Use of the microelectrode array (MEA) system to record spontaneous neuron activity from networks of cultured neurons has potential as a good risk evaluation method for drug-induced seizure events. Spontaneous electrical activity in neural networks consists of action potential spikes and organized patterns of action potential bursts. In both potentiated rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons, an epileptogenic response pattern manifests as a synchronized burst from spatially separated neurons. Here, we delineate how to perform MEA experiments using cultured neurons, and how to analyze the MEA data to detect drug-induced seizurogenic abnormalities. Usually, a drug's effects, as shown by MEA data, include changes in spike frequency, inter-spike intervals (ISI), burst frequency, burst duration, spikes in a burst, etc. Subsequently, seizurogenic events are evidenced by changes in synchronized burst phenotypes from spatially separated multiple channels in an MEA probe, such as a change in the cross correlation of the spike events from all channels in an MEA probe, or a change in histogram from the sum of ISI for all channels in a probe, etc. We attempted to depict an epileptogenic marker using a histogram of the sum of spikes for all channels in an MEA probe. Verification of these metrics for drug induced abnormalities is ongoing in various collaboration organizations, including the Consortium for Safety Assessment using Human iPS Cells (CSAHi), iPS Non-clinical Experiments for the Nervous System (iNCENS). Collaboration networks for the utilization of iPSC-derived cells during drug development are also summarized here.

　Toxicity prediction based on stem cells and tissue derived from stem cells plays a very important role in the fields of biomedicine and pharmacology. Here we report on qRT-PCR data obtained by exposing 20 compounds to human embryonic stem (ES) cells. The data are intended to improve toxicity prediction, per category, of various compounds through the use of support vector machines, and by applying gene networks. The accuracy of our system was 97.5-100% in three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs), and non-genotoxic carcinogens (NGCs). We predicted that two uncategorized compounds (bisphenol-A and permethrin) should be classified as follows: bisphenol-A as a non-genotoxic carcinogen, and permethrin as a neurotoxin. These predictions are supported by recent reports, and as such constitute a good outcome. Our results include two important features: 1) The accuracy of prediction was higher when machine learning was carried out using gene networks and activity, rather than the normal quantitative structure-activity relationship (QSAR); and 2) By using undifferentiated ES cells, the late effect of chemical substances was predicted. From these results, we succeeded in constructing a highly effective and highly accurate system to predict the toxicity of compounds using stem cells.

Mesenchymal stem cells(MSCs)are reported to have not only multipotency, but also anti-inflammatory and tissue repairing effects. Assays using animal models for osteoarthritis show that MSCs suppress the degeneration of cartilage. Based on these data, clinical researches and clinical trials have been performed for the treatment of osteoarthritis using MSCs. Results of these trials show the safety of the treatment, improvement of the symptoms, and findings indicating the cartilage repair. For the industrialization of the treatment using MSCs, application of allogenic cells has many advantages, while issues such as the immune reaction must be overcome.

Ligaments of joint have an essential role of proper mobilization and stabilization between bone and bone. Damage to ligaments caused by ageing, injury, and arthritis induce a disability of musculoskeletal system and has a problem to reduce our quality of life. To aim for the regeneration of ligaments, we have researched from the point of view of the developmet, found out that the transcription factor Mohawk has been important for the development and homeostasis of tendons and ligaments, and analyzed its function. Furthermore, we have also attempted to induce stem cells to tendon and ligament cells to produce type Ⅰ collagen fibers. In this article, we outline the mechanism of the development that has been reported including our approaches.

Induced pluripotent stem (iPS)cells have capacities of self-renewal and pluripotency. We have developed a method to differentiate human iPS cells toward chondrocytes, followed by the creation of cartilage tissue composed of chondrocytes and cartilage extracellular matrix. The mechanism through which tissue transplantation repairs cartilage defects involves the transplant itself constituting the repair tissue. Human iPS cell-derived cartilage has low immunogenicity and can be transplanted in an allogeneic manner. We are conducting pre-clinical tests on iPS cell-derived cartilage to verify efficacy and safety that will act as a basis for clinical tests.

The outcome of treatment-refractory and/or relapsed pediatric T-cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis of these cases remains poorly understood. Here, we report comprehensive profiling of 121 cases of pediatric T-ALL using RNA sequencing and/or targeted capture sequencing through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), which accounted for 3.9% (7/181) of the total examined pediatric T-ALL cases, had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell pre-commitment, establishment of T cell identity, and post-β-selection maturation with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and induced cell proliferation on constitutive expression in mouse stem/progenitor cells, resulting in a maturation block. Our findings highlight the unique role of SPI1 fusions in high-risk pediatric T-ALL.

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell lymphoma caused by the human T-cell lymphotropic virus type I. Patients with aggressive ATL show dismal prognoses, even with intensive dose-dense chemotherapy. Such patients often show chemo-refractoriness. Mogamulizumab (Moga) is a potent treatment option for patients with relapsed or refractory ATL. However, use of Moga before allo-HSCT could theoretically increase the risk of post-transplant complications like graft-versus-host disease (GVHD) as Moga depletes regulatory T-cells (Tregs). We retrospectively assessed the impact of Moga on post-transplant outcomes using data from a nationwide survey. Pre-transplant administration of Moga was associated with an increased risk of grade 3 to 4 acute GVHD and refractoriness to systemic corticosteroid for acute GVHD. The one-year cumulative incidence of non-relapse mortality was significantly higher in patients who were treated with Moga pre-transplant compared with those who were not (43.7% vs. 25.1%, P<0.01). The probability of one-year overall survival was also significantly lower in patients with pre-transplant Moga use compared to those without (32.3% vs. 49.4%, P<0.01). In summary, pre-transplant Moga was significantly associated with an increased risk of GVHD-related mortality, which supports the relevance of CCR4-expressing Tregs after allo-HSCT in humans.

A humanized mouse, which is efficiently engrafted human cells and tissues, is an important tool to mimic human physiology for biomedical researches. Since 2000s, severe combined immunodeficient mouse strains such as NOG, BRG, and NSG mice have been generated. They are great recipients to create humanized mouse models compared to previous other immunodeficient strains due to their multiple dysfunctions of innate and acquired immunity. Especially, the transfer of human hematopoietic stem cells into these immunodeficient mice has been enabled to reconstitute human immune systems, because the mice show high engraftment level of human leukocyte in peripheral blood (～50%), spleen and bone marrow (60～90%) and generate well-differentiated multilineage human immune cells including lymphoid and myeloid lineage cells. Using these mice, several human disease models such as cancer, allergy, graft-versus-host disease (GVHD), and etc. have been established to understand the pathogenic mechanisms of the diseases and to evaluate the efficacy and safety of novel drugs. In this review, I provide an overview of recent advances in the humanized mouse technology, including generation of novel platforms of genetically modified NOG (next generation NOG) mice and some applications of them to create human disease models for drug discovery in preclinical researches.

Common variable immunodeficiency (CVID) is the most frequently diagnosed congenital immunodeficiency and is characterized by dysfunctional antibody production. It often occurs at the age of ≥10 years. Here we reported a case of a 46-year-old man confirmed with adult-onset CVID. He was effectively treated with cord blood transplant (CBT). The patient was observed with repeated upper respiratory infection a few years back and was referred to our department owing to a marked decrease in neutrophil counts and progression of anemia. Laboratory tests confirmed hypogammaglobulinemia, but no autoantibodies were detected. Bone marrow aspiration showed a hypocellular marrow with predominantly mature lymphocytes. T-cell receptor excision circle assay revealed a reduction in T-cell neogenesis. Further, multicolor flow cytometry analysis revealed a low differentiation of B cells; subsequently, CVID was confirmed in the patient. The patient had a severe clinical course and therefore, received CBT for the treatment. After the transplantation, the hematopoiesis was restored and the serum immunoglobulin levels returned to normal. The patient exhibited a favorable clinical course. Nevertheless, there is no precise definition to establish the disease concept of CVID. Also, most of the potential cases are predominantly reported in adults. Therefore, further data on cases with CVID should be accumulated to establish the diagnostic criteria as well as treatment modalities.

Epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)are used as first-line treatment for patients with EGFR-mutated non-small cell lung cancer(NSCLC). Afatinib, a second-generation EGFR-TKI, is also effective against central nervous system(CNS)metastasis of NSCLC. However, patients treated with EGFR-TKI for many months may be at an increased risk of CNS metastasis due to the development of resistance in tumor cells to EGFR-TKI. The average period for development of resistance to EGFR-TKI is 8 to 10 months after the initiation of treatment. In the case presented herein, NSCLC showed a good clinical course in the 10 months following the initiation of afatinib;however, CNS metastasis progressed and presented unique findings on MR images. The lesions consisted of multiple cyst-like masses without gadolinium enhancement in the cerebellum and brain stem. The patient died within 2 months of the diagnosis of CNS metastasis. The resistance of tumor cells to afatinib may have occurred in the 10 months following the initiation of the treatment. Thus, CNS metastasis of NSCLC treated with afatinib may develop over a period of many months, exhibiting unique MRI findings, and deteriorate rapidly in some cases.

Adoptive cell therapy using tumor-infiltrating T cells has shown durable responses in patients with melanoma, and immunotherapy using genetically engineered T cells (TCR-T or CAR-T) is rapidly emerging as a promising treatment, especially for hematological malignancies. However, the progress is limited because of the lack of readily available good-quality human T cells. Although the efficacy of adoptive cell therapy correlates with the quality of infusing T cells, most antigen-specific T cells in patients with cancer have been exhausted. To overcome this, we have reprogrammed donor (or original) T cells to iPS cells (T-iPS) and differentiated these into rejuvenated antigen-specific cells (T-iPS-T). Moreover, iPS cells provide an unlimited source of genetically engineered T cells such as TCR/CAR-T or PD-1 knockout T cells. The iPS cells' potential for immune cell therapy is infinite.

Mesenchymal stem cells (MSCs) have received considerable attention in allogeneic hematopoietic cell transplantation because of their abilities to modulate immune responses and promote hematopoiesis. Because MSCs are capable of producing several cytokines and growth factors, they have been widely used in the treatment of graft-versus-host disease (GVHD). A number of clinical trials have demonstrated the safety and efficacy of MSC therapy for acute GVHD. Moreover, in Japan, allogeneic bone marrow-derived MSC product, TEMCELL®, was approved as a regenerative medicine for acute GVHD. Besides, MSCs can also produce bone marrow stroma and promote hematopoiesis, the co-transplantation of hematopoietic stem cells and MSCs have been efficiently performed in cord blood transplantation and HLA-mismatched transplantation to enhance engraftment and prevent GVHD. In this review, we provide an overview of clinical trials using MSCs in allogeneic hematopoietic cell transplantation and discuss the possibilities and optimization of MSC therapy.

Three biosimilar filgrastim products are currently available in Japan. Among these, the safety and efficacy of two imported drugs for autologous peripheral blood stem cell harvest (autoPBSCH) and autologous peripheral blood stem cell transplantation (autoPBSCT) have been studied widely; however, evidence of the safety and efficacy of domestically manufactured filgrastim is limited. Therefore, we compared the efficacy and safety of domestic biosimilar filgrastim (BF1, n=23) with those of originator filgrastim (OF, n=21) for autoPBSCH and autoPBSCT. Before autoPBSCH, the same median total dose of 3.3 mg filgrastim was administered to patients in the BF1 and OF groups. Median numbers of CD34-positive cells harvested did not significantly differ between BF1 (4.32×106/kg) and OF (4.75×106/kg) groups. After autoPBSCT, the median total doses of BF1 and OF used for neutrophil recovery were 2.7 and 3.3 mg, respectively. There were no significant inter-group differences in the time to bone marrow recovery, total transfusion units, hospitalization duration, overall survival at 1 year, or adverse events. Compared with OF, the cost of BF1 was considerably lower by 229,529 yen per transplantation case. Thus, the efficacy and safety of BF1 were comparable to those of OF, making BF1 an effective and economical alternative to OF.

Chondrocytes, which are originated from undifferentiated mesenchymal stem cells, play roles in skeletal development and growth in mammal, and smooth movement of joints. Endochondral ossification is necessary for skeletal development, and the multiple and complex biological events are precisely regulated by several hormones, cytokines, and their downstream signaling and transcriptional regulation. On the other hands, articular chondrocytes physiologically retains their features during a lifetime. Numerous molecules involved in endochondral ossification have been identified and investigation of the molecular mechanisms have amazingly progressed. Although GDF5 and Prg4 were identified as important molecules associated with articular cartilage development and its homeostasis, the molecular mechanisms are very unclear to date.

Skeletal muscle composes 30-40% of our body weight and is formed by multinuclear cells called myofibers. The formation of myofiber depends on the dynamic proliferation, differentiation and fusion of the myogenic progenitors during development. In the adult stage, the skeletal muscle exhibits excellent regeneration ability as well, depended on the muscle stem(satellite)cells that generate and repair myofibers. In this review, we would like to introduce ① the mechanisms of myogenic progenitor-dependent myofiber formation in myogenesis, ② the common fusion mechanism for myogenesis and muscle regeneration, and ③ the current status and prospects for clinical application utilizing satellite cells.

Cartilage injury remains one of the common clinical problems due to its limited regeneration capacities. Meniscectomy commonly performed for meniscus injury leads to osteoarthritis, but the indication of meniscus repair is limited. We have identified that synovial mesenchymal stem cells(MSCs)are superior to MSCs derived from other tissues in proliferation capacity and in vitro/in vivo chondrogenic potentials. When suspension of synovial MSCs was placed on the cartilage defect for 10 minutes, 60% of the cells attached to the defect site. Based on these basic researches, we started a clinical study for cartilage regeneration by arthroscopic transplantation of synovial MSCs. Additionally, we transplanted synovial MSCs for meniscus injury after meniscus repair. We obtained good clinical results of cartilage regeneration and meniscus healing without any serious side effects. Transplantation of synovial MSCs will be useful for cartilage or meniscus injuries.

The frequency of clonal hematopoiesis in humans vastly increases with aging. Older adults may develop one or several clones and this condition is called clonal hematopoiesis of indeterminate potential (CHIP). Recent genetic analyses have identified the genes inducing CHIP. These mutant genes are detected frequently in elderly people and this condition is a precursor of hematopoietic neoplasms. The prevalence of hematopoietic neoplasms in patients with CHIP is tenfold that in those without CHIP. Consequently, the mechanism of aging and leukemogenesis of hematopoietic stem cells is being understood. Furthermore, the efficacy of senolysis, selectively removing scenescent cells from tissues, has been demonstrated in mice. The clinical application of senolysis is anticipated shortly.