Exp Mol Med.  2004 Dec;36(6):515-523.

5-azacytidine induces cardiac differentiation of P19 embryonic stem cells

• Choi SC
• Yoon JH
• Shim WJ
• Ro YM
• Lim DS

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.

Keyword

aza compounds; bone morphogenetic proteins; cardiac myocytes, cell differentiation; gene expression; stem cells

MeSH Terms

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