Glycogen myophosphorylase loss causes increased dependence on glucose in iPSC-derived retinal pigment epithelium.

Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease-an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell line and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic WT controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate was increased. Oxygen consumption rate in response to physiological levels of lactate was significantly greater in WT than PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease.

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.

Neuroprotective Effects of Genome-Edited Human iPS Cell-Derived Neural Stem/Progenitor Cells on Traumatic Brain Injury.

Despite developing neurosurgical procedures, few treatment options have achieved functional recovery from traumatic brain injury (TBI). Neural stem/progenitor cells (NS/PCs) may produce a long-term effect on neurological recovery. Although induced pluripotent stem cells (iPSCs) can overcome ethical and practical issues of human embryonic or fetal-derived tissues in clinical applications, the tumorigenicity of iPSC-derived populations remains an obstacle to their safe use in regenerative medicine. We herein established a novel treatment strategy for TBI using iPSCs expressing the enzyme-prodrug gene yeast cytosine deaminase-uracil phosphoribosyl transferase (yCD-UPRT). NS/PCs derived from human iPSCs displayed stable and high transgene expression of yCD-UPRT following CRISPR/Cas9-mediated genome editing. In vivo bioluminescent imaging and histopathological analysis demonstrated that NS/PCs concentrated around the damaged cortex of the TBI mouse model. During the subacute phase, performances in both beam walking test and accelerating rotarod test were significantly improved in the treatment group transplanted with genome-edited iPSC-derived NS/PCs compared with the control group. The injury area visualized by extravasation of Evans blue was smaller in the treatment group compared with the control group, suggesting the prevention of secondary brain injury. During the chronic phase, cerebral atrophy and ventricle enlargement were significantly less evident in the treatment group. Furthermore, after 5-fluorocytosine (5-FC) administration, 5-fluorouracil converted from 5-FC selectively eliminated undifferentiated NS/PCs while preserving the adjacent neuronal structures. NS/PCs expressing yCD-UPRT can be applied for safe regenerative medicine without the concern for tumorigenesis.

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.

Development of an evaluation method for addictive compounds based on electrical activity of human iPS cell-derived dopaminergic neurons using microelectrode array.

Addiction is known to occur through the consumption of substances such as pharmaceuticals, illicit drugs, food, alcohol and tobacco. These addictions can be viewed as drug addiction, resulting from the ingestion of chemical substances contained in them. Multiple neural networks, including the reward system, anti-reward/stress system and central immune system in the brain, are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted, methods for assessing addiction have not been established. In this study, we aimed to develop an in vitro method for assessing the addiction of compounds, as an alternative to animal experiments, using human iPS cell-derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non-addictive compounds, demonstrating the ability to estimate addiction of compound. Additionally, conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine, dopamine and GABA) due to chronic administration of addictive compounds, suggesting the potential interpretation of these expression changes as addiction-like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell-derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks.

[Development of off-the-shelf universal T cell therapies from ES/iPS cells: applications in cancer and viral infection].

Methods in which patient-derived T cells are genetically modified in vitro and administered to patients have been demonstrated effective in the area of cancer immunotherapy. However, these methods have some unresolved issues such as cost, time, and unstable quality. Several groups have developed strategies to overcome these barriers by regenerating T cells from iPSCs. We have been developing a method in which specific TCR genes are introduced into iPSCs and T cells are regenerated from these iPSCs (TCR-iPSC method). We are now using starting iPSCs from the iPSC stock lines provided by CiRA-F, as the iPSC stock cells are less likely to be rejected. A study aimed at application to solid tumors demonstrated the therapeutic effect of regenerated T cells in a patient tissue xenograft model of WT1 antigen-positive renal cell carcinoma. This article will also discuss strategies by other groups to regenerate various types of T cells from iPSCs.

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells induce mixed chimerism and donor-specific allograft tolerance.

In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage- phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Sustainable Antiviral Efficacy of Rejuvenated HIV-Specific Cytotoxic T Lymphocytes Generated from Induced Pluripotent Stem Cells.

Persistence of HIV latently infected cells is a barrier to HIV cure. The "kick and kill" strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the "kick and kill" strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.

Shedding light on latent pathogenesis and pathophysiology of mental disorders: The potential of iPS cell technology.

Mental disorders are considered as one of the major healthcare issues worldwide owing to their significant impact on the quality of life of patients, causing serious social burdens. However, it is hard to examine the living brain-a source of psychiatric symptoms-at the cellular, subcellular, and molecular levels, which poses difficulty in determining the pathogenesis and pathophysiology of mental disorders. Recently, induced pluripotent stem cell (iPSC) technology has been used as a novel tool for research on mental disorders. We believe that the iPSC-based studies will address the limitations of other research approaches, such as human genome, postmortem brain study, brain imaging, and animal model analysis. Notably, studies using integrated iPSC technology with genetic information have provided significant novel findings to date. This review aimed to discuss the history, current trends, potential, and future of iPSC technology in the field of mental disorders. Although iPSC technology has several limitations, this technology can be used in combination with the other approaches to facilitate studies on mental disorders.

Regenerative medicine: current research and perspective in pediatric surgery.

The field of regenerative medicine, encompassing several disciplines including stem cell biology and tissue engineering, continues to advance with the accumulating research on cell manipulation technologies, gene therapy and new materials. Recent progress in preclinical and clinical studies may transcend the boundaries of regenerative medicine from laboratory research towards clinical reality. However, for the ultimate goal to construct bioengineered transplantable organs, a number of issues still need to be addressed. In particular, engineering of elaborate tissues and organs requires a fine combination of different relevant aspects; not only the repopulation of multiple cell phenotypes in an appropriate distribution but also the adjustment of the host environmental factors such as vascularisation, innervation and immunomodulation. The aim of this review article is to provide an overview of the recent discoveries and development in stem cells and tissue engineering, which are inseparably interconnected. The current status of research on tissue stem cells and bioengineering, and the possibilities for application in specific organs relevant to paediatric surgery have been specifically focused and outlined.

A Weakly Supervised Learning Method for Cell Detection and Tracking Using Incomplete Initial Annotations.

The automatic detection of cells in microscopy image sequences is a significant task in biomedical research. However, routine microscopy images with cells, which are taken during the process whereby constant division and differentiation occur, are notoriously difficult to detect due to changes in their appearance and number. Recently, convolutional neural network (CNN)-based methods have made significant progress in cell detection and tracking. However, these approaches require many manually annotated data for fully supervised training, which is time-consuming and often requires professional researchers. To alleviate such tiresome and labor-intensive costs, we propose a novel weakly supervised learning cell detection and tracking framework that trains the deep neural network using incomplete initial labels. Our approach uses incomplete cell markers obtained from fluorescent images for initial training on the Induced Pluripotent Stem (iPS) cell dataset, which is rarely studied for cell detection and tracking. During training, the incomplete initial labels were updated iteratively by combining detection and tracking results to obtain a model with better robustness. Our method was evaluated using two fields of the iPS cell dataset, along with the cell detection accuracy (DET) evaluation metric from the Cell Tracking Challenge (CTC) initiative, and it achieved 0.862 and 0.924 DET, respectively. The transferability of the developed model was tested using the public dataset FluoN2DH-GOWT1, which was taken from CTC; this contains two datasets with reference annotations. We randomly removed parts of the annotations in each labeled data to simulate the initial annotations on the public dataset. After training the model on the two datasets, with labels that comprise 10% cell markers, the DET improved from 0.130 to 0.903 and 0.116 to 0.877. When trained with labels that comprise 60% cell markers, the performance was better than the model trained using the supervised learning method. This outcome indicates that the model's performance improved as the quality of the labels used for training increased.

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

Involvement of kallikrein-PAR2-proinflammatory pathway in severe trastuzumab-induced cardiotoxicity.

Trastuzumab-induced cardiotoxicity interferes with continued treatment in approximately 10% of patients with ErbB2-positive breast cancer, but its mechanism has not been fully elucidated. In this study, we recruited trastuzumab-treated patients with ≥30% reduction in left ventricular ejection fraction (SP) and noncardiotoxic patients (NP). From each of these patients, we established three cases of induced pluripotent stem cell-derived cardiomyocytes (pt-iPSC-CMs). Reduced contraction and relaxation velocities following trastuzumab treatment were more evident in SP pt-iPSC-CMs than NP pt-iPSC-CMs, indicating the cardiotoxicity phenotype could be replicated. Differences in ATP production, reactive oxygen species, and autophagy activity were observed between the two groups. Analysis of transcripts revealed enhanced kallikrein5 expression and pro-inflammatory signaling pathways, such as interleukin-1ß, in SP pt-iPSC-CMs after trastuzumab treatment. The kallilkrein5-protease-activated receptor 2 (PAR2)-MAPK signaling pathway was more activated in SP pt-iPSC-CMs, and treatment with a PAR2-antagonist suppressed interleukin-1ß expression. Our data indicate enhanced pro-inflammatory responses through kallikrein5-PAR2 signaling and vulnerability to external stresses appear to be the cause of trastuzumab-induced cardiotoxicity in SP.

Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy.

Adoptive immunotherapy has emerged as a powerful approach to cure cancer and chronic infections. Currently, the generation of a massive number of T cells that provide long-lasting immunity is challenged by exhaustion and differentiation-associated senescence, which inevitably arise during in vitro cloning and expansion. To circumvent these problems, several studies have proposed an induced pluripotent stem cell (iPSC)-mediated rejuvenation strategy to revitalize the exhausted/senescent T cell clones. Because iPSC-derived cytotoxic T lymphocytes (iPSC-CTLs) generated via commonly used monolayer systems have unfavorable, innate-like features such as aberrant natural killer (NK) activity and limited replication potential, we modified the redifferentiation culture to generate CD8αß+CD5+CCR7+CD45RA+CD56--adaptive iPSC-CTLs. The modified iPSC-CTLs exhibited early memory phenotype, including high replicative capacity and the ability to give rise to potent effector cells. In expansion culture with an optimized cytokine cocktail, iPSC-CTLs proliferated more than 1015-fold in a feeder-free condition. Our redifferentiation and expansion package of early memory iPSC-CTLs could supply memory and effector T cells for both autologous and allogeneic immunotherapies.

Inhibition of Glycogen Synthase Kinase 3ß Enhances Functions of Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells in the Blood-Brain Barrier.

Brain microvascular endothelial cells (BMECs) are essential component of the blood-brain barrier (BBB). BMECs strictly regulate the entry of various molecules into the central nervous system from the peripheral circulation by forming tight junctions and expressing various influx/efflux transporters and receptors. In vitro BBB models have been widely reported with primary BMECs isolated from animals, although it is known that the expression patterns and levels of transporters and receptors in BMECs differ between humans and animals. Recently, several methods to differentiate BMECs from human induced pluripotent stem (hiPS) cell have been developed. However, the expression of P-glycoprotein (P-gp), which is a key efflux transporter, in hiPS cell-derived BMECs was detected at a relatively low level compared with primary human BMECs. In this study, we examined the involvement of the canonical Wnt signaling pathway, which contributes to the development of BBB formation, in the regulation of P-gp expression in hiPS cell-derived BMECs. We found that the barrier integrity was significantly enhanced in hiPS cell-derived BMECs treated with glycogen synthase kinase-3ß (GSK-3ß) inhibitors, which are known to positively regulate the canonical Wnt signaling pathway. In addition, our data also showed P-gp expression level was increased by treatment with GSK-3ß inhibitors. In conclusion, physiological barrier function and P-gp expression in BMECs can be enhanced by the canonical Wnt signaling pathway. Our results may be useful for promoting the development of drugs for central nervous system diseases using in vitro BBB model.

Proarrhythmia Risk Assessment Using Electro-Mechanical Window in Human iPS Cell-Derived Cardiomyocytes.

Evaluation of drug-induced cardiotoxicity is still challenging to avoid adverse effects, such as torsade de pointes (TdP), in non-clinical and clinical studies. Numerous studies have suggested that human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a useful platform for detecting drug-induced TdP risks. Comprehensive in vitro Proarrhythmia Assay (CiPA) validation study suggested that hiPSC-CMs can assess clinical TdP risk more accurately than the human ether-a-go-go-related assay and QT interval prolongation. However, there were still some outliers, such as bepridil, mexiletine, and ranolazine, among the CiPA 28 compounds in the CiPA international multi-site study using hiPSC-CMs. In this study, we assessed the effects of the positive compound dofetilide, the negative compound aspirin, and several CiPA compounds (bepridil, mexiletine, and ranolazine) on the electromechanical window (E-M window), which were evaluated using multi-electrode array assay and motion analysis, in hiPSC-CMs. Similar to previous in vivo studies, dofetilide, which has a high TdP risk, decreased the E-M window in hiPSC-CMs, whereas aspirin, which has a low TdP risk, had little effect. Bepridil, classified in the high TdP-risk group in CiPA, decreased the E-M window in hiPSC-CMs, whereas ranolazine and mexiletine, which are classified in the low TdP-risk group in CiPA, slightly decreased or had little effect on the E-M window of hiPSC-CMs. Thus, the E-M window in hiPSC-CMs can be used to classify drugs into high and low TdP risk.

Perspectives of future lung toxicology studies using human pluripotent stem cells.

The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.

Identification of Novel Regulator Involved in Differentiation of Mouse iPS Cells into Odontoblast-like Cells.

Odontoblasts differentiate from dental papilla stem cells, but the genetic changes that occur during this process remain unclear. The aim of this study was to investigate gene expression patterns during differentiation of mouse iPS cells into odontoblast-like cells. Mouse iPS cells were cultured on a collagen type-1 scaffold with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results of immunofluorescence studies for dentin sialoprotein, dentin matrix protein 1 (DMP1), and nestin were positive. A qRT-PCR analysis revealed that mRNA expression levels of neural crest marker sex determining region Y box (Sox)-10, dentin sialophosphoprotein (Dspp), and Dmp1 were up-regulated, but that mRNA expression levels of the mineralization markers bone sialoprotein and osteocalcin were down-regulated. Microarray analysis showed that 2,597 entities were up-regulated and 1,327 down-regulated among a total of 15,330 investigated. Sox11 was among the up-regulated genes identified. The Sox11 mRNA expression level with odontoblast induction after day 11 was higher than that after day 2 (p<0.05). Gene knockdown using small interference RNA (siRNA) silencing was used to characterize the function of Sox11. The Dspp mRNA expression level in Sox11 siRNA-treated cells was significantly lower than that in the control (p<0.05). These results suggest that BMP4 and RA induce mouse iPS cells to differentiate into odontoblast-like cells. The differentiation efficiency is not high, however, and many stem cells remain. The results also suggest that Sox11 is an important factor in odontoblastic differentiation.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Galectin 3-binding protein suppresses amyloid-ß production by modulating ß-cleavage of amyloid precursor protein.

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of ß-amyloid (Aß) peptides. Aß is produced from amyloid precursor protein (APP) that is sequentially cleaved by ß- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aß production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aß production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aß production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed Aß production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aß production by directly interacting with APP and thereby inhibiting APP processing by ß-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.