Contribution of ES cell-derived VPCs to tumor angiogenesis.

(A,D,G,J) Whole-mount X-gal staining of the tumor. An X-gal analog, blue gal (Nacalai), was used as a substrate for staining 5 days after the injection of undifferentiated VPCs (A), differentiated VPCs (D), VE-cadherin⁺ fraction of differentiated VPCs (G), and PDGF-BB–treated VPC (J). Undifferentiated VPCs and PDGF-BB–treated VPCs formed cell aggregates with few vascular structures (A,J). Differentiated VPCs and VE-cadherin⁺fraction of differentiated VPCs generated rich vascular-like networks (D,G). (B,C,E,F,H,I,K-O) Immunohistochemical staining of tumor sections. Differentiated VPCs were specifically incorporated as LacZ (blue)/PECAM-1 (brown) double-positive cells into developing vasculatures (E-F). Undifferentiated VPCs were often detected as LacZ⁺/PECAM-1⁻ cells (arrows; B-C). Implantation of VE-cadherin⁺Flk-1⁺ fraction of differentiated VPCs similarly generated specific vascular contribution as LacZ/PECAM-1 double-positive cells (H-I). Typical LacZ⁺PECAM-1⁺ cells (M) were observed in the differentiated VPC-injected tumor. LacZ (blue)/SMA (purple) double-positive and PECAM-1 (brown)-negative cells were also detected in the continuous sections (N-O). In the PDGF-BB–treated VPC transplantation group, some LacZ (blue)/SMA (red) double-positive cells were detected (K-L). Scale bars: panels B, E, H, K, 250 μm; panels C, F, I, L, 50 μm; panel M, 100 μm; panels N-O, 25μm.