Vascular Regeneration with Reprogrammed Endothelial Cells

Project Summary Ischemic cardiovascular diseases rank among the leading causes of morbidity and mortality worldwide. Despite advancements in therapeutic approaches, managing patients with peripheral vascular disease (PAD) remains a formidable challenge. The underlying pathology of these conditions involves vascular loss or dysfunction, alongside impaired neovascularization, processes primarily governed by endothelial cells (ECs). Consequently, therapeutic strategies should prioritize regenerating ECs. To date, conventional therapies have not successfully induced vessel growth in vivo. Thus, cell therapies utilizing stem or progenitor cells have emerged as promising options to induce neovascularization. However, the efficacy of adult stem or progenitor cell therapies has been limited. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer a potential source of ECs. Nevertheless, challenges such as complex differentiation protocols, phenotype maintenance difficulties, and concerns about tumorigenicity have hindered the clinical translation of iPSC-derived cells. In response, a novel approach leveraging lineage-specific transcription factors for direct reprogramming of somatic cells into ECs has been developed. Our research has successfully reprogrammed human dermal fibroblasts into ECs using ETV2 alone under specific culture conditions. Transplantation of these reprogrammed ECs (rECs) has demonstrated the ability to induce neovascularization, provide proangiogenic effects, and improve blood flow in animal models, with the transplanted rECs directly integrating into host blood vessels. Despite their therapeutic potential, rECs faced limited clinical applicability due to the use of lentiviral vectors, which can cause insertional mutations. To address this, we developed an adenoviral vector harboring ETV2 (Ad-ETV2), which is non-integrative and suitable for clinical applications. Using this vector, we successfully generated rECs from human fibroblasts, showcasing therapeutic potential and neovascularization capability without genomic concerns. In selecting the source cells for direct reprogramming, we focused on human urine- derived cells due to their non-invasive collection method, economic viability, and repeat sampling feasibility. Our developed culture method yields homogenous urine cells expressing mesenchymal stromal cell markers (U- cells). These U-cells were successfully reprogrammed into rECs using Ad-ETV2, termed U-rECs. The primary goal of this project is to develop U-rECs into a clinically viable product for treating severe PAD. In this phase I STTR, we aim to reprogram human U-cells into ECs with Ad-ETV2, assessing their characteristics, identity, and genome stability. By leveraging non-invasive cell collection methods and a non- integrative gene delivery system, U-rECs represent a practical and clinically compatible therapeutic option. Additionally, this product holds potential for applications in tissue engineering, disease modeling, and personalized medicine, promising a broad impact on research and therapy.