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Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non-negligible levels.We sought to establish an FC method that could prevent antibody-induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC-FC analysis of AB expression. We optimized fixatives, multicolor-staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, BThe optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co-staining of leukocyte- and erythrocyte-markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the BThe RBC-FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.

Summary A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation., Graphical Abstract, Highlights • Combination of a GSK3 inhibitor and an RAR agonist induces chondrocytes from hPSCs • The present protocol shows excellent differentiation efficiency • The potential to form hyaline cartilaginous tissues in vivo is demonstrated • RA and Wnt/β-catenin signals collaboratively regulate key genes for chondrogenesis, Saito and colleagues show that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, a GSK3 inhibitor and an RAR agonist, within 5–9 days. Genome-wide analysis of RAR and β-catenin association with DNA for sequential samples at each protocol stage demonstrates that RA and Wnt/β-catenin signaling is collaboratively involved in direct regulation of chondrocyte differentiation.

Background Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. Methods We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. Results We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. Conclusion This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients—namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)—using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Summary Oncogenic KRAS mutations in hematopoietic stem cells cause RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). KRAS plays essential roles in stemness maintenance in some types of stem cells. However, its roles in pluripotent stem cells (PSCs) are poorly understood. Here, we investigated the roles of KRAS on stemness in the context of induced PSCs (iPSCs). We used KRAS mutant (G13C/WT) and wild-type isogenic (WT/WT) iPSCs from the same RALD patients, as well as wild-type (WTed/WT) and heterozygous knockout (Δed/WT) iPSCs, both obtained by genome editing from the same G13C/WT clone. Compared with WT iPSCs, G13C/WT iPSCs displayed enforced retention of self-renewal and suppressed capacity for neuronal differentiation, while Δed/WT iPSCs showed normalized cellular characteristics similar to those of isogenic WTed/WT cells. The KRAS-ERK pathway, but not the KRAS-PI3K pathway, was shown to govern these G13C/WT-specific phenotypes, indicating the strong impact of the KRAS-ERK signaling upon self-renewal and differentiation propensity in human iPSCs., Graphical Abstract, Highlights • Isogenic wild-type and KRAS mutant iPSCs were established from RALD patients • Oncogenic KRAS enforces retention of self-renewal in human iPSCs • Oncogenic KRAS suppresses neuronal differentiation from human iPSCs • KRAS-ERK signaling pathway governs the KRAS mutant-specific phenotypes, In this article, Kubara, Yamazaki, and colleagues show that, using RALD patient-derived iPSCs, oncogenic KRAS activates mainly the MEK-ERK pathway, but not the PI3K-AKT pathway, leading to enforced retention of self-renewal and suppressed capacity for undergoing neuronal differentiation in human iPSCs.

The understanding of signaling events is critical for attaining long-term expansion of hematopoietic stem cells ex vivo. In this study, we aim to analyze the contribution of multiple signaling molecules in proliferation of hematopoietic stem cells. To this end, we design a bottom-up engineered receptor with multiple tyrosine motifs, which can recruit multiple signaling molecules of interest. This is followed by a top-down approach, where one of the multiple tyrosine motifs in the bottom-up engineered receptor is functionally knocked out by tyrosine-to-phenylalanine mutation. The combination of these two approaches demonstrates the importance of Shc in cooperation with STAT3 or STAT5 in the proliferation of hematopoietic stem cells. The platform developed herein may be applied for analyzing other cells and/or other cell fate regulation systems.

Summary Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells (DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS., Highlights • Dendritic cells were generated from iPSCs derived from CD4+ T cells (T-iPS-DCs) • Adequate numbers of functional DCs were generated from a small blood sample • The comparison between T-iPS-DCs and monocyte-derived DCs was evaluated • The functional assays of T cells in Sjögren's syndrome were analyzed by T-iPS-DCs, In this article, Sumida and colleagues demonstrate the generation of iPSCs from CD4+ T cell clones of Sjögren's syndrome (SS), which differentiated into dendritic cells (DCs) (T-iPS-DCs). Adequate numbers of functional DCs were obtained and used for the analysis of T cell functions in SS. T-iPS-DCs could be used to characterize pathogenic T cells in autoimmune diseases such as SS.

Summary Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations., Graphical Abstract, Highlights • Different BSA lots alter how HSCs respond to cytokines • RSA can replace BSA to provide HSC maintenance culture with minimal variability • By comparing the protein profiles of “good” and “bad” BSAs, HPX was identified • HPX reduces HSC intracellular reactive ROS and is expressed by BM Schwann cells, In this article, Yamazaki, Nakauchi, and colleagues demonstrate that BSA batches have unique protein profiles and vary widely in their ability to maintain mouse HSCs ex vivo. By replacing BSA with recombinant serum albumin, they developed a standardized HSC culture platform that can be used to identify novel maintenance and expansion factors.

We established three iPSC lines from postmortem-cultured fibroblasts derived following the sudden unexpected death of an 8-year-old girl with Lennox-Gastaut syndrome, who turned out to have the R551H-mutant STXBP1 gene. These iPSC clones showed pluripotent characteristics while retaining the genotype and demonstrated trilineage differentiation capability, indicating their utility in disease-modeling studies, i.e., STXBP1-encephalopathy. This is the first report on the establishment of iPSCs from a sudden death child, suggesting the possible use of postmortem-iPSC technologies as an epoch-making approach for precise identification of the cause of sudden death., Stem Cell Research, 39, art.no.101485; 2019

Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

Combination of multiple units of allogeneic HSPCs exerts radioprotective and bridging effect, allowing single-donor hematopoiesis by a principal graft., Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34+ cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34+ cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient’s age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient’s derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder characterized by defective microbial killing in phagocytes. Long-term prognosis for CGD patients is generally poor, highlighting the need to develop minimally toxic, curative therapeutic approaches. We here describe the establishment of a mouse model in which X-linked CGD can be cured by allogeneic bone marrow transplantation. Using a combination of non-myeloablative-dose total body irradiation and a single injection of anti-CD40 ligand monoclonal antibody, transplantation of whole bone marrow cells achieved long-lasting mixed chimerism in X-linked CGD mice in a haploidentical transplantation setting. Stable mixed chimerism was maintained for up to 1 year even at a low range (

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficient activity of the iduronate-2-sulfatase. This leads to accumulation of glycosaminoglycans (GAGs) in the lysosomes of various cells. Although it has been proposed that bone marrow transplantation (BMT) may have a beneficial effect for patients with MPS II, the requirement for donor-cell chimerism to reduce GAG levels is unknown. To address this issue, we transplanted various ratios of normal and MPS II bone marrow cells in a mouse model of MPS II and analyzed GAG accumulation in various tissues. Chimerism of whole leukocytes and each lineage of BMT recipients' peripheral blood was similar to infusion ratios. GAGs were significantly reduced in the liver, spleen, and heart of recipients. The level of GAG reduction in these tissues depends on the percentage of normal-cell chimerism. In contrast to these tissues, a reduction in GAGs was not observed in the kidney and brain, even if 100 % donor chimerism was achieved. These observations suggest that a high degree of chimerism is necessary to achieve the maximum effect of BMT, and donor lymphocyte infusion or enzyme replacement therapy might be considered options in cases of low-level chimerism in MPS II patients.

Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs. Electronic supplementary material The online version of this article (10.1007/s12015-017-9768-7) contains supplementary material, which is available to authorized users.

The pluripotent stem cells have a self-renewal ability and can be differentiated into theoretically all of cell types. The induced pluripotent stem (iPS) cells overcame the ethical problems of the human embryonic stem (ES) cell, and enable pathologic analysis of intractable diseases and drug discovery. The in vitro disease model using disease-specific iPS cells enables repeated analyses of human cells without influence of environment factors. Even though autoimmune diseases are polygenic diseases, autoimmune disease-specific iPS cells are thought to be a promising tool for analyzing the pathogenesis of the diseases and drug discovery in future.

Induced pluripotent stem cells (iPS) derived from disease cells are expected to provide a new experimental material, especially for diseases from which samples are difficult to obtain. In this study, we generated iPS from samples from patients with primary and secondary myelofibrosis. The primary myelofibrosis cells had chromosome 13q deletions, and the secondary myelofibrosis (SMF) cells had JAK2V617F mutations. The myelofibrosis patient cell-derived iPS (MF-iPS) were confirmed as possessing these parental disease-specific genomic markers. The capacity to form three germ layers was confirmed by teratoma assay. By co-culture with specific feeder cells and cytokines, MF-iPS can re-differentiate into blood progenitor cells and finally into megakaryocytes. We found that mRNA levels of interleukin-8, one of the candidate cytokines related to the pathogenesis of myelofibrosis, was elevated predominantly in megakaryocytes derived from MF-iPS. Because megakaryocytes from myelofibrosis clones are considered to produce critical mediators to proliferate fibroblasts in the bone marrow and iPS can provide differentiated cells abundantly, the disease-specific iPS we established should be a good research tool for this intractable disease.

Hematopoietic cell transplantation has proven beneficial for various intractable diseases, but it remains unclear how hematopoietic stem/progenitor cells (HSPCs) home to the bone marrow (BM) microenvironment, initiate hematopoietic reconstitution, and maintain life-long hematopoiesis. The use of newly elucidated molecular determinants for overall HSPC engraftment should benefit patients. Here, we report that modification of C-X-C chemokine receptor type 4 (Cxcr4) signaling in murine HSPCs does not significantly affect initial homing/lodging events, but leads to alteration in subsequent BM repopulation kinetics, with observations confirmed by both gain- and loss-of-function approaches. By using C-terminal truncated Cxcr4 as a gain-of-function effector, we demonstrated that signal augmentation likely led to favorable in vivo repopulation of primitive cell populations in BM. These improved features were correlated with enhanced seeding efficiencies in stromal cell cocultures and altered ligand-mediated phosphorylation kinetics of extracellular signal-regulated kinases observed in Cxcr4 signal-augmented HSPCs in vitro. Unexpectedly, however, sustained signal enhancement even with wild-type Cxcr4 overexpression resulted in impaired peripheral blood (PB) reconstitution, most likely by preventing release of donor hematopoietic cells from the marrow environment. We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing donor HSPCs with enhanced repopulation potential following transplantation, whilst preserving the ability to release HSPC progeny into PB for improved transplantation outcomes. Stem Cells 2014;32:1929–1942

Aim Human induced pluripotent stem (hiPS) cells are an alternative cell source of regenerative medicine for liver disease. Because variations in hepatic differentiation efficacy among hiPS cells exist, it is important to select a hiPS cell line with hepatic differentiation propensity. In addition, nuclear receptors (NR) regulate essential biological processes including differentiation and development. In this study, we identified the hiPS cell line with hepatic differentiation propensity and examined expression levels of 48 NR during this process. Methods We screened 28 hiPS cell lines, which are established from various tissues of healthy persons with various reprogramming methods, using a three-step differentiation method, and examined expression levels of 48 NR by quantitative real-time polymerase chain reaction during the differentiation process in the selected cells. Results hiPS-RIKEN-2B and hiPS-RIKEN-2F cells have hepatic differentiation propensity. Differentiation propensity towards endoderm was affected by donor origin but not by reprogramming methods or cell type of origins. Expression levels of NR were closely associated with those of hepatic differentiation markers. Furthermore, expression patterns of NR were categorized as five patterns. In particular, seven NR such as chicken ovalbumin upstream promoter transcription factor 1, retinoic acid receptor α, peroxisome proliferator-activated receptor-γ, progesterone receptor, photoreceptor cell-specific nuclear receptor, tailless homolog orphan receptor and glucocorticoid receptor were identified as the genes of which expression gradually goes up with differentiation. Conclusion These findings will be useful for not only elucidating mechanisms of hepatic differentiation of hiPS cells but also cell-based therapy for liver diseases.

Pompe disease (PD), which is also called glycogen storage disease type II (GSDII), is one of the lysosomal storage diseases (LSDs) caused by a deficiency in acid-α-glucosidase (GAA) in the lysosome and is characterized by the accumulation of glycogen in various cells. PD has been treated by enzyme replacement therapy (ERT). We generated induced pluripotent stem cells (iPSCs) from the cells of patients with infantile-type and late-onset-type PD using a retrovirus vector to deliver transgenes encoding four reprogramming factors, namely, OCT4, SOX2, c-MYC, and KLF4. We confirmed that the two types of PD-iPSCs exhibited an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. The PD-iPSCs exhibited strong positive staining with Periodic acid-Schiff (PAS). Moreover, ultrastructural features of these iPSCs exhibited massive glycogen granules in the cytoplasm, particularly in the infantile-type but to a lesser degree in the late-onset type. Glycogen granules of the infantile-type iPSCs treated with rhGAA were markedly decreased in a dose-dependent manner. Human induced pluripotent stem cell provides an opportunity to build up glycogen storage of Pompe disease in vitro. It represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies for Pompe disease.

SummaryDistinct B cell populations, designated regulatory B (Breg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified Breg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue Breg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support Breg cell function, and Breg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue Breg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that Breg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor-mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl(-/-) mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC-derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that can infect both non-proliferating and proliferating cells. Owing to its favorable safety profile, AAV is regarded as suitable for clinical purposes such as gene therapy. The target cell types of AAV depend largely on the serotype. In the retina, AAV has been used to introduce exogenous genes into photoreceptors, and photoreceptor-specific enhancers/promoters are used in most cases. Therefore, serotype specificity of AAV in retinal subtypes is unclear, particularly in vitro. We compared its infection profile in mouse and monkey retinas using EGFP under the control of the CAG promoter, which expressed the gene ubiquitously and strongly regardless of cell type. AAV1, 8, and 9 infected the horizontal cells when an embryonic day-17 retina was used as a host. Amacrine cell was also a major target of AAVs, and a small number of rod photoreceptors were infected. When adult retinas were used as a host, the main target of AAV was Müller glia. A small number of rod photoreceptors were also infected. In the adult common marmoset retina, rod and cone photoreceptors were efficiently infected by AAV1, 8, and 9. A portion of the Müller glia and amacrine cells were also infected. In summary, the infection specificity of different AAV serotypes did not differ, but was dependent on the stage of the host retina. In addition, infection specificities differed between mature marmoset retinas and mature mouse retinas.

Summary Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases., Highlights • We optimized conditioned medium preparation from L cells by lentiviral infection • We modified a protocol to differentiate iPSCs into small intestinal organoids • We succeeded in transducing a gene into organoids by 2D culture • We showed that intestinal human organoids self-proliferate in suspension culture, Takahashi et al. developed several user-friendly culture methods for human induced pluripotent stem cell (iPSC)-derived intestinal organoids. The methodological improvements include preparation of conditioned medium for organoid culture, efficient differentiation from iPSCs, gene transduction in organoids, and growth in suspension culture. This system will facilitate high-throughput screening of pathogenic factors and treatments for intestinal diseases using physiologically robust intestinal organoids.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7±4.92% donor chimerism at 16weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.