BMSCs paracrine repairs smoke inhalation injury - angiogenesis involving Notch signaling pathway Feng Zhu; Xiaoyan Yang; Junjie Wang; Yichao Jin; Xiaochen Qiu; Jiahui Li; Zhaofan Xia Online data supplement MATERIALS AND METHODS Rat Smoke Inhalation Injury Model Adult male Sprague-Dawley (SD) rats, weighing 180-200g, were provided by experimental animal center of Second Military Medical University (SMMU). Rats were housed in individual cages in a temperature-controlled room with a 12h light/dark cycle and free access to food and water. All animal experiments were approved by the SMMU in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH) (publication no. 96-01). The model was developed by using a home-made smoke generator, a specialized system. Briefly, rats, housed in a highly permeable metal cage, were exposed each time. Smoke was generated by slowly smoldering wood shavings (120g/kg body weight). We pressed button to release smoke automatically for 20s to ensure that the smoke chamber was filled with smoke. Rats exposed to successive 9min periods of smoke separated into three times by 30s exposures to ambient air. Results of blood gas analysis, inflammatory response, pathology etc confirmed that this rat smoke inhalation injury model induced by our novel self-made smoke generator could be used for acute and chronic lung injury experiments. Isolation and Culture of BMSCs Bone marrow was obtained from 2-week-old male SD rats (approximately 80-100g, provided by animal center of SMMU). The marrow was washed by phosphate-buffered saline (PBS) and plated into flasks of Dulbecco’s Modified Eagle’s Medium (DMEM) (HyClone, Utah, USA) with 10% fetal bovine serum (FBS) (HyClone, Utah, USA). After 72 hours of cultivation, the non-adherent cells were removed from the culture by changing the medium. Adherent cells were then subsequently propagated in culture with DMEM containing 10% FBS and 1% penicillin and streptomycin. Adhered cells were allowed to grow to about 80% confluency and then trypsinized and reseeded at a density of 1×105 cells/cm2. This procedure was performed for two passages. Cells were used for in vivo experiments between 8 to 11 passages. Cells surface markers-CD44, CD45 and CD90 were selected in accordance with the position statement for the minimal criteria to define an MSC, from the International Society for Cellular Therapy and detected using flow cytometry according to the manufacturer's protocol (rat monoclonal antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA). Preconditioning of BMSCs The third generation BMSCs plated into flasks in seeding density 10,000 cells/cm 2 and after 24 h was irradiated at room temperature using various dosages of Cobalt 60 (60Co) γ ray including 2 gray (Gy), 4Gy and 8Gy (All radiation dosage rate were at 165.44 cGy·min-1 ) (60Co γ ray provided by Cobalt Bomb Room of SMMU, Shanghai, China) from distance of 1 m from source. During the time of radiation, cells in non-radiation group (control BMSCs) were kept outside the machine at the same temperature as the radiated cultures. After irradiation, flasks were incubated in 5% CO 2 atmosphere under 37 ° C and cells were collected after various time for analysis. A single-cell suspension was prepared by a 5-min exposure to 0.25 trypsin EDTA solution (Sigma Aldrich), inactivation of enzyme was done by medium containing 20% FCS, and a portion of the cells was used to following culture. Preconditioned BMSCs continued to be cultured for at least 4 weeks. In the following course of culture, cells' morphology, viability, proliferation, the ability of homing, paracrine activity and differentiation potential were assessed periodically. Crystal violet staining was used for morphology assessment. Control BMSCs and preconditioned BMSCs were fixed with 0.lml l0% methanol solution for 30 seconds. Then cells were incubated with 0.3ml crystal violet solution (Aladdin, Shanghai, China) at room temperature for 20 minutes. After removal of crystal violet, PBS flushing three times and drying at 37 oC, cell morphology was assessed under microscope. Cell viability was assessed by trypan blue exclusion technique. Control BMSCs and preconditioned BMSCs were trypsinized and suspended at a density of 1×104 /mL. 9 drops of cell suspension with 1 drops of 0.4% trypan blue solution (Aladdin, Shanghai, China) was mixed at room temperature. Cell number of each passage was determined with hemocytometer within 3 minutes by excluding trypan blue-positive cells which indicates dead or no viable cells. Cell proliferation was assessed by MTT colorimetric assay. Control BMSCs and preconditioned BMSCs were incubated with 20µl MTT solution (5mg/ml) (Aladdin, Shanghai, China) at 37 oC for 4 hours. The resulting spectrophotometrical absorbance was measured at 490nm, and results are expressed as a percentage of control cell numbers. Differentiation potential of vascular endothelial cell was detected by VIII factor immunocytochemistry and assessed by counting both vascular endothelial cells and non-vascular endothelial cells per dish. It will then be expressed as the percentage of vascular endothelial cells over the total cells. Irradiated BMSCs and control cells were replated in culture medium at 3×103 cells/cm2 in six-well tissue culture plates. The following day, the culture medium was changed and the cells were cultured in 200µl conditioned medium consisting of DMEM, 10% FBS, 10ng/ml VEGF, 2ng/ml bFGF (Sigma, St. Louis, MO, USA). Medium changes were performed weekly. At week 4 cultures were assayed for VIII factor immunocytochemistry (antibody from Abcam, Cambridge, UK). Chloromethylbenzamido-1,1'-Dioctadecyl-3,3,3'3'-Tetramethylindocarbocyanine Perchlorate (CMDiI) staining was used for assessment of BMSCs’s homing ability. Preconditioned BMSCs and control BMSCs were trypsinized and suspended at a density of 106/mL. 5µL of the CMDiI solution (1:200 dilution) (Invitrogen, California, USA) supplied per mL of cell suspension was added, then incubated for 10min at 37 oC. The labeled suspension tubes was centrifuged at 1500 rpm for 5min at 37 oC. The supernatant was remove and the cells gently resuspend in warm (37 oC) medium. Repeat the wash procedure two more times. All procedures were carried out according to the Product Information. A portion of stained BMSCs reseeded in a six-well plate at appropriate density for further culture and observation. Stained BMSCs was transplanted systemically into smoke inhalation injury rats with the aim of investigating ability of homing. For CMDiIstaining detection in vivo, tissue DAPI (4',6-diamidino-2-phenylindole) (Invitrogen, California, USA) staining was performed to be as background (blue fluorescence). Red fluorochrome CMDiI-labeled BMSCs in frozen lung, liver, heart and kidney tissue sections in BMSCs and pBMSCs group were observed directly using fluorescence microscopy (Leica, Inc., Germany). The culture medium and lung tissue assay were collected in each radiation dosages groups and control BMSCs pre-and post-radiation for detection of the levels of main paracrine factors-vascular endothelial growth factor (VEGF) and fibroblast growth factor-basic (bFGF) secreted by MSC and preconditioned BMSCs using Enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK). All procedures above mentioned were carried out according to the manufacturer's instructions. The levels of main paracrine factors on angiogenesis were analyzed using an enzyme-linked immunosorbent assay reader (Spectra MAX 250; Spectra Devices, Sunnyvale, CA). The aim of preconditioning was to choose an optimal radiation dosage under which BMSCs could keep morphology, viability, proliferation, homing ability, paracrine activity but be inhibited differentiation potential for further study. Histopathology Paraffin-embedded lungs (each left upper lung lobe) were sectioned at a thickness of 5μm and stained with hematoxylin and eosin (HE). The histopathology of the lungs was evaluated and blindly scored by at least two pathologists using a light microscope. The severity of smoke inhalation injury was determined according to the following: 1) alveolar congestion; 2) hemorrhage; 3) infiltration or aggregation of neutrophils in air-spaces or vessel walls; and 4) thickness of alveolar wall/hyaline membrane formation. Each criterion was graded according to a 5-point scale. A total lung injury score was calculated as the sum of the four criteria. VIII Factor+ Cells and Notch+ Mocrovessel Immunofluorescence Detection VIII factor+ cells immunofluorescence detection: Frozen lung tissue sections were incubated with anti-VIII factor (1:150 dilution) (Abcam, Cambridge, UK) for 2hrs at 37 oC. Thereafter, tissue sections were incubated with DyLight™ 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, PA, USA) for 45min at 37 oC. Red fluorescence were monitored using a Leica TCS STED Ultra-high Resolution Laser Scanning Confocal Imaging system (Leica, Inc., RPlus software was used for calculating VIII factor-positive Germany). Image-Pro○ RPlus (VIII factor+) expression quantitatively at randomly selected field. [Image-Pro○ 6.0 software achieves results according to the principle that the amount of target protein is determined by staining shade of color (optical density) and the distribution of size. The distribution size of staining region is proportional to the amount of target protein, and the relationship of optical density and quantity of target protein is logarithmic relationship, which meets Lambert-Beer law] Immunofluorescence double staining for detection Notch+ Mocrovessel: Frozen lung tissue sections were incubated with Notch1 XP™ Goat mAb (1:100 dilution) (Cell Singaling Technology, Danvers, MA, USA) specific to Notch1 in lung for 2hrs at 37 o C initially. Then VIII factor antibody (1:150 dilution) (Abcam, Cambridge, UK) was added for another 2hrs. Finally the sections were treated with appropriate secondary antibodies mixture for 45min. The secondary antibodies were respectively Donkey Anti-Goat IgG-FITC (Santa Cruz Biotechnology, CA) binding specifically Notch1 XP™ Goat mAb (Green fluorescence excitation) and DyLight™ 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, PA, USA) binding specifically VIII factor antibody (Red fluorescence excitation). Leica TCS STED Ultra-high Resolution Laser Scanning Confocal Imaging system was RPlus software was used for calculating used for scanning. Similarly, Image-Pro○ Notch1 protein expression in microvessel quantitatively at randomly selected field. Western blot analysis Lung extracts were lysed, and the lysates were prepared according to the literature 22 with Notch1 antibody (Cell Singaling Technology, Danvers, MA, USA). Lung extracts were lysed, and the lysates were prepared according to literature. Western blot analysis was performed using primary antibodies against Notch1 (1:200 dilution) (Cell Singaling Technology, Danvers, MA, USA). The density of protein bands was assessed using a computing densitometer with Image-Pro plus software (Media Cybernetics, Inc., Bethesda, MD, USA).