Neuregulin 1 1 nrg1 b1

MSC differentiation into Schwann-like cells was accomplished according to the extensively tested protocol of Kingham and colleagues.^{18 (link)} Briefly, after two preparatory steps with ß-mercaptoethanol (Sigma-Aldrich corp., MO, USA) and all-trans-retinoic acid (Sigma-Aldrich Corp., MO, USA), the growth medium was replaced by differentiation medium containing Forskolin (Sigma-Aldrich corp., MO, USA), basic fibroblast growth factor (bFGF; PeproTech, NJ, USA), platelet-derived growth factor (PDGF-AA; PeproTech, NJ, USA) and Neuregulin-1 ß1 (NRG1-b1; R&D systems Inc, MN, USA). Successful MSC differentiation was verified by immunocytochemistry for Schwann cell marker S100 (Rabbit anti-S100; ThermoFisher Scientific, MA, USA), glial cell marker GFAP (Glial fibrillary acidic protein, mouse anti-GFAP; ThermoFisher Scientific, MA, USA) and Neurotrophin Receptor p75 (p75 NTR, rabbit anti-p75 NTR; ThermoFisher Scientific, MA, USA). Goat anti-rabbit FITC and goat anti-mouse Cyanine-3 (CY3) (both ThermoFisher Scientific, MA, USA) were used as secondary antibodies. Cell nuclei were labeled with DAPI (4’,6-diamindino-2-phenylindole, ThermoFisher Scientific, MA, USA). The fluorescent expression of the differentiated MSCs was compared to the expression of undifferentiated MSCs and Schwann cells.

MSCs were differentiated into Schwann cell-like cells using a differentiation cocktail containing 0.14% Forskolin (Sigma-Aldrich corp., MO, USA), 0.01% basis fibroblast growth factor (bFGF; PeproTech, NJ, USA), 0.005% platelet-derived growth factor (PDGF-AA; PeproTech) and 0.02% Neuregulin-1 ß1 (NRG1-b1; R&D systems Inc, MN, USA).^{16 (link)} Differentiation was assessed by immunocytochemistry for the expression of S100 (S100; ThermoFisher Scientific, MA, USA), Glial fibrillary acidic protein (GFAP, mouse anti-GFAP; ThermoFisher Scientific) and neurotrophin receptor p75 (p75 NTR, rabbit anti-p75 NTR; ThermoFisher Scientific). Goat anti-rabbit fluorescein isothiocyanate (FITC) and goat anti-mouse cyanine 3 (CY3, both ThermoFisher Scientific) were used as secondary antibodies. Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI).

MSCs were differentiated into Schwann cell-like cells according to protocol^{17 (link)}. This protocol has shown to morphologically change 81.5% of the MSCs exposed to the differentiation medium into a typical spindle-like shape^{17 (link)}. Cells were treated with ß-mercaptoethanol (Sigma-Aldrich, MO, USA) and all-trans-retinoic acid (1:1000 of stock dilution, 35 μg/mL diluted in dimethyl sulfoxide (DMSO); Sigma-Aldrich), prior to introducing a differentiation solution to the growth medium containing 0.14% Forskolin (10 mM dissolved in DMSO, Sigma-Aldrich), 0.01% basis fibroblast growth factor (bFGF; 100 mg/mL dissolved in 5 mM Tris Hydrochloride buffer, PeproTech, NJ, USA), 0.005% platelet-derived growth factor (PDGF-AA; 100 mg/mL dissolved in 10 mM acetic acid, PeproTech) and 0.02% Neuregulin-1 ß1 (NRG1-b1; 100 μg/mL reconstituted in phosphate buffered saline (PBS), R&D systems Inc, MN, USA). Successful differentiation was previously assessed by immunohistochemistry¹⁸ .
All MSC cultures were maintained at subconfluent levels in a 37 °C incubator with 5% CO2 and passaged with TrypLE (Invitrogen, UK). Growth medium was changed every 72 hours. In this experiment, passage five cells were used to seed allografts.