Human iPSCs and iPSC-Derived Endothelial Cells from PAD Patients and Healthy Donors Exhibit Comparable Characteristics and Potency: Implications for Autologous Cell Therapy in Peripheral Artery Disease

Abstract

BACKGROUND: Peripheral artery disease (PAD) can lead to amputation in advanced cases, making cell therapy using human induced pluripotent stem cells (hiPSCs) a promising therapeutic option. hiPSC-derived endothelial cells (hiPSC-ECs) have shown favorable effects in treating experimental ischemic cardiovascular disease. An autologous approach for PAD patients is preferable to avoid immunological reactions. However, it is yet unknown whether hiPSCs and hiPSC-ECs derived from PAD patients have similar characteristics and potency compared to those derived from non-PAD donors. Therefore, we explored whether there are significant differences in the characteristics and potency of hiPSCs and hiPSC-ECs between non-PAD donors and PAD patients. METHODS: We generated hiPSCs from the blood of non-PAD donors and patients with PAD. We determined the pluripotency of hiPSCs using qRT-PCR, flow cytometry, immunostaining, RNA-seq, and teratoma formation assay. The genetic stability of hiPSCs was confirmed by G-banding karyotyping. We then differentiated hiPSCs into endothelial cells (ECs) in a clinically compatible manner. We compared the characteristics and therapeutic potency of hiPSC-ECs derived from non-PAD and PAD groups using qRT-PCR, flow cytometry, immunostaining, and RNA-seq. In vitro endothelial functions were determined by nitric oxide (NO) production and capillary network formation. Therapeutic potency and incorporation of engrafted hiPSC-ECs were evaluated in a hindlimb ischemia model. RESULTS: We successfully generated hiPSCs from the blood of seven non-PAD donors and eight PAD patients. Both non-PAD and PAD-derived hiPSCs exhibited similar expression levels of pluripotency markers. All hiPSCs, regardless of the group, formed teratomas and showed normal karyotypes. RNA-seq analyses revealed similar gene expression profiles between the groups. hiPSC-ECs derived from both non-PAD and PAD donors exhibited similar expression levels of EC markers at both the gene and protein levels. RNA-seq analyses showed no significant overall differences in gene expression profiles between the groups. Functional analyses demonstrated similar endothelial characteristics and function in hiPSC-ECs from both groups. Both groups showed similar perfusion recovery, limb salvage, and vessel-forming capacity. Engrafted hiPSC-ECs from both groups also exhibited similar angiogenic and vessel-forming capabilities. CONCLUSIONS: Our study demonstrated no significant differences in hiPSCs and hiPSC-ECs derived from non-PAD donors and PAD patients in terms of molecular and cell biological characteristics, therapeutic effects, and vessel-forming capability. Our study indicates that hiPSCs and hiPSC-ECs derived from PAD patients can serve as a novel platform for autologous cell therapy.