Novel Gene-Modified Mesenchymal Stem Cell Therapy Reverses Impaired Wound Healing in Ischemic Limbs
Carlos T. Huerta*, Yulexi Y. Ortiz, Yan Li, Antoine J. Ribieras, Francesca A. Voza, Nga Le, Zhao-Jun Liu, Omaida C. Velazquez
Surgery, University of Miami, Miami, FL
Objective: Tissue loss including chronic wounds significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia (CLTI). Cell-based therapeutics utilizing mesenchymal stem/stromal cells (MSCs) hold major promise for promoting wound healing and therapeutic angiogenesis. Unfortunately, unmodified MSCs have demonstrated minimal benefits for CLTI in previous clinical trials. Here we report biologic effects of E-selectin modified MSCs, a cell-adhesion molecule capable of inducing post-natal neovascularization, on a translational murine model that creates critical hindlimb ischemia with cutaneous tissue loss. Methods: Bone marrow cells harvested from 8-10 week old FVB/Rosa26SormTmG donor mice were cultured in MesenCult™ medium to enrich MSCs. MSCs were verified as CD44+/CD73+/CD105+/CD29+/Sca-1+ by flow cytometry. Tri-lineage differentiation assay was conducted to assess differentiation capacity of transduced MSCs in vitro. MSCs were transduced with E-selectin-GFP/AAV or GFP/AAV (control). Femoral artery ligation was performed in 12-14 week old recipient FVB mice immediately followed by a 4 mm cutaneous wound in the ipsilateral limb and local injection of PBS or 1x10^6 donor GFP+/MSCs or E-selectin+-GFP+/MSCs (E-sel+/MSC). Wound closure was monitored daily for 7 days, and tissue was harvested. Confocal microscopy and whole-body Dil perfusion were conducted to evaluate wound tissue vascular density. Results: Untransduced MSCs do not express E-selectin, and E-sel+/MSCs retained MSC phenotype and trilineage differentiation capability. Mice receiving E-sel+/MSC treatment (n=10) demonstrated accelerated rates of wound closure compared to both GFP+/MSCs (n=10) and PBS (n=10) treated mice at each post-injury day (PID) with the highest degree of difference observed at PID five (94 ± 3% vs. 79 ± 10% GFP vs. 72 ± 9% PBS, p<0.001) and seven (98 ± 2% vs. 86 ± 8% GFP vs. 87 ± 6% PBS; p<0.010). Collagen deposition was more abundant in wounds treated with E-sel+/MSCs (21 ± 4% vs. 5 ± 1% GFP vs. 9 ± 1% PBS). Wound healing focused PCR-Array identified upregulation of target genes (>2-fold Wnt5a, Cxcl3, Cxcl5, Mmp9) in wound tissues responding to E-sel+/MSCs. DiI perfusion demonstrated increased vascular density in wound beds treated with E-sel+/MSCs (76 ± 15% vs. 29 ± 7% GFP vs. 9 ± 5% PBS relative vascular density) at PID 10 (Figure 1). Immunofluorescence revealed that E-sel+/MSCs exhibit stronger viability given the presence of significantly more mTmG+-E-sel+/MSCs (13.2 ± 2.6 cells/high powered field (HPF)) in the wound bed compared to GFP+/MSCs (3.4 ± 1.6 cells/HPF) and PBS (0 ± 0 cells/HPF) controls at PID 7 (both p<0.010). Conclusion: E-selectin supercharged MSC therapy promotes wound healing in a murine hindlimb ischemia model. These results demonstrate a potential role for E-selectin modified MSCs as a novel cell-based therapy in future clinical applications for delayed and non-healing wounds associated with CLTI.
Back to 2023 Abstracts