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Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising experimental tool for translational heart research and drug development. However, their usability as a human adult cardiomyocyte model is limited by their functional immaturity. Our aim is to analyse quantitatively those characteristics and how they differ from adult CMs. Methods and Results: We have developed a novel in silico model with all essential functional electrophysiology and calcium handling features of hiPSC-CMs. Importantly, the virtual cell recapitulates the immature intracellular ion dynamics that are characteristic for hiPSC-CMs, as quantified based our in vitro imaging data. The strong "calcium clock" is a source for a dual function of excitation-contraction coupling in hiPSC-CMs: action potential and calcium transient morphology vary substantially depending on the activation sequence of underlying ionic currents and fluxes that is altered in spontaneous vs. paced mode. Furthermore, parallel simulations with hiPSC-CM and adult cardiomyocyte models demonstrate the central differences. Results indicate that hiPSC-CMs translate poorly the disease specific phenotypes of Brugada syndrome, long QT Syndrome and catecholaminergic polymorphic ventricular tachycardia, showing less robustness and greater tendency for arrhythmic events than adult CMs. Based on a comparative sensitivity analysis, hiPSC-CMs share some features with adult CMs, but are still functionally closer to prenatal CMs than adult CMs. A database analysis of 3000 hiPSC-CM model variants suggests that hiPSC-CMs recapitulate poorly fundamental physiological properties of adult CMs. Single modifications do not appear to solve this problem, which is mostly contributed by the immaturity of intracellular calcium handling. Conclusion: Our data indicates that translation of findings from hiPSC-CMs to human disease should be made with great caution. Furthermore, we established a mathematical platform that can be used to improve the translation from hiPSC-CMs to human, and to quantitatively evaluate hiPSC-CMs development toward more general and valuable model for human cardiac diseases. [ABSTRACT FROM AUTHOR]

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor . Neurons formed end plate like structures with strong binding of a-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration. [ABSTRACT FROM AUTHOR]

Prolyl oligopeptidase (POP) has been connected to memory and mood through regulation of the brain levels of its biologically active peptide substrates and phosphatidylinositol system. This is the first study in a radial-arm maze of the effects of a single dose of a novel potent prolyl oligopeptidase inhibitor, KYP-2047 (5 mg/kg, dissolved in 5% Tween 80), on memory and learning of scopolamine-treated (0.4 mg/kg, dissolved in saline) rats. Habituated (days 1 and 2) and trained (days 3–11) young (3 months) and old (8–9 months) male Wistar rats were given (i) saline + Tween, (ii) saline + KYP-2047, (iii) scopolamine + Tween or (iv) scopolamine + KYP-2047 30 min. prior to testing their memory. Food rewards located in four randomly chosen arms of the maze. The rat had 10 min. to find and eat the rewards. Time spent in the maze, visits to each arm and number of eaten rewards were measured. Old rats made generally more errors, spent more time and visited fewer arms per minute in the maze than young rats. The memory- and function-impairing effects of scopolamine were also seen more clearly in old than young rats. KYP-2047 had no or only a marginal effect on memory of either age group, but when given without scopolamine, it slightly increased the maze motility of young rats and decreased the motility of old rats. In a separate locomotor activity test, KYP-2047 enhanced the motility of young rats supporting a suggested role of POP in motor functions. [ABSTRACT FROM AUTHOR]

The effects of a novel prolyl oligopeptidase (POP) inhibitor KYP-2047 on spatial memory of young (3-month-old) and old (8- to 9-month-old) scopolamine-treated rats (0.4 mg/kg intraperitoneally) was investigated in the Morris water maze. In addition, the concentrations of promnesic neuropeptide substrates of POP, substance P and neurotensin in various brain areas after acute and chronic POP inhibition were measured in young rats. In addition, inositol-1,4,5-trisphosphate (IP3) levels were assayed in rat cortex and hippocampus after effective 2.5-day POP inhibition. KYP-2047 (1 or 5 mg/kg 30 min. before daily testing) dose-dependently improved the escape performance (i.e. latency to find the hidden platform and swimming path length) of the young but not the old rats in the water maze. POP inhibition had no consistent effect on substance P levels in cortex, hippocampus or hypothalamus, and only a modest increase in neurotensin concentration was observed in the hypothalamus after a single dose of KYP-2047. Moreover, IP3 concentrations remained unaffected in cortex and hippocampus after POP inhibition. In conclusion, the behavioural data support the earlier findings of the promnesic action of POP inhibitors, but the mechanism of the memory-enhancing action remains unclear. [ABSTRACT FROM AUTHOR]

In Parkinson's disease (PD), the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta is associated with Lewy bodies arising from the accumulation of alpha-synuclein protein which leads ultimately to movement impairment. While PD has been considered a disease of the DA neurons, a glial contribution, in particular that of astrocytes, in PD pathogenesis is starting to be uncovered. Here, we report findings from astrocytes derived from induced pluripotent stem cells of LRRK2 G2019S mutant patients, with one patient also carrying a GBA N370S mutation, as well as healthy individuals. The PD patient astrocytes manifest the hallmarks of the disease pathology including increased expression of alpha-synuclein. This has detrimental consequences, resulting in altered metabolism, disturbed Ca2+ homeostasis and increased release of cytokines upon inflammatory stimulation. Furthermore, PD astroglial cells manifest increased levels of polyamines and polyamine precursors while lysophosphatidylethanolamine levels are decreased, both of these changes have been reported also in PD brain. Collectively, these data reveal an important role for astrocytes in PD pathology and highlight the potential of iPSC-derived cells in disease modeling and drug discovery. [ABSTRACT FROM AUTHOR]

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development. [ABSTRACT FROM AUTHOR]

It has remained unclear why schizophrenia typically manifests after adolescence and which neurobiological mechanisms are underlying the cascade leading to the actual onset of the illness. Here we show that the use of induced pluripotent stem cell-derived neurons of monozygotic twins from pairs discordant for schizophrenia enhances disease-specific signal by minimizing genetic heterogeneity. In proteomic and pathway analyses, clinical illness is associated especially with altered glycosaminoglycan, GABAergic synapse, sialylation, and purine metabolism pathways. Although only 12% of all 19,462 genes are expressed differentially between healthy males and females, up to 61% of the illness-related genes are sex specific. These results on sex-specific genes are replicated in another dataset. This implies that the pathophysiology differs between males and females, and may explain why symptoms appear after adolescence when the expression of many sex-specific genes change, and suggests the need for sex-specific treatments. Noise due to genetic heterogeneity potentially impacts the the discovery of genes that contribute to diseases such as schizophrenia (SCZ). In this study, authors minimize the disease-irrelevant noise between SCZ and healthy individuals by profiling transcriptional signatures among discordant monozygotic twin pairs, and demonstrate that although sexes share many of the final common pathways, the underlying primary pathophysiology of SCZ differs between males and females. [ABSTRACT FROM AUTHOR]

Background The estimated heritability of schizophrenia is 80–85%, and if one of monozygotic twins has the illness, the risk for the other twin is about 50%. It has remained unclear which factors are associated just with the increased risk, and which neurobiological mechanisms are related to the cascade leading to actual onset of illness. It has also remained a mystery why this neurodevelopmental disorder manifests itself after adolescence. Methods We used iPSC-derived neurons from 5 monozygotic twin pairs discordant for schizophrenia (3 female and 2 male pairs; all 5 affected individuals were chronically symptomatic and treated with antipsychotics, 3 with clozapine) and 6 healthy controls matched by age and sex to enhance signal by minimizing genetic heterogeneity. Diagnoses were assigned using DSM-IV criteria and SCID-I interviews, and severity of symptoms was assessed with PANSS. Skin biopsy-derived fibroblasts were reprogrammed into integration-free induced pluripotent stem cell (iPSCs) lines by Sendai virus technology. The cells were further differentiated into cortical-like neurons expressing markers of GABAergic and glutamatergic neurons. The top genes from RNAseq analysis were validated by quantitative polymerase chain reaction (qPCR). In the proteomic analysis, the 16 samples were processed through the SysQuant workflow using Tandem Mass Tag (TMT) reagents within two TMT 10plexes. A reference pool containing all samples was also included. To identify statistically significant regulated features (peptide, phosphopeptides or proteins), a LIMMA-based modified t-test was performed at the peptide, phosphopeptide and protein levels in pairwise comparisons using distribution-dependent fold change (FC) and p-value (p) thresholds. Results Transcriptomic and proteomic analyses showed very large effect sizes (differences up to 7.3-fold in gene expression and 2.7-fold in protein levels in the comparison between affected twin versus healthy twin). Among the most robust findings related to disease status were down-regulation of COL6A3, SSTR2, and LHX1 genes, and altered glycosaminoglycan, CAMK2G-related AMPA/NMDA/glutamate, GABAergic synapse, and purine metabolism pathways. While only 12% of genes were differentially expressed between healthy males and females, up to 61% of the illness-related genes were sex-specific (p-values down to 2.3 x 10E-295 for the difference). Most of the genes with the largest effect sizes were different between males and females. At gestational age of 90 days, neurons of affected twins showed altered calcium responses for NMDA-specific glutamate (p < 0.001) and for GABA exposure compared with their healthy twins, and these alterations were normalized when the neurons were treated with clozapine. Discussion Results imply that somatostatin 2 receptor defect may contribute to dysfunction of NMDA receptors in somatostatin/calreticulin GABAergic interneurons, and that clozapine might have a beneficial effect on this cascade. Consistent findings on down-regulation of N-glygan and calnexin/calreticulin pathways, affecting protein folding in endoplastic reticulum, indicate also their major role in schizophrenia. Our results imply that although both sexes share many of the final common pathways involving the same proteins, the underlying primary pathophysiology of schizophrenia may differ between males and females. This may explain why the disease typically manifests after adolescence, when the expression levels of many sex-specific genes change. [ABSTRACT FROM AUTHOR]