Modeling embryonic heart vascular plexus development and sympathetic innervation on a human heart organoid

Figures

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  Figure 1. EMOs stimulation with VEGFA and PDGFBB improves vascularization and induces EMT of epicardial cells (A) Scheme illustrates EMOs generation from hiPSC-derived myocardium and pro-epicardium organoids (MO and PEO), with VEGFA and PDGFBB stimulation from day 6 to day 10 of co-culture. (B) Percentage of EMOs area that stains for the EC marker CD31, with and without VEGFA and PDGFBB supplementation. Data were obtained from representative IF images of at least 4 independent experiments. ∗p < 0.05, ∗∗∗∗p < 0.0001. (C) Representative IF staining of V-EMOs (3D and organoid slices), highlighting the development of a CD31+ network of vascular cells surrounding the myocardium region. (D and E) Representative IF staining of V-EMOs (organoid slices), highlighting the sprouting of CD31+ cells toward the myocardium region (B), and the co-staining of the CD31 network with the ECM laminin and the pericyte marker NG2 (C). (F) Representative IF staining of the epicardium region in P-EMOs (organoid slices), highlighting the presence of two distinct layers in the epicardium region, an external mesothelium-like layer and a sub-epicardium space adjacent to the myocardial region (NKX2.5+). (G and H)…

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  Figure 2. Single-cell RNA sequencing analysis identifying cell types in V-EMOs and P-EMOs (A) t-SNE plot of filtered cells (n = 19,024) from combined V-EMOs and P-EMOs conditions, indicating eight unbiased clusters. Dot plot shows the average expression and percentage of cells expressing canonical markers for each cluster. (B) t-SNE plot (n = 501) highlights the compact myocardial-like subpopulation within the cardiomyocyte cluster. (C) Violin plots show the expression of compact myocardium markers within cardiomyocyte cluster. (D) t-SNE plot (n = 11,229) of combined Epicardium/early EPDC and epicardium-derived mesenchyme/fibroblast-like cells clusters, with pseudotime trajectories inferred from transcriptional kinetics. (E) t-SNE plot highlights the subclustering analysis of Epicardium/early EDPCs cluster. (F) Violin plots show the expression of epicardium mesothelium (cluster 1) genes within the epicardium/early EPDCs cluster. (G) Representative dot and t-SNE plots for epicardial mesothelium, early EPDCs, and epicardial mesenchyme markers, highlighting their transcriptional kinetics along pseudotime trajectory. (H) Violin plots show the expression of myogenic (cluster 2) and adipogenic…

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  Figure 3. Single-cell RNA sequencing analysis of epicardial and epicardial-derived cells in V-EMOs and P-EMOs (A and B) t-SNE plot of combined Epicardium/early EPDCs, Epicardium-derived mesenchyme/fibroblast-like cells, mural cells, endothelial cells, and proliferative fibroblast clusters (n = 16,490) (A), pseudotime trajectories inferred from transcriptional kinetics (B). Prediction data distinguished two different trajectories: the fibroblast trajectory (clusters 0, 1, and 2) and the vascular trajectory (clusters 0, 1, 3, and 4). (C) Dot plot shows the transcriptional kinetics along pseudotime of key markers through the fibroblast trajectory, and t-SNE plots for representative markers of proliferative fibroblasts. (D) Dot plot shows the transcriptional kinetics along pseudotime trajectory of key markers through the vascular trajectory, and t-SNE plots for representative markers of mural cells and ECs. See also Figure S3.

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  Figure 4. Impact of PDGFBB and VEGF stimuli on EMOs cell composition (A) t-SNE plot shows the subclustering analysis of epicardium-derived mesenchyme/fibroblast-like cells cluster. (B) Violin plots show the expression level of representative genes for each identified subcluster in epicardium-derived mesenchyme/fibroblast-like cells. (C) Relative frequency of each epicardium-derived mesenchyme/fibroblast-like cells subcluster in V-EMO and P-EMO populations. (D) t-SNE plot shows the subclustering analysis of EC cluster. (E and F) Violin plots show the expression level of representative coronary (E) and artery/venous (F) ECs markers. (G) t-SNE plots showing the subclustering analysis of the “Endothelial cells” cluster for each condition (V-EMOs and P-EMOs). (H) Representative IF staining of V-EMOs (organoid slices), highlighting the expression of DACH1, WT1, and NR2F2 in CD31+ cells, corroborating a coronary vascular artery-like phenotype for this population. (I) Representative IF staining of P-EMOs (organoid slices), highlighting the generation of two distinct layers in the epicardium region, the mesothelium (WT1+/NR2F2+/DACH1+ cells) and the sub-epicardium space (WT1−/+low, NR2F2+ cells)…

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  Figure 5. Assembly of V-EMOs and sympathetic neuron spheroids generates functional heart-innervated organoids (A) Representative BF images of SNSs and IF staining of replated SNSs (7 days after replating) after 40 days of differentiation, highlighting the expression of the sympathetic neuronal markers TH and PRPH. Scale bars, 100 μm. (B) Representative BF images of assembloids 2 days post-assembly, highlighting SNs migration from the SNS region toward the EMOs region of V-EMOs. (C and D) Representative IF z stack 3D reconstruction images of V-EMO-SNS assembloids 12-day post-assembly, highlighting SNs (Tuj1+) network in the myocardial region of the assembloids (cTnT+) (C), and the interplay with the coronary-like vascular (CD31+) network (D). See also Figure S5.