Endothelial potential of human embryonic stem cells

Abstract

Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realized. Among these obstacles is the development of a productive method of isolating endothelial cells from human embryonic stem cells and elucidating their differentiation pathway. This review will focus on the endothelial potential of human embryonic stem cells that is described in current studies, with respect to the differentiation of human embryonic stem cells to endothelial cells, their isolation, and their characterization.

Figure 1

Isolated hESC-derived endothelial cells (PECAM1⁺ cells). The cells were grown in culture for up to 7 passages. Cells are stained with VE-cad (original magnification, × 1000; 100×/1.40 oil-immersion objective lens) and VWF (original magnification, × 400; 40×/1.3 objective lens) and show the uptake of Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL; original magnification, × 200; 20×/0.5 NA air objective lens). Micrographs were taken at 25°C using a fluorescent motorized microscope (Axiovert 200; Carl Zeiss, Oberkochen, Germany) equipped with an Orca digital camera (Hamamatsu Photonics, Tokyo, Japan). OpenLab (ImproVision, Coventry, United Kingdom) and Fluoromount were used as imaging software and medium, respectively.

Figure 2

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.

Figure 3

Summary of endothelial markers and the kinetics of their expression investigated during the differentiation of hESCs or EBS. Text colors within the figure correspond with the investigations listed at the bottom of the figure. D indicates down-regulated; U, up-regulated; +, expressed by undifferentiated hESCs (day 0 of EB differentiation); −, not expressed by hESCs. Where no data are given, expression was not reported.

Figure 4

Vessellike network organization in 3D culture assay. (A,B) Dissociated EBs (8 days old) were seeded on PLGA-PLLA scaffolds and incubated for 2 weeks. The scaffold was then fixed in formalin and immunostained with anti-PECAM1 (original magnification, × 100; 10×/0.3 NA oil-immersion lens). Micrographs were taken at 25°C using an inverted microscope (Axiovert 200, Carl Zeiss) equipped with an AxioCam colored camera. AxioVision 3.1 was used as imaging software. DAB was used as fluorochrome. (C,D) A vessellike network was formed during differentiation of the EBs. hEBs obtained on day 6 (panel C) and day 13 (panel D) were fixed and stained for anti-PECAM1 to exhibit the advancement in the vascularization process and formation of complex vascular network (original magnification, × 100; 10×/0.3 NA oil-immersion lens). Micrographs were taken at 25°C using a fluorescent motorized microscope (Axiovert 200, Carl Zeiss) equipped with an Orca digital camera (Hamamatsu). OpenLab and Fluoromount were used as imaging software and medium, respectively.

Figure 5

Summary of endothelial markers and the kinetics of their expression during the differentiation of hEBs investigated/monitored over 20 days. Lines with arrows indicate the beginning and period of expression. Yellow triangles indicate the peak in expression (the time area of the peak is indicated by a thin line). Gene colors reflect the level of expression in undifferentiated hESCs: blue, low; red, high; and white, none.