Donor Age Impairs Vasculogenic Potential of hiPSC-Derived Endothelial Progenitors via Elevated Mitochondrial Reactive Oxygen Species

Background: Human induced pluripotent stem cells (hiPSCs) are a promising source for autologous vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency and hiPSC characteristics has been studied, the effect on the functionality of CD34 ⁺ hiPSC-derived endothelial progenitors (hiPSC-EPs) remains understudied. To investigate potential differences, we differentiated EPs from hiPSCs derived from three neonatal donors (ND) and three mature donors (MD) with sex- and somatic cell origin-matching between lines. This comparison revealed donor age-associated, phenotypical, functional, epigenetic, and transcriptomic differences. Notably, when cultured in 3D hydrogels, all MD-hiPSC-EPs showed markedly reduced vasculogenic potential relative to ND-hiPSC-EPs, forming vascular structures that were poorly interconnected and largely non-lumenized. DNA methylation profiling revealed that somatic cell origin was the dominant driver of variance, however, key donor age-associated differences were also present. MD-hiPSCs were hypomethylated compared to their matched pairs and we observed differentially methylated regions associated with mesoderm commitment, angiogenesis, ECM remodeling, cytoskeleton-organization and mitochondria regulation. Specifically in mitochondrial regulation genes, we found a decrease in the methylation of canonical mitochondrial reactive oxygen species (ROS) associated genes in MD-hiPSC-EPs. RNA-sequencing further highlighted a donor age-associated shift in mitochondrial regulation with a clear donor age-dependent separation in the Mitochondrial Gene Regulation pathway. We further examined mitochondrial function in a representative ND-hiPSC and MD-hiPSC pair and found that MD-hiPSC-EPs exhibited a decrease in mitochondrial membrane potential concurrent with an increase in mitochondrial ROS (mtROS). To investigate whether increased mtROS in MD-hiPSCs detrimentally impacted their functionality, we treated MD-hiPSCs with an mtROS-specific antioxidant. Once treated, we observed a shift to an ND-hiPSC-EP like state. We observed treated MD-hiPSC-EPs producing phenotypical similarities, shared methylation changes, transcriptomic grouping, and improvement in network formation, achieving levels observed in ND-hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers and solutions to translating autologous hiPSC-derived therapeutics into aged patient populations.