Computational protocol: BNIP3 induction by hypoxia stimulates FASN-dependent free fatty acid production enhancing therapeutic potential of umbilical cord blood-derived human mesenchymal stem cells

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Protocol publication

[…] All procedures involving animal were conducted following the National Institutes of Health Guidelines for Humane Treatment of Animals and with approval from the Institutional Animal Care and Use Committee of Seoul National University (SNU-140123-6). Eight-week-old male Institute for Cancer Research (ICR) mice were used. All mice were anesthetized with a 2:1:2 mixture of Zoletil™ (20 mg/kg; Virbac Laboratories, Carros, France), xylazine HCl (10 mg/kg; Rompun™, Bayer, Leverkusen, Germany), and normal saline prior to skin wound surgery. All surgery was performed by three authors who have a doctor of veterinary medicine license granted by the Ministry of Agriculture and Forestry of Republic of Korea. Every effort was made to minimize suffering during skin wound surgery. Mouse skin wound healing with stem cell transplantation was performed as previously described . The back of the anesthetized mouse was shaved, scrubbed with an organic iodine solution and 70% ethanol solution for disinfection. A 6 mm diameter circular wound was surgically created by sterile biopsy punch. BNIP3 siRNA or NT siRNA-transfected UCB-hMSCs were pretreated with hypoxia or PA for 24 h. Experimental mice were divided into six groups: mice given vehicle (group 1, n = 6); mice given NT siRNA-transfected UCB-hMSCs (group 2, n = 6); mice given NT siRNA-transfected UCB-hMSCs with hypoxia pretreatment (group 3, n = 6); mice given BNIP3 siRNA transfected UCB-hMSCs with hypoxia pretreatment (group 4, n = 6); mice given BNIP3 siRNA-transfected UCB-hMSCs with hypoxia and PA pretreatment (group 5, n = 6); and mice given BNIP3 siRNA-transfected UCB-hMSCs (group 6, n = 6). Cells (1 × 106) in 100 μL of PBS were injected to the dermis intradermally at three sites around each circular wound. All wound images were acquired at the same distance from the object (30 cm) with a digital camera system (D50; Nikon, Tokyo, Japan) at post-injection days 0, 4, 8 and 12. All wounds were covered with Tegaderm™ (3M, London, Canada). Measurement of wound area was determined by using ImageJ software. Wound closure was measured as the difference in wound area on a particular day compared to day 0. At post-injection day 12, all mice were euthanized. The skin wound samples were embedded in O.C.T. compound (cat no. 4583, Sakura Finetek, CA, USA), and stored at −80 °C. Frozen samples were sectioned to a 10 µm thickness by using a cryostat (Leica CM 1520, Leica, Wetzlar, Germany) and then mounted on SuperFrost Plus slides (Thermo Fisher) for hematoxylin and eosin (H&E) staining and immunohistochemistry assessment. Vessel intensity measurement was analyzed by using ImageJ software. [...] Skin tissue samples on slides were fixed in 80% acetone solution. Subsequently, slide samples were washed and then blocked in 5% normal goat serum (cat no. 566380, Sigma-Aldrich) in PBS for 30 min. Slides were incubated with primary (1:100 dilution) for 2 h. After washing three times with PBS, slides were incubated with Alexa 488-conjugated secondary antibody with PI (1:100 dilution) for 1 h. Slide samples were visualized by using confocal microscopy. All images were analyzed by using MetaMorph software (Universal Imaging, West Chester, PA, USA). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Chemicals Oxygen, Palmitic Acid