5-azacytidine induces cardiac differentiation of P19 embryonic stem cells.
The P19 embryonal carcinoma cell line is a useful model cells for studies on cardiac differentiation. However, its low efficacy of differentiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cells to differentiate into a high-efficacy cardiomyocytes. Embryoid-body-like structures were formed after 6 days with 1 mM of 5-aza in a P19 cell monolayer culture, beating cell clusters first observed on day 12, and, the production of beating cell clusters increased by 80.1% (29 of 36-wells) after 18 days. In comparison, the spontaneous beating cells was 33.3% (12 of 36-wells) for the untreated control cells. In response to 1 mM of 5-aza, P19 cells expressed bone morphogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac troponin I, and desmin were up-regulated in a time-dependent manner after induction of BMP signaling molecules. Immunocytochemistry revealed the expression of smooth muscle a-actin, sarcomeric a-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric a-actinin positive cells accounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces differentiation of P19 cells into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sulfoxide exposure, depending in part on alteration of BMP signaling molecules. These results suggest that 5-aza treatment could be used as a new method for cardiac differentiation in P19 cells.
Choi
Seung Cheol
SC
Department of Cardiology, Korea University Medical College, Seoul 136-705, Korea.
Yoon
Jihyun
J
Shim
Wan Joo
WJ
Ro
Young Moo
YM
Lim
Do-Sun
DS
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000276:immunology / Q000502:physiology; D000071317:Nanog Homeobox Protein; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009124:Muscle Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D014157:Transcription Factors; D001374:Azacitidine