Chir99021 chir

Pig pericyte-derived induced pluripotent stem cells (PC-iPS cells) were cultured in a modified EPS culture system [14 (link)]. Cells were cultured in LCDMV, which consisted of a base medium containing 50% (v/v) DF12 (Gibco;10,565–018) and 50% (v/v) Neurobasal™ Medium (Gibco, 21103-049). The medium also contained 10 ng/ml LIF (Peprotech, 300-05-1000), 1 μM CHIR99021 (CHIR) (Tocris, 4423), 2 μM (S)-(+)-dimethindene maleate (DIM) (Tocris, 1425), 2 μM minocycline hydrochloride (MIH) (Santa Cruz, sc-203,339), and 40 μg/mL vitamin C (Vc) (Sigma, A92902). When the PC-iPS cells reached 70% confluence, they were dissociated using StemPro™ Accutase™ Cell Dissociation Reagent (A1110501; Gibco). Electrotransfection was used to transfer plasmids into the cells. Briefly, 4 μg pST1374-NLS-flag-linker-Cas9 plasmids (Addgene, 44,758), 4 μg NANOG sgRNA plasmid, and 4 μg NANOG HMEJ donor plasmids (mass ratio 1:1:1) were co-transfected into 2.5 × 10⁶ PC-iPS cells using a Lonza AMAXA Lonza Nucleofector 2B/II (Loza, Amaxa 2B), configured to use the A030 program. To select transformants, puromycin dihydrochloride (Thermor, A1113803) (1 μg /mL) and blasticidin (Invitrogen, R210–01) (10 μg /mL) were added to the culture media 24 h post transfection. After 48 additional hours of incubation, puromycin dihydrochloride (1 μg/mL) was added again, the cells were cultured for four additional days.

Human NSCs were derived by differentiating human iPSC HEL24.3^{45 (link)}, and HEL46.11 lines using small molecule cocktail as described elsewhere^{46 (link)}, with minor adjustments. Briefly, iPSCs were detached with StemPro Accutase (Thermo Fisher Scientific) and dissociated gently into single cells suspension in hES-medium in the presence of 5 μM ROCK inhibitor (ROCKi; Y-27632, Selleckchem), 10 μM SB431542 (SB; S1067, Selleckchem), 1 μM dorsomorphin (DM; P5499-5MG, Sigma), 3 μM CHIR-99021 (CHIR; Tocris) and 0,5 μM purmorphamine (PMA; 04-0009, Stemgent) After 2 days, medium was changed to N2B27 medium (DMEM/F12:Neurobasal (1:1) supplemented with N2 and B27 without vitamin A, NEAA, PenStrep (all Thermo Fisher Scientific) and heparin (2 µg/ml; H3149-50KU, Sigma)) containing the same small molecule cocktail as above. On day 4, SB and DM were withdrawn and 150 µM ascorbic acid (AA) was added to N2B27. On day 6, the neurospheres were dissociated with 1 ml pipette and plated on Matrigel in N2B27 media containing AA, CHIR and PMA (growth media). First two passages were split at 1:3 ratio and cells were plated into growth media containing 5 μM ROCKi, which was removed next day. Later passages were split with 1:10 and 1:20 ratio using StemPro Accutase. Media were changed every other day.