Abstract

Introduction: It is estimated that about 40 to 60 million people in the United States suffer from diabetic foot ulcers (DFUs), one of the most severe DM complications. A significant proportion of affected individuals undergo amputation and are exposed to high mortality risks. Stem cell-based therapies offer a promising therapeutic avenue for these patients. Fibroblasts hold a crucial role in wound healing and angiogenesis, and we further highlighted that activated Notch signaling in fibroblasts derived from diabetic wounds leads to dysregulated fibroblast function. In this study, the potential therapeutic outcomes from genetically modified fibroblasts derived from mesenchymal stem cells (MSC-DF) in treating DFU is assessed, utilizing a unique genetic mouse model. Methods: A fibroblast-specific and inducible diabetic murine model with triple-gene engineering was created via inducible intracellular Notch1 pathway activation (RosaN1IC/Col1a2-Cre/ERT/Leprdb). MSC-DFs harvested from 10-12 week old B6db/ ROSAN1I/ / Cola2-Cre/ERT (LLRRCc) donor mice were cultured in vitro with activation versus silencing of Notch 1 signaling. Recipient LLRRCc mice (8-12-week-old) underwent a 6mm excisional dorsal skin wound and subsequent local injection of 1x10^6 donor syngeneic activated or silenced GOF Notch1 -MSC-DFs (n= 5/group). Wound closure was monitored daily for 9 consecutive days post-injury, and tissue was obtained for histological sectioning. Results: GOF Notch1 -MSC-DFs were characterized by immunostaining based on expression of markers specific to fibroblasts and a panel of Notch signaling genes. LLRRCc murine subjects receiving silenced GOF Notch1 -MSC-DFs treatment (n=5) demonstrated faster rates of wound closure over activated GOF Notch1 -MSC-DFs (n=5) treated mice. Collagen deposition was more abundant in wounds treated with silenced GOF Notch1 -MSC-DFs. Conclusion: MSC-DF deactivated Notch1 signaling therapy improves the rate of wound healing in GOF Notch Diabetic mice. These promising outcomes demonstrate a potential role for this innovative stem cell-based therapy targeting Notch-1-modified MSC-DF in future clinical applications for DFU patients through restoration of impaired fibroblast function.