GTKO/MCP/TBM transgenic pigs were developed to mitigate coagulation-mediated vascular rejection in xenotransplantation by expressing human thrombomodulin (hTBM) under the control of the endothelial-specific pICAM2 promoter. In this study, blood-derived endothelial progenitor cells (EPCs) were isolated from the pig, characterized for their endothelial properties, and then used to verify the endothelial-specific expression of the transgene.

The EPCs exhibited a typical cobblestone morphology. At passage 4, increased expression of ICAM2 was confirmed via real-time PCR. Flow cytometry analysis revealed expression of endothelial progenitor markers including CD34, CD133, CD31, vWF, VEGFR2, and VE-cadherin. The cells also demonstrated angiogenic potential in an in vitro tube formation assay and retained their morphology and marker expression up to passage 13. At this stage, 6–8% of the cells expressed the activated endothelial marker CD62E/CD62P, along with elevated levels of VEGFR2 and VE-cadherin. Notably, expression of hTBM was detected, indicating that transgene activation driven by the pICAM2 promoter is preferentially induced in activated ECs.

These findings demonstrate that blood-derived EPCs from transgenic pigs can be used to evaluate endothelial-specific transgene expression without the need for vascular tissue biopsy. This approach may offer a reliable method for molecular verification and pre-transplant evaluation in future xenotransplantation research.