Coalescence of lacZ⁺EPCs into functional blood vessels. (A) A histologic view of recruitment of lacZ⁺ bone marrow–derived EPCs to ischemic tissue starting as early as day 3 after surgery. (B) LacZ⁺ cells in the tissue at day 7 were either individual foci or cellular clusters, but did not appear as vascular structures and were not associated with vessels by CD31 staining. (C) By day 14, lacZ⁺ EPCs began to form a number of vascular-like structures. (D) Magnified histologic section at day 7 demonstrating the presence of both EPC clusters (thick arrow) and individual foci of EPCs (thin arrows). (E) Costaining of tie2/lacZ⁺ cell clusters with VWF confirmed the endothelial phenotype of lacZ-expressing cells in the tissue. (F) Day-14 histologic section demonstrating a fully formed bone marrow–derived lacZ⁺ blood vessel seen in cross-section. (G) Costaining of a tie2/lacZ⁺ vascular structure with VWF not only confirming the endothelial phenotype of lacZ-expressing blood vessels in the day-14 graft but clearly demonstrating 2 distinct blood vessels: on the left (thick arrow), a vessel composed entirely of EPCs (in blue), and on the right (thin arrow), a vessel that is not derived from EPCs. (H) Identification of EPCs and endothelial cells with antibodies against CD31 and β-gal, respectively, yielded similar findings, as shown in a high-power view of cells costaining for CD31⁺ (green) and β-gal (red). (I) (J) Colocalization of tie2/lacZ⁺ structures with the in vivo FITC-lectin stain, seen histologically and under fluorescent microscope, show perfusing vessels at day 14. Scale bar = 20 μm.