Mtesr plus supplement

HEK293T cells (ATCC Cat# CRL-3216, RRID:CVCL_0063) were cultured in high glucose (4.5 g/L) DMEM supplemented with 10% FBS, penicillin and streptomycin, and grown in a CO₂ incubator maintained at atmospheric oxygen levels and 5% CO₂. Human induced pluripotent stem cells (hiPSC) were obtained through Material Transfer Agreements from Bruce Conklin, Gladstone Institute (WTC cell line). hiPSCs were expanded on growth factor reduced Matrigel-coated plates (Corning) in mTeSR plus medium (Stemcell technologies) containing mTeSR plus supplement (Stemcell technologies), 50U penicillin and 50U streptomycin. The cell culture medium was changed every other day, and cell passaged upon reaching 70% confluence. During the first 24 hrs after passaging, 5 μM Y-27632 dihydrochloride (Tocris, 1254) was supplemented to culture medium.

Dual knockout Epi-iPSCs were subjected to neural induction to obtain an ATRT-like model. Before the induction, cells were maintained in mTeSR Plus Basal Medium (STEMCELL, Cat#: 100–0274) with antibiotics and 1x mTeSR Plus Supplement (STEMCELL, Cat#: 100–0275). On day 0, cells were dissociated by Accutase®, and 5 × 10⁴ cells were seeded per well of a low attachment 24-well plate (Corning, Cat#: 3473) in DMEM/F-12 (ThermoFisher, Cat#: 11320033) containing 2% B-27 supplement (ThermoFisher, Cat#: 17504044), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μM transforming growth factor (TGF)-beta inhibitor, SB431542, and 10 μM ROCKi, Y-27632. After 48 h, the media was changed, and 50 ng/mL FGF2 was added to promote neural induction for spheroid formation until day 7 for characterization.
For RT-PCR, day-7 spheroids were collected and centrifuged at 300×g for 5 min. Then, the total mRNA was isolated from the spheroids using RNeasy® Mini Kit (Qiagen, Valencia, CA), concentrated, and cleaned using the RNA Clean & Concentrator-5 kit (Zymo, Irvine, CA).
For immunocytochemistry, the spheroids were collected and treated with Accumax® at 37 °C for 15 min. The dissociated cells were replated on a Matrigel-coated surface for 48 h before being fixed and probed for markers of interest.