GPC were obtained by the protocol reported for Rezania et al. [15 (link)] with modifications. The protocol consists of 6 stages: stage 1, incubated for 4 days with RPMI 1640 (Gibco™. Catalog number 31800), bovine serum albumin (BSA) (Sigma-Aldrich, catalog number A9418) 2%, activin A 100 ng/mL (Prospecbio), Wnt3a 20 ng/mL (Sigma-Aldrich. Catalog number H17001), FGF2 8 ng/mL (Prospecbio. Catalog number CYT-085). Stage 2, incubated for 2 days with DMEM-F12 medium with BSA 2%, FGF7 50 ng/mL (Prospecbio. Catalog number CYT-303), Cyclopamine-KAAD 0.25 µM (SCBT, Dallas, Tx, USA. Catalog number sc-200929). Stage 3, incubation for 4 days with DMEM-F12 medium, Cyclopamin-KAAD 0.25 µM, retinoic acid (RA) 2 µM (Sigma-Aldrich. Catalog number R2625), FGF7 50 ng/mL (Prospecbio), Noggin 100 ng/mL (Prospecbio. Catalog number CYT-475). Stage 4, incubation for 3 days with DMEM:F12 medium, Inhibitor II ALK5 1 µM (SCBT. Catalog number sc-221234), Noggin 100 ng/mL, DAPT 1 µM (SCBT. Catalog number sc-201315). Stage 5, incubation for 7 days with DMEM/F12 medium, inhibitor II ALK5 1 µM. Stage 6, incubation for 7 days with DMEM F12 medium. However, we used 2% v/v FBS instead of 1% B27 as a supplement to differentiation medium during stages 3 to 6, Fig. 1b.
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