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 did not show a peak in FACS. The MSCs derived from adipose tissue, bone marrow, and tonsils showed similar morphology using microscopy (Fig. 1c).
They were labeled AD-MSC, BM-MSC, and T-MSC since they are MSCs derived from the adipose tissue, bone marrow, and tonsil, respectively (a) The process of extracting and validating MSCs from three tissues, (b) The validation result of MSCs by FACS. MSCs were positive for CD90, CD105, and CD73 and negative for CD44, (c) Microscopic image of AD-MSC, BM-MSC, and T-MSC (bar=10 µm).
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 real-time 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, f, g, and 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 co-cultured 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 × 1010 ± 5.88 × 108 particles/mL (Fig. 3i). Therefore, we observed that the three MSCs and HMEECs could be co-cultured without toxicity by crosstalk and that the EVs containing the ROS-reducing material are released in the co-culture media.
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, they should be isolated from the media and compared with single culture media. The viability of HMEECs was evaluated using EVs isolated from single culture media (AD-M, BM-M, and T-M) and EVs extracted from co-culture media (H:AD-M, H:BM-M, and H:T-M). When LPS was treated at 1 mg/mL for 24 h as EC20, the cell viability was approximately 81%. In contrast, the viability of HMEECs treated with AD-M, BM-M, and T-M for 6–48 h was 98% (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 0.5, 1, 3, 6, 24, and 48 h. EVs derived from single culture media, AD-M, BM-M, T-M, showed decreased levels of TNF-α after 1 h of exposure, which were 7.0 ± 0.64 to 4.41 ± 0.17 by AD-M, 3.41 ± 0.13 by BM-M, and 3.34 ± 0.31 by T-M (Fig. 4c). In contrast, EVs derived from the co-culture medium H:AD-M, H:BM-M, and H:TM showed decreased levels of TNF-α after 1 h of exposure, which were 2.53 ± 0.12 by H:AD-M, 0.29 ± 0.04 by H: BM-M, and 2.44 ± 0.04 by H:T-M (Fig. 4c). Interestingly, in the case of EVs derived from co-culture media, the TNF-α expression level did not increase within 1 h by H:BM-M (Fig. 4c). H:AD-M and H:T-M showed similar expression levels to AD-M and T-M. The expression level of COX-2 stimulated by LPS confirmed that BM-M and T-M showed decreased expression levels after 24 h rather than AD-M.
It was also confirmed that the expression of COX-2 was reduced by co-culture medium-derived EVs. EVs derived from single culture media, AD-M, BM-M, and T-M, showed that the expression level of COX-2 decreased after 3 h of exposure, which were 5.59 ± 0.16, 1.71 ± 0.03, 2.83 ± 002, and 1.95 ± 0.01, respectively. In contrast, EVs derived from the co-culture medium H:AD-M, H:BM-M, and H:TM decreased the expression level of COX-2 after 3 h. t was confirmed that the expression level of COX-2 also decreased significantly which showed 1.64 ± 0.08 by H:AD-M, 0.08 ± 0.34 by H: BM-M, and 1.66 ± 0.01 by H:T-M after 1 h (Fig. 4d). In addition, it was confirmed that the IL-1β expression level decreased after 3 h by BM-M and T-M, but not by AD-M. EVs derived from single culture media, AD-M, BM-M, and T-M, showed that the expression level of IL-1β decreased after 3 h exposure, which were 7.55 ± 0.73 to 3.18 ± 0.02 by AD-M, 4.08 ± 0.04 by BM-M, and 3.64 ± 0.01 by T-M (Fig. 4e). In contrast, EVs derived from the co-culture media H:AD-M, H:BM-M, and H:TM decreased the expression level of IL-1β after 3 h, which were 2.89 ± 0.75 by H:AD-M, 0.35 ± 0.611 by H: BM-M, and 3.44 ± 0.13 by H:T-M. As a result, by EVs derived from the co-culture medium, IL-1β decreased after 1 h, and in particular, H:BM-M significantly reduced the expression of IL-1b after 3 h (Fig. 4e). The expression level of IL-6 was also reduced by EVs derived from MSC culture media after stimulation by LPS. EVs derived from single culture media, AD-M, BM-M, and T-M, showed that the expression level of IL-6 decreased after 3 h exposure, which were 3.43 ± 0.18 to 1.7 ± 0.04 by AD-M, 2.88 ± 0.04 by BM-M, and 2.13 ± 0.02 by T-M (Fig. 4f). In contrast, EVs derived from the co-culture media H:AD-M, H:BM-M, and H:TM decreased the expression level of IL-6 after 3 h, which were 1.33 ± 0.42 by H:AD-M, 0.049 ± 0.15 by H: BM-M, and 1.33 ± 0.64 by H:T-M. Almost all expression levels decreased in the positive control, the AD-M, BM-M, and T-M groups showed no decrease in gene expression levels after 48 h. However, IL-6 was significantly decreased after 30 min by H:BM-M, and the expression level decreased after 3 h (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). At 1.5 times, the expression of miRNA in AD-M and H:AD-M showed an increase of 85 miRNAs and a decrease of 46 miRNAs. The expression level of miRNA in BM-M and H:BM-M showed an increase of 125 miRNAs and a decrease of 5 miRNAs. The expression levels of miRNAs in T-M and H:T-M showed an increase of 126 miRNAs and a decrease of 1 miRNA. In addition, the expression level of miRNA was analyzed as a 2-fold change. There were 69 miRNAs and 34 downregulated miRNAs in the AD-M and H:AD-M groups. There were 102 miRNAs and two decreased miRNAs in BM-M and H:BM-M. There were 117 upregulated miRNAs in the T-M and H:T-M groups.
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), and the decreased miRNA did not intersect in the TM vs H:TM samples and one was found in AD-M vs. H:AD-M and BM-M vs H:BM-M (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 the expression of APBA2-related proteins in EVs in the medium by exchanging substances with HMEEC, thereby improving the anti-inflammatory effect of HMEECs.