Identification of DNA methylation markers for lineage commitment of in vitro hepatogenesis.

Hepatocytes that have differentiated from human embryonic stem cells (hESCs) have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis-hESC, definitive endoderm and hepatocyte-using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by the MBD2 protein. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development.