PAGE 5

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Clonal efficiency assay

The assay was carried out by plating OE-MSCs (passage 7) from one representative culture per genus in 6-well plates at a density ranging from 10 to 320 cells/well in triplicate by using a 1:2 serial dilution in growth medium. After plating, the dishes were placed in an incubator (37 °C, 5% CO2) and left untouched for 7 days before being paraformaldehyde-fixed (4%, Antigenfix) during 15 min at room temperature (RT). Colonies were stained for 15 min using crystal violet and then manually counted. For each sample, clonal efficiency (% of clonogenicity) was calculated as follows: (mean number of colonies/total number of seeded cells) × 100. When too many colonies overlapped, counting was not performed.

In vitro proliferation assay

The assay was performed on OE-MSCs for each studied genus, 2 months (10 passages) and 3 months (20 pas-sages) after the initial plating. Cells from one representative culture per genus were seeded at a density of 200 cells/cm2 in 24-well plates in triplicate in growth medium, during 8, 24, 48, 72 or 96 h. After being paraformaldehyde-fixed (4%, Antigenfix) for 15 min at RT and stained with Hoechst blue (0.5 μg/ml, Sigma-Aldrich), the cells were counted for each of the 6 tested conditions, using an inverted microscope (Zeiss microscopy) and a computer procedure (ImageJ). The population doubling-time was calculated using “Doubling-Time.com” (Roth V. 2006).

In vitro mesodermal differentiation assays

Human OE-MSCs have been previously described to be able to differentiate in vitro into different types of meso-dermal cells (Murrel et al., 2005; Delorme et al., 2010). These characteristics in OE-MSCs from rat, rabbit, dog and horse were assessed. For osteogenic and chondrogenic differentiation, olfactory stem cells were grown using same techniques as previously described . To evaluate osteogenic differentiation, cells cultures were fixed in a paraformaldehyde solution (4%) for 15 min and stained with von Kossa (Bio-Optica) or Alizarin Red stain (ScienCell), according to manufacturers’ instructions. For chondrogenic differentiation, cells were grown in pellets and fixed in 10% buffered formalin (pH 7.4), routinely processed and paraffin-embedded. Four μm thick sections were cut and stained with Toluidine blue (Diapath) or Alcian blue (Bio-Optica) according to instructions.

For tenogenic differentiation, we adapted different protocols used for MSC differentiation. Thus, 30.000 OE-MSCs were grown on a 5 μg/cm2 collagen-I matrix (Sigma/Aldrich) in DMEM without FBS, 50 ng/ml GDF-5 (R&D Systems), 50 ng/ml GDF-7 (R&D Systems) and 20 ng/ml TGF-B3 (R&D Systems) for 7 days [36–38]. To evaluate tenogenic differentiation, cells were fixed as previously described and immunochemistry against Tenomodulin and Scleraxis proteins carried out using the same procedure described above, with the appropriate antibodies.

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