At a global level the analysis of DNA

At a global level, the analysis of DNA-methylation profiles provides important insights into molecular and cell growth characteristics of PSC that would otherwise be difficult to identify. Reprogramming-associated variations in X-linked CpG methylation is of particular interest because of the complex variations in degrees of XCI and X-chromosome reactivation between various hierarchical states of pluripotency. Our analysis highlighted two distinct populations of XCI female cells, with the most hypermethylated X-chromosomal PSC split between very high and absent XIST expression. Interestingly, none of the female hESC lines in this study expressed XIST, whereas many of the iPSC do. Our analyses of differentiated female PSC identified correlates between XCI and pluripotentiality that substantiate prior proposed models and provide additional candidate molecular regulators for investigation (Mekhoubad et al., 2012; Silva et al., 2008). Genes anti-correlated with XIST, principally RBBP7, share protein interactions with core pluripotency regulators, and these differences persist in the EB. This result is particularly intriguing given that RBBP7, a partner of PRC2 implicated in nucleosome binding, and SUZ12, a component of PRC2 required for Ion Channel Compound Library of the complex and EZH1/2 mediated catalytic activity, were highly enriched factors in our analysis of differentially methylated sex-associated autosomal genes. In addition to RBBP7, predicted regulation by PRC2 was recurrent in a number of our covariate analyses, including iPSC versus hESC differentially methylated probes. A growing body of literature now supports an important role for PRC2 in pluripotency, XCI, and differentiation as a recruitment tool of PRC1 (Cheng et al., 2014). Although likely not relevant in vivo, such physical protein and epigenetic interactions could be undesirable in iPSC for programmed lineage differentiation.
Experimental Procedures
Author Contributions
Acknowledgments The authors thank the PCBC line contributors in Table S1; Dr. Michael Terrin, Ling Tang, Andrea Lefever, and Liz Casher from the Administrative Coordinating Center; and Dr. Elke Grassman and Diana Nordling from the CCHMC Translational Core Laboratories for support. This work was supported by the NHLBI Progenitor Cell Biology Consortium, Administrative Coordinating Center (U01HL099997), Cell Characterization Core, Bioinformatics Core, and PCBC2012Pilot_01. Other support was provided by the National Heart, Lung, and Blood Institute (NHLBI) (U01HL099775), the National Institute for Child Health and Human Development (NICHD) (R01HD082098), the National Institute General Medical Sciences (NIGMS) (R01GM110628), and the National Eye Institute (NEI) (R01EY023962). Under a licensing agreement between Life Technologies Corporation and the Johns Hopkins University, E.Z. is entitled to a share of royalty received by the University on sales of human induced pluripotent stem cell lines. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its Conflict of Interest policies.
Introduction The WNT signaling pathway has been implicated in cell proliferation, differentiation, migration, morphological changes, and apoptosis. The canonical pathway initiates a signaling cascade through the Frizzled (Fz) transmembrane receptor and cytoplasmic Disheveled (Dsh) protein, which results in the stabilization and subsequent translocation of β-catenin to the nucleus where it associates with the DNA binding protein TCF to regulate transcription of target genes (Stamos and Weis, 2013). Notably, some of the WNT proteins have also been shown to be involved in β-catenin-independent responses (van Amerongen, 2012). Aberrant activation of the WNT pathway is one of the most frequent signaling abnormalities known in human cancers and is therefore an area of intense research (Clevers and Nusse, 2012). WNT signaling acts in the stem cell niche by maintaining self-renewal ability; however, in specific cell types, it is also involved in lineage commitment. Therefore, these signals have profound use in regenerative medicine and regulating stem cell fate in vitro. WNT molecules are lipid modified (Takada et al., 2006; Willert et al., 2003), making them highly insoluble, and in vivo they likely signal to target cells in a localized manner (Alexandre et al., 2014; Clevers et al., 2014; Farin et al., 2016; Goldstein et al., 2006; van den Heuvel et al., 1989). Currently, researchers use purified soluble WNTs, which are stored in the presence of detergents to maintain activity (Willert et al., 2003). Soluble WNT proteins are added globally to cells, and at high concentrations the detergent becomes cytotoxic. In addition, in serum-free media, the protein shows compromised stability and activity (Fuerer et al., 2010). Therefore, using a soluble source does not allow control over the spatial presentation of the protein for tissue engineering. Unlike soluble WNT, immobilization of the protein onto beads has recently been shown to promote asymmetric division of embryonic stem cells, suggesting a localized source provides a distinct signal to target cells (Habib et al., 2013). This may be a critical difference for short-range signaling activity and cell polarization within a niche. Here we report on a platform that provides a highly stable source of detergent-free active WNT molecules that can act as basal niche cues for adult and embryonic stem cells in monolayer and can contribute to the directional cues for engineering 3D tissues.