Formation of neural and epidermal lineages during the differentiation of human embryonal carcinomacells

Abstract

Within the ectoderm of the developing human embryo, cell fate determination is largely modulated by factors activated by cell-to-cell signalling. In vertebrates, ectoderm primarily differentiates into neural cells that make up the central nervous system and lining epithelium, commonly known as the skin epidermis. One of the main regulators of this process is the bone morphogenetic protein (BMP) signaling pathway. While this pathway, and its involvement in ectodermal patterning, has been well studied in lower vertebrates there is little data available for its role in human ectodermal development. The main aim of this investigation was to establish whether the human TERA2.SP12 embryonal carcinoma (EC) stem cell line may be a viable model useful in the study of this particular mechanism in humans. Pluripotent human embryonal carcinoma cells have already been shown to represent a researchable model of human embryogenesis. It has also been shown that the differentiation of the human EC cell lines can be induced in a controlled marmer using certain reagents such as retinoic acid and hexamethylene bisacetamide (HMBA). In this study it has been determined that the TERA2 EC cell sub-line TERA2.SP12 can also be induced to differentiate in a controlled manner. Retinoic acid induces cultured TERA2.SP12 cells to differentiate into a variety of cell types, including neurons and glial cells. Using molecular techniques such as flow cytometry, western analysis, immunocyto chemistry and RT-PCR these cultures were seen to express a range of proteins associated with the neural lineage. Non-neural cell types are also found in these cultures that are morphologically similar to those found when TERA2.SP12 EC cells are exposed to HMBA. Differentiation of TERA2.SP12 EC cells induced by HMBA was shown to differ to that induced by retinoic acid in that no morphologically identifiable neurons are observed in cultures grown as adherent monolayers. Through analysis of specific proteins it was noted that the expression of neural markers was significantly reduced. Interestingly, cells exposed to HMBA were found to regulate significantly higher levels of certain extra-cellular proteins such as fibronectin, laminin and cytokeratin-8. One of the mechanisms that these molecules have been previously associated with is the development of epidermis. Proteins and genes present in the BMP signalling pathway have also found to be expressed by these TERA2.SP12 differentiating cultures. These results indicate that retinoic acid induces the formation of neural tissues as well as non-neural tissues from human EC cells. Exposure to HMBA, however does not result in terminally differentiated neural phenotypes but induces the formation of a different lineage expressing high levels of markers that could be associated with developing epithelia such as immature epidermis. Accordingly, I conclude that the TERA2.SP12 EC cell system may be utilised as a potential model for cell fate specification between neural and non-neural lineages in man.