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 α2 and -α5, collagen VI α3, collagen XIV α1, collagen XVIII α1 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., α-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

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

Expression of eGFP and hepatic proteins in spheroid-derived adherent cultures.

<p>(<b>A</b>) Cell colonies outgrowing on collagen Type I matrix. Down-regulation of eGFP fluorescence during the course of the colony growth is evident. (<b>B</b>) Expression of hepatic proteins, using fluorescent immunoassay. For better view of Ecad and lst-1 expression patterns across the cells, the corresponding images are displayed at a magnification ×400. Respective staining of primary hepatocytes (panels <i>a–c</i> and <i>e</i>) or of a liver tissue section (panel <i>d</i>) are shown for comparison. Alexa Fluor® 555-conjugated IgG served as a secondary antibody; cell nuclei were visualized by Hoechst 33342 staining. Merged Alexa Fluor® 555/Hoechst 33342 images are shown. Samples treated with an isotype control antibody instead of the primary antibody stained negatively (data not shown).</p

Engrafted spheroid-derived cells within recipient liver tissue.

<p>(<b>A</b>) eGFP-fluorescent cells and cell clusters observed in liver sections one and two weeks after spheroid transplantation. (<b>B</b>) Expression of Ecad in engrafted cells. “Live” eGFP and merged Alexa Fluor® 555/Hoechst 33342 images are shown.</p

Glycogen storage and ICG up-take in spheroid-derived adherent colonies.

<p>(<b>A</b>) Glycogen-storing cells in a 12-day-old spheroid-derived adherent culture. (<b>B</b>) Cell cluster in the culture, reversibly up-taking ICG.</p

Gene expression profiles of dynamic spheroid cultures, ESCs, and fetal and adult liver tissue.

<p>Due to a big difference between expression values of single genes in particular culture and tissue types, qRT-PCR data are displayed for each gene separately using a logarithmic scale on the y-axes. Mean relative gene expression values ± standard error of the mean (SEM) are plotted. For a better overview, the mean values are additionally listed in tables below the diagrams. Abbreviations ESC, Spher d0, Spher d2, FL, and AL refer to undifferentiated ESCs, 0-day-old and 2-day-old spheroids and to fetal and adult liver, respectively. Statistical significance of the PCR data was evaluated using IBM® SPSS software. Symbols (*), (**) or (***) depict a statistically significant (p<0.05), a highly statistically significant (p<0.01) or an extremely statistically significant (p<0.001) difference in gene expression values, respectively. In control reverse transcription-negative samples and in none-template-containing blank samples, no PCR products have been detected (data not shown).</p

Spheroid formation and growth in dynamic and static conditions.

<p>(<b>A</b>) Top panels: eGFP-expressing cell aggregates immediately after their separation from EBs cultured in a rotary or in a static condition. Bottom panels: rotary and static cultures of 2-day-old spheroids. (<b>B</b>) Comparative diagrams of spheroid growth in SFs and in Petri dishes. The mean of the spheroid diameter ± standard error of the mean (SEM) are plotted. The sign (***) indicates extremely statistically significant differences (p<0.001) in spheroid size between the dynamic and static cultures.</p