Mouse skin wound healing model

All procedures for the mouse skin wound healing model were performed following the National Institutes of Health Guidelines for Humane Treatment of Animals and approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-161128-6). Eight-week-old male Institute of Cancer Research (ICR) mice were used. All mice were anesthetized with a mixture of Alfaxan™ (80 mg/kg, Jurox Pty Ltd, Rutheford, Australia) and xylazine HCl (10 mg/kg, Rompun™, Bayer, Leverkusen, Germany). The authors who have a doctor of veterinary medicine license granted by the Ministry of Agriculture and Forestry of Republic of Korea performed wound surgery. The procedure for the mouse skin wound healing surgery was performed as previously described [33]. Briefly, the back of an anesthetized mouse was shaved and scrubbed with an organic iodine solution and 70% ethanol solution for disinfection during the surgery. A wound in the back skin was made by using a 6 mm diameter circular biopsy punch. BICD1, GSK3β or NT siRNA-transfected UCB-MSCs were pretreated with normoxia or hypoxia for 24 h. Then, 1 × 10⁶ UCB-MSCs were injected into the dermis intradermally at three sites around each wound. Experimental mice groups were divided into the seven groups: mice given vehicle (group 1, n = 7); mice given NT siRNA-transfected UCB-MSCs (group 2, n = 7); mice given NT siRNA-transfected UCB-MSCs with hypoxia pretreatment (group 3, n = 7); mice given BICD1 siRNA-transfected UCB-MSCs with hypoxia pretreatment (group 4, n = 7); mice given BICD1 siRNA-transfected UCB-MSCs (group 5, n = 7); mice given GSK3β siRNA-transfected UCB-MSCs with hypoxia pretreatment (group 6, n = 7); and mice given GSK3β siRNA-transfected UCB-MSCs. All gross images were acquired at post-injection days 0, 4, 7 and 10. After the surgery, wounds were covered with Tegaderm™ (3M, London, Canada). The wound closure rate was analyzed by the ImageJ software. All mice were euthanized at post-injection day 10. Acquired skin samples were fixed with 4% PFA and then dehydrated with 20% and 30% sucrose solution. Dehydrated skin samples were embedded in optimum cutting temperature (O.C.T.) compound (Sakura Finetek, CA, USA, #4583) and stored in a deep freezer kept at –80 ℃. Frozen skin samples were sectioned to a 10 μm thickness using a cryostat (Leica CM 1520, Leica, Wetzlar, Germany) and mounted on silane-coated slides (Muto Pure Chemicals, Tokyo, Japan, #5116-20F). The vessel intensities in the skin wounds were analyzed with the ImageJ software.