi: 10.1038/ncomms 9039. PubMed PMID : 26289811 ;PMCID: PMC4560789.

18. Murayama H, Masaki H, Sato H, Hayama T,Yamaguchi T, Nakauchi H. Successful repro-gramming of epiblast stem cells by blocking nu-clear localization of beta-catenin. Stem CellReports. 2015; 4(1): 103-13. doi: 10.1016/j.stemcr.2014.12.003. PubMed PMID: 25556568; PMCID:PMC4297867.

19. Munakata S, Tashiro Y, Nishida C, Sato A,Komiyama H, Shimazu H, Dhahri D, Salama Y,Eiamboonsert S, Takeda K, Yagita H, Tsuda Y,Okada Y, Nakauchi H, Sakamoto K, Heissig B,Hattori K. Inhibition of plasmin protects againstcolitis in mice by suppressing matrix metallo-proteinase 9-mediated cytokine release frommyeloid cells. Gastroenterology. 2015; 148(3): 565-78 e4. doi: 10.1053/j.gastro.2014.12.001. PubMedPMID: 25490065.

20. Miyamoto T, Nakauchi H. [Generation of func-tional organs from pluripotent stem cells]. Rin-sho Ketsueki. 2015; 56(10): 2213-9. doi: 10.11406/rinketsu.56.2213. PubMed PMID: 26458462.

21. Masaki H, Kato-Itoh M, Umino A, Sato H, Ha-manaka S, Kobayashi T, Yamaguchi T,Nishimura K, Ohtaka M, Nakanishi M,Nakauchi H. Interspecific in vitro assay for thechimera-forming ability of human pluripotentstem cells. Development. 2015; 142(18): 3222-30.doi : 10.1242 /dev. 124016. PubMed PMID :26023098.

22. Lin HT, Masaki H, Yamaguchi T, Wada T,Yachie A, Nishimura K, Ohtaka M, NakanishiM, Nakauchi H, Otsu M. An assessment of theeffects of ectopic gp91phox expression in XCGDiPSC-derived neutrophils. Mol Ther Methods ClinDev. 2015; 2: 15046. doi: 10.1038/mtm.2015.46.PubMed PMID: 26682238; PMCID: PMC4674005.

23. Kobayashi T, Kato-Itoh M, Nakauchi H. Tar-geted organ generation using Mixl1-induciblemouse pluripotent stem cells in blastocyst com-plementation. Stem Cells Dev. 2015; 24(2): 182-9.doi: 10.1089/scd.2014.0270. PubMed PMID:25192056; PMCID: PMC4291089.

24. Kamiya A, Ito K, Yanagida A, Chikada H,Iwama A, Nakauchi H. MEK-ERK ActivityRegulates the Proliferative Activity of Fetal He-patoblasts Through Accumulation of p16/19(cdkn2a). Stem Cells Dev. 2015; 24(21): 2525-35.doi: 10.1089/scd.2015.0015. PubMed PMID:

26181762.25. Ishigaki T, Zaike Y, Nojima M, Kobayashi S,

Ohno N, Uchimaru K, Tojo A, Nakauchi H,Watanabe N. Quantification of adult T-cell leu-kemia/lymphoma cells using simple four-colorflow cytometry. Clin Chem Lab Med. 2015; 53(1):85-93. doi: 10.1515/cclm-2014-0183. PubMedPMID: 25060346.

26. Iriguchi S, Kikuchi N, Kaneko S, Noguchi E,Morishima Y, Matsuyama M, Yoh K, TakahashiS, Nakauchi H, Ishii Y. T-cell-restricted T-betoverexpression induces aberrant hematopoiesisof myeloid cells and impairs function of macro-phages in the lung. Blood. 2015; 125(2): 370-82.doi: 10.1182/blood-2014-05-575225. PubMedPMID: 25349175; PMCID: PMC4300389.

27. Iida A, Iwagawa T, Baba Y, Satoh S, MochizukiY, Nakauchi H, Furukawa T, Koseki H, Mu-rakami A, Watanabe S. Roles of histone H3K27trimethylase Ezh2 in retinal proliferation anddifferentiation. Dev Neurobiol. 2015; 75(9): 947-60. doi: 10.1002/dneu.22261. PubMed PMID:25556712.

28. Goto T, Kobayashi T, Hara H, Sanbo M, HochiS, Nakauchi H, Hirabayashi M. Knock-in of ahistone H2B-tdTomato reporter into the Rosa26locus allows visualization of cell nuclei in rats.Mol Reprod Dev. 2015. doi: 10.1002/mrd.22584.PubMed PMID: 26354179.

29. Ghosn EE, Waters J, Phillips M, Yamamoto R,Long BR, Yang Y, Gerstein R, Stoddart CA,Nakauchi H, Herzenberg LA. Fetal Hema-topoietic Stem Cell Transplantation Fails toFully Regenerate the B-Lymphocyte Compart-ment. Stem Cell Reports. 2015. doi: 10.1016/j.stemcr.2015.11.011. PubMed PMID: 26724903.

30. Chikada H, Ito K, Yanagida A, Nakauchi H,Kamiya A. The basic helix-loop-helix transcrip-tion factor, Mist1, induces maturation of mousefetal hepatoblasts. Sci Rep. 2015; 5: 14989. doi:10.1038/srep14989. PubMed PMID: 26456005;PMCID: PMC4601036.

31. Ando M, Nishimura T, Yamazaki S, YamaguchiT, Kawana-Tachikawa A, Hayama T, NakauchiY, Ando J, Ota Y, Takahashi S, Nishimura K,Ohtaka M, Nakanishi M, Miles JJ, Burrows SR,Brenner MK, Nakauchi H. A Safeguard Systemfor Induced Pluripotent Stem Cell-Derived Re-juvenated T Cell Therapy. Stem Cell Reports.2015; 5(4): 597-608. doi: 10.1016/j.stemcr.2015.07.011. PubMed PMID: 26321144; PMCID: PMC4624898.

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

Shinji Mochizuki, Yasuhiro Ebihara1, Feng Ma2,Emiko Matsuzaka, Yuji Zaike3 , HiromitsuNakauchi4, Kohichiro Tsuji5, Makoto Otsu: 1De-partment of Pediatric Hematology-Oncology, 2In-stitute of Blood Transfusion Chinese Academy ofMedical Sciences, Beijing Union Medical College,3Department of Laboratory Medicine, ResearchHospital, 4Division of Stem Cell Therapy, Centerfor Stem Cell Therapy and Regenerative Medicine,5Department of Pediatrics, National Hospital Or-ganization Shinshu Ueda Medical Center

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 withAGMS-3 stromal cells, which originate from murineaorta-gonad-mesonephros (AGM) region at 11 to 12dpc. In the co-culture, various hematopoietic pro-genitors were generated, and this hematopoietic ac-tivity was concentrated in cobblestone-like (CS)cells within differentiated human ESC or iPSC colo-nies. A fraction of CS cells expressed CD34 and re-tained a potential for endothelial cells. They alsocontained HPC, especially erythroid and multipo-tential HPC at high frequency. The multipotentialHPC abundant among the CS cells produced alltypes of mature blood cells, including adult typeglobin-expressing erythrocytes and tryptase andchymase-double positive mast cells (MC). Theyshowed neither immature properties of PSC nor po-tentials to differentiate into endoderm and ecto-derm at a clonal level. The developed co-culturesystem of hPSC can provide a novel source for he-matopoietic and blood cells applicable to cellulartherapies and drug screenings.

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


Stem Cell Bankステムセルバンク

Associate Professor Makoto Otsu, M.D., Ph.D. 准教授医学博士大津真

Stem cells represent a valuable cell source in the field of regenerative medicine.Hematopoietic stem cells provide a good example of such usefulness of stem cellresearch, showing many successful cases in both hematopoietic cell transplanta-tion and gene therapy. Pluripotent stem cells have become another possibility ofcell sources in regenerative medicine that may be utilized either for the basic re-search or to cure the diseases. Our eventual goal is to establish safe and effica-cious treatment for the patients suffering from various types of intractable dis-eases with no curative treatment available

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Yasuhiro Ebihara1, Shinji Mochizuki, Emiko Ma-tsuzaka, Yuji Zaike3, Hiromitsu Nakauchi4, Koh-ichiro Tsuji5, Makoto Otsu

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 aphenotype of CD45－, CD34－, CD14－, CD105＋,CD166＋, CD31－, and SSEA-4－, and had the abil-ity to differentiate 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.

3. Recapitulation of pathophysiological featuresof Wiskott Aldrich Syndrome using inducedpluripotent stem cells (iPSCs)

Mozhgan Khalaj Amirhosseini6, Haruna Takagi6,Chieko Konishi6, Huang-Ting Lin6, TakashiIshida6, Chen-Yi Lai6, Tomohiro Morio7, KohsukeImai7, Hiromitsu Nakauchi4, Makoto Otsu: 6Divi-sion of Stem Cell Bank, Center for Stem CellTherapy and Regenerative Medicine, 7Departmentof Pediatrics Perinatal and Maternal Medicine,Tokyo Medical and Dental University

Wiskott Aldrich syndrome (WAS) is an X-linkeddisease, which is caused by mutations in the geneencoding the WAS protein (WASp). As thrombocy-topenia is the most typical feature of this disease,often exposing patients to a significant risk of life-threatening hemorrhage, it has been the main sub-ject of study for many researchers. Because of limi-tations in disease modeling, precise mechanisms ofthe platelet abnormality remain to be elucidated.Here we established induced pluripotent stem cell(iPSC) lines from two XLT and one WAS patients

as disease models to address the issues. We firstconfirmed that these disease-specific iPSCs retainedgene mutations characteristic to each patient. Usingour differentiation culture system, we demonstratedthat numbers of both megakaryocyte (MK)s andplatelets obtainable from both XLT- and WAS-iPSCs were significantly smaller than those fromhealthy iPSCs. Detailed analysis revealed that theobserved defects were mainly due to insufficientproduction of proplatelet-bearing cells, but not toimpaired platelet production per MK. Lentiviral-mediated gene transfer led to appearance of WASpexpression in patient iPSC-derived MKs. The ex-pression of WASp, however, did not reach the nor-mal level that was seen in control-iPSC-MKs; yieldsof platelets showed some increase after gene trans-fer, but only marginally. Although further investi-gation is necessary, these results indicate the utilityof iPSC-based disease modeling for WAS. We arenow in the process of addressing how critically ex-pression levels of WASp will affect the efficacy inplatelet number recovery after gene transfer.

4. Demonstration of safe and efficacious genetherapy using X-CGD iPSCs.

Huang-Ting Lin6, Haruna Takagi6, Mozhgan Kha-raj6, Chieko Konishi6, Takashi Ishida6, Chen-YiLai6, Makoto Otsu, Hiromitsu Nakauchi4

X-linked chronic granulomatous disease (XCGD)is caused by gp91phox deficiency. This compro-mises neutrophil (NEU) killing of phagocytosedpathogens due to impaired production of reactiveoxygen species (ROS). In previous XCGD gene ther-apy clinical trials, the sustained persistence of genemarked cells could not be achieved without ad-versely triggering insertional mutagenesis. Addi-tionally cellular recovery (gp91phox and ROS) hadbeen incomplete on a per cell basis in comparisonwith healthy controls. This led to the hypothesisthat the expression of ectopic gp91phox in develop-ing NEUs could impede further differentiation intomature NEUs. To investigate this theory, a model-ing system was established by generating patientautologous XCGD-iPSCs and its differentiation intoNEUs. In this culture system, the hierarchical tran-sition of NEU differentiation could be demon-strated from developing (CD64dullCD15dull) to mature(CD64highCD15high). Alpharetroviral vectors wereused to transduce XCGD-iPSCs with the expressionof codon optimized gp91phox cDNA driven by theubiquitous EF1 short promoter. In healthy iPSC-derived NEUs, gp91phox expression and ROS pro-duction could only be detected in the mature frac-tion. In NEUs derived from transduced XCGD-iPSCs, functional recovery in the mature fractionwas incomplete. Ectopic gp91phox expression couldbe detected in the developing fraction through in-

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tracellular staining but not in healthy control cells.Most importantly, cell death was most prominentin developing NEUs ectopically expressinggp91phox. Mechanistic studies are under way to in-vestigate the role of non-physiological ROS produc-tion in inducing endoplasmic reticulum (ER) stressresulting in cell apoptosis. Therefore, affording cel-lular protection from the detrimental effects of non-physiological ROS production may improve XCGDclinical outcomes.

5. A New Strategy To Overcome The Cell DoseBarrier To Umbilical Cord Blood Transplants

Takashi Ishida6, Masaaki Higashihara8, HiromitsuNakauchi4, Makoto Otsu: 8Department of Hematol-ogy, Kitasato University School of Medicine

Umbilical cord blood (UCB) serves as a suitabledonor source in hematopoietic stem cell transplan-tation (HSCT). However, UCB has the major draw-back that is delayed engraftment due to its lowgraft cell numbers, which often limits its use inHSCT. To overcome this cell dose barrier, doubleunits UCB transplantation (UCBT) has been at-tempted, but the time to engraftment is still rela-tively delayed. Based on the report by Japan RedCross Society, the majority of UCB units remain un-used clinically because of their insufficient graft celldoses. Overall, these facts prompted us to seek fora new strategy to improve UCBT by using multiple(more than three) units. We here provide a proof offeasibility of such an approach using mouse trans-plantation models.To mimic a clinical setting of UCBT, we first es-

tablished an insufficient cell dose model by usingmouse BM KSL (c-Kit＋, Sca-1＋, lineage-marker-negative) cells. In this model, C57BL/6 (B6-Ly5.2,H2b) mice were the recipients, and congenic B6-Ly5.1 mice (H2b) were the primary donors of cellgrafts. The recipient mice were lethally irradiated,and thus could not survive with only the "insuffi-cient cell dose" of B6-Ly5.1 graft. The outcomes inHSCT were tested by the addition of mixed alloge-neic KSL cells (multi-allo HSPCs) in comparisonwith the addition of B6-Ly5.1 KSL cells (congenicHSPCs) with the equivalent doses. The effects ofmulti-allo HSPC transplants were evaluated by re-cipients' survival rate and complete blood countsover time. Detailed donor cell contribution in pe-ripheral blood and BM was also determined byflow cytometry analysis.Interestingly, addition of multi-allo HSPCs res-

cued otherwise lethal recipients as effectively ascongenic-HSPCs with the equivalent acceleration ofhematopoietic recovery. Chimerism analysis, how-ever, revealed that this "KSL cells alone" transplan-tation led to long-term existence of multi-donor he-matopoiesis, which was not ideal for a clinical set-

ting. We then replaced B6-Ly5.1 KSL grafts withthe whole BM (WBM) grafts. Titration experimentsdetermined 50,000 WBM cells as a single unit, mim-icking an "insufficient dose" of unmanipulatedUCB. Addition of multi-allo HSPCs in this modelalso showed complete protection of recipients fromlethality and enhanced early hematopoietic recov-ery. Remarkably, dominant B6-Ly5.1 chimerismwas established and maintained in this modifiedmodel. Experiments using subfractionation of theB6-Ly5.1 grafts demonstrated that small numbers ofT cells were responsible to single-donor chimerismformation possibly through graft versus graft reac-tions. Finally, to maximize transient early hema-topoietic reconstitution by multi-allo HSPCs (wecall this "bridging effect"), we tested whether cul-tured hematopoietic stem cells (HSC, defined asCD34negatvie/low KSL cells) worked better than uncul-tured KSL cells. Using our defined protocol com-patible with stem cell amplification, we demon-strated that a mixture of cultured allo-HSCs exhib-ited the bridging effect even superior to that of anuncultured KSL cell mixture.

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

Chen-Yi Lai6, Satoshi Yamazaki4, Yasuaki Iimura9,Masafumi Onodera10, Shigeru Kakuta11, YoichiroIwakura11, Hiromitsu Nakauchi4, Makoto Otsu:9Kushiro City General Hospital, 10Department ofGenetics, National Research Institute of ChildHealth and Development, 11Laboratory of Molecu-lar Pathogenesis, Center for Experimental Medi-cine and Systems,

Hematopoietic cell transplantation has provenbeneficial for various diseases, but low-level en-graftment would be problematic leading to patientmortality, requiring elucidation of the moleculardeterminant for successful engraftment. It remainsunclear how hematopoietic stem/progenitor cells(HSPCs) home to the bone marrow (BM) microenvi-ronment, initiate hematopoietic reconstitution, andmaintain life-long hematopoiesis. By monitoring thein vivo kinetics of transplanted donor cells, we dem-onstrated that modification of Cxcr4 signaling inmurine HSPCs did not affect homing/lodgingevents, but led to alteration in subsequent BM re-population kinetics, with observations confirmed byboth gain- and loss-of-function approaches. Withthe use of C-terminal truncated Cxcr4 as a gain-of-function effector, we showed that signal augmenta-tion through Cxcr4 led to favorable in vivo repopu-lation of primitive cell populations in BM. TheseCxcr4-augmented HSPCs exhibited in vitro en-hanced seeding efficiencies in stromal cell co-cul-tures and altered ligand-mediated phosphorylation

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kinetics of Extracellular signal-regulated kinases.Sustained signal enhancement, however, even withwild-type Cxcr4 overexpression resulted in poor pe-ripheral blood reconstitution due to blunted releaseof donor hematopoietic cells from BM. We thus

conclude that timely regulation of Cxcr4/CXCR4signaling will provide donor HSPCs with enhancedrepopulation potential, with preserving their abilityto release progeny into peripheral blood for im-proved transplantation outcomes.

Publications

1. Yokoi K, Akiyama K, Kaneshiro E, Higuchi T,Shimada Y, Kobayashi H et al . Effect of donorchimerism to reduce the level of glycosamino-glycans following bone marrow transplantationin a murine model of mucopolysaccharidosistype II. J Inherit Metab Dis 2015; 38(2): 333-40.

2. Yamamoto S, Otsu M, Matsuzaka E, Konishi C,Takagi H, Hanada S et al. Screening of Drugs toTreat 8p11 Myeloproliferative Syndrome UsingPatient-Derived Induced Pluripotent Stem Cellswith Fusion Gene CEP110-FGFR1. PLoS One2015; 10(3): e0120841.

3. Toriumi T, Takayama N, Murakami M, Sato M,Yuguchi M, Yamazaki Y et al. Characterizationof mesenchymal progenitor cells in the crownand root pulp of primary teeth. Biomed Res2015; 36(1): 31-45.

4. Takeuchi Y, Takeuchi E, Ishida T, Onodera M,Nakauchi H, Otsu M. Curative haploidenticalBMT in a murine model of X-linked chronicgranulomatous disease. Int J Hematol 2015; 102(1): 111-20.

5. Saito T, Yano F, Mori D, Kawata M, Hoshi K,Takato T et al. Hyaline cartilage formation andtumorigenesis of implanted tissues derived

from human induced pluripotent stem cells.Biomed Res 2015; 36(3): 179-86.

6. Otsu M, Yamada M, Nakajima S, Kida M,Maeyama Y, Hatano N et al. Outcomes in twoJapanese adenosine deaminase-deficiency pa-tients treated by stem cell gene therapy with nocytoreductive conditioning. J Clin Immunol 2015;35(4): 384-98.

7. Otsu M. [Patient-derived iPS cells as a tool forgene therapy research]. Rinsho Ketsueki 2015; 56(8): 1016-24.

8. Otsu M. Perspectives on stem cell gene therapyfor genetic disorders. 2015.

9. Nakazawa Y, Kawai T, Uchiyama T, Goto F,Watanabe N, Maekawa T et al. Effects of en-zyme replacement therapy on immune functionin ADA deficiency patient. Clin Immunol 2015;161(2): 391-3.

10. Lin H-T, Masaki H, Yamaguchi T, Wada T,Yachie A, Nishimura K et al. An assessment ofthe effects of ectopic gp91phox expression inXCGD iPSC-derived neutrophils. MolecularTherapy―Methods & Clinical Development 2015;2: 15046.

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1. Inhibition of plasmin attenuates murine acutegraft-versus-host disease mortality by sup-pressing the matrix metalloproteinase-9-de-pendent inflammatory cytokine storm and ef-fector cell trafficking

Aki Sato1, Chiemi Nishida2, Kaori Sato-Kusubata2,Makoto Ishihara1, Yoshihiko Tashiro1, IsmaelGritli2, Hiroshi Shimazu1, Shinya Munakata1,Hideo Yagita3, Ko Okumura3, Yuko Tsuda4,Yoshio Okada4 , Arinobu Tojo5 , HiromitsuNakauchi6, Satoshi Takahashi5, Beate Heissig2#,Koichi Hattori1,7#: 1Department of Stem Cell Regu-lation, The Institute of Medical Science, The Uni-versity of Tokyo, 2Department of Stem Cell Dy-namics, The Institute of Medical Science, The Uni-versity of Tokyo, 3Atopy (Allergy) Research Cen-ter, Juntendo University School of Medicine, To-kyo, 4Faculty of Pharmaceutical Sciences, KobeGakuin University, 5Department of Hematologyand Oncology, The Institute of Medical Science,The University of Tokyo, 6Department of StemCell Therapy, The Institute of Medical Science,The University of Tokyo, 7Center for Genome andRegenerative Medicine, Juntendo UniversitySchool of Medicine, Tokyo.

The systemic inflammatory response observedduring acute graft-versus-host disease (aGVHD) is

driven by proinflammatory cytokines, a 'cytokinestorm'. The function of plasmin in regulating the in-flammatory response is not fully understood, andits role in the development of aGVHD remains un-resolved. Here we show that plasmin is activatedduring the early phase of aGVHD in mice, and itsactivation correlated with aGVHD severity in hu-mans. Pharmacological plasmin inhibition protectedagainst aGVHD-associated lethality in mice. Mecha-nistically, plasmin inhibition impaired the infiltra-tion of inflammatory cells, the release of mem-brane-associated proinflammatory cytokines includ-ing tumor necrosis factor- (TNF-) and Fas-liganddirectly, or indirectly via matrix metalloproteinases(MMPs) and alters monocyte chemoattractantprotein-1 (MCP-1) signaling. We propose that plas-min and potentially MMP-9 inhibition offers anovel therapeutic strategy to control the deadly cy-tokine storm in patients with aGVHD, thereby pre-venting tissue destruction.

2. Inhibition of Plasmin Protects Against Colitisin Mice by Suppressing Matrix Metalloprotein-ase 9-mediated Cytokine Release FromMyeloid Cells

Shinya Munakata1, Yoshihiko Tashiro1, ChiemiNishida2, Aki Sato1, Hiromitsu Komiyama3, HiroshiShimazu1, Douaa Dhahri2, Yousef Salama2, Salita


Laboratory of Stem Cell Regulation幹細胞制御領域

Visiting Associate Professor Koichi Hattori, M.D, Ph.D. 客員准教授医学博士服部浩一

The goal of our laboratory is to identify novel therapeutic targets for diseases likecancer or inflammatory diseases by studying the role inflammatory and adult stemcells. Persistent inflammation is associated with diseases, including cancer, athe-rosclerosis, arthritis and autoimmune diseases. Recently, we identified plasmin asa novel therapeutic target for the treatment of inflammatory diseases like inflam-matory bowel disease, sepsis and chronic graft-versus host disease after bonemarrow transplantation, and during cancer.

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Eiamboonsert2, Kazuyoshi Takeda4, Hideo Yagita4,Yoko Tsuda5, Yoshio Okada5, Hiromitsu Nakau-chi6, Kazuhiro Sakamoto3, Beate Heissig2, KoichiHattori1,7: 1Division of Stem Cell Regulation, TheInstitute of Medical Science, The University of To-kyo, 2Division of Stem Cell Dynamics, The Insti-tute of Medical Science, The University of Tokyo,3Department of Coloproctological Surgery, Jun-tendo University Faculty of Medicine, Tokyo, 4De-partment of Immunology, Juntendo UniversityGraduate School of Medicine, Tokyo, 5Faculty ofPharmaceutical Sciences, Kobe Gakuin University,6Center for Stem Cell Biology and RegenerativeMedicine, The Institute of Medical Science, TheUniversity of Tokyo, 7Center for Genome and Re-generative Medicine, Juntendo University Schoolof Medicine, Tokyo

Activated proteases such as plasmin and matrixmetalloproteinases (MMPs) are activated in intesti-nal tissues of patients with active inflammatorybowel diseases. We investigated the effect of plas-min on progression of acute colitis. Colitis was in-duced in Mmp9－/－, Plg－/－, and C57BL/6 (control)mice by administration of dextran sulfate sodium,trinitrobenzene sulfonic acid, or CD40 antibody.Plasmin was inhibited in control mice by intraperi-toneal injection of YO-2, which blocks its active site.Mucosal and blood samples were collected andanalyzed by reverse transcription polymerase chainreaction and immunohistochemical analyses, aswell as for mucosal inflammation and levels of cy-tokines and chemokines. We showed that circulat-ing levels of plasmin were increased in mice withcolitis, compared with controls. Colitis did not de-velop in control mice injected with YO-2 or inPlg－/－ mice. Colons from these mice had reducedinfiltration of Gr1＋ neutrophils and F4/80＋macrophages, and reduced levels of inflammatorycytokines and chemokines. Colonic inflammationand colitis induction required activation of endoge-nous MMP9. Following colitis induction, micegiven YO-2, Plg－/－ mice, and Mmp9－/－ mice had re-duced serum levels of tumor necrosis factor andCXCL5, compared to control mice. In summary, inmice, plasmin induces a feedback mechanism inwhich activation of the fibrinolytic system promotesdevelopment of colitis, via activation of MMP9 orproteolytic enzymes. The proteolytic environmentstimulates influx of myeloid cells into the colonicepithelium and production of tumor necrosis factorand CXCL5. In turn, myeloid CD11b＋ cells releasethe urokinase plasminogen activator, which acceler-ates plasmin production. Disruption of the plasmin-induced chronic inflammatory circuit might there-fore be a strategy for treatment of colitis.

3. Role of mesenchymal stem cell-derived fibri-nolytic factor in tissue regeneration and can-cer progression

Beate Heissig1, Douaa Dhahri1, Salita Eiamboon-sert1, Yousef Salama1, Hiroshi Shimazu2, ShinyaMunakata2, Koichi Hattori2,3: 1Division of StemCell Dynamics, The Institute of Medical Science,The University of Tokyo, Tokyo, 2Laboratory ofStem Cell Regulation, The Institute of MedicalScience, The University of Tokyo, 3Center forGenome and Regenerative Medicine, JuntendoUniversity School of Medicine, 2-1-1 Hongo,Bunkyo-ku, Tokyo, 113-8421, Japan.

Tissue regeneration during wound healing orcancer growth and progression depends on the es-tablishment of a cellular microenvironment. Mesen-chymal stem cells (MSC) are part of this cellularmicroenvironment, where they functionally modu-late cell homing, angiogenesis, and immune modu-lation. MSC recruitment involves detachment ofthese cells from their niche, and finally MSC migra-tion into their preferred niches; the wounded area,the tumor bed, and the BM, just to name a few.During this recruitment phase, focal proteolysis dis-rupts the extracellular matrix (ECM) architecture,breaks cell-matrix interactions with receptors, andintegrins, and causes the release of bioactive frag-ments from ECM molecules. MSC produce a broadarray of proteases, promoting remodeling of thesurrounding ECM through proteolytic mechanisms.The fibrinolytic system, with its main player plas-min, plays a crucial role in cell migration, growthfactor bioavailability, and the regulation of otherprotease systems during inflammation, tissue regen-eration, and cancer. Key components of the fibri-nolytic cascade, including the urokinase plasmino-gen activator receptor (uPAR) and plasminogen ac-tivator inhibitor-1 (PAI-1), are expressed in MSC.This review will introduce general functional prop-erties of the fibrinolytic system, which go beyondits known function of fibrin clot dissolution (fibri-nolysis). We will focus on the role of the fibri-nolytic system for MSC biology, summarizing ourcurrent understanding of the role of the fibrinolyticsystem for MSC recruitment and the functional con-sequences for tissue regeneration and cancer. As-pects of MSC origin, maintenance, and the mecha-nisms by which these cells contribute to alteredprotease activity in the microenvironment undernormal and pathological conditions will also be dis-cussed.

4. Cancer therapy targeting the fibrinolytic sys-tem

Beate Heissig1, Salita Eiamboonsert1, YousefSalama1, Horoshi Shimazu1, Douaa Dhahri1, Shi-

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nya Munakata2, Yoshihiko Tashiro2, Koichi Hat-tori2: 1Division of Stem Cell Dynamics, The Insti-tute of Medical Science, The University of Tokyo,2Laboratory of Stem Cell Regulation, The Instituteof Medical Science, The University of Tokyo; Cen-ter for Genome and Regenerative Medicine, Jun-tendo University School of Medicine, Tokyo.

The tumor microenvironment is recognized as akey factor in the multiple stages of cancer progres-sion, mediating local resistance, immune-escapeand metastasis. Cancer growth and progression re-quire remodeling of the tumor stromal microenvi-ronment, such as the development of tumor-associ-ated blood vessels, recruitment of bone marrow-de-rived cells and cytokine processing. Extracellular

matrix breakdown achieved by proteases like the fi-brinolytic factor plasmin and matrix metallopro-teases is necessary for cell migration crucial for can-cer invasion and metastasis. Key components of thefibrinolytic system are expressed in cells of the tu-mor microenvironment. Plasmin can control growthfactor bioavailability, or the regulation of other pro-teases leading to angiogenesis, and inflammation.In this review, we will focus on the role of the fibri-nolytic system in the tumor microenvironmentsummarizing our current understanding of the roleof the fibrinolytic factors for the modulation of thelocal chemokine/cytokine milieu, resulting inmyeloid cell recruitment, which can promoteneoangiogenesis.

Publications

1. Munakata S, Tashiro Y, Nishida C, Sato A,Komiyama, H, Shimazu H, Dhahri D, Salama Y,Eiamboonsert S, Takeda K, Yagita H, Tsuda Y,Okada Y, Nakauchi H, Sakamoto K, Heissig B.#and Hattori, K.# Inhibition of Plasmin ProtectsAgainst Colitis in Mice by Suppressing MatrixMetalloproteinase 9-mediated Cytokine ReleaseFrom Myeloid Cells. Gastroenterology, 148: 565-578, 2015.

2. Sato A, Nishida C, Sato-Kusubata, K, Ishihara M,Tashiro Y, Gritli, I, Shimazu H, Munakata S,Yagita H, Okumura K, Tsuda Y, Okada Y, TojoA, Nakauchi H, Takahashi S, Heissig B.# andHattori K.# Inhibition of plasmin attenuatesmurine acute graft-versus-host disease mortality

by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and ef-fector cell trafficking. Leukemia, 29: 145-56, 2015.

3. Heissig B, ＊# Dhahri D, Eiamboonsert S, SalamaY, Shimazu H, Munakata S, and Hattori K#. Roleof mesenchymal stem cell-derived fibrinolyticfactor in tissue regeneration and cancer progres-sion. Cell Mol. Life Sci., 72: 4759-70, 2015.

4. Heissig B, Eiamboonsert S, Salama Y, ShimazuH, Munakata S, Tashiro Y, and Hattori K#. Can-cer therapy targeting the fibrinolytic system. Ad-vanced Drug Delivery Reviews, Adv. Drug De-liv. Res, Nov 14. pii: S0169-409X(15)00274-4. doi:10.1016/j.addr.2015.11.010, 2015.

# shared senior authorship

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

Shinji Mochizuki, Yasuhiro Ebihara1, Feng Ma2,Emiko Matsuzaka, Yuji Zaike3, Hiromitsu Naka-uchi4, Kohichiro Tsuji5, Makoto Otsu: 1Departmentof Pediatric Hematology-Oncology, 2Institute ofBlood Transfusion Chinese Academy of MedicalSciences, Beijing Union Medical College, 3Depart-ment of Laboratory Medicine, Research Hospital,4Division of Stem Cell Therapy, Center for StemCell Therapy and Regenerative Medicine, 5Depart-ment of Pediatrics, National Hospital OrganizationShinshu Ueda Medical Center

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 withAGMS-3 stromal cells, which originate from murineaorta-gonad-mesonephros (AGM) region at 11 to 12dpc. In the co-culture, various hematopoietic pro-genitors were generated, and this hematopoietic ac-tivity was concentrated in cobblestone-like (CS)cells within differentiated human ESC or iPSC colo-nies. A fraction of CS cells expressed CD34 and re-tained a potential for endothelial cells. They alsocontained HPC, especially erythroid and multipo-tential HPC at high frequency. The multipotentialHPC abundant among the CS cells produced alltypes of mature blood cells, including adult type globin-expressing erythrocytes and tryptase andchymase-double positive mast cells (MC). Theyshowed neither immature properties of PSC nor po-tentials to differentiate into endoderm and ecto-derm at a clonal level. The developed co-culturesystem of hPSC can provide a novel source for he-matopoietic and blood cells applicable to cellulartherapies and drug screenings.

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


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

Associate Professor Makoto OtsuProject Assistant Professor Shinji Mochizuki

准教授医学博士大津真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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Yasuhiro Ebihara1, Shinji Mochizuki, Emiko Ma-tsuzaka, Yuji Zaike3, Hiromitsu Nakauchi4, Koh-ichiro Tsuji5, Makoto Otsu

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 aphenotype of CD45－, CD34－, CD14－, CD105＋,CD166＋, CD31－, and SSEA-4－, and had the abil-ity to differentiate 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.

3. Recapitulation of pathophysiological featuresof Wiskott Aldrich Syndrome using inducedpluripotent stem cells (iPSCs)

Mozhgan Khalaj Amirhosseini6, Haruna Takagi6,Chieko Konishi6, Huang-Ting Lin6, TakashiIshida6, Chen-Yi Lai6, Tomohiro Morio7, KohsukeImai7, Hiromitsu Nakauchi4, Makoto Otsu: 6Divi-sion of Stem Cell Bank, Center for Stem CellTherapy and Regenerative Medicine, 7Departmentof Pediatrics Perinatal and Maternal Medicine,Tokyo Medical and Dental University

Wiskott Aldrich syndrome (WAS) is an X-linkeddisease, which is caused by mutations in the geneencoding the WAS protein (WASp). As thrombocy-topenia is the most typical feature of this disease,often exposing patients to a significant risk of life-threatening hemorrhage, it has been the main sub-ject of study for many researchers. Because of limi-tations in disease modeling, precise mechanisms ofthe platelet abnormality remain to be elucidated.Here we established induced pluripotent stem cell(iPSC) lines from two XLT and one WAS patients

as disease models to address the issues. We firstconfirmed that these disease-specific iPSCs retainedgene mutations characteristic to each patient. Usingour differentiation culture system, we demonstratedthat numbers of both megakaryocyte (MK)s andplatelets obtainable from both XLT- and WAS-iPSCs were significantly smaller than those fromhealthy iPSCs. Detailed analysis revealed that theobserved defects were mainly due to insufficientproduction of proplatelet-bearing cells, but not toimpaired platelet production per MK. Lentiviral-mediated gene transfer led to appearance of WASpexpression in patient iPSC-derived MKs. The ex-pression of WASp, however, did not reach the nor-mal level that was seen in control-iPSC-MKs; yieldsof platelets showed some increase after gene trans-fer, but only marginally. Although further investi-gation is necessary, these results indicate the utilityof iPSC-based disease modeling for WAS. We arenow in the process of addressing how critically ex-pression levels of WASp will affect the efficacy inplatelet number recovery after gene transfer.

4. Demonstration of safe and efficacious genetherapy using X-CGD iPSCs.

Huang-Ting Lin6, Haruna Takagi6, Mozhgan Kha-raj6, Chieko Konishi6, Takashi Ishida6, Chen-YiLai6, Makoto Otsu, Hiromitsu Nakauchi4

X-linked chronic granulomatous disease (XCGD)is caused by gp91phox deficiency. This compro-mises neutrophil (NEU) killing of phagocytosedpathogens due to impaired production of reactiveoxygen species (ROS). In previous XCGD gene ther-apy clinical trials, the sustained persistence of genemarked cells could not be achieved without ad-versely triggering insertional mutagenesis. Addi-tionally cellular recovery (gp91phox and ROS) hadbeen incomplete on a per cell basis in comparisonwith healthy controls. This led to the hypothesisthat the expression of ectopic gp91phox in develop-ing NEUs could impede further differentiation intomature NEUs. To investigate this theory, a model-ing system was established by generating patientautologous XCGD-iPSCs and its differentiation intoNEUs. In this culture system, the hierarchical tran-sition of NEU differentiation could be demon-strated from developing (CD64dullCD15dull) to mature(CD64highCD15high). Alpharetroviral vectors wereused to transduce XCGD-iPSCs with the expressionof codon optimized gp91phox cDNA driven by theubiquitous EF1 short promoter. In healthy iPSC-derived NEUs, gp91phox expression and ROS pro-duction could only be detected in the mature frac-tion. In NEUs derived from transduced XCGD-iPSCs, functional recovery in the mature fractionwas incomplete. Ectopic gp91phox expression couldbe detected in the developing fraction through

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intracellular staining but not in healthy controlcells. Most importantly, cell death was most promi-nent in developing NEUs ectopically expressinggp91phox. Mechanistic studies are under way to in-vestigate the role of non-physiological ROS produc-tion in inducing endoplasmic reticulum (ER) stressresulting in cell apoptosis. Therefore, affording cel-lular protection from the detrimental effects of non-physiological ROS production may improve XCGDclinical outcomes.

5. A New Strategy To Overcome The Cell DoseBarrier To Umbilical Cord Blood Transplants

Takashi Ishida6, Masaaki Higashihara8, HiromitsuNakauchi4, Makoto Otsu: 8Department of Hematol-ogy, Kitasato University School of Medicine

Umbilical cord blood (UCB) serves as a suitabledonor source in hematopoietic stem cell transplan-tation (HSCT). However, UCB has the major draw-back that is delayed engraftment due to its lowgraft cell numbers, which often limits its use inHSCT. To overcome this cell dose barrier, doubleunits UCB transplantation (UCBT) has been at-tempted, but the time to engraftment is still rela-tively delayed. Based on the report by Japan RedCross Society, the majority of UCB units remain un-used clinically because of their insufficient graft celldoses. Overall, these facts prompted us to seek fora new strategy to improve UCBT by using multiple(more than three) units. We here provide a proof offeasibility of such an approach using mouse trans-plantation models.To mimic a clinical setting of UCBT, we first es-

tablished an insufficient cell dose model by usingmouse BM KSL (c-Kit＋, Sca-1＋, lineage-marker-negative) cells. In this model, C57BL/6 (B6-Ly5.2,H2b) mice were the recipients, and congenic B6-Ly5.1 mice (H2b) were the primary donors of cellgrafts. The recipient mice were lethally irradiated,and thus could not survive with only the "insuffi-cient cell dose" of B6-Ly5.1 graft. The outcomes inHSCT were tested by the addition of mixed alloge-neic KSL cells (multi-allo HSPCs) in comparisonwith the addition of B6-Ly5.1 KSL cells (congenicHSPCs) with the equivalent doses. The effects ofmulti-allo HSPC transplants were evaluated by re-cipients' survival rate and complete blood countsover time. Detailed donor cell contribution in pe-ripheral blood and BM was also determined byflow cytometry analysis.Interestingly, addition of multi-allo HSPCs res-

cued otherwise lethal recipients as effectively ascongenic-HSPCs with the equivalent acceleration ofhematopoietic recovery. Chimerism analysis, how-ever, revealed that this "KSL cells alone" transplan-tation led to long-term existence of multi-donor he-matopoiesis, which was not ideal for a clinical set-

ting. We then replaced B6-Ly5.1 KSL grafts withthe whole BM (WBM) grafts. Titration experimentsdetermined 50,000 WBM cells as a single unit, mim-icking an "insufficient dose" of unmanipulatedUCB. Addition of multi-allo HSPCs in this modelalso showed complete protection of recipients fromlethality and enhanced early hematopoietic recov-ery. Remarkably, dominant B6-Ly5.1 chimerismwas established and maintained in this modifiedmodel. Experiments using subfractionation of theB6-Ly5.1 grafts demonstrated that small numbers ofT cells were responsible to single-donor chimerismformation possibly through graft versus graft reac-tions. Finally, to maximize transient early hema-topoietic reconstitution by multi-allo HSPCs (wecall this "bridging effect"), we tested whether cul-tured hematopoietic stem cells (HSC, defined asCD34negatvie/low KSL cells) worked better than uncul-tured KSL cells. Using our defined protocol com-patible with stem cell amplification, we demon-strated that a mixture of cultured allo-HSCs exhib-ited the bridging effect even superior to that of anuncultured KSL cell mixture.

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

Chen-Yi Lai6, Satoshi Yamazaki4, Yasuaki Iimura9,Masafumi Onodera10, Shigeru Kakuta11, YoichiroIwakura11, Hiromitsu Nakauchi4, Makoto Otsu:9Kushiro City General Hospital, 10Department ofGenetics, National Research Institute of ChildHealth and Development, 11Laboratory of Molecu-lar Pathogenesis, Center for Experimental Medi-cine and Systems

Hematopoietic cell transplantation has provenbeneficial for various diseases, but low-level en-graftment would be problematic leading to patientmortality, requiring elucidation of the moleculardeterminant for successful engraftment. It remainsunclear how hematopoietic stem/progenitor cells(HSPCs) home to the bone marrow (BM) microenvi-ronment, initiate hematopoietic reconstitution, andmaintain life-long hematopoiesis. By monitoring thein vivo kinetics of transplanted donor cells, we dem-onstrated that modification of Cxcr4 signaling inmurine HSPCs did not affect homing/lodgingevents, but led to alteration in subsequent BM re-population kinetics, with observations confirmed byboth gain- and loss-of-function approaches. Withthe use of C-terminal truncated Cxcr4 as a gain-of-function effector, we showed that signal augmenta-tion through Cxcr4 led to favorable in vivo repopu-lation of primitive cell populations in BM. TheseCxcr4-augmented HSPCs exhibited in vitro en-hanced seeding efficiencies in stromal cell co-cul-tures and altered ligand-mediated phosphorylation

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kinetics of Extracellular signal-regulated kinases.Sustained signal enhancement, however, even withwild-type Cxcr4 overexpression resulted in poor pe-ripheral blood reconstitution due to blunted releaseof donor hematopoietic cells from BM. We thus

conclude that timely regulation of Cxcr4/CXCR4signaling will provide donor HSPCs with enhancedrepopulation potential, with preserving their abilityto release progeny into peripheral blood for im-proved transplantation outcomes.

Publications

1. Yokoi K, Akiyama K, Kaneshiro E, Higuchi T,Shimada Y, Kobayashi H et al. Effect of donorchimerism to reduce the level of glycosamino-glycans following bone marrow transplantationin a murine model of mucopolysaccharidosistype II. J Inherit Metab Dis 2015; 38(2): 333-40.

2. Yamamoto S, Otsu M, Matsuzaka E, Konishi C,Takagi H, Hanada S et al. Screening of Drugs toTreat 8p11 Myeloproliferative Syndrome UsingPatient-Derived Induced Pluripotent Stem Cellswith Fusion Gene CEP110-FGFR1. PLoS One2015; 10(3): e0120841.

3. Toriumi T, Takayama N, Murakami M, Sato M,Yuguchi M, Yamazaki Y et al. Characterizationof mesenchymal progenitor cells in the crownand root pulp of primary teeth. Biomed Res2015; 36(1): 31-45.

4. Takeuchi Y, Takeuchi E, Ishida T, Onodera M,Nakauchi H, Otsu M. Curative haploidenticalBMT in a murine model of X-linked chronicgranulomatous disease. Int J Hematol 2015; 102(1): 111-20.

5. Saito T, Yano F, Mori D, Kawata M, Hoshi K,Takato T et al. Hyaline cartilage formation andtumorigenesis of implanted tissues derived

from human induced pluripotent stem cells.Biomed Res 2015; 36(3): 179-86.

6. Otsu M, Yamada M, Nakajima S, Kida M,Maeyama Y, Hatano N et al. Outcomes in twoJapanese adenosine deaminase-deficiency pa-tients treated by stem cell gene therapy with nocytoreductive conditioning. J Clin Immunol 2015;35(4): 384-98.

7. Otsu M. [Patient-derived iPS cells as a tool forgene therapy research]. Rinsho Ketsueki 2015; 56(8): 1016-24.

8. Otsu M. Perspectives on stem cell gene therapyfor genetic disorders. 2015.

9. Nakazawa Y, Kawai T, Uchiyama T, Goto F,Watanabe N, Maekawa T et al. Effects of en-zyme replacement therapy on immune functionin ADA deficiency patient. Clin Immunol 2015;161(2): 391-3.

10. Lin H-T, Masaki H, Yamaguchi T, Wada T,Yachie A, Nishimura K et al. An assessment ofthe effects of ectopic gp91phox expression inXCGD iPSC-derived neutrophils. MolecularTherapy―Methods & Clinical Development 2015;2: 15046.

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1. Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanizedmouse model.

Nakauchi Y, Yamazaki S, Napier SC, Usui JI, OtaY, Takahashi S, Watanabe N, Nakauchi H.

Graft-versus-host disease (GVHD), mediated bydonor-derived alloreactive T cells, is a major causeof nonrelapse mortality in allogeneic hematopoieticstem cell transplantation. Its therapy is not well-defined. We established allele-specific anti-humanleukocyte antigen (HLA) monoclonal antibo-dies (ASHmAbs) that specifically target HLA mole-cules, with steady death of target-expressing cells.One such ASHmAb, against HLA-A＊02:01 (A2-kASHmAb), was examined in a xenogeneic GVHDmouse model. To induce fatal GVHD, non-irradi-ated NOD/Shi-scid/IL-2R(null) mice were injectedwith healthy donor human peripheral blood mono-nuclear cells, some expressing HLA-A＊02:01, somenot. Administration of A2-kASHmAb promoted thesurvival of mice injected with HLA-A＊02:01-ex-pressing peripheral blood mononuclear cells (p <0.0001) and, in humanized NOD/Shi-scid/IL-2R(null) mice, immediately cleared HLA-A＊02:01-ex-pressing human blood cells from mouse peripheral

blood. Human peripheral blood mononuclear cellswere again detectable in mouse blood 2 to 4 weeksafter A2-kASHmAb administration, suggesting thatkASHmAb may be safely administered to GVHDpatients without permanently ablating the graft.This approach, different from those in existingGVHD pharmacotherapy, may open a new door fortreatment of GVHD in HLA-mismatched allogeneichematopoietic stem cell transplantation.

2. Myeloablative unrelated cord blood transplan-tation for Philadelphia chromosome-positiveacute lymphoblastic leukemia: comparisonwith other graft sources from related and un-related donors.

Konuma T, Kato S, Ooi J, Oiwa-Monna M, TojoA, Takahashi S.

Philadelphia chromosome (Ph)-positive acutelymphoblastic leukemia (ALL) is a distinct clinicalentity among ALL and is associated with adverseoutcomes and higher rates of relapse when conven-tional chemotherapy is used alone. Although allo-geneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for patientswith Ph＋ALL, the impact of graft sources, particu-larly cord blood transplantation (CBT), on allo-


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.

教授医学博士東條有伸准教授医学博士高橋聡

We are conducting clinical stem cell transplantation, especially using cord bloodas a promising alternative donor for clinical use and investigating optimal strate-gies to obtain the best results in this area. We are also generating pre-clinicalstudy to utilize virus-specific CTL for immune competent patients such as post-transplantation. Our goal is as allogeneic transplantation to be safer therapeuticoption and to extend for older patients.

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HSCT for patients with Ph＋ALL has yet to beclarified. We retrospectively compared clinical out-comes after unrelated CBT (n = 20), unrelated bonemarrow transplantation (n = 7), and related bonemarrow and peripheral blood stem cell transplanta-tions (n = 13) following myeloablative conditioningin 40 patients with Ph＋ALL. Although graft sourcehad no significant impact on survival or relapse,disease status at transplantation did significantly af-fect outcomes. These data suggest that unrelatedCBT is feasible and should be considered early inthe course of patients with Ph＋ALL when HLA-compatible related and unrelated donors are notavailable.

3. The Adult Myelodysplastic Syndrome andAdult Acute Myeloid Leukemia WorkingGroup of the Japan Society for HematopoieticCell Transplantation. Effect of granulocytecolony-stimulating factor combined condition-ing in cord blood transplantation for myelo-dysplastic syndrome and secondary acutemyeloid leukemia: a retrospective study in Ja-pan. Biol Blood Marrow Transplant. 2015 Sep;21 (9 ) : 1632 - 40. doi : 10.1016 / j. bbmt. 2015.05.009. Epub 2015 May 16.

Konuma T, Kato S, Ooi J, Oiwa-Monna M, Ebi-hara Y, Mochizuki S, Yuji K, Ohno N, KawamataT, Jo N, Yokoyama K, Uchimaru K, Tojo A,Takahashi S.

Granulocyte colony-stimulating factor (G-CSF) in-creases the susceptibility of dormant malignant ornonmalignant hematopoietic cells to cytarabinearabinoside (Ara-C) through the induction of cellcycle entry. Therefore, G-CSF-combined condition-ing before allogeneic stem cell transplantationmight positively contribute to decreased incidencesof relapse and graft failure without having to in-crease the dose of cytotoxic drugs. We conducted aretrospective nationwide study of 336 adult patientswith myelodysplastic syndrome (MDS) and secon-dary acute myeloid leukemia (sAML) after single-unit cord blood transplantation (CBT) who under-went 4 different kinds of conditioning regimens: to-tal body irradiation (TBI) ＞―8 Gy＋Ara-C/G-CSF＋cyclophosphamide (CY) (n = 65), TBI＞―8 Gy＋Ara-C＋CY (n = 119), TBI＞―8 Gy＋other (n = 104), orTBI < 8 Gy or non-TBI (n = 48). The TBI＞―8 Gy＋Ara-C/G-CSF＋CY regimen showed significantlyhigher incidence of neutrophil engraftment (hazardratio, 1.52; 95% confidence interval [CI], 1.10 to2.08; P = .009) and lower overall mortality (hazard

ratio,. 46; 95% CI, .26 to .82; P = .008) rates com-pared with those without a G-CSF regimen. Thisretrospective study shows that the G-CSF-combinedconditioning regimen provides better engraftmentand survival results in CBT for adults with MDSand sAML.

4. Generation of multivirus-specificT Cells by asingle stimulation of PBMCs with a peptidemixture utilizing serum-free medium.

Fujita Y, Tanaka Y, Takahashi S.

Restoration of virus-specific immunity offers anattractive alternative to conventional drugs. Re-cently, the system of rapid generation of multivi-rus-specific T cells has been reported (Gerdemann,U, 2012). With this system, polyclonal CTLs specificfor multivirus antigens can be produced after a sin-gle stimulation of PBMCs with a peptide mixturespanning the target antigens in the presence of IL4and IL7. We have introduced and verified this sys-tem to apply for clinical use in Japan. To meet therequirement for the viral infections after HSCT bybroad viral antigens and in terms of regulation bythe Japanese FDA, we generated mutivirus-specificT-cells targeting 7 viruses (CMV, EBV, AdV, HHV-6, BKV, JCV, and VZV) in serum-free medium.PBMCs were stimulated with peptide mixture span-ning the target antigens of 3 (CMV, EBV, AdV) or 7viruses as above and cultured in serum-free me-dium with IL4 and IL7 for 9-12 days. After single-stimulation and culture, the cells were analyzedwith cell number, phenotypes, TCR repertoires, andvirus-specific responses by FACS and ELISpot.Starting from 20×10^6 of PBMCs, 3 or 7 viruses'antigens stimulated cells increased to averages of92.0×10^6 and 112.7×10^6, respectively after 9-12days. These cells were mostly CD3T, which con-tained both CD4T and CD8T with a dominant phe-notype of central memory (CD62L＋CD45RO＋).Actually, the cells demonstrated specificity towardall the 7 virus antigens by IFN production orCD107a degranulation assay. Furthermore, we con-firmed the CD107a＋IFN＋ effector-cells againstspecific virus antigens displayed a wide range ofTCR diversities including several predominant TCRrepertoire populations depending on the kinds ofspecific-antigens. We could rapidly and easily gen-erate polyclonal 7 viruses-specific T cells with a sin-gle stimulation of PBMCs without using any serumproducts. This system is ready to go to the clinicaltrials after allogeneic transplantation setting.

Publications

Nakauchi Y, Yamazaki S, Napier SC, Usui JI, Ota Y, Takahashi S, Watanabe N, Nakauchi H. Effective

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treatment against severe Graft-versus-Host Dis-ease with allele-specific anti-HLA monoclonal an-tibody in a humanized-mouse model. Exp Hema-tol. 43: 79-88, 2015.

Sato A, Nishida C, Sato-Kusubata K, Ishihara M,Tashiro Y, Gritli I, Shimazu H, Munakata S,Yagita H, Okumura K, Tsuda Y, Okada Y, TojoA, Nakauchi H, Takahashi S, Heissig B and Hat-tori K. Inhibition of plasmin attenuates murineacute graft-versus-host disease mortality by sup-pressing the matrix metalloproteinase-9-depend-ent inflammatory cytokine storm and effector celltrafficking. Leukemia (2015) 29, 145-156; doi: 10.1038/leu.2014.151

Konuma T, Kato S, Ooi J, Ebihara Y, Mochizuki S,Oiwa-Monna M, Tojo A, Takahashi S. Third allo-geneic stem cell transplantation (SCT) using unre-lated cord blood for relapsed acute leukemia af-ter second allogeneic SCT. Int J Hematol. 2015Feb 6. [Epub ahead of print] PMID: 25655380

Konuma T, Kato S, Ooi J, Oiwa-Monna M, Tojo A,Takahashi S. Myeloablative unrelated cord bloodtransplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia: compari-son with other graft sources from related and un-related donors. Ann Hematol. 2015 Feb; 94(2):289-96. doi: 10.1007/s00277-014-2195-9. Epub 2014Sep 3. PMID: 25178518

Konuma T, Kato S, Ishii H, Takeda R, Oiwa-MonnaM, Tojo A, Takahashi S. HLA-DRB1 mismatch isassociated with a decreased relapse in adult acute

myeloid leukemia after single-unit myeloablativecord blood transplantation. Ann Hematol. 2015Mar 11. [Epub ahead of print] PMID: 25754345

Konuma T, Kato S, Ooi J, Ebihara Y, Mochizuki S,Ishii H, Takei T, Oiwa-Monna M, Tojo A, Taka-hashi S. Second allogeneic transplantation usingunrelated cord blood for relapsed hematologicalmalignancies after allogeneic transplantation.Leuk Lymphoma. 2015 May 25: 1-7. [Epub aheadof print]

Konuma T, Takahashi S, Uchida N, Kuwatsuka Y,Yamasaki S, Aoki J, Onishi Y, Aotsuka N, OhashiK, Mori T, Masuko M, Nakamae H, Miyamura K,Kato K, Atsuta Y, Kato S, Asano S, Takami A,Miyazaki Y: The Adult Myelodysplastic Syn-drome and Adult Acute Myeloid LeukemiaWorking Group of the Japan Society for Hema-topoietic Cell Transplantation. Effect of granulo-cyte colony-stimulating factor combined condi-tioning in cord blood transplantation for myelo-dysplastic syndrome and secondary acutemyeloid leukemia: a retrospective study in Japan.Biol Blood Marrow Transplant. 2015 Sep; 21(9):1632-40. doi: 10.1016/j.bbmt.2015.05.009. Epub2015 May 16.

Ishii H, Konuma T, Ohnuma K, Hosono O, TanakaH, Kato S, Tojo A, Takahashi S. Remission of re-mitting seronegative symmetrical synovitis withpitting edema after unrelated cord blood trans-plantation for myelodysplastic syndrome. AnnHematol. 2015 Nov 14. [Epub ahead of print]

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1. Development of new retroviral vectors.

Toshikhiko Oki, Jiro Kitaura, Yutaka Enomoto,Tomoyuki Uchida, Fumi Shibata-Minoshima, andToshio Kitamura:

We previously developed an effective retroviraltransduction system consisted of vectors named aspMXs, pMYs, pMZs and pMCs and packaging cellsnamed as PLAT-E, PLAT-A, and PLAT-F. PLAT-Eand A produce ecotropic and amphotropic retrovi-ruses, respectively, upon transient transfection ofretrovirus vectors. PLAT-F can produce retroviruseswhose envelopes are derived from feline leukemiavirus RD114 which somehow has a high-affinitybinding to human CD34. Therefore, the viruses pro-duced from CD34 can efficiently infect CD34＋cells. In fact, multiple infections of PLAT-F-pro-duced retroviruses can infect CD34＋ human bonemarrow cells with nearly 100％ infection efficien-cies. Based on this system, we developed new vec-tors including 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 regenerative

medicine

2. 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 mitotic phase, MgcRacGAP playsessential roles in completion of cytokinesis as aRho-GAP. Interestingly, Aurora B-mediated phos-phorylation of S387 converts MgcRacGAP fromRac-GAP to Rho-GAP.We have recently shown that expression of

MgcRacGAP is regulated by a cell cycle-dependentmanner: MgcRacGAP expression increases in S/G2/M phase and decreases in early G1 phase, suggest-ing that MgcRacGAP may play some roles in G1check point. In addition to the transcriptional con-trol, MgcRacGAP protein levels are controlled byubiquitin-dependent degradation, leading to its de-crease in G1 phase. Using the proteome analysis


Division of Stem Cell Signaling幹細胞シグナル制御分野

Professor Toshio Kitamura, M.D., D.M.Sc. 教授医学博士北村俊雄

Our major interest is to elucidate the mechanisms of pluripotency, self-renewaland the control of cell division and differentiation of hematopoietic stem and pro-genitor cells. We have developed the retrovirus-mediated efficient gene transferand several functional expression cloning systems, and utilized these system toour experiments. We are now conducting several projects related to stem cells tocharacterize stem cells, clarify underling mechanisms of maintenance of pluripo-tency, and differentiation.

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and retroviral transduction, we identified APC/CDH1 as an E3 ligase involved in regulation ofMgcRacGAP and the degron in MgcRacGAP. Nowwe are investigating the physiological roles of thisregulation. In summary, our results implicateMgcRacGAP in coordination of cell cycle progres-sion and cell fate determination.

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

Toshiyuki Kawashima, Susumu Goyama, AkihoTsuchiya, Toshihiko Oki, Jiro Kitaura, and ToshioKitamura:

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. In addi-tion to STAT3 inhibitors, we have started a newproject to develop STAT5 inhibitors in collaborationwith a pharmaceutical company. To this end, weare now in the process to establish a screeningmethod to search for STAT5 inhibitors.In addition to STAT3 inhibitors, we have recently

started a new project to develop STAT5 inhibitorsin collaboration with a pharmaceutical company. Tothis end, we have developed a screening method to

search for STAT5 inhibitors. In addition to STAT3/5inhibitors, we have started several collaborationswith several domestic and global pharmaceuticalcompanies to evaluate the efficacies of a variety ofmolecular targeted therapies in our establishedmouse MDS/AML/MPN models.

4. Development of G0 indicator

Toshihiko Oki, Kotarou Nishimura, Takeshi Fuku-shima, Yosuke Tanaka, Asako Sakaue-Sawano1,Atsushi Miyawaki1, Toshio Kitamura: 1Laboratoryfor Cell Function Dynamics, RIKEN, Wako, Sai-tama and ERATO Miyawaki Life Function Dy-namics Project, 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 like hematopietic stem cells and cancer stemcells with tumor initiating potentials are in G0phase.Recently we have developed the system to indi-

cate cells in G0 phase. It is a system to monitor theamount of p27, which is destructed during G0 toG1 phase and is not expressed in S/G2/M phase, us-ing the cells retrovirally trasduced with the fusionprotein between a fluorescent protein like mVenusand p27K- (a p27 mutant lacking CDK inhibitoryactivities) as a similar cell cycle indicator system,fluorescent, ubiquitination-based cell cycle indica-tor, (Fucci). mVenus-p27K- positive cells are Ki67negative quiescent cells and mVenus-p27K- signalsare enhanced when the cycling cell enter G0 phasein response to serum starvation or contactinhibi-tion.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-p27K- trasgenic micewere generated to track several kinds of tissue spe-cific stem cells in vivo. We identified muscle satel-lite and colon mVenus-p27K- positive Now we aregenerating a Rosa26-knock in mouse.

Publications

Inoue, D., Nishimura, K., Matsumoto, M., Nagase,R., Saika, M., Fujino, T., Nakayama, K-I. and Ki-tamura, T. (2016) Truncation mutants of ASXL1observed in myeloid malignancies are expressedat detectable protein levels. Exp. Hematol. 44:172-176.

Matsukawa, T., Izawa, K., Isobe, M., Takahashi, M.,Maehara, A., Yamanishi, Y., Kaitani, A., Oku-mura, K., Teshima, T., ＊Kitamura, T. and ＊Kita-

ura, J. (2016) Ceramido-CD300f binding sup-presses experimental colitis by inhibiting ATP-mediated mast cell activation. Gut 65: 777-787.

Goyama, S., Schibler, J., Gasilina, A., Shrestha, M.,Lin, S., Link, KA., Chen, J., Whitman, SP., Bloom-field, C.D., Nicolet, D., Assi, S.A., Ptasinska, A.,Heidenreich, O., Bonifer, C., Kitamura, T., Nas-sar, N.N., Mulloy, J.C. (2016) UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in hu-

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man preleukemia induced by AML1-ETO. Leuke-mia 30: 728-739.

Kitamura, T., Watanabe-Okochi, N., Enomoto, Y.,Nakahara, F., Oki, T., Komeno, Y., Kato, N.,Doki, N., Uchida, T., Kagiyama, Y., Togami, K.,Kawabata, K.C., Nishimura, K., Hayashi, Y., Na-gase, R., Saika, M., Fukushima, T., Asada, S., Fu-jino, T., Izawa, Y., Horikawa, S., Fukuyama, T.,Tanaka, Y., Ono, R., Goyama, S., Nosaka, T., Ki-taura, J., and Inoue D. (2016) Novel working hy-pothesis for pathogenesis of hematological malig-nancies: combination of mutations-induced cellu-lar phenotypes determines the disease (cMIP-DD). J. Biochem. 159: 17-25.

Inoue, D., Nishimura, K., Kozuka-Hata, H., Oyama,M. and Kitamura, T. (2015) The stability of epige-netic factor ASXL1 is regulated through ubiquiti-nation and USP7-mediated deubiquitination. Leu-kemia 29: 2257-2260.

Togami, K., Kitaura, J., Uchida, T., Inoue, D., Nishi-mura, K., Kawabata, K.C., Nagase, R., Horikawa,S., Izawa, K., Fukuyama, T., Nakahara, F., Oki,T., Harada, Y., Harada, H., Aburatani, H. and Ki-tamura, T. (2015) C-terminal mutant of C/EBP(C/EBP-Cm) down-regulates M-CSF receptorwhich is a potent accelerator in the progression

of AML with C/EBP-CmB. Exp. Hematol. 43:300-308.

Inoue, D., Kitaura, J., Matsui, H., Hou, H-A, Chou,W-C, Nagamachi, A., Kawabata, K.C., Togami,K., Nagase, R., Horikawa, S., Saika, M., Micol, J-P., Hayashi, Y., Harada, Y., Harada, H., Inaba, T.,Tien, H-F., Abdel-Wahab, O., and Kitamura, T.(2015) SETBP1 mutations drive leukemic transfor-mation in ASXL1-murtated MDS. Leukemia 29:847-857.

Miyadera, H., Ohashi, J., Lernmark, A., Kitamura,T., and Tokunaga, K. (2015) Autoimmune suscep-tible HLA alleles encode unstable proteins. J.Clin. Invest. 125: 275-291.

Goyama, S., Wunderlich, M., and Mulloy, J.C.(2015) Xenograft models for normal and malig-nant stem cells. Blood. 125: 2630-2640.

Matsuo, H., Goyama, S., Kamikubo, Y., and Adachi,S. (2015) The subtype-specific features of EVI1and PRDM16 in acute myeloid leukemia. Haema-tologica. 100: e116-117.

Goyama, S., Huang, G., Kurokawa, M., and Mulloy,J.C. (2015) Posttranslational modifications ofRUNX1 as potential anticancer targets. Oncogene.34: 3483-3492.

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1. Role of mesenchymal stem cell-derived fibri-nolytic factor in tissue regeneration and can-cer progression

Beate Heissig1, Douaa Dhahri1, Salita Eiamboon-sert1, Yousef Salama1, Hiroshi Shimazu2, ShinyaMunakata2, Koichi Hattori2,3: 1Division of StemCell Dynamics, The Institute of Medical Science,The University of Tokyo, Tokyo, 2Division of StemCell Regulation, The Institute of Medical Science,The University of Tokyo, 3Center for Genome andRegenerative Medicine, Juntendo UniversitySchool of Medicine.

Tissue regeneration during wound healing orcancer growth and progression depends on the es-tablishment of a cellular microenvironment. Mesen-chymal stem cells (MSC) are part of this cellularmicroenvironment, where they functionally modu-late cell homing, angiogenesis, and immune modu-lation. MSC recruitment involves detachment ofthese cells from their niche, and finally MSC migra-tion into their preferred niches; the wounded area,the tumor bed, and the BM, just to name a few.During this recruitment phase, focal proteolysis dis-rupts the extracellular matrix (ECM) architecture,breaks cell-matrix interactions with receptors, andintegrins, and causes the release of bioactive frag-ments from ECM molecules. MSC produce a broad

array of proteases, promoting remodeling of thesurrounding ECM through proteolytic mechanisms.The fibrinolytic system, with its main player plas-min, plays a crucial role in cell migration, growthfactor bioavailability, and the regulation of otherprotease systems during inflammation, tissue regen-eration, and cancer. Key components of the fibri-nolytic cascade, including the urokinase plasmino-gen activator receptor (uPAR) and plasminogen ac-tivator inhibitor-1 (PAI-1), are expressed in MSC.This review will introduce general functional prop-erties of the fibrinolytic system, which go beyondits known function of fibrin clot dissolution (fibri-nolysis). We will focus on the role of the fibri-nolytic system for MSC biology, summarizing ourcurrent understanding of the role of the fibrinolyticsystem for MSC recruitment and the functional con-sequences for tissue regeneration and cancer. As-pects of MSC origin, maintenance, and the mecha-nisms by which these cells contribute to alteredprotease activity in the microenvironment undernormal and pathological conditions will also be dis-cussed.

2. Cancer therapy targeting the fibrinolytic sys-tem

Beate Heissig1, Salita Eiamboonsert1, YousefSalama1, Horoshi Shimazu1, Douaa Dhahri1, Shi-


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 perform highly selective and limited cleavage of specific substrates in-cluding growth factors and their receptors, cell adhesion molecules, cytokines,apoptotic ligand and angiogenic factors. We demonstrated that the matrix metallo-proteinase-9 is activated during leukemic cell progression. In addition, the serineproteinase plasmin plays a role in the myeloid cell recruitment in inflamed tissuesduring inflammatory bowel disease, sepsis and graft versus host disease afterbone marrow transplantation and during cancer progression.

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nya Munakata2, Yoshi Tashiro2, Koichi Hattori2:1Division of Stem Cell Dynamics, The Institute ofMedical Science, The University of Tokyo, 2Divi-sion of Stem Cell Regulation, The Institute ofMedical Science, The University of Tokyo, Centerfor Genome and Regenerative Medicine, JuntendoUniversity School of Medicine.

The tumor microenvironment is recognized as akey factor in the multiple stages of cancer progres-sion, mediating local resistance, immune-escapeand metastasis. Cancer growth and progression re-quire remodeling of the tumor stromal microenvi-ronment, such as the development of tumor-associ-ated blood vessels, recruitment of bone marrow-de-rived cells and cytokine processing. Extracellularmatrix breakdown achieved by proteases like the fi-brinolytic factor plasmin and matrix metallopro-teases is necessary for cell migration crucial for can-cer invasion and metastasis. Key components of thefibrinolytic system are expressed in cells of the tu-mor microenvironment. Plasmin can control growthfactor bioavailability, or the regulation of other pro-teases leading to angiogenesis, and inflammation.In this review, we will focus on the role of the fibri-nolytic system in the tumor microenvironmentsummarizing our current understanding of the roleof the fibrinolytic factors for the modulation of thelocal chemokine/cytokine milieu, resulting inmyeloid cell recruitment, which can promoteneoangiogenesis.

3. Inhibition of plasmin attenuates murine acutegraft-versus-host disease mortality by sup-pressing the matrix metalloproteinase-9-de-pendent inflammatory cytokine storm and ef-fector cell trafficking

Aki Sato1, Chiemi Nishida2, Kaori Sato-Kusubata2,Makoto Ishihara1, Yoshihiko Tashiro1, IsmaelGritli2, Hiroshi Shimazu1, Shinya Munakata1,Hideo Yagita3, Ko Okumura3, Yuko Tsuda4,Yoshio Okada4 , Arinobu Tojo5 , HiromitsuNakauchi6, Satoshi Takahashi5, Beate Heissig2#,Koichi Hattori1,7#: 1Department of Stem Cell Regu-lation, The Institute of Medical Science, The Uni-versity of Tokyo, 2Department of Stem Cell Dy-namics, The Institute of Medical Science, The Uni-versity of Tokyo, 3Atopy (Allergy) Research Cen-ter, Juntendo University School of Medicine, To-kyo, 4Faculty of Pharmaceutical Sciences, KobeGakuin University, 5Department of Hematologyand Oncology, The Institute of Medical Science,The University of Tokyo, 6Department of StemCell Therapy, The Institute of Medical Science,The University of Tokyo, 7Center for Genome andRegenerative Medicine, Juntendo UniversitySchool of Medicine, Tokyo.

The systemic inflammatory response observedduring acute graft-versus-host disease (aGVHD) isdriven by proinflammatory cytokines, a 'cytokinestorm'. The function of plasmin in regulating the in-flammatory response is not fully understood, andits role in the development of aGVHD remains un-resolved. Here we show that plasmin is activatedduring the early phase of aGVHD in mice, and itsactivation correlated with aGVHD severity in hu-mans. Pharmacological plasmin inhibition protectedagainst aGVHD-associated lethality in mice. Mecha-nistically, plasmin inhibition impaired the infiltra-tion of inflammatory cells, the release of mem-brane-associated proinflammatory cytokines includ-ing tumor necrosis factor- (TNF-) and Fas-liganddirectly, or indirectly via matrix metalloproteinases(MMPs) and alters monocyte chemoattractant pro-tein-1 (MCP-1) signaling. We propose that plasminand potentially MMP-9 inhibition offers a noveltherapeutic strategy to control the deadly cytokinestorm in patients with aGVHD, thereby preventingtissue destruction.

4. Inhibition of Plasmin Protects Against Colitisin Mice by Suppressing Matrix Metalloprotein-ase 9-mediated Cytokine Release FromMyeloid Cells

Shinya Munakata1, Yoshihiko Tashiro1, ChiemiNishida2, Aki Sato1, Hiromitsu Komiyama3, HiroshiShimazu1, Douaa Dhahri2, Yousef Salama2, SalitaEiamboonsert2, Kazuyoshi Takeda4, Hideo Yagita4,Yoko Tsuda5 , Yoshio Okada5 , HiromitsuNakauchi6, Kazuhiro Sakamoto3, Beate Heissig2,Koichi Hattori1,7: 1Division of Stem Cell Regula-tion, The Institute of Medical Science, The Univer-sity of Tokyo, 2Division of Stem Cell Dynamics,The Institute of Medical Science, The University ofTokyo, 3Department of Coloproctological Surgery,Juntendo University Faculty of Medicine, Tokyo,4Department of Immunology, Juntendo UniversityGraduate School of Medicine, Tokyo, 5Faculty ofPharmaceutical Sciences, Kobe Gakuin University,6Center for Stem Cell Biology and RegenerativeMedicine, The Institute of Medical Science, TheUniversity of Tokyo, 7Center for Genome and Re-generative Medicine, Juntendo University Schoolof Medicine, Tokyo.

Activated proteases such as plasmin and matrixmetalloproteinases (MMPs) are activated in intesti-nal tissues of patients with active inflammatorybowel diseases. We investigated the effect of plas-min on progression of acute colitis. Colitis was in-duced in Mmp9-/-, Plg-/-, and C57BL/6 (control)mice by administration of dextran sulfate sodium,trinitrobenzene sulfonic acid, or CD40 antibody.Plasmin was inhibited in control mice by intraperi-toneal injection of YO-2, which blocks its active site.

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Mucosal and blood samples were collected andanalyzed by reverse transcription polymerase chainreaction and immunohistochemical analyses, aswell as for mucosal inflammation and levels of cy-tokines and chemokines. We showed that circulat-ing levels of plasmin were increased in mice withcolitis, compared with controls. Colitis did not de-velop in control mice injected with YO-2 or inPlg-/- mice. Colons from these mice had reducedinfiltration of Gr1＋ neutrophils and F4/80＋macrophages, and reduced levels of inflammatorycytokines and chemokines. Colonic inflammationand colitis induction required activation of endoge-nous MMP9. Following colitis induction, mice

given YO-2, Plg-/- mice, and Mmp9-/- mice had re-duced serum levels of tumor necrosis factor andCXCL5, compared to control mice. In summary, inmice, plasmin induces a feedback mechanism inwhich activation of the fibrinolytic system promotesdevelopment of colitis, via activation of MMP9 orproteolytic enzymes. The proteolytic environmentstimulates influx of myeloid cells into the colonicepithelium and production of tumor necrosis factorand CXCL5. In turn, myeloid CD11b＋ cells releasethe urokinase plasminogen activator, which acceler-ates plasmin production. Disruption of the plasmin-induced chronic inflammatory circuit might there-fore be a strategy for treatment of colitis.

Publications

<Beate Heissig Group>1. Heissig, B., ＊#Dhahri D., Eiamboonsert, S.,

Salama Y., Shimazu, H., Munakata, S., and Hat-tori, K#. Role of mesenchymal stem cell-derivedfibrinolytic factor in tissue regeneration and can-cer progression. Cell Mol. Life Sci., 72: 4759-70,2015.

2. Heissig, B., Eiamboonsert, S., Salama, Y., Shi-mazu, H., Munakata, S., Tashiro, Y., and Hattori,K#. Cancer therapy targeting the fibrinolytic sys-tem. Advanced Drug Delivery Reviews, Adv.Drug Deliv. Res., Nov 14. pii: S0169-409X(15)00274-4. doi: 10.1016/j.addr.2015.11.010, 2015.

3. Munakata, S., ＊Tashiro, Y., ＊Nishida, C., Sato,A., Komiyama, H., Shimazu, H., Dhahri, D.,Salama, Y., Eiamboonsert, S., Takeda, K., Yagita,H., Tsuda, Y., Okada, Y., Nakauchi, H.,

Sakamoto, K., Heissig, B.# and Hattori, K.# Inhi-bition of Plasmin Protects Against Colitis in Miceby Suppressing Matrix Metalloproteinase 9-me-diated Cytokine Release From Myeloid Cells.Gastroenterology, 148: 565-578, 2015.

4. Sato, A., Nishida, C., Sato-Kusubata, K., Ishihara,M., Tashiro, Y., Gritli, I., Shimazu, H., Munakata,S., Yagita, H., Okumura, K., Tsuda, Y., Okada,Y., Tojo, A., Nakauchi, H., Takahashi, S., Heissig,B.# and Hattori, K.# Inhibition of plasmin attenu-ates murine acute graft-versus-host disease mor-tality by suppressing the matrix metalloprotein-ase-9-dependent inflammatory cytokine stormand effector cell trafficking. Leukemia, 29: 145-56, 2015.

# shared senior authorship

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1. Interleukin-17B antagonizes interleukin-25-mediated mucosal inflammation.

Joseph M. Reynolds1, Young-Hee Lee2, Yun Shi2,Xiaohu Wang3 , Pornpimon Angkasekwinai4 ,Kalyan C. Nallaparaju2, Stephanie Flaherty1, SeonHee Chang2, Hiroshi Watarai5,6, Chen Dong3: 1De-partment of Microbiology and Immunology, Chi-cago Medical School, Rosalind Franklin Universityof Medicine and Science, 2Department of Immu-nology and Center for Inflammation and Cancer,MD Anderson Cancer Center, 3Institute for Immu-nology, Tsinghua University, 4Department of Medi-cal Technology, Faculty of Allied Health Sciences,Thammasat University, 5Division of Stem Cell Cel-lomics, Center for Stem Cell Biology and Regen-erative Medicine, The Institute of Medical Science,The University of Tokyo, 6Precursory Research forEmbryonic Science and Technology (PRESTO),Japan Science and Technology Agency (JST)

The pro-inflammatory functions of interleukin-17(IL-17) have been well documented. Here, we havedemonstrated further functional complexity amongthe IL-17 family of cytokines. Adding to a well-es-

tablished role in type 2 immunity, we found thatIL-25 was pathogenic in acute colitis, whereas IL-17B was conversely protective. These opposing effectswere similar in Citrobacter rodentium infection andallergic asthma. Notably, we have found a uniquerole for IL-17B in inhibiting IL-25-mediated IL-6production by CECs, making IL-17B an antagonisticand anti-inflammatory cytokine in this family.IL-25 is generally thought of as a Th2-cell-derived

factor, promoting allergic asthma responses but alsoprotecting against parasitic infection. Here, we haveshown that IL-25 in acute colitis promoted IL-6 pro-duction from CECs, which is consistent with a re-port of IL-25 promoting the release of IL-6 fromeosinophils. Throughout the course of this study,IL-25-dependent IL-6 production was the most con-sistently inhibited by IL-17B treatment. However, atthis time we cannot rule out the influence of othermediators in promoting the protective and patho-genic phenotypes observed in IL-25- and IL-17B-de-ficient mice, respectively. Most likely, IL-17B an-tagonism of IL-25 is influencing multiple facets ofthe mucosal immune response, which is the subjectof our current investigations.Our finding that IL-25 deletion protects against


Division of Stem Cell Cellomics幹細胞セロミクス分野

Project Associate Professor Hiroshi Watarai, Ph.D. 特任准教授医学博士渡会浩志

Single cell analysis has become increasingly important for cellular biologists doingbasic, translational, and clinical research. It was once believed that cell popula-tions were homogeneous, but the latest evidence shows that heterogeneity doesin fact exist even within small cell populations. Gene expression measurementsbased on the homogenized cell population are misleading averages and don't ac-count for the small but critical changes occurring in individual cells. Individual cellscan differ dramatically in size, protein levels, and expressed RNA transcripts, andthese variations are key to answering previously irresolvable questions in cancerresearch, stem cell biology, immunology, and developmental biology. We are alsotrying to develop new advanced techniques by the integration of photonics, chem-istry, electrical engineering, mechanical engineering, bioinformatics, and others.

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dextran sodium sulfate (DSS) differs from anothershowing that injecting IL-25 protects against the de-velopment of DSS colitis. Though we cannot fullyexplain the discrepancy between reports, IL-25stimulation of primary CECs in vitro promoted theproduction of IL-6, consistent with decreased in-flammation observed in Il25 ?/? mice. Indeed, whenwe injected WT mice directly with IL-25 or IL-17B,we observed phenotypes opposite to those found incytokine-deficient mice. Thus, different housingconditions or microbiota might influence results ob-tained from recombinant protein injection.IL-17B has long been a mysterious member of the

IL-17 cytokine family. Previous work has demon-strated that IL-17RB might be oncogenic whereasIL-17B might contribute to tumor cell growth. Theoriginal report characterizing IL-17B demonstratesthat this cytokine stimulates the release of TNF-and IL-1 from THP-1 cells. However, we did notobserve production of pro-inflammatory mediatorsafter IL-17B stimulation, consistent with IL-17B be-ing unable to induce IL-6 from fibroblasts. Instead,the primary effect we observed for IL-17B treatmentin CECs was direct inhibition of IL-25-mediated IL-6 production. This of course begs the question ofhow exactly IL-17B can inhibit IL-25-mediated IL-6production in CECs. One possibility is that IL-17Bcompetes with IL-25 for binding IL-17RB. Indeed,we found that IL-17B inhibited IL-25 binding to IL-17RB or IL-17RA and IL-17RB complexes. Becauseprevious work has demonstrated that IL-17A andIL-17F form heterodimers, we investigated this pos-sibility as well. However, exhaustive attempts toidentify IL-17B and IL-25 heterodimers were unsuc-cessful, suggesting that competition for IL-17RBbinding causes functional antagonism. IL-17B hadhigher activity in blocking IL-25 binding to the IL-17RA and IL-17RB heterodimer compared to IL-17RB alone. Conversely, IL-25 was capable of bindingIL-17RB in the absence of IL-17RA, suggesting thatIL-17RA was more important for signaling. Thesebinding assays have limitations considering that wecannot rule out the contribution of endogenous IL-17 receptors that might be expressed in the humanHEK cells, because we do not know whether themurine IL-17RB could form heterodimers with en-dogenous human IL-17RA. Another caveat is thatonly mouse receptors were co-expressed in thesecells and more direct studies need to be performedin the future on both mouse and human IL-17RAand IL-17RB similar to studies on IL-17 and IL-17F.Finally, we need to determine whether IL-17RB isinternalized after binding of IL-17B, IL-25, or both.Differential IL-17RB internalization could representanother mechanism governing IL-17B regulation ofIL-25.Our current model is that colon inflammation en-

hances IL-25 pro-inflammatory signaling while IL-17B is concurrently induced to restrain IL-25. IL-25

has a higher affinity for IL-17RB alone compared toIL-17B. However, treatment with IL-17B throughoutthe course of DSS colitis led to protection in WTanimals, indicating that a shift in the IL-25 to IL-17B ratio can influence the course of inflammation.Previous work has demonstrated that IL-17F insome situations antagonizes IL-17 signaling, includ-ing forming heterodimeric complexes with IL-17that have weaker activity compared to IL-17 alone.We believe that the antagonism exerted on IL-25signaling by IL-17B, however, is unique to this fam-ily because IL-17F by itself can activate signalingpathways important for inflammation but we haveyet to find a standalone function for IL-17B. More-over, IL-17B could not antagonize IL-6 productioninduced by IL-17 in CECs, demonstrating the speci-ficity of IL-17B action. Therefore, further study isneeded to determine whether IL-17B alone cantransmit a functional signal to any cell type and todetermine the precise mechanism for how IL-17B isinhibiting IL-25 binding to IL-17RB.The prospect of modulating IL-17RB signaling for

the treatment of colitis at this time seems promis-ing. Our findings that recombinant IL-17B injectionsthroughout the course of DSS administration canimprove disease outcome in WT animals furthersupports this theory. Whether IL-17B is as impor-tant in human inflammatory bowel disease (IBD) iscurrently under investigation. The production of IL-25 has been linked to human IBD even thoughpolymorphism is not believed to be a risk factor.Two recent reports have shown that IBD patientshave decreases in IL-25. These results might sug-gest that IL-25 is a protective factor in human IBD;however, IL-17B expression in these patients hasyet to be determined. Decreased IL-25 expression inhuman IBD might also be representative of a failureof IL-17B to control inflammation. Thus, further in-vestigation is necessary to determine the role of IL-17B in human IBD as well as determining whetherIL-17RB represents a viable therapeutic target.

2. In vivo and in vitro analyses of -galactosyl-ceramide uptake by conventional dendriticcell subsets using its fluorescence-labeledderivative.

Maki Ushida1, Tomonori Iyoda1, Mitsuhiro Ka-namori1, Hiroshi Watarai2,3, Kazuhiko Takahara1,Kayo Inaba1: 1Department of Animal Developmentand Physiology, Graduate School of Biostudies,Kyoto University, 2Division of Stem Cell Cellomics,Center for Stem Cell Biology and RegenerativeMedicine, The Institute of Medical Science, TheUniversity of Tokyo, 3Precursory Research forEmbryonic Science and Technology (PRESTO),Japan Science and Technology Agency (JST)

Dendritic cells (DCs) are antigen-presenting cells

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(APCs) that exist widely in the body and surveypathogen invasions. Upon recognition of pathogens,DCs present peptide antigens via major histocom-patibility complex (MHC) molecules to the T cells.DCs also present lipid antigen via nonclassicalMHC CD1 to natural killer T cells (NKT cells), in-ducing robust cytokine production.DCs are primarily divided into two populations:

conventional DCs (cDCs) and plasmacytoid DCs(pDCs). cDCs are further subdivided into twogroups, CD8＋ and CD8－ DCs, by the expressionof CD8. NKT cells are divided into two subsets bythe pattern of T cell antigen receptor (TCR) expres-sion. Type I NKT cells express invariant TCR, sothey are designated as invariant NKT (iNKT) cells.iNKT cells recognize marine sponge -galactosylce-ramide (-GalCer) on the CD1d molecule of cDCs,inducing antitumor activity via the robust produc-tion of IFN-. During interaction with cDCs, iNKTcells express CD40 ligand (CD40L), which stimu-lates cDCs via their CD40, subsequently inducingIL-12 production from CD8＋ DCs. In turn, IL-12stimulates iNKT cells, resulting in the strong pro-duction of IFN-. Furthermore, using Langerin-eGFP-diphtheria toxin receptor (DTR) mice, deple-tion of Langerin＋ cDCs decreased IL-12 and IFN-production after stimulation with injection of -Gal-Cer, suggesting that Langerin＋ CD8＋ DCs are in-volved in IFN- production of iNKT cells in vivo.However, it is unclear why CD8＋ DCs inducehigher IFN- production of iNKT cells than CD8－DCs.After i.v. injection, antigens in the blood are cap-

tured by the splenic marginal zone (MZ) cells, caus-ing a rapid immune response. MZ consists ofSIGNR1/ER-TR9＋ macrophages, MZB cells, andcDCs, forming special equipment to sense/captureantigens in the blood flowing into the sinus be-tween the MZ and marginal CD169/SER4＋ metallo-phil zone. Interestingly, Langerin＋ CD8＋ DCswere observed in the MZ, and iNKT cells migratedto the MZ after injection of -GalCer.Here, we compared -GalCer uptake and degra-

dation between CD8－ and CD8＋ DC subsets us-ing Cy5-conjugated -GalCer (Cy5--GalCer). Wealso performed histological analyses to observe theuptake of Cy5--GalCer in the spleen. Our resultssuggest that both cellular properties and localiza-tion of CD8＋ DCs enable efficient stimulation ofiNKT cells in response to -GalCer. In cDC subsets,CD8＋ DCs uptake -GalCer effectively comparedwith CD8－ DCs in vivo and in vitro. In addition,-GalCer is more stable in CD8＋ DCs than in CD8－ DCs. It is possible that these two properties ofCD8＋ DCs result in their efficient accumulationand presentation of -GalCer. Furthermore, CD8＋DCs strategically localize in the MZ, which is spe-cialized in capturing blood-borne antigens, andpreferentially uptake -GalCer injected i.v.. Further-more, CD8＋ DCs in the spleen express higher lev-els of membrane-bound CXCL16 than CD8－ DCs,possibly enhancing activation of iNKT cells. Thesecellular properties and localization of CD8＋ DCsseem to enable the robust induction of IFN- fromiNKT cells in vivo.

Publications

1. Reynolds, J.M., Lee, Y.H., Shi, Y., Wang, X., Ang-kasekwinai, P., Nallaparaju, K.C., Flaherty, S.,Chang, S.H., Watarai, H. and Dong, C. Inter-leukin-17B Antagonizes Interleukin-25-MediatedMucosal Inflammation. Immunity. 42: 692-703,2015.

2. Ushida, M., Iyoda, T., Kanamori, M., Watarai,H., Takahara, K. and Inaba, K. In vivo and in vi-tro analyses of -galactosylceramide uptake byconventional dendritic cell subsets using its fluo-rescence-labeled derivative. Immunol. Lett. 168:

300-305, 2015.3. Nagasawa, K., Meguro, M., Sato, K., Tanizaki,

Y., Nogawa-Kosaka, N. and Kato, T. The influ-ence of artificially introduced N-glycosylationsites on the in vitro activity of Xenopus laeviserythropoietin. PLoS One. 10: e0124676, 2015.

4. Watarai, H. Elucidation and control of themechanisms underlying chronic inflammationmediated by invariant natural killer T cells.Springer. Chronic Inflammation. In press.

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center for stem cell biology and regenerative …1. novel method for efficient production of...

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