Syndapin-2 mediated transcytosis of amyloid-β across the blood-brain barrier

A deficient transport of amyloid-β across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-β efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-β transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-β clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-β expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-β clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-β build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-β assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-β across the blood-brain barrier

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

<p>Abstract</p> <p>Background</p> <p>Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.</p> <p>Results</p> <p>Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.</p> <p>Conclusions</p> <p>These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.</p

Dynamic Support Culture of Murine Skeletal Muscle-derived Stem Cells Improves Their Cardiogenic Potential In Vitro

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells

Stem cells in natural product and medicinal plant drug discovery-An overview of new screening approaches

Natural products remain a rich source of new drugs, and the search for bioactive molecules from nature continues to play an important role in the development of new medicines. Also, there is increasing use of herbal medicines for the treatment of a plethora of diseases, and demands for more scientific evidence for their efficacy and safety remains a huge challenge. The propensity of stem cells to differentiate into almost every cell type not only holds promise for the delivery of cell-based therapies for currently incurable diseases or a useful tool in studying cell physiology and pathophysiology. Increasingly, stem cells are becoming an important tool in preclinical drug screening and toxicity testing. In this review, we examine the scientific advances made towards the use of pluripotent stem cells as a model for the screening of plant-based medicines. The combination of well-established in vitro electrophysiological and a plethora of toxicogenomic technologies, together with the optimisation of culture methods of herbal plants and pluripotent stem cells can be explored to establish the basis for efficacy, and tissue/organ-based toxicities of many currently used medicinal plants whose efficacies and toxicities remain unknown

Modulation of L-type Calcium Current by Intracellular Magnesium in Differentiating Cardiomyocytes Derived from Induced Pluripotent Stem Cells

Intracellular Mg2+, which is implicated in arrhythmogenesis and transient cardiac ischemia, inhibits L-type Ca2+ calcium channel current (I-CaL) of adult cardiomyocytes (CMs). We take the advantage of an in vitro model of CMs based on induced pluripotent stem cells to investigate the effects of intracellular Mg2+ on the phosphorylation or dephosphorylation processes of L-type Ca2+ channels (LTCCs) at early and late stages of cardiac cell differentiation. Using the whole-cell patch-clamp technique, we demonstrate that increasing intracellular Mg2+ concentration [Mg2+](i) from 0.2 to 5mM markedly reduced the peak of I-CaL density, showing less effect on both the activation and inactivation properties in the late differentiation stage (LDS) of CMs more so than in the early differentiation stage (EDS). Increasing the [Mg2+](i) from 0.2 to 2mM in the presence of cAMP-dependent protein kinase A significantly decreased I-CaL in LDS (70%) and in EDS (36%) CMs. In addition, the effect of forskolin was greatly attenuated in the presence of 2mM [Mg2+](i) in LDS but not in EDS CMs. The effect of forskolin was enhanced in the presence of ATP-gamma-S in LDS CMs compared with EDS CMs. The exposure of both EDS and LDS CMs to 2mM [Mg2+](i) considerably reduced the effects of iso-butylmethylxanthine (IBMX) and okadaic acid on I-CaL. Our results provide evidence for differential regulation of LTCCs activities by cytosolic Mg2+ concentration in developing cardiac cells and confirm that Mg2+ acts under conditions that favor opening of the LTCCs caused by channel phosphorylation

The influence of melatonin on the heart rhythm - An in vitro simulation with murine embryonic stem cell derived cardiomyocytes

Background: In healthy individuals, a major factor influencing the heart rate variability (HRV) is the circadian rhythm. The role of melatonin as an essential component of the circadian rhythm in the adult human organism and the beneficial effects of a treatment with melatonin during the fetal period is well described. Toxic effects of melatonin are discussed less frequently. Since pharmacological studies cannot be carried out on pregnant women, the establishment of an equivalent in vitro model is important. We therefore tested whether melatonin can influence the beat rate variability (BRV) of spontaneously beating cardiomyocytes derived from murine embryonic stem cells (mESCs) and whether melatonin exhibits toxic effects in this in vitro model. Methods: Microelectrode Arrays recorded extracellular field potentials of spontaneously beating cardiomyocytes. Melatonin was applied in a concentration range from 10(-11) M to 10(-5) M. The analysis of the BRV focused on time domain methods. Results: In line with clinical observations, melatonin decreased the beating frequency and increased the BRV. The effect of melatonin up to a concentration of 10(-)(6) M was reversible, whereas the application of higher concentrations induced an irreversible effect. Conclusion: The study underlines the potential of this in vitro model to help explore the development of circadian rhythms and their modulation by melatonin in the embryonic phase. The results imply that melatonin influences the heart rhythm as early as during the embryonic heart development. Furthermore, the results indicate a potentially toxic effect of melatonin that has not been described in detail before

The influence of light on the beat rate variability of murine embryonic stem cell derived cardiomyocytes

Background: The human heart rhythm can be quantified by analyzing the heart rate variability (HRV). A major influencing factor of the HRV is the circadian rhythm. The ocular light and the hormone melatonin play decisive roles in the circadian rhythm. The beat rate variability (BRV) is considered to be the in vitro equivalent of the HRV. Previous studies have demonstrated the influence of melatonin on cardiomyocytes. Also, the influence of light on cardiomyocytes has been described before. Nevertheless, the effect of light on the BRV of cardiomyocytes has not yet been examined. Material and methods: The BRV of spontaneously beating cardiomyocytes was measured with microelectrode arrays over a time period of 30 min. The experiments were either performed with light exposure (with and without an infrared filter) or in complete darkness. Results: The BRV was higher and the beating frequency was lower when the cardiomyocytes were exposed to the full spectrum of light, compared to the measurements in darkness as well as to the measurements with an infrared filter. In contrast, the differences of BRV between the measurements in darkness and the measurements with an infrared filter were not as distinct. Conclusions: This is the first study demonstrating the influence of light on the beating rhythm of heart tissue in vitro. The results indicate that especially the infrared spectrum of light alters the BRV. These findings could be of interest for clinical applications such as the field of optical pacing as well as in neonatal patient care

Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs

Background/Aims: Heart rate variability (HRV) refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV) that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC)-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. Methods: The Microelectrode Array (MEA) technique was used to perform short-term recordings of extracellular field potentials (FPs) of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, alpha Pig-44). Offline analysis was focused on time domain and nonlinear methods. Results: The Poincare-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 +/- 8.7% of control at 10(-9) M, p < 0.05) and increased variability measures at higher concentrations (SDNN 258.8 +/- 42.7% of control at 10(-5) M, p < 0.05). Amiodarone and Metoprolol did not alter BRV significantly. Conclusions: Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening. (C) 2016 The Author(s) Published by S. Karger AG, Base