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
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
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
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).
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
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.
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○
(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
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).