Expression of Gata4 (A), Gata62 (B), Zfpm (C), Tbx5 (D), and Nr2f2 (E) does not statistically differ in E12.5 PPFs when cultured 5 or 10 d. PDGFRa is reduced after 5 d in culture, but is not significantly reduced after 10 d in culture (F). NIH 3T3 fibroblasts have lower expression of Zfpm2 (C) and undetectable levels of Tbx5 (D). For each experiment, 3 pairs of E12.5 PPFs were pooled or 3 wells of 3T3s were pooled and then 2-5 pools were used for qPCR. Gene expression determined by qRT-PCR, normalized against 18S gene expression, and then normalized to “at harvest” value, which was set to 1. Significance tested with one-way ANOVA. Holm–Sidak post hoc tests are indicated by * p<0.05, **** p<0.0001. Error bars represent standard error of the mean (SEM).