Fig. 6: Persistent fibrotic activity in µDys-treated isogenic DMD MYOrganoids. | npj Regenerative Medicine

Fig. 6: Persistent fibrotic activity in µDys-treated isogenic DMD MYOrganoids.

From: Disease exacerbation in human DMD MYOrganoids enables gene therapy evaluation and unveils persistence of fibrotic activity

Fig. 6

A, C Volcano plot showing the differential gene expression in treated (dEx8-9-µDys) versus untreated (dEx8-9) DMD MYOrganoids (A) or DMD dEx8-9-µDys vs isogenic control (IsoCTR (C). Upregulated genes are shown in red, while downregulated genes are shown in blue. Numbers indicate the amount of downregulated and upregulated genes. B, D Gene ontology enrichment analysis of treated (dEx8-9-µDys) versus untreated (dEx8-9) DMD MYOrganoids (B) or dEx8-9-µDys vs IsoCTR (D). Normalized enrichment score (NES) and adjusted p value (p.adj) are indicated for each subcategory. X axis indicates the running enrichment score, while the y axis indicates the position in the ranked gene list. E Clustered Heat map and enlargement depicting relative transcript levels of differentially expressed genes in corrected DMD (IsoCTR) and DMD MYOrganoids transcriptomics treated or not with µDystrophin (dEx8-9 and dEx8-9 µDys high). Lower and higher expressions are depicted in blue and red, respectively. The data points above the significance threshold (p adjusted < 0.05, fold2change > 2) are marked in blue (downregulated) and red (upregulated), and others are marked in gray (not significant). F Relative gene expression of TGF-β, COL1A1, FN1 and ITGA10 in Isogenic control (IsoCTR), DMD dEx8-9 untreated and treated with empty vector and μDys at low and high dose. G Representative pictures of histological staining on longitudinal sections for phospho-SMAD (pSMAD, red), myosin heavy chain (MYHC, magenta marking the myotubes), GFP (green, marking the fibroblasts) and nuclei with DAPI in IsoCTR and µDys-treated DMD dEx8-9 MYOrganoids, and relative quantification of pSMAD3-positive nuclei in percentage in all CTR and DMD MYOrganoids. Myotube’s nuclei are circled in white, while fibroblasts’s nuclei are indicated in yellow. Scale bar = 20 µm. F, G data are presented as means ± SEM. F statistical analysis was performed with an ordinary one-way analysis of variance test with Tukey multiple comparison test (*p < 0.05, **p < 0.01, ns = not significant), while for G two-way analysis of variance test with multiple comparison and Šidák correction was performed (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 ns = not significant).

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