Shh c25ii

The cells were switched to the neural induction medium containing 1:1 mixture of N2/B27 medium containing 10 ng/ml basic fibroblast growth factor (b-FGF), 1 µM Dorsomorphin dihydrochloride (Tocris, 3093), 10 µM SB431542 (Tocris, 1614), 100 ng/ml mouse recombinant Sonic Hedgehog (SHH)-C25II (Genscript, Z03050-50), and 10 µM CHIR99021 (Sigma, SML1046). The N2 medium consisted of DMEM/F12 medium (Gibco) with 1× N2 supplement (Gibco, 17502048), 5 µg/ml insulin (Sigma, 19278), 1 mM L-Glutamine (Lonza, 17605E), 100 µM MEM-Non-Essential Amino Acid solution (NEAA) (Gibco), 100 µM 2-mercaptoethanol (Sigma, M3148), and 1:100 Penicillin-Streptomycin (Lonza, 17602E). The B27 medium consisted of a Neurobasal medium (Gibco) and 1× B27 supplement (Gibco, 17504044). The cells were washed daily using DPBS and maintained in the induction medium.

Hindbrain neuronal progenitor cells (NPCs) were generated from human induced pluripotent stem cells (iPSCs), using a protocol adapted from [19 (link)]. Human iPSCs were first cultured in DMEM/F12 (Gibco) supplemented with N2 (Gibco), B27 (Gibco), nonessential amino acids (Gibco), 1% GlutaMAX (Gibco), 2 µM SB431542 (STEMCELL Tech.), 2 µM DMH1 (Tocris), and 3 µM CHIR99021 (Tocris); collectively referred to as SDC media. Culturing in SDC media for 1 week induced human iPSC differentiation into rostral hindbrain neural stem cells (NSCs). Rostral hindbrain NSCs colonies were selected and re-plated in SDC media supplemented with 1000 ng/ml of SHH C25II (GenScript). Ventral rostral hindbrain NSC colonies were collected and re-plated in SDC + SHH media along with 10 ng/ml of FGF4 (Pepro Tech). Growth in SDC + SHH + FGF4 media-induced ventral rostral hindbrain NSC differentiation into hindbrain NPCs after 1 week. Hindbrain NPCs were GBX2, HOXA2, and HOXA4 positive as assessed via PCR to confirm hindbrain specificity at this developmental stage. All cells were grown in a 5% CO₂ humidified incubator at 37 °C.