Induction of Embryonal Teratocarcinoma Cell(P19 cell) to Differentiate into Smooth Muscle Cell

ABSTRACT

BACKGROUND: Understanding the regulatory mechanisms of the smooth muscle cells differentiation is one of the central issues in researches of atherogenesis where smooth muscle cells undergo dedifferentiation and regain embryonic phenotype. Smooth muscle myosin heavy chain isoforms(SM1,SM2) are important molecular markers to define the stage of smooth muscle cell differentiation. METHODS: In order to establish an in vitro model of smooth muscle cell differentiation using a pluripotent murine embryonal teratocarcinoma cell line(P19 cell), we first isolated cDNA clone of mouse SM1 and then tried various chemicals to induce P19 cells to differentiate into smooth muscle cells, The expression of Sm1 and alpha-smooth muscle actin was examined using RNase protection assay, Western blotting and indirect immunofluorescence. RESULTS: In the presence of luM retinoic acid, a small proportion of P19 cells could be in duced to differentiate into smooth muscle cells expressing SM1 as well as alpha-smooth muscle actin since day 8 after treatment. By blocking the Brain-2 expression, thus inhibiting neuronal differentiation, we could obtain more abundant differentiated smooth muscle cells. Sequential immunofluorescencc demonstrated that it took weveral days for smooth muscle myosin heavy chain to organize completely in differentiation smooth muscle cells. On the other hand, 10nM retinoic acid, 1% dimethyl sulfoxide or 2mM hexamethylene bisacetamide could not indduce P19 cell to differentiate to smooth muscle cells. CONCLUSION: In conclusion, P19 cells can be induced to differentiate into smooth muscle cells and this in vitro system will be useful in understanding the regulation of SM1 gene expression as well as of angiogenesis.