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Immunocytochemistry
Immunocytochemistry was carried out to assess expression of nestin protein, known to be strongly expressed in OE-MSCs [14, 34] and neural proteins GFAP and MAP2. OE-MSCs (passage 6) were plated on glass cov-erslips at a density of 15,000 cells per cm2 in growth medium for approximately 48 h. The cells were then fixed in a paraformaldehyde solution (4%, Antigenfix, MM France) for 15 min and incubated for 1 h at RT with blocking solution (3% bovine serum albumin, 5% goat serum and 0.1% Triton X-100, Sigma Aldrich) in phosphate-buffered saline (PBS) solution. Glass cover-slips were then incubated for 90 min at RT with the appropriate primary antibody diluted in the blocking solution (Table 2). For nestin detection, mouse monoclonal anti-nestin (Abcys) was used for rodent and rabbit polyclonal anti-nestin (Abcam) for all other studied gen-era. The cells were then rinsed 3 times in PBS and incubated for 60 min with the appropriate polyclonal secondary antibody (1:500, Jackson ImmunoResearch). After several washes in PBS, cells were counterstained with 0.5 μg/mL Hoechst blue ( Sigma-Aldrich) for 10 min and mounted with anti-fading medium (ProLong Diamond; Life Technologies). Negative control conditions were carried out by omitting the primary antibody. Pictures were acquired with an inverted microscope (Axio Imager, Carl Zeiss microscopy, Germany) and negative controls were used to adjust image acquisition parameters.
Flow cytometry analysis
Using flow cytometry, we analyzed expression of 3 surface markers. Two of them (CD44 and CD73) are known to be strongly expressed in human OE-MSCs while the third one, CD34, is not expressed. Cells were washed twice in PBS and then harvested using TrypLE™ Select Enzyme (Life Technologies). Then, the cells were centrifuged (300 x g, 5 min), resuspended in cold blocking solution (10% FBS in PBS) and centrifuged again. Cells were paraformaldehyde-fixed for 15 min RT (4%, Antigenfix), washed twice in blocking solution and permeabilized in cold methanol (90%, − 20 °C) 30 min at 4 °C, before being washed twice in blocking solution. Cells were then incubated 20 min RT with primary anti-bodies against CD34, CD44 or CD73 diluted in blocking solution or incubated with the corresponding isotype control (rabbit IgG, at the same concentration, as a negative control. Cells were then washed 3 times by centrifugation (600 x g, 5 min) and incubated 20 min RT in the absence of light with the corresponding secondary antibody diluted in blocking solution. After three washes, cells were immediately processed for flow cytometric analysis. Acquisitions were performed on a FACSCanto II flow cytometer (BD Bio-sciences) using BDFACSDiva software. At least 10,000 events were recorded for each analysis and measures were performed in duplicate. Percentages are presented after the subtraction of isotype background and refer to the total living population analyzed.
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