Fibroblast growth factor 2 (fgf2)

H9 hESCs (WiCell Institute, Madison, WI, USA, passages 25–55) were cultured on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) as described in a standard protocol (http://www.wicell.org). The hESC culture medium, consisting of Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco), 20% Knockout serum replacement (Gibco), 0.1 mM β-mercaptoethanol (Amresco), 1% NEAA (GIBCO), 0.5% l-glutaMAX (Gibco) and 4 ng/ml FGF-2 (Sino Biological).

All plasmids used were constructed using restriction/ligation or the Gibson assembly method. Murine genes for EGF (NM_010113.3), FGF2 (NM_008006.2), FGF7/KGF (NM_008008.4), HGF (NM_010427.4), IGF1(NM_010512.4), PDGF-B (NM_011057.3), TGF-β1 (NM_011577.1), and VEGFA (NM_001025257.3) were obtained from Sino Biological. Genes for IL-Ra (pORF9-hIL1RNa) and LL-37 (NM_004345.3) were obtained from OriGene. The luciferase reporter gene was PCR amplified from commercial plasmid firefly luciferase from pGL4.16 (Promega). For constitutive expression, soluble factors were amplified using PCR and cloned into the pcDNA3 vector on HindIII/EcoRI or KpnI/NotI site. The Tet-On 3G tetracycline-inducible gene expression systems were used (Clontech). Using the Gibson assembly, a Tet-ON 3G transactivator (pRetroX Tet3G) was cloned downstream of the CMV promoter (pcDNA3 plasmid), and genes of interest (GOI) downstream of the pTRE3GV promoter were inserted into the same plasmid. For the Tet-OFF system, a tetracycline repressor TetR (Smole et al., 2017 (link)) fused to the VPR activation domain (Lebar et al., 2019 (link)) was used. Using the Gibson assembly TetR-VPR was cloned into pcDNA3.1 downstream of the CMV promotor. In the same plasmid, six repeats of TRE binding sites following minimal promoter controlling GOI were cloned.

Neural induced differentiation Media (NGD) and neural induction medium (NIM) were prepared according to previous reports [43 (link), 44 (link)]. Compound C (Medchem Express LLC, Monmouth, UK, 2.5 μM), FGF2 (Sino Biological Inc., Beijing, China, 20 ng/mL), EGF (Sino Biological Inc., 20 ng/mL), SB431542 (Medchem Express LLC., 10 μM), DMH-1 (Medchem Express LLC., 2 μM), LIF (Sino Biological Inc., 10 ng/mL), LDN193189 (Medchem Express LLC., 0.25 μM) and insulin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, 10 ng/mL) were added to medium as described previously [6 , 43 (link)–48 (link)]. iPSCs^TLX were seeded onto a matrigel-coated 6-well plate at a density of 2.5 × 10⁴ cells/cm². The medium was changed to a differentiation medium on the next day. On day 6, cells were digested by Accutase™ for 7 min at 37 °C. Then iNSCs^TLX were seeded into a vitronectin-coated 6-well plate at a density of 2.5 × 10⁴ cells/cm² and passaged every five days.