In mammalian PGCs genetic knockout

In mammalian PGCs, genetic knockout models and short-term PGC culture experiments have implicated the growth factors BMP4, LIF, SCF, retinoic acid, and FGF in early survival and proliferation (Dolci et al., 1991, 1993; Farini et al., 2005; Matsui et al., 1991). PGCs isolated from mammalian species can only be propagated as lineage-restricted germ Ozanimod manufacturer for short periods in culture (De Felici and McLaren, 1983; Dolci et al., 1991; Durcova-Hills et al., 1998; Farini et al., 2005; Matsui et al., 1991). PGCs from male and female chicken embryos, however, have been propagated long-term in vitro while maintaining lineage specificity and germline competency (van de Lavoir et al., 2006; Song et al., 2014). Chicken PGCs that are isolated from embryonic blood during their migration to the gonad can be expanded extensively in vitro. These germline stem cells form functional gametes and offspring after re-introduction into surrogate host embryos (Choi et al., 2010; Macdonald et al., 2010, 2012). Thus, chicken PGCs potentially offer a route to both the cryopreservation, biobanking, of poultry breeds and for the introduction of targeted mutations into the chicken genome (Blesbois et al., 2008; Glover and McGrew, 2012; Park et al., 2014; Petitte, 2006; Schusser et al., 2013). The development of defined, feeder-free culture conditions will facilitate the in vitro culture of PGCs. The medium for the in vitro propagation of chicken PGCs is ill-defined, containing animal sera, conditioned medium, and a feeder cell layer (van de Lavoir et al., 2006). Here, based on defined serum-free medium conditions for embryonic stem cells (ESCs), we develop defined culture conditions for chicken PGCs and ascertain the minimal signaling pathways necessary for avian germ cell self-renewal. These culture conditions provide insight into the self-renewal of vertebrate PGCs and potential evolutionary changes in this unique population of cells.
Results
Discussion Defined culture conditions are instructive to delineate the minimal extrinsic signals needed for stem cell renewal (Ying et al., 2003). In serum-free, feeder cell-free, and physiochemically permissive medium conditions, we found that FGF2, insulin, and Activin ligands were sufficient for the derivation, expansion, and clonal growth of chicken PGCs. These ligands, signaling through their cognate receptors and downstream signaling pathways, define the signals needed for the self-renewal of a chicken PGC (Figure 6). TGF-β signaling through SMAD2/3 molecules, rather than SMAD1/5/8, appears to be more crucial to PGC self-renewal, as a constitutively active SMAD3 protein was able to rescue PGC growth in the absence of both BMP4 and Activin (Figure 5G). BMP4 can sustain self-renewal and expansion of PGC cultures, but not under clonal conditions, which suggests signaling through cell-cell interactions, reduced through lowering calcium levels, may be important for chicken PGC proliferation and survival. It is also possible that Activin A induces expression of a key downstream effector molecule that permits survival under clonal growth conditions. Nevertheless, a culture medium containing both Activin and BMP4 ligands may more generally reflect the in vivo environment, as both signaling pathways were active in migratory chicken PGCs in ovo, as evidenced by the phospho-SMAD1/5/8 and phospho-SMAD2 staining observed in the chicken embryo (Figures 1A and 1B). PGCs in birds are first found as a cluster of cells in the center of the blastoderm (Eyal-Giladi et al., 1981; Tsunekawa et al., 2000). From here, PGCs migrate to the germinal crescent anterior of the neural plate, enter the forming vascular system, are transported to the posterior lateral plate mesoderm, and finally migrate to the forming genital ridge (Nakamura et al., 2007; Nieuwkoop and Sutasurya, 1979). The complex migration path of avian PGCs may necessitate the ability to respond to multiple TGF-β-signaling ligands that vary spatially across the developing embryo.