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- Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulationPublication . Arez, Maria; Eckersley-Maslin, Melanie; Klobuar, Tajda; von Gilsa Lopes, João; Krueger, Felix; Mupo, Annalisa; Raposo, Ana Cláudia; Oxley, David; Mancino, Samantha; Gendrel, Anne-Valerie; Jesus, Bruno Bernardes De; da Rocha, Simão T.Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.
- IMPLICON : an ultra-deep sequencing method to uncover DNA methylation at imprinted regionsPublication . Klobučar, Tajda; Kreibich, Elisa; Krueger, Felix; Arez, Maria; Pólvora-Brandão, Duarte; von Meyenn, Ferdinand; da Rocha, Simão T.; Eckersley-Maslin, MelanieGenomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
- Gene reactivation upon erosion of X chromosome inactivation in female hiPSCs is predictable yet variable and persists through differentiationPublication . Raposo, Ana Cláudia; Caldas, Paulo; Jeremias, Joana; Arez, Maria; Cazaux Mateus, Francisca; Barbosa, Pedro; Sousa-Luis, Rui; Água, Frederico; Oxley, David; Mupo, Annalisa; Eckersley-Maslin, Melanie; Casanova, Miguel; Grosso, Ana Rita; da Rocha, Simão T.Female human induced pluripotent stem cells frequently undergo X-chromosome inactivation (XCI) erosion, marked by X-inactive specific transcript (XIST) RNA loss and partial reactivation of the inactive X (Xi). This overlooked phenomenon limits our understanding of its impact on stem cell applications. Here, we show that XCI erosion is frequent and heterogeneous, leading to the reactivation of several X-linked genes. These are primarily located on the short arm of the X chromosome, particularly near escape genes and within H3K27me3-enriched domains, with reactivation linked to reduced promoter DNA methylation. Interestingly, escape genes further increase their expression from Xi upon XCI erosion, highlighting the critical role of XIST in their dosage regulation. Importantly, global (hydroxy)methylation levels and imprinted regions remain unaffected, and analysis of trilineage commitment and cardiomyocyte formation reveals that XCI erosion persists across differentiation. These findings underscore the need for greater awareness of the implications of XCI erosion for stem cell research and clinical applications.