Femke Mathot

Chapter 3 46 Review Board (IACUC protocol A3053-16). Mesenchymal stem cell collection and characterization Rat MSCs were obtained from the inguinal fat pad from Lewis rats and derived as previously published. 18 The obtained MSCs were previously characterized by plastic adherence and pluripotency toward mesodermal lineages. 29 For flow cytometric assessment of stem cell surface markers, MSCs in passage five were used. The expression of MSC surface markers (CD29 and CD90) and hemapoetic cell surface markers (CD34 and CD45) were tested and compared with three control samples. Cell suspensions were pre-incubated with Fc block (Purified Mouse Anti-Rat CD32, 0.5uG per 500uL; BD PharmingenTM, CA, USA) to avoid unspecific binding. Thereafter, CD29 antibody (CD29 antibody | HM beta 1-10; Bio-rad- antibodies, CA, USA), CD90 antibody (CD90 Antibody | OX-7; Bio-rad-antibodies, CA, USA), CD45 antibody (CD45 antibody | OX-1; Bio-rad-antibodies, CA, USA) and CD34 (Mouse/Rat CD34 antibody; R&D systems Inc, MN, USA) were introduced to different samples. For the CD34-sample, the appropriate secondary reagent was added as well (Donkey Anti-Sheep IgG Fluorescein-conjugated antibody; R&D systems Inc, MN, USA). Cells were washed twice in flow cytometry buffer and centrifuged at 300g for five minutes after each wash. Finally, 7-AAD staining was added to each sample to exclude dead cells. Flow cytometry was performed with a BD FACScan flow cytometer. Mesenchymal Stem Cell differentiation into Schwann-like cells MSC differentiation into Schwann-like cells was accomplished according to the extensively tested protocol of Kingham and colleagues. 18 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. Nerve allograft processing Fifty-six sciatic nerves were obtained from 28 Sprague-Dawley rats weighing 250-350 grams and were decellularized according to the protocol of Hundepool and colleagues. 30 In this 5-day protocol, the nerve allografts are exposed to elastase, resulting in less cellular debris with preservation of the ultrastructure of the nerve. Sprague-Dawley rats were specifically selected as there is a known histocompatibility mismatch to Lewis rats. 31, 32 The nerves were