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    • In this study we investigated the

    2018-10-22

    In this study, we investigated the cellular and molecular features of p-NSCs, derived from mouse ESCs via “default mechanism”, a method that was reported to enrich p-NSC (Tropepe et al., 2001; Smukler et al., 2006; Rowland et al., 2011). We showed that p-NSCs have a decreased G2/M population compared to ESCs and similar to cells at early differentiation stages (Coronado et al., 2013); whereas d-NSCs highly resembled CNS-NSCs, showing typical d-biotin features of somatic cells (Stead et al., 2002). Furthermore, this is the first report to reveal the global expression profile of p-NSCs. Genes differentially expressed among ESC, p-NSC and d-NSC have been identified (Fig. 2A), providing new clue to unravel novel regulators in early neural induction. The global profiling analysis also provided an overview of gene expression changes during early neural induction, illustrating drastic changes occurred during p-NSC to d-NSC transition, rather than from ESCs to p-NSCs (Fig. 2F, G). Together with the cell cycle analysis, it indicated that p-NSC is an intermediate cell type that is more closely related to ESC, compared to d-NSC and CNS-NSC. Our data also supported that p-NSCs were indeed committed to differentiation (Fig. 3). 278 ESC-enriched genes were found to decrease significantly in p-NSC (Fig. 3A). These included Zscan4, Usp44, Dusp9, Foxp1 etc., which have been shown to be down-regulated upon ESC differentiation previously (Zalzman et al., 2010; Gabut et al., 2011; Fuchs et al., 2012; Li et al., 2012). On the other hand, 207 genes, including some neural-related genes Ccdc141, Hgma2, Satb2 etc. (Alcamo et al., 2008; Nishino et al., 2008; Fukuda et al., 2010), were found to be up-regulated (Fig. 3A–C). Importantly, our result showed that silencing of Ccdc141 and Hes1 severely impeded the p-NSC sphere formation (Fig. 3D, E), suggesting that their up-regulation is critical to p-NSC derivation. This data prompts to the conclusion that Ccdc141 is a potential novel regulator of early neurogenesis. Intriguingly, our study showed that p-NSCs could be converted back to ESC state after re-plating in ESC culture conditions (Fig. 4), which was supported by the decreased expression of Sox1 and Hes1, increased SSEA-1 as well as the reversible cell cycle profile. This suggested that p-NSCs represent a reversible stage of neural commitment. Our results are consistent with recent reports on mouse ESC-derived p-NSCs. Tropepe et al. and Rowland et al. have confirmed the broad developmental potential in p-NSCs through in vivo microinjection into mouse embryos and in vitro differentiation into broad range of cell types (Tropepe et al., 2001; Rowland et al., 2011). This suggested that p-NSCs have not fully committed to neural differentiation and retained some features of pluripotent cells. In addition, although some early publications reported the expression of Nestin in p-NSCs (Tropepe et al., 2001; Hitoshi et al., 2004), recent studies revealed that CNS-NSC markers Nestin and Pax6 were more likely maintained at a low level in p-NSCs (Rowland et al., 2011; Salewski et al., 2013). In line with these works, our study confirmed the expression of Oct4 and Nanog and suppression of Nestin and Pax6 in p-NSCs (Fig. 1E, G). Other than derivation of p-NSC from mouse ESCs, the induction of LIF-dependent primitive neural precursors from human ESCs has also been investigated on monolayer culture (Li et al., 2011). However, the cells obtained showed rapid down-regulation of Oct4 and Nanog with abundant expression of definitive NSC markers Nestin and Pax6 (Li et al., 2011), which were inconsistent with the features exhibited by the p-NSCs derived from mouse ESCs (Rowland et al., 2011). It is not clear whether the discrepancy was caused by profound difference between mouse and human ESCs, or due to the difference between culturing methods, i.e. attachment vs. suspension. On the other hand, another distinct early stage human NSCs were reported recently (Noisa et al., 2012). Different from SSEA4(+)/Tra-1–81(+) human ESCs and SSEA4(−)/Tra-1–81(−) mature human NSCs, the early staged human NSCs derived by Noisa et al. still express SSEA4 like human ESCs, while their partial commitment was shown by the silencing of Tra-1–81 gene (Noisa et al., 2012). Contrary to our mouse ESC-derived primitive NSCs, switching back to human ESC medium cannot reverse the partial commitment of these early human NSCs, instead, it led to the loss of both ESC and NSC identity after re-plating (Noisa et al., 2012). It is possible that these early stage human NSCs were committed to a later neural cell fate compared with our primitive NSCs, as they showed partial down-regulation of pluripotency genes, significant up-regulation of mature neural genes and increased methylation of Oct4 promoter (Noisa et al., 2012).