Fluorescence in situ hybridization (FISH) suggests that primary cell cultures derived from bovine fetal cotyledons are contaminated with maternal caruncular cells

Objectives: In the bovine epitheliochorial placenta the key sites of feto-maternal endocrine and metabolic interaction are placentomes consisting of interdigitating maternal and fetal tissue. So far, it is believed that the commonly performed manual separation prior to experiments can result in contamination of the maternal sample with fetal tissue but not vice versa. Our lab has recently developed a method to culture epitheloid cells derived from the cotyledon identifying them as trophoblast cells. Aim of this study was to validate and improve the method as the success rates were very low.

Methods: Primary cells from manually separated fetal cotyledons (n=43) and maternal caruncles (n=44) from day 90 to 270 of pregnancy were propagated and the different cell populations were compared morphologically, by immunofluorescence and FISH.

Results: Epitheloid cells present in both cultures did not differ morphologically and in the protein expression of cytokeratin (+), zonula occludens-1 (+), vimentin (+), α-smooth muscle actin (-) and desmin (-). Provided the fetus was male, FISH with a probe detecting the y-chromosome revealed that the epitheloid cells of cotyledon and caruncle were of maternal origin. In addition, a polygonally shaped cell population could be identified with cytoskeletal characteristics specific for epithelial cells. FISH confirmed the fetal origin of these cells and the lacking uptake of fluorescence conjugated low density lipoprotein (LDL) excluded the presence of endothelial cells.

Conclusion: Polygonal shaped cells isolated from the placentome were identified as the mononuclear trophoblast cells. The presence of epitheloid cells in 56% of cultures isolated from the cotyledon throughout pregnancy indicates the contamination of the fetal cotyledon with maternal tissue after manual separation. The percentage of contamination per sample could not be determined but should be considered in future experiments as it may lead to false positive results dependent on the sensitivity of the method applied.

Funded by the German Research Foundation (DFG).