Humanumbilical cord MSCs Culture and Lentiviral Transduction
Human umbilical cord MSCs were obtained from healthy donors (n = 3, ages 20-25 yrs) and cultured in a-MEM with 10% FBS and 100 U/mL penicillin-streptomycin (Gibco). MSCs in passages 3-6 were used for experiments. MSCs were incubated at 37 ℃ in humidified air with 5% CO2.
MSCs were transduced with a HIF-1a-overexpressing lentivirus (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) or a control lentivirus, named as HIF-1a-MSCs or NC-MSCs, respectively (Genechem Co. Ltd., Shanghai, China). 72 h after transduction, these cells were further confirmed by western blot analysis and fluorescence microscope.
Culture of neonatal rat cardiomyocytes and human umbilical vein endothelial cells
One-day-old (P1) S/D rats were prepared for isolation of primary cardiomyocytes. Heart ventricles were removed from neonatal pups, minced into 1-mm3 pieces, then digested with a solution containing 0.25% trypsin and 0.1% collagenase II. The dissociated cells were pre-plated at 37 °C for 1.5–2 h. Afterwards, cardiomyocytes were separated from fibroblasts through differential adhesion, followed by culture with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, USA) containing 5% fetal bovine serum (FBS, Gibco, New York, USA) and 10% horse serum (HS, Gibco, New York, USA) at 37 °C in 5% CO2 air.
Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) mixed with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, 100 mg/mL streptomycin and 110 mg/mL sodium pyruvate at 37 °C in 5% CO2 air.
Western Blot
Bradford assay (5000202; BioRad, Hercules, CA, USA) was carried out to quantify cell lysates. Western blotting was performed using antibodies including HIF-1a (36169; Cell Signaling Technology), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174; Cell Signaling Technology), TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech) and CD81 (66866, Proteintech).
Isolation of EVs
MSCs-EVs were prepared through differential ultracentrifugation, allowed to grow in the EV-free medium to a 70-80% confluency for 48 h. Cell debris was eliminated from the conditioned medium through centrifugation at 300g for 10 min, at 2000g for 10 min, and at 10,000g for 30 min. Then the EVs were filtrated with a 0.22 mm filter and harvested through ultracentrifugation at 100,000g for 70 min at 4 ℃. Having been washed and resuspended in PBS, the EVs were ultracentrifuged at 100,000g for 2 h to eliminate the contaminated proteins. After this treatment, the EVs were resuspended in PBS and stored at -80 ℃. The content of protein in the EVs was measured through a BCA protein assay kit (Promega, Madison, WI, USA). The structure of EVs was exhibited by transmission electron microscope (TEM), and their size by nanoparticle tracking analysis (NTA).
Internalization of EVs
The EVs were labeled by DiR (Beyotime, China). The H9c2 cells and HUVECs were seeded and co-cultured with DiR-labeled EVs at different concentrations. After 24 h, the cells were washed with PBS and observed using IVIS Lumina Imaging Systems. The density of BLI signals was measured as average radiance from regions of interest (ROIs).
Moreover, the EVs were also labeled by DiL (Beyotime, China) and co-cultured with receptor cells at 2 time points (2 h and 24 h). Then the cells were washed with PBS and observed under fluorescence microscope.
Calcein-AM and PI staining
To assess the effects of EVs on the survival of H9C2 cells, EVs were co-cultured with H9c2 cells for 24 h under H/SD condition and then stained with DAPI and PI, followed by fluorescence microscopy to observe the stains of red (PI) and blue (DAPI) fluorescence.
Apoptosis assays
Apoptosis was evaluated by TUNEL based on TUNEL Apoptosis Detection kit (Yisheng, Shanghai, China). Samples were mounted with mounting medium containing 4’, 6’-diamidino-2- phenylindole (DAPI; Vector Laboratories). The stained nuclei were counted with a Zeiss LSM510 META microscope. The percentage of apoptotic nuclei =the total number of TUNEL-stained nuclei divided/the total number of DAPI-positive nuclei. Apoptosis was also evaluated with caspase-3/7 cell apoptosis detection kit (Ribo, China) according to the manufacturer’s instructions. Caspase-3/7 green fluorescence, PI red fluorescence and hoechst33342 blue fluorescence could distinguish apoptotic cells, dead cells and living cells, respectively[5, 6].
HUVECs tube formation assay
To assess their angiogenic ability of EVs derived from MSCs, HUVECs were seeded (10,000 cells/well) in 96-well plates coated with growth-factor reduced Matrigel (356230; BD Biosciences, San Jose, CA, USA). Having been challenged with EVs or PBS for 8 h, photos of capillary-like tube formation were captured. Tube length and number of branches were measured with ImageJ software.
Animal experiments
All procedures were approved by the Animal Care and Use Committee in Nanjing Medical University. Sprague-Dawley rats (male, 8 weeks old) were subjected to AMI model after left anterior descending (LAD) coronary ligation as previously described[7]. MSCs-EVs (100 mg) suspended in PBS or RGD hydrogels (100 mL) was intramyocardially injected into two sites in the border area of infarcted hearts. Rats were euthanized by administration of pentobarbital at day 3 or day 28 after MI.
Echocardiography studies
At 28 days after EVs therapy, cardiac function was evaluated through transthoracic echocardiography (Ultramark 9; Soma Technology, Bloomfield, CT, USA), and dimensions calculated using MatLab R2011b software (MathWorks, Natick, MA, USA).
Masson’s trichrome and Haematoxylin-Eosin staining
Paraffin-embedded slides were stained by Masson’s trichrome. Infarcted area was measured as the percentage of fibrotic area in the total left ventricular area. The thickness of infarcted cardiac wall was calculated as the mean of three equidistant measurements on each section. Fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) was used to catch images and ImageJ software (NIH) to analyze them. The inflammation was degreed using Haematoxylin-Eosin (HE) staining.
Tissue apoptosis analysis
Apoptosis was assessed with TUNEL assay (C10625; Thermo Fisher Scientific) in vivo according to related instructions.
Immunofluorescence
Immunostaining was performed with the primary antibodies including anti–α-sarcomeric actin (α-SA, a7811, Sigma-Aldrich); anti–α-smooth muscle actin (ab5694, Abcam); anti-CD31 (ab7388; Abcam, Cambridge, United Kingdom); anti-connexin43 (3512; Cell Signaling Technology); wheat germ agglutinin (WGA, W11261, Thermofisher Scientific, USA). DAPI was used for nuclear counterstaining.
Injectable hydrogels
In the mixture of Biotin-GFFYGRGD (1.14 mg) with PBS buffer (1 mL), tthe pH value was adjusted to 7.4 through adding 2 μL of 1 M Na2CO3. Next, the compound in the suspension was completely dissolved at 95°C, then cooled (as low as room temperature) till hydrogels formed. Only 100 μM hydrogel was chosen for animal experiments. Biotin-GFFYGRGD was observed under scanning electron microscope (SEM).
In vivo tracking of EVs
In vivo, rats were LAD-ligated. EVs were stained with DIR, encapsulated in RGD hydrogels (EV/RGD-biotin), intramyocardially injected into two sites in the border area of infarcted hearts, and tracked by Gluc signals (IVIS Lumina Imaging Systems, Xenogen Corporation) at the indicated time points. The signal density of bioluminescence imaging (BLI) was measured from ROIs.
Real-Time PCR
Exosomal RNA was extracted using Trizol reagent (Life Technologies, USA). To quantify the level of miR-221-3p, cDNA was synthesized with miRNA First-Strand cDNA Synthesis Kit (by stem-loop) (Vazyme Biotech, China). Stem-loop qRT-PCR was accomplished with a FastStart Essential DNA Green Master (Roche, USA). The exosomal level of miR-221-3p was normalized to that of cel-miR-39 (C39) and calculated with the equation “relative gene expression= 2− (ΔCtsample − ΔCtcontrol)”. The primers are listed in Supplementary Table 1.
MicroRNA-inhibitor transfection
HIF-1a engineered MSCs were allowed to grow to 70%-80% confluence, then transfected with synthetic miR-221-3p inhibitor or negative control (RiboBio,Guangzhou, China) on the system of Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). After a 6-h transfection, the cells were added into EV-free FBS containing a-MEM and kept for 48 h. The conditioned medium was collected and EVs isolated as HIF-1a-EV-InhibitormiR-221 and HIF-1a-EV-InhibitorNC.
Statistical Analyses
The data were presented as mean ± SEM. The quantification was performed and graphs generated using GraphPad Prism 7 (La Jolla, CA, USA). One-way ANOVA was implemented for comparison among multiple groups and t-test for comparison between two groups. P < 0.05 was considered statistically significant.