Littérature scientifique sur le sujet « Trout-derived cell lines »

Abstract Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

Five single-cell clone lines (mRTP1B, mRTP1E, mRTP1F, mRTP1K, and mRTP2A) have been developed from adult rainbow trout pituitary glands. These cell lines have been maintained in a CO2-independent medium supplemented with 10% fetal bovine serum (FBS) for more than 150 passages. At about 150 passages, the doubling time of each single-cell clone in a CO2-independent medium supplemented with 10% FBS at 20 °C was 3.6±0.7, 2.8±0.7, 3.2±0.8, 5.5±0.6, and 6.6±0.6 days respectively. Each single-cell clone contains 60±2 chromosomes, which is within the range of the 2N chromosome numbers reported for rainbow trout. Reverse transcription-PCR analysis revealed that in addition to expressing gh, prolactin (prl), and estradiol (E2) receptor α (e2rα or esr1) genes, each single-cell clone line also expressed other pituitary-specific genes such as tsh, gonadotropin 1 (gth-1 or fshb), gonadotropin 2 (gth-2 or lhb), somatolactin (sl or smtl), proopiomelanocortin-B (pomcb), and corticosteroid receptor (cr or nr3c1). Immunocytochemical analysis showed that all the five single-cell clones produced both Gh and Prl. Furthermore, the expression of gh and prl genes in the single-cell clone lines is responsive to induction by E2, dexamethasone, and o,p′-dichlorodiphenyltrichloroethane. All together, these results confirm that each of the single-cell clones was derived from rainbow trout pituitary glands. These single-cell clone lines not only can be used to study factors that regulate the expression of pituitary hormone genes, but can also be developed as a rapid screening system for identifying environmental endocrine disruptors.

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

Infectious hematopoietic necrosis virus (IHNV) is known as a causative agent of heavy mortalities in farmed rainbow trout. However, there is limited information on its virulence for marine fish species. In this study, Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major were experimentally infected by intraperitoneal (IP) injection and immersion, with an IHNV isolate from rainbow trout in Japan, to evaluate the virulence of the virus for these fish species. The cumulative mortality for immersed rainbow trout was 15%. IHNV was isolated from all dead fish and 50% of the sequentially sampled rainbow trout. When Japanese amberjack were challenged by IP injection and immersion, the resulting cumulative mortality was 70% and 0%, respectively. The virus was isolated from all dead fish and 1 out of 3 Japanese amberjack sampled at 9 d post exposure. However, no mortality was observed in either of the red sea bream groups challenged with IHNV. IHNV was not isolated from any of the surviving red sea bream, or from any of the sequentially sampled fish. The viral titer on Japanese amberjack-derived YTF cells was in the same log range as that on FHM and RTH-149 cells, but the titers on the red sea bream cell lines SBK and GBRS were lower than the other cell lines, and were significantly different from the FHM and RTH-149 cell lines. These results suggest that Japanese amberjack has a low susceptibility to IHNV, and red sea bream has no or little susceptibility to the virus.

ABSTRACT Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to theRhabdoviridae family.

Toxic equivalent factors (TEFs) for eight polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) congeners were derived with a rainbow trout (Oncorhynchus mykiss) cell line, RTL-W1, and compared with TEFs obtained with a rat hepatoma cell line, H4IIE. Cells were exposed to a range of concentrations of the congeners which included 1,2,3,7,8-pentaCDD, 1,2,3,4,7,8-hexaCDD, 1,2,3,6,7,8-hexaCDD, 1,2,3,4,6,7,8-heptaCDD, 2,3,7,8-tetraCDF, 1,2,3,7,8-pentaCDF, 2,3,4,7,8-pentaCDF, and 1,2,3,4,7,8-hexaCDF. Ethoxyresorufin o-deethylase (EROD) activity was measured and EC50 values calculated from a dose–effect curve newly proposed for this purpose. TEFs were computed using 2,3,7,8-tetraCDD standard curves run concurrently with each assay. With the exception of 1,2,3,6,7,8-hexaCDD and 1,2,3,7,8-pentaCDF, all of the RTL-W1-derived TEFs were significantly higher (two- to eightfold higher) than the respective H4IIE TEFs. Immunoblotting analysis with the monoclonal anti-scup P4501A1 antibody was used to identify basal and induced levels of P4501A1 protein in both the RTL-W1 and H4IIE cells. It was concluded that mammalian-derived TEFs may not accurately predict the potency of PCDDs or PCDFs to fish.

The homologous genes vig1 and cig5 were identified by differential display PCR as virus-induced genes in rainbow trout and humans, respectively. These genes are significantly related to sequences required for the biosynthesis of metal cofactors, but their function remains unknown. In this study, it is shown that the mouse homologue of vig1/cig5 was induced by vesicular stomatitis virus (VSV) and pseudorabies virus (PrV) in mouse spleen cells. Among a collection of cell lines from dendritic, myeloid, lymphoid or fibroblast lineages, only the dendritic cell line, D2SC1, showed expression of mvig after virus infection. This dendritic restriction was confirmed by our finding that mvig was also induced by both VSV and PrV in CD11c++ spleen cells, separated by magnetic purification or derived from bone marrow precursor cells. Similar to the fish rhabdovirus viral haemorrhagic septicaemia virus in trout cells, VSV directly induced mvig in the dendritic cell line D2SC1, but the PrV-mediated induction required the integrity of the interferon pathway. This result indicates that mvig is interferon-inducible like its fish and human homologues. Furthermore, mvig was also induced by LPS in bone marrow-derived cells. Thus, mvig expression seems to correlate with an activated state of dendritic cells subjected to different pathogen-associated stimuli.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

To obtain an adequate comprehension of the intestinal morphology and function, I performed a detailed morphological evaluation of the intestinal epithelial cells lining the rainbow trout gut. This was obtained through histological, histochemical and immunohistochemical observation, accurate stereological analysis, morphometric measurements, and fine qualitative and quantitative characterization of goblet cells secreting activity at typical time points of in vivo feeding trials (50, 150 and 500 g). The gross anatomy of the rainbow trout gut corresponds to the general description of the organ in teleost fish. It consists of a proximal intestine with annexed pyloric caeca, easily distinguishable from the distal intestine, which presents a larger diameter, dark pigmentation, and circularly arranged blood vessels. Microscopically, the proximal intestine was characterized by simple folds whereas the distal intestine displayed a more complicated mucosa arrangement. In fact, this latter, was defined by the presence of peculiar complex folds from which other simpler originated. While only minor changes took place along the classical time points of in vivo feeding trials, two morphological and functional compartments not linearly distributed were observed along the trout gut. The first included the proximal intestine and the apical part of the complex folds of the distal portion. This was characterized by abundant and actively secreting goblet cells, no pinocytotic vacuoles, high expression of three defined functional enterocytes markers (PepT1, Sglt1 and Fabp2), low proliferation rate, few round apoptotic cells and an extended area of fully differentiated cells. The other, comprised the basal part of the complex folds and the pyloric caeca and was defined by few goblet cells with deflated mucus vacuoles, pinocytotic vacuolization, weak expression of the classical functional enterocytes markers, high proliferation and apoptotic rate and by a smaller extension of fully differentiated epithelial cells. The presence of these Chapter 2 23 distinct morphological and functional compartments suggest that digestive and absorptive function are mingled along the trout gut and that possibly in this species, the proximal intestine extend itself in the distal portion of the intestine to maximize its absorptive capacity [65]. To achieve a better knowledge of the cellular and molecular mechanisms implied in the intestinal homeostasis preservation and to further investigate the two different renewal rate previously observed, I performed a detailed qualitative description of the organization of the intestinal epithelial stem cell (IESC) niche and their regulatory molecules. To this end, the typical mouse ISCs markers (LGR5, HOPX, SOX9, NOTCH1, DLL1, and WNT3A) have been selected as target genes, and subsequently their expression have been localized along the different portions of the trout gut trough in situ hybridization. All the target genes were expressed also in rainbow trout intestine. However, considering their typical position in mouse, we highlighted substantial differences. Lgr5+ cells were rare but surprisingly in RT were located along the folds’ stroma rather than restricted in the crypt epithelium. The folds’ connective axis hosted also scattered notch1+ as well as wnt3a+ cells where they colocalized with lgr5+ cells. Interestingly, also in mouse these markers colocalize, even if this occurs in the epithelium lining the crypt base. Moreover, close to lgr5+ stromal cell population we observed elongated, epithelial cells expressing dll1. This marker in mouse is selectively expressed by Paneth cells at the crypt bottom. Despite the different spatial position of dll1+ cells in mouse and rainbow trout, it is interesting to note that in both species lgr5 and dll1 are expressed by cells topographically close each other, suggesting a functional interaction. Moreover, the epithelium lining the intestinal folds base in rainbow trout showed exclusively positivity to sox9. Particularly, here, we observed slender cells expressing sox9 at high level, strongly reminiscent of the crypt base columnar cells (CBCs) cells described in mouse intestinal crypts. Sox9 expression tended to disappeared outside del fold base to give way to hopx which was abundantly detected along the folds rather than restricted to the +4 position as described in mouse. Based on these observation, it is reasonable to assume that in this species Chapter 3 66 the functional equivalent of lgr5 is sox9, since sox9+ cells showed the typical location and shape of CBCs in mouse intestine. Whereas, lgr5 expressed by a stromal population along the folds could represent a specific mesenchymal subsets involved in the maintenance of the folds tip epithelium. To expand and integrate our previous findings describing the organization and the architecture of the RT stem cell niche, here I performed a detailed qualitative analysis of the distribution of the stromal component of the niche and in particular of telocytes (TC) as active player of the intestinal homeostasis maintenance. To this purpose, specific histological staining for the connective tissue and ultrastructural analysis have been performed. Furthermore, since the mouse is the species in which the mesenchymal component of the niche has been studied most, I selected the typical mouse TC markers (PDGFRα and FOLX1) as target genes, and I verified their topographical localization through fluorescence in situ hybridization. In addition, I studied their spatial distribution in relation with the epithelial components of the stem cell niche. Our results indicated the presence of slender elongated cells, distributed juxtaposed the enterocytes’ basement membrane creating an intricate mesh extending from the folds base to their apex. TEM analysis confirmed the identity of this cell population as TC; indeed, they possessed the distinctive features of this cell type, including a voluminous nucleus and limited cytoplasm from which long thin discontinuous branches develop. Moreover, in the close proximity of telocytes, I observed extracellular vesicles suggesting their functional implication in long distance cell-to-cell communication. In situ localization of pdgfrα transcripts highlighted two distinct pdgfrα+ cell populations, one displaying the typical morphology and position of TC, and another expressing pdgfrα at lower level and not possessing the distinctive TC morphological features and therefore considered as common fibroblasts. In situ localization of foxl1 transcripts revealed that foxl1+ cells were rare and distributed in the peri-epithelial space both at the folds base and along the folds length. In both locations, foxl1 was always co-expressed with pdgfrα, indicating the existence of a small functional telocytes subset. Basal TC were in the proximity of actively cycling intestinal stem cells, suggesting a possible interaction through short-range signaling. Their morphology and distribution at the basal and apical portion of the intestinal folds were identical to those observed in the mouse. This allows us to infer that also in RT, as in mouse, telocytes stimulate either cell proliferation or cell differentiation depending on their topographical location. Altogether, these results substantially improve our understanding of the molecular mechanism and of the cell types involved in the maintenance of intestinal homeostasis. As mentioned above, despite the complexity organization of the RT mucosa, the development of an in vitro tool able to retain similarities with the in vivo counterpart, would be a helpful tool for the rapid screen of several alternatives feeds implied in aquaculture. After several attempts, two novel cells line belonging from proximal (RTpi-MI) and distal (RTdi-MI) intestine of RT were successfully derived and characterized. Therefore, based on the extensive morphological and functional characterization of the RT mucosa in vivo, in this thesis several efforts have been also dedicated to the derivation of new stable cell lines. Then, they have been compared with the only continuous cell line that so far has been established from rainbow trout intestine (RTgutGC). Gene expression analysis demonstrated that all the cell line presented both epithelial and mesenchymal components. However, despite all of them displayed an epithelial component, this was significantly higher in the RTpiMI. Moreover, they also expressed the typical enterocytes’ functional markers (PepT1 and Sglt1) suggesting their suitability as a model of in vitro absorption. Furthermore, all the cell lines preserved both stem cells and differentiating cell types. High seeding density induced their differentiation into more mature phenotypes, as indicated by the simultaneously downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5) and upregulation of alkaline phosphatase activity. Nevertheless, among the three cell lines most parameters that we analyzed were conserved, some significant differences were observed. Therefore, this suggests that some typical features that characterize the two main intestinal portions are also preserved in vitro. Moreover, since only a mild stimulus was able to induce their differentiation it would be interesting to further explore these mechanisms using more controlled and sophisticated culture systems.