Search

Presents a study on the identification and functional characterization of cardiac subtypes during early stages of development using the embryonic stem (ES) cell in vitro differentiation system. Method used to better understand the enhanced green fluorescent protein (EGFP) labeling of ES cell-derived cardiomyocytes; Experiment performed to identify the cardiac subtype and correlate it with its EGFP fluorescence intensity; Analysis permitted by the in vitro differentiation of ES cell-derived cardiomyocytes.

Background and Purpose: Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process., Experimental Approach: Beating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias., Key Results: This system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes., Conclusions and Implications: Microelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment., (© 2011 ACEA Biosciences Inc. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). When plated on fibronectin, the selected eGFP-positive cells formed colonies, in which intensely proliferating hepatocyte precursor-like cells gave rise to morphologically differentiated cells expressing alpha-1-antitrypsin, alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte marker genes were expressed in the culture, along with the gene and protein expression of stem/progenitor markers, suggesting the features of both hepatocyte precursors and more advanced differentiated cells. When cultured in suspension, the EB-derived puromycin-selected cells formed spheroids capable of outgrowing on an adhesive substrate, resembling the behavior of fetal mouse hepatic progenitor cells. The established system based on the highly efficient selection/purification procedure could be suitable for scalable generation of ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a potential in vitro source of cells for transplantation therapy of liver diseases, tissue engineering, and drug and toxicology screening.

Progress in gene therapy has produced promising results that translate experimental research into clinical treatment. Gene modification has been extensively employed in cell transplantation. The main barrier is an effective gene delivery system. Several viral vectors were utilized in end-stage differentiated cells. Recently, successful applications were described with adenovirus-associated vectors. As an alternative, embryonic stem cell- and stem cell-like systems were established for generation of tissue-specified gene-modified cells. Owing to the feasibility for genetic manipulations and the self-renewing potency of these cells they can be used in a way enabling large-scale in vitro production. This approach offers the establishment of in vitro cell culture systems that will deliver sufficient amounts of highly purified, immunoautologous cells suitable for application in regenerative medicine. In this review, the current technology of gene delivery systems to cells is recapitulated and the latest developments for cell transplantation are discussed.

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)-derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6-10-fold because of induction of proliferation on purification. Long-term engraftment (4-5 months) was observed when co-transplanting selected ES cell-derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell-derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

The main problem in QSAR modeling from the high throughput screening (HTS) data is that by definition, it produces only a small proportion of hits against a given assay. This leads to a very small statistical significance of the hits in comparison with the "noise". Analysis based purely on the "hit" compounds removes useful information about the biological response of all the test compounds. What is needed is an analysis technique that increases the significance of the active compounds, while using the information present in the original data. In this paper we present a method for application of intelligent filtering of the data to improve statistical significance of the active compounds to generate predictive models that provide medicinal chemists with a powerful tool for both optimizing compounds and mining screening candidates in libraries.

During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the alpha-myosin heavy chain promoter (alpha-MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that specifically complex III function rather than mitochondrial ATP production is necessary for early heart cell development. When the underlying mechanism was examined, we noticed that antimycin A but not KCN lead to inhibition of spontaneous intracellular Ca++ oscillations, whereas both substances decreased mitochondrial membrane potential, as expected. We postulate that mitochondrial complex III activity is necessary for these Ca++ oscillations, which in turn are a prerequisite for cardiomyocyte differentiation.

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

Embryonic stem (ES) cells may represent an alternative source of functionally intact cardiomyocytes for the causal treatment of cardiovascular diseases. However, this requires cardiac-specific differentiation of stem cells and the selection of pure lineages consisting of early embryonic cardiomyocytes. Therefore, an understanding of the basic mechanisms of heart development is essential for selective differentiation of embryonic stem cells into cardiac cells. The development of cardiac cells from embryonic stem cells is regulated by several soluble factors and signalling molecules together with cardiac specific transcription factors such as the zinc-finger GATA proteins and Nkx-2.5. GATA-4 and Nkx-2.5 seem to be essential for heart development. The use of enhanced green fluorescent protein (EGFP) under the control of cardiac-specific promoters in combination with the ES cell system has allowed for the functional characterisation of cardiac precursor cells. Embryonic stem cell-derived cardiomyocytes developmentally express similar cardiac-specific proteins, ion channels and signalling molecules to that of adult cardiomyocytes. Furthermore, identification of growth factors and signalling molecules under cell culture conditions is crucial for the selective cardiac differentiation of embryonic stem cells. Therefore, serum-free culture conditions have to be established in order to examine the influence of different growth factors and signalling molecules on cardiac development and/or formation from ES cells. Although significant progress has been made in generating cardiac cell lineage by the combination of genetically manipulative methods with selective culture conditions for cell transplantation therapy, one of the remaining future challenges for transplantation in humans is the immunological rejection of the engrafted cardiomyocytes.

Background: Cardiomyopathy is characterized by the loss of functional cardiomyocytes resulting in heart failure. More recently, there is increasing evidence from animal studies that transplantation of cardiomyocytes may represent a valuable approach for the treatment of severe heart failure., Development of Cardiac Cells: Treatment of cardiovascular diseases using cardiomyocytes derived from embryonic stem cells prerequisites establishment of pure lineages of early embryonic cardiomyocytes from human embryonic stem cells. The development of cardiac cells from embryonic stem cells is regulated by several growth factors such as TGF-beta, IGF FGF and erythropoietin. After binding to their receptor, growth factors induce expression of a number of cardiac specific transcription factors such as the zinc finger GATA proteins and Nkx-2.5, a coactivator of the GATA-4 protein. The GATA-4 transcription factor and Nkx-2.5 are essential for heart development. In parallel to adult cardiomyocytes, embryonic stem cell-derived cardiomyocytes developmentally express cardiac specific proteins and ion channels., Generation From Embryonic Stem Cells: Recently, it has been shown that pure cardiomyocytes can be generated from genetically manipulated embryonic stem cells. In order to achieve the selective cardiac differentiation of embryonic stem cells different culture conditions are currently tested to examine in the future the influence of different growth factors. However, although significant progress has been made in generating pure cardiomyocytes, further efforts are required to avoid possible immunological rejection of the engrafted cardiomyocytes. Thus, a main challenge in the future will be the establishment of pure lineages of cardiomyocytes derived from human embryonic stem cells.

Background: Cellular cardiomyoplasty is discussed as an alternative therapeutic approach to heart failure. To date, however, the functional characteristics of the transplanted cells, their contribution to heart function, and most importantly, the potential therapeutic benefit of this treatment remain unclear., Methods and Results: Murine ventricular cardiomyocytes (E12.5-E15.5) labeled with enhanced green fluorescent protein (EGFP) were transplanted into the cryoinjured left ventricular walls of 2-month-old male mice. Ultrastructural analysis of the cryoinfarction showed a complete loss of cardiomyocytes within 2 days and fibrotic healing within 7 days after injury. Two weeks after operation, EGFP-positive cardiomyocytes were engrafted throughout the wall of the lesioned myocardium. Morphological studies showed differentiation and formation of intercellular contacts. Furthermore, electrophysiological experiments on isolated EGFP-positive cardiomyocytes showed time-dependent differentiation with postnatal ventricular action potentials and intact beta-adrenergic modulation. These findings were corroborated by Western blotting, in which accelerated differentiation of the transplanted cells was detected on the basis of a switch in troponin I isoforms. When contractility was tested in muscle strips and heart function was assessed by use of echocardiography, a significant improvement of force generation and heart function was seen. These findings were supported by a clear improvement of survival of mice in the cardiomyoplasty group when a large group of animals was analyzed (n=153)., Conclusions: Transplanted embryonic cardiomyocytes engraft and display accelerated differentiation and intact cellular excitability. The present study demonstrates, as a proof of principle, that cellular cardiomyoplasty improves heart function and increases survival on myocardial injury.

Background: Recent progress in the cardiotypic differentiation of embryonic and somatic stem cells opens novel prospects for the treatment of cardiovascular disorders. The aim of the present study was to develop a novel surgical approach that allows standardized cellular cardiomyoplasty in mouse with low-perioperative mortality., Methods: Reproducible transmural lesions were generated by cryoinjury followed by intramural injection of embryonic cardiomyocytes using a newly designed holding device and vital dye staining. This approach was validated with a transgenic mouse model, in which the live reporter gene-enhanced green fluorescent protein (EGFP) is under control of a cardiac-specific promoter., Results: The perioperative mortality was 10%. The engrafted EGFP-positive cardiomyocytes could be identified in a high percentage (72.2%, n=36) of operated animals., Conclusions: This novel approach enables reliable cellular replacement therapy in mouse and greatly facilitates the analysis of its molecular, cellular, and functional efficacy.

Primary vascular plexus originate from angioblasts through a process called vasculogenesis. The precise role of basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF) are both suggested as key regulators in vasculogenesis is still unclear. This crucial aspect was investigated by using time lapse observation of in vitro generated embryonic stem (ES) cell-derived endothelial structures which were recognizable by using the platelet cell adhesion molecule (PECAM-1) driven endothelial-specific expression of the live reporter gene enhanced green fluorescent protein (EGFP). In serum free conditions VEGF led to improved survival of angioblasts and to the formation of primitive endothelial tubes whereas bFGF alone increased their survival. Our study suggests that the complex process of vasculogenesis can be driven by VEGF alone but not by bFGF., (Copyright 2002 S. Karger AG, Basel)

Object: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD)., Methods: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation., Conclusions: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.