The influence of viral infection on cell line characteristics: Lessons learned from working with new cell lines from common carp.

Several factors influence the susceptibility of cell lines to infection by different viruses. These can be related to tissue specificity of the viruses, physiological status of the cells, their differentiation level and their capacity to mount immune responses to combat viral infection. To study the influence of cell characteristics and immune responses on their susceptibility on virus infection, newly developed cell lines from common carp brain (CCAbre), fins (CCApin), gills (CCAgill), and heart (CCAcar) and the established common carp brain (CCB) cells were exposed to the carp infecting viruses cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV), and the yet not fully characterized common carp paramyxovirus (CCPV). The susceptibility of these cells to viral infection was measured by formation of a cytopathic effect (CPE), estimation of viral particles produced by the cells and presence of viral mRNA in the cells. Viral susceptibility of the cells was compared to cell characteristics, measured by mRNA expression of the epithelial cell markers cadherin 1, occludin, and cytokeratin 15 and the mesenchymal cell marker vimentin, as well as to the level of type I interferon (IFN) responses. All cell lines were susceptible to CyHV-3 and CCPV but not to CEV infection. The cell lines had different levels of type I IFN responses towards the viruses. Typically, CyHV-3 did not induce high type I IFN responses, while CCPV induced high responses in CCAbre, CCAcar, CCApin cells but no response in CCAgill cells. Consequently, the type I IFN response modulated cell susceptibility to CCPV but not to CyHV-3. Interestingly, when the three different passage levels of CCB cells were examined, the susceptibility of one passage was significantly lower for CyHV-3 and higher for CCPV infection. This coincided with a loss of epithelial markers and lower type I IFN responses. This study confirms an influence of cell characteristics and immune responses on the susceptibility of carp cell lines for virus infection. Depending on the vulnerability of the virus to type I IFN responses, cells with a lower IFN-response can be superior for replication of some viruses. Batches of CCB cells can differentiate and thus may have significantly different levels of susceptibility to certain viruses.

Deep transcriptome analysis of the heat shock response in an Atlantic sturgeon (Acipenser oxyrinchus) cell line.

Despite efforts to restore Atlantic sturgeon in European rivers, aquaculture techniques result in animals with high post-release mortality due to, among other reasons, their low tolerance to increasing water temperature. Marker genes to monitor heat stress are needed in order to identify heat-resistant fish. Therefore, an Atlantic sturgeon cell line was exposed to different heat shock protocols (30 °C and 35 °C) and differences in gene expression were investigated. In total 3020 contigs (∼1.5%) were differentially expressed. As the core of the upregulated contigs corresponded to heat shock proteins (HSP), the heat shock factor (HSF) and the HSP gene families were annotated in Atlantic sturgeon and mapped via Illumina RNA sequencing to identify heat-inducible family members. Up to 6 hsf and 76 hsp genes were identified in the Atlantic sturgeon transcriptome resources, 16 of which were significantly responsive to the applied heat shock. The previously studied hspa1 (hsp70) gene was only significantly upregulated at the highest heat shock (35 °C), while a set of 5 genes (hspc1, hsph3a, hspb1b, hspb11a, and hspb11b) was upregulated at all conditions. Although the hspc1 (hsp90a) gene was previously used as heat shock-marker in sturgeons, we found that hspb11a is the most heat-inducible gene, with up to 3296-fold higher expression in the treated cells, constituting the candidate gene markers for in vivo trials.

Monitoring changing cellular characteristics during the development of a fin cell line from Cyprinus carpio.

The establishment and in-depth characterization of a novel continuous cell line derived from fin tissue of common carp (Cyprinus carpio), CCApin, is reported. The cells of the cell line could be propagated in Leibovitz's L-15 medium containing 15% foetal calf serum and 0.5% carp serum for >150 passages during the last 24 months, with a stable fast growth. Furthermore, antibody staining indicated that cell types obtained in primary cultures, containing the epithelial stem-cell marker tumorprotein 63, were different from cells in long-term cell cultures, containing tight junction protein zona occludens 1 and cytokeratin 7. These observations suggest a switch of dominant cell types. Molecular analysis of gene expression profiles of caudal fin tissue and CCApin cells showed that genes relevant in epithelial cells but also in mesenchymal cells were expressed. However, during cultivation of CCApin a set of very steadily expressed, primarily mesenchymal genes like collagen 1 alpha 1, fibronectin or cadherin 2 was found. In summary, the long-term cell culture could be described as a stably growing epithelial population with some mesenchymal features. There are several application possibilities, especially for virus susceptibility studies, e.g. cyprinid herpesvirus-3 (CyHV-3). The study leads to a better understanding of molecular and physiological mechanisms of in vitro fish cell cultures.

Pros and cons of fish skin cells in culture: long-term full skin and short-term scale cell culture from rainbow trout, Oncorhynchus mykiss.

Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines.

In vitro developed spontaneously contracting cardiomyocytes from rainbow trout as a model system for human heart research.

BACKGROUND/AIMS: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. METHODS: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra - and extracellular recordings. RESULTS: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. CONCLUSION: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

'Fish matters': the relevance of fish skin biology to investigative dermatology.

Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.

F-actin organization and pollen tube tip growth in Arabidopsis are dependent on the gametophyte-specific Armadillo repeat protein ARO1.

The signal-mediated and spatially controlled assembly and dynamics of actin are crucial for maintaining shape, motility, and tip growth of eukaryotic cells. We report that a novel Armadillo repeat protein in Arabidopsis thaliana, ARMADILLO REPEAT ONLY1 (ARO1), is of fundamental importance for polar growth and F-actin organization in tip-growing pollen tubes. ARO1 is specifically expressed in the vegetative cell of pollen as well as in the egg cell. ARO1-GFP (for green fluorescent protein) fusion proteins accumulate most notably in pollen tube tips and partially colocalize with F-actin in the shank of pollen tubes. ARO1 knockout results in a highly disorganized actin cytoskeleton, growth depolarization, and ultimately tube growth arrest. Tip-localized ARO1-GFP is spatially shifted toward the future site of tip growth, indicating a role of ARO1 in the signaling network controlling tip growth and regulating actin organization. After the pollen tube discharges its contents into the receptive synergid, ARO1-GFP colocalizes with emerging F-actin structures near the site of sperm cell fusion, suggesting additional participation in the mechanism of sperm cell tracking toward the female gametes. The variable localization of ARO1 in the cytoplasm, the nucleus, and at the plasma membrane, however, indicates a multifunctional role like that of beta-catenin/Armadillo and the p120 catenins.