Abstract 4140889: Preclinical study of human induced pluripotent stem cell-derived endothelial cells for peripheral artery disease

Background: Peripheral artery disease (PAD) affects approximately 230 million people globally and chronic limb-threatening ischemia (CLTI) can lead to limb amputation. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) offer a promising source for PAD treatment. However, to date, regulatory criteria for the clinical application of hiPSC-ECs have not been established yet, and there have been no reports on preclinical studies involving hiPSC-ECs. This study aims to address this gap by investigating the feasibility, safety, and efficacy of clinical-grade hiPSC-ECs through a preclinical proof-of-concept analysis. Methods and Results: Clinical-grade hiPSC lines were established from the blood of three PAD patients using episomal plasmids. Their pluripotency was confirmed by assessing their expression of pluripotency markers through qRT-PCR and immunostaining, as well as their pluripotency in a teratoma assay. Furthermore, PAD-hiPSCs exhibited normal karyotypes. Subsequently, we differentiated PAD-hiPSCs into ECs, all of which displayed a cobble-stone EC morphology and expressed EC markers as confirmed by qRT-PCR and immunostaining. PAD-hiPSC-derived ECs also expressed CDH5 at a minimum of 98.4 ± 0.2% and VWF at 94.4 ± 1.3% by flow cytometry. PAD-hiPSC-ECs maintained their normal karyotypes and copy number variation across the genome, as determined by CGH array. Additionally, they exhibited endothelial characteristics such as tube formation in Matrigel and intracellular nitric oxide production. Administration of PAD-hiPSC-ECs into ischemic hindlimbs of both female and male mice led to improved blood flow recovery (~3.3 fold), reduced risk of limb loss (~8.8 ± 0.6%), and increased vascular density (~2.7 fold) compared to control groups. These engrafted hiPSC-ECs exhibited vessel-forming capacities, thereby contributing to neovascularization. More importantly, we evaluated the toxicity, biodistribution, and tumorigenic potential of these cells in immunodeficient nude mice over one year. The results demonstrated non-detection of tumorigenic cells in twelve organs and adverse events. With all the data, the use of hiPSC-ECs was approved for a clinical trial to treat PAD. Conclusions: Our preclinical proof-of-concept findings demonstrate, for the first time, the clinical compatibility of hiPSC-ECs derived from patients for autologous cell therapy for PAD. Our study further suggests regulatory guidance for the clinical development of hiPSC-ECs.