Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles.

Properties and functions of KATP during mouse perinatal development.

Background: Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes.

Methods: Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels.

Atropine-sensitive hippocampal θ oscillations are mediated by Cav2.3 R-type Ca²⁺ channels.

Hippocampal theta oscillations are key elements in numerous behavioral and cognitive processes. Based on the dualistic theory of theta oscillations, one can differentiate between atropine-sensitive and atropine-insensitive theta subtypes. Urethane-induced atropine-sensitive theta oscillations are driven by muscarinic signal transduction pathways through G protein q/11 alpha subunit (Gα(q/11)), phospholipase β( ¼) (PLCβ( ¼), inositol trisphosphate (InsP₃), diacylglycerole (DAG), and protein kinase C (PKC).

Derivation of es Cells From Early Stage Preimplantation Embryos and Characterisation of their Cardiac Differentiation Potential in Mice.

Most murine embryonic stem cell lines have been derived from the inner cell mass of blastocysts and extensively studied in different aspects including generation of organ specific cells. However, no detailed studies have been made on cardiac specific gene expression, immunocytochemical and electrophysiological characterisation of cardiomyocytes generated from early stage (preimplantation) embryo derived embryonic stem cells in mice. In the present study, new embryonic stem cell lines were derived from early stage preimplanatation embryos in mice.

Effect of chemopreventive agents on differentiation of mouse embryonic stem cells.

Chemopreventive agents are derived from edible plants and from ancient time is a part of daily intake for many humans and animals. There are several lines of compelling evidence from epidemiological, clinical and laboratory studies that these dietary constituents are associated in reducing cancer risks. However, developmental toxicity of these natural compounds cannot be excluded.

APLP1 and Rab5A interact with the II-III loop of the voltage-gated Ca-channel Ca(v)2.3 and modulate its internalization differently.

Background: Voltage gated calcium channels (VGCCs) regulate cellular activity in response to membrane depolarization by altering calcium homeostasis. Because calcium is the most versatile second messenger, regulation of the amount of VGCCs at the plasma membrane is highly critical for several essential cellular processes. Among the different types of VGCCs, the Ca(v)2.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

Background/aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro.

Organotypic slice culture from human adult ventricular myocardium.

Aims: Cardiovascular research requires complex and functionally intact experimental models. Due to major differences in the cellular and subcellular composition of the myocardium between species, the use of human heart tissue is highly desirable. To enhance the experimental use of the human myocardium, we established methods for the preparation of vital tissue slices from the adult ventricular myocardium as well as conditions for their long-term preservation in organotypic culture.

Development of a neural teratogenicity test based on human embryonic stem cells: response to retinoic acid exposure.

The aim of this study was the development of an alternative testing method based on human embryonic stem cells for prenatal developmental toxicity with particular emphasis on early neural development. To this purpose, we designed an in vitro protocol based on the generation of neural rosettes, representing the in vitro counterpart of the developing neural plate and neural tube, and we challenged this complex cell model with retinoic acid (RA), a well-known teratogenic agent. The cells were exposed to different concentrations of RA during the process of rosettes formation.

Effective surface-based cryopreservation of human embryonic stem cells by vitrification.

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing.

Two separate Ni(2+) -sensitive voltage-gated Ca(2+) channels modulate transretinal signalling in the isolated murine retina.

Purpose: Light-evoked responses from vertebrate retinas were recorded as an electroretinogram (ERG). The b-wave is the most prominent component of the ERG, and in the bovine retina its NiCl(2) -sensitive component was attributed to reciprocal signalling by pharmacoresistant R-type voltage-gated Ca(2+) channels, which similar to other voltage-dependent Ca(2+) channels trigger and control neurotransmitter release. The murine retina has the great advantage that the effect of gene inactivation for Ni(2+) -sensitive Ca(2+) channels can be analysed to prove or disprove that any of these Ca(2+) channels is involved in retinal signalling.

Integration properties of Wharton's jelly-derived novel mesenchymal stem cells into ventricular slices of murine hearts.

Wharton's jelly (WJ) is a rich source of multiple-lineage differentiating cells, recently proposed for cell replacement therapy. However, their ability to integrate into the cardiac tissue has not been elucidated, yet. We employed in vitro cardiac transplantation models to investigate the capacity of a novel population of human WJ-derived mesenchymal stem cells (nMSCs) to integrate into both living and ischemic cardiac tissue.

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes.

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I(Ca,L)) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca(2+) channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.

Molecular and functional changes in voltage-gated Na⁺ channels in cardiomyocytes during mouse embryogenesis.

Background: Embryonic cardiomyocytes undergo profound changes in their electrophysiological properties during development. However, the molecular and functional changes in Na⁺ channel during cardiogenesis are not yet fully explained.

Methods And Results: To study the functional changes in the Na⁺ channel during cardiogenesis, Na⁺ currents were recorded in the early (EDS) and late (LDS) developmental stages of cardiomyocytes in embryonic mice.

Ca(v)2.3 Ca2+ channel interacts with the G1-subunit of V-ATPase.

Background: Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types.

Specific gene signatures and pathways in mesodermal cells and their derivatives derived from embryonic stem cells.

The vertebrate early stage embryo is consisting of the three primary germ layers ectoderm, mesoderm and endoderm, from which all organ tissues are developed. During early embryonic development, mesodermal cells become sequentially determined to more precisely defined cell types including muscle, heart, vasculature, blood, kidney, gonads, dermis and cartilage. How the prospective mesodermal cells integrate the various signals they receive and how they resolve this information to regulate their morphogenetic behavior and cell fate decisions is largely unknown.

Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy.

Concise review: induced pluripotent stem cells and lineage reprogramming: prospects for bone regeneration.

Bone tissue for transplantation therapies is in high demand in clinics. Osteodegenerative diseases, in particular, osteoporosis and osteoarthritis, represent serious public health issues affecting a respectable proportion of the elderly population. Furthermore, congenital indispositions from the spectrum of craniofacial malformations such as cleft palates and systemic disorders including osteogenesis imperfecta are further increasing the need for bone tissue.

Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing.

Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing.

Extract derived from rat brains in the acute phase following traumatic brain injury impairs survival of undifferentiated stem cells and induces rapid differentiation of surviving cells.

Dramatic cerebral responses following brain injury (TBI) comprise inflammation, cell death, and modulation of trophic factor release. These cerebral modulations might induce and/or attenuate acute neuronal damage. Here, we investigated the effect of tissue extract derived from healthy (HBE) or injured rat brain (TBE) on the differentiation of cultured embryonic stem cells in vitro.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

Background And Purpose: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals.

Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells.

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO.

Fibroblasts support functional integration of purified embryonic stem cell-derived cardiomyocytes into avital myocardial tissue.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs).

Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals.

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs.