Table 2.

Troubleshooting table

Step	Problem	Possible reason	Solution
9	Cells do not recover or grow slowly after thawing	Unhealthy cells at cryopreservation.	Cryopreserve healthy cells. Alternatively, Rho kinase inhibitor (Y-27632) can be supplemented to hESC medium at 10 μM for 24 h post thawing.
26	Cells come off the plastic during CHIR99021 phase	Cell density is too low	Plate more cells at Step 23.
37	Pellets break up during a transfer	Not enough centrifuging	Perform two sequential centrifugations of ×400g for 2 min. Rotate the tube180° before the second spin at Step 35. Up to four sequential centrifugations can be performed.
40	No nephrogenesis happens	Damaged cells due to centrifuge	If centrifugations are repeated multiple times at Step 37, reduce the number of times or duration.
		Intermediate mesoderm induction fails	Confirm FGF9 activity is intact. Use freshly thawed FGF9. Use more than 0.32 ml of FGF9/Heparin/APEL medium per cm2 at Step 27.
		Differentiation fails	Confirm if posterior intermediate mesoderm markers, HOXD11, are positive at step 29 (Fig. 3a). If they are negative, modify culture conditions for your cells; e.g. initial cell density at Step 23, CHIR99021 concentration or CHIR99021 duration at Step 25, 26.