Fig 2: A schematic illustrating the novel relationship between genes that induce bone regeneration in human bone marrow stem cells transfected with METTL7A genes.The METTL7A gene is shown to induce bone regeneration by regulating the methylation of genes involved in osteogenic differentiation and cell survival.

Fig 3: In vivo new bone formation in segmental bone defect models.A Creation of critical-size segmental long-bone defect in immunosuppressed rat. Length of the defects was 4 mm. hBMSCs were implanted with fibrin glue into segmental defects of rats created in the radius of right forelimb. B Radiographic images of segmental defects on days 28 and 56 after the implantation of hBMSCs. C MicroCT images of segmental long-bone defects at 56 days after cell implantation. D After 56 days, bone volume (BV, mm3), BV/TV (bone volume/total volume, %), and BMD (bone mineral density, mg/cc) were evaluated by MicroCT. E Goldner’s trichrome staining of healed segmental defects on day 56 post implantation of hBMSCs. Statistical processing was performed in comparison with the control group. Data were presented as mean ±standard deviation (SD, N = 4). NS: no significant difference, *p < 0.05, **p < 0.01. Normal: no surgery and no cell implantation; control: surgery only without cell implantation; MiniCircle (MC): minicircle control vector-transfected hBMSCs implantation; MC-METTL7A (MC-M7): METTL7A-transfected hBMSCs implantation.

Fig 4: Identification of the novel gene associated with enhanced survival and osteogenic differentiation in hBMSCs.A RNA-seq data analysis of hBMSCs. Analysis of hierarchical clusters of genes significantly related to each sample via RNA-seq data analysis. A heatmap was generated to visualize transcriptomic differences among undifferentiated hBMSCs cultured in control medium with 5.5 mM glucose [G(+)CM], osteo-induced hBMSCs cultured in osteogenic medium containing 5.5 mM glucose [G(+)OM] or no glucose [G(−)OM] (fold-change > 2, P < 0.05). Red indicates upregulation, while blue indicates downregulation. B Pathway analysis in Functional Annotation for significant probe list was performed using DAVID for G(+)OM and G(−)OM. An analysis of difference in signaling pathways related to metabolic process, stem cell proliferation, cell differentiation, and osteoblast development. C A selected heatmap was generated to visualize relative transcriptomic differences among transcripts from G(+)CM, G(+)OM, and G(−)OM (fold-change > 2, P < 0.05). D Relative gene expression of METTL7A was determined by RT-qPCR, and E Western blot. Data were presented as mean ± standard deviation (SD, N = 3). NS: no significant difference, *p < 0.05, **p < 0.01. G(+)CM: undifferentiated hBMSCs cultured in control medium with 5.5 mM glucose; G(+)OM: osteo-induced hBMSCs cultured in osteogenic medium containing 5.5 mM glucose; G(−)OM: osteo-induced hBMSCs cultured in osteogenic medium without glucose.

Fig 5: Cell viability and osteogenic differentiation of METTL7A gene-transfected hBMSCs.A Construction of METTL7A minicircle plasmid vector. Vector also contains RFP fluorescent genes. B Fluorescent, bright-field and merge images of minicircle control vector (MiniCircle)- or METTL7A-transfected hBMSCs (METTL7A; RFP-tagged). C Western blots of METTL7A in hBMSCs. D Cell viability of hBMSCs cultured in glucose-free osteogenic medium on days 7, 14, and 21. E Alizarin Red staining images and F quantification of hBMSCs cultured in glucose-free osteogenic medium after 7 days of culture. Data were presented as mean ± standard deviation (SD, N = 3). NS: no significant difference, *p < 0.05, **p < 0.01. hBMSCs: untransfected hBMSCs control; MC: minicircle plasmid vector (pMC)-transfected hBMSCs control; METTL7A:hMETTL7A- transfected hBMSCs.