Human Molecular Genetics Advance Access published online on April 4, 2007
Human Molecular Genetics, doi:10.1093/hmg/ddm074
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Restriction Landmark Genome Scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome
1 Division of Obstetrics and Gynaecology and Wolfson Centre for Stem Cells, Tissue Engineering and Modelling (STEM), University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK 2 Division of Human Cancer Genetics, Department of Molecular Virology, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, USA 42310 3 Hubrecht Laboratory, Netherlands Institute for Developmental Biology and the Interuniversity Cardiology Institute of the Netherlands, Uppsalalaan 8, 3584 CT Utrecht, he Netherlands 4 Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK 5 Centre for Stem Cell Biology and Developmental Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK 6 Department of Cytogenetics, Centre for Medical Genetics, Nottingham City Hospitals NHS Trust, Nottingham, NG5 1PB, UK 7 Academic & Research Applications, IT Systems, Information Services, University of Nottingham, King's Meadow Campus, Lenton Lane, Nottingham, NG7 2NA, UK
* To whom correspondence should be addressed. Tel: +44 1158230694; Fax: +44 1158230704; Email: lorraine.young{at}nottingham.ac.uk or Tel +1 6142926505; Fax: +1 6142923555; Email: plass-1{at}medctr.osu.edu
Received December 21, 2006; Revised March 10, 2007; Accepted March 20, 2007
Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modeling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of >2000 genomic loci by Restriction Landmark Genome Scanning we identified substantial inter-line epigenetic distance between four independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for 1) novel screening strategies to determine the experimental utility and biosafety of hESCs and 2) optimisation and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.
Present address: Cellular Reprogramming Laboratory, Principe Felipe Centro de Investigacion, Valencia, Spain.
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