Aggrewell eb formation medium

To form embryonic bodies (EBs) with uniform size from hiPSCs, AggreWell800 6-well plates (Stem Cell Technologies) were used. To prevent cell adhesion and promote efficient EBs formation, plates were pre-treated with anti-adherence rinsing solution (Stem Cell Technologies) and then centrifuged at 1300× g for 5 min to remove all bubbles. After washing the wells, hiPSCs suspended in AggreWellEB formation medium (#5893, Stem Cell Technologies) were added to wells, and plates were centrifuged at 100× g for 3 min to capture cells in the microwells. Plates were incubated at 37 °C with 5% CO_2, and 95% humidity and media were changed every 2 days. After 7 days, EBs were harvested using a 37-µm reversible strainer and used in subsequent experiments. EBs were identified by a decrease in pluripotent markers, including OCT4, NANOG, and DNMT3B, and an increase in three germ layer markers, including SOX1, GATA4, and T, compared to parent iPSCs. General differentiation of p53WT hiPSCs was induced by culturing cells on Matrigel-coated culture plates with DMEM containing 10% FBS, 1% NEAA, 0.1 mM β-ME, and 100 Units/mL penicillin/100 μg/mL streptomycin for 10 days. Differentiation was characterized by a decrease of pluripotent markers OCT4, NANOG, and DNMT3B compared to parent iPSCs and abbreviated as iPSC-Diff.

Culture of hESCs were dissociated into single cells using ReLeSR (Stemcell Technologies) treatment. Single cells were plated to ultralow attachment 96 well plate (Corning) with AggreWell EB Formation Medium (Stemcell Technologies) with 10 µm Y‐27632 (SelleckChem) (9000 cells per well). After 24 h, the medium was substituted by cortical organoid medium (1× DMEM/F12; 15% KOSR; 1× GlutaMax; 1× MEM‐NEAA; 1× N2 supplement; 1× N2 supplement; 1 × 2‐Mercaptoethanol; 1× Penn/Strep) supplemented with 1 µm SB‐431542, 2 µm Dorsomorphin and 2 µm XAV939 for 9 days. Next, the aggregates were embedded in Matrigel and cultured in the medium (0.5× DMEM/F12; 0.5× Neuralbasal; 1× N2; 1× MEM‐NEAA; 1× GlutaMax; 2.5 µg mL⁻¹ Insulin; 1× b‐mercaptoethanol; 1× Penn/Strep; 1 × 10 ng mL⁻¹ EGF, 10 ng mL⁻¹ FGF‐2, 10 ng mL⁻¹ NT3, and 10 ng mL⁻¹ BDNF) supplemented with 1× B27 without vitamin A for 14 days. Then the aggregates were maintained in the medium (supplemented with 1× B27 with vitamin A, 0.2 mm ascorbic acid, 0.2 mm cAMP, and 1% Matrigel) with a medium change every other day. The composition details and medium change timelines are provided in Table S1 of the Supporting Information.

Fibroblast differentiation of iPSCs was achieved by embryoid body (EB) formation assay. EBs of uniform size were generated from 1*10⁶ iPSCs as described previously^{20 (link)} using AggreWell400 plates and AggreWell EB Formation Medium (Stem Cell Technologies). After 3 weeks of suspension culture in E6 medium containing: DMEM/F12 (Invitrogen), 25 µg/mL recombinant human insulin, 10.7 µg/mL human holo-transferrin, 0.5 mg/mL sodium bicarbonate, 64 µg/mL L-ascorbic acid, 14 ng/mL sodium selenite (all Sigma, Canada), supplemented with 10 ng/mL bFGF (Peprotech, Canada) and 100 U/mL penicillium, 100 µg/mL streptomycin (Invitrogen), EBs were dissociated with TrypLE (Invitrogen) and plated in recombinant human Collagen IV-coated (Peprotech) culture dishes. Outgrowing cells were dissociated with TrypLE and replated in chemically defined fibroblast culture medium supplemented with bFGF, until confluent, passaged for five passages, characterized and cryopreserved.