Vessel formation of hESC-derived endothelial cells (ECs). (A) Vessellike structures formed in engineered skeletal muscle constructs in vitro (original magnification × 1000; 100 ×/1.40 oil-immersion objective lens). hESC-derived ECs (PECAM1⁺) were coseeded with skeletal myoblasts on PLGA-PLLA scaffold and cultured for 10 days. The construct was then fixed and immunostained for desmin (green), PECAM1 (red), and Dapi (blue). (B) The muscle constructs were implanted into murine muscle for 2 weeks, after which labeled lectin (red) was injected into the mice tail veins. Sections of the implant muscle were stained with human PECAM1 antibodies (green). The image shows functional (lectin perfused) human-derived endothelial vessels (original magnification × 1000; 100 ×/1.40 oil-immersion objective lens). Fluorescent micrographs were taken at 25°C using a fluorescent motorized microscope (Axiovert 200) equipped with an Orca digital camera (Hamamatsu). OpenLab and Fluoromount were used as imaging software and medium, respectively. (C,D) hEB differentiating cells were seeded on PLGA-PLLA scaffolds, grown for 2 weeks, and then transplanted in SCID mice. After 2 weeks, the constructs were retrieved and tissue was fixed and stained for antihuman PECAM1. Arrows indicate vessels lined by human endothelial cells. Original magnifications of panels C and D are × 1000 (100 ×/1.40 oil immersion objective lens) and × 400 (40 ×/0.6 NA air objective lens), respectively. Micrographs were taken at 25°C using an inverted microscope (Axiovert 200) equipped with an AxioCam colored camera (Carl Zeiss). AxioVision 3.1 (Carl Zeiss) was used as imaging software. DAB was used as fluorochrome.