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    • The functional roles of SOX in human pluripotency maintenanc

    2018-11-08

    The functional roles of SOX2 in human pluripotency maintenance and lineage differentiation were recently reported (Wang et al., 2012). SOX2 is not essential for hESC self-renewal. However, overexpression of SOX2 results in the promotion of neural ectodermal differentiation and the suppression of definitive endodermal differentiation in hESCs under in vitro differentiation (Wang et al., 2012). In agreement with these findings, we found that the neural program, including the expression of SOX1, LHX2, and NESTIN, was upregulated in hESCs overexpressing SOX2 (Figure S5E). Furthermore, our results demonstrated that the germline program was suppressed under the same condition, suggesting an opposing role of SOX2 in directing human neural and germ cell fate. Thus, it is tempting to speculate that PRDM1 may serve as a molecular switch to modulate the induction of neural or germline fates by turning off SOX2 during early human development (Figure 7E). The BMP4/PRDM1/SOX2 regulatory axis identified in this study may also provide a mechanistic clue to elucidate the molecular mechanisms underlying the suppression of neural development by BMP4 signaling (Hemmati-Brivanlou and Melton, 1997). The study of germline development from hESCs has been hampered by the lack of proper protocols for efficient germline induction and/or selection from differentiating hESCs. Previous approaches, including combined growth factor/cytokine stimulation (Chuang et al., 2012) and human fetal gonadal cell coculture (Park et al., 2009), have shown promising effects on germline induction of hESCs and/or hiPSCs. Furthermore, advances in generating transgenic hESCs/hiPSCs harboring germline reporters, such as VASA (Kee et al., 2006) and OCT4 (Chuang et al., 2012), have also enabled us to enrich the potential germ cell populations from differentiating PSCs. Nevertheless, the generation of germ Merimepodib from hESCs/hiPSCs by the aforementioned protocols is still not satisfactory. In this study, we provide multiple lines of evidence demonstrating that ectopic expression of PRDM1 in hESCs is sufficient to promote the germline program, resulting in the generation of increased numbers of VASA-expressing PGCs during in vitro differentiation. In addition to promoting germ cell marker expression, we detected a small number of cells capable of expressing the meiotic marker SCP3 after ectopic expression of PRDM1. Furthermore, the decreased expression of DNMT3b in the differentiated cells overexpressing PRDM1 suggests that PRDM1 may contribute to somatic-to-germline epigenomic reprogramming by promoting the erasure of DNA methylation. However, we did not observe a significantly increased number of cells exhibiting the ability to complete meiosis as judged by haploidy of two chromosome FISH probes. This suggests that, in contrast to the function of previously reported factors, such as DAZ and BOULE (Kee et al., 2006), PRDM1 is not fully competent to promote the later stages of meiosis and development of haploid gametes. These results suggest that PRDM1 acts as an early rather than late germline effector. Our studies thus provide an induction system that will be beneficial for initiating early gem cell specification and subsequent germ cell generation by hESCs/hiPSCs.
    Experimental Procedures
    Acknowledgments
    Introduction Age-related macular degeneration (AMD) (Bressler et al., 1988) is a leading cause of severe visual impairment in elderly persons in developed countries and is characterized by progressive degeneration of the retinal pigment epithelium (RPE). RPE consists of a monolayer of cells situated between the photoreceptors and the choroid, and plays essential roles in photoreceptor maintenance, such as supplying nutrition, formation of the blood-retinal barrier, and phagocytosis of mature outer rod segments shed by photoreceptors (Young and Bok, 1969), as well as in the maintenance of choroidal circulation by the secretion vascular endothelial growth factor (VEGF) from its basal aspect. Current therapies for AMD show only limited efficacy, and in recent years increasing attention has been given to the replacement of pathological RPE with healthy tissue. To date, transplantation of allogeneic fetal RPE (Algvere et al., 1994) or autologous peripheral RPE (Binder et al., 2002; van Meurs and Van Den Biesen, 2003) has been reported in AMD patients, although neither represents an ideal tissue source; fetal tissue is immunogenic, while the latter requires RPE to be harvested by an invasive procedure. Human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007) exhibit a number of clinically attractive features, including the potential to serve as an autologous cell source through reprogramming of a patient’s own somatic cells, and the ability to self-renew and expand in culture, and to differentiate into RPE.