Exosomes derived from human umbilical cord mesenchymal stem cells protect against cisplatin-induced ovarian granulosa cell stress and apoptosis in vitro

Human umbilical cord mesenchymal stem cells (huMSCs) can treat primary ovarian insufficiency (POI) related to ovarian granulosa cell (OGC) apoptosis caused by cisplatin chemotherapy. Exosomes are a class of membranous vesicles with diameters of 30–200 nm that are constitutively released by eukaryotic cells. Exosomes mediate local cell-to-cell communication by transferring microRNAs and proteins. In the present study, we demonstrated the effects of exosomes derived from huMSCs (huMSC-EXOs) on a cisplatin-induced OGC model in vitro and discussed the preliminary mechanisms involved in these effects. We successfully extracted huMSC-EXOs from huMSC culture supernatant and observed the effective uptake of exosomes by cells with fluorescent staining. Using flow cytometry (with annexin-V/PI labelling), we found that huMSC-EXOs increased the number of living cells. Western blotting showed that the expression of Bcl-2 and caspase-3 were upregulated, whilst the expression of Bax, cleaved caspase-3 and cleaved PARP were downregulated to protect OGCs. These results suggest that huMSC-EXOs can be used to prevent and treat chemotherapy-induced OGC apoptosis in vitro. Therefore, this work provides insight and further evidence of stem cell function and indicates that huMSC-EXOs protect OGCs from cisplatin-induced injury in vitro.


Typical characteristics of huMSC-EXOs.
To further obtain huMSC-EXOs, we used gradient ultracentrifugation to extract exosomes from the culture medium. Exosomes precipitated in the bottom of the tube and were light yellow in colour (Fig. 2d). The cellular lipid bilayer retracts to form multi-chambered vesicles, which results in the release of nanoscale vesicles (exosomes) in a calcium-dependent manner that bind to cell membranes. The vesicle-like morphology of exosomes was visualized via TEM, which confirmed exosome diameters of 30 to 200 nm (Fig. 2a,b and c). Fig. 2b was the simulation diagram. Western blotting analysis indicated that huMSC-EXOs expressed exosomal markers, such as CD63, CD9, Hsp70 and CD81 proteins, but did not express the endoplasmic reticulum marker calnexin or the lysosome marker Lamp 1, which showed that huMSC-EXOs isolated by the processes described above did not contain the components or pieces of the endoplasmic reticulum or lysosomes (Fig. 2e). Hence, huMSC-EXOs expressed the typical markers of exosomes and were used in the following experiments; n = 5.

Characteristics of OGCs and a cisplatin-induced cell model. The cells were adherent and grew well
after 48 h of inoculation, exhibiting polygonal and fibre-like structures ( Fig. 3a and b). After follicle-stimulating hormone receptor (FSHR) immunostaining, OGCs were dyed brown with DAB, which accounted for approximately 70-80% of the adherent cells. The brown cells stained with DAB were the OGCs, indicating that OGCs derived from rats were successfully cultured in vitro; n = 5 ( Fig. 3c and d).
Cisplatin was used in the cell model. Based on our pre-tests, 4 µg/ml was determined to be the optimal experimental concentration.
Effective uptake of huMSC-EXOs by OGCs. Using fluorescence microscopy, the protein component of huMSC-EXOs, which was labelled with the fluorescent reagent Exo-Green, could be observed by monitoring the green fluorescence, and the green fluorescence gathered in the interior of the cells. Similarly, microRNAs in huMSC-EXOs labelled with the fluorescent reagent Exo-Red could be seen as red fluorescence. The cells could effectively combine with huMSC-EXOs dyed with Exo-Green and Exo-Red (Fig. 4a).
Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to label huMSC-EXOs to quantitatively determine the uptake ratio. The detection indicator was the percentage of cells with bound CSFE-labelled huMSC-EXOs, which reflected the uptake ratio of huMSC-EXOs. In the cisplatin-negative group, the percentages of the huMSC-EXO-labelled cells analysed by guavaSoft 3.1.1 were 84.93 ± 5.23%, 85.19 ± 5.37%, 98.06 ± 1.48% and 97.67 ± 1.51% at 6 h, 12 h, 18 h and 24 h, respectively, whilst the percentages were 79.56 ± 7.00%, 89.83 ± 4.73%, 97.81 ± 2.49% and 97.68 ± 1.89%, respectively, in the cisplatin-positive group. The results showed that huMSC-EXOs were effectively taken up by OGCs, and the uptake ratio was not different when 4 µg/ml of cisplatin was used in the experiments (P < 0.05); n = 3 ( Fig. 5a and b).   All of the above-mentioned observations verified that huMSC-EXOs can effectively bind OGCs, and the uptake ratio was not different when 4 µg/ml cisplatin was used in the experiments, which produced a foundation of biological behaviours for the following experiments.

HuMSC-EXOs protect OGCs from cisplatin-induced injury and promote resistance to cell apoptosis in vitro.
OGCs cultured in six-well plates were divided into 3 groups: group A (control group), group B (cisplatin injury group) and group C (huMSC-EXO coculture group). After 48 h, the cells in groups A, B and C were observed under a microscope, and the apoptosis in group B was found to be higher than that in group C (Fig. 6a). As determined by annexin-V-FITC/PI staining and FACS analysis, the percentages of living cells, early apoptotic cells and late apoptotic cells in group A were 85.50 ± 4.45%, 5.86 ± 0.50% and 8.69 ± 2.15%, respectively. The percentages in group B were 71.37 ± 3.10%, 8.58 ± 2.04% and 17.26 ± 2.67%, respectively, and the percentages in group C were 80.09 ± 4.00%, 5.72 ± 2.15% and 10.27 ± 1.46%, respectively. The proportion of living cells between groups A and B was significantly different (P < 0.05). And the percentages of living cells in group C when compared with group B was also different (P < 0.05). No significant difference (P > 0.05) in the percentage of early apoptotic cells between groups A and B was observed, whilst group B and group C were different (P < 0.05). For the percentage of late apoptotic cells, group A was different from group B (P < 0.05), as were groups B and C (P < 0.05); n = 5 ( Fig. 6b and c).
Western blotting was used to detect changes in the expression of apoptosis-related proteins and DNA repair proteins, and the expression of Bax, cleaved caspase-3, Bcl-2 and cleaved PARP was found to be significantly different (P < 0.05) between groups A and B and groups B and C. The expression of Bax, cleaved caspase-3 and cleaved PARP in group B was increased compared with group A, whilst that of Bcl-2 was decreased. However, the expression of Bax, cleaved caspase-3 and cleaved PARP in group C was reduced compared with group B, and Bcl-2 expression was increased; n = 5 (Fig. 7).
Based on the regulation of apoptosis-related proteins, huMSC-EXOs had a robust protective effect on the cisplatin-induced damage of OGCs. Precise regulation of apoptosis was achieved by exosomes, perhaps via specific microRNAs that modified certain genes or proteins. However, cell apoptosis is a complex multi-pathway process, and thus, the effect of the exosomes on apoptosis may be not as dramatically represented in the results of the annexin-V/PI staining and FACS analysis as in the western blotting results.
Predicted target genes of microRNAs determined with a PCR array. The results indicated that microRNAs with high abundance existed in huMSC-EXOs, and some of the predicted target genes were listed to provide further evidences that these micro-RNAs had a relationship with OGC apoptosis and could participate in regulation of the apoptotic process (Table 1). U6 was considered the internal reference in the PCR assay. If the ∆Ct value was lower, the expression level of microRNAs in the exosomes was higher. We used databases (mirBase and TargetScan) to predict and analyse the potential target genes of the microRNAs with high abundance in Table 1, which we expect will be helpful in our future studies. The results predicted that microRNA-24, microRNA-106a, microRNA-19b and microRNA-25 may be closely related to apoptosis; n = 3.

Discussion
Studies have previously demonstrated that huMSCs can be used to treat POI and protect OGCs from damage by cisplatin 15 , but the exact mechanisms responsible for this protection are unclear. Therefore, we considered whether the effects of huMSCs on OGCs occurred via a paracrine secretion mechanism. In this study, we determined whether exosomes derived from huMSCs had the same therapeutic or protective effects on cisplatin-induced OGCs damage and explored the preliminary mechanisms of these effects. We demonstrated Figure 5. Quantitative uptake ratio of huMSC-EXOs and the effect of cisplatin. (a,b) In the cisplatin-negative group, the percentage of huMSC-EXO-labelled cells, analysed with guavaSoft 3.1.1, was 84.93 ± 5.23%, 85.19 ± 5.37%, 98.06 ± 1.48% and 97.67 ± 1.51% at 6 h, 12 h, 18 h and 24 h, respectively, whilst the percentage was 79.56 ± 7.00%, 89.83 ± 4.73%, 97.81 ± 2.49% and 97.68 ± 1.89%, respectively, in the cisplatin-positive group. The results showed that huMSC-EXOs were effectively taken up by OGCs, and the uptake ratio was not different in the presence of 4 µg/ml cisplatin (P < 0.05); n = 3.
that huMSC-EXOs ameliorated cisplatin-induced OGC stress and apoptosis in vitro, which provides a cytological basis for subsequent experiments in vivo.
The cellular lipid bilayer retracts to form multi-chambered vesicles, which release nanoscale vesicles (exosomes) in a calcium-dependent manner that bind to cell membranes. Exosomes are 30 to 200 nm in size, spherical in shape, and mediate local cell-to-cell communication by transferring mRNA, lncRNA, microRNA, Figure 6. HuMSC-EXOs protect against cisplatin-induced injury of OGCs and promote resistance to cell apoptosis in vitro based on FACS. (a) Groups A, B and C were cultured for 48 h, and the number of apoptotic cells in group B was higher than that in group C under the microscope (a1-a3: ×40 magnification; a4-a6: ×400 magnification). (b,c) Through annexin-V-FITC/PI double staining and FACS analysis, the proportion of living cells between groups A and B was found to be different (P < 0.05). Similarly, the proportion of living cells in group B compared with group C was also different (P < 0.05). No significant difference (P > 0.05) in the percentage of early apoptotic cells between groups A and B was observed, whilst in groups B and C, a difference was observed (P < 0.05). For the percentage of late apoptotic cells, a difference was observed between group A and group B (P < 0.05), along with groups B and C (P < 0.05); n = 5. *Group B vs. group A. # Group C vs. group B.
proteins and lipids 17 . It has been reported that CD9, CD63, Hsp70 and CD81 are frequently located on the surface of exosomes, whilst the endoplasmic reticulum marker calnexin and lysosome marker Lamp 1 are typically not present. Target cells can absorb exosomes in several ways, including membrane fusion, endocytosis and receptor binding 22 . Previous studies have shown that huMSC-EXOs promote tissue injury repair through horizontal transfer of proteins and microRNAs 27,28 . The exosomes obtained from huMSCs in this study had the same characteristics: they exhibited spheroid morphology, and TEM confirmed sizes of 30 to 200 nm. Western blotting analyses showed that the exosomes were positive for CD9, CD63, Hsp70 and CD81 expression and negative for calnexin and Lamp 1. Hence, they were used in subsequent experiments.
In the present study, we successfully cultured huMSCs and rat OGCs, isolated exosomes derived from huM-SCs and confirmed that huMSC-EXOs contain a variety of microRNAs. Moreover, by establishing an injury model in vitro, it was observed that huMSC-EXOs could be incorporated into injured OGCs, thus accelerating the recovery of OGCs from the stress and apoptosis induced by cisplatin in vitro.
First, the effective uptake of exosomes by cells was the basis for the subsequent biological effects, and two different fluorescent labelling methods were used to observe this behaviour via fluorescence microscopy and confocal microscopy. After administration of huMSC-EXOs, the level of OGC apoptosis was limited, and the number of apoptotic cells was reduced compared to the cisplatin group (group B). When cells are subjected to oxidative (b-f) The expression of Bax, cleaved caspase-3 and cleaved PARP in group B was increased compared with that in group A, whilst the expression of Bcl-2 was decreased. However, the expression of Bax, cleaved caspase-3 and cleaved PARP in group C was reduced compared with that in group B, and the expression of Bcl-2 was increased; n = 5. *Group B vs. group A. # Group C vs. group B.
stress, the level of proapoptotic proteins increases to inhibit the activity of antiapoptotic proteins, which can decrease mitochondrial activity and initiate apoptosis. It has been suggested that cisplatin-induced cell damage was associated with a rise in the level of the proapoptotic protein Bax and a reduction in the antiapoptotic protein Bcl-2. In this study, the huMSC-EXO group (group C) exhibited an evident decrease in Bax expression compared to the cisplatin alone treatment group (group B), whilst the Bcl-2 protein level was increased when huMSC-EXOs were present in vitro. In addition, cleaved caspase-3, as the executor of apoptosis, was highly expressed in the cisplatin group, whilst expression was decreased in the huMSC-EXO group. Moreover, PARP is a poly(ADP-ribose) polymerase and a DNA repair-related protein, which can be used as a substrate of caspase-3 for degradation. If the level of cleaved PARP increases, then DNA damage is severe and DNA breaks are complex, resulting in a variety of further cascades that induce apoptosis 29 . In our results, it was found that the huMSC-EXO group had an evident reduction in cleaved PARP compared to the cisplatin group. Although cisplatin has been reported to affect endocytosis of proteins 30 , it was shown through supplementary experiments that huMSC-EXOs can effectively bind to OGCs, and the uptake ratio had no significant relationship with the existence of 4 µg/ml cisplatin in our study.
However, the effects on apoptosis observed in the annexin-V/PI staining and FACS results were not as dramatic as those revealed by the western blotting results. Exosomes carry a variety of microRNAs and proteins into target cells and precisely regulate or modify certain genes or proteins, whilst cell apoptosis is a complex multi-pathway process. The expression of one of the apoptosis-related proteins changed, which indicated that the components of exosomes may regulate apoptosis-related genes or mRNAs, either directly or indirectly, but the apoptotic process also involves many other pathways and regulatory mechanisms, coupled with the repair mechanism of the cells, resulting that the final effect of exosomes on cells would likely not show up as a very significant antiapoptotic effect.
A variety of microRNAs were observed in huMSC-EXOs, and a qRT-PCR array analysis of huMSC-EXOs was conducted. MicroRNAs are a series of small noncoding RNAs (~22 nucleotides long) that regulate the expression of target genes at the post-transcriptional level. During this process, the microRNA/microRNA-induced silencing complex (miRISC) binds the 3′-UTR of target mRNA to inhibit expression via translational repression and/or mRNA degradation 24 . Databases (mirBase and TargetScan) were used to predict and analyse the potential targets of the microRNAs with high abundance in Table 1, which was expected to be helpful for subsequent studies. We predicted that microRNA-24, microRNA-106a, microRNA-19b and microRNA-25 may be closely related to apoptosis. Sang et al. identified microRNAs present in microvesicles and the supernatant of human follicular fluid, and microRNA-24 was found to regulate oestradiol concentrations and progesterone concentrations, which shows that the highly expressed microRNA-24 targets genes associated with reproductive, endocrine, and metabolic processes 31 . MicroRNA-106a is also closely related to ovarian development; some studies have shown that downregulation of the expression of microRNA-106a inhibits cell growth and metastasis of ovarian cancer cells 32 . In addition, ovarian microcirculation density reflects ovarian function, and human amnion epithelial cell  treatment enhances angiogenesis primarily through paracrine pathways in the ovaries 33 . Meanwhile, Tang et al. demonstrated that microRNA-19b plays a key role in attenuation of TNF-α-induced endothelial cell apoptosis and that this function is closely linked to the Apaf1/caspase-dependent pathway, and therefore, it can be speculated that elevated microRNA-19b may be beneficial for restoring ovarian function by increasing the antiapoptotic ability of vascular endothelial cells 34 . Moreover, microRNA-25 has an antiapoptotic role in human gastric adenocarcinoma cells, possibly via inhibition of FBXW7, thus promoting the expression of oncogenes such as CCNE1 and MYC 35 . All of these forward-looking, predictive and instructive results could provide background and ideas for our subsequent studies on microRNAs derived from huMSC-EXOs. Hence, we cultured huMSCs and rat OGCs and successfully isolated exosomes derived from huMSCs. In addition, we observed that huMSC-EXOs could become incorporated into injured OGCs, thus accelerating the recovery of OGCs after stress and apoptosis induced by cisplatin in vitro.
Next, we will focus on in vivo experiments and the protection mechanism of microRNAs contained in exosomes secreted by huMSCs in protection. In this study, the precise mechanism of how huMSC-EXOs protect cisplatin-induced OGC damage is still unclear, but it can be concluded from the results of the present study that huMSC-EXOs can promote resistance to cisplatin-induced OGC apoptosis and protect OGCs from cisplatin-induced injury in vitro.

Methods
The experiments were conducted in accordance with approved guidelines: the animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Institutional Review Board of Qilu Hospital, Shandong University (No. KYLL-2015(KS)-077), and informed consent was obtained from all patients before the study.
To observe morphology, adherent cells were stained with Wright's stain and imaged with a JEOL-1200EX transmission electron microscope, and images were recorded with a MORADA-G2 camera.
To detect typical surface markers of huMSCs, FACS was performed using the following phycoerythrin

Isolation and characterization of exosomes derived from huMSCs. HuMSCs were cultured in
BioWhittaker ultraCULTURE general purpose serum-free medium (Lonza, Basel, Switzerland) containing 2% Ultroser G serum substitute (Pall, Port Washington, NY, USA), referred to hereafter as serum-free medium. The medium was collected after 48 h. The medium was processed by 400 g centrifugation for 15 min and by 10,000 g centrifugation for 15 min at 4 °C. The supernatant was further filtered using a 0.22 µm filter (Millipore) and eventually ultracentrifuged at 100,000 g for 5 h at 4 °C. The exosome pellets were resuspended in PBS and stored at −80 °C for further use. The concentration of exosomal protein was quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

Isolation and characterization of rat OGCs and establishment of a cell model. Healthy female
Wistar rats, weighing 50-60 g, were chosen as experimental animals. Each rat was subcutaneously injected with 40 IU of FSH (Solarbio, Beijing, China). Rats were sacrificed after 48 h. Both ovaries were collected, and the surrounding fat and fascia were removed. Then, ovarian tissues were cut into cubes less than 1 cm 3 , digested with 0.25% trypsin (containing 0.01% EDTA, Life Technologies) at 37 °C for 60 min and shaken once every 10 min. The cell suspension was filtered with a 70 µm mesh filter and centrifuged at 1,000 r/min for 10 min. The collected cells were washed once with PBS and cultured in DMEM/F12 = 1:1 (HyClone) containing 10% FBS and 1% penicillin and streptomycin at 37 °C, 5% CO 2 and 100% H 2 O. Each well was inoculated with 2 × 10 5 cells in the six-well, and we chose OGCs that adhered about 50-60% plate into the subsequent experiments.
The FSHR is a specific marker of OGCs, and thus, OGCs were identified using immunohistochemistry with an FSHR rabbit antimouse polyclonal antibody (Boster, Wuhan, China). OGCs were cultured on glass coverslips. The cells were fixed in cold acetone for 15 min, immersed in 3% H 2 O 2 for 10 min at room temperature and subsequently washed 3 times with PBS. The cells were blocked in goat serum for 20 min at room temperature and then incubated with primary antibodies (dilution ratio = 1:200) overnight at 4 °C. Afterwards, the cells were washed 3 times with PBS and incubated for 70 min at 37 °C with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (dilution ratio = 1:1,000, ZSGB-BIO, Beijing, China) and HRP solution. Finally, the cells were dyed with 3,3′-diaminobenzidine (DAB) and counterstained with haematoxylin (Solarbio). Images were acquired with a microscope; n = 5.

Uptake of huMSC-EXOs by OGCs visualized with fluorescent labelling. Fluorescence micros-
copy. The fluorescent reagent Exo-Green (System Biosciences) was used to label the protein component of huMSC-EXOs, and Exo-Red (System Biosciences) was used to label the microRNAs in huMSC-EXOs. HuMSC-EXOs were labelled with Exo-Green and Exo-Red, separately, for 20 min at 37 °C, and then, the labelled huMSC-EXOs were washed with PBS and re-pelleted twice using ExoQuick-TC exosome precipitation solution (System Biosciences). Exo-Green-labelled huMSC-EXOs (100 µg, 100 µg/ml) were incubated with OGCs in a six-well plate for 2 h at 37 °C, whilst Exo-Red-labelled huMSC-EXOs were incubated for 24 h. Images were acquired with a fluorescence microscope.
Quantitative uptake ratio of huMSC-EXOs and the effect of cisplatin. CFSE (Life Technologies) was used to label huMSC-EXOs. HuMSC-EXOs were suspended in PBS and incubated for 20 min at 37 °C in 5 µM CFSE. The CFSE-labelled huMSC-EXOs were washed with PBS and re-pelleted twice using ExoQuick-TC exosome precipitation solution to remove any free dye remaining in the solution. Afterwards, huMSC-EXOs (100 µg, 100 µg/ml) were resuspended in either cisplatin-negative or cisplatin-positive serum-free medium and cultured with OGCs of each well. The cisplatin concentration was 4 µg/ml. The cells were collected at 6 h, 12 h, 18 h and 24 h and analysed with flow cytometry. The detection indicator was the percentage of cells bound with CSFE-labelled huMSC-EXOs, which reflected the uptake ratio of huMSC-EXOs. The results were analysed by guavaSoft 3.1.1 software; n = 3.

The effect of huMSC-EXOs on cisplatin-damaged OGCs. We chose OGCs that adhered about 50-60%
in the six-well plate into the subsequent experiments. OGCs cultured in six-well plates were divided into 3 groups: group A (blank control group), group B (cisplatin injury group) and group C (huMSC-EXO coculture group). Cisplatin and huMSC-EXOs were added to group C at the same time. The working concentration of cisplatin was 4 µg/ml. HuMSC-EXOs (100 µg, 100 µg/ml) were added to each well, and the plates were cultured for 48 h. The cells were collected for the following analyses.
FACS analysis. Annexin-V and propidium iodide staining (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences) were used to analyse the percentage of apoptotic cells. The experimental process was followed by the manufacturer's instruction. The results were obtained with flow cytometry and analysed with guavaSoft 3.1.1 software; n = 5.

Statistical analysis.
Data are expressed as the means ± SD. Data were analysed using one-way ANOVA or Student's t-test. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). Images were processed using Photoshop CS5 V12.0.1. A value of P < 0.05 was considered statistically significant.