Alteration of payload in extracellular vesicles by crosstalk with mesenchymal stem cells from different origin

Background The application of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) requires customized materials to target disease or cell damage. We hypothesized that EVs exert different inflammatory effects on one recipient cell, although stem cells of different origins in humans have similar payloads. Results Here, the payload of EVs released by crosstalk between MSCs and human middle ear epithelial cells (HMEECs) extracted from adipose tissue, bone marrow and tonsils significantly increased the level of anti-inflammatory factors. EVs derived from the co-culture medium decreased TNF-, COX-2, IL-1, and IL-6 levels to approximately zero within 3h in HMEECs. Expression of miR-638 and amyloid- A4 precursor protein-binding family A member 2 was analyzed using microarrays and gene ontology analysis, respectively. Conclusions In conclusion, stem cells of different origins have different payloads through crosstalk with recipient-specific cells. Inducing specific factors in EVs by co-culture with MSCs could be valuable in regenerative medicine. Graphical abstract


Background
Otitis media (OM) is one of the most common diseases affecting children [1]. Although acute OM may resolve spontaneously, oral antibiotics and corticosteroids are ineffective in the long-term treatment of recurrent and chronic OM. Bacterial resistance to antibiotics has become an emerging problem in many parts of the world. Researchers have focused on understanding the pathophysiology of OM as a replacement for antibiotic treatment, including the role of inflammatory mediators and ion homeostasis molecules [2]. Restoration of fluid homeostasis function in the middle ear epithelium could be a target for future treatment, similar to the role of the upper respiratory ciliated mucosa. The middle ear mucosa is a pseudostratified, ciliated columnar epithelium through the eustachian tube that continues to the respiratory tract. Stem cell-derived exosomes control inflammatory signaling within the airway through intercellular communication, as demonstrated by the transfer of suppressor of cytokine signaling 1 from macrophagederived exosomes to alveolar epithelial cells [3]. Human mesenchymal stem cell (hMSC)-derived extracellular vesicles (EVs) possess most of the anti-inflammatory and neuroprotective activities of MSCs in the respiratory epithelium [4,5].
Therapeutics based on stem cell technology, including stem cell-derived EVs, have emerged in recent years and can treat what were otherwise considered incurable diseases in the field of cancer or cell regeneration [5]. Studies have also been proposed to find biomarkers in EVs for specific cancers [6]. Mesenchymal stem cells (MSCs) have been found to have different sources, such as the bone marrow, fat, urine, and tonsil; although they have the same phenotype, they are reported to have different internal payloads with various efficacies of treatments [5,7]. It has been found that tumor-derived EVs play a key role in crosstalk between malignant and transformed cells and the immune system and that cancer cells suppress immune surveillance [8]. In addition, stem cell-derived EVs are involved in crosstalk with surrounding cells during development and release their molecules to them [9,10], and help the survival of surrounding cells through secretion via EVs of inflammatory, differentiation, and proliferation factors [4,5].
EVs are a heterogeneous group of secreted membranous vesicles, including microvesicles, ectosomes, and exosomes [11]. They have become valuable biomarkers in liquid biopsies [12], and existing research has focused on their characterization in different cancer types [9]. When stem cells undergo autophagy through external stimulation, exocytosis is stimulated when intracellular multivesicular bodies (MVBs) increase, resulting in a large number of extracellular vesicles [13]. Furthermore, EVs are known to be released by a variety of human cells and are important mediators in the coordination of the immune response to maintain host homeostasis [5,14]. Upon different stimulation, EVs carry different combinations of nucleic acids, proteins, and lipids to other cells [15] and have been reported to be associated with certain pathological conditions, particularly microbial infections and cancer [16]. It has also been reported that the amount of EVs released following external stimulation increases with time [13]. The ability to block the transfer of tumor-derived EVs containing oncogenic messages, such as EGFR, into recipient cells is a potential antitumor strategy. A study showed that incubation of EVs from a cell line with heparin blocked their transfer into recipient cells [17].
In this study, we evaluated the anti-inflammatory effect of EVs derived from hMSCs extracted from the adipose tissue, bone marrow, and tonsils. MSCs derived from these sources contain several factors related to cell proliferation and inflammation-reducing substances [18][19][20], but miRNA or cytokine expression and efficacy by crosstalk with new recipient cells may differ [14,21]. Various studies have reported the relief of inflammation in skin tissues and infected organs in animal models [22]. The current focus of anti-inflammatory studies using MSCderived EVs is to increase the payload of useful factors in EVs through discovery of factors through wound healing and mechanism research [23,24]; there are reports that a calcium-dependent mechanism increases the release of EVs from the cells [25]. However, the important aspect of MSC research is to observe the change in payload inside the MSC and understands its role in disease [21]. In this study, we profiled the factors related to anti-inflammatory activity in EVs derived from MSCs extracted from different origins, when co-cultured with human middle ear epithelial cells (HMEECs), which can cause OM [26]. Interestingly, the expression level of molecule payload and anti-inflammatory efficiency were different in EVs from MSCs derived from adipose tissue, bone marrow, and tonsils co-cultured with HMEECs. Therefore, we propose a technology to discover new miRNA candidates by profiling it to prove inflammation relief according to the change of EV payload by crosstalk between MSCs and target cells is altered. In addition, the technique, involving the co-culture condition, offers a new direction for MSC-derived EV research to cure diseases or cancer; studies on the mechanism underlying the crosstalk between donor and recipient cells are also needed.

Extraction of mesenchymal stem cells (MSCs) derived from adipose tissue, bone marrow, and tonsils
The human adipose tissue, bone marrow, and tonsils were obtained from the iliac crest of patients who received transplantation treatment at the Wonju Severance Christian Hospital after obtaining their written consent (IRB number: CR320104). The extraction of adipose tissuederived MSCs was performed as described by Cho et al. [18]. Briefly, the tissue samples were washed twice with phosphate-buffered saline (PBS, D8537, Sigma-Aldrich, MA, USA) to remove blood cells and incubated with 200 IU/mL collagenase (Thermo Fisher Scientific, Carlsbad, CA, USA) for 45 min at 37 °C. After incubation, the samples were centrifuged at 300×g for 7 min, the supernatant was discarded, and the remaining pellets were resuspended in MEM-alpha (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and antibiotics (anti-anti, Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were transferred to a culture plate and incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.
To extract MSCs derived from the bone marrow, aspirates were collected into Vacutainers K2 EDTA (BD Biosciences, San Jose, CA, USA). The mononuclear cells were diluted 1:5 with PBS and separated by density gradient centrifugation at 435×g for 20 min at room temperature (RT, 25 °C) using a Ficoll Hypaque (GE17-1440-02, Gibco, Grand Island, NY, USA) solution. The cell fractions were collected and cultured using MEM-alpha with 10% FBS and antibiotics at a seeding density of 5 × 10 3 cells per cm 2 . The plate was maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . To exchange the medium, the plate was washed with PBS to remove the non-adherent cells, and the medium was replaced. Upon reaching 70% confluence, the cells were passaged to 1 × 10 6 cells/plate.
Tonsil-derived MSCs were extracted as described by Bacic et al. [27]. The tonsil tissue was gently washed with ethanol and cut with surgical scissors on a plate. After washing with PBS, the tissue mixture was added to Falcon tubes and incubated with PBS, 200 IU/mL collagenase, and 10 µg/mL DNase (EN0525, Thermo Fisher Scientific, Carlsbad, CA, USA) at 37 °C in a water bath for 1 h. After filtering the suspension using a cell strainer, the monocytes were isolated from the supernatant using Ficoll Hypaque density gradient centrifugation. The cell fractions were collected and cultured using MEM-alpha with 10% FBS and antibiotics at a seeding density of 5 × 10 3 cells per cm 2 .

Flow cytometric analysis
Flow cytometry was used to assess the immune profile of MSCs using the standard for MSCs, as described by the International Society for Cellular Therapy (ISCT) [28]. The cell surface markers were analyzed using a human MSC (hMSC) analysis kit (562245, BD Biosciences, San Jose, CA, USA). According to the manufacturer's instructions, the hMSC-positive cocktail (CD90 FITC, CD105 PerCP-Cy5.5, and CD73 APC) and PE hMSC negative cocktail (CD34, CD11b, CD19, CD45, and HLA-DR) were used as the positive and negative controls, respectively. As stated in this recommendation in the manufacturer's instructions, MSCs should be positive for CD73, CD90 and CD105, but negative for CD34, CD45, CD11b or CD14, CD19 or CD79α and HLA-DR. Since MSCs are known to express numerous cell surface markers such as CD44, CD29, CD200, CD166, CD146, and CD271, we used CD44 as a representative in this study. The hMSC Positive Isotype Control Cocktail (mIgG1κ FITC, mIgG1 κ PerCP-Cy5.5, and mIgG1 κ APC) and PE hMSC Negative Isotype Control Cocktail in the kit (mIgG1 κ PE and mIgG2a κ PE) were also used as an isotype control for the analysis. The samples were analyzed through flow cytometry using a FACS Aria3 flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using the FACS Diva software.

Culture of human middle ear epithelial cells
HMEECs were provided by Dr. David J. Lim (House Ear Institutes, Los Angeles, CA, USA). The cells were immortalized with the E6/E7 genes of the human papillomavirus [29] and cultured in Dulbecco's modified Eagle's medium (Lonza, MD, USA) and bronchial epithelial basal medium (Lonza, 1:1). The medium was changed every 3 days after washing with PBS. The cells were then humidified at 37 °C with 5% CO 2 .

Isolation of extracellular vesicles
To obtain EVs, initial extraction was performed according to the MISEV guidelines as far as possible [30]. The cells were cultured in a serum-free environment for inducing EV expression, and analyzed EVs from the media by western blot and NTA. After centrifuging the cells and debris, the medium was collected and centrifuged at 2000×g for 30 min, and the total exosome isolation reagent (4478359, Invitrogen, Grand Island, USA) was used according to the manufacturer's instructions. To purify EVs from the fractions, we centrifuged the fraction at 10,000×g for 1 h at 4 °C on the next day and added 200 µL of PBS to the sunk pellets to dissolve them. The samples were centrifuged at 22,000×g for 2 h at 4 °C using a table microcentrifuge (5424R, Eppendorf, Germany) to obtain high-purity EVs.

Transwell study
Co-culture conditions employed in this study were reported by Lu et al. [31]. One million MSCs were seeded into a transwell insert with a 0.4-µm pore size (3401, Corning, NY, USA) consisting of a polycarbonate membrane. MSCs derived from adipose tissue, bone marrow, and tonsil were co-cultured with HMEECs in the inner well. In addition, HMEECs were seeded into the outer well. They were then carefully transferred and cultured in a 37 °C incubator with 5% CO 2 in a humidified atmosphere prior to the appropriate LPS treatment or isolation of EVs from the media.

Cell viability assay
Cell viability was measured using a cell counting kit according to the manufacturer's instructions (CCK-300, Seoul, Korea). The cells were seeded at 1 × 10 3 cells per well in a 96-well plate. After exposure to 1, 5, 10, and 100 µg/mL LPS for different periods of time, CCK-8 solution was added to each well and the plates were incubated for 4 h at 37 °C. Cell viability was also measured after HMEECs were co-cultured with MSCs for 24 h. The plate was mixed thoroughly using a shaker, and the optical density was measured at 450 nm using a microplate reader (Epoch, BioTek, VT, USA).

Real-time PCR
To evaluate the expression of the inflammatory markers, total RNA was isolated from HMEECs after treatment with extracellular vesicles using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed using a ReverTra Ace qPCR RT Master Mix (Toyobo Bio-Technology, Osaka, Japan). The following primers were

Confocal microscopy
Reactive oxygen species (ROS) were detected using 1 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) in HMEECs. The cells were then incubated in a humidified 5% CO 2 incubator for 30 min at 37 °C after treatment with DCFDA. In contrast, CD63, an EV marker, was observed when the MSCs were incubated in 5% normal goat serum for 1 h at RT to prevent non-specific labeling. Anti-CD63 (1:200, ab 118,307, Abcam, MA, USA) was used as the primary antibody for 1 h at 4 °C. The samples were washed with PBS three times for 5 min each time, followed by incubation with a secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor 488; 1:1000, ab150077, Abcam, MA, USA), for 1 h at RT. After washing the samples three times for 5 min with PBS, they were immobilized with a mounting solution containing DAPI (4,6-diamidino-2-phenylindole). All the samples were observed by confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany), and the images were analyzed using ZEN lite ver. 2.3.

Nanoparticle tracking analysis (NTA)
Extracellular vesicles were quantified based on the MISEV guidelines [30]. The particle concentration, size, and distribution of the isolated MSC-derived EVs were analyzed using a NanoSight NS300 (Malvern Instruments Ltd, Malvern, UK). Typically, 1 mL of a 1:100 diluted MSC-derived EV was prepared for particle visualization and recording of light scattering. Three videos, each of 60 s recordings, were analyzed and plotted to show the EV concentration, size, and distribution.

Transmission electronic microscopy (TEM)
MSCs were fixed in 2.5% glutaraldehyde for 2 h at 4 °C, and the samples were solidified in 2% agar. After washing with 0.1 M cacodylate buffer, the samples were post-fixed in 1% osmium tetroxide (OsO 4 ). The dehydration steps were performed with 50-100% ethanol, and the samples were then embedded in Epon resin. The samples were baked overnight in an oven at 65 °C, sectioned in an ultramicrotome, and examined by TEM using a field electron emission unit (JEM-1200EX-II, JEOL).

Western blotting
EVs derived from MSCs were isolated through centrifugation, and the amount of total protein was quantified using the Bradford assay. Protein samples were separated using 10% SDS-PAGE with a mini gel apparatus (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (T831.1, Merck Millipore, MA, USA). Each membrane was blocked with 3% skim milk in Tris-buffered saline containing 0.05% Tween 20. The primary antibodies anti-CD9 (1:1000, ab92726, Abcam, MA, USA), CD63 (1:1000, ab118307, Abcam, MA, USA), and anti-CD81 (1:1000, ab109201, Abcam, MA, USA) were used as primary antibodies and were incubated overnight at 4 °C. Mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz, CA, USA) was used as a secondary antibody for 1 h. The bands were visualized using enhanced chemiluminescence according to the manufacturer's instructions (Immobilon Crescendo Western HRP substrate, Millipore, Darmstadt, Germany). The band intensities were quantified using the ChemiDoc XRS + System (Bio-Rad). All the samples were developed within 10 min to obtain a band. The concentration of the sample was confirmed through quality control analysis and was confirmed to be between 0.13 and 0.28 ng/µL. To check the purity and quantity of RNA, a NanoDrop spectrophotometer was used to measure the absorbance at 260 and 280 nm. All raw data were extracted automatically using the Affymetrix data extraction protocol using the Affymetrix GeneChip ® Command Console ® Software (AGCC). The CEL files were imported, miRNA levels were normalized using the RMA algorithm, the detection above background p-values was calculated for all data, and the results were exported using the Affymetrix ® Power Tools (APT) software. Array data were filtered using species-specific annotated probes. Comparative analysis between the test and control samples was performed using fold change. All statistical testing and visualization of differentially expressed genes were conducted using the R statistical language 3.3.3 (https:// www.r-proje ct. org/). All the predicted mRNAs from the miRNAs in the EVs were analyzed using the NCBI database, ExoCarta (http:// www. exoca rta. org/), and Vesiclepedia (http:// micro vesic les. org/).

Statistical analysis
Statistical analysis was performed using the SPSS statistical package (version 21.0; SPSS Inc., USA). All graphs were plotted using GraphPad PRISM (version 5.0; Graph-Pad Inc., La Jolla, CA, USA). The descriptive results of continuous variables are expressed as mean ± standard deviation (SD) for normally distributed variables. The means were compared using a two-way analysis of variance. The level of statistical significance was set at p < 0.05.

Validation of mesenchymal stem cells derived from adipose tissue, bone marrow, and tonsil using FACS
To confirm the effects of MSCs in various locations in the human body, we obtained MSCs from three different human tissues. Adipose tissue-, bone marrow-, and tonsil-derived MSCs have been reported to have cell proliferation and inflammation alleviation efficacy in various studies [4]. We established conditions for purely separating primary cultured MSCs from fresh tissues, and all the samples were validated with specific markers using FACS (Fig. 1a). All the tissues were collected from human donors at our institute. As shown in Fig. 1b, the expression of CD markers in MSCs was demonstrated as CD90, CD105, and CD73, and CD44 in FACS. The MSCs derived from adipose tissue, bone marrow, and tonsils showed similar morphology using microscopy (Fig. 1c).

Increased levels of inflammatory markers by LPS on human middle ear epithelial cells
Anti-inflammation research using MSCs has been reported in various fields [4]. In this study, we aimed to compare the anti-inflammatory effect of MSCs derived from three different tissues; therefore, we decided to use an in vitro model that can be used for a clinical study related to inflammation. HMEECs are used in OM research because they are easy to access as an initial model to prove the effect between anti-inflammatory reactions with stem cells [26]. We selected the effective concentration (FC20) that showed 81% from 1 µg/mL viability after 24 h (Fig. 2a) and 83% after 24 h with 1 µg/ mL LPS (Fig. 2b) because HMEECs must be maintained to extract cell-derived RNA to measure inflammatory factors. Cell imaging via microscopy showed that dead cells were observed by LPS ( Fig. 2c and d). To confirm the expression of inflammatory markers, we designed four primers (COX-2, TNF-α, IL-1β, and IL-6) for realtime PCR and found that the expression levels of these markers were increased by 1 µg/mL LPS for 24 h. The expression level increased rapidly after 6 h and remained constant after 24 h (Fig. 2e-h).

Evaluated cell viability and ROS reduction in the co-culture condition between HMEEC and MSC derived from adipose tissue, bone marrow, and tonsils
Since it is difficult to directly confirm the anti-inflammatory effect of donor cells using MSCs, we tried to prove the anti-inflammatory effect using EVs generated from the co-culture medium. EVs have been reported to occur even in MSC single cultures [32], but the effect of EVs in the co-culture media has not been reported. We hypothesized that since EVs play a role in cell-cell communication, internal substances (miRNAs, proteins, cytokine etc.) change because of the crosstalk between cells [21,33]. Therefore, adipose-derived MSCs (AD-MSCs), bone marrow-derived MSCs (BM-MSCs), and tonsil-derived MSCs (T-MSCs) were cultured alone. The EVs in the single culture media were labeled as AD-M, BM-M, and T-M. In addition, AD-, BM-, and T-MSCs were cocultured with HMEECs in a transwell plate to obtain a co-culture medium. The EVs in the co-culture media between MSCs and HMEECs were labeled H:AD-M, H:BM-M, and H:T-M (Fig. 3a). All the EVs were purified using an exosome isolation kit with high centrifugation and were validated in various ways. The efficacy of the EVs was compared after inducing inflammation with LPS in HMEECs pre-treated with EVs (Fig. 3b).
Cell viability was evaluated, as shown in Fig. 3c. When HMEECs were co-cultured with AD-MSCs, BM-MSCs, and T-MSCs, the cell viability was maintained at over 98% for 3 and 24 h. In other words, it was found that HMEECs and MSCs do not cause toxicity to each other through crosstalk. Interestingly, it was also observed that the ROS levels were reduced by the three MSCs after ROS production was stimulated by LPS in HMEECs (Fig. 3d). This suggests that they can be reduced by substances generated during co-culture with MSCs; therefore, it was hypothesized that the ROS-reducing substances in the co-culture medium were EVs. In addition, the amount of protein was evaluated in EVs through specific EV markers, such as CD9, CD63, and CD81 in co-culture media, and it was observed that the protein expression level gradually increased at 3 and 24 h (Fig. 3e). When co-cultured with AD-MSCs, BM-MSCs, and T-MSCs, the ratio of the amount of protein showed a similar tendency even when the amount of protein was quantified by area (Fig. 3f ). The fluorescence images of EVs were analyzed in MSC culture media using the EV marker CD63 through immunofluorescence (IF) (Fig. 3g). Green fluorescence intensity was detected in the media, except cells, and the phenome of EVs released from the MSC surface was observed through TEM (Fig. 3h). In addition, the EVs dissolved in the media were measured by NTA; the EV size was 87.5 ± 10.1 nm, and the concentration of EV was 5.74 × 10 10 ± 5.88 × 10 8 particles/mL (Fig. 3i). Therefore, we observed that the three MSCs and HMEECs could be co-cultured without toxicity by crosstalk and it can be predicted that a substance that reduces ROS is contained in the EV.
Decreased TNF-α, COX-2, IL-1β, and IL-6 in co-culture media ROS levels were reduced by co-culture with the three MSCs, but we isolated EVs from the media. When the HMEECs were co-cultured with the three MSCs and exposed to LPS, LPS stimulated both the MSCs and HMEECs. To evaluate the efficacy of the purified EVs,  (Fig. 4a). It also showed that the viability of HMEECs was more than 97% when treated with H:AD-M, H:BM-M, and H:T-M for 6-48 h (Fig. 4b).
Since the stability of the extracted EVs was confirmed, we compared the effect of inflammatory factors in HMEECs that induced inflammation by EC20 concentration of LPS.
To evaluate the anti-inflammatory effect of EVs isolated from single and co-culture media, the RNA expression level was confirmed through real-time PCR. To confirm the anti-inflammatory effect of EVs extracted from a single culture medium and co-culture medium, the expression of RNA inflammatory factor generation was observed using real-time PCR. After treating with EVs, the HMEEC cultured media was changed and treated with LPS for 0.  (Fig. 4f ).

Increased miRNA expression level in EVs by co-culture condition
The inflammatory factors were confirmed to significantly decrease in a short time in H:BM-M; therefore, we hypothesized that it would have been reduced by the factor possessed by EVs. In particular, in H:BM-M, it was hypothesized that the payload of a specific factor increases, and some studies have reported that some of the miRNA levels may vary depending on the co-culture conditions [9,33,34].
As a result of miRNA analysis by EVs derived from cultured media in six different environments, we confirmed that a total of 161 miRNA expression levels changed (Fig. 5a). Interestingly, as shown in the heatmap, it was confirmed that the miRNA level increased significantly under co-culture conditions. The miRNA expression level was compared by a fold change value of 1.5 and 2 times (Fig. 5b)  Furthermore, to analyze the miRNAs increased by co-culture, we selected the top five miRNAs with a large increase in expression level. Comparing IAD-M and H:AD-M, the highest miRNA expression was hsa-miR-3960, hsa-miR-2115-5p, hsa-miR-320e, hsa-miR-8075, and hsa-miR-6732-5p (Fig. 5c). In addition, in the results of comparing BM-M and H:BM-M, the highest miRNA expression was hsa-miR-638, hsa-miR-5787, hsa-miR-5189-3p, hsa-miR-6732-5p, and hsa-miR-2115-It is 5p (Fig. 5d). Finally, the results of comparing T-M and H:T-M, the highest miRNA expression were hsa-miR-2115-5p, hsa-miR-5787, hsa-miR-6732-5p, hsa-miR-8075, and hsa-miR-320e (Fig. 5e). Interestingly, miRNAs that overlapped with each other were found, and a large amount of miRNA was observed to increase in H:BM-M.
To confirm the intersection of each EV sample from the three different MSCs, we performed a band diagram analysis and found that a total of 51 miRNAs were changed for overlapping factors under the three conditions (Fig. 6a). In addition, the number of upregulated miRNAs was 42 under the three conditions (Fig. 6b) (Fig. 6c).

The amyloid-based binding proteins combined to decrease inflammatory factors in H:BM-M
Following the results of miRNA analysis, we analyzed gene ontology (GO) to understand the role and biological function of proteins related to these miRNAs. In a previous experiment, we confirmed that when EVs were pre-treated with HMEEC and then induced inflammation with LPS, the result of rapidly reducing the inflammatory factor after 1 h by EVs derived from co-culture media (Fig. 4). It was assumed that there would be information on useful molecules in H:BM-M, which significantly reduced the gene expression of TNF-α, COX-2, IL-1β, and IL-6 in inflammation-induced cells. Therefore, we analyzed the expression of miR-638, which was highly expressed in H:BM-M.
First, most of the proteins expressed for miR-638 included factors that affected the development of intracellular proteins in donor cells. It is also involved in RNA transcription polymerase-related factors and cell-cell signaling. In addition, there was a small expression level related to the factors involved in differentiation (Fig. 7a).
Next, the relevance of the cellular component was analyzed, and an association with a total of 10 organelles was shown. The predicted proteins were mainly related to the intracellular, membrane, and cytoplasm (Fig. 7b). In addition, among them as a result of analyzing molecular function, it was confirmed that the factors related to protein binding significantly increased. In particular, the expression of the amyloid-beta binding protein was found to be the largest (Fig. 7c). It has been reported that amyloid-beta A4 precursor protein-binding family A member 2 (APBA2) is involved in synaptic transport and junction of neurons [35], but they are also involved in improving immunity and cell regeneration [36]. We hypothesized that the anti-inflammatory effect of HMEECs was increased by the expression of miRNA-638 in H:BM-M and APBA2 could be expected to be a candidate connection protein (Fig. 7d). APBA2 also showed an association of 0.989 with STXBP1 and a low association with NRXN1 and STX1A (Fig. 7e). Therefore, we concluded that BM-MSCs significantly increased

Discussion
Research on the development of therapeutic materials using human MSC-derived EVs has been actively reported in the field of regenerative medicine and antiinflammatory materials [5,37,38]. Representative human stem cells of various origins, such as adipose tissue, bone marrow, umbilical cord, and tonsil, have been reported [5,14]. Interestingly, some studies have reported that MSCs derived from different tissues have different payloads to communicate with recipient cells as donor cells by using EVs with a determined size of less than 100 nm [5,14]. However, there has not been a study comparing the components of MSC-derived EVs of different origins to discover therapeutic materials for inflammatory diseases or cancer diseases. We hypothesized that EVs derived from different origin influence different inflammatory effects on one recipient cell. Here, we would like to propose that two new facts.
(1) The anti-inflammatory effects of EVs derived from MSCs of different origins are indicated by differences in payloads. (2) Crosstalk between cells by co-culture can improve anti-inflammatory effects by altering the payload of EVs. MSCs were extracted from three different origin (Adipose tissue, Bone marrow, and Tonsil), and the change in payload of EVs obtained by co-culture with middle ear epithelial cells (HMEEC) in transwells was analyzed. Adipose tissue (AD)-derived MSCs express mRNAs related to transcription factors, angiogenesis, and adipogenesis [18,27]. Bone marrow (BM)-derived MSCs are widely known for their regenerative, immune stimulatory, and related factors, such as calcium signaling and cytoskeletal genes [20]. Tonsil (T)-derived MSCs have been reported as cells whose clinical therapeutic efficacy has been confirmed through studies in which the expression of inflammatory cytokines was increased through injection in acute and chronic colitis models [28,39]. In this study, using a model that causes otitis media (OM), a disease with a high incidence in children, we confirmed the efficacy of MSC-derived EV to reduce inflammatory factors (TNF-α, COX-2, IL-1β, and IL-6). The expression of inflammatory factors, induces immune regulation, mucosal changes, and inflammatory responses, leading to the development of OM [26]. Among these, TNF-α, which plays an important role in the induction of an inflammatory cascade, is known to induce OM [39]. COX-2 is the major enzyme that converts arachidonic acid to prostaglandin H2 to produce inflammatory mediators. The expression level of COX-2 was upregulated in the middle ear of patients with OM compared to that in healthy subjects [40]. It has been reported that it can damage hearing if the inflammation of HMEECs increases significantly [41]. OM is mainly focused on in clinical research, but this finding of molecules in stem cell-derived EVs can be valuable in development of a new material by reducing the death of middle ear cells and ROS reduction by inflammatory factors [41].
The viability of HMEECs was over 98% during co-culture with AD-MSCs, BM-MSCs, and T-MSCs, as verified by FACS (Fig. 1b), confirming that no toxic substances were generated. The expression levels of inflammatory markers significantly increased after 24 h of exposure to 1 µg/mL LPS, and the cell viability also decreased to less than 80%. We found that apoptosis was induced by exposure to LPS after 24 h (Fig. 2).
To analyze the change in payload in the MSC-derived EVs that we hypothesized, donor MSCs and recipient HMEECs were co-cultured in trans wells. The EVs were separated from the co-culture medium by centrifugation, and it was confirmed that ROS production induced by LPS was decreased by EVs in HMEECs (Fig. 3d). Based on these results, it was proved that the MSC-derived EVs, which are labeled AD-M, BM-M, and T-M in a single culture, contained factors capable of reducing ROS levels. Furthermore, there might be several useful factors expressed in co-culture media because large amounts of CD9, CD63, and CD81 (EV markers) were found after co-culture of 24 h (Fig. 3e and f ). In other words, the co-culture conditions showed that EV generation can be increased by the crosstalk between donor and recipient cells, suggesting that the payload can be changed.
It was confirmed that the size of EVs in the single medium and co-culture media was less than 100 nm (Fig. 3 h and i), and the levels of inflammatory factors expressed by LPS were decreased by EVs. The EVs derived from MSC single culture media (AD-M, BM-M, and T-M) reduced the mRNA expression levels of TNFα, COX-2, IL-1β, and IL6 by half (Fig. 4). Interestingly, among the EVs derived from co-culture media (H:AD-M, H:BM-M, and H:T-M), H:BM-M showed the greatest efficiency in reducing inflammatory factor expression until zero by pretreatment with HMEECs for 3 h. Therefore, we hypothesized that some molecules had been delivered to the recipient cells, which is related to reduced expression of inflammatory factors.
Interestingly, miR-638, which significantly reduced TNF-α, COX-2, IL-1β, and IL-6 ( Fig. 4) expression, showed increased expression only in H:BM-M. One of the predicted mRNAs analyzed Solute carrier family 25-member 23 (SLC25A23) and Adhesion G proteincoupled receptor E5 (ADGRE5) was reported to be involved in both adhesion and signaling processes [45]. SLC25A23 reportedly decreases mitochondrial Ca (2+) uptake and reduces cytosolic Ca (2+) clearance after histamine stimulation [51]. In addition, approximately 100 predicted mRNAs were analyzed, and we tried to select the most related proteins through gene ontology analysis. We hypothesized that miR-638-related protein inhibited the increase in inflammatory factors in HMEECs; therefore, we predicted that it was APBA2 (Fig. 7c). It is cautious to predict proteins that have organic relationships in cells, but we found that APBA2 can play a role in binding and transmitting factors related to binding to membranes containing immunity and differentiation [36]. APBA2 encodes a cell surface receptor and transmembrane precursor protein that is cleaved by secretases to form a number of peptides.
In addition, two of these peptides are antimicrobial peptides, which have been shown to have bactericidal and antifungal activities. Mutations in this gene are associated with autosomal dominant Alzheimer's disease and cerebral arterial amyloidosis (cerebral amyloid angiopathy). Several transcriptome variants have been discovered that encode several different isotypes of this gene [52]. As reported for EVs to communicate between cells, a major difference among vesicle subtypes is their origin, while microvesicles and ectosomes bud from the plasma membrane, EV formation begins on early endosomes [6,7]. After maturation in multivesicular bodies through the invagination of the endosomal membrane and formation of intraluminal vesicles (ILVs), EVs are released by the fusion of ILVs with the plasma membrane [14]. The specific proteins incorporated into ILVs are regulated mainly by the endosomalsorting complex required for transport (ESCRT), of which there are four: ESCRT-0, I, II, and III. As a result, we discussed that binding proteins, such as APBA2, can induce and stimulate the transport of inflammatory factors by connecting the cellular membrane. In a study that reported the activity of APBA2, it was reported in patients with age-related macular degeneration (AMD), and autophagy factors were identified by treating ARPE-19 cells with soluble amyloid with an oligomer [53]. Autophagy activated in ARPE-19 cells treated with APBA2 altered the expression pattern of LC3 by creating a phagocytic compartment and activating p62, thus clarifying the potential ambiguity mechanism of retinal cells. It has been reported that APBA2 is involved in the MAPK signaling pathway and it alters cell viability, migration, and invasion [52]. However, the relationship between amyloid activity and neuroinflammation has been reported [54]; the role of inflammation in auditory hearing loss relief should be explored in further studies. MSCs were extracted from three tissues and the changes in miRNA payload in EVs and the anti-inflammatory effect of MSC-derived EVs were analyzed in this study. In particular, to improve the anti-inflammatory effect of epithelial cells that induce inflammation, EVs of epithelial cells and MSC co-culture medium were extracted, and the payload was confirmed to change. This study was conducted based on the essential meaning of EV as an intercellular messenger to confirm the payload of HMEEC-derived EV and MSC-derived EV. We believed that the EVs derived from co-cultured media generated containing new molecules by the cell-to-cell communication process, so EVs in a single culture medium was used as controls. The payload in the EVs showed significantly different when co-culture between HMEEC and MSCs in Fig. 5. However, it was not possible to prove the factors expressed from different cell line in the single culture medium, we tried to explain reflecting the biological characteristics of EV for future studies. We discussed that the mechanism of cross-talk between different cell lines makes it possible to express unique biomarkers. Our study predicts that the change in the payload in EVs expressed by co-culture with stem cells can be used in various disease models. Therefore, the co-culture of stem cells into donor and recipient cells lead to the expression of new factors, which could prove valuable in the field of regenerative medicine and material development.