Introduction

Acute kidney injury (AKI) is a significant clinical problem mainly caused by renal ischemia resulting in impaired oxygen supply to the kidneys [1]. Ischemia followed by reperfusion causes further renal damage due to cytokine release and generation of free radicals and reactive oxygen species (ROS) [2]. Even though reperfusion is needed to restitute oxygen and nutrient requirements of kidney cells, it can lead to systematic inflammatory responses and tissue damage known as ischemia-reperfusion injury (IRI) [3, 4].

Recently, much research has been done on the therapeutic effect of mesenchymal stem cells (MSCs) and their secretome for IRI treatment. MSCs can reduce or restrict ischemic tissue injury via directly homing to injured tissue and regeneration of damaged cells or indirectly by paracrine/endocrine factors secretion [5]. However, systematic administration of MSCs causes localization mainly in other tissues such as the lungs [6, 7]. Also, local injection of MSCs exhibits poor delivery to ischemic tissues, attenuating their therapeutic effects [8]. On the other hand, MSC secretome or extracellular vehicles (EVs) such as exosomes are increasingly being explored in various experimental conditions, such as neurodegenerative disorders [9], bone and cartilage injuries [10], cardiovascular [11], and kidney diseases [12], wound healing [13], and tissue or organ fibrosis [14]. The sorting and packaging of cargo into exosomes are tightly regulated processes that involve various cellular pathways, including the endosomal sorting complex required for transport (ESCRT) machinery and other ESCRT-independent mechanisms [15]. Exosomes play a crucial role as mediators of cell-to-cell communication [16], due to specific membrane molecules that facilitate their interaction with cells, leading to cargo delivery through endocytosis or direct fusion with the cell membrane [17]. MSC-derived exosomes have gained significant attention for tissue repair and regenerative medicine. They are less likely to trigger an immune response compared to whole MSCs. This reduces the risk of rejection or adverse immune reaction when used for therapy [18, 19]. So, MSC-derived exosomes can home in on the injured tissue due to their small size and molecular cargo. This enables targeted delivery of therapeutic molecules to the site of IRI, potentially improving the therapeutic effect [20, 21]. Additionally, MSC-derived exosomes have anti-inflammatory and immunomodulatory properties, which can help suppress excessive inflammation associated with IRI and promote tissue healing [22]. They contain a diverse range of bioactive molecules, including growth factors, cytokines, regulatory molecules, and microRNAs [23]. These molecules can modulate various cellular processes, such as inflammation, apoptosis, and tissue repair, contributing to the mitigation of IRI. Ultimately, unlike whole MSCs, exosomes have a lower potential for tumorigenicity which is an important consideration in cell-based therapies [24].

While MSC-derived exosome exhibits therapeutic potential for various diseases, their low yield hinders their practical therapeutic use. The exosome yield is influenced by the culture condition and purification process [25]. Several reports have demonstrated that a three-dimensional (3D) culture yields a higher quantity of exosomes than the conventional two-dimensional (2D) system [26]. Additionally, when compared to exosomes generated through 2D culture, those originating from 3D culture exhibit enhanced therapeutic effects through the transfer of specific cargoes [27]. In addition, much research has shown that exosomes obtained from the culture media in which MSCs have been grown under hypoxic conditions carry molecular cargos released by the MSC in response to the environment with potential therapeutic effects in IRI [28]. Among cargos, microRNAs (miRNAs or miRs) are a member of noncoding RNA [29, 30]. These molecules are known as the transcriptional regulators of many biological processes in cell fate, such as stress response, proliferation, and death [31]. miR-210 is a major hypoxemia induced through hypoxia conditions in different cells and is illustrated as a hypoxia genetic signature [32]. Several research has shown that miR-210 has angiogenic [33,34,35] and anti-apoptotic [36, 37] effects in ischemia conditions. Therefore, by combining hypoxic preconditioning of MSC to enhance miR-210 expression and 3D culture to boost the yield of exosome secretion, we hypothesized the exosome derived from a hypoxic condition in the 3D culture of Wharton’s jelly MSCs (WJ-MSCs) would mitigate ischemic injury. To this aim, we isolated exosomes from WJ- MSCs subjected to hypoxic preconditioning and spheroid 3D system. We then assessed their capacity to load up miR-210 within HEK-293 cells and to mitigate ROS production, enhancing their survival under hypoxia-reoxygenation conditions.

Materials and methods

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.

Results

WJ-MSC isolation and characterization

WJ-MSCs grew like plastic adherent cells under in vitro culture conditions with a fibroblast-like morphology (Fig. 1a). After differentiation toward osteogenic and adipogenic lineages, the presence of calcium deposition was confirmed with Alizarin Red and lipid vacuoles was revealed by Oil Red O, respectively (Fig. 1b). According to the flow cytometry findings, WJ-MSCs expressed stromal markers (CD44 and CD90), but not hematopoietic markers (CD45 and CD34) (Fig. 1c). These findings are consistent with the literature [46]. In reality, the flow cytometry results showed that recovered cells were negative for CD45 (0.5%) and CD34 (0.24%) and positive for CD44 (91%) and CD90 (89.1%).

Fig. 1
figure 1

Characterization of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs). (a) The morphology of WJ-MSCs, (b) differentiation of WJ-MSCs to osteocytes and adipocytes, as confirmed by Alizarin Red and Oil Red O, respectively, and (c) the flow cytometry results for CD45, CD34, CD44, and CD90

Full size image

Three-dimensional (3D) WJ-MSC culture under hypoxia conditioning

Two-dimensional (2D) WJ-MSCs were cultured in a standard condition as monolayers for passaging up to P5 (Fig. 2a). Figure 2b shows WJ-MSC spheroids with approximately 1 × 106 cells formed via forced aggregation in a non-adherent flask coated by low melting agarose 1% in 24 h. To explore the effect of hypoxic preconditioning on cell viability in 3D spheroids, AO and PI double staining were employed for WJ-MSC cultured under normoxic and hypoxic conditions (Fig. 2c). AO permeates live and dead cells, producing green fluorescence upon binding to nucleated cells. Conversely, PI only enters cells with injured membranes, staining dead cells and emitting red fluorescence. When both AO and PI are applied, the fluorescence of AO is quenched by PI through fluorescence resonance energy transfer. Consequently, live nucleated cells exhibit green fluorescence, while dead nucleated cells exhibit red fluorescence. [47] The spheroid images were analyzed using the Image J software. Figure 2d shows the hypoxic condition could increase cell viability in 3D spheroids (88% vs. 62% for hypoxic precondition and normoxic, respectively).

In addition, we compared exosome secretion from 5 × 105 WJ-MSCs cultured in a conventional monolayer (2D) with that from cells cultured in 3D spheroids. After 24 h of cell seeding and spheroid formation, the medium was replaced by a serum-free medium, and the secreted exosomes were harvested after 48 h. As a preliminary test, the BCA assay was performed to determine the total exosome protein. As shown in Fig. 2e, the yield of exosomes isolated from spheroids was significantly higher than 2D culture (665 vs. 230 μg/ml) (P<0.05).

Fig. 2
figure 2

3D culture increases cell viability and exosome secretion (a) conventional 2D WJ-MSC. (b) WJ-MSC 3D culture and spheroid formation (c) AO and PI staining of cell spheroids (7500 cells per spheroid) (d) viability of spheroids according to double staining of spheroids by AO and PI and using image J software. (e) BCA assay of exosome proteins isolated from 5 × 105 WJ-MSCs seeded as 2D and 3D culture. (**P<0.01, ****P<0.0001)

Full size image

Exosome characterization

WJ-MSCs were seeded under normoxia and hypoxia conditions, and exosomes were isolated from serum-free media after 48 h incubation. Exosome markers, including CD9, CD63, and CD81 were verified by flow cytometry, confirming exosome identity. In addition, characteristic analysis illustrated that the average size of WJ-MSC-derived exosomes was 57.6 ± 2.3 nm, which was in line with the reported range of exosome size [48]. Furthermore, we assessed the exosomes with electron microscopy and then confirmed the size range of less than 100 nm with spherical morphology (Fig. 3).

Fig. 3
figure 3

Characterization of WJ-MSCs derived exosomes (a) The expression of the marker molecules CD9, CD63, and CD81 in the exosomes by flow cytometry. (b) The particle size distribution of exosomes by dynamic light scattering. (c) Morphology of exosomes observed under field emission scanning electron microscopy

Full size image

Induction of miR-210 cargo in hypoxia-preconditioned WJ-MSC-derived exosomes

The composition and functionality of exosomes are contingent upon the cell of origin, implying that intercellular communication via exosomes is a dynamic system adaptable to the conditions of the producing cell. Alterations in oxygen concentration impact several unique features of stem cells, enabling the delivery of biological information through internalization by neighboring or distant cells. Given this premise, to determine the optimal duration of hypoxic preconditioning for WJ-MSCs and its effect on cell viability before spheroid formation in a 2D culture setup using a 6-well plate, the preconditioning of WJ-MSCs at a different time (3, 6, 12, 24, 48, and 72 h) of hypoxia exposure was investigated. As shown in Fig. 4a, hypoxia treatment induced decreased viability of WJ-MSC cells in a time-dependent manner. So, 48 and 72 h hypoxia conditions were ignored because of a significantly reduced (more than 50%) cell viability (P < 0.0001). Subsequently, after spheroid formation, the miR-210 expression, as a biomarker of hypoxic condition, in exosome-derived WJ-MSCs was analyzed by RT-PCR at other times (3, 6, 12 and 24 h). As shown in Fig. 4b, the miR-210 expression significantly increased at 24 h with a 145.5 ± 14.0-fold change compared to the normoxia condition (P < 0.0001).

Therefore, 24 h hypoxia was selected for WJ-MSC preconditioning.

Fig. 4
figure 4

Hypoxia preconditioning effect on cell viability and miR-210 expression in WJ-MSCs derived exosomes: (a) viability of WJ-MSCs 2D cells in different time of hypoxia condition (3,6, 12, 24, 48, and 72 h) from the same cell seeding number (0.8 × 106 WJ-MSCs), (b) changes in miR-210 expression by hypoxia duration in exosome derived WJ-MSCs spheroids

Full size image

In vitro cellular uptake of hypoxia preconditioned WJ-MSC- derived exosomes and overexpression of miR-210 in them

The capacity for uptake and efficient internalization of exosomes by HEK-293 cells was analyzed via exosome labeling with coumarin6 (EX-C6). In accordance Fig. 5a, the green fluorescent panel illustrated that EX-C6 exhibited increased cellular uptake in HEK-293 cell lines, indicating enhanced internalization of exosomes compared to free C-6. The increased cellular uptake of exosomes may be primarily attributed to their effective cellular internalization mechanisms, involving both clathrin-mediated endocytosis and micropinocytosis [49].

Fig. 5
figure 5

Internalize of exosome derived hypoxia treated WJ-MSCs in H/R renal cells; (a) Fluorescent microscopy images in 10X of IRI HEK-293 cells treated with the coumarin-6 loaded exosomes (EX-C6) and free coumarin-6 (C6) solution after 4 h incubation at 37 °C. Green fluorescence signals indicated the presence of coumarin-6, while the blue fluorescence signals corresponded to the DAPI-stained cellular nuclei. (b) miR-210 expression in IRI HEK-293 cells treated by hypoxic and normoxic exosomes vs. untreated one; (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Full size image

H/R kidney cell viability and migration enhanced by hypoxia preconditioned WJ-MSC- derived exosomes (WJ-MSC-EXmiR-210)

The effect of WJ-MSC-EXmiR−210 on the viability of hypoxia-reoxygenation conditioned kidney cells was analyzed by MTT assay. The result showed that the percentage of cell viability and proliferation was increased in the groups treated with different concentrations of exosomes compared with the untreated control group after 24 h hypoxia and subsequent reoxygenation for 24, 48, and 72 h. As shown in Fig. 6a, our data demonstrated that the proliferation rate of conditioned HEK-293 cells has significantly enhanced by the highest concentration of 150 µg/ml WJ-MSC-EXmiR−210 for all post-hypoxia exposure time when compared with the untreated control cells (*P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001).

Cell migration plays a pivotal role in the initial stages of angiogenesis. Cells are required to move or migrate towards the location where new blood vessel formation occurs [50]. The scratch test was carried out to explore the potential effect of preconditioned WJ-MSCs-EXmiR−210 on cell migration. As illustrated in Fig. 6b & c, apparent improvement in HEK-293 migration percentage was observed following the treatment with WJ-MSC-EXmiR−210 for 72 h at 50 µg/ml (60.88% ± 1.03), 100 µg/ml (76.17%± 0.95), and 150 µg/ml (82.00% ± 0.09) concentrations compared with the untreated cells (49.02%±1.93) (P < 0.05).

Fig. 6
figure 6

WJ-MSC- EXmiR−210 alters migration and viability of renal cells: (a) Renal cell viability results via MTT test in 24, 48, and 72 h after treatment by various amounts of WJ-MSCs-EXmiR−210. (b) Increased migration of HEK-293 cells through treatment by different concentrations of WJ-MSC- EXmiR−210 (50, 100, and 150 µg/ml) at various times (0,6,24,48 and 72 h); (c) The migration rate of cells was assessed based on the distance of the selected scratch area at each time intervals of 0,6,24,48 and 72 h, and the percentage of scratch closure was determined for each time point by Image J software (n = 3, mean ± SD)

Full size image

ROS scavenging effect of WJ-MSC-EXmir-210 on the injured kidney cells

The content of ROS was measured using DCFH-DA through flow cytometry. As shown in Fig. 7, the HEK-293 cells subjected to hypoxia condition exhibited an increase in DCF fluorescence, revealing excessive ROS content (89.10% ± 0.25) compared to normal cells (6.06% ± 0.50); however, treatment by WJ-MSC-EXmiR−210 decreased the DCF fluorescence intensity and ROS percentage for 50, 100 and 150 µg/ml in a concentration-dependent manner (18.30% ± 0.25, 9.84% ± 0.37, and 6.64% ± 0.70, respectively) (P < 0.05).

Fig. 7
figure 7

In vitro reactive oxygen species (ROS) assay via DCFH-DA flow cytometry; (a) in HEK-293 cells; untreated normoxia condition (control), untreated hypoxia condition (model), hypoxia conditioned HEK-293 cells treated by different concentrations of WJ-MSC-EXmiR−210 (50,100 and 150 µg/ml, respectively). (b) the percent of DCF content in renal cells

Full size image

Anti-apoptotic and viability-enhancing effects of WJ-MSC-EXmiR-210 on H/R kidney cells

As shown in Fig. 8, compared with the normoxic control group, the apoptosis rate increased after 24 hypoxia conditions (16.90% ± 0.36 vs. 1.57% ± 0.03). Interestingly, WJ-MSCs-EXmiR−210 decreased early apoptosis in a concentration-dependent manner so that the apoptosis percentage decreased from 37.4% ± 0.36 for untreated H/R cells to 28.4% ± 0.2, 25.2% ± 0.5, and 22.7% ± 0.1 (P < 0.05) for the treated cells with 50 µg/ml, 100 µg/ml and 150 µg/ml WJ-MSC-EXmiR−210, respectively. In addition, treatment by WJ-MSCs-EXmiR−210 markedly enhanced the viability percentage in the H/R renal cells (Fig. 8), compared to untreated H/R cells (58.10% ± 0.95%), in a concentration-dependent manner (66.50% ± 0.70, 69.30% ± 0.75, 76.40% ± 1.00% for 50,100, and 150 µg/ml WJ-MSCs-EXmiR−210, respectively) (P < 0.05).

Fig. 8
figure 8

Apoptosis and cell viability assays: (a) Annexin V (FL1-H) and propidium iodide (FL3-H) staining were employed to identify distinct H/R HEK-293 cell populations. Within each panel, the lower left quadrant, characterized by FITC-Annexin V negativity and PI negativity, represents viable cells. The lower right quadrant, marked by FITC-Annexin positivity and PI negativity, indicates cells in the early stages of apoptosis. Conversely, the upper left and upper right quadrants, which are stained with PI (PI positive), signify dead cells; (b) the percentage of cell viability and apoptosis for different WJ-MSC-EXmiR−210 concentrations (50,100, and 150 µg/ml) following 24 h treatment. (****P < 0.0001)

Full size image

Discussion

In recent decades, the importance of MSCs-based cell therapies for IRI lies in their multifaceted therapeutic effects, which target various aspects of the injury and promote tissue repair and regeneration [51, 52]. Also, experimental and clinical data suggest that MSC-derived exosomes could serve as innovative cell-free therapeutic agents, offering distinct advantages over MSCs [53]. The low yield of exosome derived MSCs, poses a challenge for scaling up the manufacturing of cell-free therapies. Therefore, researchers have explored different methods to enhance the production of them, such as refining MSC growth conditions, utilizing three-dimensional culture of MSCs, and controlling the process of exosome biogenesis [25]. So, we combined advantages of hypoxic MSCs preconditioning and 3D culture to improve yield and function of WJ-MSCs derived exosomes in IRI. Reality, hypoxic preconditioning of MSCs has been shown to improve the secretion [54] and the therapeutic efficacy of MSC-derived exosomes for tissue regeneration [55, 56] and various IRI diseases, including conditions characterized by ischemia and inadequate vessel growth in different tissues [57,58,59].

Accordingly, we used a comfortable and straightforward hypoxia preconditioning of the cells method, for the first time, to introduce miR-210 cargo in the exosomes derived from WJ-MSCs. In addition, hypoxia treating of WJ-MSCs prepare them to encounter low oxygen and nutrient-limited condition in 3D culture [60] that increased cell viability in spheroids (Fig. 2c & d). Also, as shown in Fig. 2e and a, 3D culture of WJ-MSCs improved the yield of exosome secretion by about 3-fold compared to a 2D culture. Consistent with our reports, it is shown by Cao et al. that revealed exosomes derived from MSCs in a 3D culture setting yielded greater quantities. These exosomes exhibited high anti-inflammatory properties, offering protection against cisplatin-induced kidney injury [61].

Apart from the exosome yield, our study provides confirmation of the enhanced effects of hypoxia on the action of WJ-MSC secreted exosomes through overexpression of miR-210 cargo (Fig. 4b). miR-210 is recognized as the main mediator of the cellular response to hypoxic stress. A diverse range of targets regulated by miR-210 have been implicated in mitochondrial metabolism [62], oxidative stress responses [63], cell proliferation [64], and regulating cell death or apoptosis [65]. These regulatory effects have broad implications across various pathological conditions, including cancer and IRI. Remarkably, in a mouse model of renal I/R injury, miR-210 was found to be upregulated in kidney tissue [66]. Therefore, we demonstrated that exosomes with miR-210 overexpression (WJ-MSC-EXmiR−210) have a protective role in renal cells, mitigating injury and dysfunction induced by hypoxia/reoxygenation (H/R). Our findings in Fig. 5b indicated that WJ-MSC-EXmiR−210 incorporated by HEK cells and notably boost their proliferation and migration capabilities (Fig. 6b & c). Both cell migration and proliferation, which play critical roles in alleviating renal IRI and facilitating tissue repair [50], that enhanced by exosomes derived from hypoxia preconditioned MSCs [67,68,69,70]. Also, in other literatures, the elevated expression of miR-210 notably increased VEGF levels and promoted the proliferation and migration of different cells [71,72,73,74]. Moreover, in vivo studies indicated improved tissue function, and these beneficial effects were correlated with alterations in the expression of miR-210 target genes, specifically PI3K/Akt and p53 [75]. The protein p53, which directly binds to the miR-210 promoter, governs critical cellular processes like apoptosis, and it also mitigates inflammation in diverse human tissues in response to stress conditions [76]. In present work, we revealed that treatment with WJ-MSC-EXmiR−210 enhanced survival and reduced apoptosis of HEK-293 cell under H/R conditions in vitro (Figs. 6a and 8). These findings show that increased concentration of WJ-MSC-EXmiR−210, lead to elevating miR-210 expression levels in renal cells, improving he viability of injured renal cells and the incidence of apoptosis is reduced. In accordance with our results about anti-apoptotic role of miR-210, Liu et al. proved the overexpression of miR-210 resulted in a notable inhibition of the HIF-1α pathway and reduced apoptosis induced by hypoxia kidney lesions. Conversely, knockdown of miR-210 had the opposite effect, exacerbating apoptosis [77]. Zaccagnini and colleagues documented that miR-210 played a critical role in the adaptive mechanism for regulating oxidative metabolism and oxidative stress in acute peripheral ischemia [78]. Also, it is documented that miR-210 diminishes apoptosis by impeding the caspase-8 pathway in H2O2-stimulated human umbilical vein endothelial cells [79]. Our flow cytometry apoptosis/necrosis in Fig. 8 results in accordance with these investigations confirm that miR-210 overexpressed in preconditioned WJ-MSC derived exosomes exert a protective function in H/R renal cells in vitro.

Oxidative stress, recognized as a predominant pathological mechanism in IRI, denotes a disparity between the regular oxidant defense systems, encompassing enzymes like superoxide dismutase, catalase, and glutathione, and the generation of intracellular ROS. This imbalance culminates in accumulating detrimental reactive oxygen intermediates, including hydrogen peroxide (H2O2) [80]. In this work, according to the flow cytometry ROS assessment (Fig. 7), the WJ-MSCs-EXmiR−210 remarkably diminishes the ROS content in H/R kidney cells. Under typical physiological circumstances, ROS arise from routine cellular metabolic activities and are counterbalanced by antioxidants, maintaining a homeostatic equilibrium [81]. However, ROS generation is enhanced during the ischemic phase and subsequent reperfusion, exacerbating oxidative stress and cellular damage [82]. Different findings suggest that MSCs-derived exosomes may exert their effects by enhancing antioxidant mechanisms and suppressing oxidative stress within injured renal tissues. For instance, Bo Liu and colleagues propose the potential therapeutic use of human umbilical cord mesenchymal stem cells in CKD. The mechanism of action was linked to the suppression of the ROS-mediated p38MAPK/ERK signaling pathway [83]. In another study, Cao et al. demonstrated that coculturing with MSC-derived extracellular vesicles led to a reduction in ROS levels in renal proximal tubular epithelial cells. This antioxidant effect was mediated by the activation of the Keap1-Nrf2 signaling pathway [84]. Also, Mutharasan et al. found that upregulation of miR-210 decreased ROS production and cardiomyocytes death in reaction to oxidative stress, whereas the downregulation of miR-210 resulted in elevated ROS levels following hypoxia-reoxygenation [85].

Additionally, ROS signaling is pivotal in the initiation and progression of inflammatory injuries [86]. MSC-EX has demonstrated the capability to mitigate inflammatory injuries triggered by oxidative stress within tissues. Notably, in literatures were showed treatment with MSC-EVs attenuated the levels of inflammatory cytokines, indicating a potential therapeutic role in mitigating renal injury [87, 88]. Furthermore, many researches illustrate an immunomodulatory function of miR-210 [89,90,91] that improved immunosuppressive effect of MSCs EX in IRI model. For instance, Xie et al. demonstrated that mesenchymal stem cell-derived exosomes may improve kidney injury by facilitating the transition of M1 macrophages to M2 macrophages and exerting anti-inflammatory effects through M2 macrophages. This suggests that the immunomodulatory properties of MSCs-Exo play a role in the self-repair process in mice following ischemia-reperfusion injury [92].

Conclusion

Our study suggests that combined hypoxic preconditioning and 3D spheroid culture of WJ-MSCs enhances cell survival in 3D culture conditions, increasing secretion of exosomes and expression of miR-210 payload. Interestingly, WJ-MSC-EXmiR−210 increased the level of miR-210 in the H/R kidney cells. In addition, our findings revealed that WJ-MSC-EXmiR−210 can boost the protective properties against apoptosis, oxidative stress, and reduced injury induced by the H/R condition. Further research is warranted to determine WJ-MSC-EXmiR−210 impact on renal ischemia-reperfusion injury in vivo.