Ulrike Schmitz

Monoclonal Antibody CD133–2 (AC141) Against Hematopoietic Stem Cell Antigen CD133 Shows Crossreactivity with Cytokeratin 18

CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133–2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133–2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133–2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133–2/AC141 in IHC.

The first trimester human trophoblast cell line ACH-3P: A novel tool to study autocrine/paracrine regulatory loops of human trophoblast subpopulations – TNF-α stimulates MMP15 expression

BackgroundThe trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).ResultsExpression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e.g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts.ConclusionWe conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-α on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.

Extracellular pH modulates the secretion of fibronectin isoforms by human trophoblast

Differentiation of human trophoblast from the proliferative to the invasive phenotype takes place in a hypoxic and thus likely an acidic microenvironment. During differentiation, the secretion pattern of fibronectin isoforms changes. Therefore, we analysed the relation between extracellular pH, secretion of fibronectin splice variants and invasiveness. By means of immunohistochemistry and biochemistry, cellular non-oncofetal fibronectins were found in placental stroma and around extravillous trophoblast, whereas oncofetal isoforms only marked the extracellular matrix of extravillous trophoblast. In vitro, mesenchymal cells produced non-oncofetal fibronectins only, whereas choriocarcinoma cell lines, extravillous trophoblast and choriocarcinoma/trophoblast hybrid cells secreted both non-oncofetal and oncofetal isoforms. When the pH of the culture medium was either lowered or increased (between 6.0 and 8.0), the trophoblast hybrids, but not choriocarcinoma and mesenchymal cells, responded with increased secretion of fibronectins and a shift towards oncofetal isoforms. These changes were preserved after pH normalisation. Histochemical determination of local tissue acidity revealed that the site of the lowest detectable tissue pK coincided with the starting point of invasion, the proximal part of trophoblastic cell columns. Therefore, it is concluded that the local pH plays an important role as regulator of differentiation of human trophoblast as reflected by the synthesis of oncofetal fibronectins by the invasive phenotype of extravillous trophoblast.

Choriocarcinoma-trophoblast hybrid cells: Reconstructing the pathway from normal to malignant trophoblast — Concept and perspectives

Summary Extravillous trophoblast cells of normal and molar pregnancies spontaneously can transform into choriocarcinoma cells. This malignant transformation differs from that in other tissues in two respects: (a) the normal tissue of origin, extravillous trophoblast, is already invasive, and (b) both, choriocarcinoma and extravillous trophoblast, are foreign (allohaploid) to the host. Carcinogenetic transformation of trophoblast into choriocarcinoma is a multistep process in the course of which temporary intermediate geno- and phenotypes are generated; these are mixtures between trophoblast and choriocarcinoma and illustrate the process of carcinogenesis. The intermediates are either selected by the host for survival or undergo further mutations. Consequently, it remains unknown which properties are lost or acquired during the process and which of them are crucial either for control of invasion of normal extravillous trophoblast or for the malignant development of choriocarcinoma cells. In order to identify mutative trophoblast-relevant oncogenes and tumor suppressor genes, we have generated somatic cell hybrids using the choriocarcinoma cell line AC1-1 and extravillous trophoblast cells. The resulting hybrid cells were cloned, characterized cytogenetically and classified according to tumorigenicity. These hybrids represent a population of intermediates between non-tumorigenic extravillous trophoblast cells and highly tumorigenic choriocarcinoma cells, similar to those temporarily formed during carcinogenesis. The hybrids are thought to represent a suitable in vitro tool for the identification of genes and gene products involved in both control of normal invasion and malignant transformation of trophoblast cells. Analysis of the hybrid clones involves both, methods assessing phenotypic (functional) and genotypic properties.