Crl 1503

Primary MELM cells were isolated from the anterior half of the maxillary process, a developing lip region, at E11.5, as previously described [45 (link)]. The maxillary processes were dissected in sterile Dulbecco’s phosphate-buffered saline (DPBS) without Ca²⁺ and Mg²⁺ and were pooled. To isolate single-cell suspensions, the tissues were incubated with 0.25% trypsin/0.05% ethylenediaminetetraacetic acid (EDTA) for 10 min at 37 °C. MELM cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), L-glutamine, β-mercaptoethanol, and nonessential amino acids. MELM cells were passaged up to two times. Mouse cranial neural crest O9-1 cells (SCC049, Sigma-Aldrich, St. Louis, MO, USA) were cultured under a conditioned medium provided by a mouse embryonic fibroblast cell line called STO cells (CRL-1503, ATCC), as previously described [45 (link)].

As previously reported [13 (link)], undifferentiated human embryonic stem cell line, SNUhES3 (46, XY) was cultured on a mitotically inactivated STO (CRL-1503, ATCC, Manassas, VA, USA) feeder layer and passaged every 7 days by mechanical dissociation [59 (link)] under stereo-microscope (Nikon, Tokyo, Japan). The hESCs culture medium was composed of DMEM/F12 (Invitrogen, Waltham, MA, USA), 20% knockout serum replacement (KO-SR, Invitrogen, Carlsbad, CA, USA), 1% nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 50 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), 50 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and 4 ng/mL basic fibroblast growth factor (bFGF, Invitrogen, Carlsbad, CA, USA).

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.