The conversion of PGC into pluripotent stem cells

The conversion of PGC into pluripotent stem gpr119 agonist in culture is a poorly understood process. Mouse PGC are unipotent, i.e., not capable of differentiation into somatic cell types and contributing to chimeras, and acquire pluripotency only after a certain period in culture (Stewart et al., 1994; Durcova-Hills et al., 2006). For their survival and proliferation in vitro they require the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and basic fibroblast growth factor (bFGF) in the culture medium (Matsui et al., 1992; Resnick et al., 1992). As a ligand of c-kit, SCF is essential for survival of mouse PGC in vivo and in vitro by inhibiting apoptosis (Pesce et al., 1993; Sette et al., 2004), while LIF is necessary for maintenance of pluripotency. It has been shown that bFGF plays an important role for in vitro reprogramming of mouse PGC (Durcova-Hills et al., 2006). Furthermore, B lymphocyte-induced maturation protein 1 (BLIMP1/PRDM1), a transcriptional repressor critical for the specification of the germ line (Hayashi et al., 2007; Ohinata et al., 2005; Saitou et al., 2005), downregulates the transcription of c-myc and Krüppel-like factor 4 (Klf4) in mouse PGC, in addition to preventing their differentiation into the somatic lineages. Downregulation of BLIMP1 by bFGF in culture allows the expression of c-myc and Klf4 to be upregulated, which in turn leads to reprogramming of mouse PGC to attain pluripotency (Durcova-Hills et al., 2008). Both c-myc and Klf4 are transcription factors that together with Octamer binding protein 4 (Oct4) and SRY-box 2 protein (Sox2) are capable of reprogramming somatic cells into induced pluripotent stem cells (iPSC) (Takashi and Yamanaka, 2006; Takashi et al., 2007; Okita et al., 2007). Unlike in the mouse, freshly isolated porcine PGC are capable of contributing to somatic chimeras (Müller et al., 1999) and to survive and to proliferate in serum-supplemented and in serum-free culture without any growth factor supplementation (Shim et al., 1997; Petkov and Anderson, 2008). It is therefore unclear if the same mechanisms of reprogramming occur in porcine PGC when placed in culture. The EGC lines derived from these porcine cells have shown the ability to differentiate into cells from the three germ layers in vitro and also upon injection into blastocysts (Müller et al., 1999; Piedrahita et al., 1998; Shim et al., 1997). However, these EGC have been characterized for relatively few pluripotency markers and have not shown sufficient evidence for pluripotency such as germ-line chimera formation in vivo (likely due to the relatively low extent of chimeric contribution), or ability for indefinite self-renewal in vitro, two important criteria for pluripotent embryonic stem cells. Therefore, an improvement of the culture conditions for derivation and propagation of porcine EGC as well as more systematic application of all relevant methods for characterization of these cells is currently necessary. The stage of embryonic development has been shown to be a decisive factor in the derivation of EGC in the mouse, where these cells have been derived from Day 8.5–13.5 embryos (Matsui et al., 1992; Resnick et al., 1992; Labosky et al., 1994; Durcova-Hills et al., 1999; Durcova-Hills et al., 2001; Durcova-Hills et al., 2004; Tada et al., 1998; Shim et al., 2008). To date, porcine EGC have been derived from embryos at Days 24–25 (Shim et al., 1997), 25–27 (Piedrahita et al., 1998), and 26–28 (Tsung et al., 2003). Derivation of EGC lines from later stage embryos may be limited due to the fact that soon after arrival to the genital ridge, male and female PGC undergo mitotic and meiotic arrest, respectively (Buehr, 1997). In contrast, the use of younger embryos for EGC culture might help to improve the efficiency of derivation as well as the quality of the established EGC lines, since PGC at earlier stages of development may have a higher proliferative potential than those in the genital ridge. At the same time, migrating PGC are less advanced in the process of gametogenesis and therefore closer to the pluripotent epiblast from which they descend.