Table 1.

Troubleshooting

Problem	Possible reason	Solution
The iPSC lines lose colony-like morphology or grow slowly.	Cells are differentiated.	Verify whether iPSC lines have maintained an undifferentiated state by examining the expression of pluripotency markers TRA-1–60 and SSEA-4 by FACS. If cells have differentiated, restarting a new culture is recommended.
Poor attachment of iPSCs to Matrigel-coated plates.	No ROCK inhibitor was included, or a coating substrate of low quality was used.	Include a ROCK inhibitor (e.g., Y27632) in culture medium; prepare Matrigel-coated plates following the protocol and the manufacturer’s instruction.
Colonies on Matrigel-coated plates are too small.	The size of dissociated iPSC clumps at Basic Protocol 2, step 3 is too small.	At day −2, pipette the cell pellet gently to ensure that the cell clumps are larger than that during regular passaging.
Too few EBs are formed; lots of dead cells in the suspension culture.	Cell loss during feeder deletion due to poor survival, poor pluripotency status, or poor Matrigel quality.	Follow solutions for Basic Protocol 2, steps 3 to 5. Confirm expression and percentage of pluripotency markers for day −2 and day 0 iPSC lines.
Yield of myeloid progenitors is low.	The number and/or the quality of EBs is low.	Perform FACS-based characterization of day 6 and day 8 EBs to confirm the percentage of hematopoietic progenitors. Continue to culture EB and single-cell suspension in ultra-low plate for a few more days (e.g., to day 20) before harvesting for adherent culture to obtain more myeloid progenitors. Confirm the percentage of CD45+/CD18+ cells in day 15 single cells.
Low density of IPSDM at day 22.	A low seeding density was used at day 15.	Optimize the seeding density at day 15. When the day 15 seeding density is within the recommended range, low density of day 22 IPSDM is most likely due to low proliferative potential of myeloid progenitors, which may be due to poor quality of day 15 progenitors or the intrinsic variability of differentiation potential of iPSC lines.
Purity of IPSDM is low.	Purity of day 15 myeloid progenitors is low.	Characterize day 15 single cells by FACS. Perform magnetic beads or FACS-based sorting if desired. Do not allow differentiated IPSDM to grow to over 90% confluency or past day 22.