Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice

Abstract

To establish a model for adoptive transfer of endothelial cells, we transferred human umbilical vein endothelial cells (HUVECs) to immunodeficient mice (Rag 2(-/-)). HUVECs were suspended as single cells in Matrigel and injected subcutaneously in the abdominal midline. Within 10 days after injection, HUVECs expressed pseudopod-like extensions and began to accumulate in arrays. By day 20, we observed human vessels that contained erythrocytes, and on day 30 we observed perivascular cells that expressed smooth muscle actin, thus resembling mature vessels. Throughout the experimental period, HUVECs bound Ulex europaeus lectin and expressed CD31, VE-cadherin, von Willebrand factor, as well as ICAM-2. A fraction of the cells also expressed the proliferation marker Ki67. Moreover, the sialomucin CD34, which is rapidly down-regulated in cultured HUVECs, was reinduced in vivo. However, we found no reinduction of CD34 in cells cultured inside or on top of Matrigel in vitro. We also injected cells suspended in Matrigel around the catheter tip of implanted osmotic pumps. Delivery of recombinant human interferon-gamma by this route led to strong induction of MHC class II and ICAM-1 on the human vessels. In conclusion, isolated human endothelial cells can integrate with the murine vascular system to form functional capillaries and regain in vivo properties.

Figure 1.

Development of functional human vessels in Matrigel. a: H&E staining of Matrigel plug (MP) positioned between the skin (S) and abdominal muscle layer (M). b–m: Immunofluorescence staining of human and murine endothelium in the Matrigel with markers as indicated in the panels. Note close association between human and murine ECs in d, as well as apparent junction between human and murine vessels in e. Note also erythrocytes inside human (arrow) and murine (arrowhead) vessels in g, as well as Luconyl Blue (arrows) in human vessels in i (phase contrast and immunofluorescence image of identical fields). Methanol-fixed, paraffin-embedded samples were cut at 4 μm. Original magnifications: ×40 (a); ×200 [b and h (right)]; ×100 (d, e, and k); ×600 (c and m); ×400 [f, g, h (left), i, j, and l].

Figure 2.

Phenotypic analysis of human ECs and murine macrophages. Immunofluorescence staining of frozen (b) or paraffin-embedded (a and c) sections from the Matrigel plug with markers as indicated. Original magnifications, ×200.

Figure 3.

Reinduction of CD34 expression. Immunofluorescence staining of Matrigel in vivo (a–d) and in vitro (e and f) with markers as indicated (all paraffin sections). Original magnifications: ×100 (a); ×400 (b); ×600 (c); ×200 (d–f).

Figure 4.

Modulation of the EC phenotype. Immunofluorescence staining of paraffin-embedded interferon-γ-stimulated day 3 samples with markers as indicated. Original magnifications, ×600.