Sorted d4.75 populations (DN, Flk1-SP, Podxl-SP and DP) were reaggregated for 24 hr in StemPro-34 serum-free medium followed by reculture of the aggregates for 4d in the presence of VEGF and bFGF. Total and contracting colonies formed by the aggregates were scored. (A) QRT-PCR analysis of cardiac mesoderm marker expression. Total RNA was isolated from the sorted d4.75 EB populations (DN, Flk1-SP, Podxl-SP and DP) and analyzed using QRT-PCR. The expression of each gene was normalized to that of Gapdh. This experiment was performed two times, with comparable results. ESC, undifferentiated ES cells; unsorted, unsorted d4.75 EBs. (B) Results of two independent experiments are shown as number of beating colonies/number of total colonies. (C) Immunostaining of colonies formed from the Flk1-SP cell aggregates using rat anti-mouse cTnT (primary) and Alexa Fluor 488 goat anti-rat (secondary) antibodies. (Scale bars, 100 microns)