Signalling events controlling early cell fate decisions in a human embryonal carcinoma cell line, with relation to neural versus non-neural transitions m human ectoderm development

Abstract

In this study the human embryonal carcinoma cell line TERA2.cl.spl2 (TERA2.spl2) was utilised as an in vitro model in which to study mechanisms governing neural versus non- neural transitions in the human ectoderm. TERA2.spl2 cells have been previously reported to differentiate under adherent culture conditions into both neural and non neural derivatives of the ectoderm in response to treatment with retinole acid (RA). In this investigation an alternative culture method was also characterised, which markedly modulated the differ entiation fate of TERA2.spl2 cells in response to RA. Dissociated cells cultured in the presence of RA under suspension conditions generated aggregates which differentiated rapidly, and as detected by immuno-staining and Western blot analyses, exhibited marked up-regulation of neural markers without evidence of non neural differentiation. Previous work in cultured Xenopus animal cap expiants show that suspension cultures of dissociated ectoderm differentiate into neural instead of epidermal tissue as a result of the inhibition of Bone morphogenetic protein (BMP) pathway activity. Polymerase chain reaction (PCR) and Western blot analyses demonstrated that RA treated adherent cultures of TERA2.spl2 cells exhibited up-regulated expression of BMP regulated genes in comparison to cultures treated in parallel and cultured under suspension conditions. Correspondingly, addition of the BMP inhibitor Noggin to adherent cultures increased the expression of neural markers (as detected by Western blot analyses) towards that observed in suspension cultures. Both BMP and FGF mediated pathways were also manipulated in the absence of RA exposure; BMP pathway activation or FGF receptor inhibition induced similar differentiation responses, yielding a differentiated heterogeneous population. Correspondingly, FGF receptor inhibitor and BMP treatments were detected to up-regulate the expression of BMP regulated genes by Western blot analysis, supporting the hypothesis that in both treatments differentiation was driven by an up-regulation of BMP pathway activity. Treated cultures displayed positive immuno- staining for trophoblast, smooth muscle and simple epithelial endoderm derivatives, demonstrating for the first time that TERA2.spl2 cells are capable of forming non-ectodermal lineages. To further investigate the full differentiation potential of TERA2.spl2 cells, the differentiation of tumour expiants derived from TERA2.spl2 cell injection into immuno compromised mice was characterised. Although tumours exhibited markedly restricted differentiation complexity when compared with tumours derived from human embryonic stem (ES) cell lines, immuno-staining assays demonstrated that TERA2.sp12 cells formed multiple germ layer derivatives in vivo. In conclusion, neural differentiation of TERA2.spl2 cells can be modulated by active manipulation of the BMP signalling pathway, and TERA2.spl2 cells provide a suitable model in which to further investigate the effects of BMP signalling on neural induction. However, although TERA2.spl2 cells exhibit a marked propensity for ectodermal differentiation, their differentiation potential is not restricted to ectoderm derivatives and is therefore more similar to human ES cells.