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Organoids display long-term vascular network, endogenous ECM secretion, sensitive vasculature, and multipotent MSCs (A) Immunofluorescence staining of day 14 VMO cross-section demonstrates the distribution of mesenchymal stem/stromal cells (MSCs) throughout the organoid and endothelial cells forming the vasculature. The inset highlights a closer view of the vasculature and MSCs. (B) Hematoxylin and eosin (H&E) staining shows most cells located at the edges of the organoid, with large voids in the center. (C) 3D reconstruction using z stack imaging, demonstrating interconnected blood vessels within a Day 14 organoid. (D) Long-term maintenance of vasculature over 60 days, including endothelial progenitors (CD34 + ) observed through VMO immunostaining of whole mount organoids. (E) Self-secreted extracellular matrix (ECM) molecules, collagen I and laminin V, are within the organoid. (F) Analysis of <t>PECAM1</t> (qPCR normalized to housekeeping gene and control) and E-Selectin (fluorescence inside VMO normalized to control) expression in organoids cultured in inflammatory and control media, showing increased E-selectin expression inside Day 14 VMO and reduced PECAM1 levels under inflammatory conditions, consistent with tight junction relaxation during inflammation. (G) qPCR shows that organoids cultured in osteogenic medium express low levels of RUNX2 on day 14 and day 28 compared to controls and significantly higher levels of Osteopontin ( OPN) at day 28, indicating the presence of mature osteoblasts. Scale bars 50 μm (A–C, E), 20 μm (D, inset A). (F–G) show mean values ± SD with n = 4 (F) or n = 3 (G); each data point represents a replicate consisting of 16 pooled organoids. One-way ANOVA with Tukey’s multiple comparisons, and unpaired t test comparisons were used. Asterisks denote statistical significance: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. See also .
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Image Search Results


Organoids display long-term vascular network, endogenous ECM secretion, sensitive vasculature, and multipotent MSCs (A) Immunofluorescence staining of day 14 VMO cross-section demonstrates the distribution of mesenchymal stem/stromal cells (MSCs) throughout the organoid and endothelial cells forming the vasculature. The inset highlights a closer view of the vasculature and MSCs. (B) Hematoxylin and eosin (H&E) staining shows most cells located at the edges of the organoid, with large voids in the center. (C) 3D reconstruction using z stack imaging, demonstrating interconnected blood vessels within a Day 14 organoid. (D) Long-term maintenance of vasculature over 60 days, including endothelial progenitors (CD34 + ) observed through VMO immunostaining of whole mount organoids. (E) Self-secreted extracellular matrix (ECM) molecules, collagen I and laminin V, are within the organoid. (F) Analysis of PECAM1 (qPCR normalized to housekeeping gene and control) and E-Selectin (fluorescence inside VMO normalized to control) expression in organoids cultured in inflammatory and control media, showing increased E-selectin expression inside Day 14 VMO and reduced PECAM1 levels under inflammatory conditions, consistent with tight junction relaxation during inflammation. (G) qPCR shows that organoids cultured in osteogenic medium express low levels of RUNX2 on day 14 and day 28 compared to controls and significantly higher levels of Osteopontin ( OPN) at day 28, indicating the presence of mature osteoblasts. Scale bars 50 μm (A–C, E), 20 μm (D, inset A). (F–G) show mean values ± SD with n = 4 (F) or n = 3 (G); each data point represents a replicate consisting of 16 pooled organoids. One-way ANOVA with Tukey’s multiple comparisons, and unpaired t test comparisons were used. Asterisks denote statistical significance: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. See also .

Journal: iScience

Article Title: Free-floating long-term vascularized mesenchymal organoids

doi: 10.1016/j.isci.2025.114548

Figure Lengend Snippet: Organoids display long-term vascular network, endogenous ECM secretion, sensitive vasculature, and multipotent MSCs (A) Immunofluorescence staining of day 14 VMO cross-section demonstrates the distribution of mesenchymal stem/stromal cells (MSCs) throughout the organoid and endothelial cells forming the vasculature. The inset highlights a closer view of the vasculature and MSCs. (B) Hematoxylin and eosin (H&E) staining shows most cells located at the edges of the organoid, with large voids in the center. (C) 3D reconstruction using z stack imaging, demonstrating interconnected blood vessels within a Day 14 organoid. (D) Long-term maintenance of vasculature over 60 days, including endothelial progenitors (CD34 + ) observed through VMO immunostaining of whole mount organoids. (E) Self-secreted extracellular matrix (ECM) molecules, collagen I and laminin V, are within the organoid. (F) Analysis of PECAM1 (qPCR normalized to housekeeping gene and control) and E-Selectin (fluorescence inside VMO normalized to control) expression in organoids cultured in inflammatory and control media, showing increased E-selectin expression inside Day 14 VMO and reduced PECAM1 levels under inflammatory conditions, consistent with tight junction relaxation during inflammation. (G) qPCR shows that organoids cultured in osteogenic medium express low levels of RUNX2 on day 14 and day 28 compared to controls and significantly higher levels of Osteopontin ( OPN) at day 28, indicating the presence of mature osteoblasts. Scale bars 50 μm (A–C, E), 20 μm (D, inset A). (F–G) show mean values ± SD with n = 4 (F) or n = 3 (G); each data point represents a replicate consisting of 16 pooled organoids. One-way ANOVA with Tukey’s multiple comparisons, and unpaired t test comparisons were used. Asterisks denote statistical significance: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. See also .

Article Snippet: PECAM1 , Invitrogen , Hs01065279_m1.

Techniques: Immunofluorescence, Staining, Imaging, Immunostaining, Control, Fluorescence, Expressing, Cell Culture