Int J Clin Exp Pathol 2018;11(11): /ISSN: /IJCEP Dan Zhou 1,2, Wei Zhai 3, Miaomiao Zhang 1

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1 Int J Clin Exp Pathol 2018;11(11): /ISSN: /IJCEP Original Article Mesenchymal stem cell-derived extracellular vesicles promote apoptosis in RSC96 Schwann cells through the activation of the ERK pathway Dan Zhou 1,2, Wei Zhai 3, Miaomiao Zhang 1 1 School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, P. R. China; 2 Department of Laboratory Medicine, Jintan Hospital, Jiangsu University, Jintan, Jiangsu, P. R. China; 3 Department of Laboratory Medicine, Laiwu Hospital, Laiwu, Shandong, P. R. China Received September 10, 2018; Accepted September 25, 2018; Epub November 1, 2018; Published November 15, 2018 Abstract: Mesenchymal stem cells (MSCs) are known for their capacity to produce extracellular vesicles (EVs), which are key mediators of information transfer between different cells for tissue repair and regeneration. Schwann cells are the major glial cells of the peripheral nervous system and play a key role in the survival, function, and regeneration of neurons. However, the action of MSC-derived EVs (MSC-EVs) on Schwann cells remains unclear. In the present study, we investigated the effect of rat bone marrow MSC-EVs on RSC96 Schwann cells. EVs derived from Rat bone marrow MSCs were isolated by ultracentrifugation and characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The effects of MSC-EVs on RSC96 cell proliferation, migration, apoptosis, and the protein levels were analyzed using the MTT and Colony-forming assays, the Transwell and wound healing assays, flow cytometry, and western blot, respectively. We found that rat MSCs secreted nm heterogeneous small vesicles, which were defined as EVs. Incubation of RSC96 cells with rat MSC-EVs resulted in the uptake of EVs by the cells. MSC-EV treatment significantly inhibited RSC96 cell proliferation and migration, promoted their apoptosis, and activated the ERK pathway, while ERK signal repression using U0126 exhibited the opposite effects. Our data showed that MSC-EVs inhibited proliferation and migration and promoted apoptosis through the activation of the ERK pathway in RSC96 cells. Thus, the effect of BMSC-EVs on RSC96 cells may affect peripheral nerve injury and repair, as mediated by Schwann cells. Keywords: Mesenchymal stem cell, extracellular vesicles, RSC96 Schwann cells, apoptosis, ERK pathway Introduction Schwann cells (SCs), derived from neural crest cells, are the myelin-forming glial cells of the peripheral nervous system (PNS) [1]. During the past several decades, the complex, multifaceted, and crucial roles of SCs in the PNS have been revealed, including the conduction of nerve impulses along axons, nerve development and regeneration, trophic support for neurons, production of the nerve extracellular matrix, modulation of neuromuscular synaptic activity, and presentation of antigens to T-lymphocytes [2]. Moreover, SCs are the direct and/ or indirect targets of many hereditary and acquired peripheral myelin diseases; therefore, further understanding the biology and pathology of SCs has become important [3]. Mesenchymal stem cells (MSCs) are multipotent cells with self-renewal capacity and reside in various tissues; moreover, they can differentiate different mesodermal lineages, including bone, cartilage, and adipose tissues [4]. MSCs can be recruited to the site of inflammation and tissue injury/repair [5-7]. MSCs can migrate to injured sites and can thus contribute to tissue repair; however, their extent of improvement on injured tissues has not been shown to be correlated with cellular engraftment and differentiation of MSCs to tissue cells, thereby suggesting that they may play an indirect role in tissue regeneration. Growing evidence indicates that the therapeutic effect of MSCs depends primarily on their capacity to secrete soluble factors [8]. Recent

2 studies have found that MSCs secrete extracellular vesicles (EVs), such as microvesicles (MVs) and exosomes. They are small, spherical membrane fragments that are involved in cell-to-cell communication and can alter the cell fate and phenotype of recipient cells; moreover, they are proposed as key mediators of information transfer between different cells for tissue repair and regeneration [9]. EVs derived from MSCs (MSC-EVs) have been shown to mimic the therapeutic effects of MSCs in kidney, cardiac, and brain injuries [10, 11]. MVs from human bone marrow MSCs (BM-MSCs) stimulate the proliferation and apoptosis resistance of tubular epithelial cells in vitro [12, 13]. Microvesicles from MSCs protect ischemia/reperfusion-induced and lethal cisplatin-induced acute kidney injuries [13, 14]. Furthermore, exosomes produced by MSCs significantly reduce infarct size in pig and mouse models of myocardial infarction [15]. However, the therapeutic effects of MSC-EVs on neurological disease and injury are still sparse. Lopez-Verrilli et al. recently reported that dorsal root ganglia neurons and cortical neuron culture cells react differently to treatments with bone narrow (BM)-, umbilical cord blood (UCB)-, chorion (ChoSC)-, and human menstrual fluid (MenSC) MSC-derived exosomes. Only MenSC exosomes have been able to enhance neuritic outgrowth in cortical neuron cultures, whereas ChoSC exosomes decreased the total neuron branch numbers. Moreover, BM- and MenSCderived exosomes increased the rate of neuritic growth in dorsal root ganglia neuron culture compared with control cells [16]. Del Fattore et al. demonstrated that BM- and UCB-derived MVs decreased the proliferation rate of glioblastoma cell lines, whereas MVs released by adipose tissue MSCs (AT-MSCs) had the opposite effect. Furthermore, MVs from BM- and UCB-MSCs induced apoptosis of neoplastic cells, whereas AT-MSC MVs had no effect [17]. However, the actions of MSC-EVs on Schwann cells, which mediate nerve regeneration and repair, remain unclear. In this study, we identified and isolated EVs from rat BM-MSCs and assessed the effects of MSC-EVs to RSC96 Schwann cells on proliferation, apoptosis, and migration in vitro. Our data shows that MSC-EVs can be taken up by RSC96 cells and inhibit proliferation and migration. MSC-EVs increased RSC96 cell apoptosis through the activation of the ERK pathway, thereby affecting cell proliferation and migration. Therefore, the effects of MSC-EVs on RSC96 cells may affect peripheral nerve injury and repair, as mediated by Schwann cells. Methods Ethics statement Animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals ( ), and efforts were exerted to minimize the suffering of the animals. All animal procedures for the use of laboratory animals were approved by the Institutional Animal Care and Use Committee of Jiangsu University (Permit Number: JSU ). Cell lines and culture The rat Schwann cell line RSC96 was obtained from Hanbio (Shanghai, China) and cultured in high-glucose Dulbecco s Modified Eagle s Medium (Gibco BRL Co., Ltd., USA) supplemented with 10% fetal bovine serum at 37 C in a humidified atmosphere of 5% CO 2 and 95% air. Isolation and characterization of MSCs MSCs were isolated from the bone marrow of rats and cultured as previously described [18]. In brief, after male Sprague-Dawley (SD) rats ( g) were sacrificed, the femora and tibias were removed and cleaned. The cells were pushed out by phosphate-buffered saline (PBS), collected, and resuspended in low-glucose Dulbecco s Modified Eagle s Medium (L-DMEM; Gibco BRL Co., Ltd., USA) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 U/ml). The cells were plated in culture dishes and cultured at 37 C in a humidified atmosphere of 5% CO 2 and 95% air. Flow cytometry analysis was performed to identify the phenotype of MSCs. Briefly, the cells were incubated with PE anti-cd19, PE anti- CD29, PE anti-cd90, and fluorescein isothiocyanate (FITC) anti-cd105 mab (Becton 5158 Int J Clin Exp Pathol 2018;11(11):

3 Dickinson, San Jose, CA, USA). PE- and FITCconjugated IgG1 were used as isotype controls. Osteogenic and adipogenic differentiation assays The multipotent differentiation ability of MSCs was induced according to previous reports [19]. In brief, for osteogenic differentiation, the cells were plated into 24-well plates with a density of cells/well and cultured with osteogenic induction medium, composed of L-DMEM supplemented with 100 nm/l dexamethasone (Sigma-Aldrich), 10 mm/l β-glycerophosphate (Sigma-Aldrich), 4 μg/ml basic fibroblast growth factor (bfgf) (Sigma-Aldrich), and 50 μg/ml ascorbic acid (Wako Chemicals, Neuss, Germany) for 21 days. The medium was changed every three days. Calcium deposits were observed using Alizarin Red staining. For adipogenic differentiation, the cells were plated into 24-well plates with a density of cells/well and cultured with an adipogenic induction medium, consisting of L-DMEM supplemented with 10% FBS, 10 μg/ml insulin (Sigma-Aldrich), 0.5 mm/l isobutylmethylxanthine, 200 μm/l indomethacin, and 1 μm/l dexamethasone for 21 days. The medium was replaced every three days. Lipid droplets were observed using Oil Red O staining as described previously [19]. Isolation and characterization of MSC-EVs EVs were purified from supernatants of MSCs according to a previously described protocol with slight modifications [12]. Briefly, 80% confluent MSCs were cultured overnight without a serum culture medium for 48 h. Subsequently, the medium was collected and centrifuged at 2000 g for 20 min to remove cells and debris. Cell-free supernatants were ultracentrifuged at g (Beckman Coulter Optima L-90K ultracentrifuge) for 1 h at 4 C, washed in serum-free medium 199 to remove macromolecular protein complexes, and submitted to a second ultracentrifugation in the same conditions with the EV pellets. The protein concentration of the EVs was quantified by the Bradford method (Bio-Rad, Hercules, USA). Isolated EVs were characterized for particle size distribution and quantified by NanoSight LM100 (Nano- Sight). The data were processed using Nanoparticle Tracking Analysis (NTA) 2.2 analytical software. MSC-EVs were stored at -80 C. MSC-EVs were characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For TEM analysis, MSC-EVs were fixed with 2% paraformaldehyde and absorbed for 20 min to a Formvar/carboncoated grid. After rinsing, the samples were transferred to a drop of 1% glutaraldehyde for 5 min and then washed, dried, and negatively stained with 2% uranyl acetate for 5 min. The grids were dried and observed under a Tecnai 12 TEM (Philips) at 80 kv. For SEM analysis, MSC-EVs were fixed in Karnovsky fixative, dehydrated in alcohol, dried on a glass surface, and sputter coated with gold. The samples were observed on a S4800 II scanning electron microscope (Hitachi). The images were acquired via secondary electron at a working distance of mm and an accelerating voltage of kv. MSC-EVs CM-Dil labeling The MSC-EVs were labeled using the CM-Dil Red Fluorescent Cell Linker Kit (Invitrogen) according to the manufacturer s instruction and washed thrice with PBS. The labeled MSC- EVs were incubated with RSC96 cells for 4 h on coverslips in a 24-well plate and viewed on a Nikon Eclipse 80i Fluorescence Microscope. RSC96 cell proliferation assay RSC96 cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RSC96 cells were treated with 10 or 20 μg/ml MSC-EVs or medium for 24 h. In certain experiments, RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Subsequently, the cells were collected and seeded into 96-well plates with a density of cells/well for 48 h. MTT (20 μl) was added to each well for an additional 4 h. When the reaction was terminated, the reagent was discarded, and dimethyl sulfoxide (150 μl) was added to each well. The optical density was determined at 492 nm using an FLx800 Fluorescence Microplate Reader (Biotek, VT, USA) Int J Clin Exp Pathol 2018;11(11):

4 Figure 1. Morphology and differentiation potential of rat BM-MSCs. A. A representative plot shows the spindly and fibroblast-like morphology of rat BM-MSCs after three passages. B. Osteogenic differentiation capacity of rat MSCs determined by Alizarin Red staining. Representative images were taken at 100 magnification. C. Adipogenic differentiation capacity of rat BMSCs confirmed using Oil Red O staining. Representative images were taken at 200 magnification. D. Cell surface markers were analyzed using flow cytometry. The numbers represent the percent expression of each marker. Histograms are representative of three independent experiments. PE- and FITC-conjugated IgG1 were used as isotype controls. Colony-forming assay RSC96 cells were treated with 10 or 20 μg/ml MSC-EVs or a medium for 24 h and then collected and seeded into 0.01% poly-l-lysinecoated 6-well plates with a density of cells/well for six days. The cells were fixed with 4% formaldehyde in PBS for 30 min and stained using 0.1% crystal violet followed by colony counting. Wound healing assay A wound healing assay was performed using Culture-Insert as previously described [20]. Briefly, RSC96 cells were treated with 10 or 20 μg/ml MSC-EVs or medium for 24 h, then harvested and seeded into 6-well plates, and cultured until 100% confluent. The confluent monolayers of the cells were scratched through their central axes using sterile pipette tips, and the drop cells were washed away using PBS thrice. Wound healing was monitored and photographed at 0 and 48 h at multiple sites, and representative images were captured. The degree of motility 48 h after the confluent cells were scratched was expressed as the percentage of wound closure, calculated as follows: (distance of scratch at 0 h - distance of cell 5160 Int J Clin Exp Pathol 2018;11(11):

5 Figure 2. Characterization of rat MSC-derived EVs. A. Size contributions of rat MSC-derived EVs were identified using NTA. B. Representative micrographs of TEM obtained on collected and purified EVs (scale bar: 200 nm). C. Release of EVs from the surface of MSCs by SEM (scale bar: 2 μm). D. Representative micrographs of SEM of purified MSCderived EVs (scale bar: 0.2 μm). migration at 48 h/distance of scratch at 0 h) 100%. Cell migration assay The migration assays were carried out following the manufacturer s instructions (Corning Inc., Corning, NY, USA) with slight modifications. The RSC96 cells were treated with 10 or 20 μg/ml MSC-EVs or medium for 24 h. In certain experiments, RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. A total of 100 μl of serumfree L-DMEM containing resuspended RSC96 cells ( ) was seeded in the upper chamber of the transwells of the 24-well plate. Before adding 600 μl L-DMEM containing 10% FBS into the lower chambers, the cells were allowed to migrate at 37 C in humidified 5% CO 2. The cells were cultured for 16 h, and the upper surface of each membrane was wiped off with cotton swabs. Cells that adhered to the bottom surface of each membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Three low-magnification areas (magnification 100 ) were randomly selected in each chamber to observe the cells. Cell Counter (Borland Software Corporation, Scotts Valley, CA, USA) was used to count the migrated cells. Cell apoptosis assay Cells stages of apoptosis were detected using an Annexin V-FITC Apoptosis Detection Kit, according to the manufacturer s protocol. In brief, RSC96 cells were treated with 10 or 20 μg/ml MSC-EVs or medium for 24 h. In certain experiments, the RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Subsequently, the cells were stained with PI and Annexin V-FITC for 20 min at RT in the dark after being washed 5161 Int J Clin Exp Pathol 2018;11(11):

6 Figure 3. MSC-derived EVs are internalized by RSC96 cells. RSC96 cells incubated with CM-Dil-labeled rat MSCderived EVs for 4 h. EVs are internalized by RSC96 cells (scale bar: 20 μm). twice with cold PBS. Finally, the cells were analyzed using BD FACSCanto II Flow Cytometer by FCAP Array TM (BD Bioscience). Western blot analysis The RSC96 cells were treated with 10 or 20 μg/ ml MSC-EVs or medium for 24 h. In certain experiments, the RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. The total cellular proteins of the cells were extracted using a modified radioimmunoprecipitation assay lysis buffer (Vazyme, Nanjing, China) and supplemented with phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China). The protein concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, MA, USA). Equal amounts of protein (200 μg) were electrophoresed in 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Beyotime). Non-specific binding was blocked with Tris-buffered saline/0.05% Tween- 20 (TBST) containing 5% defatted milk powder at room temperature for 1 h and incubated with anti-erk1/2 (1:500), anti-p-erk1/2 (1:500), anti-bcl-2 (1:500), anti-bax (1:500), and GA- PDH (ImmunoWay Biotechnology Company, Tennyson Pkwy, USA) overnight at 4 C. The blots were washed with TBST and incubated with HRP-linked anti-mouse IgG secondary antibodies (ImmunoWay Biotechnology Company, Tennyson Pkwy, USA). Finally, immunoblot signals were visualized using chemiluminescence (ECL) reagents (Beyotime) and then imaged and quantitated using Scion Image (Scion Corporation, USA). Statistical analysis Data are expressed as the mean ± SD. Statistically significant differences between the groups were assessed using one-way ANOVA with a post-hoc two-sided Dunnett t-test adjustment. The statistical analyses were performed using GraphPad Prism 5, and statistical significance was considered at P < Results Morphology and differentiation potential of rat BM-MSCs A homogenous population of BM-MSCs was obtained from SD rats after three passages in vitro. The cells displayed a spindly and fibroblast-like morphology (Figure 1A). The differentiation of MSCs was apparent after two or three weeks of induction under specific differentiation culture media. The MSCs were capable of differentiating into osteocytes and adipocytes, as shown by the positive staining of Alizarin Red (Figure 1B) and Oil Red O (Figure 1C). The majority of the cells showed the prominent presence of MSC surface markers CD29 (98.2%), CD90 (98.3%), and CD105 (98.9%). Minimal, if any, CD19 expression was detected (0.2%) (Figure 1D). Characterization of rat MSC-derived EVs The EVs were isolated from the MSCs through a series of ultracentrifugation steps of serumfree conditioned media as described in the materials and methods section. Zetasizer anal Int J Clin Exp Pathol 2018;11(11):

7 Figure 4. MSC-derived EVs inhibit the proliferation of RSC96 cells. A. MTT assay for the proliferating ability of RSC96 cells with or without MSC-EV treatment. Data are expressed as the means ± SD of three experiments performed in triplicate (*P < 0.05; ***P < 0.001). B. Clone formation assay for the proliferating ability of RSC96 cells with or without MSC-EV treatment. Data are expressed as the means ± SD of three experiments performed in triplicate (*P < 0.05; **P < 0.01). C. One representative experiment of clone formation images of RSC96 cells with or without MSC-EV treatment. yses determined that the sizes of the EVs ranged from nm, with a mean value of 167 nm (Figure 2A). TEM showed their round or oval morphology and confirmed their sizes. EVs were aggregated in clusters (Figure 2B). Using SEM, isolated on the surface of the cell membrane of EVs revealed the same morphological and size characteristics, as detected by TEM (Figure 2C). Electron microscopy randomly captured the different stages of EV formation and release processes (Figure 2D). These results indicated that EVs originated from the rat MSCs. MSC-EVs were taken up by RSC96 cells We first examined whether EVs secreted from BMSCs could be taken up by RSC96 cells. EVs labeled with CM-Dil were incubated with RSC96 cells, as shown by confocal microscopy. As shown in Figure 3, most RSC96 cells exhibited intracellular red fluorescence after incubation in the MSC-EVs, and the CM-Dil-labeled EVs were localized in the cytoplasm, suggesting that MSC-EVs could be taken up by RSC96 cells. MSC-EVs inhibit the proliferation of RSC96 cells To determine the effects of MSC-EVs on RSC96 cell growth, we evaluated the viability of the cells treated with 10 or 20 μg/ml MSC-EVs for 24 h. As shown in Figure 4A, MSC-EVs inhibited RSC96 cell growth in a dose-dependent manner by the MTT assay. This finding was further confirmed by the colony counting results (Figure 4B and 4C). Thus, the results suggested that BMSC-EVs inhibit the proliferation of RSC96 cells Int J Clin Exp Pathol 2018;11(11):

8 Figure 5. MSC-derived EVs inhibit the migration of RSC96 cells. A. Wound healing assay for the migration ability of RSC96 cells with or without MSC-EV treatment. RSC96 cells treated with different concentrations of EVs (10 and 20 μg/ml) were subjected to a wound healing assay for 48 h ( 100). B. Percentage of closure of the wounded areas is measured. Data are expressed as the means ± SD of three experiments performed in triplicate (***P < 0.001). C. Transwell migration assay for the migration ability of RSC96 cells with or without MSC-EV treatment for 12 h ( 100). D. Cells that migrated to the bottom are counted. Data are expressed as the means ± SD of three experiments performed in triplicate (***P < 0.001). MSC-EVs suppress the migration of RSC96 cells To analyze whether the migratory ability of RSC96 cells was affected by MSC-EVs, RSC96 cells were incubated with 10 or 20 μg/ml MSC- EVs for 48 or 12 h. Cell migration was determined by wound healing and transwell assays. As shown in Figure 5, compared with the control groups, MSC-EV treatment significantly decreased the relative wound closure rate and cell count. Thus, the results suggested that MSC-EVs suppress the migration of RSC96 cells. MSC-EVs induce cellular apoptosis of RSC96 cells To further study the effects of MSC-EVs on RSC96 cell apoptosis, the cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. As shown in Figure 6A and 6B, compared with the control group, 10 and 20 μg/ml EV treatment increased the percentage of Annexin V and PI double-positive cells. Thus, MSC-EVs promote the apoptosis of RSC96 cells. To understand the effects of MSC-EVS on apoptosis, we evaluated the expression of apoptotic protein Bcl-2 and Bax of the RSC96 cells treated with MSC-EVs. As shown in Figure 6C and 6D, the protein levels of Bcl-2 were down-regulated, and the Bax levels were up-regulated after exposure of RSC96 cells to MSC-EV treatment (Figure 5C). The Bcl-2/Bax ratio is a common method to represent the degree of apoptosis. As shown in Figure 5D, compared with the control group, 10 and 20 μg/ml EV treatment significantly reduced the Bcl-2/Bax ratio. However, no statistically significant difference was observed among the RSC96 cells after treatment with the two concentrations of EVs. Therefore, MSC-EVs induce apoptosis in RSC96 cells. ERK pathway is involved in MSC-EVs to induce apoptosis of RSC96 cells ERK activity appears to be involved in regulating cell survival [21]. To evaluate the role of the ERK pathway in the induction of apoptosis, the expression of the p-erk of the RSC96 cells following MSC-EV treatment was studied. Furthermore, U0126, which is a specific inhibitor 5164 Int J Clin Exp Pathol 2018;11(11):

9 Figure 6. MSC-derived EVs induce cellular apoptosis of RSC96 cells. A. RSC96 cells were treated with or without MSC-EVs, and late apoptotic cells (Annexin-V + PI + ) were detected by FCM. One representative experiment of apoptosis of the RSC96 cells from three individual experiments is shown. Values indicate the percentage of events in the indicated quadrant. B. The percent of late apoptotic (annexin-v + PI + ) RSC96 cells is shown. Data are expressed as the means ± SD of three experiments performed in triplicate (**P < 0.01; ***P < 0.001). C. Western blot analysis of Bcl-2 and Bax levels in RSC96 cells with or without MSC-EV treatment. D. Densitometric analysis of the Bcl-2 and Bax bands using Scion Image software The Bcl-2/Bax ratio was then analyzed. Data are expressed as the means ± SD of three experiments performed in triplicate (***P < 0.001). of the ERK pathway, was used to determine whether ERK activation is required for MSC-EVinduced apoptosis. As shown in Figure 7A and 7B, ERK activity increased upon MSC-EV treatment of RSC96 cells; however, U0126 prevented its activation. The down-regulation of Bcl-2 was increased, whereas the up-regulation of Bax was decreased when RSC96 cells were pretreated with U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Quantitation of apoptotic cells demonstrated that the increase of apoptotic cells was decreased when treated with MSC-EVs and U0126 (Figure 7C and 7D). Thus, the results suggested that activation of the ERK pathway participates in the induction of apoptosis by MSC-EVs. ERK pathway involvement in MSC-EVs inhibits the proliferation and migration of RSC96 cells To determine whether the ERK pathway affects the proliferation and migration of RSC96 cells, we further tested cell growth and cell count after MSC-EV and U0126 treatment by MTT and transwell assay, respectively. As shown in Figure 8A and 8B, consistent with the above reported observations that MSC-EVs inhibit RSC96 cell growth and counting; however, prior to the addition of U0126, they significantly reverse the MSC-EVs-induced suppression of RSC96 cell proliferation and migration. Thus, the results suggest that the ERK pathway is involved in the inhibition of the proliferation and migration of RSC96 cells using the MSC-EV treatment. Discussion The development of cell-free therapeutics based on the use of MSC-EVs is a safe and effective alternative to cell-based approaches and has already shown promise for treating various disorders in a number of animal models, including neurological diseases [10]. Fur Int J Clin Exp Pathol 2018;11(11):

10 Figure 7. The ERK pathway is involved in MSC-EVs to induce apoptosis of the RSC96 cells. A. The RSC96 cells were treated with or without MSC-EVs, and then p-erk and ERK levels were determined using a Western blot analysis. B. RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Next, p-erk, ERK, Bcl-2, and Bax levels were determined using a Western blot analysis. C. The late apoptotic (annexin-v + PI + ) RSC96 cells were detected by FCM. One representative experiment of apoptosis of the RSC96 cells from three individual experiments is shown. Values indicate the percentage of events in the indicated quadrant. D. The percent of late apoptotic (annexin-v + PI + ) RSC96 cells is shown. Data are expressed as the means ± SD of three experiments performed in triplicate (**P < 0.01; ***P < 0.001). thermore, Schwann cells are the major glial cells of the PNS and play a key role in the survival, function, and regeneration of neurons. However, how MSC-EVs affect Schwann cells is not yet known. In this study, we aimed to test the impact of rat BM-MSC-EVs on Schwann cells. Consistent with a previous study [22], we successfully isolated and identified the rat MSCs, and the MSCs differentiated into adipocytes and osteoblasts. Subsequently, we successfully isolated EVs from rat MSCs, and their size and shape are consistent with those observed in a previous study [23]. Membrane dye labeling showed that rat MSC-EVs could be incorporated into RSC96 Schwann cells. The incorporation of EVs may affect the biological effects of RSC96 cells. Schwann cell proliferation is crucial in successful nerve regeneration [24]. Furthermore, immature Schwann cells differentiate into a myelinating phenotype that packages the axon of neurons in the PNS, and they proliferate and migrate into the injured nerve areas to support axonal re-growth [25]. Therefore, we detected the RSC96 cell proliferation, migration, and apoptosis after the MSC-EV treatment. In contrast to the microvesicles derived 5166 Int J Clin Exp Pathol 2018;11(11):

11 Figure 8. The ERK pathway is involved in MSC-EV-suppressed proliferation and migration of RSC96 cells. A. RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Next, an MTT assay was performed to assess the proliferating ability of the RSC96 cells. Data are expressed as the means ± SD of three experiments performed in triplicate (*P < 0.05; ***P < 0.001). B. The RSC96 cells were pretreated with 10 µm U0126 for 30 min prior to the addition of 10 and 20 μg/ml EVs. Next, the migration ability of RSC96 cells was determined using a transwell migration assay. Data are expressed as the means ± SD of three experiments performed in triplicate (**P < 0.01; ***P < 0.001). C. One representative experiment of the transwell migration assay of the RSC96 cells from three individual experiments. from human BM-MSCs [12] and liver stem cells [26], which stimulate the proliferation and apoptosis resistance of tubular epithelial cells or hepatocytes, respectively, we found that rat MSC-EVs inhibited the proliferation and migration and induced the apoptosis of RSC96 cells. These results are consistent with a previous study that demonstrated the toxicity of the MSC-conditioned medium (MSC-CM) to hippocampal slice cultures and that it aggravated cell death induced by reactive oxygen species and neuroinflammation; however, the authors 5167 Int J Clin Exp Pathol 2018;11(11):

12 did not specifically indicate that the MSC-CM was EVs [27]. However, EVs are well known to originate from CM and are a significant part of the MSC secretome. Schwann cell damage could lead to the reduction of cellular viability and ultimately apoptotic or autophagic death; moreover, the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 are important regulators in these processes [28]. As shown in our data, the Bcl-2/ Bax ratios were decreased in RSC96 cells because of the down-regulated Bcl-2 and upregulated Bax expression levels after MSC-EV treatment. This finding is in contrast to other studies, in which rat inguinal adipose-derived MSCs significantly reduced the apoptosis of Schwann cells in diabetic rats [29]. However, MSC-EVs have functions similar to those of MSCs [30, 31], and bone marrow-derived MSCs are known to exert anti-apoptotic effects [32]. Furthermore, in vivo studies confirmed that MSC-EVs may promote the proliferation and survival of cells by reducing apoptosis in brain or kidney injury models. Xin et al. reported [33] that systemic administration of EVs generated from BM-MSCs significantly increased axonal density and synaptophysin-positive areas in the ischemic cortex and the striatum of rats with middle cerebral artery occlusion (MCAO). BM-MSC-derived EV treatment also increased the newly formed double cortin-positive cells (neuroblasts) and improved the functional recovery of rats with stroke compared with the PBS-treated controls. Gatti et al. showed that microvesicles derived from human adult MSC protected against ischemia/reperfusion-induced acute and chronic kidney injuries, and the mechanism of protection has mainly been ascribed to the stimulation of cell proliferation and the inhibition of apoptosis [14]. Moreover, the inhibition of in vitro apoptosis of cisplatintreated tubular cells was associated with the down-regulation of genes involved in the execution phase of cell apoptosis (caspase 1 and 8) and with the up-regulation of anti-apoptotic genes (Bcl-xL and Bcl2) [14]. The importance of the MAPK signaling pathways in regulating apoptosis in stress conditions has been widely investigated. Many studies have supported the general view that the activation of the ERK pathway delivers a survival signal that counteracts pro-apoptotic effects associated with JNK and p38 activation [34, 35]. However, in this study, we confirmed that the activation of ERK is important for the induction of MSC-EV-induced apoptosis in RSC96 cells. MSC-EVs treatment resulted in the increased activation of ERK in RSC96 cells; moreover, the down-regulation of ERK using U0126 led to an inhibition of MSC-EV-induced apoptosis, thereby reversing MSC-EV-mediated proliferation and migration suppression in RSC96 cells. This finding is in contrast to other studies, in which human MSC-derived exosomes inhibited 5-FU-induced apoptosis by activating the CaM-Ks/Raf/MEK/ERK pathway [36]; furthermore, the human umbilical cord MSC-derived exosomes suppressed cisplatininduced apoptosis and promoted proliferation in NRK-52E cells by activating the ERK pathway [37]. Whether these differences were due to different cells, disease models, or treatment remain to be investigated in future studies. However, the intrinsic role of BMSC-EVs on RSC96 cells is unclear, and further studies are necessary to understand their functional role during peripheral nerve injury and repair. Conclusions In conclusion, our study demonstrates that rat MSC-EVs inhibit proliferation and migration and induce apoptosis in RSC96 cells by activating the ERK pathway. MSC-EVs represent a novel cell-free therapeutic strategy that can overcome the limitations and risks related to cellbased therapies; however, caution should be exercised in using them for peripheral nerve injury and repair. Acknowledgements The current study was supported by grants from the key research and development plan of Jiangsu Province (BE ), the Natural Science Foundation of Jiangsu Province (BK ), the clinical science and technology development foundation of Jiangsu University (JLY ), Social Development Projects of Zhenjiang (SH ), Key Medical Personnel of Zhenjiang, LiuGeYi Projects of Jiangsu Province (LGY ), and the Affiliated Hospital of Jiangsu University (jdfyrc ). Disclosure of conflict of interest None Int J Clin Exp Pathol 2018;11(11):

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