Problem	Solution
Poor DE induction	Adjust cell density (very important) Optimize BMP4 concentration Remove any differentiated colonies from hPSCs before setting up the DE differentiation Protect cells from heat shock Use fresh growth factors in every differentiation, if possible Sometimes switching cell lines may cause variations in DE generation and hence optimization is necessary
Monolayer breaks down after adding hindgut induction medium	This may be caused by initial DE differentiation that had poor efficiency. If this occurs in all of the wells, discard the plate and optimize the DE differentiation step first This may be caused when pipetting media. Dispense the medium on the side of the wells not directly in the center as this may disrupt the monolayer
Low spheroid number	This may be caused by initial DE differentiation that had poor efficiency. See above
Organoids growing in 2D rather than 3D after splitting on Day 21	This occurs when the organoids are dispensed and move through the Matrigel® to the bottom of the dish and adhere to the tissue culture plastic. While passaging, discard any organoids that attach to the plastic and grow in 2D Avoid the long-term storage of Matrigel® at 4°C
Organoids grow in 3D but lack the expression of SATB2 on Day 35	We recommend assessing the expression levels of HOXA13 and HOXD13 by qPCR on Day 10 to ensure proper patterning by BMP2. The levels of these two genes are normally 10–20 times more in HCOs than those in HIOs Patterning growth factors may not be active. Repeat experiment and use freshly thawed growth factors aliquots or freshly purchased growth factors