ESC were depleted of MEFs for at least three passages, dissociated and 500 cells were aggregated to embroid bodies for 72hrs via hanging drops in complete media without LIF and with 10% FBS. For analysis of Otx2, Hand1 and GATA4 expression, EBs were dissociated, plated onto eight chamber culture slides (Corning) and processed for immunocytochemistry after 3 more days of culture. For analysis of Nestin, AFP and Brachyury expression, two EBs were per plated per well in four chamber culture slides (Corning) and EB outgrowths were processed for immunocytochemistry after 3 more days of culture.
Maass K., Shekhar A., Lu J., Kang G., See F., Kim E., Delgado C., Shen S., Cohen L, & Fishman G.I. (2015). Isolation and Characterization of ESC-Derived Cardiac Purkinje Cells. Stem cells (Dayton, Ohio), 33(4), 1102-1112.
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