Objective To Explore the therapeutic effects of HGF modRNA-engineered human mesenchymal stromal cells

in treating lower limb ischemia in mice. Methods First， HGF modRNA was prepared and electroporated into human

mesenchymal stromal cells. After 24 hours， the cell supernatant was collected for ELISA detection. Subsequently， the cell  supernatant was used to culture human umbilical vein endothelial cells. Cell migration ability was assessed using a scratch assay， tube formation ability was evaluated through a tube formation assay， and cell proliferation ability was measured using an EdU assay with smooth muscle cells. Further， the pro-angiogenic effects in vivo were analyzed via a subcutaneous Matrigel plug assay. After establishing a mouse hindlimb ischemia model， PBS （PBS group）， the cells’ suspension of hMSCs after Luciferase modRNA electroporation （hMSCs Luc group）， or hMSCs after HGF modRNA electroporation （hMSCs HGF group） were respectively injected intramuscularly. Laser Doppler flowmetry was employed to assess hindlimb blood perfusion. On day 14， the ischemic gastrocnemius muscle was harvested for tissue sectioning. HE and Masson staining were performed to evaluate tissue damage and fibrosis， while immunofluorescence staining was used to assess tissue angiogenesis and apoptosis. Results After electroporation of HGF modRNA into human mesenchymal stromal cells for 24 hours， ELISA detection showed sustained expression of HGF protein for more than 3 days， peaking at 24 hours. Human umbilical vein endothelial cells cultured with cell supernatant exhibited higher migration rates and tube formation effects， as well as stronger abilities to promote smooth muscle cell proliferation， with more significant subcutaneous angiogenesis effects. The lower limb blood flow detection results showed that the hMSCs HGF group had significantly better blood flow reperfusion compared to the control group. Histological staining results indicated that the hMSCs HGF group effectively protected against tissue damage caused by lower limb ischemia， demonstrating superior angiogenesis effects and anti-apoptotic capabilities. Conclusion HGF modRNA engineered human mesenchymal stromal cells significantly protect against ischemic limb injury in mice and promote the recovery of limb blood flow

Angiogenesis, Lower limb ischemia, Mesenchymal stromal cells, Hepatocyte growth factor, modRNA