Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent. According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.