Fluorescence Activated Cell Counting

To achieve single cell suspension, PP clusters were incubated in a 0.5% trypsin/EDTA solution (Sigma) for 20 minutes at 37°C under gentle shaking (Sigma). The reaction was stopped by the addition of 3× volume of knockout DMEM supplemented with 10% FBS. To minimize cell aggregation, the dissociated EBs were then passed several times through a 25 gauge needle and filtered through 0.45 μm cell strainers (BD Biosciences) that have been prewashed in PBS containing 0.5% FBS. Cell viability was determined by the Trypan Blue assay and cell suspensions were subjected to the β‐galactosidase fluorescence assay. At the end of the reactions, cells were resuspended in PBS containing 5% FBS and analyzed using a BD FACS Aria III (BD Biosciences). The median fluorescence intensity (MFI) was calculated for the FITC⁺ gated populations using the corresponding functions of FlowJo (FLOWJO, LLC).