A 72-year-old man visited our hospital in July 2009 with a major complaint of lightheadedness. Based on bone marrow aspiration, myelodysplastic syndrome (MDS), refractory anemia with excessive blast-2 was diagnosed. Complete remission (CR) was achieved after low-dose cytarabine and aclarubicin therapy. After two courses of low-dose cytarabine therapy, at the first CR, cord blood transplantation (CBT) was performed after reduced-intensity conditioning in January 2010. However, recurrence was found in September 2011. Azacitidine (AZA) was administered subcutaneously daily for either 7 or 5 days and repeated every 4 weeks at doses of 100 mg/day. During nine cycles of AZA treatment, no graft-versus-host disease was observed and no transfusions of red cells/platelet concentrate were required. As of 1 year after the relapse was detected, the patient remains alive with stable disease. As there are few reports on AZA treatment for patients with MDS who experience relapse after CBT, the efficacy of this approach remains unclear. Further clinical trials including dose, duration, and number of cycles of AZA for MDS patients who relapse after transplantation are required.

The rate of severe late adverse effects has decreased with the highly accurate administration of radiation therapy; however, the total number of patients who suffer from late effects has not decreased because of the increased total number of patients and better survival rates. Late adverse effects, occurring more than a few months after irradiation, include the extension and collapse of capillaries, thickening of the basement membrane, and scarring of tissue due to loss of peripheral vessels. The main causes of these late effects are the loss of stromal cells and vascular injury. This is in contrast to early reactions, which occur mainly due to the reorganization of slow-growing non-stem cell renewal systems such as the lung, kidney, heart, and central nervous system. In addition, the patient's quality of life is impaired if acute reactions such as mouth or skin dryness are not alleviated. Most adverse effects are radiation dose dependent, and the thresholds differ according to the radiosensitivity of each organ. These reactions occur with a latency period of a few months to more than 10 years. Understanding the clinical and pathological status, through discussion with radiation oncologists, is the essential first step. Some of the late effects have no effective treatment, but others can be treated by steroids or hyperbaric oxygen therapy. An appropriate decision is important.

Endothelial progenitor cells (EPC) play an important function in vasculogenesis and acquire a capability for vascular repair and formation as they differentiate. We examined the kinetics of EPCs from the viewpoint of EPC biology by creating oxygen-induced retinopathy (OIR) in a mouse model imitating retinopathy of prematurity (ROP), which causes pathological retinal neovascularization. Delayed differentiation, mobilization, and tissue recruitment of EPCs were obvious in the vaso-oblitelative phase, but they were promoted in the vascular proliferative state. Moreover, pathological retinal vasculature in OIR was repaired by the intravitreally transplanted definitive EPCs. These results suggest that delay of bone marrow derived EPC differentiation is a factor of morbid retinal blood vessels' formation in OIR. Aberrant kinetics of bone marrow derived EPC contribute to the formation of the retinal blood vessels, and it was proved that understanding and improving EPC differentiation can constitute a diagnostic and remedial base concept in the diagnosis of ROP.

Rett syndrome (RTT) is a neurodevelopmental disorder characterized by normal early psychomotor development followed by the loss of psychomotor and acquired purposeful hand skills and the onset of stereotyped movement of the hands and gait disturbance. The causative gene was discovered, and the disease was found to be caused by a mutation of methyl-CpG-binding protein 2. However, in many ways this clinically peculiar condition remains a mystery. I review the current status of clinical and basic research on RTT including data on the neurophysiology of the disease, neurotransmitter involvement, neuroimaging and neuropathology findings, molecular biology, animal models, regenerative medicine including ES cells and iPS cells, and other interventions and therapeutic trials.

A 75-year-old woman was admitted to our hospital with elevated serum hepatic enzyme levels. After evaluation with imaging studies, she was diagnosed as having gallbladder cancer, which had invaded the liver and hepatic artery, with lymph node metastases. The tumor was considered unresectable, and the patient received chemotherapy with gemcitabine (GEM)alone. Six months later, computed tomography(CT)indicated shrinkage of the gallbladder tumor and disappearance of lymph node metastases. Surgical resection was planned. However, liver metastasis was suspected on the basis of macroscopic findings, and the patient underwent gallbladder bed resection. Pathological examination indicated that almost all of the tumor cells in the gallbladder were viable; however, there were no tumor cells in the liver nodule and lymph node. The postoperative course was uneventful. The patient received adjuvant chemotherapy with GEM and was alive without recurrence 17 months after tumor resection. Immunohistochemical analysis showed that 80.9% of the tumor cells were positive for CD133, a cancer stem cell marker. This case illustrates a possible relationship between cancer stem cells and chemoresistance.

It is generally accepted that the difficulty in obtaining a sufficient number of functional dendritic cells (DCs) poses a serious problem in DC-based immunotherapy. Therefore, we used induced pluripotent stem (iPS) cell-derived DCs (iPSDCs) instead. If the therapeutic efficacy of iPSDCs was equivalent to that of bone marrow-derived DCs( BMDCs), then the above-mentioned problems may be solved. In this study, we generated iPSDCs from iPS cells and compared their capacity to mature and migrate to the regional lymph nodes with that of BMDCs. We adenovirally transduced the hgp100 gene, which codes for a natural tumor antigen, into the DCs and immunized the mice with these genetically modified DCs. The cytotoxic activity of CD8( +) cytotoxic T lymphocytes( CTLs) was assayed using a 51Cr-release assay. The therapeutic efficacy of the vaccination was examined in a subcutaneous tumor model. Our results demonstrated that iPSDCs equaled BMDCs in terms of their maturation and migration capacity. Furthermore, hgp100-specific CTLs were generated in mice that were immunized with the genetically modified iPSDCs. These CTLs exhibited a high level of cytotoxicity against B16 cells, which is similar to that exhibited by CTLs generated in BMDCs immunized mice. Moreover, vaccination with genetically modified iPSDCs elicited a high level of therapeutic efficacy equaling that of vaccination with BMDCs. This study clarified experimentally that genetically modified iPSDCs are equivalent to BMDCs in terms of tumor-associated antigen-specific therapeutic antitumor immunity. This vaccination strategy may therefore be useful for future clinical application as a cancer vaccine.

Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) characterized by the inability of phagocytes to produce reactive oxygen intermediates (ROIs) due to a defect in the NADPH oxidase complex. Recent studies have revealed that ROIs are involved in inflammatory signaling in phagocytes, illuminating the underlying mechanisms of hyper-inflammation in CGD. CGD patients frequently suffer from CGD-associated bowel inflammation, granuloma, and life-threatening infections. Based on the discovery of the regulatory function of ROIs in the immune response, therapeutic methods for excessive inflammation focusing on inflammatory cytokines are being developed for CGD. Although hematopoietic stem cell (HSC) transplantation (HSCT) is a curative therapy for CGD, successful transplants greatly depend on HSC source selection and the degree of matching of potential donors. Gene therapy trials for PID have been performed on over 120 patients with no HLA identical donor for HSCT, and have demonstrated clinical benefits. Genotoxicity in HSC gene therapy trials has expanded our knowledge on the mechanisms of vector-associated clonal expansion of gene-modified cells, which will advance gene therapy development using self-inactivating retrovirus and lentivirus vectors. We will discuss the complications of HSCT for CGD. We will then outline the status of gene therapy approaches in the treatment of CGD.

In this study, we have standardized experimental protocols to evaluate the possibility of using cells differentiated from human induced pluripotent stem cells (hiPSCs) in the pre-clinical studies for the drug approval processes. Cells differentiated from hiPSC, especially cardiomyocytes, neurons and hepatocytes, are expected to be used as new pharmacological and toxicological assay tools. Current preclinical test methods have limitations for predicting clinical adverse drug reactions. This is because of the so-called 'problem of species difference'. Drug-induced arrhythmia, cognitive impairment and hepatotoxicity which can't be predicted in pre-clinical studies are major causes of the high rate attrition of new-drug candidates in clinical studies and of withdrawal of products from the market. The development of new pre-clinical test methods using cells differentiated from hiPSCs would resolve these problems, in addition to solving the issue of "the replacement, refinement and reduction (3Rs)" of animal experiments. From 2010 to 2011, we surveyed companies belonging to the Japan Pharmaceutical Manufacturers Association (JPMA) and academic researchers about the usage of differentiated cells in their laboratories. We found that studies were performed using differentiated cells from different cell lines of hiPSC with laboratory-specific differentiation methods. The cells were cultured in various conditions and their activities were measured using different methods. This resulted in a variety of pharmacological responses of the cells. It is therefore impossible to compare reproducibility and ensure reliability of experiments using these cells. To utilize the cells in the drug approval processes, we need robust, standardized test methods to accurately reproduce these methods in all laboratories. We will then be able to compare and analyze the obtained results. Based on the survey, the Ministry of Health, Labor and Welfare funded our study. In our study, we standardize pharmacological methods among several laboratories, including our laboratory, to develop robust tests, using the same lot of cells, the same culture conditions, reference compounds, experimental protocols, and analysis methodology. In conclusion, to standardize robust test methods, we need a consistent supply of high-quality differentiated cells. Further, indexes to quantify the quality of the differentiated cells will be needed for their effective usage in the pre-clinical safety studies.

Regenerative medicine is regarded as innovative therapy for severe diseases and damages caused by tissue loss and functional impairment. In Japan, regenerative medicine is one of the most important subjects issued by Council for Science and Technology Policy and also referred to in Medical Innovation of New Growth Strategy. Cell/tissue-processed products are living cells, which have been manipulated or processed for the purpose of regenerative medicine, and are extensively developing. Human somatic cells, somatic stem cells, embryonic stem cells, and induced pluripotent stem cells are cell sources used for regenerative medicine. Since we lack in experiences with cell/tissue-processed products, technical development of safety and quality assessment is urgently needed. National Institute of Health Sciences has carried out a mission of Regulatory Science and worked on safety assessment of pharmaceuticals and medical devices and their guideline development. The objective of our study is to develop safety and quality assessment methods for cell/tissue-processed products derived from stem cells, based on recent progresses in life science. We are currently developing methods to evaluate products as follows; a) useful and quantitative tumorigenicity tests to detect contamination of undifferentiated and/or abnormal cells in products, b) quality assessment by gene expression analysis and detection of genetic stability in a manufacturing process, and c) analysis of quality attributes associated with propensity of undifferentiated cells to set acceptable criteria of cell banks. We will be able to provide indicators to control the quality, efficacy and safety of stem cell-processed products and support efficient and economical promotion of the products. Especially, this study would help translate stem cell science into therapeutic products to patients with severe and life-threatening diseases, consequently contributing to administrative policy of Ministry of Health, Labor and Welfare.

Current therapies for cartilage injury remain some issues such as the quality of regenerated cartilage and its invasiveness. We have been trying to develop a low invasive treatment for cartilage regeneration with synovial mesenchymal stem cells (MSCs) . Here we introduce our preclinical study with miniature pigs whose knee joints are similar to those of humans in terms of size and cartilage metabolism. Cartilage defect was created at the weight bearing area of both porcine knee joints. Synovial MSCs were transplanted by delivering a synovial MSC suspension onto the cartilage defect of the one side and the knee was kept immobilized for 10 minutes. Sequential arthroscopic and histological observations showed the contribution of synovial MSCs after transplantation, and a better hyaline cartilaginous-tissue regeneration in the MSC-treated knees than in the non-treated control knees at 12 weeks. Based on this and other preclinical studies, we have started a clinical study for cartilage regeneration with autologous synovial MSCs.

  The goal of pharmaceutical sciences is to deliver effective and safe medicinal products to patients. To achieve this goal, we need to ensure the efficacy, safety and quality of the products. Currently, many attempts are made to utilize human induced pluripotent stem cells (hiPSCs) in regenerative medicine/cell therapy. There are significant obstacles, however, preventing the clinical use of hiPSC-derived products. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. Therefore, the assessment and control of the tumorigenicity of hiPSC-derived products is essential in order to prevent tumor development by residual pluripotent stem cells after implantation. We recently examined three in vitro assay methods to detect undifferentiated cells: soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of LIN28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10(4) RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived RPE cells for their clinical use.

We participated as physiatrists in Honmou et al's study that designed to assess feasibility and safety of transplantation of autologous human mesenchymal stem cells in patients with cerebral infarction, and tried to detect improvements that were distinguishable from usual course of rehabilitation. Improvements of motor function were found in rather small functional units, such as movements in one of the fingers, toes, and a single joint of an extremity. In the methods of evaluation, Brunnstrom stage may detect gross changes, while the more detailed scales were necessary for assessment of recovery after transplantation of stem cells. In the investigator-initiated clinical trial of stem cell therapy for stroke, we are going to include fine-grained evaluation methods and investigate the most suitable technique of rehabilitation for patients after treatment.

Intravenous transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been reported to ameliorate functional deficits in several CNS diseases. The objectives of this presentation were to show feasibility and safety of cell therapy using auto serum-expanded autologous MSCs in stroke. Twelve (male and female) patients with stroke were enrolled. Cryopreserved MSCs were thawed and injected intravenously into patients over 30 min. Serial evaluations showed no severe adverse cell-related effects. In patients with cerebral infarcts, the intravenous administration of autologous MSCs appears to be feasible and safe, and merits further study as a therapy that may improve functional recovery.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which motor neuron (MN) loss in the spinal cord leads to progressive paralysis and death. Cytosolic aggregations in ALS MNs are composed of Tar DNA-binding protein-43 (TDP-43). Genetic analysis has identified more than twenty mutations of TDP-43 in ALS cases. Although accumulating evidence provides several hypotheses of disease mechanism, it is still needed to discover effective cure for ALS. We aimed to reveal cellular phenotypes in ALS MNs for identifying a drug-screening target for ALS using patient-specific induced pluripotent stem cells (iPSCs). To generate patient-specific iPSCs, dermal fibroblasts were obtained by biopsy from ALS patients carrying mutant TDP-43. The fibroblasts were reprogrammed by retrovirus or episomal vectors. Disease-specific iPSCs were differentiated into MNs expressing HB9 and SMI-32. Despite short culture period, ALS MNs recapitulated several disease phenotypes including detergent-insoluble TDP-43, shortened neurites and cellular vulnerability that observed in patient and animal models. Anacardic acid treatment reverted those phenotypes. Disease-specific iPSCs might provide a first step for drug-screening platform for ALS using patient-specific iPSCs.

Recently, several stem cell-based approaches have been considered as a novel treatment for stroke, such as delivery of non neural stem cells, stimulation of endogenous neural stem cells, and transplantation of neural stem cells. Among them, transplantation of neural stem cells is thought to have an advantage for functional recovery through neuronal replacement rather than other mechanisms. We demonstrated that human iPS cells derived neural stem cells form functional neurons and improve recovery after grafting in stroke-damaged rodents without tumor formation. Here we discuss about the potential and clinical application of iPS cells for stroke therapy.