Building blood vessels--stem cell models in vascular biology

Abstract

Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell-based models for the study of human vascular development.

Figure 1.

Outline of 2D and 3D EB models for vasculogenesis and angiogenesis. Stem cells are trypsinized (d 0), (A) and aggregated to create EBs, in drops hanging from the lid of a Petri dish (B). Aggregation can also occur spontaneously by seeding ESCs in suspension in a nonadhesive Petri dish, resulting in EBs of variable size. After 4 d, EBs are seeded on a tissue culture slide (2D), (C) or alternatively embedded in a 3D collagen gel (D). Addition of VEGF induces formation of a peripheral vascular plexus in 2D (C and E) and endothelial cell sprouts (“angiogenesis”) in 3D (D and F) (bottom panel in F adopted from Magnusson et al., [2005]). Whole-mount stainings for CD31 of 2D (E) or 3D (F) EBs at d 10 of differentiation, untreated (Ctrl, top) or induced with VEGF (bottom). Bars, 500 μm.

Figure 2.

Angiogenic sprouts invade the surrounding matrix. (A) Features of blood vessel sprouts formed in 3D collagen matrix in response to VEGF. Expression is shown of the endothelial cell marker CD31/platelet-endothelial cell adhesion molecule (PECAM; red), the pericyte markers αSMA (green, top), and NG2 (green, bottom middle). Hoechst 33342 was used to indicate nuclei (blue). Lumen formation is evident in larger vessels (left, cross section [z-stack] of a sprout [d 18] generated by confocal microscopy). The tip cell at the front of growing sprouts send out filopodia to sense growth factor gradients. Occasional filopodia are also detected on stalk cells that lack pericyte coverage. Bars, 10 μm. (B) Knockout EBs and the assembly of chimeric EBs. Cells deficient in production of HS (Ndst1/2 ^−/−) or lacking VEGFR-2 (vegfr2 ^−/−) do not form vascular sprouts in the EB model. However, chimeric EBs generated by mixing of the two ESC lines before EB formation respond to VEGF and form sprouts. In the chimeras, the endothelial cells (CD31; red) are derived from Ndst1/2 ^−/− cells expressing VEGFR-2, whereas functional HS is provided by pericytes (αSMA; green) lacking VEGFR-2 (Jakobsson et al., 2006). Bar, 300 μm.