For cell analysis, cells were dissociated with TrypLE and pelleted. A 100-μl fluorescent-conjugated antibodies cocktail (5 μl of SSEA-4 330408 and 5 μl of TRA-1-60 330610; BioLegend) in staining media (1× PBS and 2% FBS) was used to resuspend the pellet and incubated 30 min on ice. Cells were then resuspended in excess of staining media, span down and resuspended in staining media, filtered through a cell strainer, and kept on ice. Cells were analyzed using the BD Biosciences LSR Fortessa. The analysis was done using the FlowJo software. For directed mesodermal and endodermal differentiation experiments, a similar workflow was used with the exceptions of using accutase to dissociate the cells and using saponin buffer [saponinin PBS (1 mg/ml) + 1% BSA (bovine serum albumin)] to permeabilize the cells before incubating with fluorescent-conjugated SOX17 (BD Biosciences, 562594) or brachyury (Fisher Scientific, IC2085P) in saponin buffer.

For cell sorting, a similar procedure was followed. Cells were eventually resuspended in their media supplemented with rock inhibitor and sorted accordingly using the BD Biosciences InFlux sorter (fig. S7B). Cells were then transferred to appropriate dishes for culture and kept on rock inhibitor during the next 24 hours.

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