Human umbilical cord mesenchymal stem cells-derived exosomes exert anti-inflammatory effects on osteoarthritis chondrocytes

Culture and identification of hUC-MSCs

Isolation and culture of hUC-MSCs

The Ethics Committee of the Second Hospital of Jilin University and the Ethics Committee of the Qingyuan People's Hospital granted approval for human umbilical cord collection and all experimental procedures (approval no.: 2019-142, IRB-2023-017).

After obtaining approval and patient consent, umbilical cord was obtained from a healthy newborn at term. For the detailed method of hUC-MSCs isolation, refer to the previous literature [14]. Umbilical cord tissues were rinsed 3 times with phosphate buffer (PBS; ThermoFisher) containing 5% Penicillin/Streptomycin (P/S; ThermoFisher). Umbilical vein and umbilical artery were removed, and Wharton’s jelly tissue was retained. Wharton's jelly tissue was cut into small pieces (about 0.5×0.5mm), attached to petri dishes, and dropped into medium containing 10 ng/mL basic fibroblast growth factor (bFGF; PeproTech), 1% P/S, 10% fetal bovine serum (FBS; ThermoFisher) and 89% Dulbecco's modified eagle medium nutrient mixture F-12; (DMEM/F12; ThermoFisher) for culture. When the confluence of the cells reached approximately 90%, the cells were rinsed with PBS and then digested with 0.25% EDTA-trypsin (ThermoFisher). The cells were centrifuged at 300× g and subculture. The fifth passage of cells was used for identification and exosome collection experiments.

Immunofluorescence staining

An immunofluorescence staining assay was conducted to identify surface markers. The primary antibodies mainly include: anti-CD31 (1:100; MA1-26196, ThermoFisher), anti-CD44 (1:1000; MAB7045, R&D Systems, UK), anti-CD45 (1:100; 16-0451-85, ThermoFisher), anti-CD73 (1:100; 41-0200, ThermoFisher), anti-CD90 (1:100; PA5-82115, ThermoFisher) and anti-CD105 (1:100; MA5-17041, ThermoFisher). hUC-MSCs were incubated overnight with primary antibodies and, after rinsing, incubated with secondary antibodies (Alexa Fluor 488; 1:1000; CST). Hoechst 33342 (10 μg/mL; ThermoFisher) is utilized to stain the nucleus of hUC-MSCs. Images of stained hUC-MSCs were acquired by fluorescence microscope. The negative control consisted of cells stained only with the secondary antibody.

Differentiation ability

Multilineage differentiation potential of hUC-MSCs was examined by chondrogenic, adipogenic and osteogenic differentiation assays. For chondrogenic differentiation, 20 μL suspension drop the fifth passage of hUC-MSCs (8 × 10^6 cells/mL) were cultured into chondrospheres Subsequently, these spheres were cultured in chondrogenic induction medium consisting of high glucose-Dulbecco's Modified Eagle Medium (HG-DMEM; ThermoFisher) supplemented with 10% FBS, 50 nM of ascorbate-2-phosphate (Sigma-Aldrich), 10 ng/mL transforming growth factor-beta 1 (PeproTech, USA) and 6.25 μg/mL insulin. The induction medium was changed at 3-day intervals. After three weeks of chondrogenic induction and histopathological manipulation the tissue sections of cartilage spheres were stained with toluidine blue (Dingguo, China) according to the manufacturer's protocol. When the fusion degree of the fifth passage of hUC-MSCs was about 80%, adipogenic induction solution, which contained HG-DMEM supplemented with 10% FBS, 1% P/S, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 10 μM insulin (Sigma-Aldrich), 200 μM indomethacin (Sigma-Aldrich) and 1 μM dexamethasone (Sigma-Aldrich) was added. The induction medium was changed at 3-day intervals. After continuous culture for 2 weeks, the lipid inclusion vacuoles were detected using oil red O (Sigma-Aldrich) according to the previous methods of our research group [15]. When the fusion degree of the fifth passage of hUC-MSCs was about 60%, osteogenic induction solution, which contained HG-DMEM supplemented with 10% FBS, 1% P/S, 50 μM ascorbate-2-phosphate (Sigma-Aldrich), 0.1 μM dexamethasone and 10 nM β-glycerophosphate (Sigma-Aldrich) was added. The induction medium was changed at 3-day intervals. After continuous culture for 3 weeks, the calcium salt was stained with alizarin red S (Sigma-Aldrich) according to the previous methods of our research group [15]. Images were recorded using an inverted fluorescence microscope.

Isolation and identification of hUC-MSCs-Exos

Isolation of hUC-MSCs-Exos

Ultracentrifugation was used to obtain hUC-MSCs-Exos [16]. When hUC-MSCs had grown to approximately 50% confluence, they were rinsed once with PBS and replaced with exosomes-free medium for 48 hours (containing 89% DMEM/F-12, 10 ng/mL bFGF, 10% FBS without exosomes (SBI, USA) and 1% P/S). The hUC-MSCs culture supernatant was centrifuged at 300 ×g for 10min at 4° C in order to remove the cells. The hUC-MSCs culture supernatant from which the cells were removed was centrifuged for 10min at 2,000×g in order to remove debris of cells. Subsequently, supernatants collected from the above experiments were centrifuged at 10,000×g for 30min at 4° C in order to remove organelles. After serial centrifugations, the supernatant of hUC-MSCs culture medium was centrifuged by ultracentrifugation at 120,000 ×g for 90 min at 4° C using a SW32Ti rotor (Beckman Coulter, USA) to obtain hUC-MSCs-Exos. The hUC-MSCs-Exos were rinsed once with PBS and centrifuged by ultracentrifugation to remove the mixed proteins. The hUC-MSCs-Exos were dissolved in 400ul PBS, packaged separately, and stored at -80° C in a refrigerator for future use.

Identification of hUC-MSCs-Exos

To identify hUC-MSCs-Exos, a series of experiments were conducted. The number of hUC-MSCs-Exos was detected utilizing ExoELISA-ULTRA (CD81 detection; System Biosciences, USA) according to the instructions. The size distribution of hUC-MSCs-Exos was detected utilizing ZETASIZER Nano series-Nano-ZS (Malvern Instruments, UK). The data was analyzed utilizing zetasizer software (Malvern Instruments). Additionally, the morphology of the hUC-MSCs-Exos was observed by transmission electron microscopy (TEM). Briefly, the hUC-MSCs-Exos were dropped-added to the copper mesh, fixed by adding 2% uranyl acetate (Sigma-Aldrich) and allowed to air dry at room temperature. The images of hUC-MSCs-Exos were acquired through TECNAI 12 TEM (FEI, USA). The markers of exosome were assessed using western blotting, with antibodies against CD81 (1:1000; ab79559, Abcam), CD9 (1:1000; 13403S, Cell Signaling), TSG101(1:1000; AF8262, Beyotime) and CD63 (1:1000; ab271286, Abcam, UK). Expression ratio of exosome markers were quantified by flow cytometry (BD Bioscienes, USA). hUC-MSCs-Exos were incubated with CD81-antibody-PE (1:5; BD) and CD63-antibody-FITC (1:5; BD) for 2 h on ice. hUC-MSCs-Exos without treatment were used as the negative control.

Anti-inflammatory effects of hUC-MSCs-Exos on human chondrocytes

The effect of hUC-MSCs-Exos on IL-1β-induced human chondrocyte

Human knee chondrocytes (PromoCell, Germany) were cultured using chondrocyte growth medium (PromoCell, Germany), and the third passage chondrocytes were used for subsequent experiments. The method used to replicate the cartilage inflammation model was previously reported in the literature [17]. After 24 hours of cell seeding, the chondrocyte growth medium was added with IL-1β (10 ng/mL; PeproTech, USA). The chondrocyte growth medium containing hUC-MSCs-Exos (4 × 10^7 particles/mL) or without were changed. After 48 hours, Chondrocytes were harvested and examined accordingly. Normal cultured chondrocytes were used as control.

hUC-MSCs-Exos co-culture with macrophages and chondrocytes in transwell systems

Chondrocytes were co-cultured with macrophages using 0.4 μm permeable cell culture chambers (Corning, USA) Briefly, THP-1 cells were seeded on the upper chambers of the permeable cell culture chambers at a density of 5 × 10^5 cells/chamber in 12-well with 25 ng/mL PMA for 24 h, followed by polarization to M1 macrophages with 100 ng/mL LPS for 24 h. Meanwhile, chondrocytes were seeded in the lower chamber of the permeable cell culture chambers at a density of 5 × 10^4 cells/chambers for 24 h. For further incubation with hUC-MSCs-Exos, macrophages chambers were washed with PBS, moved into chondrocytes chamber, added fresh hUC-MSCs-Exos in the upper chamber for 48 h. The co-culture system with LPS and hUC-MSCs-Exos were supplemented as the experimental group, the one without hUC-MSCs-Exos was used as the control group, and the one without LPS and hUC-MSCs-Exos were used as the blank control group.

RT-qPCR

Total RNA was extracted applying trizol (ThermoFisher, USA) according to the manufacturer's instructions. cDNA was synthesized utilizing Titan One Tube RT-PCR kit (Roche, Switzerland) and according the manufacturer’s guidelines. qPCR was conducted using FastStart Universal SYBR Green Master (Roche) according to the manufacturer's instructions on CFX Connect Real-Time System (Bio-Rad, Germany). The expression abundance of the detected mRNA was calculated using the 2^(-ΔΔCT) method. RT-qPCR primers targeting GAPDH (NM_001256799.2), IL-1β (NM_000576.2), ARG1(NM_000045.2), IL-10 (NM_000572.2), COL2A1 (NM_033150.2), MMP13 (NM_002427.3) and TNF-α (NM_001286813.1) were procured from Guangzhou GeneCopoeia Co., Ltd (China).

Immunofluorescence staining

Chondrocytes were incubated with antibodies against MMP13 (1:1000; MAB511, R&D Systems) and COL2A1 (1:100; MA5-12781, ThermoFisher), followed by corresponding secondary antibody (Alexa Fluor 488, 1:1000). Hoechst 33342 is utilized to stain the nucleus of chondrocytes. Images of stained chondrocytes were acquired by fluorescence microscope.

TUNEL staining

Chondrocytes were washed with PBS, fixed with 4% paraformaldehyde for 30min, and permeabilized with 0.1% TritonX-100 for 15min. And then, chondrocytes were stained with In Situ Cell Death Detection Kit (Roche) according to the instructions. Chondrocytes were visualized under an inverted fluorescence microscope after counterstaining with hoechst 33342 (10 μg/mL).

Exos small RNA sequencing and data analysis

Exosomal miRNA sequencing and bioinformatics analysis of miRNA target genes

Exosomal miRNA sequencing was performed by Ribobio Co. Ltd (China). Exosomes derived from three volunteers' hUC-MSCs were employed.

The reads that remained after filtering were mapped to the miRbase database (v22) to quantify their expression against known miRNAs. The read count for each miRNA was obtained by performing comparison and correction. The expression abundance of hUC-MSCs exosomes in the top 50 miRNAs was screened. Target gene of the top 50 miRNAs prediction and bioinformatics analysis were performed. The most significant terms/pathways for the three primary categories (molecular functions, biological processes and cell components) in the Gene Ontology (GO) database, as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were displayed.

Chondrocyte RNA sequencing and data analysis

After treatment by IL-1β with and without hUC-MSCs-Exos for 48 h, the total RNA of chondrocytes was extracted applying Trizol. mRNA sequencing was performed by Ribobio Co. Ltd (China). The genes up-regulated in IL-1β treated chondrocyte, while genes down-regulated after being treated with hUC-MSCs-Exos and relative to OA target genes (GeneCards database, https://www.genecards.org/), were take intersection by Venn diagrams and used to further data analysis. KEGG and GO analyses were carried out on the intersection mRNAs.

Statistical analysis

All results were repeated at least three times. Data was presented as the mean ± standard deviation. GraphPad Prism 7.0 software was used to analyze the data. Independent-sample t-test was used to compare two different groups, while ANOVA was employed for statistical analysis of multiple groups of data. P < 0.05 was considered statistically significant.

Availability of data and material

The datasets generated during the current study are available.

Consent for publication

Consent for publication was obtained from all participants of this study.

Isolation and identification of hUC-MSCs

Cells emerged from the Wharton’s jelly tissue between days 7 and 14 and propagated in cell-culture dish, as revealed in Figure 1A. Cells derived from Wharton’s jelly tissue displayed a spindle-shaped morphology, as depicted in Figure 1B. The fifth passage cells were characterized and utilized in downstream experiments. The cells derived from Wharton’s jelly tissue that expressed the MSCs markers CD90, CD44, CD105 and CD73, but did not express vascular endothelial marker CD31 and leukocyte marker CD45. As revealed in Figure 1C–1F, the cells possessed multipotent differentiation potentials towards chondrocytes, adipocytes, and osteoblasts. Therefore, the cells derived from Wharton’s jelly tissue were considered as hUC-MSCs.

Figure 1. Culture and identification of hUC-MSCs. (A) On day 12, hUC-MSCs migrated from the Wharton’s jelly tissue. Scale bar=200 μm. (B) Images of long-scaled hUC-MSCs cultured for the fifth passage. Scale bar=200 μm. (C) Immunofluorescence staining of hUC-MSCs with surface markers CD44, CD73, CD90, and CD105 (green fluorescence). Hoechst 33342 (blue) was utilized to stain the nucleus of hUC-MSCs. Scale bar=50 μm. (D) Toluidine blue staining was used to detect the ability of hUC-MSCs to differentiate into chondrocytes. Scale bar=200 μm. (E) Oil red O staining was used to detect the ability of hUC-MSCs to differentiate into adipocytes. Scale bar=200 μm. (F) Alizarin red S staining was utilised to test the capacity of hUC-MSCs to differentiate into osteoblasts. Scale bar=200 μm.

Identification of hUC-MSCs-Exos

The oval vesicle of 30-200 nm diameter with a typical membrane structure of exosomes from hUC-MSCs was revealed by TEM, as depicted in Figure 2A. The results of nanoparticle showed that 82.5% of the hUC-MSCs-Exos had distribution rang 30-200 nm, as shown in Figure 2B. The western blotting results showed that the hUC-MSCs-Exos highly expressed the markers of exosome CD9, CD63, CD81 and TSG101 (Figure 2C). Flow cytometry confirmed some markers of exosome CD63 (71.3%) and CD81 (79.2%) (Figure 2D). Based on the aforementioned findings, it can be concluded that the isolation of hUC-MSCs-Exos was successful.

Figure 2. Characterization of hUC-MSCs-Exos. (A) Representative TEM images of hUC-MSCs-Exos. Scale bar=200 nm. (B) The particle size distribution of hUC-MSCs-Exos was detected by ZETASIZER Nano series-Nano-ZS. (C) Western blot detected the expression of exosomal markers (CD9, CD63, CD81 and TSG101). (D) Flow cytometry detected the proportion of expression of exosomal markers CD63 (71.3%) and CD81 (79.2%).

hUC-MSCs-Exos reverse IL-1β-induced chondrocyte inflammatory response

The inflammatory factor IL-1β has a constructive role in the disease progression of OA. IL-1β can induce chondrocytes to produce excess of matrix metalloproteinases (MMP), which causes degradation of extracellular matrix components of chondrocytes, including COL2A1 and aggrecan. In vitro, IL-1β is commonly employed to induce inflammation in chondrocytes of OA [18]. Therefore, agents targeting the IL-1β-induced chondrocyte inflammatory response can potentially attenuate the progression of OA [19]. As revealed in Figure 3A, IL-1β downregulated the gene expression of COL2A1 and upregulated that of MMP13, successfully establishing an IL-1β-induced chondrocyte inflammation model. Notably, supplementation with hUC-MSCs-Exos in chondrocyte inflammation model reversed the protein expression of MMP13 and COL2A1, consistent with results of RT-qPCR (Figure 3B). To conclude, hUC-MSCs-Exos demonstrated the ability to alleviate the inflammation of chondrocytes provoked by IL-1β in vitro model.

Figure 3. The anti-inflammatory effect of hUC-MSCs-Exos was evaluated chondrocytes inflammation model. (A) The expressions of COL2A1 and MMP13 mRNA of chondrocytes were assayed by qRT-PCR. This experiment was repeated three times. **P < 0.01, ***P < 0.001. (B) Immunofluorescence staining was used to detect the protein expression of COL2A1 and MMP13 (green) in chondrocytes. Hoechst 33342 (blue) is utilized to stain the nucleus of chondrocytes. Scale bar=50 μm.

hUC-MSCs-Exos inhibit M1 macrophage damage to chondrocytes

Activation and polarization of synovial macrophages to M1 phenotypes play a major role in OA progression [20]. M1 macrophages is activated by LPS, which secretes high levels of pro-inflammatory cytokines, such as IL-1β, TNF-α and low levels of anti-inflammatory factors of ARG1 and IL-10, that lead to inflammation of the chondrocytes [21]. In order to investigate whether hUC-MSCs-Exos inhibit M1 macrophage damage to chondrocytes. A co-culture system of M1 macrophages and chondrocytes was established as shown in Figure 4A. The addition of hUC-MSCs-Exos and LPS to macrophages in the upper chamber resulted in down-regulation of the expression of TNF-α and IL-1β genes and up-regulation of ARG1 and IL-10 genes compared to macrophages with LPS alone (Figure 4B). Chondrocytes co-cultured with macrophages added with hUC-MSCs-Exos and LPS showed upregulated expression of the chondrocyte marker COL2A1 gene and protein and downregulated expression of the chondrocyte catabolic enzyme marker MMP13 gene and protein compared to chondrocytes co-cultured with M1 macrophages (Figure 4C, 4D). M1 macrophages were able to induce apoptosis in chondrocytes. However, when hUC-MSCs-Exos were added, the number of apoptotic chondrocytes was significantly reduced compared to chondrocytes co-cultured with M1 macrophages (Figure 4E). The results demonstrated that hUC-MSCs-Exos had a protective effect against M1 macrophage-induced chondrocyte damage and apoptosis.

Figure 4. The anti-inflammatory effect of hUC-MSCs-Exos was evaluated by co-culture system of macrophages and chondrocytes. (A) Graphical abstract of co-culture system of chondrocytes and macrophages. (B) The expressions of IL-1β, TNF-α, IL-10 and ARG1 mRNA of macrophages were assayed by qRT-PCR. This experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The expressions of COL2A1 and MMP13 mRNA of chondrocytes were assayed by qRT-PCR. This experiment was repeated three times. *P < 0.05, **P < 0.01. (D) Immunofluorescence staining was used to detect the protein expression of COL2A1 and MMP13 (green) in chondrocytes. Hoechst 33342 (blue) is utilized to stain the nucleus of chondrocytes. Scale bar=100 μm. (E) TUNEL (green) staining of chondrocytes. Hoechst 33342 (blue) is utilized to stain the nucleus of chondrocytes. Scale bar=100 μm.

hUC-MSCs-Exos miRNA sequencing and bioinformatic analysis

Exosomes are rich in a variety of miRNAs, which regulate the biological behavior of cells after binding to target genes. The top 50 high-abundance miRNAs in hUC-MSCs-Exos were screened according to the total read count and presented in Figure 5A. Previous studies have shown there was a high abundance of miRNAs of hUC-MSCs-Exos, such as miRNA-21-5p and miR-146a-3p which play important roles in regulating inflammation of chondrocytes [22, 23]. In chondrocytes, up-regulation of miRNA-21-5p can increase the expression of COL2A1, decrease the expression of MMP13 and ADAMTS5, and regulate the ECM balance of chondrocytes. miRNA-21-5p may be a key miRNA in OA as it promotes hyaline cartilage production [22]. miRNA-146a plays a key role in inflammatory diseases [23]. Furthermore, miRNA-146a induces downregulation of M1 markers and upregulation of M2 markers in macrophages, regulating macrophage polarization [24]. In addition, functional enrichment analysis was able to predict the dominant pathway controlled by miRNA of hUC-MSCs-Exos [23]. The MAPK signaling pathway, metabolic pathway, PI3K-Akt signaling pathway and cancer pathway were the primary KEGG pathway enrichments (Figure 5B). GO analysis depicted that the biological process component was dominated by “single-organism process” and “cellular process”, while the cellular component was characterized by “cell part” and “intracellular.” Additionally, the molecular function component was enriched in “protein binding” and “organic cyclic compound binding,” as demonstrated in Figure 5C–5E. In summary, these conclusions obtained by bioinformatics analysis indicate a possible contribution of miRNAs of hUC-MSCs-Exos in regulating inflammation of chondrocytes.

Figure 5. Sequencing and bioinformatics analysis of miRNAs in hUC-MSCs-Exos. (A) Top 50 highly expressed hUC-MSCs-Exos miRNAs. (B–E) KEGG and GO (GO molecular function, GO biological process and GO cellular component) analysis of genes regulated by the top 50 highly expressed miRNAs of hUC-MSCs-Exos. The color of bars indicates p values, and p< 0.05 was considered significant.

Chondrocyte mRNA sequencing and bioinformatic analysis

As sequencing results showed, there were 1675 genes up-regulated in IL-1β-induced chondrocytes compared to normal chondrocytes, there were 275 genes down-regulated in hUC-MSCs-Exos-treated chondrocyte by induced IL-1β compared to IL-1β-induced chondrocytes (Figure 6A). The osteoarthritis-related genes in the GeneCards database were intersected with the up- and down-regulated genes in the sequencing results, and 22 genes were finally screened (Figure 6B, 6C). KEGG pathway enrichment analysis was performed, and a total of 6 pathways were obtained (Figure 6D). Among them, MAPK and PI3K-Akt is an important signaling pathway for hUC-MSCs-Exos to exert anti-inflammatory effects in OA. GO analysis (Figure 6E–6G) showed that “regulation of cell development process” and “regulation of lipid metabolic process” in the biological process component, “extracellular matrix” and “external encapsulating structure” in the cellular component, “signaling receptor activator activity” and “signaling receptor regulator activity” in the molecular function component had the high significance.

Figure 6. Sequencing and bioinformatic analysis of mRNA in chondrocytes. (A) Volcano plot of genes differentially expressed by sequencing of chondrocytes from different groups. (B) Venn diagram of 1675 genes up-regulated in chondrocytes treated with IL-1β relative to normal chondrocyte, 275 genes downregulated in chondrocytes treated with IL-1β and hUC-MSCs-Exos relative to chondrocytes treated with IL-1β, and 3095 genes related to osteoarthritis in GeneCards database. (C) Heatmap of the screened 20 differentially expressed genes. (D–G) KEGG and GO (GO biological process, GO cellular component and GO molecular function) analysis of the screened 20 differentially expressed genes. The color of bars indicates p values, and p< 0.05 was considered significant.