[Show abstract][Hide abstract] ABSTRACT: A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.
[Show abstract][Hide abstract] ABSTRACT: SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health.The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels:(i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological mode-of-actions leading to adverse outcomes (addressing mainly liver toxicity);(ii)adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions);(iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing.
[Show abstract][Hide abstract] ABSTRACT: Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1b (NRG-1b) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem cells (iPS) and assessed the regenerative effects of these CMs on cardiac tissue following acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from α-MHC-GFP mice were derived and differentiated into CMs through NRG-1b and/or DMSO treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, qRT-PCR, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium-control were injected into the peri-infarct region of hearts following coronary artery ligation, and functional and histology changes assessed from 1-8 weeks post-transplantation. Upon differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1b and DMSO compared to DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing and adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1b and DMSO leads to efficient differentiation of iPS into mature ventricular-like cardiac cells, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated Ca(2+) channels (VGCCs) are ubiquitous in excitable cells. These channels play key roles in many physiological events like cardiac regulation/pacemaker activity due to intracellular Ca(2+) transients. In the myocardium, the Cav1 subfamily (L-type: Cav1.2 and Cav1.3) is the main contributor to excitation-contraction coupling and/or pacemaking, whereas the Cav3 subfamily (T-type: Cav3.1 and Cav3.2) is important in rhythmically firing of the cardiac nodal cells. No established cardiac function has been attributed to the Cav2 family (E-/R-type: Cav2.3) despite accumulating evidence of cardiac dysregulation observed upon deletion of the Cav2.3 gene, the only member of this family so far detected in cardiomyocytes. In this review, we summarize the pathophysiological changes observed after ablation of the E-/R-type VGCC and propose a cardiac mechanism of action for this channel. Also, considering the role played by this channel in epilepsy and its reported sensitivity to antiepileptic drugs, a putative involvement of this channel in the cardiac mechanism of sudden unexpected death in epilepsy is also discussed.
Reviews of physiology, biochemistry and pharmacology. 10/2014;
[Show abstract][Hide abstract] ABSTRACT: Abstract Background/Aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF 1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and I f currents as well as functional hormonal regulation and sensitivity to I f channel blockers. Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed I f current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.
Cellular Physiology and Biochemistry 09/2014; 34:1199-1215. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective
Skeletal myoblasts fuse to form functional syncytical myotubes as an integral part of the skeletal muscle. During this differentiation process expression of proteins for mechanical and electrical integration is seized, which represents a major hindrance towards the application of skeletal myoblasts in cardiac regenerative cell therapy, as global heart function is dependent on intercellular communication.
We epicardially transplanted mechanically preconditioned engineered tissue constructs, containing neonatal mouse skeletal myoblasts. Y-chromosomal specific PCR was undertaken up to 10 weeks after transplantation to confirm the presence of grafted cells. Histological and electrophysiological analyses were carried out one week after transplantation.
Cells within the grafted construct expressed connexin 43 at the interface to the host myocardium, indicating electrical coupling, confirmed by sharp electrode recordings. Analyses on maximum stimulation frequency (5.65 ± 0.37 Hz), conduction velocity (0.087 ± 0.011 m/s) and sensitivity for pharmacological conduction block (0.736 ± 0.080 mM 1-heptanol) revealed effective electrophysiological coupling between graft and host cells, although significantly less robust than in native myocardial tissue (maximum stimulation frequency: 11.616 ± 0.238 Hz, p < 0.001; conduction velocity: 0.300 ± 0.057 m/s; p < 0.01; conduction block: 1.983 ± 0.077 mM 1-heptanol; p < 0.001).
While untreated skeletal myoblasts cannot couple to cardiomyocytes, we confirm that mechanical preconditioning enables transplanted skeletal myoblasts to functionally interact with cardiomyocytes in vivo and, thus, reinvigorate the concept of skeletal myoblast based cardiac cell therapy.
Journal of Thoracic and Cardiovascular Surgery 09/2014; · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts – MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3�2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment.The implications of these findings are critically discussed.
Frontiers in Cellular Neuroscience 09/2014; · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract (300 words max)/ Résumé (300 mots max)
Objective: The present study was undertaken to evaluate the effect of Erythrina senegalensis stem bark aqueous extract (EAES) on the mechanical and electrical activity as well as the embryotoxicity on cardiomyocytes (CMs) derived from mouse Embryonic Stem cells. Methods: Murine Embryonic Stem cell (ESC) line D3 (-pig44), engineered to express eGFP under control of -MHC promoter, to specifically detect cardiomyocytes during the developmental process were used. In vitro differentiation of ESC into cardiomyocytes (CMs) was performed using the mass culture protocol. Embryoid bodies (EBs) formed by 1 x 106 ESC were cultured in absence and in presence of various concentrations of EAES (1ng/ml – 1mg/ml). In another set of experiment, action potentials (APs) of spontaneously beating CMs were recorded by the whole-cell current-clamp technique before and after treatment with 1 µM isoproterenol (ISO), 1 µM carbachol (CCh) or 50 µg/mL EAES. EAES was also evaluated in presence of Iso or Cch. Using florescence activated cell sorting, the effect of EAES was evaluated on CMs differentiation. Results: EAES and ISO demonstrated similar positive chronotropic effect in CMs derived ESC. Likewise, coapplication of carbachol (10 µM) led to a comparable blockade of the ISO and EAES effects suggesting that both ISO and EAES may share similar binding pathways. EAES at 1mg/ml showed potent embryotoxicity on 8 days old EBs while lower concentrations (≤ 100 µg/ml) were without any effect on the life and percentage of active EBs. At the concentrations ranging 1 to 100 µg/ml EAES did not affect the cardiomyocytes differentiation. Conclusion: EAES shows weak embryotoxicity and positive chronotropic effect on cardiomyocytes derived from ES cells. This extract may be beneficial in cell therapy alternative for heart stroke.
Key words (4 max) : Stem cell, Positive chronotropic, action potential, embryotoxicity, Erythrina senegalensis
Fourth Life Sciences Conference, University of Dschang; 08/2014
[Show abstract][Hide abstract] ABSTRACT: Brillantaisia nitens Lindau (Acanthaceae) leaves are commonly used in traditional medicine in Africa for the treatment of many disorders including heart diseases and malaria. In this study, we therefore evaluated the effect of the methylene chloride/methanol leaf extract of Brillantaisia nitens on the proliferation of mouse pluripotent stem cells and their cardiomyocytes derivatives.
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.
Stem Cell Reviews and Reports 07/2014; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-β (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.
Cell Death & Disease 07/2014; 5:e1320. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.
Journal of Cardiovascular Pharmacology and Therapeutics 06/2014; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main achievements and results of the ESNATS project (Embryonic Stem Cell-based Novel Alternative Testing Strategies) were presented at the final project conference that was held on 15 September 2013, the day before the traditional EUSAAT (European Society for Alternatives to Animal Testing) Congress in Linz, Austria. The ESNATS project was an FP7 European Integrated Project, running from 2008 to 2013, the aim of which was to develop a novel toxicity testing platform based on embryonic stem cells (ESCs), and in particular, human ESC (hESCs), to accelerate drug development, reduce related R&D costs, and propose a powerful alternative to animal tests in the spirit of the Three Rs principles. Altogether, ESNATS offered the first proof of concept that hESCs can be used to create robust, reproducible and ready-to-use test assays for predicting human toxicity. In the end, essentially five test systems were developed to an adequate level for entering possible pre-validation procedures. These methods are based on hESCs, and can be combined to study the possible effects, on the human embryo, of exposure to a chemical during the early stages of development. In addition to the presentations by the main project partners, external speakers were invited to give lectures on relevant topics, both in the field of neurotoxicity and, more generally, on the applicability of hESCs in the development of advanced in vitro tests.
[Show abstract][Hide abstract] ABSTRACT: Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration. This metabolic switch of differentiating stem cells is required to cover the energy demands of the different organ-specific cell types. Among other metabolic signatures, amino acid and carbohydrate metabolism is most prominent in undifferentiated embryonic stem cells, whereas the fatty acid metabolic signature is unique in cardiomyocytes derived from embryonic stem cells. Identifying the specific metabolic pathways involved in pluripotency and differentiation is critical for further progress in the field of developmental biology and regenerative medicine. The recently generated knowledge on metabolic key processes may help to generate mature stem cell-derived somatic cells for therapeutic applications without the requirement of genetic manipulation. In the present review, the literature about metabolic features of stem cells and their cardiovascular cell derivatives as well as the specific metabolic gene signatures differentiating between stem and differentiated cells are summarized and discussed.
Circulation Research 04/2014; 114(8):1346-60. · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is debatable whether a local inflammatory tissue response caused by herniated disc material contributes to sciatic pain and/or sensorimotor deficits. The impact of inflammatory changes on local tissue remodelling, the healing process and the clinical course of disease remains unclear.
In this prospective observational study, we included a total of 31 patients with a single-level, unilateral lumbar disc herniation. The diagnosis was confirmed by magnetic resonance imaging (MRI)±gadolinium. The presence of peridiscal contrast enhancement was correlated with the extent of inflammatory reactions in the herniated fragments as confirmed by immunohistochemistry; clinical symptoms, including the duration of radicular pain; and the incidence of sensorimotor deficits.
Peridiscal contrast enhancement was found in 17 patients (55%) and was encasing the adjacent rootlet in 4 cases. There was no significant correlation between gadolinium uptake and the presence of sensorimotor deficits or the duration of radicular symptoms. Degenerative changes were observed in all 31 disc specimens. Overall, 18 cases exhibited increased cellularity in the marginal areas, which were mostly populated by CD68(+) macrophages and fibroblasts. Additionally, these areas displayed a limited number of CD3(+) T-lymphocytes and different degrees of concomitant neovascularisation, which represented a chronic and unspecific immune response. Peridiscal contrast enhancement on MRI was significantly correlated with the histopathological characteristics of tissue inflammation. However, no correlation was found between the histological evidence and the degree of inflammation and neurological symptoms.
Gadolinium-enhanced MRI is a sensitive method to detect unspecific inflammatory reactions in therapy-naïve disc herniations. However, the neuroradiological and histological evidence of peridiscal inflammation was not correlated with the severity of pain or sensorimotor deficits in our patients. Additional research is needed because the occurrence of local inflammation may indicate an ongoing degradation of herniated fragments and thus be helpful in therapeutic decision-making.
Clinical neurology and neurosurgery 04/2014; 119:28-34. · 1.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular Mg<sup>2+</sup> which is implicated in arrhythmogenesis and transient cardiac ischemia, inhibits L-type Ca<sup>2+</sup> channels current ( I <sub>CaL</sub>) of adult cardiomyocytes. We take the advantage of an in vitro model of cardiomyocytes based on induced pluripotent stem (iPS) cells to investigate the effects of intracellular Mg<sup>2+</sup> on the phosphorylation or dephosphorylation processes of L-type Ca<sup>2+</sup> channel (LTCCs) at early and late stages of cardiac cell differentiation. Using the whole-cell patch-clamp technique, we demonstrate that increasing intracellular Mg<sup>2+</sup> concentration [Mg<sup>2+</sup>]<sub>i</sub> from 0.2 mmol/L to 5 mmol/L markedly reduced the peak of I <sub>CaL</sub> density, showing less effect on both the activation and inactivation properties in the late developmental stage (LDS) of CMs differentiation more so than in the early developmental stage (EDS). Raising the [Mg<sup>2+</sup>]<sub>i</sub> from 0.2 mmol/L to 2 mmol/L in the presence of cAMP-dependent protein kinase A (PKA) significantly decreased I <sub>CaL</sub> in LDS (70%) and in EDS (36%) CMs. In addition, the effect of forskolin was greatly attenuated in the presence of 2 mmol/L [Mg<sup>2+</sup>]<sub>i</sub> in LDS but not in EDS cardiomyocytes. The effect of forskolin was enhanced in the presence of ATP-γ-S in LDS cardiomyocytes compared with EDS cardiomyocytes. The exposure of both EDS and LDS cardiomyocytes to 2 mmol/L [Mg<sup>2+</sup>]<sub>i</sub> considerably reduced the effects of isobutylmethylxanthine (IBMX) and okadaic acid (OA) on I <sub>CaL</sub>. Our results provide evidence for differential regulation of LTCCs activities by cytosolic Mg<sup>2+</sup> concentration in developing cardiac cells and confirm that Mg<sup>2+</sup> acts under conditions that favor opening of the LTCCs caused by channel phosphorylation.
Stem cells and development 02/2014; · 4.15 Impact Factor