Fig 1: In vivo antifibrotic effects of various liposome preparations in the CDAHFD-induced fibrosis model.a Schematic of the CDAHFD-induced fibrosis model and treatment timelines for the various liposomes. b Representative images of liver sections stained for FAP (green), aSMA (red), and DAPI (blue) (n = 5 biologically independent mice per group). Scale bar: 50 µm. c, d FAP expression levels on HSCs from normal or CDAHFD-induced fibrotic liver were evaluated by flow cytometry (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PCDAHFD(+) - Normal < 0.0001). Results are presented as mean ± S.D.. e, f Representative Sirius red-stained liver images for the evaluation of liver fibrosis and quantification of the Sirius red-positive area (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal < 0.0001, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. g Top panel shows representative Masson’s trichrome-staining of liver. Bottom panel displays pseudocolored images by InForm 2.2.1 analysis, distinguishing fibrotic regions (blue) from normal tissue (red) (n = 5 biologically independent mice per group). Scale bar: 50 µm. h Quantification of Masson’s trichrome-stained connective tissue area calculated from five randomly selected fields per sample (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal < 0.0001, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum–maximum. i Collagen content of fibrotic liver samples, as measured using hydroxyproline assays. Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test PPRL–normal = 0.1877, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL = 0.0001). Results are presented as mean ± S.D. Blood was collected for serum analysis of AST (j) and ALT (k) (n = 5 biologically independent mice per group). Significant differences were assessed using a one-way ANOVA with Tukey test (Tukey’s test AST: PPRL–normal = 0.7672, PPRL–untreated = 0.0002, PPRL–PL = 0.0002, PPRL–ML = 0.0005, PPRL–SCL = 0.0003, ALT: PPRL–normal = 0.9835, PPRL–untreated < 0.0001, PPRL–PL < 0.0001, PPRL–ML < 0.0001, PPRL–SCL < 0.0001). Results are presented as mean ± S.D. CDAHFD, a choline-deficient, L-amino acid-defined high-fat diet; HSC hepatic stellate cell; UT untreated group. PL plain PEGylated liposomes; ML maleimide-activated PEGylated liposomes; SCL scrambled cys-promelittin peptide-tagged liposomes; PRL promelittin-modified liposomes; CD26 cluster of differentiation 26; CD31 cluster of differentiation 31; Ck7 cytokeratin7; aSMA alpha-smooth muscle actin; ALT alanine transaminase; AST aspartate transaminase. Source data are provided as a Source Data file.
Fig 2: The AGM Contains Two HSC Types in Explant Culture(A) Percentage donor cell chimerism in peripheral blood (PB) of adult irradiated transplant recipients at 4 months after injection of unsorted cells from E11 AGM explants (AGMex) cultured in serum-containing (+) or serum-free (-) medium with BMP4 and/or Shh, as shown below the graph. On to three AGM embryo equivalents (ee) were transplanted per recipient (n = 2 or 5; 1 or 4 mice transplanted/experiment). See Table S2. Each dot represents one recipient mouse. p = 0.0005 by z test for proportions.(B) Percentage donor cell chimerism in the PB of adult irradiated transplant recipients at 4 months after injection of E11 AGMex BRE GFP+ (+) or BRE GFP- (-) cells (2–4 ee transplanted/recipient; 1 or 2 mice transplanted/experiment; n = 7, 4, or 3). See Table S2. Culture conditions with cyclopamine and/or VEGF are indicated below the graph. p = 0.06 and p = 0.02 by z test for proportions. For (A) and (B), positive repopulation was considered to be >5% chimerism, as denoted by the gray dashed line.(C) qRT-PCR results for Ihh, Gli1, and Vegf expression in unsorted cells from AGMin (white bars) and AGMex (black bars). Error bars show ±SEM, with p value by t test (n = 3).(D) Gli1 transcript levels (FPKMs) in AGM cell fractions as detected by RNA sequencing. E11 AGM BRE GFP hematopoietic progenitor/stem cells (HPSC; CD31+cKit+), endothelial cells (EC, CD31+cKit-), and “other” non-HPSC, non-EC cells (OC; CD31-) were sorted by flow cytometry into GFP+ and GFP- fractions from AGMin (white bars), AGMex (black bars), and AGMex,cyclopamine (gray bars).
Fig 3: Pathway analysis using WGCNA reveals interesting new genes differentiating vWAT and scWAT responses to HFD. a Log2 Gene expression level of Nos2 and Ass1 in vWAT and scWAT of controls or HFD-treated mice for 1, 8 and 20 weeks (n = 6). b Heatmap of the WGCNA output. Each row represents a module containing the number of genes reported in parenthesis. Each column represents a contrast as indicated below the heatmap, from left to right: (1) tissue differences between CTR vWAT and CTR scWAT, (2) HFD-dependent effects in scWAT after 1 week, (3) HFD-dependent effects in vWAT after 1 week, (4) HFD-dependent effects in scWAT after 8 weeks, (5) HFD-dependent effects in vWAT after 8 weeks, (6) HFD-dependent effects in scWAT after 20 weeks, (7) HFD-dependent effects in vWAT after 20 weeks. c Zoom on the Chocolate4 module. d Pathway enrichment analysis performed on the genes contained in the Chocolate4 module (n = 321 genes). The dots are colored based on the significance of each pathway and the size represents the ratio between the number of genes in the cluster and in the pathway. Black underlined pathways are related to angiogenic processes, while the red ones are in common with pathways in Fig. 5. e Log2 Gene expression levels of the angiogenesis and epithelial cell-related genes Vegfa, Cdh5 and Pecam1 in vWAT (top) and scWAT (bottom) of controls or HFD-treated mice after 1, 8 and 20 weeks (n = 6). For a, e data are represented as mean ± SD. Significance between the indicated groups was calculated using a two-tailed Student’s t test. *(p value < 0.05), **(p value < 0.01), ***(p value < 0.001), ****(p value < 0.0001)
Fig 4: In vivo engraftment of 3D bioprinted and bulk hydrogels. (a) Implants after 15 days of engraftment, just before explantation (left column). TNN1 immunofluorescence staining used to detect CM and to study their orientation (middle column). The presence of vasculature was identified by vWF labelling (red) while to distinguish between host’s and engineered vasculature we performed Lamin A/C staining that univocally identifies capillaries originated from human endothelial cells (left column). Polar graphs represent the orientation of CM, with 0° corresponding to the fibers deposition direction (printed samples) or the X-axis of the image (bulk sample). Scale bars represent 50 µm and 100 µm. (b) Scatter plots indicating the percentage of CD31+ cells after the digestion of subcutaneous transplants with different geometries.
Fig 5: Transcriptome Analysis of E11 AGM Cell FractionsTranscriptional differences between BRE GFP+ and GFP- HPSCs (CD31+cKit+) as shown in a pie chart of significantly enriched GO categories for genes with >2-fold higher FPKMs in HPSC AGMex (A) GFP+ compared with GFP- and (B) GFP- compared with GFP+.(C) Gene set enrichment analysis of genes upregulated (>2 fold higher FPKM) in AGMex HPSC GFP+ fraction compared with GFP- in which the same genes are downregulated.(D) Gene set enrichment analysis of genes with >2-fold upregulated expression in AGMex HPSC GFP- fraction and downregulated in the GFP+ fraction. Gene set analysis was performed using the Enrichr web tool. Gene sets significantly enriched in GFP+ (2,229) or GFP- (1,876) (most highly significant gene sets are selected). Shown are transcription factor and drug gene sets with a consistent enrichment pattern, for which upregulated targets are enriched in one fraction while the downregulated targets are enriched in the opposite fraction.(E and F) Pie charts of significantly enriched GO and reactome gene categories with >2-fold higher expression in AGMex (E) other cells (OC; CD31-) and (F) endothelial cells (EC, CD31+cKit-) compared with the AGMin EC and OC, respectively. In (C) and (D), the Fisher exact test was performed for each gene set by comparing the number of GFP+ or GFP- genes and their overlap to each other. The calculated p values from Fisher exact tests were corrected for multiple testing using the FDR method. In all other panels, FDR correction for multiple testing was performed on chi-squared test outcomes from the Enrichr web tool.
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