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  • The expression of germ line

    2018-11-12

    The expression of germ-line-specific genes was also detected in the putative EGC, showing a consistent increase compared with pFF. All of our analyzed PGC-derived cell lines expressed BLIMP1 during the entire period of culture (over 10 passages), even in the presence of bFGF at concentrations as high as 10ng/ml (data not shown). Interestingly, BLIMP1 did not downregulate the expression of c-myc and Klf4 in our porcine PGC-derived cell lines. As this downregulation is observed in mouse, it suggests a slightly different mode of action of BLIMP1 in the porcine species as compared with mouse. One possible explanation for this difference is the cytoplasmic localization of this gene in our putative EGC. It has been shown in mouse PGC that prior to their arrival in the genital ridges Blimp1 is localized in the nucleus but translocates to the cytoplasm afterward, thereby losing its function as a transcriptional repressor (Ancelin et al., 2006). It is therefore possible that the porcine PGC have continued their germ-line differentiation program in vitro, characterized by the loss of nuclear BLIMP1 localization and associated with it MYC and KLF4 repression. Elements of the LIF signaling pathway, namely LIFr, gp130, and STAT3, were detected in all of the tested cell lines. Similarly, we detected transcripts for FGF receptors 1 and 2. This suggests that these pathways may be active in porcine PGC; however, as the use of hrLIF and hrbFGF apparently did not facilitate the isolation of pluripotent cells, additional factors might be required to maintain pluripotency in porcine EGC. On the contrary, the use of hrSCF can be questioned since the expression of C-KIT was absent after P0 in all of the tested cell lines. Finally, TDH was shown to be expressed in our putative EGC lines at P0, but was not detected at P3 (data not shown) or P6–7. A recent publication (Wang et al., 2009) showed that mouse ESC and embryos are critically dependent on threonine and on the function of the Tdh gene for their fast proliferation. Therefore, the downregulation of TDH may explain the typical slow proliferation of porcine putative EGC. Porcine EGC have been reported by us and other groups to be capable of forming simple EB (Piedrahita et al., 1998; Shim et al., 1997; Tsung et al., 2003; Petkov and Anderson, 2008). In our hands, the putative EGC derived from younger embryos formed EB-like transcription factors that were indistinguishable from those pictured in these reports. Characteristically, these aggregates have a simple structure and differ from mouse and human EB, which are larger and contain multiple differentiated cell types. When allowed to attach on gelatin-treated plastic surfaces, they proliferated extensively and formed different cell types, such as epithelial-like, mesenchymal-like, and neuronal-like cells. It should be noted, however, that the differentiation was limited in scope, and cells in a more advanced stage of differentiation (such as beating cardiac muscle cells) were not observed, despite the adaptation of differentiation protocols applied in mouse and human ESC differentiation (results not shown). When injected in immunodeficient mice, ESC form teratomas that contain differentiated cells from the three germ layers. In contrast, injection of our putative porcine EGC into nude mice did not result in teratoma formation. Interestingly, human EGC usually fail to form teratomas when injected as undifferentiated cell population (Shamblott et al., 2000; Turnpenny et al., 2005); however, EB-derived cells resulting from in vitro differentiation of human EGC have been able to differentiate into neurons and glia (Teng et al., 2009) and liver cells (Chen et al., 2007) when injected into the brain or liver of immunodeficient mice. The reasons for this discrepancy are not currently known. As a result of our injections, the experimental mice developed tumors that clearly show the presence of STO feeders together with the host mouse cells. This is in agreement with a report where tumors of mouse origin have formed as a result of ovine EGC injection (Ledda et al., 2010). One possible explanation for our results would be that the feeder cells were not completely inactivated by the mitomycin C treatment. However, our control assessments of the mitotic inactivation of the mitomycin C-treated feeder cells before and after the injection experiments have indicated that they do not proliferate in culture. Moreover, injection of up to 2×106 STO cells alone did not result in tumor formation within 14weeks after injection. This may suggest that even though the injected porcine cells did not form teratomas, they might have influenced the proliferation of the neighboring STO and/or host cells. Taking into account that even a small fraction of mitomycin C-treated feeder cells may contribute to tumor formation, complete removal of the feeder cells may be necessary when injecting cells for teratoma analysis. In addition, our experience obviates the need for verification of the species origin of the teratomas, especially if the tested cells have been cultured on feeder layers.