Joris van Dongen

313 Adipose ECM hydrogels to release paracrine factors propagated in medium with 250 µg/ml G418 for a second round of selection. Stable cell lines were propagated and stored in liquid nitrogen. For the current study, clone iADSC13 was used and characterized. Tagging with CMV promoter-driven reporter genes respectively EGFP (green fluorescence) or dTomato (red fluorescence) was with third generation VSV-pseudotyped replication-deficient lentiviruses. Up to three rounds of transduction were done to increase the fraction of reporter-expressing ASCs. These were named iADSC13EGFP and iADSC13dTomato respectively. Additionally, transfected cells were FACS-sorted, propagated and cryopreserved in liquid nitrogen. Between 80 and 95%of the sortedASCs showed reporter expression thatwas detectable with a fluorescence inversion microscope, which sufficed for the experiments. Characterization and differentiation potential of immortalized ASC Flow cytometry Immortalized ASC, lentivirally tagged with either EGFP or dTomato, were analysed using low cytometry (FACS) for CD surface marker expression. Cells were stained with the following anti-human monoclonal antibodies: CD31‐ phycoerythrine/cyanine7 (Pe/Cy7; eBioscience, Vienna, Austria), CD45‐fluorescein isothiocyanate (FITC; IQ Products) and CD90‐allophycocyanin (APC; BD Bioscience, San Jose, CA). CD29‐ APC (eBiosience), CD44‐FITC (BD Bioscience) and CD105‐Pe/Cy7 (eBiosience). For controls we used the following monoclonal antibodies: Mouse IgG1 kappa‐Pe/Cy7, Mouse IgG1 kappa‐APC (both eBioscience), IgG2b FITC (BD Bioscience) and IgG1 FITC (IQ products). Cells were mixed with the antibodies and incubated for 30 mins. Adipogenic, osteogenic and smooth muscle cell differentiation assay Basal medium DMEM (BioWhittaker Walkersville, MD) containing 10% foetal bovine serum (FBS), 1% penicillin/streptomycin and 1% glutamine was used. Immortalized ASC, tagged with EGFP or dTomato, were cultured to confluency and medium was changed to promote differentiation. For adipogenic differentiation this was basal DMEM plus 0.1 µM dexamethasone, 1 nM insulin, 0.5 mM isobutymethylxanthine. For osteogenic differentiation this was basal DMEM plus 0.1 µM dexamethasone, 10 mM β ‐glycerophosphate and 0.05 mM ascorbic acid. Finally, smooth muscle cell differentiation was in basal DMEM plus with 10ng/ml TGF‐ β 1. After 14 days culture in the differentiation medium, cells were fixed with 2% PFA and stained for Oil Red O (Sigma‐Aldrich, St. Louis, MO) for adipogenic differentiation, Alizarin Red (Sigma Aldrich) for osteogenic differentiation and Phalloidin‐FITC (Invitrogen, Thermo Fisher Scientific) in DAPI for 30 minutes for smooth muscle cell differentiation.