Composition of expansion and differentiation media is given in Table 1 . After surgical en bloc resection, the dentate gyrus was micro-dissected and dissociated mechanically followed by enzymatic digestion as described previously (Hoehn et al., 2002 (link)). Of the obtained cells, haNSC from passage 5 to 11 were cryopreserved, shipped to the primary investigation site and used for all experiments. Cells were plated on poly-L-ornithine (250 μg/mL)-/laminin (15 μg/mL, both Sigma-Aldrich, Munich, Germany)-coated cell culture dishes (Sarstedt, Nuembrecht, Germany) and grown in expansion medium at 37°C and 5% CO2. Medium was changed every second day. Cells were detached with accutase (PAA, Cölbe, Germany) at 80% confluence, and split. Culturing over at least five passages selected for proliferating cells.
Previously cryopreserved mouse embryonic stem cells (mESCs, CRL-1934, ATCC, Manassas, United States) were cultured in expansion medium (Table 1 ) at 37°C and 5% CO2 on mouse fibroblasts (CRL-1503, ATCC, Manassas, United States) inactivated by mitomycin C (Sigma-Aldrich). Stem cells were separated from the fibroblast layer at 60% confluence by detaching them with accutase, and transferred to gelatin-coated cell culture dishes with daily medium changes. mESCs were detached and transferred every other day. A fibroblast-free mESCs culture was obtained after three passages.
Previously cryopreserved mouse embryonic stem cells (mESCs, CRL-1934, ATCC, Manassas, United States) were cultured in expansion medium (