Each of the billions of the cells in our body exhibits their identity with unique gene expression profile. Recent advances in single cell transcriptomics enable to conduct cell taxonomy identifying new cell types and to re-arrange cells in order of pseudo-time course describing differentiation status of each cell. Even though the cost is still high, the single cell transcriptomics now becomes one of the conventional assays. We have applied the single cell gene expression analysis to dissect human development. In this article, we show our recent progress on a study describing early development of the kidney using human iPS cells by the single cell transcriptomics.

The central nervous system (CNS) is composed of three major cell types, neurons, astrocytes, and oligodendrocytes, which differentiate from common multipotent neural stem/precursor cells (NS/PCs). However, NS/PCs do not have this multipotentiality from the beginning: neurons are generated first and astrocytes are later during CNS development. This developmental progression is observed in vitro by using human (h) NS/PCs derived from pluripotent cells, such as embryonic- and induced pluripotent-stem cells (ES/iPSCs), however, in contrast to rodent's pluripotent cells, they require quite long time to obtain astrocytic differentiation potential. Here, we show that hypoxia confers astrocytic differentiation potential on hNS/PCs through epigenetic alteration for gene regulation. Furthermore, we found that these molecular mechanisms can be applied to functional analysis of patient' iPSC-derived astrocytes. In this review, we summarize recent findings that address molecular mechanisms of epigenetic and transcription factor-mediated regulation that specify NS/PC fate and the development of potential therapeutic strategies for treating astrocyte-mediated neurological disorders.

In allogeneic hematopoietic stem cell transplantation (HSCT), ascites may develop owing to several causes, including sinusoidal obstruction syndrome, infections, malignancies, and malnutrition. However, it is often difficult to determine its precise cause. Here, a 59-year-old male developed chylous ascites three months post allogeneic bone marrow transplantation for relapsed acute myeloid leukemia. None of the attempted treatments resulted in improvement. Lymphangioscintigraphy revealed a lymphatic flow disorder at the level of the cisterna chyli. Autopsy revealed no leukemic cell infiltration or graft-versus-host disease of the liver or pancreas. The pancreatic specimen revealed parenchymal fibrosis and infiltration of plasma cells, suggesting chronic inflammation in addition to pathological changes caused by acute pancreatitis. These findings indicate that acute or chronic pancreatitis caused a lymphatic flow disorder that developed into refractory ascites. Although we could not diagnose pancreatitis while the patient was alive, it is important to recognize that asymptomatic pancreatitis can develop after HSCT. Furthermore, one should attempt to make an accurate diagnosis as early as possible.

This review reflects back over almost 40 years of the author's basic research conducted at Graduate School of Pharmaceutical Sciences, Osaka University, Japan. After performing postdoctoral research in USA, the author became a research associate at Prof. Yoshiharu Miura's lab and started research on Biochemical Engineering in 1984. At that time, the main research purpose was to solve global environmental issues for maintaining human health. The author's achievements included novel useful material production system under inorganic conditions and genetically engineered whole-cell bacterial sensors detecting arsenite by naked eye without a detecting device. Another theme in the lab was to construct bioartificial liver support system. Various scaffolds for hepatocytes were newly prepared for constructing the compact reactor. Besides the bioreactor study, the author conducted cell transplantation research for the treatment of chronic liver diseases. It was shown that mesenchymal stem cells derived from third molars (wisdom teeth) could differentiate into hepatocytes and exhibit therapeutic effects in liver-damaged animals. After 2006, the lab started research on drug delivery systems, including noninvasive delivery of drugs such as peptides and nucleic acids by regulating epithelial tight junctions. Many substances enabling drug delivery through "paracellular" route were newly prepared. The author started basic research on Biochemical Engineering in the 1970s. Although these studies eventually shifted into the pharmaceutical field, the underlying concept was based on "engineering" throughout a 40-year research period. The author cordially thanks all colleagues for supporting engineering research in our lab.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease that develops with a skin lesion and is often accompanied by leukemic transformation. The normal counterparts of BPDCN tumor cells are progenitors of plasmacytoid dendritic cells, whereas the origins are thought to be hematopoietic stem cells. Approximately 10%-20% of BPDCN patients develop other hematologic malignancies, including chronic myelomonocytic leukemia (CMML). Mutations in epigenetic regulators are frequently observed in both BPDCN and CMML tumors. Azacitidine, a drug that targets epigenetic dysregulation, is known to be an effective treatment for CMML. However, it has been used in few BPDCN patients. Here, we report a BPDCN patient with skin lesions, bone marrow infiltration, and lymphadenopathy. CMML also developed during the course of BPDCN. Azacitidine had positive effects on CMML; however, BPDCN aggressively relapsed during treatment. Two TET2 mutations were found in both BPDCN and CMML tumors; one of which was commonly identified in both tumors.

Osteoporosis increases the risk of bone fractures (e.g., the femur), reduces a person's activities of daily living (ADL) and increases the likelihood of being bedridden. Therapeutic drugs for osteoporosis include oral bisphosphonates and intravenous receptor activator of nuclear factor-κB ligand (RANKL) antibodies, both of which suppress osteoclast activity, as well as the subcutaneously administered recombinant human parathyroid hormone (PTH), which activates osteoblasts. However, there is currently no oral osteogenesis-promoting drug. In the present study, we found a low-molecular-weight compound, KY-273, with osteogenesis promoting effects. KY-273 induced osteoblast differentiation in ST2 cells and in rat bone marrow-derived mesenchymal stem cells at a dose of 0.1 μM. On the other hand, KY-273 did not clearly exert differentiation effects in osteoclasts, chondrocytes, adipocytes, or myoblasts. In ovariectomized rats, KY-273 clearly increased serum bone alkaline phosphatase (ALP) by at a dose of 3 mg/kg for 8 weeks, and increased both the cortical bone volume and medullary volume of the diaphyseal and epiphyseal regions of femoral bone, but did not affect trabecular bone. Although alendronate (used to decrease bone loss) increased trabecular bone, it did not have any significant effects on cortical bone. PTH increased epiphysis cortical and trabecular bone volume, and reduced medullary volume. KY-273 also displayed good oral absorption in rats. In conclusion, KY-273 is a promising candidate for use as an oral anti-osteoporosis drug with osteogenesis promoting effects.

Various types of stresses account for the dysregulation of the self-renewal activity of stem cells, resulting in the functional failure of tissues or tumorigenesis promotion. Although diets also affect our health, the effect of harmful dietary stresses on the tissue or stem cell homeostasis remains unclear. Recent research has revealed that Spred1, which negatively regulates RAS-MAPK signaling, protects hematopoietic stem cell (HSC) homeostasis against high-fat diet (HFD) -induced systemic stress. In steady-state conditions, Spred1 negatively regulates HSC self-renewal in a manner supported by the Rho kinase (ROCK) activity. In addition, Spred1 deficiency in mice mitigates HSC dysfunction induced by aging or lipopolysaccharide treatment, enhances the HSC self-renewal capacity, and prolongs HSC lifespan, but does not induce leukemia because of the compensatory upregulation of Spred2-the other Spred family member. Conversely, HFD triggers ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in HSC dysfunction, severe anemia, and the development of lethal myeloproliferative neoplasm-like disease. The depletion of the gut microbiota by antibiotics restored myeloproliferation, anemia, and HSC reconstitution ability in HFD-fed Spred1-deficient mice, suggesting that HFD-induced hematopoietic abnormalities were partially because of alterations in the gut microbiota composition. Thus, HFD-induced systemic stress affects the regulation of HSC self-renewal, and Spred1 safeguards HSC homeostasis against the diet-induced systemic stress.

A 19-year-old male with therapy-related myelodysplastic syndrome underwent allogeneic bone marrow transplantation with reduced-intensity conditioning from his HLA-identical sibling whose ABO blood type exhibited major incompatibility with the patient. After post-transplantation 1 month, chimerism analysis of the bone marrow revealed mixed chimerism with 30% of recipient cells, and after post-transplantation 3 months, complete remission was maintained; however, recipient granulocytes were elevated up to 50% per the chimerism analysis. Next, pancytopenia developed following the rapid discontinuation of the immunosuppressive agent. Although neutrophils and platelets spontaneously recovered, anemia progressed. Based on severe erythroid hypoplasia in the bone marrow and the elevation of anti-ABO isohemagglutinin against donor-derived red blood cells, the patient was diagnosed with pure red cell aplasia (PRCA) following hematopoietic cell transplantation. Because complete chimerism was attained at the PRCA onset even for B cells, we decided to conservatively manage PRCA with only red blood cell transfusion. Notably, after 2 months of the PRCA onset, anemia improved. This case suggests that the therapeutic strategy for PRCA following hematopoietic cell transplantation should be determined by considering the status of each patient, including chimerism.

Blood vessels in the central nervous system (CNS) limit the material exchange between blood and parenchyma by blood brain barrier (BBB). At present, no appropriate in vitro BBB models are available for the investigation whether or not the candidate compounds for new drugs could be delivered to the CNS. This causes huge difficulties of the development of CNS drugs and prediction of CNS adverse effects. In this review, I first outline the structures and functions of BBB, together with the parameters used for the quantification of BBB functions. I also introduce the history of in vitro BBB models used in the drug development so far, i.e., the transition from non-cell models to the models using primary culture of rodent cells, porcine, bovine, cell lines, etc. More recently, the application of human cells differentiated from human induced pluripotent stem cells and microfluidic engineering have already started. BBB is essential for the maintenance of brain homeostasis and the mechanisms of the BBB development will be clarified by reproducing functional BBB on the dish. The new in vitro models and the data may provide accurate prediction of drug delivery to the CNS and the improvement of the evaluation system for toxicity and safety, thereby leading to successful launch of new drugs on the market.

Recipients of hematopoietic stem cell transplantation suffering from severe immunosuppression and immune dysregulation because of GVHD or other causes are at higher risk of viral infection. Often, cytomegalovirus and EB virus are reactivated from their latent state in these patients ; viral infections thus remain a primary cause of severe morbidity and mortality. Since the 1990s, virus-specific T cells have been generated by different methods using immunogenic epitope peptides or EBV-LCL from transplant donors, and studies have demonstrated the efficacy of these methods. However, establishing such order-made, virus-specific T cells requires 8-10 weeks, which is not feasible for application in the frontline of clinics. Recent advances, such as direct donor T cell selection using peptide-HLA multimers or cytokine capture method and virus-specific T cells bank generated using third-party donors, facilitated the broadening of the applicability of this method. The clinical development of virus-specific T-cell therapy is considered as the prototype of future T-cell immunotherapy for various infections or malignant diseases.

Mesenchymal stem cells (MSCs) perform multiple functions, such as immunomodulation and tissue repair, and they are also capable of differentiation into bone, cartilage, and fat cells. Furthermore, an MSC culture method has been established, and clinical safety is guaranteed; therefore, MSCs can be clinically applied for the treatment of many diseases. MSC treatment for hematological diseases is expected to be effective against refractory acute graft-versus-host disease (GVHD). It is presently used for treating chronic GVHD, preventing GVHD, promoting the engraftment of hematopoietic stem cells, and treating refractory aplastic anemia. However, owing to the cellular properties of MSCs, there are some concerns including increases in relapse, the deterioration of infectious diseases, and tumor formation or malignant transformation of MSCs. In the present review, I describe the present situation, problems, and prospects of the clinical application of MSCs for treating hematological diseases, including recent topics such as placental-derived decidual stromal cells and highly purified MSCs.

In other countries, unrelated donor peripheral blood stem cell transplantation (PBSCT) is more prevalent than bone marrow transplantation; however, in Japan, it was introduced in 2010 after confirming the safety of donors. PBSCT does not require blood donation, general anesthesia, or frequent bone marrow aspiration of the donor. After PBSCT, numerous hematopoietic cells can prompt blood recovery and engraftment, which has enabled reduced intensity transplantation in elderly patients and patients with concurrent diseases, such as infection. In addition, GVL effect by a large number of donor lymphocytes is expected, however, chronic GVHD is a major concern. When introducing PBSCT in Japan, manuals were drafted considering the short-term safety of donors, and data were collected on the occurrence of long- and short-term adverse events. A randomized trial reported no difference in the survival rate between bone marrow transplantation and PBSCT at 5 years; however, it revealed that QOL was better in the former. PB is a essential transplant source option, and attempts are being made to overcome chronic GVHD. PBSC contains abundant stem cells, progenitor cells, and immunocompetent cells and is indispensable for the development of cell therapy for blood diseases in the future.

Cure rates achieved with allogeneic hematopoietic cell transplantation (allo-HCT) for nonremission leukemia have been largely unsatisfactory (10-20%). Cord blood (CB) transplantation has long been thought to have a low propensity for graft-versus-leukemia (GVL) effect because of the low incidence of graft-versus-host disease (GVHD). However, several retrospective studies have revealed that GVL effect of CB transplantation is comparable to or even greater than that of peripheral blood (PB) or bone marrow (BM) transplantation. The underlying mechanism of this phenomenon remains unclear; however, basic research has revealed some unique characteristics of immune cells in CB which are distinct from those of PB or BM. In clinical transplantation, the observed systemic immune-mediated manifestations in the pre-engraftment phase are also specific to CB transplantation. These findings suggest that CB is a potential donor source for patients in nonremission due to its unique immunological potential as well as rapid availability for nearly all patients in the near future.

CAR T-cell therapy is a novel cancer immunotherapy targeting cancer-specific cell-surface antigen. CD19-CAR T cells have been demonstrated to be highly efficacious in treating B-cell leukemia/lymphoma. Currently, several researchers are developing CAR T cells for multiple myeloma. In some early-phase clinical trials, CAR T cells targeting B-cell maturation antigen have exhibited promising efficacy. Recently, we reported that CAR T cells targeting the activated integrin 7 can selectively eradicate MM cells including CD19+ clonotypic B cells; a clinical trial for further assessment regarding this study is in process.

Tyrosine kinase inhibitors (TKIs) markedly improve the prognosis of patients with chronic myelogenous leukemia (CML) by potentially helping to achieve a deep molecular response (DMR). In many clinical trials, beginning with the French Stop Imatinib (STIM1) trial, approximately 40%-60% patients with chronic CML who sustained a long DMR could discontinue TKI therapy and achieve long-term treatment-free remission (TFR). These trials have proposed many predictive factors for successful TKI therapy discontinuation, including deeper molecular response, longer duration of DMR and lack of TKI resistance prior to the discontinuation, and greater numbers of natural killer (NK) cells during the discontinuation. However, further investigations are necessary because only 20%-30% patients with chronic CML could achieve TFR. Recent studies have suggested that the recovery and reconstitution of immune effector cells against CML, including NK cells, and the elimination of CML cells using aberrant clones that are resistant to TKI are crucial for successfully achieving DMR and subsequent TFR. CML stem cells are not sensitive to TKI and could potentially interfere with TFR. Therefore, therapies targeting CML stem cells are being investigated, and the results are highly anticipated.

Lifelong, self-renewing, and, multilineage-differentiating hematopoietic stem cells (HSCs) gradually divide in steady-state bone marrow (BM). Conversely, in cases of hematopoietic stress, including infection and inflammation, hematopoiesis is highly demanded due to massive cell consumption in the stressed tissues and involves HSC recruitment to fulfil the hematopoietic demand. Accumulating evidence indicates that infection-related inflammation acts on blood-forming HSCs and progenitors within the BM to facilitate hematopoiesis for self-defense. In this review, we discuss the mechanisms used by various inflammatory responses involving not only HSCs but also the niche cells in the BM, a site that has long been considered an immune-privileged organ.

Mesenchymal stromal/stem cells (MSCs) exhibit various biological characteristics, including immunomodulation, tissue regeneration, anti-inflammation, and hematopoietic support. Extensive efforts have been made toward the development of cell therapy using MSCs to treat various diseases. In Japan, off-the-shelf allogeneic human bone marrow-derived MSC products from third-party donors are clinically available for treating patients with steroid-resistant/refractory acute graft-versus-host disease (GVHD). In this manuscript, we present an overview of the current status of clinical development of MSC-based cell therapy and the basic understanding of MSCs. We also introduce a novel approach to obtain the anti-GVHD effect utilizing bone marrow MSC-derived extracellular vesicles.

Platelet transfusion products derived from induced pluripotent stem cells (iPSCs) have been pursued as a blood donor-independent and genetically manipulative measure to complement or as an alternative to current platelet products. Platelets are enucleate blood cells indispensable for hemostasis. Thus, platelet transfusions have been clinically established to treat patients with severe thrombocytopenia. However, current blood products face issues in the balance of supply and demand, alloimmune responses, and infections and are expected to meet the shortage of donors in aging societies. iPSc-derived platelet products are qualitatively and quantitatively approaching a clinically applicable level, owing to advances and novel findings in expandable megakaryocyte cell lines, turbulence-incorporating bioreactors, and reagents that enable feeder cell-free production and improve platelet quality. Currently, the establishment of guidelines to assure the quality of iPSC-derived blood products for clinical application is in process. Considering the low risk of tumorigenicity and the large demand, ex vivo production of iPSC-derived platelets could lead to iPSC-based regenerative medicine becoming a common clinical practice and the development of a future system in which anyone can safely receive a platelet transfusion in their time of need.

It has long been considered that hematopoietic system development is based on hematopoietic stem cell differentiation with a gradual loss of multipotency. However, recent studies have indicated that hematopoietic stem cells are composed of subsets that exhibit lineage skewing, thereby comprising a heterogeneous population. Therefore, the present hierarchical development model of the hematopoietic system may be reconsidered. Appropriate transcription regulators and epigenetic influences are required for cell differentiation. To this end, transcription factors, such as GATA1 for erythropoiesis and PU.1 for myelopoiesis, have been believed to play key roles at the initiation of hematopoietic cell differentiation. However, the mechanisms controlling the expression of these transcription factors are controlled, and the points at which the lineage commitments occur from hematopoietic stem cells through to progenitor cells are not fully elucidated. As per the findings of this study, we propose that the regulation of transcription and epigenetic factors are central events in hematopoietic cell differentiation.

Hematopoietic stem cell (HSC) biology is a current topic of interest having significant implications for clinical HSC transplantation and basic HSC research. It was long believed that the most primitive HSCs in mammals, including those in mice and humans, were CD34 antigen-positive (CD34+) cells. However, Nakauchi et al. reported that murine long-term lymphohematopoietic reconstituting HSCs were lineage marker-negative (Lin-) c-kit+Sca-1+CD34-low/negative (CD34low/-), known as CD34low/-KSL cells. We have previously identified very primitive human cord blood (CB) -derived CD34-negative (CD34-) severe combined immunodeficiency (SCID) -repopulating cells (SRCs) utilizing an intra-bone marrow injection method and have proposed a new concept that CD34-SRCs (HSCs) reside at the apex of human HSC hierarchy. Recently, we developed an ultra-high-resolution purification method using the two positive/enrichment markers CD133 and GPI-80 and succeeded in purifying CD34+/-HSCs on a single-cell level. On the basis of these data, we propose a revised roadmap for the commitment of human CD34-HSCs. This review updates the concept of the stem cell nature of human CB-derived primitive CD34+/-HSCs.