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Cologne, Germany

Stoelzle S.,Nanion Technologies GmbH | Obergrussberger A.,Nanion Technologies GmbH | Bruggemann A.,Nanion Technologies GmbH | Haarmann C.,Nanion Technologies GmbH | And 3 more authors.
Frontiers in Pharmacology

Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing. © 2011 Stoelzle, Obergrussberger, Brüggemann, Haarmann, George, Kettenhofen and Fertig. Source

MicroRNA (miRNA) plays a critical role in a wide variety of biological processes. Profiling miRNA expression during differentiation of embryonic stem cells will help to understand the regulation pathway of differentiation, which in turn may elucidate disease mechanisms. The identified miRNAs could then serve as a new group of possible therapeutic targets. In the present paper, miRNA expression profiles were determined during cardiomyocyte-specific differentiation and maturation of murine embryonic stem (ES) cells. For this purpose a homogeneous cardiomyocyte population was generated from a transgenic murine ES cell line. Two high throughput array platforms (Affymetrix and Febit) were used for miRNA profiling in order to compare the effect of the platforms on miRNA profiling as well as to increase the validity of target miRNA identification. Four time points (i.e. day 0, day 12, day 19 and day 26) were chosen for the miRNA profiling study, which corresponded to different stages during cardiomyocyte-specific differentiation and maturation. Fifty platform and pre-processing method-independent miRNAs were identified as being regulated during the differentiation and maturation processes. The identification of these miRNAs is an important step for characterizing and understanding the events involved in cardiomyocyte-specific differentiation of ES cells and may also highlight candidate target molecules for therapeutic purposes. © 2011 Gan et al. Source

Gan L.,RWTH Aachen | Schwengberg S.,Axiogenesis | Denecke B.,RWTH Aachen
Gene Expression Patterns

The differentiation to cardiomyocytes is a prerequisite and an important part of heart development. A good understanding of the complicated cardiomyocyte differentiation process benefits cardiogenesis study. Embryonic stem cells (ESCs), cell lines with infinite ability to proliferate and to be differentiated into all cell types of the adult body, are important research tools for investigation of differentiation and meanwhile good models for developmental research. In the current study, genome-wide gene expression of ESCs is profiled through high throughput platform during cardiomyocyte-specific differentiation and maturation. Gene expression patterns of undifferentiated ESCs and ESC-derived cardiomyocytes provide a global overview of genes involved in cardiomyocyte-specific differentiation, whereas marker gene expression profiles of both ESC-related genes and cardiac-specific genes presented the expression pattern shift during differentiation in a pure ESC-derived cardiomyocyte cell culture system. The differentiation and maturation process was completed at day 19 after initiation of differentiation, according to our gene expression profile results. Functional analysis of regulated genes reveals over-represented biological processes, molecular functions and pathways during the differentiation and maturation process. Finally, transcription factor regulation networks were engineered based on gene expression data. Within these networks, the number of identified important regulators (Trim28, E2f4, Foxm1, Myc, Hdac1, Rara, Mef2c, Nkx2-5, Gata4) and possible key co-regulation modules (Nkx2-5 - Gata4 - Tbx5, Myc - E2F4) could be expanded. We demonstrate that a more comprehensive picture of cardiomyocyte differentiation and its regulation can be achieved solely by studying gene expression patterns. The results from our study contribute to a better and more accurate understanding of the regulation mechanisms during cardiomyocyte differentiation. © 2014 Elsevier B.V. All rights reserved. Source

Berkessel A.,University of Cologne | Seelig B.,University of Cologne | Schwengberg S.,Axiogenesis | Hescheler J.,University of Cologne | Sachinidis A.,University of Cologne

A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosine heavy chain (α-MHC) promoter (pα-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80% increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Source

Provided are assay systems for determining the therapeutic or toxic effect of a putative drug based on assaying its activity in cells which have been differentiated in vitro from stem cells, and induced to display a phenotype that resembles a disease to be treated.

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