393 CHARACTERIZATION OF PORCINE EMBRYONIC GERM CELL LINES DERIVED FROM DANISH LANDRACE AND YUCATAN MINI PIG EMBRYOS AT DAYS 20 TO 24 OF PREGNANCY

The establishment of pluripotent embryonic stem cells (ESC) in farm animals would benefit animal health and provide meaningful models for regenerative therapies and for studying embryonic development. However, long-term porcine ESC with preserved stem cell characteristics have not yet been established. Porcine embryonic germ cells (pEGC), which are pluripotent stem cell-like cells derived from primordial germ cells, have been established with high efficiency and, therefore, are an alternative to porcine ESC. Here we report the establishment of pEGC from Danish Landrace and Yucatan mini pig embryos at Day 20-24 of pregnancy, an embryonic stage that is significantly younger than reported previously (Day 25-28). The derived pEGC lines were characterized by morphological evaluation, scanning and transmission electron microscopy, RT-PCR for the expression of pluripotency markers, and immunostaining. In addition, molecular signaling pathways that might be involved in maintenance of pluripotency were investigated. We were successful in the derivation of pEGC lines from embryos at Day 20-21 (2/2 pooled, 9/15 individual; lines derived/PGC cultured from pooled or individual embryos, respectively) and Day 23-24 (3/3 pooled, 9/11 individual), but not from Day 17-18 (0/1 pooled, 0/14 individual) in the Danish Landrace. In addition, we successfully derived pEGC lines fromYucatan mini pig embryos at Day 23-24 of pregnancy (9/9 individual). All of the established pEGC lines proliferated robustly and were maintained for at least 8 passages (maximum 11 passages) without showing signs of senescence. The pEGC colonies had ESC-like morphology, and electron microscopy revealed the presence of cilia on the cell surfaces, similar to human ESC. The cells expressed at various levels OCT4, SOX2, NANOG, TERT, C-MYC, KLF4, CDH1, REX1, and other pluripotency markers. Receptors for leukemia inhibitory factor (LIF) (LIFr and gp130) and basic fibroblast growth factor (bFGF) (FGFr1 and FGFr2, isoform 2) were present at passages 0-7, whereas C-KIT was expressed at high levels only in primary cultures and down-regulated by passage 6. To determine which growth factors are relevant in maintaining pluripotency gene expression, we established and maintained pEGC lines for 10 passages in serum-free conditions in medium supplemented either with LIF, stem cell factor (SCF), or bFGF. Compared with nontreated controls, OCT4, SOX2, NANOG, and STAT3 were significantly up-regulated in LIF-treated pEGC lines (P < 0.01). In SCF-treated pEGC, NANOG and STAT3 were significantly higher than in control lines (P < 0.01), whereas in bFGF-treated pEGC, only STAT3 was significantly up-regulated (P < 0.01). Our results suggest that the LIF signaling pathway is active in pEGC; therefore, it is currently under further investigation. The pluripotency of 12 established cell lines is being tested by teratoma formation. These results will help to improve the culture of pluripotent stem cells in the pig.