Table 2.

Troubleshooting

Step	Problem	Possible reasons	Solution
4	Poor attachment of hPSCs on Matrigel- or Synthemax-coated plates	No ROCK inhibitor included in step 3 or coating substrate of low quality used	Include a ROCK inhibitor, (e.g. Y27632) in thawing medium; use qualified substrate
6	Cell death or detachment of hPSCs after exposure to CHIR99021 for 24 hr	Initial cell seeding density is not optimal or too high of a CHIR99021 concentration is used	Optimize initial cell seeding density in Step 3 or pretreat cells with low dose CHIR99021 (e.g. 1 μM) on day -1 in Step 4; optimize CHIR99021 (3–12 μM) for your specific hPSC lines or CHIR99021 lot.
13, 15 A (v), 15 D (ii), Box 1	Detachment of cardiac progenitors or epicardial cells	Cell seeding density is too low or cells need more albumin or FBS	Increase cell seeding density in Steps 12, 15 A (iv), and Box 1; Increase concentration of albumin or FBS (up to 20%)
14	Low percentage of WT1+ cells at day 12	Initial cardiac progenitor purity is too low or cell seeding density is not optimal, or CHIR99021 addition dose and/or time is not optimal	Make sure Isl1+Nkx2.5+ cells are over 50% on day 6 or optimize initial cardiac progenitor cell seeding density in Step 9; Optimize CHIR99021 concentration on day 7–9 in Step 13
15 A (vi)	Slow proliferation or differentiation of WT1+ cells after passage	The concentration of TGFβ inhibitor is not optimal; the initial cell seeding density is too low	Optimize TGFβ inhibitor concentration; Increase the initial cell seeding density.