B27 without retinoic acid

Our PSC-CM differentiation protocol was adapted from multiple previously published protocols. The day prior to initiation of differentiation protocol (D-1), cells were changed to expansion media with 1000 U Lif/mL with no CHIR99021 or PD0325901. On Day 0 of differentiation, cells were dissociated with TrypLE (Thermo Fisher) and suspended in a serum-free differentiation media (SFD). SFD was composed of ¾ volume IMDM to ¼ volume Ham’s F12 media, with 0.5% v/v N2 supplement (Gibco), 1% v/v B27 without retinoic acid (Gibco), 0.5% v/v BSA (Sigma) in PBS, 0.75% v/v glutamine (Gibco), 0.75% v/v penicillin-streptomysin (Gibco), 50 ug/mL ascorbic acid (Sigma), and 0.039 ul/mL 1-thiogylcerol. For the first four days of differentiation, differentiating cells were maintained as embryoid bodies. On Day 2, media was replaced with fresh SFD plus 3uM CHIR99021 (Selleckchem) and 2.5 ng/mL BMP4 (R&D Systems). On Day 4, cells were dissociated with TrypLE and replated as a confluent monolayer on gelatin-coated flasks. At this time, cells were cultured with SFD with 0.1% v/v XAV939. On Day 6, the media was changed to SFD. From Day 10 to Day 13, lactate selection was performed by culturing the cells in DMEM without glucose plus lactate. Subsequently, the cells were cultured in SFD, with media changes every two days.

SMB55 cells were a gift of R. Segal (Dana Farber Cancer Institute, Harvard Medical School). SMB55 cells were cultured in DMEM/F-12 without L-Glutamine (Corning) supplemented with 2% B-27 without retinoic acid (Gibco) and 1% penicillin/streptomycin (HyClone). Cells were maintained as neurospheres in 3-D culture in untreated 96 well plates (Corning), changing media every 2 days. Cells were dissociated every 6 days with Accutase (Sigma-Aldrich) and plated at 150,000 cells/mL. All experiments, except for the live imaging experiments, were performed in 3-D culture. Cells were frequently tested to confirm that growth was dependent on Hh pathway activity using SANT-1 treatment and EdU incorporation as described below. SMB55 cells were genotyped to confirm the absence of the wild-type Ptch1 allele using primers recognizing the wild-type (Ptch-WT3, Ptch-WT4) and mutant alleles (NeoF3, PtchR3) as described in [12 (link)]. Genotyping PCR was performed using Taq polymerase on genomic DNA extracted from SMB55 cells using ZymoBead™ Genomic DNA kit (Zymo). Genomic DNA from Ptch1^−/− mouse embryonic fibroblasts and a wild-type mouse were used as a control for the mutant and wild-type alleles respectively. All cells were grown in a humidified incubator at 37 °C, 5% CO₂.