until day 24. For the generation of hSS, the medium was supplemented with additional small molecules during the first 23 days in culture; a schematic showing the recipes is presented in Supplementary Fig. 4a). The hSS-IS condition involved the addition of the Wnt pathway inhibitor IWP-2 (5 μM; Selleckchem) from day 4 until day 24, and the SHH pathway agonist SAG (smoothened agonist; 100 nM; Selleckchem) from day 12 to day 24. The hSS-ISA condition also included IWP-2 (inhibitor of WNT production–2) and SAG (smoothened agonist) with the addition of allopregnanolone (AlloP 100 nM; Cayman Chemicals) from day 15 to day 23, while the hSS-ISRA condition included AlloP (100nM) from day 15–23, and a brief exposure (day 12–15) to retinoic acid (RA 100 nM; Sigma). From day 25 to 42, the NM for both the hCS and hSS conditions, was supplemented with the growth factors BDNF (20 ng/ml; Peprotech) and NT3 (20 ng/ml; Peprotech) with medium changes every other day. From day 43 onwards, hCS and hSS were maintained in unsupplemented NM with medium changes every four to six days.
