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  • All PGCs differentiated from ESCs

    2018-10-20

    All PGCs differentiated from ESCs in our study, regardless of differentiation approach or sorting strategy, were capable of implementing ICC erasure at the Snprn ICC. However, unlike E9.5 PGCs from the embryo, the hypomethylated state at this ICC was unstable, and in every case re-methylation was initiated within 24 hr. This cannot be explained by the natural course of germline development, as wild-type XY germline hippo pathway in vivo never re-methylate the Snprn ICC following erasure, and all ESC lines used in this study were XY. Our hypothesis is that germline cells differentiated from mouse ESCs in vitro exhibit a lower barrier to EGC reversion and this is accompanied by abnormal expression of genes encoding proteins required for DNA methylation. The RNA-seq analysis supports this hypothesis given that both iPGCs and PGCLCs enter the 7F medium experiment with a transcriptional program that is more closely related to EGCs. However, we cannot rule out the possibility of heterogeneity in the EGC derivation system, leading to selective growth of cells with particular methylated signatures. One way to address this in the future would be to perform single-cell methylation sequencing. Correct erasure and removal of methylation from ICCs in the germline needs to be seriously considered if the entire process of PGC differentiation into gametes is to be successfully attempted in vitro. Male and female PGCLCs have been differentiated successfully into terminally differentiated germ cells capable of fertilization, but only after transplantation of the PGCLCs into a gonadal niche (Hayashi et al., 2011, 2012). The differentiation of functional germline cells using this approach is a remarkable achievement and one of the few in-vitro-differentiated cell types proven successful in functional assays. However, not all PGCLCs are capable of differentiation within the niche after transplantation, with many germ cells dying for unknown reasons (Hayashi et al., 2011, 2012). Furthermore, offspring born after fertilizing PGCLC-derived male germ cells are not always healthy, with some mice dying prematurely with head and neck tumors (Hayashi et al., 2011). In the case of PGCLC-derived oocytes, a high proportion do not extrude the second polar body upon fertilization, leading to abnormal 3-pronuclear embryos (Hayashi et al., 2012). Therefore, there is still much to be understood about the process of differentiating high-quality gametes in vitro and in vivo, and our study would suggest that methylation erasure at ICCs is one of the vulnerable developmental transitions prone to error. In future studies it will be important to determine whether gonadal niche cells protect in-vitro-differentiated germline cells from aberrant acquisition of DNA methylation and to suppress the potential for EGC reversion, which could lead to germ cell tumors following transplantation. Finally, our data highlight the importance of initiating PGC differentiation from ESCs with a normal epigenome. Abnormal hypermethylation of the H19/Igf2 ICC has been reported in Rhesus macaque ESC lines as well as human ESCs (Mitalipov et al., 2007; Park et al., 2009). Our work demonstrates that abnormal hypermethylation of the H19/Igf2 ICC in undifferentiated ESCs is not erased in the ESC-to-PGC differentiation model, and failure to correctly erase methylation in the germline will pose a significant problem to the generation of high-quality gametes in the future.
    Experimental Procedures
    Author Contributions
    Acknowledgments
    Introduction Ever since the first generation of induced pluripotent stem cells (iPSCs) using retroviral vectors for Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M, respectively) (Takahashi and Yamanaka, 2006), many researchers have focused on understanding the reprogramming mechanism for a more efficient and rapid generation of iPSCs. To this end, it is essential to understand the molecular roadmaps toward successful reprogramming, to identify bottlenecks, and to develop strategies to overcome these obstacles. Recently, a few detailed reprogramming roadmaps have been described from time course gene expression analyses as well as cell-surface-marker-based reprogramming intermediate subpopulation analyses (Hussein et al., 2014; O’Malley et al., 2013; Polo et al., 2012). In this context, we have reported that monitoring expression changes of CD44, ICAM1, and a Nanog-GFP reporter enables the tracking of the successful progression of reprogramming using a 2A-peptide-linked MKOS polycistronic reprogramming cassette (M-K-O-S in this order) (O’Malley et al., 2013). Reprogramming intermediates isolated based on their CD44/ICAM1/Nanog-GFP profile demonstrated an increasing probability to reach a pluripotent state concordant with the stage of progression defined by the markers. However, whether the roadmaps change when different reprogramming systems that have distinct reprogramming efficiencies are used has not been addressed yet.