Table 2.

TROUBLESHOOTING

Step	Problem	Possible cause	Solution
4 and 5	Poor Attachment of hPSCs to tissue culture plates	Variability between lines	Add Rho kinase inhibitor Y- 27632 in the medium during passaging and for the next 24 hours after passaging.
6	Decrease of pluripotency following passaging	Break-up of hPSC colonies is not optimal	If clumps are too big (>3000 cells), the middle of the colony differentiates disturbing the downstream endoderm specification. This can be easily avoided by vigorous pipeting of the clumps during passaging. If clumps are too small (<200 cells), hPSC will differentiate becoming unsuitable for downstream liver differentiation. This can be avoided by decreasing enzymatic treatment and by gently dissociating clumps during passaging.
7, 8 and 9	Suboptimal differentiation	Variability between lines	DE differentiation is the most important part of the protocol. Indeed, artificial hepatocytes are systematically obtained as long this step of the protocol works efficiently. Thus optimisation of the first part of the protocol is essential.. For that, we recommend to define the most efficient concentration of FGF and CHIR in step 1. For that, cells should be differentiated in increasing/decreasing dose of CHIR and / or bFGF and then DE differentiation can be monitored using QPCR/Immuno and flow cytometry analyses. At least 60% of the cells should express those markers to ensure an efficient hepatic differentiation
11	Suboptimal differentiation	BMP 4 effect	BMP4 might have a negative effect on hepatoblast specification in some hPSC lines. Thus, step 7 can be replaced by extending step 6 for 3 additional days (i.e. 3 additional days in RPMIB27 supplemented with Activin). Importantly, increasing or decreasing dose of BMP4 has no effect on hepatoblast specification in vitro.
12	Suboptimal maturation	Variability between lines	Hepatoblast derived hPSCs respond differentially to a variety of basal cell culture media and thus optimisation might be necessary for specific cell lines. Hepatozyme can be used instead of HBM.