Adhesive and Degradative Properties of Human Placental Cytotrophoblast Cells in Vitro Susan J
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Adhesive and Degradative Properties of Human Placental Cytotrophoblast Cells In Vitro Susan J. Fisher, Tian-yi Cui, Li Zhang, Lynn Hartman, Kim Grahl, Zhang Guo-Yang, John Tarpey, and Caroline H. Damsky Departments of Stomatology, Anatomy, and Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143 Abstract. Human fetal development depends on the ponents into the medium. No similar degradative ac- embryo rapidly gaining access to the maternal circula- tivity was observed when second or third trimester tion. The trophoblast cells that form the fetal portion cytotrophoblast cells, first trimester human placental of the human placenta have solved this problem by fibroblasts, or the human choriocarcinoma cell lines transiently exhibiting certain tumor-like properties. BeWo and JAR were cultured on radiolabeled matrices. Thus, during early pregnancy fetal cytotrophoblast To begin to understand the biochemical basis of this cells invade the uterus and its arterial network. This degradative behavior, the substrate gel technique was process peaks during the twelfth week of pregnancy used to analyze the cell-associated and secreted pro- and declines rapidly thereafter, suggesting that the teinase activities expressed by early, mid, and late highly specialized, invasive behavior of the cytotropho- gestation cytotrophoblasts. Several gelatin-degrading blast cells is closely regulated. Since little is known proteinases were uniquely expressed by early gesta- about the actual mechanisms involved, we developed tion, invasive cytotrophoblasts, and all these activities an isolation procedure for cytotrophoblasts from could be abolished by inhibitors of metalloproteinases. placentas of different gestational ages to study their By early second trimester, the time when cytotropho- adhesive and invasive properties in vitro. Cytotropho- blast invasion rapidly diminishes in vivo, the pro- blasts isolated from first, second, and third trimester teinase pattern of the cytotrophoblasts was identical to human placentas were plated on the basement mem- that of term, noninvasive cells. These results are the brane-like extracellular matrix produced by the PF first evidence suggesting that specialized, temporally HR9 teratocarcinoma cell line. Cells from all tri- regulated metalloproteinases are involved in tropho- mesters expressed the calcium-dependent cell adhesion blast invasion of the uterus. Since the cytotrophoblasts molecule cell-CAM 120/80 (E-cadherin) which, in the from first trimester and later gestation placentas main- placenta, is specific for cytotrophoblasts. However, tain for several days the temporally regulated degrada- only the first trimester cytotrophoblast cells degraded tive behavior displayed in vivo, the short-term the matrices on which they were cultured, leaving cytotrophoblast outgrowth culture system described large gaps in the basement membrane substrates and here should be useful in studying some of the early releasing low molecular mass 3H-labeled matrix corn- events in human placentation. ORMATION of the human placenta requires a remark- 1976; Turtle et al., 1985). This invasive activity peaks during able series of events involving highly specialized fetal the twelfth week of pregnancy and declines rapidly there- F and maternal ceils. Once the blastocyst adheres to the after. The result is formation of the human hemochorial uterus, the fetal cytotrophoblast cells rapidly penetrate the placenta, in which blood from the maternal circulation con- endometrium. Soon thereafter, mononuclear cytotropho- stantly bathes the fetal chorionic villi. blasts and multinuclear syncytiotrophoblasts are found min- Although several species have hemochorial placentas, gled with maternal decidual cells throughout much of the there are important features of human placentation that are placental bed, a condition that persists during the remainder unique. First, there is generalized mixing of maternal and fe- of the pregnancy (see Fig. 1). In addition, groups of cytotro- tal cells in the placental bed such that there is no definitive phoblasts migrate through the decidua, invade the walls of boundary between these components. This is in contrast to the spiral arterioles, and replace the endothelial lining as far the mouse where a well-defined boundary exists between the as the myometrial segments of these vessels (Brosens and fetal trophoblast cells and the decidua. Second, human Dixon, 1966; Boyd and Hamilton, 1970; Ramsey et al., trophoblast invasion is extensive, reaching the first third of © The Rockefeller University Press, 0021-9525/89/08/891/12 $2.00 The Journal of Cell Biology, Volume 109, August 1989 891-902 891 the myometrium. Invasion in even closely related primates systematic investigation of the developmental regulation of is limited to the endometrium (Ramsey et al., 1976). These plasminogen activators or other trophoblast proteinases that differences suggest that certain of the mechanisms used by might contribute to the observed limitation of trophoblast in- human trophoblast cells to penetrate the uterus are unique. vasion to early gestation. Currently, most of what we know about these processes in In vitro models have also been used to study human vivo has been learned from morphological observations of trophoblast outgrowth. Hatched, human blastocysts have early human implantation sites recovered surgically (Hertig been cultured on monolayers of human endometrial epithe- et al., 1956; O'Rahilly, 1973; Enders, 1976). More recently, lium. This morphological study suggested that, in contrast additional information has been obtained using in vitro to many other species including the mouse, cytotrophoblast models. cells mediate the initial stages of human implantation (Lin- Trophoblast outgrowth models have been used to study denberg et al., 1986). Work from our laboratory has shown certain aspects of the dynamic processes thought to occur that isolated first trimester human chorionic villi can adhere during implantation in the rodent. Blastocysts have been cul- to and rapidly degrade the ECMs on which they are grown tured on plastic, glass, or collagen (Mintz, 1964; Cole and (Fisher et al., 1985). The timetable of invasive behavior ob- Paul, 1965; Gwatldn, 1966a,b; Sherman and Barlow, 1972; served in vitro paralleled that which occurs in vivo in that Spindle and Pedersen, 1973; Nilsson, 1974; Sherman, second trimester villi could adhere to, but not invade, the 1975a,b). Trophoblast outgrowth on more complex sub- same matrices. Morphological evidence suggested that ma- strates such as extracellular matrices (ECMs) ~ (Glass et al., trix degradation was mediated by cytotrophoblast cells that 1983) and on cell monolayers (Cole and Paul, 1965; Salo- migrated from the villi. mon and Sherman, 1975; Sherman and Salomon, 1975; To demonstrate conclusively that cytotrophoblasts can ac- Sherman, 1975a,b; Glass et al., 1979) has also been studied. count for the developmentally regulated degradative be- These systems offer the advantage of providing a more bio- havior of human chorionic villi, we developed a method for logically relevant, three-dimensional substrate for tropho- isolating cytotrophoblast cells that could be applied to blast migration. Investigators using such systems have con- placentas from both first and second trimester. The method sistently found that the outgrowing cells can invade and of Kliman et al. (1986) was used to isolate cytotrophoblasts migrate through ECMs (Glass et al., 1983), uterine stromal from term placentas. Two molecules that, within placental cells (Salomon and Sherman, 1975; Sherman and Salomon, villi, are specific for cytotrophoblasts were used as markers 1975; Sherman, 1975a,b; Glass et al., 1979), or various to verify the identity of the isolated cells from all three other cell types (Cole and Paul, 1965; Salomon and Sher- trimesters. These were cytokeratin and cell-CAM 120/80 man, 1975; Sherman and Salomon, 1975; Glass et al., 1979). (hereafter referred to as E-cadherin [Damsky et al., 1983; It has been suggested that the clear halo that sometimes Yoshido-Noro et al., 1984; Takeichi, 1988]), a calcium- forms slightly ahead of the outgrowth may be the result of dependent cell adhesion molecule expressed on differen- cytolytic enzymes (Salomon and Sherman, 1975; Sherman tiated epithelia (also known as uvomorulin [Peyreiras et al., and Salomon, 1975). 1983] and in avian cells as L-CAM [Gallin et al., 1983]). Consistent with this interpretation are experiments sug- Culturing the cells on ECM showed that cytotrophoblasts gesting that exogenous proteinase inhibitors can prevent from first, but not second and third, trimester placentas blastocyst attachment in vitro (Blackwood et al., 1968). could degrade a complex ECM substrate. The stage-specific Kubo et al. (1981) used the proteinase inhibitor approach to behavior of these cells suggests that this short-term culture demonstrate that in the mouse, the process of blastocyst at- system will be useful in the study of some of the adhesive and tachment requires a trypsin-like activity and the process of invasive mechanisms required for successful human placen- trophoblast outgrowth requires both plasminogen activator tation. As an example of the use of this system, we compared and trypsin-like activities. Plasminogen activators have been the repertoire of gelatin-degrading proteinases produced by implicated in developmental and pathological processes that invasive, early gestation cytotrophoblasts with those pro-