Journal: Molecular Therapy
Article Title: High Local Concentrations of Intradermal MSCs Restore Skin Integrity and Facilitate Wound Healing in Dystrophic Epidermolysis Bullosa
doi: 10.1038/mt.2015.58
Figure Lengend Snippet: Mesenchymal stromal cells (MSCs) stably express and secrete C7. (a) Quantitative real time polymerase chain recation of COL7A1 expression in normal human fibroblasts (NHFs) and MSCs with GAPDH as reference gene. n ≥ 9 different donors per group in three independent experiments. Values are mean ± SEM. (b) Western blots of representative experiments. NHFs and MSCs were seeded in six-well plates and grown to 80% confluence before adding serum-free culture medium + 50 µg/ml ascorbic acid for 48 hours. Conditioned medium was precipitated with ammonium sulfate and analyzed by western blotting, cells and matrix were extracted and processed for western blot analysis, too. Western blots were run under reducing conditions. Blots were probed with antibody against C7 and β-actin as loading control. (c) Densitometric quantification of three independent western blots of cell and matrix lysates. n ≥ 9 different donors per group in three independent experiments. Values represent mean ± SEM of C7 expression in NHFs and MSCs normalized to β-actin expression. (d) C7 expression in MSCs in correlation to passage number and donor age. A representative western blot of cell and matrix lysates of the same donor in a low (passage 3) and a high (passage 12) passage number and between a young (12 years) and an older (42 years) donor are shown. Western blots were run under reducing conditions. Similar C7 expression was detectable in all presented settings. (e) Limited trypsin digestion assay of secreted C7. Conditioned medium was pelleted as described above (b); the pellets were resuspended in TBS and digested with 0.05% trypsin at 30 °C for 10 minutes. Positive controls were boiled prior to trypsin digestion. Western blotting was performed as described above and probed with antibody recognizing the P1 fragment of C7. MSC-derived C7 was resistant to trypsin proteolysis suggesting stable triple helical configuration. (f) Representative images of NHFs and MSCs from different donors seeded on collagen I coated glass slides for 48 hours in the presence of 50 µg/ml ascorbic acid and stained for human C7 (green). In MSCs, a mix of C7 high (white arrows) and low (white arrowheads) expressing cells was detected. Nuclei (blue), bar = 50 µm.
Article Snippet: The following antibodies were used: rat anti-CD11b (BD Biosciences, Heidelberg, Germany), Cy3-conjugated mouse anti–α-smooth muscle actin (SMA) (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti–COL7A1 (Millipore, Schwalbach, Germany), mouse anti–β-actin (Sigma-Aldrich), mouse anti- COL7A1 (Sigma-Aldrich), rabbit anti- COL7A1 (Eurogentec, Cologne, Germany), rabbit anti-C3 (Dako, Hamburg, Germany), biotinylated hamster anti-CD3e (BD Pharmingen, Heidelberg, Germany), rabbit anti-NC2-10 for C7 P1 fragment.
Techniques: Stable Transfection, Expressing, Western Blot, Trypsin Digestion Assay, Derivative Assay, Staining
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