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    • Unfortunately exploration of the biochemical

    2018-11-08

    Unfortunately, exploration of the biochemical mechanisms that underlie germ cell specification and early PGC formation in the mammalian embryo is hampered by the scarcity of neutrophil elastase inhibitor at these early embryonic time points. In vitro derivation of PGCs from embryonic stem cells (ESCs) allows the generation of sufficient cell numbers to perform robust biochemical analysis of protein-protein and protein-mRNA interactions (Hübner et al., 2003; Toyooka et al., 2003; Geijsen et al., 2004; Hayashi et al., 2011). To explore the role of DAZL during PGC development, we have generated a Dazl-GFP reporter ESC line that allows the prospective isolation and biochemical analysis of in vitro developing PGCs. Using this in vitro assay, as well as conformational analysis in vivo, we demonstrate that DAZL acts as a translational repressor to simultaneously suppress pluripotency, somatic differentiation, and apoptosis in nascent PGCs.
    Results
    Discussion In this study, we utilized in vitro generation of PGC-like cells from ESCs and identified a network of DAZL-interacting proteins and mRNAs that plays a central role in controlling pluripotency, differentiation, and apoptosis. We demonstrate that the in vitro PGC-like cells recapitulate many aspects of in vivo PGC development, including the developmental timing of germ cell marker expression (Stella versus GFP). In vitro PGC-like cells are clearly distinct from the ESCs in terms of their gene expression profile as well as the subcellular localization of DAZL, which is homogeneously distributed in the cytoplasm in ESCs but localized to P-bodies in nascent PGCs, both in vitro and in vivo. Nonetheless, the in vitro-generated PGCs exist outside the context of the embryonic gonad and as such lack control by the stromal microenvironment, which may limit their in vitro development to stages beyond the PGC state. The lack of stromal control may also account for the fact that the in vitro PGC-like cells are developmentally more heterogeneous than their in vivo counterparts. We therefore call these cells PGC-like, to reflect this important difference with in vivo PGCs. Nonetheless, the in vitro generation of large numbers of PGC-like cells from ESCs provides an important tool to perform robust biochemical analysis on this otherwise inaccessible cell population.
    Experimental Procedures
    Author Contributions H-H.C., M.W., and N.G. conceived work and wrote the manuscript. H.-H.C., M.W., and N.G. designed the experiments and analyzed the data. H.-H.C. and M.W. performed the majority of the experiments. D.B.B. helped with the analysis of the P-body complexes. J.M. and A.J.H. analyzed the protein interaction data. E.M and R.W. helped confirm the translational-suppressive effect of the Dazl complex. X.C. and C.T. assisted with the in vitro differentiation experiments. J.W. performed in silico analysis of DAZL binding sites. A.Y., Y.-F.C., and C.B. assisted with the generation of Dazl-GFP mouse. A.K. supervised the in silico analysis. N.G. supervised all aspects of the project.
    Acknowledgments
    Introduction The potential of stem cell-derived cardiomyocytes for disease modeling has been enhanced by the realization that cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) can be obtained also with disease-specific genotypes and phenotypes (Park et al., 2008). These cells are suggested to have many of the properties of authentic cardiomyocytes, and their phenotypes provide validation that characteristics of the disease can be reproduced in vitro (Park et al., 2008). The initial focus for using hESC-CMs or hiPSC-CMs was modeling acute cardiac responses, with the aim of producing models of contractile impairment, contractile frequency, or arrhythmias or for using cells as a screen to identify cardiotoxicity of experimental or clinical compounds. An important goal is now to extend this to modeling of longer-term disease processes. Hypertrophy is an obvious target for investigation, given its central role in the transition to heart failure. Intense studies in animal models and human myocardium have revealed hypertrophic networks with complex interdependence and redundancies (Ryall et al., 2012), which makes the design of therapies challenging. The high-throughput capabilities of the hESC-CM/hiPSC-CM system are ideally placed to dissect these pathway interactions by systems approaches and then to translate into a drug discovery platform. Our earlier data have revealed the ability of hESC-CMs to respond to canonical pathological and physiological hypertrophic stimuli (Földes et al., 2011). In the present study, we extend these observations using newly designed assays on a number of automated platforms and show how these approaches can identify new targets. Although the field of modeling of genetic diseases has advanced rapidly, researchers have started to evaluate more critically hiPSCs relative to hESCs (Ma et al., 2014) and have made an effort to better understand how these cell populations differ from one another. We present here data showing that hiPSC-CMs diverge systematically from hESC-CMs and investigate the reason for the difference at multiple levels from receptor expression to kinase effector pathways.