My research started in 1979 to investigate counter-regulatory secretion and action of insulin and glucagon on glucose metabolism in pancreas auto/allografts using mongrel dogs. I then had the opportunity to study pancreas islet transplantation in 1984, when I worked as a research fellow with Prof. A. P. Monaco in New England Deaconess Hospital. The first success of establishment of islet isolation method in mouse facilitated research process on the studies of islet immunology and tolerance induction. Specific results are summarized as follows. 1) Stationary digestion of the pancreas following intraductal collagenase injection is a method to isolate reproducibly high yields of islets from rodent pancreata, 2)handpicked islets are less immunogenic compared with crude islets, 3)the number of transplanted islets is critical to induce long-term graft survival in nonimmunosuppressed recipients, 4) further reducing immunogenicity could be obtained by using multiple donors with diverse histocompatibilities, 5)antilymphocyte serum has unique and potent actions on inducing tolerance through manipulation of donor and recipient immune system, 6) immunogenicity of islet graft can be altered by gamma-irradiation. Even today these results are fresh in my memory. I always believe in the words that nature does not tell a lie just as an exquisite wine served together with a matching appetizer. I conclude my talk with special thanks to distinguished researchers who have been involved in ongoing government-supported clinical islet transplantation trials in Japan(UMIN000003977).

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Organ replacement regenerative therapy, but not stem cell transplantation regenerative therapy for tissue repair, is expected to enable the replacement of a dysfunctional organ with a bioengineered organ reconstructed by three-dimensional cell manipulation in vitro. We previously reported a novel bioengineering method for reconstituting regenerated organ germs from teeth and hair follicles as a three-dimensional cell processing method(Nature Methods 2007). Recently, we further reported a successful replacement with fully functioning bioengineered organ replacement such as teeth and hair follicles through the transplantation of bioengineered organ germ into an adult environment (PNAS 2009, Nature communication 2012). Furthermore, we provided that the transplantation of a bioengineered mature organ involving teeth and hairs led to immediately perform of the full functions in vivo(PLoS ONE 2011, Scientific Reports 2012). In our current study, we achieved fully functional secretory organ regeneration such as salivary gland and lachrymal gland through orthotopic transplantation of a bioengineered secretory gland germ reconstituted using the organ germ method. This technology further demonstrates the potential for the regeneration of a wide range of defective organs. Our models provide a substantial advance for future organ replacement regenerative therapy.

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Since the current Pharmaceutical Affairs Law(PAL)system has been in force since 2005, there are lots of changes in the system, especially for the medical sector. The concept of the risk base is the key for the review process as well as license issue. Usually class Ⅱ, Controlled medical devices, were reviewed by the Government but according to the revised PAL, if the relevant technical standard has been set, those devices may reviewed by the third party which is newly appointed. We so called those medical devices as the Designated controlled medical devices. According to the revised PAL system, all medical devices required to meet the essential principle, then the checklist for those Designated controlled medical devices are published and publically available. Therefore, such as the organ preservation container except the solution has been set as this designated controlled medical device, so it may handle by the third party. Here you may find the risk base concept for the review process and some key standard such as JIS_T0993 based on ISO14971.

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Tacrolimus(TAC), an immunosuppressive agent, requires therapeutic drug monitoring(TDM)for the dose adjustment because of large individual variation in the pharmacokinetics associated with adverse effects and drug-drug interaction. This review describes the impact of drug metabolizing enzyme (CYP3A) and concomitant drugs on TAC pharmacokinetics. Gene polymorphism of CYP3A5, a metabolic enzyme for TAC on liver and intestine, is one of the factors for individual variation of blood TAC concentration when TAC is orally administered. Gastrointestinal absorption of TAC is higher in the patients with CYP3A5＊3/＊3 compared with the CYP3A5＊1 carriers. Concomitant drugs such as voriconazole (VRCZ) and lansoprazole (LAN), an inhibitor and substrate for CYP3A4, respectively, are associated with drug interaction, where blood TAC elevation is observed. Drug interaction of TAC and VRCZ is remarkable, when VRCZ is given orally. Drug interaction of TAC and LAN is frequently observed in the patients carrying CYP2C19 mutant alleles, which provide higher blood LAN concentration due to poor metabolic activity of LAN. Thus, assessing CYP activity in TAC TDM is quite useful to predict the individual variation or fluctuation of blood TAC concentration. Assay problem for the TDM is also discussed in the issue of falsely positive and negative concentration of blood TAC.

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Despite recent advances in the field of transplantation, there remains a large discrepancy between the number of patients that could benefit from transplantation and the number of available donor organs. Recently, techniques for reprogramming adult cells by gene transduction to produce pluriopotent stem cells have renewed interest in the possibility of tissue regeneration for organ repair while avoiding ethical issues associated with embryonic cell use. However, this technology needs significant development before preclinical testing. Therefore, xenotransplantation of solid organs utilizing innovative gene KO swine donors as well as use of marginal donor organs with strategies to repair damaged-organ function remains at the forefront of the search for a solution to the organ shortage. Translational research utilizing relevant pre-clinical species is necessary before the powerful therapeutic implications of this murine research can be applied in a clinical setting. CLAWN miniature swine that have been developed in Kagoshima University, were MHC inbred miniature swine. The preclinical large animals were particularly useful in assessing the effects of MHC matching on rejection and／or tolerance induction. Our research laboratory, Organ Replacement and Xenotransplantation Surgery, Advanced Biomedical Science and Swine Research, Kagoshima University, aims to develop innovative strategies to overcome organ shortages in preclinical large animal models；(1)xenotransplantation in GalT-KO pigs to non-human primates,(2)allotransplantaton, kidneys, lungs, islets and small intestine in MHC inbred CLAWN miniature swine and (3)ischemic reperfusion(IRI)models in CLAWN miniature swine. In this lecture, we introduce our recent data, especially focus on(1)preclinical IRI lung／kidney models and(2)recent attempts to reduce／inhibit IRI with CO inhalation or anti-HMGB1 antibody in CLWAN miniature swine.

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Although inhibition of activated hepatic stellate cells(HSCs)using Rho-kinase(ROCK)inhibitor well improves ischemia-reperfusion(IR)injury on steatotic liver, administration of ROCK inhibitor induced severe side effect such as hypotension. We developed the new liposomal drug carrying ROCK inhibitor which targeted to activated HSCs. ROCK-inhibitor contained by liposome inhibited activated HSCs compared with one hundredth concentration of Y-27632 in vitro study. Additionally, this new drug well accumulated hepatic stellate cells on rat with steatotic liver,therefore hypotension after intravenous injection was not observed. Because of increase of inhibition on ROCK and selectivity to activated HSCs, this liposomal ROCK inhibitor may become an effective therapy for IR injury on steatotic liver.

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ameliorate ischemia-reperfusion injury of lung grafts in a mouse model. The left lung of wild type Lewis rat was transplanted into GFP transgenic Lewis rat following 24 hours of lung preservation. GFP＋/VE-cadherin＋cell were observed in intima of small vessels in the lung grafts harvested at day 21 after transplantation. The left lung of C57BL/6 J mouse infused with MSC was synergistically transplanted into recipient mouse following 24 hours of lung preservation. Infusion of MSC reduced protein concentration and cell counts in bronchoalveolar lavage fluid collected at 6 hours after transplantation.

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Ischemia reperfusion injury(IRI)during intestinal transplantation(ITx)causes bacterial translocation and leads to morbidity and mortality. Hydrogen is noted as an antioxidant material, which can reduce IRI. Thus, we examined the effect of hydrogen-rich solution by intraluminal injection in intestinal IRI model of rats. Intraluminal injection of hydrogen-rich solution significantly reduced oxidative stress and decreased intestinal IRI. Towards clinical application, we are going to proceed to swine ITx model by using nondestructive hydrogen dissolver for intestinal graft preservation. Graft injury was ameliorated by this preservation method in spite of 18-24 hours of cold ischemic time. Hydrogen-rich solution could be a next-generation and promising preservation solution for clinical ITx.

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Beta-cell replacement is a promising therapy for type 1 diabetes. But shortage of pancreas donor is serious problem in Japan. A number of successful results have been reported about induction beta-cell from embryonic stem cells or induced pluripotent stem(iPS) cells. However, risk of teratoma formation is still an obstacle for clinical application. Here we show epidermal growth factor receptor ligands can dedifferentiate pancreatic acini derived from adult mice. Nestin positive cells can be obtained and they show stem cell like characters. In addition, recent literatures concerning this emerging field were introduced.

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In case of heart and lung procurement, multiple non-thoracic organs, such as liver, pancreas,kidney and intestine are also procured from the donor in the same time. Therefore, we need to pay attention to and collaborate with other procurement teams as well as medical staffs in the procurement hospital. To perform smooth procurement operation, pre-procurement meeting is held by procurement transplant coordinators, anesthesiologists, and transplant surgeons to discuss and determine how to manage the donor, what organ to be procured, how to procure each organ collaboratively, and how to transport each organ. In this article, standard techniques and pitfalls in procurement of thoracic organs are described.

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The organ procurement team must evaluate whether the organs such as liver, small intestine, pancreas, kidney can be procured or not by the donor conditions, past history, present illness, blood chemistry and ultrasonography. Before donor operation, multiorgan procurement teams should discuss about the order of organ procurement, crossclamp, blood drainage, perfusion technique and the division of the vessels. Donation of the abdominal organs from brain-dead donor is performed with the procedure of multiorgan donation technique. In the abdominal organs, small intestine is first extracted followed by the extraction of the liver and pancreas separately or en bloc. When the liver is solitary procured, en bloc procurement of pancreas and kidneys is popular in Japan. In the back table, the pancreas was isolated from the kidney grafts followed by the separation of both kidney grafts. Both kidneys are divided by the division of the aorta, the vena cava and the connective tissues. The kidney grafts are perfused with UW solution before packing on ice.

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Due to the critical shortage of diseased donor grafts, use of donation after circulatory death (DCD) donor is one important way of resolution. However, the ischemic damage of those DCD grafts jeopardizes organ viability during cold storage. Maintaining organ viability after donation until transplantation is important for optimal graft function and survival. However, the implementation of such strategy requires the development of novel preservation methods to recover from damages due to warm ischemia. Simple cold storage is the most widely used form of preservation in clinical practice even for DCD grafts. However, it is questionable whether this method is able to prevent deterioration of organ quality in the present era. This review describes machine perfusion preservation, an effective preservation method for DCD liver graft and discuss the concepts to reduce ischemia reperfusion injury. Although hypothermic machine perfusion may have some advantages to static cold preservation, DCD livers are exposed to the risks of hypothermia induced damage. Recently, some groups have proposed the beneficial effects of normothermic machine perfusion as a new perspective in DCD liver preservation. We introduce our own experimental results which is focused on rewarming preservation up to 22°℃ with porcine DCD liver that suffered from 60 min warm ischemia. Rewarming machine perfusion is expected to facilitate the recovery and resuscitation of DCD liver grafts.

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To improve the performance of transplantation procedures, an effective method of organ preservation is important. Recently, there have been many reports of several medical gases with organ- and/or cell-protective effects. It has become evident that carbon monoxide(CO)functions on mitochondria and controls metabolism with exerts vasoactive, anti-proliferative, antioxidant, anti-inflammatory and anti-apoptotic effects. We recently succeeded in resuscitating an extracted rat heart following 48-hour preservation using a high-pressure preservation with CO and oxygen (O2)-mixed gas(PCO＝4,000 hPa＋PO2＝3,000 hPa)at 4℃. This review mentions the current status of medical gas for organ perseveration and the high-pressure preservation method with CO and O2-mixed gas.

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We developed Ep4 solution for lung perfusion and preservation in lung transplantation in 1987. Here we studied the result of 47 clinical lung transplantations with Ep4 solution which enables 48 hours lung preservation in animal lung transplant models. In this study, the longest graft ischemic time was 14 hours and the transplantation was succeeded. On the other hand, we had an experience of primary graft failure, the ischemic time of which was less than 7 hours. We showed possibility of long preservation with Ep4 solution more than 10 hours. But various donor and recipient conditions in clinical transplantation complicated the evaluation of Ep4 solution.

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It is no doubt that a new methodology to repair grafts from extended criteria donor(ECD)is necessary in a clinical settings of organ transplantation. The superiority of organ perfusion, to cold storage, is generally accepted in ECD grafts. However, it is still difficult to resuscitate grafts by oxygenated perfusion regardless of perfusion temperature when the grafts were subjected to warm ischemic insult and subsequent cold storage. Therefore, to establish a reliable method to resuscitate DCD grafts by oxygenated perfusion, reduction of cold preservation injury is one of the important issues as well as seeking for the optimal conditions of perfusion. We reviewed the efficacy and roles of simple cold storage and organ perfusion from the viewpoints of clinical use, with special reference to DCD grafts.

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Despite many reports describing functional cell differentiation from pluripotent stem cells (PSCs), for over decades, researchers have failed to generate a complex vascularised organ as an alternative to living／cadaveric organ transplantation. To hurdle this, we focused on the most primitive process of organogenesis. By recapitulating endothelial and mesenchymal interactions during bud development, three-dimensional liver buds(iPSC-LB)were successfully created from iPSCs, resembling in vivo liver buds. Formation of functional vasculatures stimulated the maturation of iPSC-LB into adult liver-like tissues with human specific-functions in vivo. We here propose a new paradigm of(re)generative medicine through the in vitro-derived bud transplant.

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The efficacy of regenerative cell therapy as strategy of cardiac functional recovery in patients with end-stage heart failure has been recently reported, and clinical applications of autologous skeletal myoblast cell transplantation has been already started in Europe and US. We developed cell sheet technology with a temperature-responsive culture dish, which enabled to retain cell-to-cell junction, and started a clinical study of cardiac regenerative therapy using autologous skeletal myoblast cell sheet implantation.

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Toward the establishment of next generation therapy for liver diseases and diabetes mellitus,approaches based on cell transplantation and tissue engineering have been actively investigated. Our group has established several innovative approaches to create functional liver and islet tissues in vivo using isolated mature parenchymal cells(hepatocytes and islet cells). To achieve sustained survival of the cells followed by expressing higher functionality, cells need to be strategically aligned in vivo. Our group has particularly focused attention on aligning the cells to mimic microstructure of the liver and islets. This review highlights morphology and function of the ectopically engineered liver and islet tissues in animal models.

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Islet transplantation is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care. Islet transplantation has advanced significantly on several fronts, including pancreatic ductal protection, improved pancreas preservation systems, a new islet purification method, and culture／preservation of isolated islets. These techniques make it feasible to use these pancreata efficiently for islet transplantation in patients with type 1 diabetes. The promising results afforded by islet transplantation, coupled with the shortage of cadaver pancreata relative to the potential demand, have lent strong impetus to the search for new sources of insulin-producing cells. The formation of new b cells from pancreatic stem／progenitor cells(pancreatic duct cells), embryonic stem(ES)cells, and induced pluripotent stem(iPS)cells is an active area of investigation. Recent progress in the search for new sources of b cells has opened up several possibilities for the development of new treatments for diabetes.

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With the aim of hepatocyte transplantation for the patients with hyperammonemia due to congenital metabolic disorder, cell banking is in progress. In the course of cell processing, there are a number of problems that may be solved by technological approach. Isolated hepatocytes suffer from various stress such as ischemia, shear stress, cryodamage, temperature change, etc. Among them,shear stress is the most important but remains to be investigated yet. This issue must be studied by medical and technological cooperation.

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Immunosuppressive therapy is indispensable for patients undergoing liver transplantation(LT). Accumulating experiences have disclosed various issues to be solved in the use of immunosuppressant. In the present review, we summarize immunosuppressive therapy for ABO-identical／compatible LT, ABO-incompatible LT, and HCV-positive recipients. Moreover, we refer to future perspectives focusing on side effects and usefulness of immunosuppressant.

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Immunosuppressive regimen for pancreas transplantation is currently almost the same as that for kidney transplantation in Japan. A number of clinical trials, such as avoidance or withdrawal of steroids, use of mTOR inhibitor, etc, have been carried out abroad. T-cell depleting antibody has been commonly used as an induction therapy. For islet transplantation, various clinical trials in immunosuppressive therapy have been carried out after Edmonton protocol. Minnesota group reported long-term graft survival using antithymocyte globulin (rATG), TNF-a inhibitor,cyclosporine and everolimus. In Japan, a new clinical trial has started using calcineurin inhibitor, MMF rATG and TNF a inhibitor

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Recent advances in immunosuppression(IS)have improved the outcomes in pediatric liver transplantation(LT). Particular attention should be paid to several difference between children and adults in IS for LT. Better understanding of the pharmacokinetics of immunosuppressants in children is one of the most important issues, although data on this aspect are still insufficient. Controling alloreactivity by appropriate IS is crucial for the success of pediatric LT. In our series of pediatric living donor LT, younger children suffering from acute liver failure often encountered steroid-resistant rejection after LT. The selection of immunosuppressive agents must be clues to overcome steroid-resistant rejection. Epstein-Barr virus(EBV)is also one of the major topics in pediatric LT. Monitoring of EBV-viral DNA load and lymphocyte surface markers may be effective to control EBV infection after LT. Basic and clinical epoch-making data related to immune tolerance have been reported in pediatric recipients undergoing living donor LT in Kyoto, which may bring significant benefits to pediatric LT in the near future.

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Establishment of strong immunosuppressive therapy and advance in postoperative care has improved the short-and mid-term outcomes of organ transplant in recent years. However, long-term allograft outcomes remain inadequate and have reached plateau due to complications of chronic rejection, infection, cancer, cardiovascular disease and unresolved issues. The challenge is to improve long-term outcomes and the expected answer to this challenge is mTOR inhibitor. Everolimus, which was developed as a derivative of sirolimus, has been approved for the prophylaxis of rejection of kidney allograft in Japan since December 2012. It has become possible to make a new immunosuppressive therapy to improve long-term outcomes.

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The risk of developing deep-seated mycosis has been rapidly increasing, and its immediate and appropriate treatment affects the prognosis. Epidemiological surveys in Japan since 2005 have shown a decrease in the percentage of patients with mixed infection with Aspergillus and Candida and an increase in those with mixed infection with Aspergillus and Zygomycosis. During the past 10-year period, new, excellent antimycotic drugs have also been clinically used in Japan. However,deep-seated mycosis is still an intractable infection. In the future, for more effective antimycotic treatment, administration schedules based on PK／PD will be important. In particular, the metabolism of azole antimycotic drugs is mediated by CYP2C19, and the incidence of complete or partial genetic defects of this enzyme in Japanese is 4-5 times that in Westerners. In such patients, the risk of adverse effects associated with an increase in the blood antimycotic drugs concentration increases, requiring caution.

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Recent advancement of science and technology reveals the cause of disease, identifies their targets for new drugs, builds new disease models, and discovers new biomarkers. Biomarkers are indispensable for the research and development of innovative drug for new indication. Pharmacokinetic biomarkers and predictive biomarkers are discussed in translational medicine and clinical settings. Possible changes of pharmaceutical development scheme by means of biomarkers are also discussed.

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