In human fetuses of week 16 of pregnancy the various epithelia already contain intermediate-sized filaments of the cytokeratin type and desmoplakin-rich desmosomal plaques, as demonstrated by immunofluorescence microscopy of sections through frozen fetal tissues. When cells present in amniotic fluids obtained by amniocentesis during weeks 16–18 of pregnancy are allowed to grow in vitro, monolayer culture colonies of different morphology and cytoskeletal composition are obtained. We have examined such cells by electron microscopy and immunofluorescence microscopy, using antibodies to intermediate-filament proteins (cytokeratins, vimentin, desmin) and to desmoplakin, the major protein of the desmosomal plaque. Of the four major types of cell colonies regularly observed in such cultures, one morphotype ( ED) is characterized by a cobblestone-like pattern of closely spaced, small cells which contain filaments stained with diverse cytokeratin antibodies, including those raised against epidermal prekeratin, as well as desmoplakin-positive sites at cell-to-cell-boundaries. In such colonies only a few individual cells have been detected which also express vimentin filaments. Colonies of morphotype E are formed by larger cells which often leave variously-spaced gaps between each other and contain filaments decorated by diverse cytokeratin antibodies as well as vimentin filaments but reveal desmosomal staining only in a certain subpopulation of cells. AF-colonies contain cells which do not grow in epithelial-like layers but in irregular arrays. These cells react with antibodies to cytokeratins and vimentin but are heterogeneous, even within the same colony, with respect to their reactions with certain antibodies to epidermal prekeratin and to desmoplakin. The fourth major type of colony is formed by elongated ‘fibroblastoidal’ cells ( F-cells) which are stained by antibodies to vimentin and to some cytokeratins, but not by certain antibodies to epidermal prekeratins. F-cells do not reveal junctions stained by desmoplakin antibodies but do contain, like a certain proportion of AF cells, intracellular accumulations of desmoplakin-positive material. In cells of E-, F-, and AF-morphology double immunofluorescence microscopy has revealed bundles of intermediate-sized filaments stained with both antibodies to cytokeratins and antibodies to vimentin, besides other fibrils which are stained only with cytokeratin antibodies. Desmin filaments have not been detected in any of these colonies. Cells positive for vimentin but negative for all cytokeratin antibodies have only rarely been detected and are not regular components of such cultures. Gel electrophoretic analyses of cytoskeletal proteins of colonies of cell morphotypes have shown the presence of cytokeratins Nos. 7, 8, 18, and 19, together with some vimentin, in E, AF, and F colonies, but an absence of basic cytokeratin polypeptides. Desmin has not been detected. These results emphasize the importance of non-morphologic markers in the identification and classification of cultured cells. Specifically, they show that all four major morphotypes of cell colonies which are routinely used for prenatal diagnosis consist of cells of epithelial origin and that, in normal fetuses, mesenchymally derived cells (fibroblasts, astrocytes, etc.) do not make a considerable contribution to such cultures. The different epithelial morphotypes which can be distinguished in such cultures could be due to their derivation from different epithelia or from cells of different degrees of differentiation in the same epithelium. An alternative explanation, which we consider more likely on the basis of the biochemically identical cytokeratin patterns, is that such morphotypes may represent different cell clones from the same epithelium varying in their response to the culture conditions. In general, culturing seems to promote in these cells the expression of vimentin filaments, in addition to cytokeratin filaments, and the reduction of desmosomes. In this sense, ED cells would be the most conservative in terms of maintenance of epithelial character, whereas AF- and F-cells are grossly altered to the extent that they are no longer readily identified as epithelial cells because of their altered morphology, their negative reactions with certain antibodies to some epidermal keratins, and the sparsity, if not absence, of desmosomes in many cells. The identification of the same cytokeratin polypeptides in all four morphotypes strongly suggests that the differences among the different cell types in their reactivity with different antibodies to cytokeratins do not reflect differences of expression of cytokeratins but rather differences of the arrangement of these cytokeratins in the filaments of the specific cell type. Possible fetal epithelia from which these cell colonies might have originated are discussed.
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Oct 1, 2024
Cecilia Arriagada + 3
Open Access