The aim of the current study was to compare the expression of microRNAs (miRNAs) in exosomes derived from human bone mesenchymal stem cells (hBMSCs) with and without chondrogenic induction. Exosomes derived from hBMSCs were isolated and identified. Microarray analysis was performed to compare miRNA expression between exosomes derived from hBMSCs with and without chondrogenic induction, and quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to verify the differentially expressed miRNAs. hBMSCs were transfected with miRNA mimic to extract miRNA‐overexpressed exosomes. The results showed that most exosomes exhibited a cup‐shaped or round‐shaped morphology with a diameter of approximately 50‐200 nm and expressed CD9 and CD63. We detected 141 miRNAs that were differentially expressed with and without chondrogenic induction by over a twofold change, including 35 upregulated miRNAs, such as miR‐1246, miR‐1290, miR‐193a‐5p, miR‐320c, and miR‐92a, and 106 downregulated miRNAs, such as miR‐377‐3p and miR‐6891‐5p. qRT‐PCR analysis validated these results. Exosomes derived from hBMSCs overexpressing miR‐320c were more efficient than normal exosomes derived from control hBMSCs at promoting osteoarthritis chondrocyte proliferation, down‐regulated matrix metallopeptidase 13 and up‐regulated (sex determining region Y)‐box 9 expression during hBMSC chondrogenic differentiation. In conclusion, we identified a group of upregulated miRNAs in exosomes derived from hBMSCs with chondrogenic induction that may play an important role in mesenchymal stem cell‐derived exosomes in cartilage regeneration and, ultimately, the treatment of arthritis. We demonstrated the potential of these modified exosomes in the development of novel therapeutic strategies.

The aim of this work was to determine whether miR-455-3p regulates DNA methylation during chondrogenic differentiation of hMSCs. The expression of miR-455-3p and de novo methyltransferase DNMT3A was assessed in micromass culture of hBMSCs, which induced chondrogenic differentiation in vitro, and in E16.5 mice in vivo. A luciferase reporter assay was used to confirm whether miR-455-3p directly targets DNMT3A by interaction with the 3′-UTR. Using an Illumina Infinium Methylation EPIC microarray, genome-wide DNA methylation of hBMSCs with or without overexpressed miR-455-3p was examined for 28 days during induced chondrogenic differentiation. Here, we showed that miR-455-3p was more expressed during the middle stage of hBMSC chondrogenic differentiation, and less expressed in the late stage. DNMT3A was less expressed in the middle stage and more expressed in the late stage, and was also more expressed in the palms of miR-455-3p deletion mice compared to those of wild-type mice. The luciferase reporter assay demonstrated that miR-455-3p directly targets DNMT3A 3′-UTR. miR-455-3p overexpression inhibits the degenerate process during chondrogenic differentiation, while deletion of miR-455-3p in mice accelerated cartilage degeneration. Genome-wide DNA methylation analysis showed miR-455-3p overexpression regulates DNA methylation of cartilage-specific genes. GO analysis revealed PI3K-Akt signaling pathway was most hypomethylated. Our data show that miR-455-3p can regulate hMSC chondrogenic differentiation by affecting DNA methylation. Overexpression of miR-455-3p and DNA methylation inhibitors can thus potentially be utilized to optimize chondrogenic differentiation.

Collagen type II (CII) induced arthritis (CIA) in mice is an experimental model for rheumatoid arthritis. Induction with non-self (e.g. human) CII induces severe arthritis whereas the mice are less susceptible to induction with self CII (i.e. mouse). To analyse whether an autoimmune response to human CII can develop and is pathogenic the authors have established transgenic mice expressing human CII in cartilage and backcrossed them into two different gene backgrounds susceptible to CIA (DBA/1 and C3H.Q). The transgenic human CII expression was restricted to cartilage and did not disturb cartilage morphology or lead to chondrodystrophy. In addition, development of stress-induced arthritis was not affected by the transgene. The cartilage specific expression of human CII reduced, but did not eliminate, the susceptibility to CIA irrespective of the species source (human, bovine, chick, rat) of CII used for immunization. A common denominator between these heterologous CII in comparison with mouse CII is the previously defined CII 256-270 epitope. An expression level dependent T-cell tolerance was seen in this epitope as well as to the entire CII. However, all human transgenic mouse lines could still mount significant autoreactive T-and B-cell responses. Approximately 10% of the transgenic mice developed arthritis after immunization with human CII. These findings show, therefore, that cartilage-located human CII induce tolerance but can nevertheless be a target for development of arthritis.