Validation and establishment of cell culture models to study invasion and feto-maternal interaction in the bovine placentome
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A unique feature of bovine synepitheliochorial placenta is the occurrence of restricted trophoblast invasion characterized by the migration and fusion of trophoblast giant cells (TGC) with single maternal epithelial cells. Unlike tumor invasion, this process is limited to the depth of the caruncular epithelium suggesting the bovine placenta to be an ideal model to study mechanisms regulating cellular invasion. In-vitro models of the reproductive tissues involved are lacking though these models would also allow the assessment of pathogenesis of pregnancy-associated diseases und thus reduce the number of animal experiments. The thesis presented discusses an interconnection of three articles published in peer reviewed journals ([1] Theriogenology, 2007; 68 (4):592-603; [2] Placenta, 2007; 28 (11-12):1110-1117; [3] Biology of Reproduction, 2008; in press) focusing on the characterization and functional assessment of primary caruncular epithelial cells as well as the development of a cell line. Primary caruncular epithelial cells were successfully isolated and cultured from manually separated caruncles from the third month of gestation onwards. Fluorescence in-situ hybridization (FISH) not only confirmed the maternal origin of these cells but also demonstrated a contamination of cotyledonary-derived cultures with maternal epithelial cells. An extensive phenotypic and functional characterization defined these cells to be of epithelial origin forming an intact epithelial monolayer in culture. The cells grew as a monolayer, and via transmission and scanning electron microscopy a polarized morphology with apical microvilli and junctional complexes was demonstrated. Expression of epithelial-specific cytokeratin and Zonula occludens-1 protein as well as vimentin but not alpha-smooth muscle actin and desmin was shown by immunofluorescence. Furthermore, transepithelial electrical resistance measurements (TEER) of cultures grown on Transwell® inserts confirmed the presence of an intact epithelial barrier. The expression and co-localization of integrin subunit beta1 and associated signaling molecules (alpha-actinin, focal adhesion kinase, phosphotyrosine and talin) showed that caruncular epithelial cells actively participate in integrin-mediated inside-out and outside-in signaling pathways. Culture on dishes coated with proteins of the extracellular matrix, fibronectin in particular, resulted in increased proliferation and enhanced the expression of integrin and integrin-associated signaling molecules. By continuous subculture and spontaneous immortalization of the first caruncular epithelial cell line (BCEC-1) derived from a pregnant cow was established. The cells maintained their epithelial properties as shown for primary cells for up to 32 passages. In addition, the cells were tested negative for the BVDV but remained susceptible to infection. In order to introduce this model for commercial use (i.e. screening essays), BCEC-1 has been patented (PCT/DE 2007 001274). In conclusion, the thesis demonstrates the importance of appropriate cell characterization and identification especially when cells are isolated from epitheliochorial placentae. The phenotypic and functional properties of primary caruncular epithelial cells and BCEC-1 suggest that both models provide excellent potential for further functional studies, i.e. co-culture invasion assays as well as the physiology and pathology of feto-maternal interaction and pregnancy-associated diseases. In-vitro models, cell cultures in particular cannot completely replace experiments in-vivo however may be more efficient in generating preliminary results, and subsequently less animal experiments will be required. Supported by a grant of the German Research Foundation (DFG).Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler 2008