Concomitant with its suppressive effect on blast colony form

Concomitant with its suppressive effect on blast colony formation, the inclusion of WNT3A, or the WNT agonist BIO, in methylcellulose cultures promoted the emergence of colonies we termed “mesospheres” (Figure 7H). These colonies were capable of osteogenic, adipogenic, and smooth muscle differentiation and were enriched for mesoderm markers including APLNR, PDGFRA, PDGFRB, FOXF1, HAND1, SNAI2, and CDH11, reminiscent of the mesenchymal stromal SM-164 (MSCs) differentiated from hESCs reported by a number of laboratories (Karlsson et al., 2009). In contrast to mesospheres, that emerged in serum-free MC in response to WNT3A, most hESC-MSCs were derived in medium supplemented with serum or serum replacer plus FGF2. It is interesting to speculate on the relationship between mesosphere-forming cells and a mesoderm-derived precursor cell, the mesenchymoangioblast, that gives rise to endothelium and mesenchymal stem cells (Slukvin and Vodyanik, 2011; Vodyanik et al., 2010). Interestingly, formation of mesenchymal cells was inhibited by VEGF, which has been postulated to block the necessary endothelial-mesenchymal transition (EndMT) (Medici and Olsen, 2012; Slukvin and Vodyanik, 2011). In contrast, mesosphere development in methylcellulose cultures occurred in the presence of VEGF. A plausible hypothesis linking the WNT3A dependence of mesospheres with the prior findings of Slukvin and colleagues, is that WNT3A stimulated the endothelial-mesenchymal transition from a mesenchymoangioblast-derived VEGF-dependent angiogenic precursor, thus overcoming the inhibitory effect of VEGF on EndMT. There is evidence for involvement of WNT signaling in both epithelial-mesenchymal transitions (Wu et al., 2012) and EndMT (von Gise and Pu, 2012), with canonical WNT signaling required for the endocardial-mesenchymal transition during endocardial cushion formation in mice (Liebner et al., 2004). However, an alternative hypothesis would postulate that mesospheres represent the expanded progeny of a distinct mesenchymal progenitor cell that did not pass through an endothelial precursor stage.
Experimental Procedures
Acknowledgments
Introduction In sexually reproducing organisms germ cells provide the continuous link between the generations, delivering the genetic and epigenetic information required to construct a new organism (Surani, 2007). Primordial germ cells (PGCs) represent the founder cells of the germline lineage. In mice, they are induced from Oct4- (also known as Pou5f1) positive pluripotent epiblast cells at the onset of gastrulation. By E7.5, PGCs are said to be specified, coincident with the expression of Stella (also known as Dppa3, Pgc7) (McLaren and Lawson, 2005; Saitou et al., 2002). During normal development, PGCs behave as unipotent progenitors and produce only germ cells. Yet, they express pluripotency genes until after colonization of the genital ridges (Surani et al., 2007). Significantly, PGCs can give rise to pluripotent tumors in ectopic sites and they can serve as the cell of origin of testicular teratocarcinomas (Stevens, 1967). Ex vivo PGCs can directly give rise to pluripotent stem cell lines known as embryonic germ (EG) cells (Matsui et al., 1992; Resnick et al., 1992). Like embryonic stem (ES) cells, EG cells are genetically normal and are capable of contributing to chimeras (Labosky et al., 1994; Stewart et al., 1994). The process by which PGCs convert to pluripotency is erratic and poorly characterized. Three growth factors are reported to play key roles; stem cell factor (SCF), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Individually, each factor positively influences PGC proliferation and/or survival (Dolci et al., 1991; Godin et al., 1991; Matsui et al., 1991), but in combination they facilitate conversion to EG cells. Only LIF plays a role in subsequent self-renewal of EG cells. bFGF is important during the first day of culture but not thereafter, suggesting it may trigger the conversion process (Durcova-Hills et al., 2006). bFGF appears to act though the PI3K/AKT pathway because it is not required for EG cell formation from Pten deletion mutants (Kimura et al., 2003) or if AKT is hyperactivated (Kimura et al., 2008). Retinoic acid (RA) and forskolin (FK), two potent PGC mitogens, can substitute for bFGF in EG cell derivation (Koshimizu et al., 1996), as can the histone deacetylase inhibitor trichostatin A (Durcova-Hills et al., 2008). However, whether the activity of these factors is direct or mediated through induction of FGFs or other factors remains unclear due to the complex culture conditions, which include serum, feeders, and heterogeneous somatic cells. Previously, we showed that addition of two small molecule inhibitors of mitogen-activated protein kinase (MAPK) signaling and glycogen synthase kinase 3 (GSK3) (2i) (Ying et al., 2008) enables reliable generation of EG cells from mouse and rat PGCs (Leitch et al., 2010; Blair et al., 2012). However, undefined components should be eliminated to delineate the individual contributions of signaling molecules and pathways that mediate the derestriction of PGCs to pluripotency. Here, we develop a defined culture system and exploit this to clarify pathway requirements and in addition to track the PGC to EG cell conversion at the single cell level.