Fig. 3

Vascular organoids transplanted to chick CAM. (A) Schematic protocol of CAM transplantation. Eggs were incubated to day 7, the CAM was dropped, and the shell was windowed for access. Organoids were implanted onto the CAM with a pre-incubated matrix. Transplants were reincubated for 11 days. Created with Biorender.com. (B-D) Organoids were harvested, stained, and imaged to analyze changes to vascular morphology. Organoids showed angiogenic remodeling and patterning after CAM implantation. Vessels displayed a branched hierarchy with a greater number of associated NG2⁺ cells, indicating mural cell progenitors or immature pericytes. Large diameter vessels wrapped with αSMA⁺ cells formed within the organoid (solid arrowheads). The endothelial layer of these vessels is organoid-derived (PECAM-1⁺), while the αSMA⁺ source cannot be determined from staining. (B’-B’’’ and C’-C’’’) Individual markers further show mural cell covered organoid vessels. (D-E) Anastomoses between the chick host and organoid implant were observed. Double positive vessels arose from organoid-derived cells (solid arrowheads) while αSMA⁺ vessels with no underlying PECAM-1 staining are chick derived (open arrowheads), as PECAM-1 does not stain chick ECs. Double arrowheads identify anastomoses, where αSMA⁺ cells continuously envelop both chick (PECAM⁻) and organoid (PECAM⁺) vessels. (D’, D’’, E’, E’’) Single staining images depict these anastomoses. (F) Analysis of organoid vasculature after implantation showed larger average diameters compared with in vitro and close association with αSMA⁺ mural cells. Data are mean ± SD, N = 4 organoids, n = 49 images for average diameter analysis, unpaired t-test, ****p < 0.0001.