FUNCTIONAL-HETEROGENEITY OF HUMAN TERM CYTOTROPHOBLASTS REVEALED BY DIFFERENTIAL SENSITIVITY TO EXTRACELLULAR CA2+ AND NUCLEOTIDES

We have prepared purified cytotrophoblasts from human term placentas a nd examined the sensitivity of fura-2 loaded cells to the nucleotides ATP and UTP and to changes in extracellular Ca2+ concentration ([Ca2+] (o)). Purified cytotrophoblasts were obtained by collagenase digestion and separation according to density using self-generated Percoll grad ients. The cytotrophoblast fraction was free of red cell and largely f ree of white cell contamination (as assessed by uniformly negative sta ining for vimentin and the failure of >90% of fura-2 loaded cells to r espond to the chemotactic peptide fMet-Leu-Phe). Purified cells secret ed progesterone in a linear fashion over several hours in the presence of 25-hydroxycholesterol. The cells ranged in size from approximately 75 to 50 mu m in diameter as described previously for purified cytotr ophoblasts, and an analysis of cells for sensitivity to [Ca2+](o) or n ucleotides suggested functional heterogeneity within the cytotrophobla st population. Small cells (7.5-10 mu m) were negative for cytokeratin -8 and, after loading with fura-2, were insensitive to extracellular n ucleotides but sensitive to elevations in [Ca2+](o). Medium-sized cell s (12-20 mu m) were largely cytokeratin-positive (70% of cells) and se nsitive to both ATP and UTP but largely insensitive to [Ca2+](o). Larg e cells (25-50 mu m) were uniformly cytokeratin-positive (100% of cell s) and, after fura-2 loading, sensitive to both [Ca2+](o) and extracel lular ATP or UTP. We examined the likely origin of small, medium and l arge cytotrophoblasts using an immunomagnetic cell sorting procedure t hat separates villous cytotrophoblasts (which do not express major his tocompatibility class I antigens) from extravillous cytotrophoblasts. This procedure resulted in the selective sedimentation of almost all m edium and large cells, leading to the conclusion that the small cells were villous cytotrophoblasts whereas medium and large cells were pred ominantly extravillous in origin. The data suggest that small, medium and large cytotrophoblasts have distinct roles in the function of the term placenta.