Dorsomorphin

hiPSCs were routinely cultured in 6-well plates on 1:75 diluted Matrigel™ HC (Corning #354263), in FTDA medium [10 (link)]. FTDA consisted of DMEM/F12, 1× PenStrep/l-glutamine, 1× defined lipids (Life Technologies #21331020, #10378016, and #11905031, respectively), 0.1 % human serum albumin (Biological Industries #05-720-1B), 1× ITS (BD #354350), 10 ng/ml FGF2 (PeproTech #100-18B), 0.2 ng/ml TGFβ1 (eBioscience #34-8348-82), 50 nM Dorsomorphin (Santa Cruz #sc-200689), and 5 ng/ml Activin A (eBioscience #34-8993-85). Cells were routinely passaged as single cells or, initially, as clumps of cells. For single cell splitting, cells were grown to full confluence (until cultures seemingly appeared syncytial), digested for 10–15 min using Accutase™ (Millipore #SCR005) with 10 µM Y27632 (abcamBiochemicals #ab120129), and replated in the presence of 10 µM Y27632 at 400,000–600,000 cells per well of a 6-well plate. hiPSCs reached confluence after 3 days under these conditions and were subsequently harvested as above, for continuous maintenance or for the induction of differentiation. hiPSCs were kept in culture for a maximum of 30 passages. Cell lines were tested negative for mycoplasma.

We modified the protocol for neuronal induction shown in [7 (link)]. Briefly, for preparing dual SMAD inhibitor-treated hPSCs, cells were cultured in Stem Fit/AK02N containing 1 µM SB431542 (Merck, Darmstadt, Land Hessen, Germany), 1 µM Dorsomorphin (Santa Cruz Biotechnology, Dallas, TX, USA) and 1 µg/ml Y27632 from day 1 to 7. On day 7, cells were dissociated into single cells using TrypLE Select and cultured in KBM Neural Stem Cell (KOHJIN BIO, Sakado, Saitama, Japan) containing B27 supplement with 2 µM SB431542, 2 µM IWP-2 (Merck, Darmstadt, Land Hessen, Germany) and 10 µM Y27632 at 4% O₂/5% CO₂. On day 14, cells were dissociated again, followed by culture in the same conditions as day 7. On day 20, cells were dissociated into single cells using TrypLE Select and plated on culture plates coated with Matrigel (Corning, Steuben County, NY, USA) and cultured in a neuronal medium (Neurobasal plus medium containing 2% B27 plus supplement, 1% Culture One supplement, 1% Glutamax, 200 µM L-ascorbic acid, 300 µM dbcAMP, 20 ng/ml BDNF, 20 ng/ml GDNF, 20 ng/ml NT-3 and 0.5 µg/ml iMatrix-511) at the normal (20%) O₂ concentration/5% CO₂. The medium was replaced every 3 days.

WT hESCs (HuES6^{29 (link)} background, obtained from Harvard University), WT hiPSCs (F1 background, fetal liver fibroblast-derived^{21 (link)}), and their genetically modified derivatives were maintained on 6-well dishes coated with 1 ml/well 1:75 diluted Matrigel™ HC (Corning #354263), in defined FTDA medium^{30 (link)}. FTDA was composed of DMEM/F12, 1 × PenStrep/Glutamine, 1 × defined lipids (Thermo), 1 × ITS (Corning), 0.1% human serum albumin (Biological Industries), 10 ng/ml FGF2 (PeproTech #100-18B), 0.2 ng/ml TGFβ1 (eBioscience #34-8348-82), 50 nM Dorsomorphin (Santa Cruz), and 5 ng/ml Activin A (eBioscience #34-8993-85). Fully confluent hPSC cultures were harvested by a 15–20 min incubation with Accutase™ (Sigma) containing 10 µM ROCK inhibitor Y-27632 (abcamBiochemicals) and seeded out for passaging into new 6-well plates at 400,000–500,000 cells per well, in FTDA + ROCKi. Cells were split every 3–4 days and kept in culture for a maximum of 30 passages. Short-term signaling stimulation experiments were carried out using semiconfluent cultures.