Small Extracellular Vesicles Derived From Human Ipsc-Derived MSC Ameliorate Tendinopathy-Related Acute Pain Through Inhibiting Mast Cells Activation

Renzhi Gao Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Teng Ye Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Zhaochen Zhu Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Qing Li Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Juntao Zhang Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Ji Yuan Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University Bizeng Zhao Shanghai Jiao Tong University A liated Sixth People' Hospital Zongping Xie (  x91034@qq.com ) Shanghai Jiao Tong University A liated Sixth People' Hospital Yang Wang Shanghai Jiao Tong University A liated Sixth People' Hospitaliated to Shanghai Jiaotong University

Background Pain is de ned as an unpleasant sensory and emotional experience of actual or potential tissue damage or an experience expressed in such terms (1), which can be acute or chronic. Persistent stimulation by acute pain would cause neural plasticity remodeling in pain coding pathways and develop into chronic pain (2). Thus, the key to preventing chronic pain is to control the progression of pain during the acute phase. Tendinopathy is a prevalent musculoskeletal disease characterized by pain, swelling, and limited joint movement (3). It was reported that the prevalence and incidence rates of lower extremity tendinopathy were 11.83 and 10.52 per 1000 person-years (4). Chronic pain derived from tendinopathy would limit the movements of joints, even resulting in disability. To date, most studies related to tendinopathy focus on promoting tendon regeneration without considering relieving acute pain. Clinically, the effectiveness of current treatments for acute pain in tendinopathy remains ambiguous. Though corticosteroid injection has been a mainstay treatment for tendon-related disorders for many years, its effectiveness remains controversial (5). In conclusion, it is essential to explore a new therapeutic strategy to relieve acute pain derived from tendinopathy.
Mesenchymal stem cells (MSCs) have been well investigated in regulating immune response and tissue regeneration(6, 7). Recently, we have generated MSCs from induced pluripotent stem cells (iPSCs)(8). The MSCs derived from iPSCs (iMSCs) have a more robust proliferation and differentiation potential than adult BM-MSCs (9). Accumulating studies have indicated that the e cacy of MSCs is attributed to the paracrined small extracellular vesicles (sEVs), lipid bilayer nanoparticles containing proteins, lipids, nucleic acid, and other biomolecules (10,11). Our previous research has shown that sEVs derived from iMSCs (iMSC-sEVs) could attenuate osteoarthritis by alleviating in ammation and promoting chondrocyte proliferation (12,13). Besides, previous studies have demonstrated that BMSC-EVs could promote tendon healing by suppressing in ammation and apoptotic cell accumulation and increasing the proportion of tendon-resident stem/progenitor cells (14,15). Nevertheless, the therapeutic potential of iMSC-sEVs for alleviating acute pain in tendinopathy has barely been reported so far.
As proin ammatory cells and the immune system's rst responders (16), mast cells are localized in proximity to afferent bers (17). The proximity of mast cells to afferent nerve bers potentiates critical molecular crosstalk, contributing to initiating and developing pain responses(18). They are a critical link between the nervous system and the immune system (19). Studies have shown that mast cells play a vital role in pain transmission in tendinopathy (20)(21)(22)(23)(24), indicating that suppressing the in ltration and activation of mast cells may be a target for relieving acute pain in tendinopathy. In addition, Cho et al. Accordingly, we hypothesized that iMSC-sEVs could ameliorate pain by inhibiting mast cell activation in tendinopathy.
In the present study, we rst found that iMSC-sEVs could relieve acute pain and inhibit in ammation in a rat tendinopathy model. Then we showed that iMSCs-sEVs could inhibit mast cells activation and interaction with nerve bers in vivo. Moreover, iMSC-sEVs inhibited substance P-induced activation of mast cells in vitro. Mechanically, iMSC-sEVs might regulate the HIF-1 signaling pathway in mast cells.
Herein, we demonstrated for the rst time that iMSC-sEVs possess the therapeutic potential to ameliorate pain in tendinopathy by stabilizing mast cells, at least in part, via the HIF-1 signaling pathway.

Derivation and characterization of induced MSCs
The local ethics committee approved using human iPSC in this study of the Shanghai Sixth People's Hospital a liated with Shanghai Jiao Tong University. The generation of mesenchymal stem cells from human induced pluripotent stem cells as previously described (27). Flow cytometry was used to detect phenotypical markers of iMSCs. The cells were incubated with 1% (w/v) bovine serum albumin (BSA) (Gibco) to block the non-speci c antigens. Then, 1 × 10 6 cells were stained with the following conjugated mouse monoclonal antibodies: CD24

Isolation of iMSC-sEV
The iMSC-sEV were isolated from the cell culture medium of iMSCs by differential ultracentrifugation protocols. Brie y, the obtained medium was centrifuged at 300g for 10 min and 2000g for 10 min. After centrifugation at 10,000g for 1 h, the supernatant was ltered through a 0.22-µm lter sterilize Steritop™ (Millipore) to remove cellular debris and microvesicles. The collected medium was further ultracentrifuged at 100,000g for 70 min twice. After removal of the supernatant, the pellet was resuspended in PBS.

Size distribution and particle concentration of iMSC-sEVs
The size and concentration of the iMSC-sEVs were assessed using nano-ow cytometer (N30 Nano ow Analyzer, NanoFCM Inc., Xiamen, China) as previously described(28). Brie y, isolated iMSC-sEVs diluted with 100-fold PBS (for a nanoparticle concentration of approximately 10 8 /mL) were loaded to the nanoow to measure the side scatter intensity (SSI). The concentration of iMSC-sEVs was calculated according to the ratio of SSI to particle concentration in the standard polystyrene nanoparticles. The size distribution of iMSC-sEVs sample was calculated according to the standard cure generated by standard silica nanoparticles.

Western blot analysis
To identify sEV using western blot analysis, three positive markers of iMSC-sEV, including CD9, TSG101, and CD63, and one negative marker GM130, were evaluated. Cells or iMSC-sEV proteins were harvested using RIPA lysis buffer (Beyotime biotechnology, China, P0013C) supplemented with protease inhibitor cocktail (Beyotime biotechnology, China, ST505). Lysates were cleared by centrifugation at 12,000 g for 20 min. The supernatant fractions were used for western blot analysis. Protein extracts were resolved by Previous studies have established a rat tendinopathy model by carrageenan (29). Hence, we injected 100ul 4% (w/v) carrageenan into the peritendon space of the quadriceps tendon under ultrasound guidance to induce a rat tendinopathy model while sham rats received a PBS injection. One week later, injection of iMSC-sEVs was performed in the sEVs group and PBS in the control group once a week for 4 weeks. Pain-related behaviors were analyzed one week after the administration of iMSC-sEVs or PBS. Reversal (%) of pain-related behaviors was calculated as follows: Reversal(%) = 100 × (posttreatvalue, tendinopathyrats) − (pretreatvalue, tendinopathyrats) (avg. pretreatvalueshamrats) − (pretreatvalueintendinopathyrats) where "value" represents the values for static weight-bearing or hind paw withdrawal threshold.
Pain assessment

Hind-Paw Withdrawal Threshold
Hind-paw withdrawal thresholds were measured as described previously (30). The electronic von Frey instrument (model BIO-EVF4; Bioseb, Vitrolles France) was used to vertically stimulate the center of the rat hind paw with increasing intensity. The probe tip was gently placed perpendicularly into the mid-plantar surface of the paw, and steadily increasing pressure was applied until the hind paw was rst lifted. Until the withdrawal reaction was positive, and there were 3 positive withdrawal reactions within the 5 consecutive stimuli, the value was de ned as PWT and was expressed in grams (g).

Static Weight Bearing
The static weight-bearing (SWB) distribution over the right and left knee was assessed by measuring the postural balance between the injected and non-injected leg (30). Brie y, a rat was placed in the chamber of a weight-bearing measuring device (model #BIO-SWB-TOUCH-M; Bioseb). The force applied through each hind limb to the paw resting on the oor of the chamber was measured in grams (g), and an SWB index was calculated as follows: For each rat, the test was given at least three times at each assessment period.

Gait analysis
Dynamic pain-related behavior was measured by the gait of the rats(31). The Catwalk system objectively quanti es behavioral gait adaptation after daily use of a painful limb, automatically documenting paw placements on a surface and related parameters of inter-limb coordination (32). Brie y, rats were placed on a walkway apparatus (Shanghai Mobiledatum Information Technology, Shanghai, China). A camera below the walkway captures and digitally records footprint images. These paw print placements and gait parameters were collected and further analyzed by WalkAnalysator (Shanghai Mobiledatum Information Technology). The CatWalk gait test was administered at weeks 4 after treatment. Print area, swing speed, duty cycle and max contact mean intensity were recorded and analyzed as Right/Left.

Histology and Immunohistochemistry
For histological analysis, the rat tendon samples were xed en bloc in 4% PFA for 24 hours, dehydrated with a graded ethanol series, embedded in para n, and sectioned (5 µm thick) parallel to the long axis of the tendon. The sections were prepared for hematoxylin and eosin (H&E) and immunohistochemical analysis.

Uptake of iMSC-sEVs by mast cells in vitro
To determine the uptake of iMSC-sEVs into RBL-2H3 cells in vitro, we labeled iMSC-sEVs with Dil uorochrome (Thermo Fisher, USA) under room temperature for 15 min, followed by ultracentrifugation at 100,000g in PBS to get rid of the unlabeled dye. Next, Dil-labeled sEV were incubated with RBL-2H3 cells for 12 hours. And then, the culture medium was discarded, and the cells were rinsed twice with PBS before image capture under the uorescence microscope (Leica, DM6B, Germany).
β-Hexosaminidase release assay The RBL-2H3 cells were conducted β-Hexosaminidase release assay as previously described with a small modi cation to determine the degranulation activity (34). Brie y, after being stimulated by SP, the RBL-2H3 cells were treated with iMSC-sEVs (10 9 /ml) or vehicle for different time (6h/ 9h/ 12h/ 24h) at 37°C in 5% CO2 atmosphere. The supernatants (15 µl Real-time quantitative polymerase chain reaction (RT-qPCR) analysis The expression of targeted genes was analyzed by RT-qPCR. Brie y, The total RNA of samples was extracted using EZ-press RNA Puri cation Kit (EZBioscience, USA). RNA quantity and purity were con rmed with a Nanodrop spectrophotometer (Thermo Scienti c, Wilmington, DE). A 4× Reverse Transcription Master Mix (EZBioscience, USA) was used for reverse transcription reaction. PCR reactions were run using the ABI Prism 7900HT Real-Time System (Applied Biosystems, Carlsbad, CA) with 2× SYBR Green qPCR Master Mix (EZBioscience, USA). The primer sequences used in this study are listed in Additional le 1: Table S1.
Additional le 1: Table S1. The primer sequences were used in this study.

Enzyme-linked Immunosorbent Assays (ELISA)
The supernatant collected at 18h after different treatment was evaluated for proin ammatory molecules and NGF by ELISA. IL-1β, TNF-α, IL-6, IL-10, and NGF concentrations were measured by using a rat ELISA kit (Shanghai Westang Bio-Tech Co., LTD., Shanghai, China) according to the manufacturer's instructions. The absorbance was measured by a microplate reader (Thermo Fisher Scienti c, Waltham, MA, USA) at 450 nm.

Statistical analysis
Data were presented as mean ± SD. Student t-test was used to assess the difference between two groups, and the one-way ANOVA with the Bonferroni post hoc test was applied for comparisons among multiple groups. All experiments were independently performed at least three times. Statistical analysis was performed using GraphPad Prism software (version 8.0). The signi cant difference was considered to be P-value < 0. 05.
iMSC-sEVs alleviated the tendinopathy-related pain in a rat model Firstly, we established the tendinopathy model by injecting carrageenan into the quadriceps tendon, and then iMSC-sEVs (1 × 10 9 particles) or PBS were administrated. (see Additional le 2: Fig. S1). Then, we performed H&E and IHC staining on the quadriceps tendon. H&E staining revealed that tendons in the iMSC-sEVs group showed more continuous and regular arrangement than disorganized tendons in the vehicle group ( Fig. 2A). Besides, Movin score in the iMSC-sEVs group was signi cantly lower than the vehicle group (Fig. 2B). IHC staining showed that proin ammatory cytokine expression signi cantly decreased in the iMSC-sEVs group compared with the vehicle group at 2 weeks and 4 weeks ( Fig. 2A, 2B).
Our results suggested that iMSC-sEVs could reduce proin ammatory cytokines production and repair the injured tendon.
Pain is a dominant character of in ammation. Therefore, we evaluated whether iMSC-sEVs could relieve the pain in tendinopathy. We accessed static weight-bearing (SWB) and hind-paw withdrawal threshold (PWT) after iMSC-sEVs administration for 4 weeks. For the static state, the reversals(%) of PWT and SWB in the iMSC-sEVs group were signi cantly increased compared to the vehicle group (Fig. 2C, D). CatWalk tests were applied to examine whether iMSC-sEVs improve gait and motor function at 4 weeks after treatment. The lower limbs of the vehicle group showed less coordination than that of the iMSC-sEVs treatment group during walking (Fig. 2E). Speci cally, iMSC-sEVs signi cantly elevated the right /left hind values ratio in the print area, swing speed, and max contact mean intensity compared with the vehicle group (Fig. 2F). In addition, He et al. reported that BMSC-sEVs could reduce the expressions of CGRP (neuropathic pain marker) and iNOS (in ammatory marker) in OA rats' dorsal root ganglion (DRG) tissues (35). Therefore, we performed immuno uorescence staining to detect the expressions of CGRP and iNOS. The result showed that the expression of these two proteins was signi cantly downregulated in DRG of the iMSC-sEVs group compared with the vehicle group (see Additional le 3: Fig. S2A, B). These results indicated that iMSC-sEVs could mitigate tendinopathy-related pain.

iMSC-sEVs inhibited mast cells in ltration and interactions with nerve bers in the quadriceps tendon
Previous reports have demonstrated that mast cells play a vital role in tendinopathy-related pain (20)(21)(22)(23).
We conducted double immuno uorescence staining on tendon sections for tryptase and PGP9.5 to assess the number of activated (tryptase+) mast cells and the anatomical interaction between mast cells and nerve bers. The results showed that compared to the sham group, tryptase+ mast cells increased markedly in the tendinopathy group, as evidenced by the mean gray value of the tryptase staining area (Fig. 3A, B). Besides, the number of tryptase+ mast cells closed ( 5 µm) to PGP9.5+ nerve bers signi cantly also increased (Fig. 3A, B).
It is well known that NGF is a prominent role that mediates interactions among mast cells and nerve bers(36). As expected, IHC staining of NGF showed a signi cantly increased expression compared to the sham group (Fig. 3C, D). Therefore, these data supported the assumption that enhanced activation of mast cells after model establishment.
We then investigated whether iMSC-sEVs could regulate activated mast cells in ltration and interaction with nerve bers. Double immuno uorescence staining for tryptase and PGP9.5 was applied as described above. Compared with vehicle treatment, iMSC-sEV treatment signi cantly reduced the in ltration and interaction, as re ected by the signi cantly decreased number of tryptase+ mast cells and that near PGP9.5+ nerve bers (Fig. 3A, B). Additionally, iMSC-sEV treatment signi cantly decreased the positive area of NGF compared to vehicle treatment (Fig. 3C, D). Altogether, these results suggested that iMSC-sEVs treatment could stabilize mast cells under in ammatory conditions and impede their crosstalk with nerve bers in a tendinopathy model.

iMSC-sEVs restrained Substance P-induced activation of mast cells
To further investigate the effect of iMSC-sEVs on the function of mast cells, substance P (SP) was applied to activate RBL-2H3 cells ( a widely used mast cell line (37)) in vitro. First of all, we determined whether iMSC-sEVs could be internalized by RBL-2H3 cells. iMSC-sEVs were labeled with Dil uorescent dye and added to the culture medium. After 12 h of incubation, Dil-labeled iMSC-sEVs were e ciently uptaken by RBL-2H3 cells (Fig. 4A). RT-qPCR results showed that iMSC-sEVs reduced the mRNA expression of IL-1β, IL-6, TNF-α and NGF from SP-stimulated RBL-2H3 cells in a dose-dependent manner (Fig. 4B).
Therefore, iMSC-sEVs with the dose of 1 × 10 9 particles/ml were chosen for the following experiments. β-hexosaminidase release assay showed that iMSC-sEVs signi cantly reduced the degranulation of SPstimulated RBL-2H3 cells, especially after 12 h incubation (Fig. 4C). Toluidine blue staining showed that iMSC-sEVs signi cantly reduced the percentage of degranulated mast cells compared with vehicle treatment (Fig. 4D, E). In addition, the expression of proin ammatory cytokines in the supernatant was signi cantly declined in the iMSC-sEVs group as determined by ELISA (Fig. 4F). Collectively, these data indicated that iMSC-sEVs restrained SP-induced activation of mast cells in vitro.
iMSC-sEVs modulated the gene expression pattern of mast cells To elucidate the underlying molecular mechanism by which iMSC-sEVs restrained the degranulation of mast cells, we performed RNA-seq analysis to pro le the gene expression patterns in SP-stimulated RBL-2H3 cells treated with vehicle and iMSC-sEVs. We identi ed 768 up-regulated genes (> 2-fold, p < 0.05) and 530 down-regulated genes (< 0.5-fold, p < 0.05) after iMSC-sEVs treatment (Fig. 5A, B). Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the downregulated genes in the iMSC-sEVs group were enriched for functional annotations related to the HIF-1 signaling pathway and other metabolic pathways ( Figure. 5C). Similarly, Gene Ontology (GO) analysis revealed that the downregulated genes in the iMSC-sEVs group were enriched for biological processes, such as regulation of apoptotic cell clearance and positive regulation of IL−1β secretion and response to type I interferon (Fig. 5D). Subsequently, the heatmap veri ed the expression of speci c genes in the HIF-1 signaling pathway analysis and biological processes like positive regulation of IL−1β and response to interferon, which showed a signi cant difference between groups (fold change >2) (Fig. 5E). Furthermore, RT-qPCR analysis con rmed that the expression of the HIF-1 signaling pathway-related genes was downregulated after iMSC-sEVs treatment (Fig. 5F). Therefore, these results suggested that iMSC-sEVs restrained mast cell activation through regulating the HIF-1 signaling pathway.

Discussion
In this study, we demonstrated for the rst time that the application of sEVs isolated from iPS-MSCs (iMSC-sEVs) signi cantly alleviated acute pain in a rat tendinopathy model. We found that iMSC-sEVs could notably inhibit mast cell activation and suppress in ammation in vivo. Further in vitro study illustrated that iMSC-sEVs could restrain SP-induced mast cells activation via the HIF-1 signaling pathway in part.
Tendinopathy describes a spectrum of changes in damaged and diseased tendons, resulting in pain and dysfunction(38). The pathogenesis of tendinopathy is multifactorial and complex. It is widely accepted that overuse and in ammation contribute to tendinopathy (39,40). This study focused on the pathogenicity of in ammation. Pain is an essential manifestation of in ammation, but pain can generally be classi ed into nociceptive pain, in ammatory pain and neuropathic pain according to the pathogenesis. Nociceptive and in ammatory pain are adaptive and protective, while neuropathic pain occurs after damage to the nervous system. In tendinopathy, in ammatory mediators evoke pain via direct activation and sensitization of nociceptors (41). In contrast, persistent nociceptive input results in the growth of central sensitization and neuropathic pain, characterized by the hyperactivity and hyperexcitability of neurons in the brain and spinal cord (42). Besides, previous studies suggest that tendinopathy-related pain is a combinational form of in ammatory and neuropathic pain (43,44). Calcitonin gene related peptide (CGRP) is generally involved in transmitting nociceptive information and pain sensitization in the peripheral and spinal cords (35). Consistent with these, we determined the increased expression of CGRP and iNOS in dorsal root ganglion of tendinopathy rats. Meanwhile, iMSC-sEVs treatment relieved the pain in tendinopathy, thereby reducing central sensitization and neuropathic pain, as re ected by decreased expression of CGRP and iNOS.
After injuries occur in local tissue, mast cells and other immune cells are activated and release in ammatory mediators such as bradykinin, prostaglandin, protease and histamine to stimulate adjacent nociceptor afferent bers (45,46). In turn, affected afferent bers of nociceptors also release neuromodulators, such as substance P (SP), calcitonin-producing peptide, and vasoactive intestinal protein to activate mast cells. Consequently, an in ammatory cascade reaction of mast cell activation and peripheral neuro-hypersensitivity is formed, further amplifying pain and in ammation(46-48) (49). Scott et al. revealed that the number of mast cells in the patellar tendon specimens of patients with patellar tendinopathy was signi cantly increased (21). Consistently, in this study, we found that the number of activated mast cells and those closed ( 5 µm) to PGP9.5+ nerve bers signi cantly increased in the tendon after model establishment in rats. Therefore, mast cells may be a target for relieving acute pain in tendinopathy.
MSCs have been proved to possess anti-in ammatory, analgesic and regenerative capacities (50).
However, current methods for the large-scale preparation of MSC face several limitations and challenges. MSCs derived from iPSCs (iMSCs) can avoid the ethical problem and immune rejection, and iMSC-sEVs production offers several advantages for applications of MSCs (11). The anti-in ammatory and analgesic effects of iMSCs are mediated by paracrine action, which is dominated by sEVs containing various nucleic acids, DNA, and proteins. Previous studies have reported that MSC-sEVs could inhibit mast cells activation via a PGE2-dependent mechanism(26). Our present research found that iMSC-sEVs dependently down-regulate SP-induced release of proin ammatory cytokines and degranulation of RBL-2H3 cells (a mast cell line (37)) in vitro. Besides, in vivo study showed that iMSC-sEVs decreased the expression of proin ammatory cytokines, the activation of mast cells and the distance with nerve bers, con rming the ability of iMSC-sEVs in suppressing mast cells activation. As expected, our in vivo study showed that iMSC-sEVs treatment increased reversals(%) of PWT and SWB, improved gait performance and motor function, revealing the analgesic effect of iMSC-sEVs on tendinopathy in vivo. Overall, these results demonstrate that iMSC-sEVs alleviate pain derived from tendinopathy partially through inhibiting the activation of mast cells.
RNA-seq and bioinformatics analysis of sequencing data identi ed differentially expressed genes involved in several signaling pathways. Interestingly, the expression of genes in metabolism-related signaling pathways like glycolysis/gluconeogenesis, biosynthesis of amino acids, and carbon metabolism upregulated signi cantly in the vehicle group. It might be attributed to the activation of mast cells caused by SP. So far, an increasing number of studies have reported that the HIF-1 signaling pathway gures prominently in regulating mast cells. Mast cells-derived HIF-1a signi cantly contributes to regulating mast cell function, which promotes the development of colorectal cancer(51). Abebayehu et al. (52) found that lactic acid could suppress IL-33-mediated mast cell in ammatory responses via HIF-1αdependent miR-155 suppression. In addition, Yan et al. (53) discovered that SP could upregulate the level of HIF-1α in gingival broblasts and participate in periodontitis. According to the KEGG analysis, 17 genes in the HIF-1 signaling pathway downregulated signi cantly after iMSC-sEVs treatment and RT-qPCR con rmed it. Thus, our study suggests that iMSC-sEVs could module the activation and function of mast cells by regulating the HIF-1 signaling pathway.

Conclusions
In summary, our study report for the rst time that iMSC-sEVs treatment relieves acute pain derived from carrageenan-induced rat tendinopathy by modulating neuro-immune interactions via the suppression of mast cells. Besides, iMSC-sEVs could suppress SP-stimulated activation in mast cells partly through regulating the HIF-1 signaling pathway. These ndings unravel molecular mechanisms underlying the application of iMSC-sEVs on mast cells and provide a novel treatment strategy for pain derived from tendinopathy.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate Animal care and experimental procedures were approved by the Animal Research Committee of the Shanghai Jiao Tong University A liated Sixth People's Hospital (approval code: DWLL2021-0910).

Consent for publication
Not applicable Figure 1 Characterization of iMSCs and iMSC-sEVs. A Identi cation of iMSCs by ow cytometry. B Representative image of iMSC-sEVs observed by TEM. Scale bar = 100 nm. C Size distribution of iMSC-sEVs measured by nano-ow cytometer. D The expression of exosomal markers including CD9, TSG101, and CD63, but not the negative marker GM130 measured by western blot. E Evaluation of iMSC-sEVs yield in terms of particle concentration, n=3. F Evaluation of iMSC-sEVs yield in terms of protein concentration, n=3. Data were expressed as mean ± SD.  D Quanti cation of immunohistochemical staining, n=5 per group. Data were expressed as mean ± SD. *P 0.05. **P 0.01. ***P 0.001. #P 0.0001. Figure 4 iMSC-sEVs restrained SP-induced degranulation of mast cells. A Representative immuno uorescence image of RBL-2H3 cells cultured with Dil labeled iMSC-sEV (red). Scale bar = 100μm. B RT-qPCR was performed to measure the expression of IL-1β, IL-6, TNF-α and NGF in RBL-2H3 cells. C The degranulation of RBL-2H3 cells was measured by β-hexosaminidase release assay. D Toluidine blue staining was performed to measure the number of degranulated RBL-2H3 cells (yellow arrows). Scale bar = 100μm. E Quanti cation of the percent of degranulated mast cells. F ELISA assay was performed to detect the level of IL-1β, IL-6, TNF-α and NGF in the supernatant. Data were expressed as mean ± SD. *P 0.05. **P 0.01. ***P 0.001. #P 0.0001. ns, no signi cant (P 0.05). All experiments were repeated at least three biological replicates independently. Figure 5