212 Increased expression of YAP/TAZ encourages outgrowth establishment and three-dimensional colony formation and boosts plasticity of parthenogenetic stem cells

Abstract

Cell proliferation, apoptosis, and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs), which are able to proliferate maintaining pluripotency and, at the same time, give rise to all populations belonging to the three germ layers in response to specific stimuli. ESCs are, therefore, considered a well-established invitro model to study the complexity of these processes. We previously generated porcine and human diploid parthenogenetic embryonic stem cells (ParthESC) that showed many features and regulatory pathways common to biparental ESCs. However, we observed that monoparental cells demonstrated a greater ability to form outgrowths and generate stable three-dimensional (3D) spheroid colonies, which are distinctive signs of high plasticity (Brevini et al. 2010 STCR 6; https://doi.org/10.1007/s12015-010-9153-2). In the present study, we compared porcine and human biparental ESCs and ParthESC. Pluripotency gene expression was analysed and ELISA tests for global DNA methylation and total YAP levels were performed. Our results showed that ParthESCs showed significantly higher expression levels of YAP and TAZ than biparental ESCs. In contrast, the analysis of the upstream genes involved in the Hippo signalling pathway revealed no differences. We demonstrated that YAP remains in a dephosphorylated form, encouraging its nuclear compartmentalization and direct interaction with the nuclear transcription factors TEADs and SMADs, that are also upregulated and maintain pluripotency, repressing differentiation processes (Beyer et al. 2013 Cell Rep. 5, 1611-1624; https://doi.org/10.1016/j.celrep.2013.11.021). These complex regulatory interactions accompanied the overexpression of the pluripotency-related genes OCT4, NANOG, REX1, SOX2, UTF1, and TERT. In parallel, we observed a decrease of DNMT3 and DNMT4 activity that resulted in a global DNA hypomethylation and a chromatin high-permissive state. Altogether, our results demonstrate a significant upregulation of YAP/TAZ activity in monoparental cell lines. This may account for their greater ability to form outgrowths and generate 3D spheroid colonies and increased plasticity compared with biparental counterparts. It is interesting to note that YAP mRNA is supplied and expressed by the oocyte and is maternally inherited (Yu et al. 2016 Cell Res. 26, 275-287; https://doi.org/10.1038/cr.2016.20). Although further clarifications are needed, we hypothesise that its higher expression in ParthESC may be related to the strictly maternal origin of these cells. This study was supported by the Carraresi Foundation. Authors are members of the COST Actions CA16119.