5-azacytidine induces cardiac differentiation of P19 embryonic stem cells.
- Author:
Seung Cheol CHOI
1
;
Ji hyun YOON
;
Wan Joo SHIM
;
Young Moo RO
;
Do Sun LIM
Author Information
1. Department of Cardiology Korea University Medical College Seoul 136-705, Korea. dslmd@kumc.or.kr
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
aza compounds;
bone morphogenetic proteins;
cardiac myocytes, cell differentiation;
gene expression;
stem cells
- MeSH:
Animals;
Azacitidine/*pharmacology;
Bone Morphogenetic Proteins/genetics/metabolism;
Cell Differentiation/drug effects/genetics;
Cell Line, Tumor;
Cell Proliferation/drug effects;
DNA-Binding Proteins/genetics/metabolism;
Embryo/cytology;
Gene Expression;
Homeodomain Proteins/genetics/metabolism;
Mice;
Muscle Proteins/analysis/genetics/metabolism;
Myocytes, Cardiac/*cytology/immunology/physiology;
Research Support, Non-U.S. Gov't;
Stem Cells/*drug effects/metabolism;
Transcription Factors/genetics/metabolism
- From:Experimental & Molecular Medicine
2004;36(6):515-523
- CountryRepublic of Korea
- Language:English
-
Abstract:
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 micrometer 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 micrometer 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.
