1. Cytokine profiles of pre-engraftment syn-drome after single-unit cord blood transplan-tation for adult patients.

Konuma T1, Kohara C2, Watanabe E3, MizukamiM4, Nagai E4, Oiwa-Monna M2, Tanoue S1, IsobeM1, Kato S1, Tojo A1,2,5, Takahashi S1,2,5: 1Depart-ment of Hematology/Oncology, IMSUT Hospital,2Division of Molecular Therapy, 3IMSUT clinicalflow cytometry laboratory, 4Department of Labo-ratory Medicine, IMSUT Hospital, 5Division ofStem Cell transplantation

Clinical manifestation of high-grade fever andskin rash before neutrophil engraftment, termedpre-engraftment syndrome (PES) or pre-engraft-ment immune reaction, has been frequently ob-served after cord blood transplantation (CBT). Thepathophysiology of PES is poorly understood, butcytokine storm during the early phase of CBT isthought to be 1 of the main cause of PES. However,the cytokine profiles of PES after CBT are unclear.Therefore, we examined the relationship betweenserum cytokine profiles and PES in 44 adult pa-tients who received CBT in our institution betweenFebruary 2013 and June 2016. Serum levels of 21cytokines, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-

22, IL-23, IL-33, monocyte chemoattractant protein-1, IFN-a, IFN-g, and TNF-a, were measured bymultiplex bead assays using a flow cytometer. Themedian time until the absolute neutrophil countwas >.5×109/L was 21 days (range, 15 to 41 days).The cumulative incidence of PES was 79.6％ (95％confidence interval, 63.3％ to 88.5％) at 60 days af-ter CBT. Serum levels of IL-5 (P=.009) and IL-6 (P=.01) at 2 weeks were significantly higher in pa-tients who developed PES compared with thosewho did not develop PES. The conversion from na-ïve to effector or central memory phenotype of Tcells was observed in PES. These data indicate thatelevations of IL-5 and IL-6 around the time of clini-cal manifestation may be possible biomarkers forPES after CBT.

2. Cryopreserved CD34＋ cell dose, but not totalnucleated cell dose, influences hematopoieticrecovery and extensive chronic GVHD aftersingle-unit cord blood transplantation in adultpatients.

Konuma T1, Kato S1, Oiwa-Monna M2, Tanoue S1,Ogawa M2, Isobe M1, Tojo A1,2,3, Takahashi S1,2,3:1Department of Hematology/Oncology, IMSUTHospital, 2Division of Molecular Therapy, 3Divisionof Stem Cell transplantation

Center for Stem Cell Biology and Regenerative Medicine

Division of Stem Cell Transplantation幹細胞移植分野

Professor Arinobu Tojo, M.D., D.M.Sc.Associate Professor Satoshi Takahashi, M.D., D.M.Sc.

教 授 医学博士 東 條 有 伸准教授 博士（医学） 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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Low cryopreserved total nucleated cell (TNC)dose in a cord blood (CB) unit has been shown tobe associated with engraftment failure and mortal-ity after single-unit cord blood transplantation(CBT) in adults. Although CB banks offer specificcharacteristics of cryopreserved cell dose, such asTNC, CD34＋ cells, and colony-forming unit forgranulocyte/macrophage (CFU-GM), the impact ofeach cell dose on engraftment and outcomes aftersingle-unit CBT in adults remains unclear. We ret-rospectively analyzed the results of 306 CBTs for261 adult patients in our institution between 1998and 2016. The median age was 43 years (range, 16to 68), the median actual body weight (ABW) was56.2kg (range, 36.2 to 104.0), the median ideal bodyweight (IBW) was 62.3kg (range, 39.7 to 81.3), themedian TNC dose was 2.46×107/ABW kg (range,1.07 to 5.69), the median CD34＋ cell dose was .91×105/ABW kg (range, .15 to 7.75), and the medianCFU-GM dose was 24.46×103/ABW kg (range, .04to 121.81). Among patients who achieved engraft-ment, the speed of neutrophil, platelet, and redblood cell engraftment significantly correlated withCD34＋ cell dose, but not with TNC and CFU-GMdose, based on both ABW and IBW. In multivariateanalysis, the incidence of extensive chronic graft-versus-host disease (GVHD) was significantlyhigher in patients receiving the highest CD34＋ celldose, based on both ABW and IBW. Nevertheless,no cell dose was associated with survival, trans-plantation-related mortality, and relapse. In conclu-sion, cryopreserved CD34＋ cell dose was the bestpredictor for hematopoietic recovery and extensivechronic GVHD after CBT. The cryopreserved CD34＋ cell dose should be used for unit selection crite-ria in single-unit CBT for adults.

3. Severe infusion-related toxicity after a secondunrelated cord blood transplantation.

Tanoue S1, Konuma T1, Kato S1, Oiwa-Monna M2,Isobe M1, Tojo A1,2, Takahashi S1,2: 1Department ofHematology/Oncology, IMSUT Hospital, 2Divisionof Molecular Therapy

Several reports have demonstrated that severeand life-threating infusion-related toxicities, such ascardiac arrhythmias or cardiopulmonary events,rarely occur after cryopreserved CB infusion. In ourpatient, the main symptoms of infusion-related tox-icity were hypoxemia and hypotension. Althoughurticaria was not observed, the rapid onset ofsymptoms after infusion and severe hypotension,which were probably not due to cardiogenic, hypo-volemic and septic shock, suggested that the infu-sion-related toxicity in our patient was consistentwith an anaphylactic reaction. Although the causeof infusion-related toxicities of cryopreserved cellswas unclear, cryoprotective agents, such as DMSO

and dextran, might be causative agents. Therefore,an initial sensitizing exposure to these agents afterthe first CB infusion might contribute to the devel-opment of anaphylactic reaction after a second CBinfusion. t.

4. Alloimmune hemolysis due to major RhE in-compatibility after unrelated cord blood trans-plantation.

Isobe M1, Konuma T1, Abe-Wada Y2, Hirata K2,Ogami K2, Kato S1, Oiwa-Monna M3, Tanoue S1,Nagamura-Inoue T2, Takahashi S1,3, Tojo A1,3.

Extension to donors other than HLA-matchedsiblings following advanced immunosuppressivetreatment has resulted in the emergence of viral in-fections as major contributors to morbidity andmortality after hematopoietic stem cell transplanta-tion (HSCT). The degree of risk for infection is dic-tated by the degree of tissue mismatch between do-nor and recipient, and the resultant degree of im-munosuppression. Reactivation of latent virusessuch as cytomegalovirus (CMV), Epstein-Barr virus(EBV), herpes simplex and herpes zoster are com-mon and often cause symptomatic disease. Respira-tory viruses such as adenovirus, influenza and res-piratory symptomatic virus also frequently causeinfection. While pharmacological agents are stan-dard therapy for some, they have substantial toxici-ties, generate resistant variants, and frequently inef-fective and costly. Moreover, immune reconstitutionis necessary for long-term protection especially afterHSCT. As the delay in recovery of virus-specificcellular immune response is cleary associated withviral reactivation and disease, cellular immunother-apy to restore virus-specific immunity offers an at-tractive alternative to conventional drugs. Adoptivetransfer of virus-specific lymphocytes (VSTs) fromstem cell donors has been proved to be safe and ef-fective to treat viral infection.Manufacture of VSTs requires preparation of spe-

cialized antigen presenting cells (APCs), uses vi-ruses or viral vectors to provide vial antigens topresent on APCs. Recent report from a group ofBaylor College of Medicine has introduced a newmethod to generate multiple VSTs by direct stimu-lation of peripheral blood mononuclear cells(PBMCs) with peptides to replace the complex andlengthy process above.By this method, VSTs can be prepared by single

stimulation with non-viral products and containpolyclonal mixture of T cells specific for a largenumber of epitopes in a multiple pathogenic vi-ruses, which reduces the risk of immune escape byviral escape mutants and meet the requirement totreat viral infection after HSCT which occurs bybroad pathogens. Moreover this method is as sim-ple and fast as possible and takes only 10-14 days

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for preparation which makes it clinically useful.With this method, polyclonal CTLs specific for mul-tivirus antigens can be produced after single stimu-lation of PBMCs with a peptide mixture spanningthe target antigens in the presence of IL4 and IL7.We have introduced and verified this system to

apply for clinical use in Japan.The culture media for T cell expansion used in

the studies reported by Baylor College of Medicineare supplemented with fetal bovine serum (FBS).While these serum products are traditionally usedto expand T cells to promote cell growth and vi-

ability, there are some countries which the use ofserum product is not allowed. Also, uncharacter-ized elements contained in the serum products maycause inconsistency in results from batch to batch.Cell expansion in serum-free media would there-fore be preferable.To meet the requirement for the viral infections

after HSCT by broad viral antigens and in terms ofregulation by the Japanese FDA, we established themethod to generate multivirus-specific T cells tar-geting 7 viruses (CMV, EBV, AdV, HHV-6, BKV,JCV, and VZV) in serum-free medium.

Publications

Yasu T, Konuma T, Kato S, Kurokawa Y, TakahashiS, Tojo A. Serum C-reactive protein levels affectthe plasma voriconazole trough levels in alloge-neic hematopoietic cell transplant recipients.Leuk Lymphoma. 2017 Mar 17:1-3. doi: 10.1080/10428194.2017.1300897. [Epub ahead of print] Noabstract available.

Konuma T, Kato S, Oiwa-Monna M, Tanoue S,Ogawa M, Isobe M, Tojo A, Takahashi S. Cryo-preserved CD34＋ cell dose, but not total nucle-ated cell dose, influences hematopoietic recoveryand extensive chronic GVHD after single-unitcord blood transplantation in adult patients. BiolBlood Marrow Transplant. 2017 Apr 5. pii: S1083-8791(17)30387-7. doi: 10.1016/j.bbmt.2017.03.036.[Epub ahead of print]

Tanoue S, Konuma T, Takahashi S, Watanabe E,Sato N, Watanabe N, Isobe M, Kato S, Ooi J, TojoA. Long-term persistent donor-recipient mixedchimerism without disease recurrence aftermyeloablative single-unit cord blood transplanta-tion in adult acute myeloid leukemia followingmyelodysplastic syndrome. Leuk Lymphoma.

2017 Dec;58(12):2973-2975. doi: 10.1080/10428194.2017.1318440. Epub 2017 May 16

Tanoue S, Konuma T, Kato S, Oiwa-Monna M, Iso-be M, Tojo A, Takahashi S. Severe infusion-re-lated toxicity after a second unrelated cord bloodtransplantation. Cytotherapy. 2017 Aug;19(8):1013-1014. doi: 10.1016/j.jcyt.2017.05.004. Epub2017 Jun 20.

Isobe M, Konuma T, Abe-Wada Y, Hirata K, OgamiK, Kato S, Oiwa-Monna M, Tanoue S, Nagamura-Inoue T, Takahashi S, Tojo A.. Alloimmunehemolysis due to major RhE incompatibility afterunrelated cord blood transplantation. Leuk Lym-phoma. 2017 Jul 20:1-4. doi: 10.1080/10428194.2017.1352095. [Epub ahead of print]

Miwa Y, Yamagishi Y, Konuma T, Sato T, Narita H,Kobayashi K, Takahashi S, Tojo A. Risk factorsand characteristics of falls among hospitalizedadult patients with hematologic diseases. Journalof Geriatric Oncology. J Geriatr Oncol. 2017 Sep;8(5):363-367. doi: 10.1016/j.jgo.2017.07.003. Epub2017 Jul 22

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1. Dissection of signaling events downstream ofthe c-Mpl receptor in murine hematopoieticstem cells via motif-engineered chimeric re-ceptors

Koichiro Saka, Chen-Yi Lai, Masanori Nojima,Masahiro Kawahara, Makoto Otsu, HiromitsuNakauchi, Teruyuki Nagamune

Hematopoietic stem cells (HSCs) are a valuableresource in transplantation medicine. Cytokines areoften used to culture HSCs aiming at better clinicaloutcomes through enhancement of HSC reconstitu-tion capability. Roles for each signal moleculedownstream of receptors in HSCs, however, remainpuzzling due to complexity of the cytokine-signal-ing network. Engineered receptors that are non-re-sponsive to endogenous cytokines represent an at-tractive tool for dissection of signaling events. Wehere tested a previously developed chimeric recep-tor (CR) system in primary murine HSCs, targetcells that are indispensable for analysis of stem cellactivity.

2. Evaluation of the Utility of Induced Pluripo-tent Stem Cells as a platform for modelingWiskott Aldrich Syndrome

HONDA-OZAKI Fumiko, LIN Huan-Ting, OKU-MURA Takashi, LAI Chen-Yi, OTSU Makoto

Wiskott Aldrich syndrome (WAS) is an X-linkeddisease, caused by mutations in the gene encodingthe WAS protein (WASp). Thrombocytopenia is themain feature of this disease, often threatening pa-tients to a significant risk of serious hemorrhage. Ithas been an issue of debate whether defective pro-platelet release from megakaryocytes (MKs)(i.e.,production) and/or destruction of platelets (PLTs)by the spleen macrophages (i.e., consumption) con-stitutes the major cause of thrombocytopenia. Dueto the limitations inherent to classical experimentalmodels, there have been conflicting results pub-lished; precise mechanisms causing thrombocy-topenia in WAS therefore remain to be elucidated.To have an appropriate model in our hands and toelucidate mechanism(s) of thrombocytopenia inWAS to improve treatment of WAS patients in fu-ture, we here evaluated the utility of iPSCs as anemerging disease model for WAS. WASp-deficientiPSCs produced MKs and PLTs with similar yieldsas did control cells when differentiated from sortedMK progenitors. Interestingly, however, WASp-de-ficient iPSCs yielded only fewer MKs and PLTsthan did healthy counterparts when differentiatedfrom unsorted HPCs. In addition, we found that

Center for Stem Cell Biology and Regenerative Medicine

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

Associate Professor Makoto Otsu, M.D., Ph.D.Project Assistant Professor Chen-Yi Lai, Ph.D.

准教授 博士（医学） 大 津 真特任助教 博士（生命科学） 頼 貞 儀

Stem cells represent a valuable cell source in the field of regenerative medicine.Hematopoietic stem cells represent a valuable cell source for transplantationmedicine, with which many diseases including primary immunodeficiency and he-matologic malignancies can expect life-long cure by reconstitution of healthy he-matopoiesis. Our eventual goal is to establish safe and efficacious transplantationstrategies in a form of either allogeneic transplantation or gene therapy using au-tologous hematopoietic stem cells after gene correction.

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WASp-deficiency variously affected phagocytosisfunction of iPSC-monocytes in a context specificmanner, depending on what targets they wouldface. These data imply that not just MKs and PLTsbut also other lineage cells such as phagocytic cellsmay play a role for WAS-thrombocytopenia. Weare now trying to establish an in vitro co-culturesystem of iPSCs-derived monocytes and -plateletsas a new experimental platform to clarify accurateroles of each cellular compartment.

3. The functional analysis of dendritic cells de-veloped from T-iPS cells from a single CD4＋T cell of Sjögren's syndrome

Mana Iizuka-Koga, Hiromitsu Asashima, MikiAndo, Chen-Yi Lai, Shinji Mochizuki, MahitoNakanishi, Toshinobu Nishimura, Hiroto Tsuboi,Tomoya Hirota, Hiroyuki Takahashi, Isao Matsu-moto, Makoto Otsu, Takayuki Sumida

Though it is important to clarify the pathogenicand/or regulatory functions of the T cells in humansamples, their examination is frequently restrictedin opportunity because the acquisition of sufficientquantity of dendritic cells (DCs), used as antigenpresenting cells, might not be available, especiallyin autoimmune diseases. In this study, we devel-oped the generation of mature DCs from inducedpluripotent stem cells derived from T cells (T-iPSCs). We reprogramed only a single CD4＋ T cellof a patient with Sjögren's syndrome (SS) into iPScells, which were differentiated into DCs (TiPS-DCs) via TiPS-Sacs. Moreover, we examined theircharacterizations and functions. Under the micro-scope, the mature TiPS-DCs were dendritic cells-like morphology. The expression of CD11b, CD11c,HLA-ABC and CD40 was detected, and the CD80,CD86 and HLA-DR was also increased. The matureTiPS-DCs were able to produce TNF-a and IL-6through stimulation. As compared with monocytes-derived DCs, TiPS-DCs was equal to the ability ofantigen processing, and also the induction of robustproliferative response of allogeneic T cells in thedependent manner of cell numbers.In conclusion, we succeeded to obtain an ade-

quate amount of functional DCs from a single CD4＋ T cell by way of iPS cells. This method shouldshed light on the characterization of pathogenic Tcells for not only SS but also other diseases to eluci-date the function of autoreactive T cells. Themethod shown in the study will enable us to iden-tify autoreactive T cells in autoimmune diseasesalong with elucidate the function and pathogenicityof them.

4. Mutant iPS cells derived from patients withRALD show significance of KRAS for self-re-newal and differentiation propensity

Kenji Kubara, Kazuto Yamazaki, Yasuharu Ishi-hara, Huan-Ting Lin, Masashi Ito, Kappei Tsuka-hara, Masatoshi Takagi, and Makoto Otsu

KRAS is widely known as a proto-oncogene andhas been reported to play essential roles instemness maintenance in some types of stem cells,including cancer stem cells. However, the roles ofKRAS in pluripotent stem cells (PSCs) are largelyunknown. Recently, somatic gain-of-function muta-tions of KRAS or NRAS in hematopoietic stem cellshave been reported to cause RAS-associated auto-immune lymphoproliferative syndrome-like disease(RALD). Here, we investigated the roles of KRASon stemness maintenance in the context of humanPSCs using isogenic KRAS mutant (G13C/WT) andwild-type (WT/WT) induced PSCs (iPSCs), gener-ated from the same RALD patients with the so-matic KRAS mutation. Using the isogenic iPSClines from two patients, we revealed that G13C/WTiPSCs displayed self-renewal and differentiationcharacteristics distinct from those of WT/WT iPSCs:expression of stemness markers including POU5F1and NANOG, which is indicative of an undifferen-tiated state, was maintained at high levels in G13C/WT iPSCs under bFGF depletion; neuronal differen-tiation was clearly blunted from G13C/WT iPSCs.In addition, we generated wild-type (WTed/WT)and heterozygous knockout (Ded/WT) iPSCs fromthe same G13C/WT clone using gene-editing tech-niques. As expected, the G13C/WT-specific pheno-types were normalized in WTed/WT iPSCs. Inter-estingly, Ded/WT iPSCs showed lower potential tomaintain undifferentiation status under bFGF de-pletion, with a higher tendency to differentiate intoneuronal lineage than WTed/WT iPSCs. Biochemi-cal analysis indicated hyper-activation of KRAS andsubsequent increased phosphorylation levels ofERK in G13C/WT iPSCs. Pharmacological studiesusing specific kinase inhibitors demonstrated thatthe features compatible with enhanced stemnessmaintenance were canceled in mutant cells by pan-RAF and MEK inhibitors, but not by PI3K inhibi-tors. In addition, neuronal differentiation was im-proved in G13C/WT iPSCs with the MEK inhibitortreatment. These observations suggested that theKRAS-ERK pathway plays more critical roles thanthe KRAS-PI3K pathway in G13C/WT iPSCs. Col-lectively, the analyses on the isogenic and genome-edited iPSCs from the RALD patients revealed thecrucial roles of the KRAS-ERK signaling on thestemness maintenance, having a strong impact onself-renewal and differentiation propensity in hu-man iPSCs.

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Publications

1. Takahashi, Y., Sato, S., Kurashima, Y., Lai, C.Y.,Otsu, M., Hayashi, M., Yamaguchi, T., Kiyono,H. Reciprocal Inflammatory Signaling BetweenIntestinal Epithelial Cells and Adipocytes in theAbsence of Immune Cells. EBioMedicine. 23: 34-45, 2017.

2. Saka, K., Lai, C.Y., Nojima, M., Kawahara, M.,Otsu, M., Nakauchi, H., Nagamune, T. Dissec-tion of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic StemCells Via Motif-Engineered Chimeric Receptors.Stem Cell Rev. 2017.

3. Natsumoto, B., Shoda, H., Fujio, K., Otsu, M.,Yamamoto, K. Investigation of the pathogenesisof autoimmune diseases by iPS cells. Nihon Rin-sho Meneki Gakkai Kaishi. 40: 48-53, 2017.

4. Ishida, T., Suzuki, S., Lai, C.Y., Yamazaki, S.,Kakuta, S., Iwakura, Y., Nojima, M., Takeuchi,Y., Higashihara, M., Nakauchi, H., Otsu, M. Pre-

Transplantation Blockade of TNF-alpha-Medi-ated Oxygen Species Accumulation Protects He-matopoietic Stem Cells. Stem Cells. 35: 989-1002,2017.

5. Iizuka-Koga, M., Asashima, H., Ando, M., Lai, C.Y., Mochizuki, S., Nakanishi, M., Nishimura, T.,Tsuboi, H., Hirota, T., Takahashi, H., Matsu-moto, I., Otsu, M., Sumida, T. Functional Analy-sis of Dendritic Cells Generated from T-iPSCsfrom CD4＋ T Cell Clones of Sjogren's Syn-drome. Stem Cell Reports. 8: 1155-1163, 2017.

6. Ieyasu, A., Ishida, R., Kimura, T., Morita, M.,Wilkinson, A.C., Sudo, K., Nishimura, T., Ohe-hara, J., Tajima, Y., Lai, C.Y., Otsu, M., Naka-mura, Y., Ema, H., Nakauchi, H., Yamazaki, S.An All-Recombinant Protein-Based Culture Sys-tem Specifically Identifies Hematopoietic StemCell Maintenance Factors. Stem Cell Reports. 8:500-508, 2017.

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1. Developing Analysis Tools for Cell Cycle andCell Division of Hematopoietic Stem Cells:MgcRacGap - hmKusabiraOrange 2 ( MRG -hmKuO2) fusion protein for midbody marker.

Yosuke Tanaka, Tsuyoshi Fukushima, ToshihikoOki, Kotarou Nishimura, Asako Sakaue-Sawano1,Atsushi Miyawaki1, Toshio Kitamura: 1Laboratoryfor Cell Function Dynamics, RIKEN, Wako, Sai-tama and ERATO Miyawaki Life Function Dy-namics Project, JST.

Previously, we reported that MgcRacGap is amarker for midbody and that MgcRacGap-mVenusfusion protein visualized asymmetric inheritanceand release of midbody during cytokinesis(Nishimura et al., 2013). We retrovirally introducedMRG-hmKuO2 into hematopoietic stem cells(HSCs), in order to examine whether midbodyasymmetric inheritance and release is involved withasymmetric division of HSCs. HSCs showed highfrequency of midbody release during cytokinesis inculture. Interestingly, one daughter cell releasingmidbody differentiated earlier than the otherdaughter cell inheriting midbody. We generatedCre-inducible MRG-hmKuO2 mouse line. Briefly,the MRG-hmKuO2 fusion gene is inserted into Rosa26 locus following a loxP-NEO-STOP-loxP cassette,in order to visualize asymmetric inheritance and re-

lease of midbody in vivo without retroviral infec-tion. Crossing MRG-hmKuO2 mice with Vav-Cremice, MRG-hmKuO2 nicely marked midbodyasymmetric inheritance and release in HSCs in cul-ture. We are planning to do paired-daughter assayusing HSCs from MRG-hmKuO2 mice to examinewhether inheritance and release of midbody link toasymmetric division of HSCs. Given that someproblems were found in this new mouse line, weare planning to establish surrogate experimentalmodels for this.

2. Developing Analysis Tools for Cell Cycle andCell Division of Hematopoietic Stem Cells: Anovel G0 marker, mVenus-p27K- and itstransgenic mouse

Tsuyoshi Fukushima, Yosuke Tanaka, ToshihikoOki, Kotarou Nishimura, 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 hematopoietic stem cells and cancer stemcells with tumor initiating potentials are in G0

Center for Stem Cell Biology and Regenerative Medicine

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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phase.We have developed a novel G0 marker, mVenus-

p27K- (Oki et al, 2013). The mVenus-p27K- clearlymarked G0 and very early G1 in NIH3T3 cells. Toexamine G0 status in HSCs, we generated a Cre-in-ducible mVenus-p27K- mouse line that carriedmVenus-p27K- fusion gene in Rosa26 locus follow-ing a loxP-NEO-STOP-loxP cassette. After crossingwith Vav-Cre mice, we analyzed mVenus-p27K- ex-pression in HSCs. We expected most of the HSCsare mVenus-p27K- positive because most of theHSCs are dormant. However, surprisingly, threedifferent populations (mVenus-p27K-high (70％),mVenus-p27K-low (20％), mVenus-p27K-negative(10％)) were given from HSC fraction (CD150＋CD48-cKit＋Sca-1＋Lineage-). These three populationswere in G0 stage judged by Pyronin Y/Hoechstdouble-staining. To examine difference among thesethree populations, we performed bone marrow re-constitution assay. To our surprise, only mVenus-p27K-high population showed high bone marrow re-constitution ability. Moreover, mVenus-p27K-high

population was only detected in HSC fraction inthe bone marrow, not in other hematopoietic frac-tions. This indicates that our mVenus-p27K- markercould be a good marker for isolation of transplant-able HSCs. To characterize differences among thethree fractions in HSCs, we performed single-cellRNASeq (scRNASeq) analysis. The tSNE analysisfrom scRNASeq data showed that mVenus-p27K-high single-cells and mVenus-p27K-low single-cellswere plotted into similar cluster, whereas mVenus-p27K-negative single-cells were plotted into a dif-ferent cluster from other two. However, HSC-re-lated genes, such as Lyl1, Hlf, Hhex, were highlyexpressed in mVenus-p27K-high single-cells thanmVenus-p27K-low single-cells. Other new unknowngenes were also plotted in the same position, sug-gesting that those genes are strong candidate to de-fine mVenus-p27K-high HSCs. Small-cell Mass Specanalysis and metabolome analysis for these cellfractions are ongoing. We also plan to examinewhere mVenus-p27K-high HSCs locate in bonemarrow using 4D imaging.

Publications

Shiba, E., Izawa, K., Kaitani, A., Isobe, M., Mae-hara, A., Uchida, K., Maeda, K., Nakano, N.,Ogawa, H., Okumura, K., Kitamura, T., Shimizu,T., and Kitaura, J. (2017) Ceramide-CD300f bind-ing inhibits lipopolysaccharide-induced skin in-flammation. J. Biol. Chem. 292:2924-2932.

Inoue, A., Komatsu, N., Nishitoh, H., Kataoka, H.,and Nunoi, H. (2017) Mouse model identifies ma-jor facilitator superfamily domain containing 2aas a critical transporter in the response to tunica-mycin. Human Cell 30: 88-97.

Sakurai, H., Harada, Y., Ogata, Y., Kagiyama, Y.,Shingai, N., Doki, N., Ohashi, K., Kitamura, T.,Komatsu, N., and Harada, H. (2017) Overexpres-sion of RUNX1 short isoform has an importantrole in the development of myelodysplastic/myeloproliferative neoplasms. Blood Adv. 1:1382-1386.

Maehara, A., Kaitani, A., Izawa, K., Shiba, E., Na-gamine, M., Takamori, A., Isobe, M., Uchida, S.,Uchida, K., Ando, T., Keiko, M., Nakano, N.,Voehringer, D., Roers, A., Shimizu, T., Ogawa,H., Okumura, K., Kitamura, T., and Kitaura, J.Role of the ceramide-CD300f interaction in gram-negative bacterial skin infections. J. Invest. Der-matol, in press.

Isobe, M., Izawa, K., Sugiuchi, M., Sakanishi, T.,Kaitani, A., Takamori, A., Maehara, A., Mat-sukawa, T., Takahashi, M., Yamanishi, Y., Oki, T.,Uchida, S., Uchida, K., Ando, T., Maeda, K.,Nakano, N., Yagita, H., Takai, T., Ogawa, H.,Okumura, K., Kitamura, T., and Kitaura, J. TheCD300e molecule in mice is an immune-activat-

ing receptor. J. Biol. Chem. in press.Goyama, S., Shrestha, M., Schibler, J., Rosenfeldt,L., Miller, W., O'Brien, E., Mizukawa, B., Kita-mura, T., Palumbo, JS., and Mulloy, J.C. (2017)Protease-activated receptor 1 inhibits proliferationbut enhances leukemia stem cell activity in acutemyeloid leukemia. Oncogene 36:2589-2598.

Goyama, S., and Kitamura, T. (2017) Epigenetics innormal and malignant hematopoiesis: An over-view and update 2017. Cancer Science 108:553-562.

Yonezawa, T., Takahashi, H., Shikata, S., Liu, X.,Tamura, M., Asada, S., Fukushima, T., Fuku-yama, T., Tanaka, Y., Sawasaki, T., Kitamura, T.,and Goyama, S. (2017) The ubiquitin ligase STUB1 regulates stability and activity of RUNX1 andRUNX1-RUNX1T1. J. Biol. Chem. 292:12528-12541.

Kawabata, K.C., Hayashi, Y., Inoue, D., Meguro, H.,Sakurai, H., Fukuyama, T., Tanaka, Y., Asada, S.,Fukushima, T., Nagase, R., Takede, R., Harada,Y., Kitaura, J., Goyama, S. Harada, H., Aburatani,H. and Kitamura, T. (2018) High expression ofABC-G2 induced by EZH2 disruption plays piv-otal roles in MDS pathogenesis. Leukemia 32:419-428.

Hisaoka, T., Komori, T., Kitamura, T., and ＊

Morikawa, Y. (2018) Abnormal behaviours rele-vant to neurodevelopmental disorders in Kirrel3-knockout mice. Sci Rep 8: 1408.

Miyashita, K., Kitajima, K., Goyama, S., Kitamura,T. and Hara, T. Overexpression of Lhx2 sup-presses proliferation of human T cell acute lym-

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phoblastic leukemia-derived cells, partly by re-ducing LMO2 protein levels. Biochem. Biophys.Res. Commun. (2018) 495: 2310-2316.

Maeda, S., Morita, M., Shimada, S., Taniguchi, J.,Kashiwazaki, G., Bando, T., Naka, K., Kitamura,T., Nakahata, T., Adachi, S., Sugiyama, H., andKamikubo, Y. RUNX1 transactivates BCR-ABLexpression in Philadelphia chromosome positiveacute lymphoblastic leukemia. Sci Rep in press.

Kitamura, T. ASXL1 mutations gain a function.Blood 131: 274-275.

Nagase, R., Inoue, D., Pastre, A., Fujino, T., Hou,H-A, Yamasaki,N., Goyama, S., Saika, M., Kanai,A., Sera, Y., Horikawa, S., Ota, Y., Asada, S.,

Hayashi, Y., Kawabata, K.C., Takeda, R., Tien, H.F., Honda, H., Abdel-Wahab, O. and Kitamura T.Expression of mutant Asxl1 perturbs hematopoi-esis and promotes susceptibility to leukemictransformation. J Exp Med. in press.

Inoue, D., Fujino, T., Sheridan, P., Zhang, Y-Z., Na-gase, R., Horikawa, S., Li, Z., Matsui, H., Kanai,A., Saika, M., Yamaguchi, R., Kozuka-Hata, H.,Kawabata, K.C., Yokoyama, A., Goyama, S., In-aba, T., Imoto, S., Miyano, S., Xu, M., Yang, F-C.,Oyama, M., and Kitamura, T. A novel ASXL1-OGT axis plays roles in H3K4 methylation andtumor suppression in myeloid malignancies. Leu-kemia in press.

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1. The angiogenic factor Egfl7 alters thymo-genesis by activating Flt3 signaling

Yousef Salama, Koichi Hattori, Beate Heissig

Thymic regeneration is a crucial function that al-lows for the generation of mature T cells aftermyelosuppression like irradiation. However mo-lecular drivers involved in this process remain un-defined. Here, we report that the angiogenic factor,epidermal growth factor-like domain 7 (Egfl7), isexpressed on steady state thymic endothelial cells(ECs) and further upregulated under stress likepost-irradiation. Egfl7 overexpression increased in-trathymic early thymic precursors (ETPs) and ex-panded thymic ECs. Mechanistically, we show thatEgfl7 overexpression caused Flt3 upregulation inETPs and thymic ECs, and increased Flt3 ligandplasma elevation in vivo. Selective Flt3 blockadeprevented Egfl7-driven ETP expansion, and Egfl7-mediated thymic EC expansion in vivo. We pro-pose that the angiogenic factor Egfl7 activates theFlt3/Flt3 ligand pathway and is a key moleculardriver enforcing thymus progenitor generation andthereby directly linking endothelial cell biology tothe production of T cell-based adaptive immunity.

2. The role of plasmin in the pathogenesis ofmurine multiple myeloma

Salita Eiamboonsert, Yousef Salama, HiroshiWatarai, Douaa Dhahri, Yuko Tsuda, YoshioOkada, Koichi Hattori, Beate Heissig

Aside from a role in clot dissolution, the fibri-nolytic factor, plasmin is implicated in tumorigene-sis. Although abnormalities of coagulation and fi-brinolysis have been reported in multiple myelomapatients, the biological roles of fibrinolytic factors inmultiple myeloma (MM) using in vivo models havenot been elucidated. In this study, we established amurine model of fulminant MM with bone marrowand extramedullar engraftment after intravenousinjection of B53 cells. We found that the fibrinolyticfactor expression pattern in murine B53 MM cells issimilar to the expression pattern reported in pri-mary human MM cells. Pharmacological targetingof plasmin using the plasmin inhibitors YO-2 didnot change disease progression in MM cell bearingmice although systemic plasmin levels was sup-pressed. Our findings suggest that although plas-min has been suggested to be a driver for diseaseprogression using clinical patient samples in MMusing mostly in vitro studies, here we demonstratethat suppression of plasmin generation or inhibitionof plasmin cannot alter MM progression in vivo.

Center for Stem Cell Biology and Regenerative Medicine

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

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

Proteases 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. The goal of our laboratory is to identifynovel therapeutic targets for diseases like cancer or inflammatory diseases bystudying the role of proteases and growth factors during inflammation, tissue re-generation, and in stem cell biology.

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3. Pharmacological targeting of plasmin pre-vents lethality and tissue damage in a murinemodel of macrophage activation syndrome

Hiroshi Shimazu, Shinya Munakata, YoshihikoTashiro, Yousef Salama, Salita Eiamboonsert,Yasunori Ota, Haruo Onoda, Yuko Tsuda, YoshioOkada, Hiromitsu Nakauchi, Beate Heissig, KoichiHattori

Macrophage activation syndrome (MAS) is a life-threatening disorder characterized by a cytokinestorm and multiorgan dysfunction due to excessiveimmune activation. Although abnormalities of co-agulation and fibrinolysis are major components ofMAS, the role of the fibrinolytic system and its keyplayer, plasmin, in the development of MAS re-mains to be solved. We established a murine modelof fulminant MAS by repeated injections of Toll-like receptor-9 (TLR-9) agonist and d-galactosamine(DG) in immunocompetent mice. We found plas-min was excessively activated during the progres-sion of fulminant MAS in mice. Genetic and phar-macological inhibition of plasmin counteractedMAS-associated lethality and other related symp-toms. We show that plasmin regulates the influx ofinflammatory cells and the production of inflamma-tory cytokines/chemokines. Collectively, our find-ings identify plasmin as a decisive checkpoint inthe inflammatory response during MAS and a po-tential novel therapeutic target for MAS.

4. Plasminogen activator inhibitor-1 regulatesmacrophage-dependent postoperative adhe-sion by enhancing EGF-HER1 signaling inmice

Kumpei Honjo, Shinya Munakata, YoshihikoTashiro, Yousef Salama, Hiroshi Shimazu, SalitaEiamboonsert, Douaa Dhahri, Ichimura, A., Taka-shi Dan, Toshio Miyata, Kazuyoshi Takeda,

Kazuhiro Sakamoto, Koichi Hattori, Beate Heissig

Adhesive small bowel obstruction remains acommon problem for surgeons. After surgery,platelet aggregation contributes to coagulation cas-cade and fibrin clot formation. With clotting, fibrindegradation is simultaneously enhanced, driven bytissue plasminogen activator-mediated cleavage ofplasminogen to form plasmin. The aim of thisstudy was to investigate the cellular events andproteolytic responses that surround plasminogenactivator inhibitor (PAI-1; Serpine1) inhibition ofpostoperative adhesion. Peritoneal adhesion was in-duced by gauze deposition in the abdominal cavityin C57BL/6 mice and those that were deficient in fi-brinolytic factors, such as Plat－/－ and Serpine1－/－ Inaddition, C57BL/6 mice were treated with the novelPAI-1 inhibitor, TM5275. Some animals weretreated with clodronate to deplete macrophages.Epidermal growth factor (EGF) experiments wereperformed to understand the role of macrophagesand how EGF contributes to adhesion. In the earlyphase of adhesive small bowel obstruction, in-creased PAI-1 activity was observed in the perito-neal cavity. Genetic and pharmacologic PAI-1 inhi-bition prevented progression of adhesion and in-creased circulating plasmin. Whereas Serpine1－/－

mice showed intra-abdominal bleeding, mice thatwere treated with TM5275 did not. Mechanistically,PAI-1, in combination with tissue plasminogen acti-vator, served as a chemoattractant for macrophagesthat, in turn, secreted EGF and up-regulated the re-ceptor, HER1, on peritoneal mesothelial cells, whichled to PAI-1 secretion, further fueling the viciouscycle of impaired fibrinolysis at the adhesive site.Controlled inhibition of PAI-1 not only enhancedactivation of the fibrinolytic system, but also pre-vented recruitment of EGF-secreting macrophages.Pharmacologic PAI-1 inhibition ameliorated adhe-sion formation in a macrophage-dependent manner.

Publications

<Beate Heissig Group>1. Salama, Y., Hattori K., Heissig, B. The angiogenic

factor Egfl7 alters thymogenesis by activating Flt3 signaling. Biochem Biophys Res Commun.19,490(2): 209-216, 2017.

2. Eiamboonsert, S., Salama, Y., Watarai, H.,Dhahri, D., Tsuda, Y., Okada, Y., Hattori, K., He-issig, B. The role of plasmin in the pathogenesisof murine multiple myeloma. Biochem BiophysRes Commun. 24: 488(2): 387-392, 2017

3. Shimazu, H., Munakata, S., Tashiro, Y., Salama,Y., Eiamboonsert, S., Ota, Y., Onoda, H., Tsuda,Y., Okada, Y., Nakauchi, H,. Heissig, B., Hattori,

K. Pharmacological targeting of plasmin preventslethality and tissue damage in a murine modelof macrophage activation syndrome. Blood, Jul 6;130(1): 59-72, 2017.

4. Honjo, K., Munakata, S., Tashiro, Y., Salama, Y.,Shimazu, H., Eiamboonsert, S., Dhahri, D.,Ichimura, A., Dan, T., Miyata, T., Takeda, K.,Sakamoto, K., Hattori, K., Heissig, B. Plasmino-gen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancingEGF-HER1 signaling in mice. FASEB J, Jun; 31(6): 2625-2637, 2017.

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1. The role of plasmin in the pathogenesis ofmurine multiple myeloma.

Salita Eiamboonsert1, Yousef Salama1, HiroshiWatarai2, Douaa Dhahri1, Yuko Tsuda3, YoshioOkada3, Koichi Hattori4, Beate Heissig1: 1Divisionof Stem Cell Dynamics, Center for Stem Cell Biol-ogy and Regenerative Medicine, The Institute ofMedical Science, The University of Tokyo, 2Divi-sion of Stem Cell Cellomics, Center for Stem CellBiology and Regenerative Medicine, The Instituteof Medical Science, The University of Tokyo, 3Fac-ulty of Pharmaceutical Sciences, Kobe GakuinUniversity, 4Center for Genome and RegenerativeMedicine, Juntendo University School of Medicine.

Aside from a role in clot dissolution, the fibri-nolytic factor, plasmin is implicated in tumorigene-sis. Although abnormalities of coagulation and fi-brinolysis have been reported in multiple myelomapatients, the biological roles of fibrinolytic factors inmultiple myeloma (MM) using in vivo models havenot been elucidated. In this study, we established amurine model of fulminant MM with bone marrowand extramedullar engraftment after intravenous

injection of B53 cells. We found that the fibrinolyticfactor expression pattern in murine B53 MM cells issimilar to the expression pattern reported in pri-mary human MM cells. Pharmacological targetingof plasmin using the plasmin inhibitors YO-2 didnot change disease progression in MM cell bearingmice although systemic plasmin levels was sup-pressed.Our findings suggest that although plasmin has

been suggested to be a driver for disease progres-sion using clinical patient samples in MM usingmostly in vitro studies, here we demonstrate thatsuppression of plasmin generation or inhibition ofplasmin cannot alter MM progression in vivo.

2. IL-22BP dictates characteristics of Peyer'spatch follicle-associated epithelium for anti-gen uptake.

Toshi Jinnohara1,2, Takashi Kanaya1,2, Koji Hase3,4,Sayuri Sakakibara1, Tamotsu Kato1, Naoko Tachi-bana1, Takaharu Sasaki1, Yusuke Hashimoto1,2,Toshiro Sato7, Hiroshi Watarai5, Jun Kunisawa6,8,Naoko Shibata6, Ifor R. Williams9, Hiroshi Ki-yono6,10, Hiroshi Ohno1,2: 1Laboratory for Intestinal

Center for Stem Cell Biology and Regenerative Medicine

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 博士（医学） 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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Ecosystem, RIKEN Center for Integrative MedicalSciences, 2Department of Medical Life Science, Di-vision of Immunobiology, Graduate School ofMedical Life Science, Yokohama City University,3Division of Biochemistry, Faculty of Pharmacy,Keio University, 4Division of Mucosal Barriology,The Institute of Medical Science, The University ofTokyo, 5Division of Stem Cell Cellomics, The Insti-tute of Medical Science, The University of Tokyo,6Division of Mucosal Immunology, The Institute ofMedical Science, The University of Tokyo, 7De-partment of Gastroenterology, Keio UniversitySchool of Medicine, 8Laboratory of Vaccine Mate-rials, National Institutes of Biomedical Innovation,Health and Nutrition, 9Department of Pathology,Emory University School of Medicine, 10Core Re-search for Evolutional Science and Technology,Japan Science and Technology Agency.

Interleukin-22 (IL-22) acts protectively and harm-fully on intestinal tissue depending on the situ-ation; therefore, IL-22 signaling needs to be tightlyregulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intes-tinal and lymphoid tissues, although its precise dis-tribution and roles have remained unclear. In thisstudy, we show that IL-22BP is highly expressed byCD11b＋CD8a－ dendritic cells in the subepithelialdome region of Peyer's patches (PPs). We foundthat IL-22BP blocks IL-22 signaling in the follicle-as-sociated epithelium (FAE) covering PPs, indicatingthat IL-22BP plays a role in regulating the charac-teristics of the FAE. As expected, FAE of IL-22BP-deficient (Il22ra2－/－) mice exhibited altered proper-ties such as the enhanced expression of mucus andantimicrobial proteins as well as prominent fuco-sylation, which are normally suppressed in FAE.Additionally, Il22ra2－/－ mice exhibited the de-creased uptake of bacterial antigens into PPs with-out affecting M cell function. Our present studythus demonstrates that IL-22BP promotes bacterialuptake into PPs by influencing FAE gene expres-sion and function.

3. Analyses of a mutant Foxp3 allele revealBATF as a critical transcription factor in thedifferentiation and accumulation of tissueregulatory T cells.

Norihito Hayatsu1, Takahisa Miyao1, MasashiTachibana1, Ryuichi Murakami1,6, Akihiko Kimu-ra1, Takako Kato1, Eiryo Kawakami2,5, Takaho A.Endo3, Ruka Setoguchi4, Hiroshi Watarai7, TakeshiNishikawa1, Takuwa Yasuda4, Hisahiro Yoshida4,Shohei Hori1,6: 1Laboratory for Immune Homeosta-sis, RIKEN Center for Integrative Medical Sci-ences, 2Laboratory for Disease Systems Modeling,RIKEN Center for Integrative Medical Sciences,3Laboratory for Integrative Genomics, RIKEN

Center for Integrative Medical Sciences, 4Labora-tory for Immunogenetics, RIKEN Center for Inte-grative Medical Sciences, 5Disease Biology Group,RIKEN Medical Sciences Innovation Hub Pro-gram, 6Laboratory of Immunology and Microbiol-ogy, Graduate School of Pharmaceutical Sciences,The University of Tokyo, 7Division of Stem CellCellomics, Center for Stem Cell Biology and Re-generative Medicine, The Institute of Medical Sci-ences, The University of Tokyo.

Foxp3 controls the development and function ofregulatory T (Treg) cells, but it remains elusive howFoxp3 functions in vivo. Here, we establishedmouse models harboring three unique missenseFoxp3 mutations that were identified in patientswith the autoimmune disease IPEX. The I363V andR397W mutations were loss-of-function mutations,causing multi-organ inflammation by globally com-promising Treg cell physiology. By contrast, theA384T mutation induced a distinctive tissue-re-stricted inflammation by specifically impairing theability of Treg cells to compete with pathogenic Tcells in certain nonlymphoid tissues. Mechanisti-cally, repressed BATF expression contributed tothese A384T effects. At the molecular level, theA384T mutation altered Foxp3 interactions with itsspecific target genes including Batf by broadeningits DNA-binding specificity. Our findings identifyBATF as a critical regulator of tissue Treg cells andsuggest that sequence-specific perturbations ofFoxp3-DNA interactions can influence specific fac-ets of Treg cell physiology and the immunopatholo-gies they regulate.

4. A novel mouse model of iNKT cell deficiencygenerated by CRISPR/Cas9 reveals a patho-genic role of iNKT cells in metabolic disease.

Yue Ren1,2, Etsuko Sekine-Kondo1, Risa Shibata1,3,Megumi Kato-Itoh4, Ayumi Umino4, Ayaka Yana-gida4,9, Masashi Satoh5, Komaki Inoue6, TomoyukiYamaguchi4, Keiichi Mochida6, Susumu Nakae3,Luc Van Kaer7, Kazuya Iwabuchi5, HiromitsuNakauchi4,8, Hiroshi Watarai1: 1Division of StemCell Cellomics, Center for Stem Cell Biology andRegenerative Medicine, Institute of Medical Sci-ence, University of Tokyo, 2The Neurological Insti-tute of Jiangxi Province, Department of Neurol-ogy, Jiangxi Provincial People's Hospital, 3Labora-tory of Systems Biology, Center for ExperimentalMedicine and Systems Biology, Institute of Medi-cal Science, University of Tokyo, 4Division of StemCell Therapy, Center for Stem Cell Biology andRegenerative Medicine, Institute of Medical Sci-ence, University of Tokyo, 5Department of Immu-nology, Kitasato University School of Medicine,6Cellulose Production Research Team, RIKENCenter for Sustainable Resource Science, 7Depart-

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ment of Pathology, Microbiology and Immunology,Vanderbilt University School of Medicine, 8Insti-tute for Stem Cell Biology and Regenerative Medi-cine, Department of Genetics, Stanford UniversitySchool of Medicine.

iNKT cells play important roles in immune regu-lation by bridging the innate and acquired immunesystems. The functions of iNKT cells have been in-vestigated in mice lacking the Traj18 gene segmentthat were generated by traditional embryonic stemcell technology, but these animals contain a biasedT cell receptor (TCR) repertoire that might affectimmune responses. To circumvent this confoundingfactor, we have generated a new strain of iNKTcell-deficient mice by deleting the Traj18 locus us-ing CRISPR/Cas9 technology, and these animalscontain an unbiased TCR repertoire. We employedthese mice to investigate the contribution of iNKTcells to metabolic disease and found a pathogenicrole of these cells in obesity-associated insulin-resis-tance. The new Traj18-deficient mouse strain willassist in studies of iNKT cell biology.

5. Ultrafast confocal fluorescence microscopybeyond the fluorescence lifetime limit.

Hideharu Mikami1, Jeffrey Harmon1, HirofumiKobayashi1, Syed Hamad1, Yisen Wang1,2, OsamuIwata3, Kengo Suzuki3, Takuro Ito4, Yuri Aisaka5,Natsumaro Kutsuna5, Kazumichi Nagasawa6, Hiro-shi Watarai6, Yasuyuki Ozeki7, Keisuke Goda1,4,8:1Department of Chemistry, The University of To-kyo, 2College of Precision Instrument and Opto-electronic Engineering, Tianjin University,3Euglena Co., Ltd., 4Japan Science and TechnologyAgency, 5LPixel Inc., 6Division of Stem Cell Cel-lomics, Center for Stem Cell Biology and Regen-erative Medicine, Institute of Medical Science, TheUniversity of Tokyo, 7Department of Electrical En-gineering and Information Systems, The Univer-sity of Tokyo, 8Department of Electrical Engineer-ing, University of California.

Laser-scanning confocal fluorescence microscopyis an indispensable tool for biomedical research byvirtue of its high spatial resolution. Its temporalresolution is equally important, but is still inade-quate for many applications. Here we present aconfocal fluorescence microscope that, for the firsttime to our knowledge, surpasses the highest possi-ble frame rate constrained only by the fluorescencelifetime of fluorophores (typically a few to severalnanoseconds). This microscope is enabled by inte-grating a broadband, spatially distributed, dual-fre-quency comb or spatial dual-comb and quadratureamplitude modulation for optimizing spectral effi-ciency into frequency-division multiplexing withsingle-pixel photodetection for signal integration.

Specifically, we demonstrate confocal fluorescencemicroscopy at a record high frame rate of 16,000frames/s. To show its broad biomedical utility, weuse the microscope to demonstrate 3D volumetricconfocal fluorescence microscopy of cellular dy-namics at 104 volumes/s and confocal fluorescenceimaging flow cytometry of hematological and mi-croalgal cells at 2 m/s.

6. Generation and validation of novel anti-bo-vine CD163 monoclonal antibodies ABM-1A9and ABM-2D6.

Yoshinori Shimamoto1, Junko Nio-Kobayashi4, Hi-roshi Watarai5, Masashi Nagano6, Natsuko Saito2,Eiki Takahashi7, Hidetoshi Higuchi3, Atsushi Ko-bayashi6, Takashi Kimura6, Hiroshi Kitamura2:1Laboratory of Animal Therapeutics, Departmentof Veterinary Science, Rakuno Gakuen University,2Laboratory of Veterinary Physiology, Departmentof Veterinary Medicine, Rakuno Gakuen Univer-sity, 3Laboratory of Animal Health, Department ofVeterinary Medicine, Rakuno Gakuen University,4Laboratory of Histology and Cytology, Depart-ment of Functional Morphology, Graduate Schoolof Medical Sciences, Hokkaido University, 5Divi-sion of Stem Cell Cellomics, The Institute of Medi-cal Science, The University of Tokyo, 6Laboratoryof Theriogenology and Laboratory of Pathology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine, Hok-kaido University, 7Research Resources Center,RIKEN Brain Science Institute.

The scavenger receptor CD163 is widely used asa cell signature of alternatively active "M2" macro-phages in mammals. In this study, we generatedtwo monoclonal antibodies, namely ABM-1A9 andABM-2D6, against the extracellular region of bovineCD163. Conventional Western blotting using theantibodies gave immune reactive bands correspond-ing to the transmembrane (～150 kDa) and soluble(～120 kDa) forms of bovine CD163 when testedusing spleen protein lysate extracted from cattlewith mastitis. The minimum limit of detectable con-centration of both antibodies was relatively lower(5.0 ng/mL) than that of the anti-human CD163monoclonal antibody AM-3K (> 1.0 mg/mL), whichhas been used previously for the detection of bo-vine CD163. An immunohistochemical study usingformalin-fixed paraffin-embedded sections indi-cated that ABM-1A9 and ABM-2D6 clearly stainedsome Iba1＋ macrophages in the lymph nodes ofcattle with mastitis. Moreover, the CD163-stainedmacrophages were frequently observed engulfingleukocytes. ELISA using ABM-2D6 distinguishedlevels of circulating soluble CD163 between healthycattle (< 16.9 pmol/mL) and cattle with mastitis (>33.7 pmol/mL). These new monoclonal antibodies

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can be used for the diagnosis and evaluation ofprognosis of inflammatory diseases in cattle, using

immunohistological analysis and blood test applica-tions.

Publications

1. Eiamboonsert, S., Salama, Y., Watarai, H.,Dhahri, D., Tsuda, T., Okada, Y., Hattori, K., He-issig, B. The role of plasmin in the pathogenesisof murine multiple myeloma. Biochem. Biophys.Res. Commun. 488(2): 387-392, 2017. doi: 10.1016/j.bbrc.2017.05.062. PMID: 28501622.

2. Jinnohara, T., Kanaya, T., Hase, K., Sakakibara,S., Tachibana, N., Hashimoto, Y., Sato, T., Wata-rai, H., Kunisawa, J., Shibata, N., Williams, I.R.,Kiyono, H., Ohno, H. IL-22BP dictates character-istics of Peyer's patch follicle - associatedepithelium for antigen uptake. J. Exp. Med. 214(6): 1607-1618, 2017. doi: 10.1084/jem.20160770.PMID: 28512157.

3. Hayatsu, N., Miyao, T., Tachibana, M., Mu-rakami, R., Kimura, A., Kato, T., Kawakami, E.,Endo, T.A. , Setoguchi, R. , Watarai, H. ,Nishikawa, T., Yasuda, T., Yoshida, H., Hori, S.Analyses of a mutant Foxp3 allele reveal BATFas a critical transcription factor in the differentia-tion and accumulation of tissue regulatory Tcells. Immunity. 47(2): 268-283, 2017. doi: 10.1016/j.immuni.2017.07.008. PMID: 28778586.

4. Ren, Y., Sekine-Kondo, E., Shibata, R., Kato-Itoh,M., Umino, A., Yanagida, A., Satoh, M., Inoue,K., Yamaguchi, T., Mochida, K., Nakae, S., VanKaer, L., Iwabuchi, K., Nakauchi, H., Watarai, H.A novel mouse model of iNKT cell-deficiencygenerated by CRISPR/Cas9 reveals a pathogenicrole of iNKT cells in metabolic disease. Sci. Rep.7(1): 12765, 2017. doi: 10.1038/s41598-017-12475-4.PMID: 28986544.

5. Mikami, H., Harmon, J., Kobayashi, H., Hamad,S., Wang, Y., Iwata, O., Suzuki, K., Ito, T.,Aisaka, Y., Kutsuna, N., Nagasawa, K., Watarai,H., Ozeki, Y., Goda, K. Ultrafast confocal fluores-cence microscopy beyond the fluorescence life-time limit. Optica. 5(2): 117-126, 2018. doi:10.1364/OPTICA.5.000117.

6. Shimamoto, Y., Nio-Kobayashi, J., Watarai, H.,Nagano, M., Saito, N., Takahashi, E., Higuchi,H., Kobayashi, A., Kimura, T., Kitamura, H.Generation and validation of novel anti-bovineCD163 monoclonal antibodies ABM-1A9 andABM-2D6. Vet. Immunol. Immunopathol. 198(4): 6-13, 2018. doi: 10.1016/j-vetimm.2018.02.004.

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1. Dissection of signaling events downstream ofthe c-Mpl receptor in murine hematopoieticstem cells via motif-engineered chimeric re-ceptors

Koichiro Saka, Chen-Yi Lai, Masanori Nojima,Masahiro Kawahara, Makoto Otsu, HiromitsuNakauchi, Teruyuki Nagamune

Hematopoietic stem cells (HSCs) are a valuableresource in transplantation medicine. Cytokines areoften used to culture HSCs aiming at better clinicaloutcomes through enhancement of HSC reconstitu-tion capability. Roles for each signal moleculedownstream of receptors in HSCs, however, remainpuzzling due to complexity of the cytokine-signal-ing network. Engineered receptors that are non-re-sponsive to endogenous cytokines represent an at-tractive tool for dissection of signaling events. Wehere tested a previously developed chimeric recep-tor (CR) system in primary murine HSCs, targetcells that are indispensable for analysis of stem cellactivity.

2. Evaluation of the Utility of Induced Pluripo-tent Stem Cells as a platform for modelingWiskott Aldrich Syndrome

HONDA-OZAKI Fumiko, LIN Huan-Ting, OKU-MURA Takashi, LAI Chen-Yi, OTSU Makoto

Wiskott Aldrich syndrome (WAS) is an X-linkeddisease, caused by mutations in the gene encodingthe WAS protein (WASp). Thrombocytopenia is themain feature of this disease, often threatening pa-tients to a significant risk of serious hemorrhage. Ithas been an issue of debate whether defective pro-platelet release from megakaryocytes (MKs)(i.e.,production) and/or destruction of platelets (PLTs)by the spleen macrophages (i.e., consumption) con-stitutes the major cause of thrombocytopenia. Dueto the limitations inherent to classical experimentalmodels, there have been conflicting results pub-lished; precise mechanisms causing thrombocy-topenia in WAS therefore remain to be elucidated.To have an appropriate model in our hands and toelucidate mechanism(s) of thrombocytopenia inWAS to improve treatment of WAS patients in fu-ture, we here evaluated the utility of iPSCs as anemerging disease model for WAS. WASp-deficientiPSCs produced MKs and PLTs with similar yieldsas did control cells when differentiated from sortedMK progenitors. Interestingly, however, WASp-de-ficient iPSCs yielded only fewer MKs and PLTsthan did healthy counterparts when differentiated

Center for Stem Cell Biology and Regenerative Medicine

Stem Cell Bankステムセルバンク

Associate Professor Makoto Otsu, M.D., Ph.D.Project Assistant Professor Chen-Yi Lai, Ph.D.

准教授 博士（医学） 大 津 真特任助教 博士（生命科学） 頼 貞 儀

Stem cells represent a valuable cell source in the field of regenerative medicine.Pluripotent stem cells are newly emerging types of stem cells that may be utilizedeither for the basic research or to cure the diseases. In this laboratory, we havebeen focusing especially on the utilization of induced pluripotent stem cells as aresearch platform to elucidate pathophysiology of intractable diseases based ontheir proper modeling. Our eventual goal is to establish safe and efficacious treat-ment for the patients suffering from various types of diseases currently with nocurative treatment available

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from unsorted HPCs. In addition, we found thatWASp-deficiency variously affected phagocytosisfunction of iPSC-monocytes in a context specificmanner, depending on what targets they wouldface. These data imply that not just MKs and PLTsbut also other lineage cells such as phagocytic cellsmay play a role for WAS-thrombocytopenia. Weare now trying to establish an in vitro co-culturesystem of iPSCs-derived monocytes and -plateletsas a new experimental platform to clarify accurateroles of each cellular compartment.

3. The functional analysis of dendritic cells de-veloped from T-iPS cells from a single CD4＋T cell of Sjögren's syndrome

Mana Iizuka-Koga, Hiromitsu Asashima, MikiAndo, Chen-Yi Lai, Shinji Mochizuki, MahitoNakanishi, Toshinobu Nishimura, Hiroto Tsuboi,Tomoya Hirota, Hiroyuki Takahashi, Isao Matsu-moto, Makoto Otsu, Takayuki Sumida

Though it is important to clarify the pathogenicand/or regulatory functions of the T cells in humansamples, their examination is frequently restrictedin opportunity because the acquisition of sufficientquantity of dendritic cells (DCs), used as antigenpresenting cells, might not be available, especiallyin autoimmune diseases. In this study, we devel-oped the generation of mature DCs from inducedpluripotent stem cells derived from T cells (T-iPSCs). We reprogramed only a single CD4＋ T cellof a patient with Sjögren's syndrome (SS) into iPScells, which were differentiated into DCs (TiPS-DCs) via TiPS-Sacs. Moreover, we examined theircharacterizations and functions. Under the micro-scope, the mature TiPS-DCs were dendritic cells-like morphology. The expression of CD11b, CD11c,HLA-ABC and CD40 was detected, and the CD80,CD86 and HLA-DR was also increased. The matureTiPS-DCs were able to produce TNF-a and IL-6through stimulation. As compared with monocytes-derived DCs, TiPS-DCs was equal to the ability ofantigen processing, and also the induction of robustproliferative response of allogeneic T cells in thedependent manner of cell numbers.In conclusion, we succeeded to obtain an ade-

quate amount of functional DCs from a singleCD4＋ T cell by way of iPS cells. This methodshould shed light on the characterization of patho-genic T cells for not only SS but also other diseasesto elucidate the function of autoreactive T cells. Themethod shown in the study will enable us to iden-tify autoreactive T cells in autoimmune diseasesalong with elucidate the function and pathogenicityof them.

4. Mutant iPS cells derived from patients withRALD show significance of KRAS for self-re-newal and differentiation propensity

Kenji Kubara, Kazuto Yamazaki, Yasuharu Ishi-hara, Huan-Ting Lin, Masashi Ito, Kappei Tsuka-hara, Masatoshi Takagi, and Makoto Otsu

KRAS is widely known as a proto-oncogene andhas been reported to play essential roles instemness maintenance in some types of stem cells,including cancer stem cells. However, the roles ofKRAS in pluripotent stem cells (PSCs) are largelyunknown. Recently, somatic gain-of-function muta-tions of KRAS or NRAS in hematopoietic stem cellshave been reported to cause RAS-associated auto-immune lymphoproliferative syndrome-like disease(RALD). Here, we investigated the roles of KRASon stemness maintenance in the context of humanPSCs using isogenic KRAS mutant (G13C/WT) andwild-type (WT/WT) induced PSCs (iPSCs), gener-ated from the same RALD patients with the so-matic KRAS mutation. Using the isogenic iPSClines from two patients, we revealed that G13C/WTiPSCs displayed self-renewal and differentiationcharacteristics distinct from those of WT/WT iPSCs:expression of stemness markers including POU5F1and NANOG, which is indicative of an undifferen-tiated state, was maintained at high levels in G13C/WT iPSCs under bFGF depletion; neuronal differen-tiation was clearly blunted from G13C/WT iPSCs.In addition, we generated wild-type (WTed/WT)and heterozygous knockout (Ded/WT) iPSCs fromthe same G13C/WT clone using gene-editing tech-niques. As expected, the G13C/WT-specific pheno-types were normalized in WTed/WT iPSCs. Inter-estingly, Ded/WT iPSCs showed lower potential tomaintain undifferentiation status under bFGF de-pletion, with a higher tendency to differentiate intoneuronal lineage than WTed/WT iPSCs. Biochemi-cal analysis indicated hyper-activation of KRAS andsubsequent increased phosphorylation levels ofERK in G13C/WT iPSCs. Pharmacological studiesusing specific kinase inhibitors demonstrated thatthe features compatible with enhanced stemnessmaintenance were canceled in mutant cells by pan-RAF and MEK inhibitors, but not by PI3K inhibi-tors. In addition, neuronal differentiation was im-proved in G13C/WT iPSCs with the MEK inhibitortreatment. These observations suggested that theKRAS-ERK pathway plays more critical roles thanthe KRAS-PI3K pathway in G13C/WT iPSCs. Col-lectively, the analyses on the isogenic and genome-edited iPSCs from the RALD patients revealed thecrucial roles of the KRAS-ERK signaling on thestemness maintenance, having a strong impact onself-renewal and differentiation propensity in hu-man iPSCs.

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Publications

1. Takahashi, Y., Sato, S., Kurashima, Y., Lai, C.Y.,Otsu, M., Hayashi, M., Yamaguchi, T., Kiyono,H. Reciprocal Inflammatory Signaling BetweenIntestinal Epithelial Cells and Adipocytes in theAbsence of Immune Cells. EBioMedicine. 23: 34-45, 2017.

2. Saka, K., Lai, C.Y., Nojima, M., Kawahara, M.,Otsu, M., Nakauchi, H., Nagamune, T. Dissec-tion of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic StemCells Via Motif-Engineered Chimeric Receptors.Stem Cell Rev. 2017.

3. Natsumoto, B., Shoda, H., Fujio, K., Otsu, M.,Yamamoto, K. Investigation of the pathogenesisof autoimmune diseases by iPS cells. Nihon Rin-sho Meneki Gakkai Kaishi. 40: 48-53, 2017.

4. Ishida, T., Suzuki, S., Lai, C.Y., Yamazaki, S.,Kakuta, S., Iwakura, Y., Nojima, M., Takeuchi,Y., Higashihara, M., Nakauchi, H., Otsu, M. Pre-

Transplantation Blockade of TNF-alpha-Medi-ated Oxygen Species Accumulation Protects He-matopoietic Stem Cells. Stem Cells. 35: 989-1002,2017.

5. Iizuka-Koga, M., Asashima, H., Ando, M., Lai, C.Y., Mochizuki, S., Nakanishi, M., Nishimura, T.,Tsuboi, H., Hirota, T., Takahashi, H., Matsu-moto, I., Otsu, M., Sumida, T. Functional Analy-sis of Dendritic Cells Generated from T-iPSCsfrom CD4＋ T Cell Clones of Sjogren's Syn-drome. Stem Cell Reports. 8: 1155-1163, 2017.

6. Ieyasu, A., Ishida, R., Kimura, T., Morita, M.,Wilkinson, A.C., Sudo, K., Nishimura, T., Ohe-hara, J., Tajima, Y., Lai, C.Y., Otsu, M., Naka-mura, Y., Ema, H., Nakauchi, H., Yamazaki, S.An All-Recombinant Protein-Based Culture Sys-tem Specifically Identifies Hematopoietic StemCell Maintenance Factors. Stem Cell Reports. 8:500-508, 2017

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Yumiko Ishii, Sayaka Yamane, Azusa Fujita

Center for Stem Cell Biology and Regenerative Medicine

FACS Core LaboratoryFACSコアラボラトリー

Associated Professor Makoto Otsu, M.D., Ph.D. 准教授（兼務） 博士（医学） 大 津 真

The FACS Core Laboratory provides high quality, cost effective state-of-art flowcytometry services for internal and external researcher. The facility has three BDFACSAria cell sorters, SONY SH800 cell sorter for sorting, BD FACSCalibur, BDFACSVerse for analysis. We offer assistance in the following areas, (1) initial pro-ject planning (2) antibody panel design and optimization (3) instrument operationand maintenance (4) data analysis.

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