Journal: Stem Cell Research & Therapy
Article Title: Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing
doi: 10.1186/s13287-025-04501-4
Figure Lengend Snippet: Combining dual inhibition of TP53 and DNAme maintenance during Cas9-mediated NHEJ improves the efficiency on reactivating XIST in female hPSCs. a Experimental scheme for assessing the effects of TP53 and DNAme inhibition on XIST reactivation in female hPSCs. For DNAme inhibition, the DNMT1-specific inhibitor GSK3685032 was applied. Cells were pre-treated with (for 24 h) or without GSK3685032 before transfection, and those in the DNAme inhibition group were maintained in GSK3685032 for 48 h post-transfection prior to GFP sorting. Sorted cells were then assessed for H3K27me3 foci (XCI hallmark) by immunofluorescence (IF). The representative image on the right shows H3K27me3 foci. In transfection, a vector expressing a dominant-negative form of TP53 (mmTP53) was co-transfected with Cas9-EGFP and gRNA vectors targeting the XIST promoter region. b Representative images of H3K27me3 and OCT4 staining, along with quantification results. Each dot represents the percentage of H3K27me3 foci cells with OCT4 expression within individual colonies, with at least 218 cells analyzed per colony. Statistical analysis was conducted using one-way ANOVA with post-hoc Tukey’s HSD test. Scale bar: 100 μm. c Experimental scheme for optimizing the timing of GSK3685032 treatment. d Quantitative results showing the percentage of H3K27me3 foci-positive cells within colonies. Only OCT4-positive cells were analyzed, with at least 383 cells counted per colony. e XCI status assessment for sub-cloned XIST -reactivated 253G1-derived lines (4e, 5d, and 6f). (Upper panel) Representative images of H3K27me3, H2AKub119 foci, and HUWE1 expression status. IF was used for H3K27me3 and H2AKub119 analysis, and RNA-FISH was used for HUWE1 expression. In FISH assay, DNA was stained with DAPI. Scale bar: 20 μm. (lower panel) Quantification results based on analysis of over 316 cells in IF and 139 cells in FISH per line. f Scalable expansion of XCI-rescued lines. Day 0 represents the day of transfection. XCI-rescued lines were cultured to reach over 10⁷ cells in the presence of RI and assessed for XCI status by IF of H3K27me3. Only OCT4 positive cells were counted. (right graph) Quantification of XCI-positive cells. At least 584 cells were analyzed per line to confirm XCI restoration. g Model of dual inhibition approach for XIST reactivation. Transient inhibition of TP53 and DNMT1 during the NHEJ process boosts genome editing efficiency and prevents DNA methylation maintenance, thereby enhancing the efficiency of XIST reactivation
Article Snippet: hPSCs were treated with GSK3685032 (#HY-139664; MedChemExpress, NJ, USA) for indicated periods and concentrations in each figure.
Techniques: Inhibition, Transfection, Immunofluorescence, Plasmid Preparation, Expressing, Dominant Negative Mutation, Staining, Clone Assay, Derivative Assay, Cell Culture, DNA Methylation Assay
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