Free-floating long-term vascularized mesenchymal organoids

Abstract

The vasculature is essential for tissue function and pathology. Spheroid co-cultures of endothelial and marrow/mesenchymal stromal/stem cells (MSCs) form consistent structures, but the vascular components are short-lived. iPSC-derived vascular organoids can establish complex vasculature but often have variable cell maturation and low reproducibility. This article presents consistently formed, free-floating, long-term vascularized mesenchymal organoids (VMOs), by co-culturing human umbilical vein endothelial cells (HUVECs) and MSCs in a pre-gelled minimal Matrigel scaffold. VMOs support 60-day stable vasculature, exhibiting tissue maturation involving inflammation, extracellular matrix remodeling, and endothelial development. Compared to traditional spheroids, VMOs showed enhanced vascular complexity, sustained extracellular matrix production, and higher cell viability. The system preserved MSC heterogeneity including perivascular cell types, offering physiological relevance. Engraftment of breast cancer cells revealed stromal-tumor niches, enabling modeling of bone marrow metastasis. This robust platform offers an alternative model for studying vascular biology, stromal dynamics, and cancer progression, with potential applications in drug testing.

Keywords: Stem cells research; Tissue Engineering; Vascular remodeling.

Graphical abstract

Figure 1

VMOs formed at different EC:MSC ratios support long-term culture and preserve morphology (A) Schematic of the vascular mesenchymal organoid (VMO) formation process, combining human umbilical vein endothelial cells (HUVECs) and mesenchymal stem/stromal cells (MSCs) at 25:75, 50:50, and 75:25 ratios with Matrigel to generate organoids, no-Matrigel spheroid aggregates were cultured for comparison. (B) Time-lapse images show organoid formation during the first 3 days across different endothelial:MSC ratios. Organoids are fully formed by day 3 (D3) in all tested ratios. (C) Whole mount immunofluorescence analysis of D14 VMOs with different seeding ratios, demonstrating the presence of both VE-Cadherin⁺ endothelial cells and CD90⁺ MSCs. (D) Circularity measurements of organoids over 60 days of culture, showing stable (>0.8) circularity across all ratios. (E) Quantification of the cross-sectional area of organoids at different seeding ratios, showing that the 50:50 ratio produces the largest organoid area. (F) Analysis of luminescent cell viability signal over a 60-day culture period, revealing that the 50:50 ratio exhibits the highest proliferative capacity during the first 35 days, while the 25:75 ratio had the fewest cells throughout the culture duration. Two-way ANOVA with Šidák’s multiple comparisons test was used, and asterisks denote statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Scale bars 50 μm (C) and 100 μm (B). (D–F) show mean values ± SD with n = 16 individual VMO. See also Figure S1.

Figure 2

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 Figure S2.

Figure 3

VMOs exhibit enhanced proliferation, improved vascularization and ECM assembly, and an inflammatory phenotype compared to spheroids (A) Comparison between D14 VMO and spheroid architecture using whole mount staining, showing a mesh-like vasculature in the VMO and elongated vasculature in the spheroid. Spheroid vessels range from 4 to 8 μm, while VMO vessel diameters are up to 20 μm. (B) Vascularized area comparison with the D14 VMO, exhibiting a larger vascularized area. Values were normalized to the fraction of organoid or spheroid imaged. (C) Circularity measurements over 60 days, showing significant differences in circularity only at D14, with similar circularity between both structures during the rest of the culture time. (D) Cross-sectional area comparison between VMO and spheroid, with VMOs displaying a larger area. (E) Viable cell density over 60 days, demonstrating enhanced survival in VMOs compared to spheroids throughout the culture period. (F) Heatmap shows differential gene expression in D14 VMOs versus spheroids. (G) Top 10 gene ontology (GO) terms in D14 VMOs and spheroids, showing that VMOs upregulate angiogenesis, blood vessel development, and extracellular matrix (ECM) pathways. (H) Top 25 enriched pathways in VMOs identified by reactome analysis reveal the upregulation of ECM-related pathways, Notch signaling, RhoA GTPases, and IL2 signaling, indicating ECM remodeling, RhoA-associated cellular stretching, and inflammation. (I) Summary of the main differences between VMO and spheroids. Scale bars 50 μm (A). n = 8 individual VMO and n = 6 individual spheroid (B), n = 16 individual replicates (C–E). (B–E) Mean values ± SD. 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 Figure S3 and Tables S1, S2, and S3.

Figure 4

VMOs preserve cellular diversity with endothelial cells and heterogeneous MSC subpopulations (A) UMAP plot identifies 8 clusters derived from single-cell RNA sequencing of D3 and D14 VMOs. (B) Cluster annotation using canonical markers, assigning each cluster to a specific cell type. (C) Heatmap of top 5 GO enriched pathways per cluster. (D) Dot plot of canonical markers for bone marrow arteriolar and sinusoidal endothelial cells, showing a lean toward sinusoidal type in the endothelial cells from day 14. (E) Dot plot for the top 10 upregulated genes in the MSC fibroblastic cluster, revealing the enrichment of pathways related to ECM remodeling and cell adhesion. (F) Organoid mid-section staining identified MSC fibroblastic cells by their expression of fibronectin, a canonical marker, with localization at the edges of the organoid. (G) Localization of the MSC proliferative cluster based on Ki67 expression on organoid mid-section, Ki67+ indicates active proliferation. (H) Dot plot of the top 10 upregulated genes in the myofibroblast cluster, highlighting genes associated with ECM remodeling and soluble factor secretion. (I) Immunofluorescence staining of the organoid midsection for α-smooth muscle actin (α-SMA) confirmed the perivascular localization of myofibroblast cells. (J) Trajectory plot demonstrates that MSC-derived transitional cluster 1 connects to endothelial cells, suggesting an intermediate state between endothelial and MSC states. Scale bars 5 μm (E, G, I). See also Figures S4–S6 and Tables S4 and S5.

Figure 5

Organoids develop dynamic communication networks driving angiogenesis and ECM remodeling (A) Interaction analysis compares D3 versus D14 cell-cell communication, showing a higher number of interactions at D14. (B) Venn diagram compares pathways activated at D3 and D14 and showing common pathways between the two time points. D14 is associated with more upregulated angiogenic, sprouting, and ECM pathways compared to D3. Angiogenic/sprouting pathways upregulated at D14 are shown in red, common angiogenic pathways in green, ECM pathways upregulated at D14 in brown, and common ECM pathways in blue. (C) Top 5 angiogenic pathways, including PDGF, EGF, and Notch, which are present at both D3 and D14. While angiogenic pathways are predominantly active in MSC clusters at D3, endothelial cells and VSMC begin to participate more significantly in these pathways at D14. (D) Top 3 ECM pathways, revealing that at D3, ECM signaling is mainly driven by MSC clusters, whereas at D14, endothelial cells also play a key role in ECM-related pathways. See also Figures S7–S9.

Figure 6

VMOs cultured over 60 days exhibit temporal shifts in inflammation, ECM organization and endothelium development pathways (A) Volcano plots show differential expression of genes (DEGs) comparing different time points in the organoid culture: D3 vs. D14, D14 vs. D28, and D28 vs. D60. D3 vs. D14 and D28 vs. D60 exhibit the largest number of DEGs. (B) GO analysis of the top 10 upregulated and downregulated pathways between D3 and D14 shows the upregulation of ECM-related pathways and the downregulation of hypoxic pathways and endothelium development. (C) Top 10 differentially regulated biological processes identified by KEGG analysis in D3 versus D14 VMOs. Signaling pathways were upregulated while inflammation-related pathways showed downregulation. (D) GO analysis of the top 10 upregulated and downregulated pathways between D28 and D60 shows the upregulation of inflammation and the downregulation of vasculature and ECM development. (E) Top 10 differentially regulated biological processes identified by KEGG analysis in D28 versus D60 VMOs confirm the upregulation of inflammation and downregulation of cell adhesion and ECM-related pathways. See also Figures S10 and S11 and Tables S6, S7, S8, S9, S10, S11, and S12.

Figure 7

Applying VMOs as an in vitro platform to study breast cancer invasion in bone marrow (A) Representative images show the engraftment of MCF7 cells (green) into 3D organoid and spheroid models during the first 3 days after cancer cell seeding. Both models were derived from the same endothelial cells and MSCs. (B) Representative whole mount images of MCF7 tumors in VMO and spheroid cultures at D14 and D21 after seeding. VMOs maintain their structural integrity, while spheroids are overtaken by cancer cells by D21. (C) Intra-cellular flow cytometry staining for Ki67 in MCF7 cells cultured for 7 days in 2D (monolayer) or 3D (VMO) culture systems. A significant reduction in cell proliferation is observed in organoids compared to 2D culture. (D) Comparison of MCF7 cell fluorescence intensity (GFP) in 2D and 3D organoid cultures shows a significantly lower MFI in VMOs. (E) Schematic of the doxorubicin (DOX) dose-response experiment used to assess chemotherapy resistance in VMO and spheroid models, with DOX concentrations ranging from 0.01 μM to 100 μM. (F) Impact of DOX treatment on MCF7 cells grown in VMO and spheroid cultures measured by fluorescence intensity normalized to PBS vehicle-treated controls. VMOs demonstrate significantly increased protection from the effects of chemotherapy compared to spheroids across the DOX dose range (p < 0.05). (G) Decreased GFP fluorescence indicates decreased MCF7 cells in VMOs and spheroids treated with increasing concentrations of DOX. Scale bars 100 μm (A), 50 μm (B, G). (D, F) Mean values ± SD, n = 3 with each point representing cells pooled from 32 organoids (D) and n = 18 individual organoids (F). Unpaired t test and two-way ANOVA with Šidák’s multiple comparisons tests were used, and asterisks denote statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.