Femke Mathot

Chapter 4 62 Table 1. Experimental design. MSCs = mesenchymal stromal cells qPCR analysis = quantitative polymerase chain reaction analysis Mesenchymal stromal cell collection and characterization Rat mesenchymal stromal cells were obtained from the inguinal fat pad of isogenic Lewis rats and derived as previously described. 16 Lewis rat MSCs were previously characterized by adherence to plastic, pluripotency towards mesodermal lineages, the presence of stem cell surface markers CD29 and CD90 and the absence of hemapoetic cell surface markers CD34 and CD45. 37, 38 MSCs were cultured 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, NY, USA), 1% GlutaMAX (GlutaMAX Supplement 100X; Life Technologies Corporation, NY, USA) and 0.2% Heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; Fresenius Kabi, IL, USA). Mesenchymal Stem Cell differentiation into Schwann-like cells The differentiation protocol of Kingham and colleagues was used to differentiate MSCs into Schwann-like cells. 16 This protocol describes MSC-culture in differentiation medium that consists of 70 m L forskolin (Sigma-Aldrich corp., MO, USA), 5 m L of basis Fibroblastic Growth Factor (bFGF; PeproTech, NJ, USA), 2.5 m L of Platelet Derived Growth Factor (PDGF-AA; PeproTech, NJ, USA), and 10 m Lof Neuregulin 1- b 1 (NRG1-b1; R&D systems Inc, MN, USA) per 50mL normal growth medium. In accordance with the protocol, differentiation of MSCs was confirmed by immunocytochemistry for Schwann cell markers S100 (Rabbit anti-S100; ThermoFisher Scientific, MA, USA), GFAP (Glial fibrillary acidic protein, mouse anti-GFAP; ThermoFisher Scientific, Waltham, MA, USA) and neurotrophin Receptor p75 (p75 NTR, rabbit anti-p75 NTR; ThermoFisher Scientific, Waltham, MA, USA). Differentiation markers were visualized with two different secondary antibodies (goat anti-mouse Cyanine-3 and goat anti- rabbit FITC; ThermoFisher Scientific, Waltham, MA, USA) and cell nuclei were labeled with DAPI (4’,6-diamindino-2-phenylindole, ThermoFisher Scientific, Waltham, MA, USA). Immuno- Group Description Time points N Outcome measurements I Undifferentiated MSCs + nerve allograft T- = processed nerve allografts only Tc = cells only T0 = directly after seeding T1 = 1 day after seeding T2 = 3 days after seeding T3 = 7 days after seeding T4 = 14 days after seeding T5 = 21 days after seeding 5 samples per group for each time point qPCR analysis • Neurotrophic genes • NGF, GDNF, PTN, GAP43, PMP22 • Angiogenic genes • CD31, VEGF1 • ECM genes • COL1A1, COL3A1, FBLN1, LAMB2 • Regulatory cell cycle genes • CASP3, CCBN2 • Reference gene • GAPDH II Differentiated MSCs + nerve allograft