Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei.

The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first 24 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham's/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration. While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15- to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.