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

Abstract

Background Parental imprinting is an epigenetic mechanism that leads to monoallelic expression of a subset of genes depending on their parental origin. Imprinting disorders (IDs), caused by disturbances of imprinted genes, are a set of rare congenital diseases that mainly affect growth, metabolism and development. To date, there is no accurate model to study the physiopathology of IDs or test therapeutic strategies. Human induced pluripotent stem cells (iPSCs) are a promising cellular approach to model human diseases and complex genetic disorders. However, aberrant hypermethylation of imprinting control regions (ICRs) may appear during the reprogramming process and subsequent culture of iPSCs. Therefore, we tested various conditions of reprogramming and culture of iPSCs and performed an extensive analysis of methylation marks at the ICRs to develop a cellular model that can be used to study IDs. Results We assessed the methylation levels at seven imprinted loci in iPSCs before differentiation, at various passages of cell culture, and during chondrogenic differentiation. Abnormal methylation levels were found, with hypermethylation at 11p15 H19/IGF2:IG-DMR and 14q32 MEG3/DLK1:IG-DMR, independently of the reprogramming method and cells of origin. Hypermethylation at these two loci led to the loss of parental imprinting (LOI), with biallelic expression of the imprinted genes IGF2 and DLK1, respectively. The epiPS™ culture medium combined with culturing of the cells under hypoxic conditions prevented hypermethylation at H19/IGF2:IG-DMR (ICR1) and MEG3/DLK1:IG-DMR, as well as at other imprinted loci, while preserving the proliferation and pluripotency qualities of these iPSCs. Conclusions An extensive and quantitative analysis of methylation levels of ICRs in iPSCs showed hypermethylation of certain ICRs in human iPSCs, especially paternally methylated ICRs, and subsequent LOI of certain imprinted genes. The epiPS™ culture medium and culturing of the cells under hypoxic conditions prevented hypermethylation of ICRs in iPSCs. We demonstrated that the reprogramming and culture in epiPS™ medium allow the generation of control iPSCs lines with a balanced methylation and ID patient iPSCs lines with unbalanced methylation. Human iPSCs are therefore a promising cellular model to study the physiopathology of IDs and test therapies in tissues of interest.

Epigenetic Reprogramming in Mice and Humans: From Fertilization to Primordial Germ Cell Development

“…An approach by Pham et al, enabled the generation of iPSC lines with balanced methylation for control purposes and iPSC lines from ID patients with unbalanced methylation. Human iPSCs thus represent a valuable cellular model for investigating the underlying mechanisms of IDs and assessing potential therapeutic strategies in relevant tissues [103].…”

Imprinted small nucleolar RNAs: Missing link in development and disease?

“…The effect may be due to the antioxidant property of vitamin C; however, a study by Stadtfeld et al confirmed that the addition of ascorbic acid (vitamin C) to the culture media inhibited the loss of imprinting at the DLK1‐DIO3 locus (Esteban et al, 2010; Habib et al, 2014; Stadtfeld et al, 2012). Moreover, adding vitamin C and culturing the iPSCs in hypoxic (5% oxygen) conditions prevented hypermethylation of paternally methylated ICRs such as IG‐DMR, thereby maintaining a normal expression of imprinted genes and pluripotency (Pham et al, 2022). A decade‐old report implicated the importance of the 14q32.2 cluster, specifically SNORD114‐3 and MEG3 , in embryonic stem cells' initial and early passage.…”

Stem cell models of Angelman syndrome

“…In the forthcoming years, the AS stem cell model portfolio will likely expand, hopefully encompassing the full spectrum of (epi)genetic variability observed in this condition. Current concerns in the epigenetic and genetic fidelity of human stem cells are being tackled ( Pham et al, 2022 ) and hopefully will be solved in the near future. Also, there are grand expectations for further development of stem cell-based disease modeling with the potential to revolutionize biomedical research, drug development, and, ultimately, patient care.…”