Characterization of injected ES cell-derived VPCs and transplantation of VPCs in tumor angiogenesis model.

(A-C) Flow cytometric sorting and analysis of Flk-1⁺undifferentiated VPCs. CCE/nLacZ ES cells were cultured on type IV collagen-coated dishes in the absence of leukemia inhibitory factor (LIF). (A) After 4 days of differentiation, Flk-1⁺VPCs were sorted by flow cytometry (undifferentiated VPCs). (B-C) Flk-1⁺ undifferentiated VPCs did not express other EC markers (VE-cadherin, PECAM-1, and CD34). (D-G) After a 3-day incubation of Flk-1⁺ undifferentiated VPCs, differentiated VPCs were induced. Flow cytometric analysis revealed that about 30% were Flk-1⁺VE-cadherin⁺PECAM-1⁺CD34⁺ECs and 70% were Flk-1⁻VE-cadherin⁻PECAM-1⁻CD34⁻(D-E). (F-G) Double staining of differentiated VPCs was performed as previously reported.1 About 70% of cells that lost Flk-1 expression were positive for SMA (brown) and surrounded PECAM-1⁺ EC sheets (blue). (F) The expressions of PECAM-1 and SMA were exclusive for differentiated VPCs (scale bar, 100 μm). (G) Fluorescent immunostaining of Flk-1 (red) and SMA (green) revealed that they were expressed exclusively (scale bar 20 μm). (H-I) Tumor angiogenesis model of nude mice. Subcutaneous transplantation of VPCs to the growing tumor 7 days after C6 glioblastoma cell implantation was performed (H). Vascularized tumor was obtained 5 days after VPC implantation (I). Monoclonal antibody (MoAb) for murine E-cadherin (epithelial cadherin; ECCD2), Flk-1 (AVAS12), and vascular endothelial (VE)-cadherin (VECD1) have been described previously.6 7Fluorescein isothiocyanate (FITC)-conjugated MoAb for murine PECAM-1 (Mec13.3) and FITC-conjugated MoAb for CD34 (RAM34) were purchased from Pharmingen (San Diego, CA). MoAbs for SMA 1A4, CCA7 were obtained from Neo Markers (Fremont, CA) and ENZO Diagnostics (Farmingdale, NY).