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Breast cancer is the most common invasive cancer in women, affecting more than 10% of women worldwide. Microscopic analysis of a biopsy remains one of the most important methods to diagnose the type of breast cancer. This requires specialized analysis by pathologists, in a task that i) is highly time- and cost-consuming and ii) often leads to nonconsensual results. The relevance and potential of automatic classification algorithms using hematoxylin-eosin stained histopathological images has already been demonstrated, but the reported results are still sub-optimal for clinical use. With the goal of advancing the state-of-the-art in automatic classification, the Grand Challenge on BreAst Cancer Histology images (BACH) was organized in conjunction with the 15th International Conference on Image Analysis and Recognition (ICIAR 2018). A large annotated dataset, composed of both microscopy and whole-slide images, was specifically compiled and made publicly available for the BACH challenge. Following a positive response from the scientific community, a total of 64 submissions, out of 677 registrations, effectively entered the competition. From the submitted algorithms it was possible to push forward the state-of-the-art in terms of accuracy (87%) in automatic classification of breast cancer with histopathological images. Convolutional neuronal networks were the most successful methodology in the BACH challenge. Detailed analysis of the collective results allowed the identification of remaining challenges in the field and recommendations for future developments. The BACH dataset remains publically available as to promote further improvements to the field of automatic classification in digital pathology., Accepted for publication at Medical Image Analysis (Elsevier). Publication licensed under the Creative Commons CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

© 2016 American College of Cardiology Foundation Background Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder. Objectives The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). Methods This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs. Results BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. Conclusions Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen–dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.

Breast cancer is one of the leading cancer-related death causes worldwide. Analysis of histology whole-slide and microscopy images is essential for early diagnosis. However this task is time consuming and has to be performed by specialists. Computer-aided Diagnosis can accelerate the process and reduce cost. Candy Cane, a system for performing pixel-wise labelling of whole-slide images in four classes: normal tissue, benign lesion, in situ carcinoma and invasive carcinoma is introduced. It uses fully convolutional Densenet architecture which performs successive convolutions and downsampling on input image followed by successive upsampling and transpose convolution to predict the label image. This allows the system to examine both microscopic low level details as well as high level features and organization of the tissue to make its pixel labeling decision. Accuracy s score of 0.501 are achieved in four-class pixelwise labeling of images on test set.

Summary There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts., Graphical Abstract, Highlights • Innate immunity is crucial in rejection of minor HA mismatched grafts • Stem cell alloimmune responses modeled with an “allogenized mouse” • Humanized mice are unable to fully model immune responses to stem cell allografts • Splenocytes and graft-infiltrating lymphocytes display an exhausted phenotype, Kooreman et al. use various types of humanized mice for the modeling of pluripotent stem cell alloimmunity. They report the development of a wasting disease-like syndrome within these mice over time, limiting their functionality, and provide ways to address this by using an immune reconstituted “allogenized” mouse model.

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_ AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/ AKT/ mTOR (mammalian target of rapamycin) pathway or GSK3 beta inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.

Defects in urothelial integrity resulting in leakage and activation of underlying sensory nerves are potential causative factors of bladder pain syndrome, a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. Herein, we identified the microRNA miR-199a-5p as an important regulator of intercellular junctions. On overexpression in urothelial cells, it impairs correct tight junction formation and leads to increased permeability. miR-199a-5p directly targets mRNAs encoding LIN7C, ARHGAP12, PALS1, RND1, and PVRL1 and attenuates their expression levels to a similar extent. Using laser microdissection, we showed that miR-199a-5p is predominantly expressed in bladder smooth muscle but that it is also detected in mature bladder urothelium and primary urothelial cultures. In the urothelium, its expression can be up-regulated after activation of cAMP signaling pathways. While validating miR-199a-5p targets, we delineated novel functions of LIN7C and ARHGAP12 in urothelial integrity and confirmed the essential role of PALS1 in establishing and maintaining urothelial polarity and junction assembly. The present results point to a possible link between miR-199a-5p expression and the control of urothelial permeability in bladder pain syndrome. Up-regulation of miR-199a-5p and concomitant down-regulation of its multiple targets might be detrimental to the establishment of a tight urothelial barrier, leading to chronic pain.

Summary Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca 2+ ) imaging indicated dysregulation of Ca 2+ cycling and elevation in intracellular Ca 2+ ([Ca 2+ ] i ) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca 2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies.

We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM.

What’s known on the subject? and What does the study add? In this paper we established the mRNA expression profiles of selected genes involved in bladder contractility and epithelial permeability in the bladder dome and trigone in order to evaluate the use of cold cut biopsies for comparative quantitative studies reflecting the anatomical differences between the bladder regions. Using QPCR and immunofluorescence we demonstrated differences in the amount of smooth muscle and the structure of urothelium of the dome and trigon. Our mRNA and immunofluorescence data show that cold cut biopsies from the bladder dome have a higher relative SM content compared to the trigone, reflecting a well-developed network of sub-urothelial myofibroblasts and muscularis mucosae present in the bladder dome. An up-regulation of the genes encoding the tight junction proteins in the bladder trigone is independent of the urothelium content, and might imply further discrepancy between these regions. OBJECTIVE • To establish the mRNA expression profiles of selected genes involved in bladder contractility and epithelial permeability in the bladder dome and trigone in order to evaluate the use of cold-cut biopsies for comparative quantitative studies into the anatomical differences between these two bladder regions. PATIENTS AND METHODS • After informed consent, cold-cut biopsies from the bladder dome and trigone were obtained from eight asymptomatic subjects. • RNA was extracted from muscle biopsies, and the expression levels of selected genes were analysed using TaqMan real-time PCR-based gene expression assays. • Protein levels and localization were investigated by immunofluorescence. RESULTS • mRNA levels of NK2 receptor, P2X1, ASIC1a and muscarinic cholinergic receptors M2, and M3 were significantly higher in the dome than in the trigone (P < 0.05). In contrast, the mRNA levels of cellular adhesion and tight junction proteins were up-regulated in the trigone. • There were no significant differences in expression levels of NK1R, and TRPV1 between the dome and trigone. • Although the mRNA levels of uroplakin UP2 were similar in both sample groups, the smooth muscle (SM) markers were up-regulated in the dome biopsies, indicating the higher SM content of these biopsies. • Consistent with these observations, when normalizing for the SM content, there were no significant differences in the levels of SM-specific markers between the two sample groups. In contrast, occludin, junctional adhesion molecule 1, claudins 1 and 4, γ-catenin and E-cadherin were up-regulated in the trigone. • These observations were confirmed by immunofluorescence labelling, showing differences in the amount of SM and in the structure of urothelium of the dome and trigone. CONCLUSIONS • Our mRNA and immunofluorescence data show that cold-cut biopsies from the bladder dome have a higher relative SM content compared with the trigone, reflecting a well-developed network of suburothelial myofibroblasts and muscularis mucosae present in the bladder dome. • An up-regulation of the genes encoding the tight junction proteins in the bladder trigone is independent of the urothelium content, and might imply further discrepancy between these regions.

PURPOSE We examined the possible role of H(+) activated acid sensing ion channels in pain perception. We characterized expression in bladder dome biopsies from patients with bladder pain syndrome and controls in cultured human urothelium and in urothelial TEU 2 cells. MATERIALS AND METHODS Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with bladder pain syndrome symptoms. Acid sensing ion channel expression was analyzed by quantitative real time polymerase chain reaction and immunofluorescence. Channel function was measured by electrophysiology. RESULTS Acid sensing ion channel 1a 2a and 3 mRNA was detected in the human bladder. Similar amounts of acid sensing ion channel 1a and 3 were detected in detrusor smooth muscle while in urothelium acid sensing ion channel 3 levels were higher than levels of acid sensing ion channel 1a. Acid sensing ion channel 2a mRNA levels were lower than acid sensing ion channel 1a and 3 levels in each layer. Acid sensing ion channel currents were measured in TEU 2 cells and in primary cultures of human urothelium. Activated acid sensing ion channel expression was confirmed by quantitative real time polymerase chain reaction. TEU 2 cell differentiation caused acid sensing ion channel 2a and 3 mRNA up regulation and acid sensing ion channel 1a mRNA down regulation. Patients with bladder pain syndrome showed up regulation of acid sensing ion channel 2a and 3 mRNA but acid sensing ion channel 1a remained unchanged. In contrast transient receptor potential vanilloid 1 mRNA was down regulated during bladder pain syndrome. All differences were statistically significant (p

In skeletal muscle of patients with clinically diagnosed statin-associated myopathy, discrete signs of structural damage predominantly localize to the T-tubular region and are suggestive of a calcium leak. The impact of statins on skeletal muscle of non-myopathic patients is not known. We analyzed the expression of selected genes implicated in the molecular regulation of calcium and membrane repair, in lipid homeostasis, myocyte remodeling and mitochondrial function. Microscopic and gene expression analyses were performed using validated TaqMan custom arrays on skeletal muscle biopsies of 72 age-matched subjects who were receiving statin therapy (n = 38), who had discontinued therapy due to statin-associated myopathy (n = 14), and who had never undergone statin treatment (n = 20). In skeletal muscle, obtained from statin-treated, non-myopathic patients, statins caused extensive changes in the expression of genes of the calcium regulatory and the membrane repair machinery, whereas the expression of genes responsible for mitochondrial function or myocyte remodeling was unaffected. Discontinuation of treatment due to myopathic symptoms led to a normalization of gene expression levels, the genes encoding the ryanodine receptor 3, calpain 3, and dystrophin being the most notable exceptions. Hence, even in clinically asymptomatic (non-myopathic) patients, statin therapy leads to an upregulation in the expression of genes that are concerned with skeletal muscle regulation and membrane repair.

Background: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown. Methods: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury. Results: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10× the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3). Interpretation: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

Cardiomyocytes derived from human induced pluripotent stem cells (hIPS-CM) are useful to understand basic structural and functional characteristics of normal and diseased human heart cells. We investigated mechanical properties of hIPS-CM derived from unaffected and affected members of a family harboring a dilated cardiomyopathy (DCM) mutation in cardiac troponin T (cTnT), the tropomyosin binding unit of troponin. Patients with a cTnT point mutation (R173W) develop DCM, which commonly leads to diastolic and systolic dysfunction and progressive heart failure. To study the shortening and shortening velocity of normal and cTnT mutant hIPS-CM cells, cells were plated on substrates with a relatively soft stiffness (160 kPa) molded from polydimethylsiloxane and measured using a line scan method. Data were collected using a Zeiss 710 confocal microscope. Visual observation of cells after 5 days maturity on the PDMS substrates indicate a smaller percent of mutant (8%) compared to normal (87%) hIPS-CM were spontaneously beating. Immunohistochemistry showed that myofibril structure was better developed in normal compared to cTnT mutation cells. Day 5 line scans revealed normal hIPS-CM cells shorten more (0.47 μm) than mutant cTnT cells (0.32 μm); shortening velocity was faster in normal (1.42 μm/s) compared to mutant cTnT cells (0.67 μm/s). Acute treatment (1 dose with a 10 min activation dwell time) with omecamtiv mecarbil (200nM), a cardiac myosin activator, increased the shortening and shortening velocity of normal (0.78 μm at 1.85 μm/s) but not cTnT mutant (0.32 μm at 0.70 μm/s) hIPS-CM cells. Although more studies are necessary, these results suggest that cTnT may affect development of sarcomeres and the regulation of contractility. Furthermore, the myosin activator omecamtiv mecarbil may not be sufficient to rescue dysfunction in the cTnT mutation R173W. [Funding: NIH T32HL07692, PO1HL62426]