Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Robotic search for optimal cell culture in regenerative medicine.

Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that obtained by the pre-optimized culture in terms of the pigmentation scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.

Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion.

Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor ß signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.

A Variable Scheduling Maintenance Culture Platform for Mammalian Cells.

Cell culturing is a basic experimental technique in cell biology and medical science. However, culturing high-quality cells with a high degree of reproducibility relies heavily on expert skills and tacit knowledge, and it is not straightforward to scale the production process due to the education bottleneck. Although many automated culture systems have been developed and a few have succeeded in mass production environments, very few robots are permissive of frequent protocol changes, which are often required in basic research environments. LabDroid is a general-purpose humanoid robot with two arms that performs experiments using the same tools as humans. Combining our newly developed AI software with LabDroid, we developed a variable scheduling system that continuously produces subcultures of cell lines without human intervention. The system periodically observes the cells on plates with a microscope, predicts the cell growth curve by processing cell images, and decides the best times for passage. We have succeeded in developing a system that maintains the cultures of two HEK293A cell plates with no human intervention for 192 h.

Depth inversion with a 3D structure influences brightness perception.

Whether or not depth perception influences brightness and/or lightness perception has been repeatedly discussed, and some studies have emphasized its importance. In addition, a small number of studies have empirically tested and shown the effect of depth inversion, such as seen in the Mach card illusion, on perceived lightness, and they interpreted such results in terms of lightness constancy. However, how perceived brightness changes contingent on depth inversion remains unexplained. Therefore, this study used the matching method to examine changes in brightness perception when depth inversion is observed. We created and used a three-dimensional (3D) concave object, composed of three sides made of card stock, which could be perceived as having two different shapes in 3D; it could be perceived as a horizontal concave object, corresponding to its actual physical structure, and as a convex standing object, similar in shape to a building. Participants observed this object as both a concave object and as a convex object, and judged the brightness of its surfaces during each observation. Our results show that the perception of the brightness of the object's surfaces clearly changed depending on the perception of depth. When the object was seen as convex, one part of the surface was perceived as darker than when the object was seen as concave, but the other part of the surface remained unchanged. Here we discuss the relationship between depth perception and brightness perception in terms of perceptual organization.

CD147/Basigin Deficiency Prevents the Development of Podocyte Injury through FAK Signaling.

Podocytes, which are susceptible to injury by various stimuli and stress, are critical regulators of proteinuric kidney diseases, regardless of the primary disease and pathogenesis. We further confirmed a significant correlation between urinary CD147/basigin (Bsg) levels and proteinuria in patients with focal segmental glomerulosclerosis. However, the molecular mechanism of podocyte injury involving Bsg is not fully understood. Here, the involvement of Bsg in the pathogenesis of podocyte injury was elucidated. Healthy podocytes rarely express Bsg protein. In two independent mouse models, including adriamycin-induced nephropathy and Nω-nitro-l-arginine methyl ester (l-name)-induced endothelial dysfunction, Bsg induction in injured podocytes caused podocyte effacement, which led to development of proteinuria. Bsg silencing in cultured podocytes exposed to transforming growth factor-ß suppressed focal adhesion rearrangement and cellular motility via the activation of ß1 integrin-focal adhesion kinase-matrix metallopeptidase signaling. In addition, induction of vascular endothelial growth factor and endothelin-1, which are implicated in podocyte-to-endothelial cross-communication, was lower in the supernatants of cultured Bsg-silenced podocytes stimulated with transforming growth factor-ß. In this setting, Bsg may be involved in a physiological positive feedback loop that accelerates podocyte cell motility and depolarization. The current study thus suggests that Bsg silencing via suppression of ß1 integrin-focal adhesion kinase-matrix metallopeptidase signaling may be an attractive therapeutic strategy for the maintenance of podocytes in patients with proteinuric kidney diseases.

miR-146a targeted to splenic macrophages prevents sepsis-induced multiple organ injury.

Development of a novel agent against life-threatening sepsis requires the in-depth understanding of the relevant pathophysiology and therapeutic targets. Given the function of microRNAs (miRNAs) as potent oligonucleotide therapeutics, here we investigated the pathophysiological role of exogenously applied miRNA in sepsis-induced multiple organ injury. In vitro, miR-16, miR-126, miR-146a, and miR-200b suppressed the production of pro-inflammatory cytokines in RAW264.7 macrophage cells after lipopolysaccharide (LPS) stimulation. Of these, miR-146a displayed the most highly suppressive effect, wherein the transcriptional activity of nuclear factor kappa B (NF-κB) was decreased via targeting of interleukin 1 receptor-associated kinase 1 and tumor necrosis receptor-associated factor 6. Sepsis was induced in mice via cecal ligation and puncture (CLP) and an intravenous injection of a complex of miR-146a-expressing plasmid and polyethyleneimine. Treatment with this complex significantly decreased the level of serum inflammatory cytokines, attenuated organ injury including kidney injury, and led to increased survival from polymicrobial sepsis induced by CLP. miR-146a-expressing plasmid was abundantly distributed in splenic macrophages, but not in renal parenchymal cells. CLP mice treated with miR-146a displayed significantly decreased NF-κB activation and splenocyte apoptosis. Splenectomy diminished the anti-inflammatory effects of miR-146a. The collective results support the conclusion that the induction of miR-146a expression in splenic macrophages prevents excessive inflammation and sepsis-induced multiple organ injury. This study establishes a novel and critical pathophysiological role for splenic macrophage interference in sepsis-related organ injury.

Therapeutic efficacy of rituximab for the management of adult-onset steroid-dependent nephrotic syndrome: a retrospective study.

BACKGROUND: Recent reports have described the efficacy of rituximab in treating steroid-dependent nephrotic syndrome (SDNS) in pediatric patients. However, few reports describe data regarding adult-onset SDNS. We investigated the efficacy of rituximab for the management of adult-onset SDNS. METHODS: We performed a retrospective cohort study investigating eight patients with adult-onset SDNS who were treated with rituximab. Clinical data were obtained at the initiation of rituximab therapy. The primary outcomes evaluated were successful suppression of relapses and CD19+ cells after rituximab treatment. The corticosteroid- and immunosuppressant-sparing effect and adverse events were additionally evaluated. RESULTS: All eight patients were diagnosed with minimal change nephrotic syndrome and received immunosuppressants in addition to corticosteroid. Total number of relapses was 10.5 times as a median value. Rituximab administration was repeated in two patients, whereas six received single-dose rituximab. Three of eight (37.5%) patients showed relapse after rituximab therapy. A rituximab-induced depletion in CD19+ cells noted initially was followed by their reappearance in all patients. There were cases with no relapse after the reappearance of CD19+ cells. The median relapse time pre- and post-rituximab therapy showed a decrease from 1 time/year (interquartile range [IQR] 1-3 times/year) to 0 time/year (IQR 0-1 time/year). Rituximab treatment induced a significant reduction in the required doses of corticosteroid and cyclosporine (P < 0.01). No serious adverse events were observed. CONCLUSION: Rituximab treatment was effective not only in childhood-onset but also in adult-onset SDNS. Further studies are needed to establish optimal treatment regimens.

The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

The clinical relevance of plasma CD147/basigin in biopsy-proven kidney diseases.

BACKGROUND: Precise understanding of kidney disease activity is needed to design therapeutic strategies. CD147/basigin is involved in the pathogenesis of acute kidney injury and renal fibrosis through inflammatory cell infiltration. The present study examined the clinical relevance of CD147 in biopsy-proven kidney diseases that lead to the progression of chronic kidney disease. METHODS: Kidney biopsy specimens and plasma and urine samples were obtained from patients with kidney diseases, including IgA nephropathy (IgAN), Henoch-Schönlein purpura nephritis (HSPN), diabetic kidney disease (DKD), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN), who underwent renal biopsy between 2011 and 2014. Plasma and urinary CD147 levels were measured and evaluated for their ability to reflect histological features. Disease activity of IgAN tissues was evaluated according to the Oxford classification and the Japanese histological grading system. RESULTS: In biopsy tissues, CD147 induction was detected in injured lesions representing renal inflammation. Plasma CD147 values correlated with eGFR in patients with inflammation-related kidney diseases such as IgAN, HSPN, and DKD. Particularly in IgAN patients, plasma CD147 levels were correlated with injured regions comprising more than 50% of glomeruli or with tubular atrophy/interstitial injury in biopsy tissues. Proteinuria showed a closer correlation with urinary values of CD147 and L-FABP. Of note, plasma and urinary CD147 levels showed a strong correlation with eGFR or proteinuria, respectively, only in DKD patients. CONCLUSION: Evaluation of plasma and urinary CD147 levels might provide key insights for the understanding of the activity of various kidney diseases.

Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk+/+) mice showed more severe glomerular injury than MK-deficient (Mdk-/-) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk-/- mice, the frequency of splenic CD69+ T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk+/+ mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4+ T cells in vivo and in vitro. MK induced activated CD4+ T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4+ T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4+ T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.

Off Target, but Sequence-Specific, shRNA-Associated Trans-Activation of Promoter Reporters in Transient Transfection Assays.

Transient transfection promoter reporter assays are commonly used in the study of transcriptional regulation, and can be used to define and characterize both cis-acting regulatory sequences and trans-acting factors. In the process of using a variety of reporter assays designed to study regulation of the rhodopsin (rho) promoter, we discovered that rhodopsin promoter-driven reporter expression could be activated by certain species of shRNA in a gene-target-independent but shRNA sequence-specific manner, suggesting involvement of a specific shRNA associated pathway. Interestingly, the shRNA-mediated increase of rhodopsin promoter activity was synergistically enhanced by the rhodopsin transcriptional regulators CRX and NRL. Additionally, the effect was cell line-dependent, suggesting that this pathway requires the expression of cell-type specific factors. Since microRNA (miRNA) and interferon response-mediated processes have been implicated in RNAi off-target phenomena, we performed miRNA and gene expression profiling on cells transfected with shRNAs that do target a specific gene but have varied effects on rho reporter expression in order to identify transcripts whose expression levels are associated with shRNA induced rhodopsin promoter reporter activity. We identified a total of 50 miRNA species, and by microarray analysis, 320 protein-coding genes, some of which were predicted targets of the identified differentially expressed miRNAs, whose expression was altered in the presence of shRNAs that stimulated rhodopsin-promoter activity in a non-gene-targeting manner. Consistent with earlier studies on shRNA off-target effects, a number of interferon response genes were among those identified to be upregulated. Taken together, our results confirm the importance of considering off-target effects when interpreting data from RNAi experiments and extend prior results by focusing on the importance of including multiple and carefully designed controls in the design and analysis of the effects of shRNA on transient transfection-based transcriptional assays.

Chimpanzees can visually perceive differences in the freshness of foods.

Colour vision in primates is believed to be an adaptation for finding ripe fruit and young leaves. The contribution of the luminance distribution, which influences how humans evaluate the freshness of food, has not been explored with respect to the detection of subtle distinctions in food quality in non-human primates. We examined how chimpanzees, which are closely related to humans, perceive the freshness of foods. The findings suggest that chimpanzees were able to choose fresher cabbage based on both colour and grey-scale images. Additional tests with images of novel cabbage, spinach, and strawberries revealed that one chimpanzee could detect the freshness of other fruits and vegetables. The critical factor in determining the judgements of freshness made by the chimpanzees was the spatial layout of luminance information. These findings provide the first known evidence that chimpanzees discriminate between images representing various degrees of freshness based solely on luminance information.

Eleven-month-old infants infer differences in the hardness of object surfaces from observation of penetration events.

Previous studies have shown different developmental trajectories for object recognition of solid and non-solid objects. However, there is no evidence as to whether infants have expectations regarding certain attributes of objects, such as surface hardness, in the absence of tactile information. In the present study, we examined infants' perception of the hardness of object surfaces from visually presented penetration events using the familiarization-novelty preference procedure. Experiment 1 showed that by 11 months old infants distinguished a relatively soft surface from a crusty surface based on changes in the velocity of a moving object as the moving object penetrated the surface of the target object. Experiment 2 ruled out the possibility that infants were merely sensitive to differences in the velocity changes in the stimuli.

Gene regulation: it matters who you hang out with.

How broadly expressed transcription factors contribute to tissue-specific gene expression is not well understood. Andzelm, Cherry et al. (2015) demonstrate that myocyte enhancer factor 2D (MEF2D) binds and activates retina-specific regulatory regions by cooperative interaction with the tissue-specific transcription factor CRX.

Midkine Regulates BP through Cytochrome P450-Derived Eicosanoids.

The effects of endothelium-derived hyperpolarizing factors have been attributed to cytochrome P450-derived epoxyeicosatrienoic acids (EETs), but the regulation and role of EETs in endothelial dysfunction remain largely unexplored. Hypertension is a primary risk factor for renal dysfunction, which is frequently accompanied by various systemic diseases induced by endothelial dysfunction in the microcirculation. We previously reported that the endothelial growth factor midkine (MK) enhances hypertension in a model of CKD. Here, we investigated the hypothesis that MK regulates EET activity and thereby BP. MK gene-deleted mice were resistant to hypertension and developed less glomerulosclerosis and proteinuria after administration of a nitric oxide synthase (NOS) inhibitor in the setting of uninephrectomy. The hypertension observed in uninephrectomized wild-type mice after NOS inhibition was ameliorated by anti-MK antibody. MK-deficient mice produced higher amounts of EETs, and EETs dominantly regulated BP in these mice. Furthermore, MK administration to MK-deficient mice recapitulated the BP control observed in wild-type mice. EETs also dominantly regulated renal blood flow, which may influence renal function, in MK-deficient mice. Taken together, these results suggest that the MK/EET pathway is physiologically engaged in BP control and could be a target for the treatment of hypertension complicated by endothelial dysfunction.

Material perception of a kinetic illusory object with amplitude and frequency changes in oscillated inducer motion.

The magnitude of the phase difference between inducers' oscillation of a kinetic illusory surface influences visual material impressions (Masuda et al., 2013). For example, impressions of bending or waving motions on a surface tend to occur at a 30- or 90-deg. phase difference, respectively. Here, we elucidate whether amplitude and frequency changes in an inducer's oscillation influence the visual impressions of an illusory surface's hardness, elasticity, and viscosity. Nine participants were asked to use an analog scale to judge their visual impressions relative to a standard pattern with no damping and no frequency change for each phase difference. Results revealed that hardness ratings were greater when amplitude decayed with time only in the 30-deg. phase difference. Elasticity ratings were greater when the frequency of oscillation had a large increase in the 90-deg. phase difference. In the 30-deg. phase difference, similar tendencies were only observed with no damping and ample damping. Viscosity ratings were greater when the frequency of oscillation decreased in both phase differences and when the amplitude decayed with time in the 30-deg. phase difference. These findings suggest that amplitude and frequency changes in an inducer's oscillation are significant factors for material perception derived from motion.

The transcription factor GTF2IRD1 regulates the topology and function of photoreceptors by modulating photoreceptor gene expression across the retina.

The mechanisms that specify photoreceptor cell-fate determination, especially as regards to short-wave-sensitive (S) versus medium-wave-sensitive (M) cone identity, and maintain their nature and function, are not fully understood. Here we report the importance of general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1) in maintaining M cone cell identity and function as well as rod function. In the mouse, GTF2IRD1 is expressed in cell-fate determined photoreceptors at postnatal day 10. GTF2IRD1 binds to enhancer and promoter regions in the mouse rhodopsin, M- and S-opsin genes, but regulates their expression differentially. Through interaction with the transcription factors CRX and thyroid hormone receptor ß 2, it enhances M-opsin expression, whereas it suppresses S-opsin expression; and with CRX and NRL, it enhances rhodopsin expression. In an apparent paradox, although GTF2IRD1 is widely expressed in multiple cell types across the retina, knock-out of GTF2IRD1 alters the retinal expression of only a limited number of annotated genes. Interestingly, however, the null mutation leads to altered topology of cone opsin expression in the retina, with aberrant S-opsin overexpression and M-opsin underexpression in M cones. Gtf2ird1-null mice also demonstrate abnormal M cone and rod electrophysiological responses. These findings suggest an important role for GTF2IRD1 in regulating the level and topology of rod and cone gene expression, and in maintaining normal retinal function.

Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.