Table 1.

Troubleshooting Guide for iPSC Differentiation into Keratinocytes

Problem	Possible Cause	Solution
iPSC do not attach to plate	Insufficient coating of plate	Ensure plates are coated for at least 1 hr with Vitronectin XF™
	Inappropriate media	Ensure Rock inhibitor has been added to media
	Inappropriate Accutase™ treatment	Ensure iPSC were passaged as small clumps, not as single cells
	Equipment failure	Check appropriate temperature and CO2 concentration of cell culture incubator
Poor detachment of iPSC during dissociation	Incomplete Accutase™ treatment	Wash cells with PBS (Ca2+ and Mg2+‐free) Extend Accutase™ treatment up to 10 min. Ensure Accutase™ is prewarmed.
EBs do not attach to Collagen IV‐coated plate	Inappropriate media	Ensure 2% FBS and Rock inhibitor were added to the media
	Inappropriate plate coating	Ensure plates were coated for at least 1 hr
Large number of non‐ keratinocyte colonies in day 20‐26 cultures	Media issues	Ensure BMP4 and RA are not expired and at the correct concentration
	Insufficient cleaning of cultures	Manually remove non‐keratinocyte colonies and cells. Remove non‐keratinocytes by incubating with Accutase™ for 2 min and discarding cells that detach. After that, a normal split can be performed.
Poor attachment of keratinocytes to Collagen IV‐coated plates	Media issue	Add 10 μM Rock inhibitor to media
	Plate coating issue	Coat plates at least 1 hr with Collagen IV
Heterogeneous keratinocyte cultures	Ineffective differentiation	Enrich keratinocytes by FACS using cell surface antigens such as α6 integrin and β4 integrin