Intracellular Ca2+ Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells

Early (E9.5–E11.5) embryonic heart cells beat spontaneously, even though the adult pacemaking mechanisms are not yet fully established. Here we show that in isolated murine early embryonic cardiomyocytes periodic oscillations of cytosolic Ca2+ occur and that these induce contractions. The Ca2+ oscillations originate from the sarcoplasmic reticulum and are dependent on the IP3 and the ryanodine receptor. The Ca2+ oscillations activate the Na+-Ca2+ exchanger, giving rise to subthreshold depolarizations of the membrane potential and/or action potentials. Although early embryonic heart cells are voltage-independent Ca2+ oscillators, the generation of action potentials provides synchronization of the electrical and mechanical signals. Thus, Ca2+ oscillations pace early embryonic heart cells and the ensuing activation of the Na+-Ca2+ exchanger evokes small membrane depolarizations or action potentials

Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

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

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

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 mu m are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode's solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 degrees C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode

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

A IncRNA Perspective into (Re)Building the Heart

Our conception of the human genome, long focused on the 2% that codes for proteins, has profoundly changed since its first draft assembly in 2001. Since then, an unanticipatedly expansive functionality and convolution has been attributed to the majority of the genome that is transcribed in a cell-type/context-specific manner into transcripts with no apparent protein coding ability. While the majority of these transcripts, currently annotated as long non coding RNAs (IncRNAs), are functionally uncharacterized, their prominent role in embryonic development and tissue homeostasis, especially in the context of the heart, is emerging. In this review, we summarize and discuss the latest advances in understanding the relevance of IncRNAs in (re)building the heart

ESNATS Conference - The Use of Human Embryonic Stem Cells for Novel Toxicity Testing Approaches

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

Analyzing murine electrocardiogram with PhysioToolkit

Quantitative electrocardiogram (ECG) analysis is a very important tool in cardiovascular and neuroedocrine research. It is useful in clinical trials with human beings, as well as in animals such as rabbits, rats, and mice, for example, in studying knockout models. The species of interest differ in their typical baseline heart rate and therefore in the sampling rate in ECG detection. However, for obvious reasons, there are no available analysis programs adjusted to each species. We demonstrate how to use PhysioToolkit, an open source software developed by Massachusetts Institute of Technology for physiologic signal processing and analysis in humans, with murine ECG signals, with full control over analysis options. The procedure can be transferred on any other species in an analogue way. (C) 2010 Elsevier Inc. All rights reserved