Skeletal muscle atrophy reduces quality of life and increases mortality. However, there are few available drugs for the treatment of muscle atrophy. Recently, cell signaling pathways involved in skeletal muscle atrophy or hypertrophy have been determined. To develop drugs for skeletal muscle atrophy, we have studied compounds which modulate pathways of myogenic differentiation, a pivotal step for the maintenance of skeletal muscle mass. First, we examined a K+ channel opener on myogenic differentiation, since hyperpolarization is a trigger for skeletal muscle differentiation. 5,6-Dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), an opener of the small/intermediate conductance Ca2+ activated K+ (SKCa/IKCa) channels, increases myogenic differentiation in C2C12 mouse skeletal myoblasts. This effect was inhibited by TRAM-34, an IKCa channel blocker. This suggests that K+ channels in skeletal muscle stem cells are potential targets for an anti-muscle atrophy drug. Next, we searched for drugs which prevent sepsis-induced muscle atrophy. Lipopolysaccharide (LPS), an inducer of sepsis, attenuates myogenic differentiation in C2C12 myoblasts. LPS also increases the protein expression of myostatin and activates NFκB during differentiation. The TLR4 signal inhibitor TAK-242, and an anti-TNFα neutralizing antibody, reduce these inflammatory responses. Our data suggest that LPS inhibits myogenic differentiation via the NFκB/TNFα pathway. This pathway may be involved in the development of muscle wasting caused by sepsis.

　Tissues of the small intestine are crucial to understanding drug disposition because these tissues regulate the bioavailability of drugs. However, no evaluation system is currently available for precise and comprehensive analysis of intestinal pharmacokinetics. To address this, functional intestinal epithelial cells were generated from human induced pluripotent stem (iPS) cells for use in pharmacokinetic studies. An improved intestinal differentiation method was established by screening a variety of small molecule compounds against cells during differentiation. The mRNA expression levels of intestinal markers, drug transporters, and CYP3A4 were found to increase following treatment with compounds that act as inhibitors of mitogen-activated protein kinase, DNA methyltransferase, and transforming growth factor-β. Therefore, we inferred that these compounds enhanced differentiation into intestinal epithelial cells. The differentiated intestinal epithelial cells in the presence of these compounds possessed drug-metabolizing enzyme activities, such as those of CYPs, UDP-glucuronosyltransferase, and sulfotransferase. In addition, these cells had the ability to induce CYP3A4 in the presence of 1α,25-dihydroxyvitamin D3. The differentiated intestinal epithelial cells seeded on cell culture inserts formed loose-tight junctions, similar to those in the human small intestine, rather than Caco-2 cells. The cells exhibited polarity, such as apical and basal sides. We also demonstrated that the uptake and efflux transport activities in the cells occurred via peptide transporter and breast cancer resistance protein, respectively. Taken together, it was suggested that human iPS cell-derived intestinal epithelial cells are pharmacokinetically functional, and represent a promising model system for pharmacokinetic studies of drug candidates.

Post allogeneic hematopoietic stem cell transplantation, CD4＋CD25＋Foxp3＋ regulatory T cells (Tregs) play a central role in the maintenance of self-tolerance and immune homeostasis. In recent years, various immune-modulating agents, including cyclophosphamide, mogamulizumab, and antibodies to inhibitory co-stimulating molecules (CTLA-4 and PD-1), have been used to control donor-derived immunity or host-tumor burden during the peritransplant period; however, the underlying impact of these agents on Treg homeostasis is yet to be characterized. In particular, recent retrospective analyses suggested that blockade (an inhibitory molecule) before and after HSCT was associated with abnormal Treg homeostasis and the development of severe acute GVHD and other immune-mediated complications. Our recent studies, using murine model and clinical sample analyses, aim to define the effects of immune-modulating agents on the reconstitution of each lymphocyte subset, particularly in terms of Treg homeostasis. An enhanced understanding of the reconstituting mechanisms and the target-specificity of post-HSCT GVL response combined with novel immunotherapy will enable the development of a safe and efficient therapeutic strategy.

Multiple myeloma (MM) cells acquire dormancy and drug resistance via their interaction with bone marrow stroma cells (BMSCs) in a hypoxic microenvironment. In this study, we found a positive expression of CD180/MD-1 complex (a non-canonical toll-like receptor) on MM cells, which was markedly up-regulated under adherent and/or hypoxic conditions. Bacterial lipopolysaccharide (LPS) enhanced the growth of MM cells via the activation of MAP kinases, an effect which showed a positive correlation with the expression levels of CD180. LPS administration significantly increased CD180/CD138 double-positive cell number in a murine xenograft model after the inoculation of MM cells directly attached to BMSCs. Notably, the shRNA-mediated knockdown of CD180 terminated the LPS response in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the CD180 gene, whose transcription was activated via cell adhesion and hypoxia by increasing Ikaros expression and its binding to the promoter region. Pharmacological targeting of Ikaros with lenalidomide ameliorated the response of MM cells to LPS in a CD180-dependent manner in vitro and in vivo. CD180/MD-1 pathway may represent a novel mechanism for the regulation of the growth of MM cells in BM milieu and may serve as a therapeutic target to prevent the regrowth of dormant MM cells.

Paroxysmal nocturnal hemoglobinuria (PNH) arises as a consequence of clonal expansion of hematopoietic stem cells that have acquired a somatic mutation in the PIGA gene. The resulting hematopoietic cells have deficiencies in the GPI-anchored complement regulatory proteins CD55 and CD59, which account for the intravascular hemolysis that is the primary clinical manifestation of PNH. Thromboembolism is a major cause of morbidity and mortality in PNH, particularly in Caucasian patients. In a previous report on the clinical course of PNH patients in the United States and Japan, we showed that thrombosis was significantly more prevalent in white PNH patients than in Asian PNH patients. The pathophysiological mechanisms underlying thrombosis in PNH have not been fully clarified, and multiple factors are likely to be involved. Eculizumab, a humanized monoclonal antibody, targets the terminal complement protein C5 and inhibits terminal complement-mediated hemolysis associated with PNH. Brodsky et al. reported that eculizumab treatment reduces the risk of clinical thromboembolism in patients with PNH. These facts strongly suggest that the main cause of thrombosis in PNH is complement activation and/or hemolysis. In this review, the pathophysiology of thrombosis in PNH is discussed in the context of observations in PNH patients treated with eculizumab.

Extranodal natural killer (NK) /T-cell lymphoma (ENKL), nasal-type, is an aggressive lymphoma that is relatively common in Asia and is resistant to anthracycline-based chemotherapy. Antigen-specific cytotoxic T-lymphocyte (CTL) therapy can induce durable remission in selected tumors, such as melanomas and virus-related tumors. ENKLs are commonly infected with the Epstein-Barr virus (EBV) ; therefore, CTL therapy can be effective for these lymphomas. However, CTLs that are continuously exposed to viral or tumor antigens usually get exhausted. Antigen-specific CTLs generated from iPSCs have a higher proliferative capacity and longer telomeres than original CTLs and are functionally rejuvenated (rejuvenated T cell, rejT). For clinical translation, the tumorigenic potential of iPSCs and the malignant transformation of differentiated iPSCs are major safety concerns. To address these issues, we introduced inducible caspase-9 (iC9) -based safeguard system into iPSCs. iC9-rejTs exert strong anti-tumor effects against EBV-infected tumors in vivo, and the iC9 suicide gene system provides a reliable safeguard for rejT therapy. We initiated a preclinical study using this novel immunotherapy for EBV-associated lymphoma. We believe that rejT therapy can be a potential safe approach as an "off-the-shelf" therapy for refractory ENKL in the future.

All blood cells are produced by hematopoietic stem cells (HSCs) in the adult bone marrow. However, prior to the emergence of the first HSCs at embryonic day (E) 11, mouse embryos present several hematopoiesis waves, such as primitive hematopoietic cells, definitive erythro-myeloid progenitors, lymphoid potent cells, and multi-potent progenitors. Non-HSC-derived hematopoiesis is called HSC-independent hematopoiesis and has been considered a transient wave that is diminished after birth. Recent reports have shown that tissue-resident macrophages are derived from the yolk sac (YS) and are HSC-independent. Similarly, the presence of a developmental pathway for innate-like B-1a cells, independent of fetal liver HSCs, has been shown, although the question of whether B-1a cells are produced by fetal liver HSCs remains controversial. The present review focuses on the process of HSC development and introduces recent information regarding HSC-independent tissue macrophages and B-1 lymphocytes.

Stem cells are self-renewing, either multipotent or unipotent, and they offer opportunities for stem cell-based therapies in the clinical setting. The mechanism underlying the division patterns of hematopoietic stem cells (HSCs) is one of the most fundamental biological questions. However, to date, analyses of individual HSC cell fate decisions have been restricted by the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate. Comprehensive research accompanied with genetic models, metabolomics analyses, and single-cell approaches have highlighted the critical contributions of metabolic control to HSC homeostasis. Consequently, the roles of mitochondrial metabolism in the HSC division symmetry have become a central focus of the current research. Nevertheless, we are only beginning to comprehend the metabolic requirements of stemness. This review summarizes the recent advances in our understanding of the intriguing relationship between mitochondrial metabolism and HSC fate decisions. In addition, this study highlights our recent findings regarding the contributions of the mitochondrial quality control by autophagy to HSC division balance. Elucidation of the metabolic cues governing HSC fate decisions should lead to new techniques of metabolic manipulation, which can shift the division balance of HSCs and offer effective targets in strategies against leukemia and will, thus, be of high clinical importance.

A 73-year-old woman diagnosed with symptomatic multiple myeloma (MM; IgG-κ type, D&S: IIIA, ISS: 2) was administered bortezomib plus dexamethasone (BD) therapy. Post BD therapy, although autologous hematopoietic stem cell transplantation and thalidomide, lenalidomide, and melphalan/prednisolone/thalidomide (MPT) therapies were also performed, the patient remained unresponsive. However, the disease relapsed, and she eventually developed pantalgia. Therefore, the patient was admitted to our hospital and was administered pomalidomide and dexamethasone (Pd) therapy. The serum amylase (AMY) and urine AMY levels were 6,329 and 6,098 IU/l, respectively, which were salivary gland-type amylase (S-AMY). Notably, the markedly high levels immediately decreased after the first course of the Pd therapy; additionally, the pantalgia also disappeared. The S-AMY level in the supernatant from cultured bone marrow mononuclear cells was higher than that observed in a normal control. In addition, AMY was high when MM previously relapsed, suggesting the presence of AMY-producing MM. Although AMY-producing MM was first reported by Hata et al. in 1988, few cases have been reported in the new-drug era. In conclusion, AMY-producing MM frequently, including in our case (as the patient was refractory to treatment), is difficult to treat. However, our patient positively responded to the novel next-generation drugs such as pomalidomide and carfilzomib.

Three-dimensional (3D) cardiac myoblast tissues derived from iPS cells were constructed by cell coating technology with nanometer-sized extracellular matrix. Vascularized 3D-cardiac tissues were also fabricated by employing cardiac endothelial cells. These 3D-cardiac tissues are expected to apply to pharmaceutical assays.

Monocytes give rise to macrophages and dendritic cells (DCs) under steady-state and inflammatory conditions, thereby contributing to host defense and tissue pathology. Inflammation triggers the differentiation of tissue-infiltrating monocytes into monocyte-derived macrophages and DCs, which are associated with homeostatic host defense reactions and inflammatory diseases. In mice, monocytes are divided into classical Ly6chi- and non-classical Ly6clo-expressing subsets. Ly6clo monocytes are present only in the blood; however, Ly6chi monocytes are found in blood and other tissues (wherein they differentiate into macrophages and DCs). In this context, most Ly6clo monocytes are derived from Ly6chi monocytes. In humans, monocytes comprise major CD14+CD16- and other CD14+CD16+ and CD14loCD16+ monocytes. A monocyte lineage-restricted common monocyte progenitor (cMoP) was previously identified in mice; herein, we introduce human cMoP, which was identified as a CLEC12AhiCD64hi subpopulation of conventional granulocyte-monocyte progenitors (cGMPs) in umbilical cord blood and bone marrow. The human cMoP produced monocyte subsets without showing any potential of differentiating into myeloid or lymphoid cells ex vivo. Within the cGMP population, we also identified revised GMPs that completely lacked DC and lymphoid potential, which sequentially produced cMoPs, pre-monocytes, and monocytes. Collectively, our findings enhance the current understanding of human myeloid cell differentiation pathways.

Under stress conditions such as infection, inflammation, and hematopoietic recovery following chemotherapy or transplantation, the hematopoietic system is required to meet the increasing demands, especially from myeloid cells. Therefore, an understanding of the molecular mechanism underlying stress hematopoiesis is clinically imperative. We previously showed that C/EBPβ, which is a transcription factor required for emergency granulopoiesis, plays a pivotal role at the level of hematopoietic stem/progenitor cells under stress conditions. Upon exposure to stress, the C/EBPβ protein is upregulated in the hematopoietic stem cells. A close examination of C/EBPβ knockout mice revealed that C/EBPβ regulates the proliferation and differentiation of hematopoietic stem cells at the cost of the self-renewing activity. Further elucidation of the functions and regulation of C/EBPβ in hematopoietic stem cells will facilitate an understanding of stress hematopoiesis.

Tyrosine kinase inhibitors (TKIs) have markedly improved the prognosis of patients with chronic-phase chronic myeloid leukemia (CML). However, the development of a novel therapeutic strategy that can target and eradicate CML stem cells remains an important requirement to achieve a complete cure for CML because these stem cells cause relapse and drug resistance even in patients receiving TKI treatment. Interferon-α (IFNα) has long been used for the treatment of chronic-phase CML, and its efficacy is now being re-evaluated as a therapeutic option. Some clinical studies have demonstrated the additive efficacy of IFNα in CML patients treated with TKI. However, the mechanism of action is not fully understood. In this review, we introduce our recent findings on the effects of IFNα on CML stem cells via the transcription factor CCAAT/enhancer binding protein β, which regulates stress-induced hematopoiesis.

Acquired aplastic anemia (AA) is an autoimmune disease caused by T cells specific to hematopoietic stem cells (HSCs). The presence of HLA allele-lacking leukocytes due to uniparental disomy of the short arm of chromosome 6 (6pUPD) or allelic mutations strongly indicates the involvement of such cytotoxic T cells in the pathogenesis of AA. Attempts to improve treatment outcomes by intensification of immunosuppressive therapy (IST) have been unsuccessful. Eltrombopag (EPAG), a thrombopoietin receptor agonist, has recently emerged as a novel therapeutic agent for AA. EPAG directly acts on HSCs and stimulates proliferation, thereby achieving remission in approximately 40% AA patients refractory to IST. However, some cases develop chromosomal aberrations during treatment. Because somatic mutations are common in patients with AA, verifying whether EPAG induces clonal proliferation or evolution of mutant HSCs is critical.

We report the case of a 42-year-old female with neurosarcoidosis who was hospitalized in year 2017 for gait disturbance. In 2011, she suddenly had vertigo that lasted for a few days. In 2013, she noticed left hemiplegia. A brain MRI revealed an acute infarction on the right side of the upper pons extending longitudinally from the ventral surface. In 2017, she again had left lower limb paralysis. A Brain MRI showed another infarction on the right side of the mid-pons. Hydrocephalus and brainstem atrophy were also noted. The patient was referred to our hospital. Upon neurological examination, she presented with down beat nystagmus, muscle weakness on the left side, and a broad-based spastic gait. CSF findings included an increased number of cells and protein levels with decreased glucose levels. A contrast-enhanced MRI revealed basilar meningitis causing hydrocephalus. A contrast CT scan revealed inguinal lymph node swelling, and scintigram found gallium accumulation. We diagnosed sarcoidosis via a lymph node biopsy. We speculate that chronic basilar meningitis obstructed the patient's branching penetrating arteries inducing infarction together with obstruction of the spinal fluid flow causing hydrocephalus and cerebral atrophy.

Ten years have passed since Yamanaka et al. reported the establishment of human iPS cells, which became one of the triggers to make national efforts in Japan to promote research and translation of regenerative medicine and cell therapy (regenerative medicine etc.). However, it has been unreasonable in many cases to directly apply the existing regulation to cells processed for the purpose of use in regenerative medicine etc., which have quite different properties from conventional pharmaceuticals and medical devices. For this reason, in recent years, drastic reforms of various regulations of medical and pharmaceutical affairs have been vigorously pursued for efficient translation of regenerative medicine etc. Regarding medical affairs, "The Act on the Safety of Regenerative Medicine" was established for the purpose of providing safe regenerative medicine etc. to patients promptly and smoothly, establishing standards for regenerative medicine providing agencies and cell culture processing facilities. Regarding pharmaceutical affairs, a new chapter and an early approval system (conditional/term-limited approval system) for "regenerative medical products", which consists of cellular and gene therapy products, were introduced into "Pharmaceuticals and Medical Devices Act", a revised and renamed version of "Pharmaceutical Affairs Law". In this review article, we overview the current perspectives of regulations and challenges for translation of regenerative medicine etc. in Japan.

A current broad spectrum of genomic studies on acute myeloid leukemia (AML) has demonstrated that a gene encoding for DNA methyltransferase, specifically DNA methyltransferase 3 alpha (DNMT3A) is frequently mutated. However, DNMT3A variants are present in elderly healthy individuals and patients with AML in complete remission, which suggests that DNMT3A mutations may contribute to pre-leukemic clonal hematopoiesis. Although DNMT3A mutation has been thought to play a pivotal role in AML pathogenesis through the loss of DNA methylation functionality, other potential disease-related mechanisms are poorly understood. We identified that DNMT3A Arg882 mutation has two distinct mechanisms for developing clonal hematopoiesis and leukemia: (1) DNA methylation-dependent effect, which caused up-regulation of the anterior Hoxb cluster and many hematopoietic stem cell (HSC) -related genes, with hypo-methylation of the promoter-associated CpG island; and (2) DNA methylation-independent effect with enhanced recruitment of polycomb repressive complex 1 (PRC1) that subsequently suppressed the expression of an array of differentiation-associated genes. Through these mechanisms, DNMT3A mutations were shown to inhibit the differentiation of HSCs and leukemic cells. These results identified PRC1 as a promising candidate for the development of therapeutic strategies in mutant DNMT3A-associated AML. Here, we review recent studies on AML with a focus on the clinical features and functional roles of DNMT3A mutations, and discuss future challenges to effectively cure DNMT3A mutation-associated hematopoietic disorders.

Chronic graft-versus-host disease (cGVHD) occurs in approximately 40% of patients who undergo allogeneic hematopoietic cell transplantation. It affects various organs and causes significant morbidity and mortality. The manifestations of cGVHD resemble those of autoimmune diseases. Inflammation, cellular immunity, humoral immunity, and fibrosis are implicated in cGVHD pathogenesis. The 2005 NIH consensus criteria for cGVHD have set standards for designing and reporting clinical trials, and the criteria were revised in 2014 to incorporate accumulated evidence and questions. The criteria are ready to be applied to design clinical trials aimed at identifying drugs for the treatment of cGVHD. Recent preclinical cGVHD trials have revealed the central roles of regulatory T cells, Th17 cells, Tc17 cells, follicular helper T cells, and follicular regulatory T cells as well as B cell signaling and fibrosis-promoting factors. Based on these advances, clinical trials targeting the specific pathogenic pathways of cGVHD are rapidly emerging, awaiting the approval of effective drugs to improve patient outcomes.

A 38-year-old woman in the first remission of mixed-phenotype acute leukemia underwent unrelated bone marrow transplantation from an HLA-DR-mismatched donor in the host-versus-graft (HVG) direction with myeloablative conditioning. Neutrophil engraftment was achieved and complete donor chimera was obtained on days 21 and 29 after transplantation, respectively. Subsequently, with delayed blood cell recovery, continuous transfusion was needed to replace platelets. In the CD3 peripheral blood chimerism test, the percentage of recipient cells on days 50, 63, and 80 was 27.3%, 90%, and 95% or higher, respectively. With no relapse of leukemia observed on bone marrow examination, secondary graft failure associated with autologous hematopoietic recovery was diagnosed. Bone marrow transplantation from the patient's HLA-haploidentical sister was performed because of graft failure on day 111 after the initial transplant using post-transplant cyclophosphamide (PTCy). Neutrophil engraftment was achieved and complete donor chimera was obtained on days 14 and 21 after the second transplantation, respectively. With no serious complications or acute graft versus host disease, the patient was discharged with symptomatic improvement. According to our results, retransplant using PTCy obtained from an HLA-haploidentical sibling donor is a potential treatment for graft failure.

The only curative treatment for chronic active Epstein-Barr virus infection (CAEBV) is hematopoietic stem cell transplantation. For young female patients, ovulation induction and oocyte cryopreservation may be performed prior to transplantation to provide for future pregnancies. However, the effects of this ovum treatment on CAEBV and EBV infections have not been reported. Attempts were made to collect ova from three female CAEBV patients before transplantation conditioning, but this was only successful in two cases. Ovarian stimulation did not induce disease progression, and there was no change in the peripheral blood EBV DNA load. In one patient, 460 copies/ml of EBV DNA were detected in the follicular fluid by real-time PCR. Red blood cells were also present in the follicular fluid but not mononuclear cells. EBV protein mRNA was not detected in the RNA extracted from the same fluid, suggesting that the EBV DNA resulted from peripheral blood contamination. Moreover, there were no EBV-infected cells in the follicular fluid. Therefore cryopreservation of oocytes from CAEBV patients is possible and may be used to provide for future pregnancies.