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. 2018 Jun 28;13(6):e0199996. doi: 10.1371/journal.pone.0199996

Fig 1. Experimental design.

Fig 1

Cells were cultured for several days in expansion (XP) medium. At day minus 3 (D-3) sonic hedgehog (SHH) and CHIR99021 were added to the culture medium. Cells were subcultured into XP medium that contained SHH and CHIR99021 (0.5 μM) at D-1. At D0 the culture medium was changed to Pre-differentiation medium and was supplemented with CHIR99021 (0.4 μM), LDN193189 (100 nM) and DAPT (5 μM). At D+2, the culture medium was changed to Differentiation medium that was supplemented with NT-3 and 1% FBS. SHH and the three small molecules were also added. At D+4, SHH and CHIR99021 were removed. At D+6, the culture medium was changed to Differentiation medium, that is in the absence of NT-3 and FBS. HGF (100 ng/ml) was added on D+12. The culture medium was exchanged every 48 hours.