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After 4 d, generated EBs were collected and embedded in 50 μl (around 40 EBs) of growth factor–reduced Matrigel (BD Biosciences), plated in 12-well plates, and treated for 3 d with 1 mg/ml doxycycline (Sigma-Aldrich), to stimulate Nkx2-1 and Pax8 transgene expression