Hamilton syringe

The human glioma cell line U87MG was obtained from LGC Standards (Wesel, Germany) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Sigma-Aldrich, Taufkirchen, Germany). Six to eight week old male NMRI nude mice were injected stereotactically with 50.000 U87MG-tdTomato cells in 2 μl PBS. Implantation was performed into the right hemisphere, 1 mm rostral and 2 mm lateral from the Bregma at a depth of 500 μm (n = 5 mice, Charles River, Sulzfeld, Germany) using a Hamilton syringe, driven by a fine step motor. Animals were anesthetized with ketamine/xylazine and unresponsive to stimuli during the intracranial injection. MRI was performed on day 16 and 28 post tumor cell implantation.

The pluripotency of 3-D grown cells was assessed by teratoma formation assays performed in triple experiments. ESCs (1 × 10⁶) were dissociated into single cells following Accutase treatment, resuspended in PBS, mixed with an equal volume of Matrigel (BD Biosciences, San Jose, CA, USA), and injected subcutaneously into the flanks of 4-week-old immunocompromised Fox Chase Severe Combined Immunodeficiency (SCID) Beige mice (Charles River, Wilmington, MA, USA) using a Hamilton syringe. Animals were monitored daily and humanely euthanized by CO₂ overdose following teratoma formation at 10–12 weeks post-injection. Explanted teratomas were either fixed for histological analysis or flash frozen for RNA isolation and assessed by immunohistochemistry and quantitative real time-polymerase chain reaction (qRT–PCR) for germ layer marker expression, respectively. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Oakland University (IACUC protocol number: 17,031).