FIG. 1.
Inhibition of cardiomyocyte differentiation by overexpression of noggin. (A) P19CL6 cells and P19CL6noggin cells were cultured in growth medium (a, b, e, and f) or in differentiation medium containing 1% DMSO (c, d, g, and h). Both parental P19CL6 cells (a and b) and P19CL6noggin cells (e and f) grew well and remained undifferentiated in growth medium. However, parental P19CL6 cells differentiated into beating cardiomyocytes when cultured in medium containing 1% DMSO. On day 14, most P19CL6 cells had differentiated into mononucleated contracting cardiomyocytes (c [magnification, × 1,000] and d [magnification, ×200]). On the other hand, P19CL6noggin cells did not differentiate into beating cardiomyocytes after treatment with DMSO (g). Overexpression of BMP-2 with adenovirus induced differentiation of P19CL6noggin cells into cardiomyocytes (h), suggesting the essential role of BMPs in the differentiation of P19CL6. The cells were stained with anti-sarcomeric MHC antibody (MF20) (a, c to e, g, and h) or Hoechst dye (b and f). (B) Quantification of the areas stained by MF20 in P19CL6 and P19CL6noggin cells. Like overexpression of BMP-2 with adenovirus, addition of BMP protein to the culture medium at the concentration of 100 ng/ml but not 10 ng/ml partially induced differentiation of P19CL6noggin cells into cardiomyocytes (columns 5 to 7). The areas of at least five fields were measured for each cell line under the same conditions. The results are expressed as the mean percents ± standard deviations.