In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20–24 of gestation

Abstract

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency. RT-PCR showed that gene expression among all 31 obtained cell lines was very similar, and only minor changes were detected during in vitro passaging of the cells. Genome-wide RNA-Seq expression profiling showed no expression of the core pluripotency markers OCT4, SOX2, and NANOG, although most other pluripotency genes were expressed at levels comparable to those of mouse embryonic stem cells (ESC). Moreover, germ-specific genes such as BLIMP1 retained their expression. Functional annotation clustering of the gene expression pattern of the putative EGC suggests partial differentiation toward endo/mesodermal lineages. The putative EGC were able to form embryoid bodies in suspension culture and to differentiate into epithelial-like, mesenchymal-like, and neuronal-like cells. However, their injection into immunodeficient mice did not result in teratoma formation. Our results suggest that the PGC-derived cells described in this study are EGC-like, but seem to be multipotent rather than pluripotent cells. Nevertheless, the thorough characterization of these cells in this study, and especially the identification of various genes and pathways involved in pluripotency by RNA-Seq, will serve as a rich resource for further derivation of porcine EGC.

Recent Advances in Stem and Germ Cell Research: Implications for the Derivation of Pig Pluripotent Cells

“…These cells could be maintained for 35 passages; however, they did not express OCT-4, NANOG and SOX-2, and did not generate teratomas (Petkov et al 2011). No EG cell lines were obtained from day 17 to 18 foetuses (Petkov et al 2011). Thus, it appears that early pig PGCs may have different culture requirements compared to their early gonadal counterparts.…”

“…A recent report shows that such lines can be produced from 20-to 24-day-old foetuses. These cells could be maintained for 35 passages; however, they did not express OCT-4, NANOG and SOX-2, and did not generate teratomas (Petkov et al 2011). No EG cell lines were obtained from day 17 to 18 foetuses (Petkov et al 2011).…”

Porcine pluripotent stem cells: progress, challenges and prospects

“…They show a large, flat and round (polygonal in rare cases) shape with compact colonies and distinct borders, with relatively small diameters and a high nucleus-to-cytoplasm ratio, a single nucleus with multiple nucleoli, and are sensitive to tryptase. These cells grow more slowly and have limited capability to be integrated into host blastocysts [92,120,122,134,141,143] . Another type of porcine PSCs was shown to have a mouse ESCs morphology, i.e., small, not flat and with a compact, glistening, doom shaped appearance.…”

Pluripotent cells in farm animals: state of the art and future perspectives

“…The culture of porcine EGCs in medium supplemented with knock-out serum (KSR) and LIF, SCF and bFGF led to cells with a morphology similar to mouse ESC, which expressed stem-cell markers such as AP, SSEA-1, SSEA-3, SSEA-4 and TRA-1-81, had a normal karyotype, proliferated for up to 54 passages and differentiated in vitro into cells from the three germ layers (Petkov and Anderson 2008). Recent in-depth characterisation of putative porcine EGCs by functional and molecular studies, including RNA sequencing (RNAseq), indicates that the cells are rather multipotent than pluripotent and do not show higher expression levels of some of the key pluripotency transcription factors than porcine embryonic fibroblasts (Petkov et al 2011). EGCs can potentially be cultured in vitro indefinitely in the undifferentiated state, and thus have the potential to overcome the limitations encountered with the short lifespan of somatic cells and to facilitate the production of transgenic animals (Rui et al 2004(Rui et al , 2006Ahn et al 2007).…”