Table 3.

Troubleshooting

Step	Problem	Possible Reason	Solution
8 (d-2, i)	hPSCs are not dissociated into small clusters or single cells.	Cells have not been exposed to EDTA for enough time.	Avoid additional pipetting and increase the duration of EDTA treatment.
8 (d-2, ii)	Confluency of hPSCs is lower than 80%.	Not enough hPSCs were collected from maintenance plates or passaging involved excessive cell death.	Increase passaging ratio to 5:4 (i.e all cells from 5 wells of maintenance plate to 4 wells of a new 6-well plate)
8 (d8, vii)	Monolayer is detaching from the well surface.	Forceful feeding has separated monolayer cells from the well surface.	When adding fresh medium, tilt plate and gently add medium to the lower side and corner of the well.
8 (d10, viii)	NC cells are detached from underlying monolayer and have spontaneously formed free-floating spheroids.	Accelerated and efficient ENC induction.	Since free-floating spheroids contain the desired ENC cells, do not aspirate away these cells with old medium. Collect these cells with a P1000 micropipette and add them back into well once Accutase has been added.
15 (ENC spheroid phase)	ENC free-floating spheroids have conjoined to form large, irregularly shaped bodies.	Elevated confluency of ENC added to low-attachment well.	Gently separate large, irregularly shaped bodies into smaller spheroids by pipetting them up and down 2–3 times with a P1000 micropipette.