title: Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells.
abstract: The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes.
authors.affiliation: Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., 675-1, Fujisaki, Okayama 702-8006, Japan. fcch@hayashibara.co.jp
authors.forename: Kazue
authors.initials: K
authors.lastname: Tsuji-Takayama
authors.forename: Toshiya
authors.initials: T
authors.lastname: Inoue
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ijiri
authors.forename: Takeshi
authors.initials: T
authors.lastname: Otani
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Motoda
authors.forename: Shuji
authors.initials: S
authors.lastname: Nakamura
authors.forename: Kunzo
authors.initials: K
authors.lastname: Orita
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000166:cytology* / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D008745:Methylation; D051379:Mice; D016133:Polymerase Chain Reaction; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D013447:Sulfites / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013447:Sulfites; D000469:Alkaline Phosphatase; D001374:Azacitidine