Hitoshi Funayama

EXTRACELLULAR MATRIX COMPONENTS OF THE PLACENTAL EXTRAVILLOUS TROPHOBLAST : IMMUNOCYTOCHEMISTRY AND ULTRASTRUCTURAL DISTRIBUTION

Invasive extraiillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, ‘i”. Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaiclike patches of structurally and immunocytochemically different comparments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shown reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen “i” was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectinpositive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with “i” in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

Parent cells for trophoblast hybridization I: Isolation of extravillous trophoblast cells from human term chorion laeve

Summary Extravillous trophoblast cells of human placenta produce matrix-type fibrinoid, an oncofetally modified extracellular matrix. Matrix-type fibrinoid is linked to differentiation of extravillous trophoblast cells. It is involved in anchoring of the placenta to the uterine wall, at the same time enabling the trophoblast cells to migrate and to invade maternal tissues. Biochemical isolation of matrix-type fibrinoid is impossible. In vitro production is hampered by the lack of proliferation of differentiated extravillous trophoblast cells. Therefore, we want to immortalize the capability for production of matrix-type fibrinoid. In order to avoid any introduction of non human regulative genes into the trophoblastic genome, we have chosen the hybridoma technique for immortalization. Negatively selectable choriocarcinoma cell lines are available as trophoblast-related malignant parent line for fusion. Here we describe a rapid isolation protocol for differentiated term extravillous trophoblast cells. With chorion laeve as source we avoid any accidental admixture of trophoblast cells of villous origin. Immunomagnetic isolation of extravillous trophoblast cells was performed using placental alkaline phosphatase as highly trophoblast specific positive marker molecule. The relatively simple method yields preparations of differentiated extravillous trophoblast cells of high purity, which are used as second parental cell population for generation of hybridomas.

Parent cells for trophoblast hybridization II: AC1 and related trophoblast cell lines, a family of HGPRT-negative mutants of the choriocarcinoma cell line JEG-3

Summary To gain analytical and productive access to the oncofetally modified molecules in the extracellular matrix of human non-proliferative invasive trophoblast cells, we intend to immortalize these cells together with their productive capacity by fusion with their malignant trophoblast counterpart. To generate a suitable malignant fusion partner we have selected hypoxanthine-guanosin-phosphoribosyl-transferase-negative mutants of the choriocarcinoma cell line JEG-3. We obtained the cell line AC1 and its subclones AC1-1, AC1-5 and AC1-9. AC1-1 is our preferred candidate for fusion with matrixproducing invasive trophoblast cells since this subclone homogenously did not react with antibodies recognizing trophoblast matrix molecules.

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid.

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Comparative study of placental protein 19, human chorionic gonadotrophin and pregnancy-specific ?1-glycoprotein as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease

SummaryPP19, a new placental tissue protein, has α1-β1 electrophoretic mobility, a molecular weight of 36 500 and 3.9% carbohydrate. To study immunocytochemical PP19 localization in extravillous trophoblast, we obtained formalin-fixed specimens from extravillous tubal pregnancy at gestational weeks (GW) 7–9 (12 blocks); four early intrauterine pregnancies at GW 7–13 (12 blocks); four late pregnancies at GW 28–38 complicated with intramural uterine myoma, placenta increta and abruptio placenta (8 blocks); four invasive complete moles (9 blocks); and seven primary and metastatic gestational choriocarcinomas (12 blocks). Immunohistochemical staining was done for PP19, pregnancy-specific β1-glycoprotein (SP1) and human chorionic gonadotrophin (hCG) using the indirect-labeled antibody method [purified PP19 (Lot no. 225/242) and antibody against PP19 (Lot no. 632ZA) prepared by H. Bohn, antibodies against hCG (Behringwerke, Marburg, FRG) and SP1 (Dakopatts, Copenhagen, Denmark)]. In both early and late intrauterine pregnancies, the extravillous syncytiotrophoblastic cell (XST) showed positive staining for hCG and SP1 in the cytoplasm, as well as for PP19, which stained more intensively in the nucleus than in the cytoplasm. The three proteins were not seen in the evtravillous cytotrophoblastic cell (XCT) in the trophoblastic cell column and shell. The interstitial cytotrophoblast-like cell (ICT), which infiltrated into the decidua and myometrium, and their blood vessels, was immunoreactively positive for PP19 but negative for hCG and SP1 with the exception of SP1-positive ICT in the myometrium in late pregnancy. XST and ICT in the endosalpinx of tubal pregnancy stained for all three proteins. In invasive complete mole, XST stained for the three proteins, but ICT infiltrating into the decidua and myometrium stained more intensively for PP19 than for either hCG or SP1. XCT did not stain for the three proteins. Staining for the three proteins in gestational choriocarcinoma resembled that in invasive mole. By PP19 staining, XST and ICT infiltrating into surrounding tissue were clearly distinguishable from other cells of similar shape. PP19 staining thus can be a useful histochemical marker in assessing the cell viability of the trophoblastic tumor after chemotherapy.

Choriocarcinoma-trophoblast hybrid cells: Reconstructing the pathway from normal to malignant trophoblast — Concept and perspectives

Summary Extravillous trophoblast cells of normal and molar pregnancies spontaneously can transform into choriocarcinoma cells. This malignant transformation differs from that in other tissues in two respects: (a) the normal tissue of origin, extravillous trophoblast, is already invasive, and (b) both, choriocarcinoma and extravillous trophoblast, are foreign (allohaploid) to the host. Carcinogenetic transformation of trophoblast into choriocarcinoma is a multistep process in the course of which temporary intermediate geno- and phenotypes are generated; these are mixtures between trophoblast and choriocarcinoma and illustrate the process of carcinogenesis. The intermediates are either selected by the host for survival or undergo further mutations. Consequently, it remains unknown which properties are lost or acquired during the process and which of them are crucial either for control of invasion of normal extravillous trophoblast or for the malignant development of choriocarcinoma cells. In order to identify mutative trophoblast-relevant oncogenes and tumor suppressor genes, we have generated somatic cell hybrids using the choriocarcinoma cell line AC1-1 and extravillous trophoblast cells. The resulting hybrid cells were cloned, characterized cytogenetically and classified according to tumorigenicity. These hybrids represent a population of intermediates between non-tumorigenic extravillous trophoblast cells and highly tumorigenic choriocarcinoma cells, similar to those temporarily formed during carcinogenesis. The hybrids are thought to represent a suitable in vitro tool for the identification of genes and gene products involved in both control of normal invasion and malignant transformation of trophoblast cells. Analysis of the hybrid clones involves both, methods assessing phenotypic (functional) and genotypic properties.

Placental Protein 19 in Clinical Blood Coagulation Disorder

The human placenta is a highly functional organ able to synthesize a wide variety of biologically active proteins, including hormones, enzymes, proenzymes, activators, inhibitors, immunoregulatory factors, transport and storage proteins, receptors, and structual proteins [1], Moreover, many proteins of unknown function have been discovered and isolated in placental extracts.