Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Abstract

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.

Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation

“…4C). Likewise, the presence or absence of XIST, as well as the degree of erosion, does not affect methylation patterns at imprinted regions as depicted by IMPLICON, a targeted amplicon-seq-based method to measure DNA methylation at imprinted regions (Klobučar et al, 2020; Arez et al, 2022). IMPLICON was employed across two sets of isogenic XIST+ and XIST-hiPSCs, along the isogenic pair, CE and CD, exhibiting varying degrees of erosion.…”

Harshening stem cell research and precision medicine: The states of human pluripotent cells stem cell repository diversity, and racial and sex differences in transcriptomes

“…Nonetheless, male and female pluripotent stem cells differ in autosomal gene expression as evident by transcriptomics analysis, albeit their comparable ground state of stemness ( Ronen and Benvenisty, 2014 ). Cellular sex difference is a key factor in dimorphic pathophysiology, which is caused by both sex hormones and chromosomal genotype ( Walker et al, 2021 ), as well as sex-dependent different regulations in genomic imprinting ( Arez et al, 2022 ). Some genes that were differentially expressed were sex-specific; for example, Zinc Finger Y-linked ( ZFY ) and Taxilin Gamma ( TXLNGY ) ( Figure 2 ).…”

Maintenance of methylation profile in imprinting control regions in human induced pluripotent stem cells

“…However, they did not examine the methylation levels at other ICRs. More recently, Arez et al have shown that the addition of ascorbic acid during the reprogramming of murine cells stabilizes the methylation of H19/Igf2 and Dlk1 - Dio3 regions which were hypermethylated without this addition [ 37 ]. Moreover, although it has long been accepted that hypoxia enhances the generation of iPSCs, its combination with ascorbic acid has never been reported [ 38 ].…”