In 1992, we started a program on living-donor liver transplantation(LDLT)at Keio University that aimed to apply basic research to clinical practice. Basic research focused on microcirculation, free radicals, and cytokines to elucidate the mechanism and treatment of ischemiareperfusion injury. Experimental liver transplantations were performed on pigs in preclinical trials,which showed that intraportal administration of prostaglandin E1(PGE1)mitigated ischemiareperfusion injury and helped maintain positive graft liver circulation and function. In addition, in vivo fluorescence microscopy was used to visualize the beneficial role of PGE1 in the suppression of leukocyte adhesion to the vascular endothelium and in the improvement of microcirculation in the liver. This research was translated to the clinical practice of adult LDLT and intraportal infusion therapy for ABO-incompatible liver transplantation. The first LDLT at Keio University Hospital was performed on April 3, 1995. The patient developed jaundice after the surgery and had to be hospitalized for approximately 3 months, but was later discharged uneventfully and is now in good health. In November 1998, the first adult ABO-incompatible LDLT was performed. To prevent single organ DIC that occurs in the graft, intraportal infusion of PGE1, gabexate mesilate, and steroids was decided based on the aforementioned basic research. In my 11 years at Keio, I performed 119 LDLTs in 116 patients, achieving a 5-year survival rate of 84.3% overall, 87.2% in children, and 82.3% in adults, which were all higher than the corresponding national averages. For the 19 patients (12 adults, 7 children) in whom I performed ABO-incompatible liver transplantationLDLTs, the 5-year survival rate was 78.9%, which is not significantly different from the 83.1% for compatible cases. At Tokyo Medical University, I successfully resumed the LDLT program in 2012 with the assistance of the staff, which had been put on hold.

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Half a century ago, the sedative thalidomide caused one of the worst notorious drug disasters in history, with more than 10,000 babies born with deformities. The drug is now used in the treatment of multiple myeloma. Recently new thalidomide derivatives called immunomodulatory drugs (IMiDs) have been developed. Among them, lenalidomide and pomalidomide have excellent anti-cancer activity. However, the use of them is limited due to its potent teratogenic activity. The mechanism by which IMiDs including thalidomide induce birth defects and therapeutic effects was a long-standing question. Using an affinity beads technology we originally developed, we have identified cereblon(CRBN)as a primary target of IMiDs. CRBN forms an E3 ubiquitin ligase complex. IMiDs alter the activity and induce various biological effects such as teratogenicity, anti-cancer and immunomodulation.Understanding IMiDs and CRBN may lead to uncover new therapeutic pathways for overcoming refractory cancer diseases.

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Our new technology of Bio-Texture Modeling by multi-material 3D printing system enabled manufacturing patient-specific bio-elastic 3D organ models by simultaneous jetting of different types of model materials and compounding water-soluble synthetic resins. Such realistic tangible organ models can be soaked in water to look and feel closer to real organs. We evaluated its feasibility in navigation and simulation for organ transplantation biology.

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The role of organ preservation solution is fundamentally important in the transplantation therapy. We have established the effectiveness of the transplantation of mesenchymal stem cell on the alleviation of graft ischemia reperfusion injury in experimental rat model. Since it is well known that organ deterioration during preservation period exacerbate ischemia reperfusion injury, fresh graft is preferably used in transplantation. Thus, we examined whether the factors secreted by mesenchymal stem cells have the potency of elongation of storage time and improvement of graft survival when added to preservation solution.

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Hydrogen(H2), used as a treatment to counter adverse events associated with organ transplantation, is valued not only as an anti-oxidant, anti-inflammatory and anti-apoptotic agent, but also possibly as a mitochondrial protector. Although we previously demonstrated that administration of exogenous hydrogen using gas bubbling could potentially cause adverse events, including increased risk of bacterial contamination and possible mechanical injury due to bubbling, we have been investigating novel, feasible, and more secure hydrogen delivery methods for transplantation. Using a rat cardiac transplant model, we evaluated the efficacy of a hydrogen-rich water bath, a novel and portable graft preservation method that can provide H2 to stored organs during cold preservation safely, effectively, aseptically, and continuously. Elder cardiac grafts(60- to 70-week-old Lewis rats)or allografts(12-week-old Brown Norway rats)were harvested and packed with Celsior preservation solution. Packed grafts were stored at 4℃ in the cold water bath equipped with／without an electrolyzer to saturate the water with H2. Cold ischemia time was six hours for elder grafts or eight hours for allografts. Cardiac grafts then were heterotopically engrafted into Lewis rat recipients. The hydrogen level of cardiac tissue stored in the hydrogen-rich water bath significantly increased six hours after cold preservation(12.9±0.53 pmol／mg), whereas that in the control group stayed at basal level(0.87±0.26 pmol／mg). In both experimental settings, severe increases in serum injury markers for cardiac injury(CPK, troponinⅠ), neutrophil infiltration, and upregulation of mRNAs for pro-inflammatory cytokines／chemokines in grafts were observed three hours after reperfusion in the control group. Cold ischemic damage, including mitochondrial damage, myocardial fiber injury, and endothelial injury was ultrastructurally observed in the grafts without H2 treatment after preservation. Myocardial injury and inflammatory events were significantly attenuated by organ storage in the hydrogen-rich water bath. The grafts preserved in the hydrogen-rich water bath also exhibited higher hemeoxygenase (HO)-1 expression. Furthermore, tissue ATP content and mRNA levels of mitochondrial biogenesis-related factors increased in the grafts stored in the hydrogen-rich water bath. Continuous administration of H2 during cold preservation ameliorated myocardial injury in heart grafts pre- and post-transplantation. Hydrogen-rich water bath can be a useful method for cardiac preservation.

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Donor shortage has been an unresolved problem in clinical transplantation. It is difficult to enlarge donor pool to the expanded criteria donor(ECD)by simple cold storage, due to the severe ischemia and reperfusion injury (IRI). Resuscitation of the susceptible grafts by extracorporeal perfusion has been studied, but there has been no gold standard yet. In this review, we summarized the efficacy of novel medical gases, hydrogen and helium, for the use as an antioxidant and anti-inflammatory agents. Since hydrogen gas alone could not improve ATP content, cytoskeletal integrity, homeostasis of cytosolic ions including Na^＋, K^＋, and Ca^2＋, possiblesupplemental actions of heavy water were briefly described.

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Oxygenation condition during organ preservation is one of most important consideration issues to develop a machine perfusion system for transplantation. It is necessary to investigate the suitable preservation condition for the donor grafts from donors after cardiac death(DCD). However oxygen consumption during the machine preservation has not been explained yet in detail. In this report, the oxygen consumption of the DCD liver graft were discussed during the hypothermic machine perfusion(HMP)and the newly developed rewarming machine perfusion(RMP).

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In order to develop an effective organ/cell preservation method and to monitor/control post-transplant graft function continuously and non-invasively, an innovative optic technology to visualize/control cell/organ function seems to be useful. Recently, we have developed molecular probes to visualize redox states and cellular stresses, pH and cellular antigens in deeper lesions of the organ, and trying to activate intracellular key molecules by optical irradiation. In the hepatic ischemia/reperfusion model of mice, we successfully imaged liver oxidative stress and apoptosis by caspase-3 activity non-invasively and chronologically in a single mouse. We also developed a unique tool for visualizing intracellular pH and succeeded in imaging dynamic changes of pH in a mouse posterior limb ischemia/reperfusion model. Furthermore, we are now developing new tools to control intracellular molecules by blue light. We used plant-derived proteins to activate Akt/PKB molecule, essential for cell survival. By irradiating blue light, Akt in hepatocytes were phosphorylated(Ser and Thr)and activated. These tools will definitely provide a new avenue toward cell/organ transplantation in the future.

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We assessed cardiac death(CD)kidney tissue by BOLD(blood oxygenation-level dependency)MRI and diffusion MRI. BOLD and diffusion MRI successfully and non-invasively imaged and quantified red cell congestion and edema in CD grafts respectively. They can predict organ damage, and therefore the outcome, of transplanted CD kidney grafts.

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ABO incompatible renal transplant recipients were administered immunosuppressive agents from one month before operation as desensitization immunosuppressive therapy. Lymphocyte-immunosuppressant sensitivity test(LIST)can predict the pharmacological efficacy for renal transplant recipients at just before transplantation. However, in case of ABO incompatible renal transplantation, the evaluation point of LIST has been unclear either at the beginning of desensitization immunosuppressive therapy or just before renal transplantation. Therefore, we evaluated pharmacological efficacies of tacrolimus(Tac), cyclosporine(CyA), and mycophenolic acid(MPA)by LIST, as well as ATP amounts in peripheral blood mononuclear cells(PBMCs)of patient origin, at both the beginning of desensitization immunosuppressive therapy and just before renal transplant operation. The study includes patients treated by Tac without basiliximab(Bax) (n=8), patients treated by Tac with Bax(n=10), and patients treated by CyA with Bax(n=11) immunosuppressive therapy. In the recipients treated by Tac without Bax, the rate of acute rejection episodes of the Tac high sensitivity group was significantly higher than that of the low Tac sensitivity group at only just before operation(p=0.022). However, the rate of cytomegarovirus (CMV)infection did not significantly correlate with Tac sensitivity. The rate of acute rejection episode or CMV infection was not significantly different between the above two patient subgroups at the beginning of desensitization immunosuppressive therapy. From these observations, we concluded that LIST should be carried out for the evaluation of Tac pharmacological efficacy at just before operation to predict occurrence of acute rejection episodes in renal transplantation.

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Mesenchymal stem cells(MSCs)are known to possess anti-inflammatory effect. We investigated whether intratracheal administration of MSCs ameliorate ischemia-reperfusion injury of prolonged cold ischemia in a mouse model. The mouse lungs were perfused with low-potassium dextran glucose solution and preserved at 4℃ for 18 hours prior to transplantation. Cultured MSCs isolated from healthy human were administered into the left bronchus of the lung graft. Orthotopic left lung transplantation was performed. The recipient mice were euthanatized 6 hours after transplantation. Bronchoalveolar lavage fluid(BALF)from the left lung was collected and the total protein concentration, cell counts and the concentrations of pro-inflammatory cytokines were measured. The other lung grafts were histologically examined. The results indicated that intratracheal administration of MSCs ameliorated ischemia-reperfusion injury.

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Only limited treatment options are available now in end-stage organ failure, including replacement therapy such as hemodialysis or organ transplantation. In addition, the outcome of these therapeutic approaches is often challenging because of the requirement for longterm technical support, or complicated surgical techniques and life-long immune suppression, respectively. Moreover, donor shortage provides only limited outcome to patients who are in long waiting list. Therefore, new methods to facilitate recovery from such organ failure are highly desirable. Regenerative therapy could be an option in this regard. Recent progress in the field of tissue engineering has opened attractive approaches for clinical applications of regenerative medicine, including the use of microfluidics, bioreactors or organoid constructs. Of these, tissue decellularization technology, which retains all the necessary cues for cell maintenance and homeostasis, such as the three dimensional structure of the organ and its extracellular matrix(ECM) components, has recently been applied to whole organs. It has demonstrated efficacy for generating an engineered graft which is transplantable by vascular anastomosis. In this review, we focus on tissue decellularization as a new bioengineering approach, most recently for whole organs, that has been applied for liver fabrication.

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Regeneration therapy using derivatives of induced pluripotent stem cells(iPSCs)is a promising treatment for advanced cardiac failure. We herein document our studies for allogeneic transplantation therapy using iPSCs-derived cardiac myocytes(CMs), which were prepared as a scaffold-free cell-sheet form, for treating chronic cardiac failure in a large animal model. Cardiomyogenic differentiation was induced in iPSCs of human origin in vitro, showing that more than 80% of the cells were positive for cardiac specific markers. Scaffold-free cell-sheets of the iPSC-CMs, which were prepared by thermoresponsive culture dish, were then transplanted into the cardiac surfac e of the immune-suppressed pigs which were subjected to myocardial infarction 4 weeks prior to the treatment.As a result, global and regional cardiac function was significantly improved until 8 weeks after the treatment, as assessed by echocardiography and multi-slice CT scanning, without generating arrhythmia or tumor. The transplanted cells expressing cardiacspecific contractile proteins remained in the heart for 8 weeks.In conclusion, transplantation of the iPSC-CM cell-sheets into the pig chronic myocardial infarction model was feasible, safe and effective. Although pre-clinical proof-of-concept established, this treatment needs to be developed by further basic studies to ensure safety and enhance therapeutic efficacy to launch clinical study.

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Bilioenteric anastomosis is a commonly used procedure for biliary tract reconstruction in a variety of hepatobiliary and pancreatic surgery. However, the procedure is associated with loss of physiological function of the duodenal papilla of Vater and allows reflux of bowel fluid into bile ducts, resulting in anastomotic stricture and cholangiocarcinoma. The procedure is not ideal, so we are investigating the procedure which preserve physiological function of the duodenal papilla of Vater. In this review, we introduce a regenerative therapy of bile duct using tissue engineering technique and mention future prospects of the therapy in clinical settings.

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Hepatocyte transplantation (HCT) is a promising option for the non-invasive treatment of acute liver failure and urea cycle disorder. A problem to solve in HCT is the limited availability of cell donor. Here, we review the possibility of use of remnant liver of hyper reduced left lateral segment(HRLLS)graft from living donor liver transplantation as the cell source. Because liver transplantation for low body weight infants often needs reduction of an adult liver of which quality is assured to be compatible to transplantation. Thus, the remnant liver tissues provide the best opportunity of hepatocytes banking. In Japan, the research use of organs from brain dead donor is not allowed legally and the remnant liver of HRLLS graft of living donor is only invaluable resource. Indeed, the quality of cells isolated by ourselves showed comparable to that reported by oversea researchers. We propose that the remnant liver of HRLLS graft from living donor could be an important cell source for HCT.

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Mesenchymal stem cells (MSCs), including adipose tissue-derived mesenchymal stem cells(ADSC), are multipotent and can differentiate into various cell types possessing unique immunomodulatory features. Several clinical trials have demonstrated the safety and possible efficacy of MSCs in organ transplantation. Thus, stem cell therapy is promising for tolerance induction. In this study, we assessed the reprogramming capacity of murine ADSCs and found that CD90(Thy-1), originally discovered as a thymocyte antigen, could be a useful marker for cell therapy. Murine ADSCs were isolated from B6 mice, sorted by selection of CD90Hi or CD90Lo, and then transduced with four standard factors (Oct4, Sox2, Klf4, and c-Myc). Among unsorted, CD90Hi-, and CD90Lo- murine ADSCs, reprogrammed CD90Hi ADSCs showed increased numbers of alkaline phosphatase-positive colonies compared with reprogrammed CD90Lo ADSCs. The relative reprogramming efficiencies of unsorted, CD90Hi-sorted, and CD90Lo-sorted ADSCs were 100%, 116.5%, and 74.7%, respectively. CD90Hi cells were more responsive to reprogramming. CD90Hi ADSCs had greater reprogramming capacity than CD90Lo ADSCs, suggesting that ADSCs have heterogeneous subpopulations. Thus, CD90Hi selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell-based tolerance induction therapy.

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Short-term outcome of renal transplantation has dramatically improved due to advance in immunosuppressive agents. However effective treatments for chronic renal graft injury have not been established, and long-term outcome remained unsatisfactory. Transplanted kidney toxicity of side-effect of calcineurin inhibitors, and antibody-mediated rejection by anti-HLA antibodies are major factors hindering the long-term graft survival. To overcome chronic renal graft injury, we currently underway to establish tailor-made immunosuppressive therapy in renal transplantation, by grouping renal transplant recipients with immunological and non-immunological risk factors. Here we report our protocol and short-term results.

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Pancreatic islet transplantation is performed as a curative treatment of type 1 diabetes mellitus, however, this approach is significantly limited due to the critical shortage of islet source. Recently, a number of publications have developed protocols for directed β-cell differentiation of pluripotent cells such as embryonic stem(ES), or induced pluripotent stem(iPS) cells. By recapitulating the molecular developmental cues, decades of studies reported the modified protocols with improved efficiency by combining various growth factors and small molecules. In many cases, the differentiated pancreaticβ-cells have been shown to rescue experimentally induced diabetes animal models in vivo, however, the final step of directed differentiation into functional, mature pancreatic β-cells with sufficient quantities has yet to be achieved in vitro. Here, we summarize recent progress in the directed differentiation into the pancreaticβ-cells with a focus on two-dimensional (2D) and three-dimensional (3D) differentiation settings. We also discuss the limitations of current methods with the use of stem cells for diabetes therapy, and predict the future directions of pancreaticβ-cells differentiation.

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In this study, we herein examined the effects of the exosomes secreted from a stable hepatocellular carcinoma (HCC) cell line (HCC-exosomes) on the lumen formation of human umbilical vein endothelial cells(HUVECs). The uptake of HCC-exosomes by HUVECs and lumen formation by the HUVECs that included the exosomes were observed. The degree of lumen formation of HUVECs was dependent on the number of the HCC-exosomes. HCC-exosomes expressed a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor(VEGF)receptor. In addition, the exosomes contained several microRNAs (miRNAs)reported to exist in the serum of HCC patients. The changes in the expression levels of miRNAs associated with angiogenesis were detected in the HUVECs treated with HCC-exosomes. Moreover, the abilities of the exosomes maintained after the treatment of cryopreservation. These results suggest that the HCC-exosomes play an important role in the angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.

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The clinical utility of pharmacogenetic information during warfarin induction therapy was assessed in Asians. A prospective randomized trial in Chinese patients was performed to compare responses of warfarin between genotype-guided vs. standard dosing groups. Population pharmacokinetic-pharmacodynamic(PK-PD)analyses were conducted in an attempt to predict the time-courses of the plasma S-warfarin concentration, Cp(S), the normal prothrombin concentration (NPT)as a coagulation marker and anticoagulation response(INR). We estimated the clearance of S-warfarin, CL(S), IC50 in the relationship between Cp(S)and NPT and the non-linear indexλin the relationship between NPT and INR. Population analyses accurately predicted the time-courses of Cp(S), NPT and INR. Multivariate analyses revealed that CYP2C9 ＊3 mutation and body surface area were covariates of CL(S), that VKORC1 and CYP4F2 polymorphisms were covariates of IC50, and that baseline NPT was a covariate of λ. To clarify PK-PD factors associated with the overanticoagulation response, obtained PK-PD parameters were compared between the patient groups with and without INRB4. CL(S)andλwere significantly lower in patients with INRB4 than in those with INR＜4 (P＜0.01). These results indicate that factors associated with the reduced metabolic activity of warfarin represented by CL(S), might be critical determinants of the overanticoagulation response during warfarin initiation in Asians.

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Genetic information, which is useful for understanding and describing large interindividual differences in the pharmacokinetics and pharmacodynamics of clinically useful drugs, is continuously increasing. Genetic polymorphism in drug metabolizing enzymes and drug transporters has been focused on currently, especially for cytochrome P-450s (CYPs, such as CYP2C9, CYP2C19, and CYP2D6), and organic anion transporting polypeptides 1B1(OATP1B1)and pglycoprotein, respectively, are available in various clinical situations. Currently in Japan, pharmacogenomic(PGx)observations are incorporated into drug labels(for various types of drugs such as proton pump inhibitors(PPIs), anti-cancer drugs and NSAIDs), highly advanced medical technology(e. g., CYP2C19 genotyping for eradication of H. pylori by PPIs), and national healthcare insurance for the genotyping test(e. g., UGT1A1 genotyping in irinotecan therapy). Nevertheless, the clinical uptake of PGx knowledge has been limited. Concise and reproducible evidences, the genotyping cost, and education of PGx utility for medical staffs should be considered for the future medicine.

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Cartilage tissue is characterized by its limited regenerative potential because of the poor cellularity and perfusion by blood vessels. Replenishing cellular constituents can be one of the promising strategies for regeneration of defective cartilage. Synovial mesenchymal stem cells are superior as a cell source for cartilage regeneration to those from other tissues due to its higher colony formation rate, proliferative potential with autologous serum, and in vitro／vivo chondrogenic potentials. Approximately 60% of synovial mesenchymal stem cells placed on cartilage defects attached to the defect within 10 minute, and the addition of magnesium further enhanced the ratio of attached cells and resulted in better regeneration. Based on the accumulated evidences from basic researches, we have started clinical study for cartilage defect by transplantation of synovial stem cells arthroscopically. No problematic adverse events have been confirmed so far, and both regeneration of cartilage defect and improvement of the symptom have been recognized in most patients.

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Lymphocyte immunosuppressant-sensitivity test (LIST) is useful to predict the pharmacological efficacy for renal transplant recipients just before transplantation. Generally, IC50 rate and bottom levels based on peripheral blood mononuclear cells(PBMCs)blastogenesis are used for evaluation of the efficacy of immunosuppressants. Kuzuya et al. reported the strong positive correlation bottom level of both cyclosporine(CyA)and tacrolimus(Tac)by flow cytometry method. In our institute, we have used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)method for LIST to date. In this study, we have evaluated the relationship between IC50 level and bottom level of CyA and Tac by MTT method in 21 healthy volunteers. All the participantsʼ CyA bottom levels were below 0％, so we thought 2-sigmoid curve pattern(blastogenesis and killing cells). On the other hands, Tac bottom levels were over 0％, so we thought 1-sigmoid curve pattern(blastogenesis). We made IC50 rate and bottom levels by least squares method. We have found statistically significant correlation between CyA-Tac bottom levels(r＝0.48, p＝0.027), however, there were no positive correlation were indicated in any other combinations. In order to evaluate the immuno-compromised status, in accordance with patientsʼ clinical courses, we have compared cytomegalovirus(CMV)reactivation/infection status and these pharmacological parameters. CMV reactivation/infection was diagnosed by CMV antigenemia method. However, there was neither positive correlation between IC50 levels nor bottom levels and CMV reactivation status after kidney transplantation〔CyA(CMV＋: n＝11, CMV−: n＝2)IC50: p＝0.344, CyA bottom: p＝0.927, Tac (CMV＋: n＝9, CMV−: n＝6) IC50: p＝0.141 bottom: p＝0.629〕. In CMV＋patient, CyAʼs average trough were 144.81±60.00 ng/mL(n＝11), and Tacʼs average trough were 4.58±1.16 ng/mL(n＝9). In future, we would like to examine what kind of phrarmacological parameter is useful for evaluating adequate immunosuppression status after kidney transplantation.

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Certain kinds of antibiotics were known to have biological activities other than their original antimicrobial activity. However, precise action mechanisms of these antibiotics on immune cells of human origin have not been well-understood. In this study, we examined inhibitory effects of valinomycin, azithromycin and nigericin on the production of several cytokines from T cell mitogen-stimulated peripheral blood mononuclear cells(PBMCs)obtained from healthy subjects. Concentrations of interferonγ(IFN-γ), tumor necrosis factorα(TNF-α), interleukin(IL)-2, IL-4, IL-6, IL-10 and IL-17 in the culture supernatant of concanavalin A-stimulated PBMCs were determined with cytometric bead array procedures. Valinomycin and nigericin at 0.01-100μmol/L significantly decreased the production of these cytokines. Azithromycin also decreased the cytokine production, while its effects were weaker than that of valinomycin or nigericin. Immunosuppressive antibiotics may have benefits for prevention of acute rejection and opportunistic infection in organ transplant recipients. Thus, our present data valuate further study for the efficacies of valinomycin, azithromycin, and nigericin as immunosuppressive agents.

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Part ⅠHistory of Marginal Donors in the World／Part ⅡManagement of Extended Criteria Donors／Part Ⅲ Heart Transplantation／Part Ⅳ Lung Transplantation／Part Ⅴ Liver Transplantation／Part Ⅵ Kidney Transplantation／Part Ⅶ Pancreas Transplantation／Part Ⅷ Islet Transplantation／Part Ⅸ Small Intestine Transplantation／Part Ⅹ ABO-Incompatible Donor

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