PLVAP promoter knock-in iPS cells for tracking endothelial-like differentiation and angiogenic activity in ischemic models

Differentiating human induced pluripotent stem cells (iPS) into endothelial cells is now technically feasible, but it remains difficult to follow, in a quantitative manner, when and how these cells acquire mature endothelial characteristics. In this study, we investigated whether inserting a reporter driven by the plasmalemma vesicle–associated protein (PLVAP) promoter into human iPS could be used to visualize endothelial-like differentiation and pro-angiogenic activity. We generated a PLVAP promoter reporter knock-in iPS line and directed these cells toward an endothelial lineage to obtain induced endothelial cells (iEC). Endothelial identity and function were examined by qRT-PCR, protein-level characterization, in vitro Matrigel tube formation assays, functional NO production assays, and an in vivo Matrigel plug assay. We further evaluated the therapeutic performance of iEC in a murine hindlimb ischemia (HLI) model. Protein-level characterization further confirmed that iEC express and exhibit a pharmacologically regulated, eNOS-dependent NO response to the antagonist L-NAME and the agonist VEGF-A. Matrigel plugs containing iEC showed markedly higher red blood cell infiltration than plugs containing undifferentiated iPS. In the HLI model, iEC transplantation improved perfusion recovery and reduced limb loss compared with iPS injection. Histology revealed increased capillary density and elevated angiogenic cytokine expression in iEC-treated muscles, and transplanted iEC displayed endothelial-like phenotypes within ischemic tissue, consistent with in vivo vascular incorporation. The PLVAP promoter knock-in reporter provides a platform to enrich and functionally evaluate iPSC-derived endothelial-like populations with strong angiogenic and regenerative activity. These findings suggest that a PLVAP promoter–driven reporter can be used to refine and functionally evaluate iPS-derived endothelial-like cell products with strong pro-angiogenic properties in ischemic models and to support the development of cell-based therapies for ischemic cardiovascular disease.