hESC colonies were cultured on Matrigel-coated six-well plates for 3 to 4 days, and AP staining was performed with the Alkaline Phosphatase Kit, as directed by the manufacturer’s instructions. Briefly, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times with 1× tris-buffered saline with Tween 20 (TBST); then, 30 μl of FRV-alkaline solution and 30 μl of sodium nitrite solution were mixed for 2 min at room temperature and added to the wells with 1350 μl of water and 30 μl of Naphthol AS-BI. The cells were incubated in the dark at room temperature for 30 min, rinsed with TBST, covered with PBS, and observed under a microscope.

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