N2 and b27

Parental human iPSC line (NCRM 1, NIH Common Fund Regenerative Medicine Program) was genetically altered by CRISPR-mediated targeting of AAVS1 locus to introduce floxed-stop CAG-boosted tdTomato donor DNA construct. EF1-alpha promotor-driven Cre recombinase was introduced episomally to generate pan-tdTomato + human iPSC line (pan-red line). The pan-red line was differentiated toward ventral telencephalic fate using and adaptation of previously described methods (Bagley et al., 2017 (link); Xiang et al., 2017 (link)). In brief, pan-red hiPSCs were plated into ultra-low attachment u-bottom 96-well plates (9000 cells/well) in neural induction medium containing LDN-193189 (100 mM), SB431542 (10 mM), and XAV939 (10 mM) to form organoids for 10 days. From days 10 to 17, cells changed to neuronal differentiation medium containing N2 and B27 (Invitrogen) with IWP2 (2.5 μM) and SAG (100 nM). From day 18 onward, neuronal differentiation medium also contains BDNF (20 μg/mL), cAMP (125 mM), and ascorbic acid (200 μg/mL). At day 35, SPARC (50 ng/mL) and SerpinE1 (20 ng/mL) are added for 14 days. At day 49, organoids are gently dissociated in EDTA for 5 min, then Acutase 15 min at 37°C for downstream experiments (in vitro migration assays and xenotransplants).

Fibroblasts from humans and nonhuman primates were reprogrammed to iPSCs using Yamanaka retroviral vectors expressing reprograming genes (Klf4, Oct4, Sox2 and cMyc) and differentiated into NPCs as previously described (Marchetto et al., 2013 (link); Marchetto et al., 2010 (link)). Established iPSC colonies were kept in feeder-free conditions indefinitely and passed using mechanical dissociation. To obtain NPCs from iPSCs, EBs were formed by mechanical dissociation of iPSC clusters and plated into low-adherence dishes in DMEM/F12 plus N2 and B27 (Invitrogen) medium in the presence of Noggin (R and D) for forebrain induction for approximately seven days. Then, floating EBs were plated onto poly-ornithine/laminin (Sigma)-coated dishes in DMEM/F12 supplemented with N2 and B27 (Invitrogen) and Noggin. Rosettes were collected after seven days. Rosettes were then dissociated with Accutase (Chemicon) and plated again onto coated dishes in DMEM/F12 supplemented with N2 and B27 and 10 ng/ml of FGF2 (R and D). Homogeneous populations of NPCs were obtained after one to two passages with Accutase under the same conditions. To obtain neurons, NPCs were cultured in DMEM/F12 supplemented with N2 and B27, laminin (1 µg/ml), BDNF (20 ng/ml), GDNF (20 ng/ml) and cyclic AMP (500 µg/ml) for up to eight weeks.

Approximately 10–20 × 10⁴ cells from either normal intestinal murine crypts (organoids) or dissociated intestinal adenomas (spheroids) from Apc^Min/+ mice were seeded in 50 μL Matrigel (BD Biosciences) in 24-well plates. After polymerization, 500 μL of complete spheroid medium [DMEM/F12 plus penicillin (100 U/mL) and streptomycin (100 μg/mL) (Biological Industries); N2 and B27 (Invitrogen); 140 nM ROCK inhibitor, 100 ng/mL Noggin, and 20 ng/mL bFGF (Peprotech); 100 ng/mL R-spondin (R&D Systems) and 50 ng/mL EGF (Sigma)] was added. Cultures were maintained at 37 °C, 5% CO₂ and medium changed every 2 days. According to standard protocols, we also added Wnt3 (R&D Systems) at the start of the organoids cultures to facilitate their formation, and then removed it. Notch inhibitor DAPT (Calbiochem) was used at 50 μM. Inducible deletion of Jag1 in the preformed Apc^Min/+;Jag1^lox/lox;β-actin-Cre-ERT spheroids was achieved by treating the cultures with the indicated doses of 4-hydroxytamoxifen up to 72 h.
For whole-mount immunostaining analysis, organoids and spheroids were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 (Pierce). Primary antibodies were incubated overnight. Secondary antibodies were Alexa Fluor from Molecular Probes and were incubated for 2 h at room temperature at a 1:1000 dilution. Slides were mounted in VectaShield with DAPI (Vector).

Glioblastoma stem cells (GSCs) were generated from tumors by enzymatic digestion into single cells and subsequent growth in NBE medium, comprised of Neurobasal-A medium (Invitrogen, Carlsbad, CA) supplemented with N2 and B27 (Invitrogen), bFGF and epidermal growth factors (R&D Systems, Minneapolis, MN) as previously described [27 (link),42 (link)].

The human GBM cell line U87 was purchased from the Chinese Academy of Science (Shanghai, China) and was verified using short tandem repeat assays by GENEWIZ (Suzhou, China). All cells were grown in DMEM (Hyclon, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit-Haemek, Israel), 1% penicillin and streptomycin (P/S; Hyclon). To initiate stem-like induction, the cells derived from U87 xenografts were cultured in a defined serum-free neural stem cell (NSC) medium containing 20 ng/ml of basic fibroblast growth factor (bFGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/ml of epidermal growth factor (EGF, Peprotech), N2 and B27 (Invitrogen). TMZ, Doxorubicin (DOX), Etoposide (Eto), Cis-platinum (CIS), and 5-Fluorouracil (5-Fu) were acquired from Selleck Chemicals.