Diethyldithiocarbamate-mediated zinc ion chelation reveals role of Cav2.3 channels in glucagon secretion.

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.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

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.

Scalable selection of hepatocyte- and hepatocyte precursor-like cells from culture of differentiating transgenically modified murine embryonic stem cells.

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.

Genetic modification of cells for transplantation.

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.

Effect of cardioactive drugs on action potential generation and propagation in embryonic stem cell-derived cardiomyocytes.

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 Ca(2+) 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.

Gene expression in acute Stanford type A dissection: a comparative microarray study.

BACKGROUND: We compared gene expression profiles in acutely dissected aorta with those in normal control aorta. MATERIALS AND METHODS: Ascending aorta specimen from patients with an acute Stanford A-dissection were taken during surgery and compared with those from normal ascending aorta from multiorgan donors using the BD Atlas Human1.2 Array I, BD Atlas Human Cardiovascular Array and the Affymetrix HG-U133A GeneChip. For analysis only genes with strong signals of more than 70 percent of the mean signal of all spots on the array were accepted as being expressed. Quantitative real-time polymerase chain reaction (RT-PCR) was used to confirm regulation of expression of a subset of 24 genes known to be involved in aortic structure and function. RESULTS: According to our definition expression profiling of aorta tissue specimens revealed an expression of 19.1% to 23.5% of the genes listed on the arrays. Of those 15.7% to 28.9% were differently expressed in dissected and control aorta specimens. Several genes that encode for extracellular matrix components such as collagen IV alpha2 and -alpha5, collagen VI alpha3, collagen XIV alpha1, collagen XVIII alpha1 and elastin were down-regulated in aortic dissection, whereas levels of matrix metalloproteinases-11, -14 and -19 were increased. Some genes coding for cell to cell adhesion, cell to matrix signaling (e.g., polycystin1 and -2), cytoskeleton, as well as several myofibrillar genes (e.g., alpha-actinin, tropomyosin, gelsolin) were found to be down-regulated. Not surprisingly, some genes associated with chronic inflammation such as interleukin -2, -6 and -8, were up-regulated in dissection. CONCLUSION: Our results demonstrate the complexity of the dissecting process on a molecular level. Genes coding for the integrity and strength of the aortic wall were down-regulated whereas components of inflammatory response were up-regulated. Altered patterns of gene expression indicate a pre-existing structural failure, which is probably a consequence of insufficient remodeling of the aortic wall resulting in further aortic dissection.

A new method for optimizing multiplex DNA microsatellite analysis in low quality archival specimens.

BACKGROUND: Testing microsatellite instability seems to be a useful tool for the initial screening of putative non-polyposis colorectal cancer (HNPCC), preceding analysis of germ-line mutations of DNA mismatch repair genes. However, diagnosis of microsatellite instability becomes complicated when highly-damaged DNA from formalin-fixed paraffin-embedded tissue specimens has to be investigated. MATERIALS AND METHODS: A new methodical approach was established based on special multiplex PCR regimes (e.g., on touch-up cycling conditions), allowing both sufficient, as well as specific, amplification of the Bethesda reference panel loci with low quality DNA as template. RESULTS: By applying our new method, microsatellite instability could be analyzed successfully in 75 out of 84 investigated tumors (89%), whereas, by using standard PCR protocols, microsatellite analysis failed in 36% of the investigated cases. CONCLUSION: Our new methodical approach should be recommended for the use of archival material since it allows an efficient and accurate amplification of the Bethesda marker fragments and is less dependent on the DNA quality.