While the vast majority of mammalian autosomal genes are expressed from both parental alleles, a number express in a parent-of-origin-specific manner due to genomic imprinting. This has been shown to be regulated by gender-specific acquisition of DNA methylation in the germline at imprinting control regions (ICRs) that is maintained after fertilization, determining allele-specific expression of nearby genes. Dysregulation of ICR methylation, known as loss of imprinting (LOI), disrupts this parent-of-origin expression and has been shown to lead to developmental abnormalities. In keeping with the important developmental role of imprinted genes, ICR methylation has been thought to be very stable in normal physiological contexts, however the commonly used tools to assess genomic imprinting have been based on bulk and/or retrospective analysis, limiting resolution of rare populations of cells. In contrast to imprint stability in vivo, we and others have observed a susceptibility to LOI in cultured pluripotent stem cells, highlighting the need for further investigation.
To explore imprint stability in different contexts, we used the Dlk1-Dio3 imprinted locus as a paradigm and developed a dual-reporter system to enable visualization of cells that have LOI and those that retain their parent-of-origin ICR methylation, known as maintenance of imprinting (MOI). We found that while Dlk1-Dio3 imprinting is maintained during in vivo mouse development, LOI was readily observed in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We linked this susceptibility to mouse genetic background and found that C57BL/6J pluripotent cells develop LOI in culture and during iPSC reprogramming, while 129S2 and C57BL/6N have MOI. With Quantitative Trait Locus (QTL) analysis we identified a region on chromosome 13 that is responsible for regulating this susceptibility. Furthermore, we found that addition of ascorbic acid (AA) to the media during culture and/or reprogramming of these cells attenuates Dlk1-Dio3 imprint instability and preserves developmental potential of unstable lines. Our observations identify a strong genetic determinant of locus-specific epigenetic abnormalities in pluripotent cells and provide a non-invasive way to suppress them. This highlights the importance of considering genetics in conjunction with culture conditions for assuring the quality of pluripotent cells for biomedical applications.
|Commitee:||Nance, Jeremy, Sfeir, Agnel, Small, Stephen|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 82/1(E), Dissertation Abstracts International|
|Subjects:||Developmental biology, Genetics, Cellular biology|
|Keywords:||DNA methylation, Embryonic stem cells, Epigenetics, Genomic imprinting, Induced pluripotent stem cells, Pluripotency|
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