ISOLATION AND CHARACTERIZATION OF HUMAN PLACENTAL TROPHOBLAST SUBPOPULATIONS FROM FIRST-TRIMESTER CHORIONIC VILLI

A method for the simultaneous preparation of highly enriched human pla cental trophoblast populations (villous and eutravillous) from first-t rimester placental villi (5 to 12 weeks) bg using sequential trypsiniz ation, percoll gradient centrifugation, and negative selection viith a nti-CD9 immunomagnetic separation is described, The purification metho d resulted in the isolation of four distinct trophoblast populations i dentified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophohlast cells which, through differentiation, become c ommitted to syncytium formation; (ii) an extravillous trophoblast popu lation which appeared as a ''crazy pavement'' and, with subsequent sub culturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleate d 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 extravi llous trophoblast. Short-term cultures of the freshly isolated cell fr actions consisted of heterogeneous trophoblasts at different different iation stages as determined by their varied biochemical and morphologi cal properties, All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-ce ll adhesion molecule E-cadherin, The isolated villous trophoblasts in culture expressed integrins alpha 6 and beta 4 and reduced levels of b eta 1 subunits, whereas the proliferating extravillous trophoblast cul tures expressed alpha 1, alpha 3, and alpha 5 and high levels of pi in tegrin subunits, vitronectin receptor (alpha V beta 3/beta 5), and maj or histocompatibility complex class 1 molecules. Furthermore, the isol ated trophoblast populations secreted metalloproteases (such as type T V collagenases [mainly 72- and 92-kDa enzymes, i,e,, gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate ge l zymography, This method of isolation should facilitate in vitro stud ies of trophoblast proliferation, differentiation, invasion, virus int eractions, cytokenesis, and immunology.