The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Extracellular recordings of spontaneous electrical activity in contracting cardiac clusters differentiated from murine embryonic stem cells enable to study electrophysiological features of this in-vitro cardiac-like tissue as well as effects of pharmacological compounds on its chronotropy and electrical conduction. To test if the microelectrode array (MEA) system could serve as a basis for development of a pharmacological screening tool for cardioactive drugs, we used spontaneously beating outgrowths of three-dimensional ES cell aggregates (“embryoid bodies”, EBs) plated onto substrate-integrated MEAs. The effects of the L-type Ca²⁺ channel antagonist verapamil and Na⁺ and K⁺ channel blockers (tetrodotoxin, 4-aminopyridine, and sparfloxacin) on the deduced interrelated cardiac network function were investigated. Application of 10^-6 M verapamil led to arrhythmic spiking with a burst-like pattern; at a higher concentration (10^-5 M) the drug caused a sustained negative chronotropy up to complete stop of beating. In the presence of tetrodotoxin a conduction block was observed. Since modulation of K⁺ channel activity can cause anti- or proarrhythmic effects, the influence of K⁺ channel blockers, namely 4-aminopyridine and sparfloxacin, was investigated. 4-aminopyridine (2x10^-3 M) significantly stabilized beating frequency, while the field potential duration (FPD) was concentration-dependently prolonged up to 2.7-fold. Sparfloxacin (3x10^-6 M) stabilized the beating frequency as well. At a higher concentration of sparfloxacin (3x10^-5 M), a significant prolongation of the spike duration was registered; application of the drug caused also early afterdepolarizations. The results demonstrate a suitability of the studied in-vitro cardiac cell model for pharmacological drug testing in cardiovascular research.

Background: We compared gene expression profiles in acutely dissected aorta with those in normal control aorta.

ABSTRACT

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Peptide-hormone secretion is partially triggered by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) and gene inactivation of Zn2+-sensitive Cav2.3-type VGCCs is associated with disturbed glucose homeostasis in mice. Zn2+ has been implicated in pancreatic islet cell crosstalk and recent findings indicate that sudden cessation of Zn2+ supply during hypoglycemia triggers glucagon secretion in rodents. Here we show that diethyldithiocarbamate (DEDTC), a chelating agent for Zn2+ and other group IIB metal ions, differentially affects blood glucose and serum peptide hormone level in wild-type mice and mice lacking the Cav2.3-subunit. Fasting glucose and glucagon level were significantly higher in Cav2.3-deficient compared to wild-type mice, while DEDTC Zn2+-chelation produced a significant and correlated increase of blood glucose and serum glucagon concentration in wild-type but not Cav2.3-deficient mice. Glucose tolerance tests revealed severe glucose intolerance in Zn2+-depleted Cav2.3-deficient but not vehicle-treated Cav2.3-deficient or Zn2+-depleted wildtype mice. Collectively, these findings indicate that Cav2.3 channels are critically involved in the Zn2+-mediated suppression of glucagon secretion during hyperglycemia. Especially under conditions of Zn2+ deficiency, ablation or dysfunction of Cav2.3 channels may lead to severe disturbances in glucose homeostasis.

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.

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