This study investigates the complex interplay between gut microbiome dysbiosis and systemic inflammation as a critical risk factor in the pathogenesis of osteoarthritis (OA). Furthermore, it elucidates the role of gut microbiota (GMB) dysbiosis in driving OA progression. A refined “double-hit” murine model was developed to explore this relationship. The first intervention involved inducing gut microbiota dysbiosis through the administration of colistin and Escherichia coli, followed by surgical destabilization of the medial meniscus (DMM) to induce joint instability. The composition of the gut microbiota was analyzed using 16 S rRNA sequencing. Gut permeability was assessed via RT-PCR and immunofluorescence (IF), while mRNA sequencing was employed to examine alterations in gene expression. Treatment with colistin and E. coli significantly altered the gut microbiota composition, characterized by a marked increase in the absolute abundance of Firmicutes and a concomitant reduction in Bacteroidota and the Bacteroidota/Firmicutes (B/F) ratio. At the genus level, the absolute abundances of Muribaculaceae, Rikenellaceae_RC9_gut_group, and Roseburia were significantly diminished. GMB dysbiosis led to the downregulation of intestinal tight junction proteins, including ZO-1 and Occludin, resulting in increased intestinal permeability. Consequently, serum levels of lipopolysaccharide (LPS) were significantly elevated, indicating LPS translocation from the gut into systemic circulation. Notably, GMB dysbiosis markedly exacerbated OA progression, as evidenced by accelerated cartilage degeneration, increased osteophyte formation, and reduced bone mineral density (BMD). The OARSI scoring system revealed that OA severity in both colistin and E. coli treatment groups was significantly higher than in the control group. Additionally, GMB dysbiosis promoted the expression of inflammation-related genes in the synovium and induced M1 polarization of macrophages, demonstrated by the upregulation of CD86 and an elevated CD86/CD206 ratio. Correlation analyses indicated that Bacteroidota and the B/F ratio were positively associated with intestinal barrier integrity and negatively correlated with OA progression. In contrast, Firmicutes exhibited a positive correlation with inflammation and OA deterioration. These findings collectively underscore the critical role of GMB dysbiosis in modulating intestinal permeability, systemic inflammation, and OA pathogenesis. The protective effects of Bacteroidota and the B/F ratio, as well as the detrimental impact of Firmicutes, highlight potential therapeutic targets for mitigating OA progression through GMB modulation.

Genetic perturbations in nicotinamide adenine dinucleotide de novo (NAD) synthesis pathway predispose individuals to congenital birth defects. The NADSYN1 encodes the final enzyme in the de novo NAD synthesis pathway and, therefore, plays an important role in NAD metabolism and organ embryogenesis. Biallelic mutations in the NADSYN1 gene have been reported to be causative of congenital organ defects known as VCRL syndrome (Vertebral-Cardiac-Renal-Limb syndrome). Here, we analyzed the genetic variants in NADSYN1 in an exome-sequenced cohort consisting of patients with congenital vertebral malformations (CVMs). A total number of eight variants in NADSYN1, including two truncating variants and six missense variants, were identified in nine unrelated patients. All enrolled patients presented multiple organ defects, with the involvement of either the heart, kidney, limbs, or liver, as well as intraspinal deformities. An in vitro assay using COS-7 cells demonstrated either significantly reduced protein levels or disrupted enzymatic activity of the identified variants. Our findings demonstrated that functional variants in NADSYN1 were involved in the complex genetic etiology of CVMs and provided further evidence for the causative NADSYN1 variants in congenital NAD Deficiency Disorder.

Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUCMSCs) have emerged as promising therapeutic candidates for osteoarthritis (OA). Sirtuin 6 (SIRT6), a class III histone deacetylase, exerts protective effects by preventing cellular senescence, reducing inflammation, and promoting longevity. This study aims to generate immortalized hUCMSCs with SIRT6 and telomerase reverse transcriptase (TERT) overexpression and investigate the therapeutic potential of their secreted EVs (EVs@SIRT6) in OA. hUCMSCs were genetically engineered using lentiviral transduction to overexpress SIRT6 and TERT, referred to as SIRT6/TERT-hUCMSCs. Natural EVs and EVs@SIRT6 were isolated via differential ultracentrifugation and characterized by morphology, size distribution, marker expression, and proteomic signature. Their effects on IL-1β stimulated chondrocytes were assessed in vitro, including cell viability, apoptosis, scratch closure, inflammatory cytokine secretion, and oxidative stress. Transcriptomic alterations were analyzed by RNA sequencing. Therapeutic efficacy in vivo was evaluated in a rat anterior cruciate ligament transection (ACLT) model via micro-computed tomography, histological analyses, and immunohistochemistry. SIRT6/TERT-hUCMSCs preserved mesenchymal identity, trilineage differentiation potential, and sustained proliferative capacity up to passage 60, accompanied by longer telomeres and reduced senescence compared to parental cells. EVs@SIRT6 maintained typical EV features but were enriched in SIRT6 protein and displayed a distinct proteomic signature, comprising 1,440 unique proteins associated with nuclear/chromatin repair. In vitro, EVs@SIRT6 more effectively enhanced chondrocyte viability and wound healing while reducing apoptosis compared with natural EVs. EVs@SIRT6 also markedly alleviated inflammatory response, promoted anabolism, suppressed catabolism, and mitigated oxidative stress in chondrocytes. The protective effects of EVs@SIRT6 on chondrocytes were associated with the suppression of the JAK-STAT signaling pathway. Studies in a rat ACLT model further confirmed that EVs@SIRT6 outperformed natural EVs in attenuating subchondral osteosclerosis, reducing osteophyte formation, and mitigating cartilage damage. Engineered EVs@SIRT6 outperform natural EVs in preserving chondrocytes homeostasis and reducing OA progression, establishing an efficient platform for preparing engineered EVs for clinical application.

TBX6-associated congenital scoliosis (TACS) is a genetic subtype of congenital scoliosis caused by reduced TBX6 gene dosage, most commonly through compound inheritance of a rare loss-of-function allele and a hypomorphic T-C-A haplotype. However, the mechanisms linking TBX6 dysfunction to impaired human skeletal development remain unclear. Here, we established a patient-derived induced pluripotent stem cell (iPSC) model of TACS and recapitulated key stages of axial skeletal development through stepwise differentiation into paraxial mesoderm, nascent somites, sclerotome, and subsequently osteogenic and chondrogenic lineages. TACS-derived cells showed impaired differentiation, with reduced mineralized matrix deposition and diminished cartilage matrix formation. Bulk transcriptomic profiling revealed marked alterations in both lineages. Notably, mu-cin-type O-glycan biosynthesis was consistently enriched among downregulated genes in osteogenic and chondrogenic cells and was also significantly represented among genes commonly downregulated across both lineages. qRT-PCR further confirmed reduced expression of GALNT3, GALNT5, GALNT7, GALNT10, GCNT1, and ST6GALNAC2, which participate in distinct steps of mucin-type O-glycan biosynthesis. Consistent with these transcriptional alterations, Tn-antigen staining showed reduced Tn-associated O-GalNAc signals in TACS-derived osteogenic and chondrogenic cells. In silico promoter analysis identified candidate sequences matching the TBX6-binding motif within the upstream regions of these genes, providing potential regulatory regions for future experimental investigation. Collectively, these results establish a human iPSC model for TACS and identify coordinated dysregulation of a mucin-type O-glycan biosynthesis-related transcriptional program, accompanied by reduced Tn-associated O-GalNAc signals, as molecular features associated with impaired osteochondral differentiation in this model. These findings should be interpreted within the context of a single family-based iPSC model, and validation in additional TACS lines and isogenic TBX6-corrected models will be required.

Mammary morphogenesis is a highly coordinated process involving cellular differentiation, proliferation, and organization to form a bilayered epithelial network of ducts and lobules within the stromal matrix. Here, we identified the DDB1 and CUL4 associated factor 8-like 1(DCAF8L1) as a novel regulator of mammary morphogenesis. To investigate its role, we established stable DCAF8L1-expressing MCF10A cell lines, which normally lack DCAF8L1 expression. Overexpression of DCAF8L1 enhanced cell proliferation, migration, and induced branching morphogenesis and vacuolar structure assembly in three-dimensional (3D) culture. Subsequently, transcriptomic and proteomic analyses identified the discoidin domain receptor tyrosine kinase 1 (DDR1) as a key downstream effector of DCAF8L1. Further investigation revealed that DCAF8L1 upregulates DDR1, leading to activation of the Notch signaling pathway. These findings suggest that DCAF8L1 drives mammary branching morphogenesis and vacuolar structure assembly via DDR1-mediated Notch activation in 3D-cultured MCF10A cells.

Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases, and there is no approved pharmacological therapy that can reliably reverse cartilage degeneration or restore cartilage structure. Allogeneic mesenchymal stromal cells (MSCs) have shown therapeutic potential for OA, but inflammatory conditions can enhance HLA expression and increase allogeneic immune recognition risk. Here, we generated B2M and CIITA double-knockout induced pluripotent stem cells (dKO-iPSCs) using CRISPR/Cas9 and subsequently differentiated them into dKO-iMSCs. B2M/CIITA double-knockout did not affect iPSC pluripotency, karyotype, or teratoma formation. dKO-iMSCs retained characteristic MSC phenotypes, exhibited greater proliferative capacity than tissue-derived MSCs, and could be expanded to passage 18. Notably, HLA-ABC and HLA-DR expression remained undetectable in dKO-iMSCs even after IFN-γ stimulation. In co-culture assays with PHA-L-stimulated allogeneic PBMCs, dKO-iMSCs suppressed PBMC and T-cell proliferation as effectively as wild-type iMSCs, indicating preservation of their immunomodulatory activity. Intra-articular administration of dKO-iMSCs significantly reduced cartilage degeneration in the destabilization of the medial meniscus (DMM) mouse model of OA. The treatment showed greater therapeutic efficacy than wild-type iMSCs and tissue-derived MSCs and did not cause evident systemic organ toxicity. Single-cell RNA sequencing of BM-MSCs and dKO-iMSCs after in vitro IFN-γ stimulation revealed enhanced extracellular matrix-associated cell–cell communication and markedly reduced IL1B expression in dKO-iMSCs, indicating a less pro-inflammatory and more matrix-supportive cellular state under inflammatory conditions. Collectively, B2M/CIITA double-knockout enables sustained reduction in HLA expression while preserving the MSC phenotype and immunomodulatory activity, and enhances the therapeutic efficacy of iMSCs in the mouse OA model, providing a potential strategy for developing iPSC-derived MSCs with reduced HLA expression for allogeneic cell therapy.