Identification of 3i target molecules and their involvement in porcine pluripotency networks

Recent studies have shown that the use of specific inhibitors for signaling pathways known to drive murine embryonic stem cell (ESC) differentiation, may represent a tool to derive and maintain pluripotent cell lines. The application of this novel approach could provide a new strategy to overcome the limitations still existing for the derivation of ESC in large animal species. These molecules, also known as 3i factors, include CHIR99021 (GSK3 inhibitor), PD173074 (FGF inhibitor) and PD0325901 (MAPK/ERK kinase or MEK inhibitor). However only scattered information are available on the involvement of these pathways in the maintenance of pluripotency in domestic animals. Aim of this study was to investigate the presence of the receptors for these inhibitors in porcine ICMs and to isolate and culture porcine pluripotent lines in a serum free medium supplemented with the 3i factors, without any additional growth factor. Ovaries were collected at the local abattoir and cumulus-oocyte complexes (COCs) were aspirated from antral follicles. In vitro maturation was then performed for 46 hours. Frozen-thawed spermatozoa were purified and live spermatozoa were co-cultured with denuded oocytes for 24 hours. Putative embryos were cultured in NCSU-23 medium until the blastocyst stage and then subjected to immuno-surgery. Isolated ICMs were analyzed by RT-PCR. Poly(A)+RNA was extracted using Dynabeads® mRNA DIRECT Micro-kit (Invitrogen) and immediately reverse-transcribed with Superscript- ™ II Reverse Transcriptase (Invitrogen). Specific primers were designed for FGF4, FGFR-1, FGFR-2, FGFR-4, GSK3 and MEK genes. PCR amplified products were sequenced and aligned using ClustalW. RT-PCR results showed that porcine ICMs actively transcribe for GSK3, MEK, FGFR-2, FGFR-4, FGF4 genes, while no signal was detectable for FGFR-1 gene. Based on these observations IVF -derived ICMs were plated onto inactivated STO feeder cells and cultured in N2B27 medium supplemented with 3μm CHIR99021, 100nM PD173074 and 0.4μM PD0325901. Outgrowth formation was monitored and cells were passaged to a new STO monolayer every seven days, as previously described (Brevini et al., Stem Cell Rev. 2010). Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 15 passages. The results obtained indicate that porcine cells cultured in 3i medium, without additional growth factors, expressed pluripotency markers, namely OCT4, NANOG, SOX2 and REX1, preserving their pluripotent state over time. Our data indicate that porcine ICMs express 3i factor target molecules. In agreement with this, the use of 3i medium allows the establishment and proliferation of pluripotent cell lines. Altogether these findings suggest the involvement of the GSK3, FGF, and MEK pathways in porcine pluripotency network and advocate the use of the 3i medium as an efficient tool for ESC derivation in porcine.