Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase

This study addresses a major functional barrier to utilizing patient-specific induced pluripotent stem cell-derived endothelial cells for organ engineering and other clinical applications. These stem cell-derived vascular cells lose their mature phenotype rapidly in culture due to a loss of mitophagy and accumulation of mitochondria. This can be overcome by increasing autophagy activity, rendering them stable and longer lived for implementation in clinical applications. This removes a blockade in vascularizing more complex organs and in generating more advanced personalized therapies for vascular disease Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.

Automated summary

This study addresses the limitations of using patient-specific induced pluripotent stem cell-derived endothelial cells in clinical and research applications. By increasing autophagy activity, the stability and longevity of these cells can be improved. This has significant implications for organ engineering and personalized therapies for vascular disease.