Small extracellular vesicles (sEVs) derived from different osteoarthritic (OA) tissues regulate OA-related biological processes through transporting their content (proteins, miRNAs, etc.) to recipient cells. This study aimed to characterize the miRNA profile of synovial fibroblasts-derived small EVs (FS_OA_sEVs) and investigate their role in inflammation in chondrocytes. Chondrocytes were isolated from macroscopically preserved and lesioned OA cartilage (C_OAmin and C_OAmax) and synovial fibroblasts from OA synovium. Synovial fibroblasts-derived small EVs (FS_OA_sEVs) were characterized according to ISEV guidelines and used for miRNA profiling and bioinformatics analysis. miR-21-5p was identified as one of the most abundant, and its target genes, such as KLF6, were enriched in OA-related processes including inflammation. Treatment of C_OAmin chondrocytes with FS_OA_sEVs resulted in decreased expression of COL2A1 and ACAN and an increase in catabolic markers MMP-3 and MMP-13. Moreover, C-OAmin receiving FS_OA_sEVs exhibited increased levels of inflammatory markers and miR-21-5p expression, resembling chondrocytes’ phenotype from lesioned OA cartilage, whereas miR-21-5p inhibition reversed their expression of inflammatory markers and miR-21-5p. Compared to C_OA min, C_OAmax chondrocytes exhibited increased miR-21-5p and inflammatory markers expression and decreased KLF6 expression. miR-21-5p inhibition in C_OAmax led to KLF6 upregulation and suppression of inflammatory mediators, whereas co-treatment with siRNA against KLF6 negated this effect, confirming a potential direct regulatory relationship between miR-21-5p and KLF6. Our results provide novel insights into the FS_OA_sEV-mediated inflammation axis, highlighting FS_OA_sEV-derived miR-21-5p as a driver of OA progression via regulating inflammation in chondrocytes.

Although MSCs grant pronounced potential for cell therapies, several factors, such as their heterogeneity restrict their use. To overcome these limitations, iMSCs (MSCs derived from induced pluripotent stem cells (iPSCs) have attracted attention. Here, we analyzed the transcriptome of MSCs, iPSCs and iMSCs derived from healthy individuals and osteoarthritis (OA) patients and explored miRNA-mRNA interactions during these transitions. We performed RNA-seq and gene expression comparisons and Protein-Protein-Interaction analysis followed by GO enrichment and KEGG pathway analyses. MicroRNAs’ (miRNA) expression profile using miRarrays and differentially expressed miRNA’s impact on regulating iMSCs gene expression was also explored. Our analyses revealed that iMSCs derivation from iPSCs favors the expression of genes conferring high proliferation, differentiation, and migration properties, all of which contribute to a rejuvenated state of iMSCs compared to primary MSCs. Additionally, our exploration of the involvement of miRNAs in this rejuvenated iMSCs transcriptome concluded in twenty-six miRNAs that, as our analysis showed, are implicated in pluripotency. Notably, the identified here interactions between hsa-let7b/i, hsa-miR-221/222-3p, hsa-miR-302c, hsa-miR-181a, hsa-miR-331 with target genes HMGA2, IGF2BP3, STARD4, and APOL6 could prove to be the necessary tools that will convey iMSCs into the ideal mean for cell therapy in osteoarthritis.

Background/Objectives: Emerging evidence shows long non-coding RNAs (lncRNAs) as critical regulators of osteoarthritis (OA) progression, often acting in complex networks with microRNAs (miRNAs). In our study, we investigated the potential regulatory function of the lncRNA MEG3/miR-21-5p axis in the OA phenotype of chondrocytes. Methods: Differential gene expression analysis in damaged vs. intact cartilage was performed, re-analyzing existing public RNA-seq data. MiRTarBase, LncRNADisease, and Open Targets databases were utilized to identify miR-21-5p target genes and OA-associated lncRNAs and genes. Functional enrichment analysis and protein–protein interaction (PPI) network construction were performed using the DAVID and STRING databases, respectively. MEG3, miR-21-5p, SOX5, COL2A1 and ACAN mRNA expressions were assessed by qRT-PCR. The role of the MEG3/miR-21-5p axis in OA chondrocytes was examined using transfection experiments. Results: Eighty-one lncRNAs displayed significant differences in expression between damaged and intact cartilage, including MEG3. Bioinformatic analysis indicated that MEG3 interacts with miR-21-5p, while SOX5 was identified to be a putative target of miR-21-5p. MEG3 and SOX5 expression levels were significantly downregulated in OA chondrocytes, whereas miR-21-5p expression was upregulated. Silencing of MEG3 resulted in increased miR-21-5p levels in chondrocytes. Conversely, inhibition of miR-21-5p led to increased SOX5 expression and anabolic markers COL2A1 and ACAN. Notably, MEG3 silencing significantly reduced SOX5 expression, an effect that was reversed upon miR-21-5p inhibition. Conclusions: Our findings highlight a potential regulatory role of the dysregulated MEG3/miR-21-5p axis in modulating the anabolic phenotype of chondrocytes through regulation of SOX5 expression. This novel lncRNA/miRNA/mRNA regulatory network may represent a candidate therapeutic axis for knee osteoarthritis.

Background/Objectives: Osteoarthritis (OA) is strongly associated with mitochondrial dysfunction, oxidative stress, and the accumulation of mitochondrial DNA (mtDNA) alterations, which contribute to impaired cellular homeostasis and reduced regenerative potential. In this study, we systematically characterized mtDNA heteroplasmy and variant distribution across bone marrow-derived mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs) and induced MSCs (iMSCs) derived from OA pa-tients and healthy donors. Methods: Ultra-deep mitochondrial genome sequencing was integrated with transcriptomic and miRNome analyses to investigate mitochondrial remodeling during cellular reprogramming and its interaction with nuclear-encoded mitochondrial pathways. Results: OA-derived MSCs exhibited a markedly increased heteroplasmic burden and accumulation of non-synonymous variants, particularly within OXPHOS-related genes, including MT-ND1, MT-ND2, MT-ND3, MT-ND5, MT-ATP8, MT-CO1, and MT-RNR1/2. Reprogramming into iPSCs and subsequent differentiation into iMSCs resulted in the progressive elimination of pathogenic mtDNA variants, in-cluding m.7913C>T and m.7821G>A, as well as substantial reduction of heteroplasmic hotspots, indicating a purifying mitochondrial remodeling process. Multi-omic analysis further revealed coordinated deregulation of mitochondrial-associated nuclear genes and competing endogenous RNA regulatory networks involving lncRNAs MEG3 and SNHG14, along with multiple mitochondria-related miRNAs. These findings suggest post-transcriptional regulation of mitochondrial adaptation in iMSCs. Conclusion: Col-lectively, our findings demonstrate that cellular reprogramming promotes mitochondrial genomic reorganization and indicates a potential metabolic recalibration. These results support the utility of iMSCs as a relevant model for studying OA-associated mitochondrial alterations and as a potential tool for regenerative approaches in OA.

Background/Objectives: Osteoarthritis (OA) is strongly associated with mitochondrial dysfunction, oxidative stress, and the accumulation of mitochondrial DNA (mtDNA) alterations in several joint-associated cell types, including chondrocytes, synoviocytes, and mesenchymal stromal cells (MSCs). These alterations contribute to impaired cellular homeostasis and reduced regenerative potential. In this study, we systematically characterized mtDNA heteroplasmy and variant distribution across bone marrow MSCs, induced pluripotent stem cells (iPSCs) and induced MSCs (iMSCs) derived from OA patients and healthy donors. Methods: Ultra-deep mitochondrial genome sequencing was integrated with transcriptomic and miRNome analyses to investigate mitochondrial remodeling during cellular reprogramming, encompassing changes in mtDNA heteroplasmy, variant distribution, mtDNA copy number and associated transcriptomic adaptations of nuclear-encoded mitochondrial pathways. Results: OA-derived MSCs exhibited a markedly increased heteroplasmic burden, followed by decrease in mtDNA copy number and accumulation of non-synonymous variants, particularly within OXPHOS-related genes, including MT-ND1-5, MT-ATP8, MT-CO1, and MT-RNR1/2. Reprogramming into iPSCs and subsequent differentiation into iMSCs were associated with an increase in mtDNA copy number and a progressive reduction in heteroplasmic variants predicted to have pathogenic potential, including m.7913C>T and m.7821G>A, as well as substantial reduction in heteroplasmic variant burden within several mitochondrial genes, suggesting mitochondrial genome remodeling during cellular reprogramming. Multiomic analysis further revealed coordinated deregulation of mitochondrial-associated nuclear genes and ceRNA regulatory networks involving lncRNAs MEG3 and SNHG14, along with multiple mitochondria-related miRNAs. These findings suggest post-transcriptional regulations associated with mitochondrial adaptation in iMSCs Conclusion: Collectively, our findings suggest that cellular reprogramming is associated with mitochondrial genomic reorganization in the donor-matched cell populations examined. These exploratory observations support the utility of iMSCs as a relevant model for studying OA-associated mitochondrial alterations and as a potential tool for regenerative approaches in OA.