WJ-MSCs isolation and culture

WJ-MSCs were isolated and expanded according to the previously reported methods [38, 39]. Human umbilical cord samples (n = 3) were aseptically collected from the Obstetric Department affiliated with Shiraz University of Medical Sciences (SUMs), Shiraz, Iran. Infectious samples were excluded by performing hepatitis C and B virus (HCV, HBV) and human immunodeficiency virus tests. The tissues were stored in PBS solution supplemented with 1% antibiotic–antimycotic solution on ice. After removing blood vessels, the Wharton’s jelly was scraped from the amnion and dispensed into 2 to 3-mm pieces. These tissue pieces were explanted and cultured in DMEM-F12 supplemented with 10% FBS and 1% antibiotic-antimycotic solution at 37 °C with 5% CO2.

Commercial osteocyte and adipocyte differentiation media (Bonyakhteh, Iran) were used for evaluating the cell differentiation potential. After 14 and 21 days, the samples were fixed and stained with Alizarin Red and Oil Red O staining to confirm the cell differentiation potential.

To determine the phenotype of cell-surface antigens, cells from the third passage were stained with FITC-conjugated antibodies (BioLegend, San Diego, CA, USA) specific for hematopoietic lineage markers CD34 and CD45, and stromal surface markers CD90 and CD44. The stained cells were resuspended in PBS before being analyzed with a FACSCalibur flow cytometer (Becton Dickinson, USA). At least 10,000 events were recorded for each sample as similarly reported elsewhere [40].

WJ-MSC spheroid formation and hypoxia conditioning

We used a straightforward method for hypoxic preconditioning of WJ-MSCs by standard incubators (37 °C with 5% CO2) [41]. Accordingly, WJ-MSCs (passage number 2–5) were isolated from Wharton jelly and seeded in 4 wells of a 6-well plate at the density of 2 × 105 cells/well. When cells were grown to 70–80% confluence, we put an oxygen absorber pack (Bihava, Iran, Tehran) in one of the empty wells and in the other empty well, an oxygen indicator (Anaerotest Strips, Merck, Germany) was located. The color change on the reaction zone of the indicator test strip provides a clear visual indication of the oxygen levels (< 1%) in the environment, making it easy to justify hypoxia conditioning. For exchanging gas inside the 6-well plate, two plastic spacers were placed in parallel crossing the top of any three wells. Then, the assembled cell culture plate was put into a vacuum bag. Finally, the bag containing the cell culture plate was evacuated and sealed by a plastic sealing machine. This package was inserted in a standard incubator for different times (3, 6, 12, and 24 h). After hypoxic preconditioning, WJ-MSCs were trypsinized and immediately used to form spheroids at a density of 1 × 106 cells per 25 cm2 flask coated by 1% low melt agarose for 24 h [42]. For determining cell viability under hypoxia conditioning, the spheroids were subjected to the acridine orange (AO) / propidium iodide (PI) double staining assay [43]. Briefly, the cell spheroids, formed by the hanging drop method (7500 cells/drop) under normoxia and hypoxia conditions for 24 h, were washed with PBS, and then 10 µl fresh AO/PI stain solution (10 µg/ml AO, 50 µg/ml PI) were added. After 15 min, the spheroid was washed with PBS and examined under fluorescence microscopy (FV1000 viewer Olympus, Japan).

Exosome isolation and characterization

After 24 h following spheroid formation, the medium was replaced by a serum-free culture medium to exclude FBS supplementation. After incubation of spheroids in serum-free medium for 48 h, the supernatant was collected by centrifuging at 3,000 rpm for 10 min by Hettich Centrifuge (Model Rotofix 32, Germany), and exosomes were separated using Exocib exosome isolation kit (Cibbiotech, Tehran, Iran) according to the manufacturer instruction. Briefly, the cell culture supernatant was centrifuged at 300 g for 10 min to remove residual debris. Subsequently, it was filtered via a 0.22 μm sterile syringe filter (CA syringe filter, single pack, USA) and Exocib reagent A was then added to the supernatant. After 5 min vortex and overnight incubation at 4 °C, the mixture was centrifuged at 3,000 g for 40 min at 4 °C. Finally, the separated exosome pellets were resuspended in Exocib reagent B and stored at −70 °C before use.

The total protein level of lysed exosome (0.5% triton x-100) was analyzed by the BCA assay kit (Kiazist, Iran). The average exosome yield was 1790 µg/ml from 50 ml of culture supernatant. Also, the isolated exosomes were subjected to morphological assessment using field emission scanning electron microscopy (FESEM; QUANTA FEG-450, FEI, USA). To prepare a sample for FESEM imaging, 20 µl of exosome suspension was air-dried at room temperature and subsequently coated with a thin layer of gold. Then, it was examined using an accelerating voltage of 20 keV. Additionally, isolated exosome size distribution was determined by a dynamic light scattering (DLS) nanoparticle analyzer (Horiba, sz-100, Japan). For sample preparation, 50 µl of sample suspension was diluted to 1 ml phosphate buffer (pH 7.4) before analysis. The CD markers of exosomes such as CD9, CD63, and CD81 protein percent were detected by flow cytometry assay with a FACSCalibur flow cytometer (Becton Dickinson, USA).

In-vitro hypoxia-reoxygenation (H/R) treatment

HEK-293 cells were plated in a 6-well plate at a density of 3.5 × 105 cells/well in a high glucose DMEM medium containing 10% fetal bovine serum. After 70% confluence was reached, an oxygen-glucose deprivation (OGD) experiment was conducted to simulate ischemia in vitro. Briefly, the medium was replaced by low glucose DMEM, and the cells were incubated in a hypoxic condition for 12 h. Then, the vacuum bag was opened, and the cells were transferred to normal conditions during reoxygenation for 24 h. During reoxygenation, the cells were treated with different preconditioned WJ-MSC-derived exosomes (WJ-MSC-EXmiR−210) (50, 100, and 150 µg/ml). Cell viability was determined by trypan blue staining. The cells cultured in various hypoxic times were suspended and stained with 0.4% trypan blue solution. Then, the number of living and dead cells was counted by hemocytometry.

In vitro cellular uptake of coumarin-6 labeled exosomes (Ex-C6)

To evaluate how well the exosomes were taken up by cells, a process involving fluorescent labeling was conducted using coumarin-6, a lipophilic fluorescent dye [44, 45]. Briefly, equal volumes of exosomes dispersed in phosphate-buffered saline (PBS) and ethanolic coumarin-6 solution at a concentration of 250 µg/ml were mixed and incubated at 37 °C for 30 min. Both the coumarin-6-labeled exosomes (Ex-C6) and a solution of free ethanolic coumarin-6 were then incubated with IRI model HEK-293 cells in a 6-well plate, with a total of 2 × 105 cells per well for 4 h. Following the incubation period, the cells were washed with PBS and then fixed with a 4% paraformaldehyde solution. After another rinse with PBS, the cells were stained with DAPI, a fluorescent nuclear stain. Imaging of the cells was then performed using fluorescence microscope (FV1000 viewer Olympus, Japan). Finally, the images obtained were analyzed using Image J software.

RNA extraction and quantitative real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from exosomes and cell specimens using TRIzol reagent (Invitrogen, Paisley, UK). Briefly, 3 µg of total RNA was converted to cDNA according to the manufacturer’s instructions (Royan stem cell, Tehran, Iran). The Real-time PCR was conducted using 1X SYBR® Green PCR Master Mix (Applied Biosystems, California, USA). Finally, the expression of assessed genes was normalized relative to U6 (Seq (5 − 3) AAGGATGACACGCAAAT) as a reference gene for miR-210 (Seq (5 − 3) CCTGTGCGTGTGACAG), and quantification of gene expression was analyzed via ABI Step One System (Applied Biosciences, Foster City, CA). The superscript method was used to calculate the relative expression.

For assessing miR-210 expression in HEK 293 cells, RNA was extracted from 106 cells treated with exosomes isolated from normoxic or hypoxic WJ-MSCs and compared to untreated control cells. Subsequently, cDNA was synthesized, and RT-PCR was conducted as previously explained.

MTT cell viability assay

Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Briefly, OGD-conditioned HEK cells (104 cells/well) were seeded in a 96-well plate and treated with different concentrations of WJ-MSC-EXmiR−210 for 24, 48, and 72 h. After treatment of hypoxia and subsequent normoxia renal cells by WJ-MSC-EXmiR−210 for 24 h, the MTT solution (0.5 mg/ml, 20 µl) was added, and cells were incubated for 4 h at 37 °C. Subsequently, 100 µl DMSO was added to each well to solubilize the formazan reaction product with gentle shaking for 5 min. The optical density (OD) was read at 570 nm on a microplate reader (BioTek, USA). Experiments were conducted in triplicate and survival of the untreated control group was defined as 100%, and that of the H/R group was expressed as the percentage of the control group.

Scratch test

The HEK293 cells were seeded at 3.5 × 105 cells/well into 6-well plates, and when they reached a confluence of 80%, a hypoxia condition was applied. Subsequently, a scratch line was made across each well using a sterile plastic of 100 µl micropipette tip. After washing cells twice with PBS, serum-free media containing different concentrations of WJ-MSC-EXmiR−210 was added to wells. Images for each scratch were taken at 0, 6, 12, 24, 48, and 72 h. Cell migration was measured by calculating the percent change in wound area over time compared to the wound area at zero time by Image J software (open source).

Intracellular ROS assay

Cellular reactive oxygen species (ROS) levels were quantified using 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma). This dye, a stable nonpolar compound, easily permeates cells, converting to DCFH. In the presence of peroxidase, intracellular ROS transforms DCFH into the highly fluorescent DCF. The resulting fluorescence intensity is thus indicative of cellular ROS production. For this assay, H/R renal cells were treated with different exosome concentrations. After 24 h, cells were incubated with 10 mM H2DCFDA for 30 min at 37 °C. Then, cells were washed twice with PBS and immediately evaluated using a flow cytometer in the emission wavelength of 538 nm (BD Biosciences, USA).

Apoptosis assay

Cell apoptosis was assessed using the Annexin V-FITC/PI apoptosis detection kit (Thermo Fisher Scientific). Briefly, the hypoxia/reoxygenation-conditioned HEK-293 cells were treated with three concentrations of WJ-MSC-EXmiR−210 (50,100, and 150 µg/ml) for 24 h. Then, the treated cells, as well as untreated hypoxia/reoxygenation-conditioned and normoxic control cells were trypsinized (Bioidea, Tehran, Iran) and collected. After washing with PBS, each tube of cells was suspended in 200 µl of binding buffer containing 5 µl of Annexin V-FITC, and 5 µl of PI. Subsequently, tubes were incubated in the dark for 20 min. Finally, stained cells were analyzed using Flow Cytometry (BD Biosciences, USA).

Statistical analysis

All data are expressed as mean ± SD from a minimum of three independent experiments. Statistical analyses were conducted using GraphPad Software 7.0. Student’s t-test was employed for comparisons between two groups, while one-way or two-way ANOVA with Tukey’s test was used for multiple comparisons. A significance level of P < 0.05 was considered statistically significant.