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 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.
Highlights
The pluripotent P19 embryonal carcinoma (EC) cell line is derived from a teratocarcinoma formed in C3H/He mice, and this cell line has the ability to differentiate into all three germ layers (Martin, 1975; McBurney, 1993)
We have demonstrated that 5-aza induces cardiac differentiation of P19 cells in a confluent monolayer culture even in the absence of prior embryoid body (EB) formation and dimethyl sulfoxide (DMSO) exposure; this was confirmed by the up-regulation of BMP signaling molecules and cardiac marker genes, and the increase of beating cell clusters after 5-aza exposure
To investigate how P19 cells can be differentiated into cardiac cells by 5-aza treatment with the absence of prior EB formation and without exposure to DMSO, we examined the morphological changes of the P19 cells in the presence of 1 μM of 5-aza
Summary
The pluripotent P19 embryonal carcinoma (EC) cell line is derived from a teratocarcinoma formed in C3H/He mice, and this cell line has the ability to differentiate into all three germ layers (Martin, 1975; McBurney, 1993). 5-azacytidine (5-aza), a cytosine analog, was originally developed and tested as a nucleoside antimetabolite for acute myelogenous leukemia (Sorm et al, 1964; Cihak, 1974) This compound is an effective DNA hypomethylating agent, and it is capable of altering the expressions of certain genes (Mohandas et al, 1981; Branch et al, 1996) or regulating cell. We have demonstrated that 5-aza induces cardiac differentiation of P19 cells in a confluent monolayer culture even in the absence of prior EB formation and DMSO exposure; this was confirmed by the up-regulation of BMP signaling molecules and cardiac marker genes, and the increase of beating cell clusters after 5-aza exposure
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