Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi

Abstract

A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a ''crazy pavement'' and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule Ecadherin. The isolated villous trophoblasts in culture expressed integrins ␣6 and ␤4 and reduced levels of ␤1 subunits, whereas the proliferating extravillous trophoblast cultures expressed ␣1, ␣3, and ␣5 and high levels of ␤1 integrin subunits, vitronectin receptor (␣V␤3/␤5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72-and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.

Placental Expression and Chromosomal Localization of the Human Gcm 1 Gene

“…Trophoblast invasion of the maternal uterus during human placentation is a highly complex process that involves cell proliferation, migration, and cell differentiation. Several known factors have been described in human syncytiotrophoblasts, including the Wnt genes (Nusse and Varmus 1992), the Hox gene (Oudejans et al 1990;Taylor et al 1997), and the integrin family (Yang et al 1995;Aboagye-Mathiesen et al 1996). In this study we have characterized a member of the human Gcm gene family and have demonstrated that the h Gcm 1 gene, like its mouse homologue, is also expressed in placenta.…”

A microarray‐based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal disease

“…This is because such samples are likely to contain multiple other cells types, in addition to trophoblasts including fibroblasts and vascular cells. One alternative to our approach would be to selectively purify trophoblasts from placental villus tissue (Aboagye-Mathiesen et al, 1996). However, we were concerned that such procedures involved would severely limit the availability of DNA for down stream molecular analysis and we also wished to avoid potential process-dependent modification of the epigenetic signature.…”

Leukemia inhibitory factor promotes human first trimester extravillous trophoblast adhesion to extracellular matrix and secretion of tissue inhibitor of metalloproteinases-1 and -2

“…Normal placental development depends on the ability of EVTs to attach to and then invade into the maternal decidua and the first third of the myometrium. This occurs in order to anchor the fetus to the maternal endometrium and then invade the maternal spiral arteries creating large-diameter, low resistance vessels that provide a means of steady perfusion of the floating villi with maternal blood ( Genbacev et al ., 1992 ; Irving et al ., 1995 ; Aboagye-Mathiesen et al ., 1996 ). Insufficient trophoblast invasion of the spiral arteries has been associated with pre-eclampsia and fetal growth restriction ( Hustin et al ., 1990 ; Kadyrov et al ., 2006 ).…”