209 Overexpression of WAVE1 activates pluripotency-related genes in porcine somatic cells

Abstract

Despite extensive efforts, cellular reprogramming in livestock species has had limited success. Induced pluripotent stem cells (iPSC) have been established; however, these cells often show incomplete reprogramming status, and constitutive expression of exogenous reprogramming factors is required due to inactivation of endogenous pluripotency-related genes. A previous study reported that overexpression of the Xenopus egg-derived WAVE1 gene assists reprogramming of murine somatic cells into the pluripotent state. The WAVE1 gene is also required for oocyte-mediated reprogramming by transcriptional activation of embryonic genes. In this study, we investigated the role of porcine WAVE1 in cellular reprogramming by inducing overexpression of WAVE1 in porcine fetal fibroblasts (PFF). Previously, we cloned the coding sequences (CDS) of porcine WAVE1 using porcine expressed sequence tags (EST) and predicted porcine WAVE1 sequences. The WAVE1 CDS, derived from porcine mature oocytes, was overexpressed in PFF by transfection using the Neon system. Then, G418-based antibiotic selection was performed to enrich cells constitutively overexpressing WAVE1. After cell culture for 4 weeks, RNA was extracted from the WAVE1 transfected and control PFF, and cDNA was synthesised from the RNA using random hexamers. The cDNAs were used for quantitative reverse transcription PCR to analyse the expression pattern of pluripotency- and reprogramming-related genes: POU5F1, NANOG, KLF2, SOX2, DPPA3, ZFP42, ESRRB, TET1, TET2, and TET3. The expression of target genes were normalized to GAPDH level and the ^ΔΔCt algorithm was used for analysis. Three technical replications and 4 biological replications were performed. Student’s t-test was used for the comparison and P-values < 0.05 were considered significant. On average, a 20-fold increase of WAVE1 was observed in the transfected cells compared with control cells. Interestingly, overexpression of WAVE1 activated some of the pluripotency-related genes in porcine PFF. Specifically, transcript levels of NANOG, KLF2, and SOX2 were increased compared with those in the control cells (P < 0.05). In addition, levels of POU5F1 and DPPA3 tended to be higher in WAVE1-overexpressing cells compared with those in the control cells (P < 0.1). However, transcript levels of other pluripotency-related genes (ZFP42, DPPA3, and ESRRB) did not change in WAVE1-overexpressing cells. The expression level of TET family (TET1, TET2, and TET3), which is enriched in pluripotent stem cells and a key regulator of DNA methylation, was not changed in WAVE1-overexpressing cells. These results indicate that WAVE1 can be a novel factor in porcine cellular reprogramming. Considering that a key defect of current porcine iPSC generation is insufficient expression of endogenous pluripotency genes, application of WAVE1 may enhance quality of porcine iPSC. We intend to evaluate expression of pluripotency markers at the protein level in WAVE1-overexpressing cells and investigate mechanisms underpinning WAVE1-mediated reprogramming process in future studies.