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Isolation and expansion of OE-MSCs
Cultures of OE-MSCs were carried out as previously described by the authors [34]. Following biopsy, pieces of olfactory mucosa were washed twice in DMEM/ F12 medium. The biopsies were then dissected into small pieces using 25-gauge needles (< 1 mm2). Each explant was plated on a 2 cm2 culture well coated with poly-L-lysine (5 μg/cm2 in sterile water, Sigma-Aldrich). Except for rodents, the mucosal pieces were covered with a sterile glass coverslip to facilitate and accelerate the adhesion of the explants at the bottom of the culture wells. The wells were filled with 250 μl of the culture medium described above. One week later, concentrations of antibiotics were reduced (100 units/mL of penicillin and to 100 μg/mL of streptomycin), Amphotericin B and Ketoconazole were removed and Plasmocin Treatment was replaced by 1.25 μg/ml Plasmocin Prophylactic (InvivoGen). This medium is referred as growth medium throughout the manuscript. Culture medium was gently renewed every 2 to 3 days. After an additional week, the cells began to grow out of the explants and invaded the culture dish. When confluency was reached, the cells were detached using a trypsin-EDTA solution (0.05%, Life Technologies), pooled and centrifuged at 300 x g for 5 min and replated without exceeding a 1:3 cell split ratio. When several individuals of a species were biopsied, cells from different cultures were compared.
Generation of spheres
For generating spheres, cells were counted in duplicate using an automated counter (Scepter, Millipore) and plated at a density ranging from 15,000 cells/cm2 to 30,000 cells/cm2 (rabbit: 15,000 cells/cm2; mouse & rat:
30,000 cells/cm2; all other genera: 20,000 cells/cm2), into poly-L-lysine coated dishes (5 μg/cm2) and fed with serum-free DMEM/ F12 culture medium supplemented with insulin, transferrin, selenium (ITS-X, 1% Life Tech-nologies), Epidermal Growth Factor (EGF, 50 ng/mL; R&D Systems) and Fibroblast Growth Factor 2 (FGF2, 50 ng/mL; R&D Systems). This culture medium was changed every two days. After 10 days of treatment, spheres were harvested by gently shaking the medium and pooled in one culture well (2 cm2) for imaging.
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