E-selectin Gene Modified Mesenchymal Stem Cell Therapy Reverses Impairments in Wound Healing in Ischemic Limbs

Abstract

Tissue loss is a major source of morbidity and substantially worsens the risk of extremity amputation in patients with chronic limb-threatening ischemia (CLTI). Although cell-based therapies utilizing mesenchymal stem cells (MSC) hold significant promise to augment wound healing and therapeutic angiogenesis, unmodified MSCs have demonstrated only minor benefits for CLTI in previous clinical trials. Here we report the effects of MSCs engineered to overexpress E-selectin (E-sel), a cell adhesion molecule capable of inducing neovascularization, on a translational murine model recapitulating hindlimb ischemia with cutaneous tissue loss. MSCs were harvested from bone marrow of 8- to 10-week-old FVB/Rosa26SormTmG donor mice and cultured in MesenCult medium. MSCs were verified as CD44+/CD73+/CD105+/CD29+/Sca-1+ by flow cytometry. MSCs underwent viral transduction with E-sel-GFP/AAV or GFP/AAV. Femoral artery ligation was performed in 12- to 14-week-old recipient FVB mice followed by a 4-mm acute cutaneous wound in the ipsilateral limb and local injection of PBS or 1 × 106 donor GFP+/MSCs or E-sel+/MSCs (E-sel+/MSC). Wounds were monitored daily for 7 days, at which point tissue specimens were harvested for microscopy and whole-body DiI perfusion studies. Unmodified MSCs do not express basal levels of E-sel, and E-sel+/MSCs retained MSC phenotype and trilineage differentiation capability. Mice receiving E-sel+/MSC treatment (n = 10) demonstrated accelerated wound closure rates compared to both GFP+/MSCs (n = 10) and phosphate-buffered saline (PBS) (n = 10)-treated mice at each postoperative day (POD) with the highest degree of difference observed at POD 5 (94 ± 3% vs 79 ± 10% GFP vs 72 ± 9% PBS; P < .001) and 7 (98 ± 2% vs 86 ± 8% GFP vs 87 ± 6% PBS; P < .010). Collagen deposition was more abundant in wounds treated with E-sel+/MSCs (21 ± 4% vs 5 ± 1% GFP vs 9 ± 1% PBS). DiI perfusion at POD 10 demonstrated greater vessel density in wounds treated with E-sel+/MSCs (76 ± 15% vs 29 ± 7% GFP vs 9 ± 5% PBS relative vascular density). Wound healing focused polymerase chain reaction array identified upregulation of target genes in wound tissue responding to E-sel+/MSCs. E-sel+/MSCs demonstrated enhanced viability more mTmG+-E-sel+/MSCs (13 ± 3 cells/high powered field [HPF] vs GFP+/MSCs 3 ± 2 cells/HPF vs PBS 0 ± 0 cells/HPF; P < .010) observed at POD 7 on microscopy. Cell-based therapy using E-sel-overexpressing MSCs reverses impairments in wound healing of ischemic limbs. These data exhibit the potential role for E-sel-modified MSCs as a novel therapeutic alternative in future clinical applications for delayed and nonhealing wounds associated with CLTI.