No.1, University Road Tainan, Tainan, Taiwan (Republic of China)
Abstract: Endothelial progenitor cells (EPCs) are integral to neovascularization and vascular repair, offering considerable therapeutic promise for ischemic disorders. Here, we present a streamlined protocol to generate and expand EPCs from induced pluripotent stem cells (iPSCs) without strict reliance on a detailed time-course. The procedure begins with iPSCs plated on Geltrex®-coated surfaces, followed by an initial mesoderm-inductive phase mediated by the GSK-3β inhibitor CHIR-99021. After mesodermal specification, FGF2 is introduced to reinforce lineage commitment, and VEGF plus BMP4 guide the transition toward early EPCs. Once cells reach an appropriate differentiation stage, the culture is supplemented with the Rho-associated kinase inhibitor Y-27632, the TGF-β inhibitor A83–01, and additional CHIR-99021 to bolster proliferation and maintain the EPC phenotype. Subsequent TrypLE™ replating eliminates non-EPC contaminants, yielding a highly enriched and functionally competent EPC population. Gene expression analyses reveal a clear progression from pluripotency to the endothelial lineage, as evidenced by downregulation of Oct4, transient upregulation of the mesoderm marker TBXT, and an increase in MMP1, indicative of angiogenic activity. Phenotypic assessment confirms that early EPC markers (e.g., CD105) are eventually superseded by late-stage markers (e.g., CD133) as the cells mature. By leveraging targeted small-molecule supplementation, this protocol consistently produces high-purity EPCs capable of robust expansion. The resulting cells exhibit strong angiogenic potential and hold significant promise for scalable therapeutic applications in regenerative medicine, particularly in managing ischemic conditions and enhancing vascular repair.
