Animal experiments have occupied an important position in the safety assessment of chemicals. However, due to the rise in animal welfare as seen in the ban of animal experiments in European cosmetic development, the development of alternative methods for animal experiments has become very important in recent years. Development of in vitro tests for local toxicity such as irritation and sensitization tests is preceded. Meanwhile, alternative tests for systemic toxicity such as chronic and developmental toxicities are under development. In developing alternative methods using cultured cells, we have been focusing on pluripotent stem cells such as ES and iPS cells and studying alternatives to developmental toxicity and neurotoxicity. As an alternative test of developmental toxicity, we developed the Hand 1-Luc EST, which is a simple test utilizing cardiomyocyte differentiation process of mouse ES cells, and Tubb 3- and Reln-Luc ESTs using nerve differentiation process. Recently, it was clarified that the combination of the Hand 1-Luc EST and the Tubb 3- and Reln-Luc ESTs improves the prediction of the developmental toxicity. In the study of in vitro neurotoxicity test using neurons derived from mouse ES cells, evaluation methods for neurite outgrowth using high-content imaging technology and for neural function using multi-electrode arrays were developed. In addition, we introduce differentiation methods for retinal tissues from human ES/iPS cells, which are the results as the collaboration with RIKEN and the present state of an in vitro phototoxicity test using retinal pigment epithelial cells (RPE) derived from human ES cells.

Hematopoietic stem cells(HSCs)in the bone marrow(BM)are maintained in distinct microenvironments called niches. Technological advances in in vivo imaging have enabled dynamic analyses of BM cells. This in vivo imaging can be a key tool to elucidate the mechanisms underlying HSC maintenance in the BM through analysis of HSC motility and evaluation of the relationship between HSCs and their niche factors over time. Furthermore, application of this imaging technology to leukemia research can lead to new discoveries in leukemic stem cell maintenance and the development of novel antileukemic drugs. In this review, we summarize recent advances in dynamic analyses of HSCs and leukemic cells in the BM by intravital imaging.

In bone tissues, there are various kinds of cell types, such as osteoclasts, osteoblasts, monocytes, granulocyte, lymphocytes, mesenchymal cells and hematopoietic stem cells. They form a network with each other, and play critical roles in our life activities. The recent development of intravital two-photon imaging has enabled us to visualize the in vivo behavior of bone marrow cells in living bone tissues. This technique facilitates investigation of cellular dynamics in the physiology and pathogenesis of bone disorders in vivo, and would thus be useful for evaluating the efficacy of novel drugs. In this review, we summarize the basis of intravital bone imaging, and also discuss its further application.

Steroid-refractory graft-versus-host disease (SR-GVHD) is one of the most important complication post allogeneic hematopoietic stem cell transplantation. Mesenchymal stem cells (MSCs) possess characteristic immunomodulatory features which are beneficial in the salvage treatment for SR-GVHD. Following the first case report in 2004, numerous clinical trials have shown encouraging results with MSC infusions for treating SR-GVHD. In Japan, two clinical trials have achieved favorable results, and subsequently in September 2015, MSC infusion became the first approved allogeneic cell therapy for SR-GVHD. Currently, MSCs are available at limited institutes, and all patients infused with MSCs have been registered in the post marketing survey. Based on the results of this survey, the establishment of guidelines for the proper use of MSCs is anticipated. Moreover, several groups have reported the efficacy of MSCs for chronic GVHD or GVHD prophylaxis. In the present review, current issues regarding the use of MSCs in the management of GVHD are summarized.

Here we report a case of secondary graft failure that was effectively treated with donor lymphocyte infusion (DLI). A 64-year-old female patient with acute myeloid leukemia obtained partial remission with azacitidine therapy and subsequently underwent unrelated allogeneic bone marrow transplantation (BMT). After confirming successful engraftment and achieving complete remission with incomplete blood count recovery, she was subsequently followed up at an outpatient clinic. A routine test performed by day 110 after BMT revealed the presence of pancytopenia. A bone marrow aspirate did not reveal any evidence of disease relapse or hemophagocytic syndrome but demonstrated hematopoietic insufficiency. Donor chimerism also declined over time; thus, the patient was diagnosed with secondary graft failure. Supportive treatment, including granulocyte-colony stimulating factor and blood transfusion, failed to improve the blood parameters. Because the patient refused a second BMT, we performed DLI on day 147 after BMT (CD3+ cells: 1.0×107/kg, single dose). Consequently, the blood cell count improved promptly and dramatically without adverse events. Following this, we discussed the case and analyzed the related literature.

  Osteoclasts are differentiated from precursors of the monocyte/macrophage lineage originated from bone marrow hematopoietic stem cells and are the sole bone-resorbing cells in the body. Osteoclast differentiation is thought to require M-CSF (macrophage colony-stimulating factor) and RANKL (receptor activator of nuclear factor kappa-B ligand) signaling. However, it has recently been proposed that under chronic inflammatory conditions, such as systemic autoimmune diseases (e.g., rheumatoid arthritis), an increase in inflammatory cytokine levels within joints induces pathological osteoclast differentiation, causing excessive bone resorption. In addition, the authors have reported that stimulating mouse bone marrow monocytes and human CD14+ monocytes with combination of TNFα and IL-6 can induce differentiation of osteoclast-like cells, which are cells with bone resorption activity. In the present article, we discuss the mechanism of osteoclast differentiation of RANKL-independent bone-resorbing cells, using both data from the aforementioned report as well as the latest findings. Understanding the mechanisms underlying RANKL-independent, cytokine-mediated osteoclast differentiation could facilitate the development of novel therapies for inflammatory joint diseases.

  Bone is a component of the skeletal-locomotor system but also functions as an immunological organ that harbors hematopoietic stem and progenitor cells. Since the immune and skeletal systems are closely related through a number of shared regulatory molecules including cytokines and receptors, bone can be affected in various immune disorders. Rheumatoid arthritis is a typical disease in which the immune system affects the bone metabolism. The enhanced activity of osteoclasts by the activation of Th17 cells causes the joint destruction in rheumatoid arthritis. Studies on bone destruction associated rheumatoid arthritis have highlighted the importance of the interplay between the immune and bone systems, and promoted the new interdisciplinary field of "osteoimmunology". Furthermore, recent studies have suggested that regulation of bone tissues by IL-17 is more complicated than we had expected. IL-17-prodcuing cells contribute to new bone formation at the enthesis in ankylosing spondylitis, and IL-17-producing γδ T cells promote bone regeneration by acting on the mesenchymal stem cells in bone fracture healing. It would be necessary to comprehensively understand the interplay between the immune and bone systems for elucidation of the molecular mechanisms underlying the pathogenesis of various diseases that involves the two systems.

Congenital leukemia is a rare subgroup of childhood leukemia. Lineage switches in leukemic cells are relatively rare events, which have been occasionally reported in congenital leukemia. To the best of our knowledge, the survival of congenital leukemia patients with lineage switch has not been previously documented. This lack of documentation may be attributable to extremely poor prognosis of these patients. We describe a case of a newborn female with initial diagnosis of MLL-AF4 positive B-precursor acute lymphoblastic leukemia, who developed lineage switch to acute monocytic leukemia following the induction therapy. Although morphological remission was temporary, she received an HLA-haploidentical bone marrow transplant from her father with non-remission status because of an early relapse at the age of 4 months. Despite many difficulties such as graft-versus-host disease, growth impairment, and psychomotor retardation, she remained in remission for 3 years and 7 months after the transplant. This successful outcome suggests that the graft-versus-leukemia effect was potentially accomplished in the patient. Taken together, early HLA-haploidentical stem cell transplant following remission is required for congenital leukemia patients with lineage switch, and it may be an effective alternative for refractory patients.

Although peripheral blood stem cell (PBSC) transplantations in an unrelated transplant setting have been performed since 2010, prior harvesting and cryopreservation of PBSCs from unrelated donors has not been approved in Japan. There are no restrictions with regard to related donors. Therefore, in April 2015, we conducted a nationwide survey and obtained written answers from 123 transplant hospitals throughout Japan. Our survey revealed that as much as 81.3% of transplant hospitals routinely perform prior harvesting and cryopreservation of PBSCs from related donors and that both cell processing and quality management of cryopreserved products have been appropriately conducted in line with domestic guidelines, although post-thaw quality control and storage period setting require further improvements. Moreover, informed consent obtained from both patients and donors with regard to cryopreservation of PBSCs was not always sufficient in some hospitals. We found that the average number of unused or discarded cryopreserved PBSCs is 1.09 per hospital, and the overall nonuse or discard rates of cryopreserved PBSCs were estimated to be as low as 2.67%.

Genome editing technology can alter the genomic sequence at will, contributing the creation of cellular and animal models of human diseases including hereditary disorders and cancers, and the generation of the mutation-corrected human induced pluripotent stem cells for ex vivo regenerative medicine. In addition, novel approaches such as drug development using genome-wide CRISPR screening and cancer suppression using epigenome editing technology, which can change the epigenetic modifications in a site-specific manner, have also been conducted. In this article, I summarize the current advances and future prospects of genome editing technology in the field of biomedicine.

Glucocorticoid(GC)is widely used to treat a variety of autoimmune, inflammatory and allergic diseases. GC causes a number of adverse effects and among them osteoporosis and degenerative bone fracture are major complications of GC therapy. GC inhibits differentiation of mesenchymal stem cells to osteoblasts, induces apoptosis of osteoblasts and osteocytes and prolongs lifespan of osteoclast, resulting in low bone mass and micro-architectural deterioration of bone tissue, which leads to severe osteoporosis, increased bone fragility and susceptibility to bone fracture. Physicians who prescribe GC have to manage patients according to guidelines on the management and treatment of GC-induced osteoporosis.

Veno-occlusive disease(VOD)is a fatal coagulopathy, which is caused by an endothelial disorder among patients who undergo hematopoietic stem cell transplantation(SCT). Some protective agents, such as administration of fresh frozen plasma(FFP), have been evaluated for both prophylactic and targeted therapy.

Blood products derived from iPS cells have been pursued as a blood donor-independent and genetically manipulative measure to complement or alternate current transfusion products. Erythrocytes and platelets are anucleate blood cells that are indispensable for oxygen delivery and hemostasis, respectively. Consequently, blood transfusions have been clinically established to treat severe anemia and thrombocytopenia. However, current blood products exhibit issues with regard to supply-demand imbalance and alloimmune responses and infections, and they also face a future shortage of donors in aging societies. While the production of erythrocytes from iPS cells has challenges to overcome, such as their differentiation into an adult-type phenotype and scalable production, platelet products are qualitatively and quantitatively approaching a clinically applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Currently, the establishment of guidelines that assure the quality of iPSC-derived blood products for clinical application is in progress. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, the ex vivo production of iPSC-derived blood cells can be expected to lead iPSC-based regenerative medicine to become common clinical practice.

Recently, post-transplant cyclophosphamide (PTCy) -based HLA-haploidentical stem cell transplantation (PTCy-haploSCT) has been increasingly performed. The mechanism of GVHD prevention by PTCy is selective depletion of proliferating alloreactive T cells, while preserving non-alloreactive T cells. Several studies have reported that the incidence of non-relapse mortality, relapse, disease-free survival, and overall survival after PTCy-haploSCT are equivalent to those after HLA-matched SCT. Furthermore, the incidence of GVHD, especially chronic GVHD, after PTCy-haploSCT was lower than that of HLA-matched SCT. In Japan, we have conducted several prospective, multicenter, phase II studies since 2013. To reduce relapse, several strategies have been developed. In the first study (Haplo13), we used peripheral blood stem cells and added busulfan (BU) to the original non-myeloablative regimen developed by a Johns Hopkins group. In the second study, we used a BU- or TBI-based myeloablative conditioning regimen (Haplo14 MAC). In the third study, we reduced the dose of PTCy (Haplo16 RIC). In the latest study (Haplo17), we reduced the dose and/or duration of MMF and tacrolimus. We hope that these studies improve the outcome of PTCy-haploSCT.

In Japan, the latest trends in induction therapy for patients newly diagnosed with multiple myeloma are multi-drug combinations, including bortezomib, lenalidomide, and thalidomide. Patients <65 years old and those <70 years old who have normal cardiac and lung functions without any serious complications are good candidates for high-dose L-PAM with autologous stem cell transplantation. For successful stem cell collection, anti-cancer drugs that have a negative impact on stem cell mobilization are usually excluded from induction therapies. It has been reported that bortezomib and thalidomide do not affect mobilization of stem cells; however, extended exposure to lenalidomide may have a potential risk of poor mobilization. Under these circumstances, PAD, VTD, VCD, and RVD are recommended as first induction therapies for transplant-eligible patients. On the other hand, two- or three-drug combinations are effective as induction therapies for transplant-ineligible patients >65-70 years old and <65 years old with serious complications or insufficiencies of cardiac or lung functions as well as for any patients who refuse transplantation strategy. For these patients, VMP, MPT, and Rd are usually recommended. For elderly patients >75 years old, it is important that the dosage of each drug be properly reduced according to the frailty of the patient.