Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells.
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.
Tsuji-Takayama
Kazue
K
Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., 675-1, Fujisaki, Okayama 702-8006, Japan. fcch@hayashibara.co.jp
Inoue
Toshiya
T
Ijiri
Yoshihiro
Y
Otani
Takeshi
T
Motoda
Ryuichi
R
Nakamura
Shuji
S
Orita
Kunzo
K
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
D004791:Enzyme Inhibitors; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013447:Sulfites; D000469:Alkaline Phosphatase; D001374:Azacitidine