Spontaneous embryoid body-induced differentiation

Embryoid bodies [28] (EBs) were made by either passaging feeder-dependent colonies with a StemPro EZPassage™ (Thermo Fisher) tool or passaging feeder-free colonies with Accutase. One to three wells of a six-well plate, depending on density, were passaged into one well of an ultralow attachment six-well plate. These cells were fed with 50:50 medium every other day for 7 days and then transferred to a plate coated with 0.1% gelatin for seven more days to continue the differentiation. The cells were subsequently fixed for immunocytochemistry [ICC] or trypsinized, rinsed, and frozen at −80°C in Dulbecco's phosphate buffered saline (DPBS) for RNA extraction and analysis.