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(A) Schematic overview of the experimental design for CHARM editing, tracking and sorting of CD34 + CD45RA − CD90 + cells, and subsequent megakaryocytic differentiation. (B) Top, representative <t>CFSE</t> dilution profiles of CD34 + CD45RA − CD90 + cells at days 2 and 7 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Bottom, targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in the sorted CD34 + CD45RA − CD90 + cells. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (C) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from sorted CD34 + CD45RA − CD90 + cells following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (D) Schematic overview of the experimental design for extended cytokine-free culture and subsequent megakaryocytic differentiation following CHARM editing of HSPCs. (E) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs at day 11 and day 29 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (F) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from HSPCs after extended cytokine-free culture following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (G) Schematic overview of the experimental design for serial CFU replating and targeted bisulfite sequencing following CHARM editing of HSPCs. (H) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs after CHARM editing and in cells derived from primary (1°), secondary (2°), and tertiary (3°) CFU replatings following targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. All data are presented as mean ± SD, significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, or n.s. not significant.
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(A) Schematic overview of the experimental design for CHARM editing, tracking and sorting of CD34 + CD45RA − CD90 + cells, and subsequent megakaryocytic differentiation. (B) Top, representative CFSE dilution profiles of CD34 + CD45RA − CD90 + cells at days 2 and 7 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Bottom, targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in the sorted CD34 + CD45RA − CD90 + cells. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (C) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from sorted CD34 + CD45RA − CD90 + cells following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (D) Schematic overview of the experimental design for extended cytokine-free culture and subsequent megakaryocytic differentiation following CHARM editing of HSPCs. (E) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs at day 11 and day 29 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (F) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from HSPCs after extended cytokine-free culture following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (G) Schematic overview of the experimental design for serial CFU replating and targeted bisulfite sequencing following CHARM editing of HSPCs. (H) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs after CHARM editing and in cells derived from primary (1°), secondary (2°), and tertiary (3°) CFU replatings following targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. All data are presented as mean ± SD, significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, or n.s. not significant.

Journal: bioRxiv

Article Title: Epigenome editing of human hematopoietic stem cells enables sustained and reversible thrombosis prevention

doi: 10.64898/2026.03.27.714536

Figure Lengend Snippet: (A) Schematic overview of the experimental design for CHARM editing, tracking and sorting of CD34 + CD45RA − CD90 + cells, and subsequent megakaryocytic differentiation. (B) Top, representative CFSE dilution profiles of CD34 + CD45RA − CD90 + cells at days 2 and 7 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Bottom, targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in the sorted CD34 + CD45RA − CD90 + cells. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (C) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from sorted CD34 + CD45RA − CD90 + cells following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (D) Schematic overview of the experimental design for extended cytokine-free culture and subsequent megakaryocytic differentiation following CHARM editing of HSPCs. (E) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs at day 11 and day 29 following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. (F) Left, representative flow cytometry histograms of CD61 ( ITGB3 ) surface expression in megakaryocytes differentiated from HSPCs after extended cytokine-free culture following CHARM targeting of the ITGB3 promoter or AAVS1 as a control. Right, quantification of the percentage of CD61 + cells among differentiated megakaryocytes. (G) Schematic overview of the experimental design for serial CFU replating and targeted bisulfite sequencing following CHARM editing of HSPCs. (H) Targeted bisulfite sequencing displaying CpG methylation patterns near the ITGB3 TSS in HSPCs after CHARM editing and in cells derived from primary (1°), secondary (2°), and tertiary (3°) CFU replatings following targeting of the ITGB3 promoter or AAVS1 as a control. Each circle represents an individual CpG site; filled circles indicate methylated CpGs. All data are presented as mean ± SD, significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, or n.s. not significant.

Article Snippet: CFSE fluorescence was assessed by flow cytometric analysis on a Cytek Aurora flow cytometer at day 0 and day 5 after labeling.

Techniques: Control, Methylation Sequencing, CpG Methylation Assay, Methylation, Flow Cytometry, Expressing, Derivative Assay