Immunosuppressive therapy (IST) is the first-line treatment for young patients with severe aplastic anemia (AA) when a human leucocyte antigen (HLA)-matched related donor (MRD) is unavailable. Fulminant AA (FAA) is defined as AA with a complete absence of neutrophils at presentation and no response to granulocyte-colony stimulating factor (G-CSF) treatment. Here we report a 38-year-old male FAA patient who underwent allogeneic stem cell transplantation from an HLA haplotype-mismatched donor as first-line treatment. The patient had no remarkable disease history and was referred to our hospital because of a peritonsillar abscess and severe pancytopenia. Bone marrow biopsy revealed marked hypocellularity without dysplasia. His neutrophil count remained 0.0×109/l following G-CSF administration, and he was diagnosed with FAA. His siblings were not MRDs, but his sister had haploidentical HLAs. After administering a conditioning regimen, the patient received a transplant of peripheral blood stem cells donated by his sister. Neutrophil engraftment was observed on post-transplant day 16, and he experienced acute graft-versus-host disease (grade I, skin stage 1), but no other complications were observed. Hematopoietic stem cell transplantation from an HLA haplotype-mismatched related donor may be a viable option for first-line treatment of FAA when an MRD is unavailable.

Thrombopoietin (Thpo) is a hematopoietic cytokine that regulates the production of megakaryocyte/platelet lineage cells and maintains hematopoietic stem and progenitor cells (HSPCs). While Thpo directly stimulates the proliferation of HSPCs, it also maintains HSCs in quiescence to form a reserve pool of HSCs in the bone marrow. Moreover, Thpo activates mitochondria and induces HSC differentiation to megakaryocyte/platelet lineage cells. Being void of instigating anti-Thpo antibody formation in vivo, the use of Thpo receptor agonists (Mpl agonists) transcends the use of recombinant Thpo in the treatment of immune thrombocytopenia. Since its invention, the therapeutic indication of Mpl agonists has extended to the treatment of bone marrow failure in aplastic anemia. As the clinical application of Mpl agonists expands, a detailed investigation of the function and effect of Mpl agonists on physiological HSCs and bone marrow failure is necessary.

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which T cells are regenerated from induced pluripotent stem cells (iPSCs) that were originally derived from T cells, and succeeded in regenerating cytotoxic T lymphocytes (CTLs) specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. We recently have extended our strategy to solid tumors. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of renal cell carcinoma, demonstrating the feasibility of our strategy against solid tumors.

NKT cells are innate lymphocytes that express an invariant T cell receptor. Since activated NKT cells exert strong anti-tumor responses, NKT cells have been intensively studied for the purpose of their application to cancer immunotherapeutic approaches. Although human peripheral blood contained a very low fraction of NKT cells, and decreased number of NKT cells was also demonstrated in cancer-bearing patients, peripheral blood NKT cells can be activated by ligand-pulsed antigen presenting cells, and can produce a large amount of interferon-γ upon activation. The clinical trials of adoptive transfer of autologous NKT cells were already performed in patients with non-small cell lung cancer, and with head and neck cancer at Chiba University to show its effectiveness and limitation. Meanwhile, RIKEN reported NKT cell regeneration using iPS cell technology in mice, and subsequently established a protocol for regenerating NKT cells from human peripheral blood NKT cells using iPS cell technology. It was confirmed that the iPS cell-derived NKT cells (iPS-NKT) have sufficient expansion c apacity and potent direct and indirect cytotoxic activity in the humanized mice models, which suggests their therapeutic competence. We are currently planning an investigator-initiated clinical trial of allogeneic iPS-NKT cell therapy for head and neck cancer.

Seminal studies by Dr. Shinya Yamanaka revealed that reprogramming technology was able to convert differentiated somatic cells to self-renewing and pluripotent stem cells. Although reprogramming process does not require changes in the genome information, cellular reprogramming elicits dynamic changes of epigenetic regulation. Therefore, reprogramming technology is a powerful tool for the modifying epigenetic regulation. Previous studies have reported that epigenetic regulation plays a critical role on both the development and maintenance of cancer cells. Taking advantage of reprogramming technology, previous studies have actively modified the epigenome of cancer cells and revealed the importance of the coordinated interactions between genetic abnormalities and epigenetic regulation in cancer cells. In this review, we describe advances and challenges in the use of reprogramming technology for studying cancer biology.

We have developed cell sheet-based regenerative medicine, in which cell sheets are fabricated with temperature-responsive culture surfaces. We succeeded in clinical translation and large animal model experiments of cell sheet-based regenerative medicine to treat various complications of cancer therapy including esophageal stricture after esophageal early cancer endoscopic submucosal dissection(ESD)and lung air leaks. We would like to continue development of cell sheet-based regenerative medicine to treat frail, sarcopenia, and cancer cachexia after surgery, chemotherapy, and radio therapy by supplying stem cells and paracrine effects.

Many studies in recent years have reported cell therapies using embryonic stem cells, induced pluripotent stem cells, and bone marrow-derived mononuclear cells for cerebral ischemia. However, obtaining these cells is challenging, and these cell therapies require complicated procedures to prepare cells for administration. Notably, peripheral blood mononuclear cells (PBMCs) are a useful cell source for clinical applications because cell collection is easier. In this review, we report the therapeutic effects of PBMCs preconditioned by oxygen-glucose deprivation (OGD-PBMCs) on cerebral ischemia. Cell therapies using tissue-protective OGD-PBMCs might be a simple and ideal therapeutic strategy against ischemic stroke.

Graft-versus-host disease (GVHD) is a potentially life-threatening complication associated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Although prophylaxis and GVHD treatment using immunosuppressants are essential for a successful allo-SCT, profound immunosuppression could lead to an infection and/or the recurrence of malignant diseases. Recently, the concept of tissue tolerance has emerged. This concept has been identified as a means to suppress GVHD by relying on tissue-intrinsic mechanisms that are independent from those underlying immune tolerance. Thus, GVHD prophylaxis and treatments targeting the mechanisms that promote tissue tolerance could help suppress GVHD and maintain leukocyte-mediated immune responses against tumors and infectious pathogens. Epithelial regeneration from LGR5-positive intestinal stem cells and epithelial maintenance mediated by metabolites from the intestinal microbiota are representative mechanisms that promote tissue tolerance in the gut. However, these protective mechanisms are weakened by GVHD and conditioning regimens. This review focuses on the pathophysiology of acute GVHD and tissue-intrinsic mechanisms that promote tissue tolerance.

Expanded human bone marrow-derived mesenchymal stromal/stem cells (MSC) have been used in the treatment of acute graft-versus-host disease (GVHD). It is currently accepted that the use of allogeneic off-the-shelf MSC has therapeutic efficacy with no apparent serious early-onset adverse effects; however, further development would be needed to overcome the current situation of MSC therapy for intractable GVHD. In the emerging recognition of the importance of extracellular vesicles (EV) as modulators of cell-cell communication physiologically and pathologically, we recently revealed that human bone marrow MSC-derived EV ameliorate GVHD clinically and pathologically through the preservation of peripheral naïve T-cell populations in a murine model. In this article, we summarize future perspectives on MSC-based therapy for GVHD.

Dimethyl sulfoxide (DMSO) is used as a cryoprotectant for peripheral blood stem cells (PBSC) preservation. Dimethyl sulfide (DMS) is a metabolite of DMSO secreted through patients' breath after PBSC infusion. It possesses malodor causing an unpleasant environment. We evaluated the efficacy of a photocatalyst environment purifier, which has the potential to lyse toxic substances, in reducing DMS malodor. High DMS concentration in the air after PBSC infusion rapidly decreased after operating the device. Our results suggest that photocatalytic reaction has the potential to reduce the DMS odor associated with PBSC infusion.

Various artificial cells and artificial tissues can be generated from induced pluripotent stem cells (iPS cells). There is now an urgent need to standardize the quality evaluation and management of iPS cells. Recently, artificial intelligence (AI) technology such as machine learning is providing evaluation method for the quality of iPS cells and iPS cell-derived somatic cells based on optical microscopy. Light, which is the principle of optical microscopy, has an interesting and important feature. There are various kinds of interaction between light and molecule, and the scattered light includes internal information of the molecule. Raman scattering inheres all the vibration mode of molecular bonds composing a molecule, and second harmonic generation (SHG) light, which is one of second-order non-linear scattering light, is derived from electric polarizations in the molecule, in other words, carries structural information within the protein. While states of a cell are usually defined by protein/gene expression patterns, we have proposed to apply Raman spectra for cellular fingerprinting as an alternative for identifying the cell state, and now succeeded in predicting gene-expression of antibiotic resistant bacteria in combination with machine learning technology. Meanwhile, SHG microscopy has been used to visualize fiber structures in living specimens, such as collagen, and microtubules as a label-free modality. By utilizing the feature that SHG senses protein structure change, we developed a new method to measure actomyosin activity in cardiac cells. The most important advantage of the use of the scattering light is their non-labeling and non-invasive capability.

Deep learning technology is rapidly advancing, and is now used to solve complex problems. induced pluripotent stem cells (iPSCs) can be used for several purposes such as regenerative medicine, disease modeling study and drug screening. It is inevitable to identify iPSC-derived differentiated cells in microscopy for any use. Here, we used deep learning to establish an automated method to identify endothelial cells derived from iPSCs, without the need for immunostaining or lineage tracing.

It is reported that the incidence of atrial arrhythmias has been increasing year by year and it might increase from now on. Although not only aging but pharmaceutical drug treatments might relate to atrial arrhythmias, experimental method to detect drug-induced atrial arrhythmias has not been established so far. Therefore, we induced differentiation of atrial-like cardiomyocytes from human induced pluripotent stem (iPS) cell, and clarified their characteristics and verified their dug responsiveness. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their character was compared to RA-untreated cardiomyocytes. In gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have the characters of atrial or ventricular like cardiomyocytes, respectively. In addition, it was also confirmed that atrial-like cardiomyocytes induced reentry-like conduction disorder, which is atrial arrhythmias. Furthermore, as a result of examining the responsiveness of various ion channel inhibitors using these cells, the inhibition of ultra-rapid delayed rectifier potassium current (IKur) specifically existed in atrial muscle induced prolongation of PWD30cF (membrane potential duration at 30% depolarization corrected by Fridericia formula) only in atrial-like cardiomyocytes. In addition, ventricular-like cardiomyocytes alone exhibited an early after depolarization by treatment of rapid rectifier potassium current (IKr) inhibitor which induced ventricular arrhythmia in clinical situation. Based on above evidences, current evaluation systems using human iPS cell-derived atrial-like cardiomyocytes might be a valuable tool for drug-induced atrial arrhythmias.

Disease-specific iPS cells have been considered and used as platforms for disease modeling and drug discovery for intractable diseases. In the field of cardiovascular medicine, iPS cells have been generated from patients with heart diseases including inherited cardiomyopathy. The disease-specific iPS cells showed the certain parts of phenotype of the disease on culture dishes in in vitro systems, but the cells do not necessarily recapitulate patients' clinical properties, particularly those of physiological-/pathophysiological aspects. The point should be solved to establish disease reliable platforms. The discrepancy may be attributed to the lack of developmental process during culture procedure. To settle the problems, various techniques have been attempted such as culture dishes with specific structures. This review describes issues to be solved to recapitulate "heart diseases on culture dishes", introducing the phenotype of disease specific iPS-cells from patients with cardiomyopathy.

Human induced pluripotent stem cell-derived neurons (hiPSC-neurons) have potentials to improve the predictability of the adverse effects (AEs) in the human central nervous system (CNS). We have succeeded in reproducing the human neural networks stably on dish. This is important because most of the CNS AEs were caused not by the neuronal death but by the abnormal neural network activities. Our group have been attempting to establish the in vitro assay system to detect abnormal neural network activities by microelectrode array system (MEA). Furthermore, the algorithm that can determine the synchronized burst firing (SBF) objectively was developed in the collaboration of our iPS Non-clinical Experiments for Nervous System (iNCENS) project and Consortium for Safety Assessment using Human iPS Cells (CSAHi). iNCENS and CSAHi are also involved in the international Life Sciences Institute (ILSI): Health and Environmental Sciences Institute (HESI) HESI NeuTox MEA sub team, and are participating in the pilot study using 12 compounds. In the big flow to develop physiological CNS safety assessment system, we have to show Japan initiative based on our innovative iPS technology we have developed so far.

Development of an in vitro drug efficacy and safety assessment based on the function of the neural network is required in preclinical studies. A microelectrode array (MEA), which can simultaneously measure the electrical activity of a human induced pluripotent stem cell-derived neural network at multiple points, is an effective assay system. In this study, we focused on seizure liability and clarified the responsiveness to seizure-positive compounds depending on the excitatory and inhibitory balance (E/I balance) of each evaluation sample. In addition, it has been shown that multivariate analysis and AI analysis methods are effective for detecting toxicity and predicting drug mechanisms of action. The future challenge is to approach in vitro-to-in vivo extrapolation (IVIVE) for in vitro assessment. An assessment using brain organoids and low-frequency component analysis, in which enable comparison with in vivo ECoG are effective approaches to IVIVE. MEA can be applied to the central nervous system and the peripheral nervous system; therefore, MEA is also expected to become a highly useful assessment tool for peripheral neuropathy.

In the drug development in pharmaceuticals, development of drugs may be discontinued due to the toxicity and clinical side effect, therefore, safety assessment is one of the important factors in drug development. Consortium for Safety Assessment using Human Cells (CSAHi) has been launched for developing and standardizing a toxicity evaluation system for development of drug using human iPS cell differentiated cells. CSAHi focuses on hepato-, cardio-, and neuro-toxicities as important toxicity organs which are attributed to the causes of discontinuation of drug development. In neurotoxicity, seizure is an important finding because of high frequency expression in nonclinical. Multi-electrode array (MEA) systems have recently attracted attention as useful for evaluating seizure risk because they can non-invasively measure the electrophysiological activities of neural networks. We are evaluating the electrophysiological responses to several seizure compounds using MEA in cultured hiPSC-derived neurons. It is important to establish an analytical method to detecting seizure-like activities. We have focused the establish of the effective analysis parameters for detecting seizure risk. We identify to be separate the responses between seizure-positive and seizure-negative compounds using principal component analysis of 10 analysis parameters. In addition, we could separate the mechanism of action of the seizure-positive compounds by principal component analysis and cluster analysis using 10 parameters. It is considered that principal component analysis or cluster analysis could not only assess the seizure risk but also classify mechanism of action by in vitro MEA system using human iPS cell-derived neurons.

Multiple myeloma (MM) is among the most intractable of malignancies and is characterized by uncontrolled growth of malignant plasma cells in the bone marrow (BM). Elucidation of the mechanisms underlying cell adhesion-mediated drug resistance (CAM-DR) may prolong remission and ultimately improve the survival of MM patients. Toward this goal, we identified trimethylation of histone H3 at lysine-27 (H3K27me3) as a critical histone modification associated with CAM-DR. Cell adhesion counteracted drug-induced hypermethylation of H3K27 via inhibiting phosphorylation of enhancer of zeste homolog 2 (EZH2), and promoted sustained expression of anti-apoptotic genes. In addition, we found that CD180, a non-canonical lipopolysaccharide (LPS) receptor, was markedly up-regulated in response to adherence and/or hypoxic conditions. Bacterial LPS enhanced the growth of MM cells both in vitro and in vivo, correlating with expression of CD180. Promoter analyses identified Ikaros (IKZF1) as a pivotal transcriptional activator of the CD180 gene; expression of CD180 was activated via cell adhesion- and/or hypoxia-mediated increases in IKZF1 expression. Administration of lenalidomide prevented the LPS-triggered activation of MM cells by targeting CD180. Taken together, our results suggest that lenalidomide-mediated prevention of LPS-triggered disease progression may be an effective means for prolonging survival in patients with MM.

Ring sideroblasts show abnormal mitochondrial iron accumulation, and their emergence in the bone marrow is a characteristic of sideroblastic anemias (SAs). SAs are a group of heterogeneous congenital and acquired disorders. Congenital SA is a rare disease caused by gene mutations involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur after exposure to certain drugs or alcohol and due to copper deficiency (secondary SA). Furthermore, SAs are associated with myelodysplastic syndrome (idiopathic SA), strongly correlating with specific somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery. Recent reports have indicated that common defects in iron/heme metabolism underlie in the mechanisms of ring sideroblast formation in congenital and acquired SAs. Current understanding of SA pathophysiology, including the mechanisms of ring sideroblast formation, is discussed in this review.

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.