Tiam Mana Saffari

191 FUNCTIONAL MOTOR RECOVERY OF STEM CELLS DELIVERED TO NERVE GRAFTS 9 Stem cell preparation and differentiation MSCs were derived from the inguinal fat pad of inbred Lewis rats according to protocol 16 . Cells were previously characterized by plastic adherence, pluripotency towards mesodermal lineages, the expression of mesenchymal stem cell markers CD29 (88.2%) and CD90 (88.3%) and the absence of hematopoetic cell markers CD34 (91.1% absent) and CD45 (86.0% absent) 26-28 . Both cell-types were cultured in an incubator at 37°C (5%CO 2 ) and the growthmedium was changed every 72 hours. Passage six MSCs were used in this experiment for both differentiated and undifferentiated MSCs. MSC culture The stromal cell pellet was re-suspended in normal growth medium consisting of a-MEM (Advanced MEM (1x); Life Technologies Corporation, NY, USA), 5% platelet lysate (PLTMax®; Mill Creek Life Sciences, MN, USA), 1% Penicillin/Streptomycin (Penicillin-Streptomycin (10.000 U/mL; Life Technologies Corporation), 1%GlutaMAX (GlutaMAX Supplement 100X; Life Technologies Corporation) and 0.2% Heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; Fresenius Kabi, IL, USA). MSC differentiation 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 . 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 f luorescein 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).