PEDF-enriched Urine-derived Stem Cell Sheets for Corneal Wound Healing in Mustard Gas Injury

About This Grant

PROJECT SUMMARY Sulfur mustard (SM)-induced chronic corneal injuries pose a significant public health challenge, often leading to vision impairment or blindness. Current treatments are limited, underscoring the need for innovative therapies. While stem cell-based tissue regeneration holds promise, existing cell sources such as limbal stem cells (LSCs) and bone marrow- or adipose-derived mesenchymal stem cells have limitations, including invasive harvesting and limited cell yield. Induced pluripotent stem cells and embryonic stem cells also carry tumorigenic risks. Urine- derived stem cells (USCs) offer a non-invasive, readily accessible alternative to traditional stem cell sources, exhibiting regenerative and immunomodulatory properties without reported tumorigenicity. We have previously demonstrated the ability to generate autologous stratified epithelial sheets from USCs, characterized by tight junctions, epithelial marker expression, and a lack of in vivo inflammation. Furthermore, pigment epithelium- derived factor (PEDF) is a multifunctional protein crucial for corneal homeostasis, promoting epithelialization via LSC regeneration and enhancing wound healing by mitigating reactive oxygen species production, inflammation, neovascularization, fibrosis, and collagen deposition. However, the short half-life of native PEDF limits its therapeutic potential, necessitating a sustained delivery strategy. In addition, while genetically engineering stem cells via viral transduction with PEDF can significantly increase the PEDF expression and improve its regenerative capacity, the potential risks of cell and viral integration as well as the possibility of insertional mutagenesis raise significant safety concerns. This proposal investigates a novel regenerative approach using PEDF-expressing autologous rabbit USC (rUSC) sheets to repair SM-induced chronic corneal injury in a rabbit model. We hypothesize that SM depletes PEDF and that PEDF-expressing rUSC sheets will effectively deliver PEDF, promoting corneal wound healing. We propose the following specific aim, comprised of two parts: A) Development and optimization of PEDF delivery by autologous rUSC sheets. This part of the project will optimize PEDF expression in USCs via mRNA or plasmid transfection, comparing transfection efficiency and PEDF production. Engineered rUSC sheets will be characterized in vitro by assessing controlled release of PEDF (peak value and duration), tight junction formation, barrier function, and their impact on LSC function (stemness, migration, and corneal epithelial differentiation); B) Evaluation of the therapeutic efficacy of PEDF-expressing, autologous rUSC sheets in a rabbit model of SM-induced chronic corneal injury, we will assess their impact on corneal function recovery (epithelialization, LSC regeneration, and neurogenesis) and on wound-healing processes. Our findings will establish autologous PEDF-USC sheets as an effective, safe, and cost-effective approach for treating SM-induced corneal injuries, leveraging their virus-free, DNA-integration-free, and immunoreaction-free characteristics. This project may transform treatment paradigms for SM-induced chronic corneal injuries and broaden the application of USC-based therapies for various corneal diseases.