PPARα mediates hypoxia-induced metabolic reprogramming to confer a redox-balanced synthetic phenotype in hiPSC-VSMCs for cell sheet-based vascular grafts

Functional small-diameter tissue-engineered vascular grafts (TEVGs) require seed cells with robust proliferation and extracellular matrix (ECM) synthetic capacity. Human induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) are promising but often functionally immature. While physiological hypoxia is a key developmental signal, its effects on hiPSC-VSMC phenotype, metabolism, and redox homeostasis remain unclear, and whether hypoxia-preconditioned cells can facilitate scaffold-free TEVG fabrication is unexplored. HiPSCs were differentiated into VSMCs under normoxic or hypoxic conditions. Integrated transcriptomic, metabolomic, and functional assays were employed to evaluate phenotypic, metabolic, and redox changes. Peroxisome proliferator-activated receptor alpha (PPARα) was identified as a potential central hub by bioinformatics and its activity was modulated using agonist (WY14643) and antagonist (GW6471). A cell sheet (CS) platform was established and optimized by screening medium additives and seeding density. Optimized CSs were then rolled and matured within a bioreactor to generate scaffold-free TEVGs, followed by decellularization to produce ECM scaffolds evaluated for decellularization efficiency and ECM integrity. Physiological hypoxia (5% O₂) promoted hiPSC-VSMC proliferation and synthetic switching, with multi-omics revealing lipid downregulation and suggesting fatty acid β-oxidation activation. Hypoxia reduced oxidative stress, enhanced antioxidant defenses, and improved mitochondrial function, with PPARα emerging as a key mediator. Using optimized conditions (ITS + Ultroser™ G; 3.0 × 10⁵ cells/cm²), we generated robust CSs, which were rolled into scaffold-free TEVGs and decellularized to yield ECM scaffolds with DNA < 50 ng/mg and preserved ECM composition, meeting decellularization standards. Physiological hypoxia (5% O₂) activates PPARα, accompaning with enhanced hiPSC-VSMC proliferation, ECM synthesis, and redox homeostasis. Leveraging this insight, we established a simple, chemically defined strategy for CS fabrication and subsequent assembly of intact, decellularizable scaffold-free TEVG, providing a mechanistically validated proof-of-concept platform for future studies on scaffold-free vascular graft engineering.