Vasculogenic potential of peripheral blood (PB)– versus cord blood (CB)–derived endothelial progenitor cells (EPCs). PB-EPCs and CB-EPCs were mixed with 10T1/2 cells in a collagen gel, and implanted into cranial windows in severe combined immunodeficient (SCID) mice. Images were taken at periodic time points with multiphoton laser scanning microscope for in vivo dynamics of vascularization by the implanted endothelial cells. PB-EPCs formed vascular-like structure 4 days after implantation and some of them became perfused at day 11. The PB-EPC–derived blood vessels were transient and almost completely disappeared by day 21 (A). There was no significant difference in the mean (± SEM) density of functional vessels derived from PB-EPCs between groups implanted with PB-EPCs only and PB-EPCs with 10T1/2 cells (B) (n = 4 for each group and experiments were performed with 3 different batches of adult peripheral blood). In some animals, there were still some sparse but functional blood vessels 27 days after implantation (C). In contrast, CB-EPCs formed a uniformly dense network of functional blood vessels (D). Implantation of CB-EPCs alone led to a rapid regression of the implanted cells, while coimplantation of CB-EPCs and 10T1/2 cells resulted in a stable and functional vasculature (E) (n = 4 for each group and experiments were performed with 3 different batches of human umbilical cord blood). The CB-EPC–derived vascular network was stable and functional for more than 119 days in vivo (F). Green indicates PB- or CB-derived endothelial cell expressing enhanced green fluorescent protein (EGFP); red, functional blood vessels contrast-enhanced by rhodamine-dextran. Scale bars represent (A,D) 50 μm; (C,F) 100 μm.

Characterization of CB-EPC–derived vascular networks in vivo. Whole mount staining of the implanted collagen gel revealed that the CB-EPCs (EGFP+) at day 87 after implantation maintained the expression of CD31 (A) and had intact basement membrane of collagen type IV (B) in vivo (EGFP, green; CD31 and collagen type IV, red). CB-EPCs implanted alone at high cell density (5 million cells/mL at high density vs 1 million cells/mL at normal density) were not able to form long-lasting blood vessels (C). The blood-derived endothelial cells were exposed to serum-free media for 48 hours, and the cells were then stained and quantified for TUNEL, a marker for apoptotic cells (D). CB-EPCs exhibited a higher resistance to serum-free medium–induced apoptosis. The vascular permeability of the CB-EPC–derived blood vessels to albumin was measured (Table 1). CB-EPC–derived blood vessels had low vascular permeability with values similar to those of normal mouse pial blood vessels. Blood flow rate as measured by red blood cell (RBC) velocity was comparable between the CB-EPC–derived vessels and mouse brain vessels (E). The number of rolling leukocytes on CB-EPC–derived blood vessels was increased after induction of systemic inflammation with intraperitoneal injection of 100 ng IL-1β for 4 hours (F). #P < .001; *NS; **P < .05. Scale bars (A,B) represent 50 μm.