Systematic analysis of imprinting defects in female and male mouse iPSCs

Reprogramming of somatic cells into Induced Pluripotent Stem Cells (iPSCs) provided a major leap towards more personalized cellular models for disease modelling and offers great prospects for regenerative medicine. Despite their considerable success, we still lack the ability to control the state of iPSCs fully, especially at the epigenetic level. This can be appreciated at chromosomal regions regulated by genomic imprinting which provides a good read-out for epigenetic fidelity in iPSCs. Indeed, imprinting defects have already been reported in both mouse and human iPSCs. However, the extent, nature, causes and consequences of these defects still remain to be analysed in a systematic manner. To fill this gap in knowledge, we used a controlled secondary reprogramming system whereby the donor mouse embryonic fibroblasts (MEFs) contain a doxycycline (DOX)-inducible Yamanaka cassette and also Single Nucleotide Polymorphisms (SNPs) to distinguish between the two parental alleles. Several female and male isogenic mouse iPSCs (miPSCs) were obtained using independent culture conditions (defined versus serum) and screened by allelic-specific IMPLICON, a targeted next-generation method to measure DNA methylation at imprinted regions with unprecedented genomic coverage of 1000-fold. Our results showed that imprinting defects are remarkably common in miPSCs. Interestingly, we found important differences in gender responses to culture conditions. In particular, female iPSCs exhibit widespread hypomethylation defects regardless of culture conditions, while male miPSCs show tendency for hypomethylation defects under defined conditions, and hypermethylation defects under serum conditions in particular loci. As expected, methylation defects at imprinted regions resulted in dysregulation of monoallelic expression of imprinted genes. Our results are of utmost importance to devise future reprogramming strategies to generate epigenetic error-free iPSCs Overall design: RNA profiles of parental MEFs, female and male miPSCs, and mESCs were generated by deep sequencing, in triplicates, using Illumina HiSeq platform. Methylation profile of specific genomic regions in female and male miPSCs generated in two different culture conditions, parental MEFs and mESCs, using Illumina MiSeq platform.