Figure 1. Generation of an in vitro model of vessel organoids (VOr).

(A) Schematic view of the methods for generating VOrs from GFP-hESC. EBs: embryonic bodies; VPs: vascular progenitors; VOr: vessel organoid; hESC: human embryonic stem cell. (B) Different developmental stages of VOrs from day (D) 4 to D20. Top, right field; bottom, GFP. Scale bar, 200 μm. (C) qPCR analysis for expression of stem markers (NANOG, OCT4) and vessel markers (PECAM1, VE-Cadherin, VWF, VEGFR1, VEGFR2, PDGFR) in developing VOrs, using GAPDH as internal control. Data are presented as mean ± SEM (n = 3 independent experiments), error bars indicate SEM. (D) Immunostaining of GFP and CD31 in D40 VOrs. Scale bar, 200 μm. Bottom: Imaris reconstruction of VOrs showing integrated vasculature structures. (E) Immunostaining of GFP and CD31 for the vascular structures in VOrs. Scale bar, 20 μm. Top right: section view in VOr showing the lumen structure. (F) Immunostaining of CD31 and PDGFRβ for endothelial cells and pericytes, respectively. Scale bar, 50 μm.

Figure 1—figure supplement 1. Vessel organoids (VOrs) recapitulate human vessel development.

(A) Quantification of the VOr diameter from day (D) 4 to D40. Data are mean ± SEM of 10–21 organoids at each time points. (B) Morphological appearance of VOrs at D40. Scale bar, 500 μm. (C) Flow cytometry plots of temporal development of CD31⁺ GFP⁺ cells in differentiating VOrs. (D) Immunostaining of GFP and CD31 for the vascular angiogenesis structures. Scale bar, 20 μm. (E) Quantification of the average vessel length (E1), total number of junctions (E2), and mean E lacunarity (E3) for D40 VOrs. Error bars indicate SEM. (F) Uptake of acetylated low-density lipoprotein (DiI-Ac-LDL) of VOrs at indicated time points. Scale bar, 20 μm.

Figure 1—video 1. Phosphate-buffered saline (PBS) fluid was microinjected into the vessel-like lumen in day (D) 40 vessel organoid (VOr) with continuous pressure, showing liquid flow and vessel wall expansion without leakage.

Download video file ^{(4.5MB, mp4)}

Electrode inner diameter: 500 nm. Scale bar, 2 μm.