A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are (1) fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; (2) epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; (3) negative for the stage-specific embryonic antigen-1 (SSEA-1); and (4) capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.
Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.
A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the materanl syncytial layer. Immunoprecipitation of I-labelled microvilli by SBU-3 characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.
This paper describes the identification and characterization of a new peptide growth factor. The peptide was isolated from trophoblastic brush border membranes of human placenta. The purified preparation was homogeneous and consisted of a single polypeptide of Mr 34 000 with a pI of about 6.0. This peptide stimulated DNA replication in cultured fibroblasts. The following association was seen between activity and protein: During DEAE-cellulose chromatography, both the 34-kilodalton (kDa) protein and the mitogenic activity displayed identical binding and salt dependence of elution. Nondenaturing electrophoresis at pH 8.3 revealed a comigration of the 34-kDa protein and the DNA replication stimulatory activity. Identical electrophoretic mobilities were displayed for both activity and protein at pH 7.0. These results demonstrate that the preparation is homogeneous and show that growth factor activity is intrinsic to the 34-kDa polypeptide. Binding of the 125I-labeled 34-kDa mitogen to target fibroblastic cells was specific; i.e., nanomolar concentrations of the unlabeled 34-kDa protein competed effectively with the labeled protein, whereas a variety of well-characterized growth factors and hormones were unable to compete even at micromolar levels. Thus the 34-kDa protein interacts with target cells through highly specific surface receptors. Chemical cross-linking techniques were used to investigate the identity of the receptor for the 34-kDa mitogen. Cross-linking of fibroblastic cells containing bound 125I-labeled 34-kDa protein generated a radiolabeled complex of 86 kDa in all four cell types examined.