S TEM C ELL T ECHNOLOGY :E PIGENETICS ,G ENOMICS ,P ROTEOMICS , AND M ETABONOMICS Vitamin C Promotes Widespread Yet Specific DNA Demethylation of the Epigenome in Human Embryonic Stem Cells TUNG-LIANG CHUNG, a,b,c ROMULO M. BRENA, d GABRIEL KOLLE, e SEAN M. GRIMMOND, e BENJAMIN P. BERMAN, d PETER W. LAIRD, d MARTIN F. PERA, f ERNST JURGEN WOLVETANG a a Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia; b Australian Stem Cell Centre, Melbourne, Victoria, Australia; c Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia; d USC Epigenome Center, Keck School of Medicine, University of Southern California, Los Angeles, California, USA; e Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia; f Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, USA Key Words. Human embryonic stem cells • Epigenetic changes • DNA hypomethylation • CpG shores ABSTRACT Vitamin C (ascorbate) is a widely used medium supple- ment in embryonic stem cell culture. Here, we show that ascorbate causes widespread, consistent, and remarkably specific DNA demethylation of 1,847 genes in human em- bryonic stem cells (hESCs), including important stem cell genes, with a clear bias toward demethylation at CpG island boundaries. We show that a subset of these DNA demethylated genes displays concomitant gene expression changes and that the position of the demethylated CpGs relative to the transcription start site is correlated to such changes. We further show that the ascorbate-deme- thylated gene set not only overlaps with gene sets that have bivalent marks, but also with the gene sets that are demethylated during differentiation of hESCs and during reprogramming of fibroblasts to induced pluritotent stem cells (iPSCs). Our data thus identify a novel link between ascorbate-mediated signaling and specific epige- netic changes in hESCs that might impact on pluripo- tency and reprogramming pathways. STEM CELLS 2010;28:1848–1855 Disclosure of potential conflicts of interest is found at the end of this article. INTRODUCTION Human embryonic stem cells (hESCs) and human induced plu- ritotent stem cell (iPSCs) display both indefinite self-renewal as well as the ability to differentiate into most, if not all, cell types of the human body. Because of these remarkable properties these cells have attracted great interest as they can serve both as useful model systems for human embryogenesis and disease and as the cell of choice in regenerative medicine approaches. Both the regulation of lineage-specific differentiation as well as the reprogramming of somatic cells into iPSCs is controlled through the concerted temporally controlled action of specific sets of transcription factors in conjunction with epigenetic remodeling of the stem cell genome. Indeed, comparison of the methylome of fibroblasts, iPSCs, and hESCs has revealed that DNA methylation and demethylation of CpG and non-CpG nu- cleotides outside of classical CpG islands (CGIs), in so-called CpG shores, are important determinants for tissue-specific gene expression, cancer, and reprogramming [1]. Here, we investi- gate the role of the antioxidant vitamin C, a commonly used medium supplement for the culture of hESCs under serum-free conditions (knock-out serum replacement [KOSR] medium, mTeSR1 medium, StemPro medium) on global DNA methyla- tion using the Infinium DNA methylation array platform. We show that vitamin C, at the concentration used in commercial media formulations, promotes widespread yet remarkably spe- cific DNA demethylation of the hESC epigenome that are bi- ased toward demethylation of shore CGIs and that significantly overlaps with bivalent domains and gene sets that are demethy- lated during reprogramming. Our data identify a novel link between vitamin C and specific epigenetic changes in hESCs that have the potential to directly impact on the differentiation of hESCs and reprogramming of somatic cells. Author contributions: T.-L.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; R.M.B.: conception and design, collection and/or assembly of data, and data analysis and interpretation; G.K.: conception and design, collection and/or assembly of data, microarray data analysis and interpretation; S.M.G.: conception and design, and financial support; B.P.B.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; P.W.L.: conception and design, and financial support; M.F.P.: conception and design, data analysis and interpretation, and manuscript writing; E.W.: overall planning and design, financial support, and provision of study material or patients, data analysis and interpretation, manuscript writing, and final approval of manuscript. Correspondence: Ernst Jurgen Wolvetang, PhD, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Rds (Bldg 75), The University of Queensland, Brisbane Qld 4,072 Australia. Telephone: 61-7-33463894; Fax: 61-7-33463973; e-mail: e.wolvetang@uq.edu.au Received March 11, 2010; accepted for publication July 22, 2010; first published online in STEM CELLS EXPRESS August 4, 2010. V C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.493 STEM CELLS 2010;28:1848–1855 www.StemCells.com