A83 01

U87 cells untreated or pretreated with TGF-β (T) for 96 h in the presence or absence of the TGF-β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF-β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands). A total of 5 animals per condition were used, and 3 × 10⁵ GG5 cells (1 × 10⁵/μl PBS) were injected in the striatum of the animals using a stereotactic frame. Then, 5 × 10⁵ cells of GG14 and GG16 were injected (3 animals/cell line) to determine tumorigenicity and invasive growth. Mice were monitored and killed when they presented with neurological signs or after being 6 months in the experiment, following which the brains were harvested and fixed in 4% paraformaldehyde for 48 h and embedded in paraffin and prepared for IHC. These experiments were approved by the committee for Animal care and conducted in compliance with the Animal Welfare Act Regulations.

Human blastoids were generated according to a previously described method^{15 (link)} with a few modifications. WIBR3 human ES cells were grown in HENSM for at least 3 passages in feeder-free conditions (1% Matrigel- or Cultrex-coated plates) and were used for generating human blastoids. After 3 days of growth, naive ES cells were collected and counted, and 55 cells were seeded per microwell (total of 66,000 cells were seeded per 24-well in 1 ml of medium) AggreWell 400 24-well (Stem Cell Technologies, 34415) in N2B27-BSA aggregation medium supplemented with 10 µM of the ROCK inhibitor Y27632. The next day, medium was changed to PALLY, which consisted of N2B27 base with 1 µM of the MEK1/2 inhibitor PD0325901 (Axon Medchem, 1408), 1 µM of the TGFR inhibitor A83-01 (Axon Medchem, A83-01), 1 µM LPA (Tocris, 3854), human LIF (10 ng ml^–1), and 10 µM of the ROCK inhibitor Y2763. This medium was repeated on day 2, but on day 3, the medium was changed for LY (1 µM LPA (Tocris, 3854) and 10 µM of the ROCK inhibitor Y27632) for another 48 h. Afterwards, human blastoids were manually selected and collected for further analysis. The entire procedure was conducted under 37 °C, 5% O₂ and 5% CO₂ conditions. PALY medium has the same composition of PALLY but without including LIF.