Background: To standardize stem cell-based therapeutic angiogenesis for peripheral artery disease, a transplantation strategy to achieve efficient and sustainable therapeutic effect is required. In this study, we have investigated therapeutic effects of human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and vascular mural cells (MCs) incorporated with gelatin sponge (GS) scaffold in a murine hindlimb ischemia model.
Methods: A hindlimb ischemia model was established in male 8-week-old NOD/SCID mice. We induced ECs and MCs from hiPSCs using a 2-dimensional high-density culture, respectively. We prepared GS by the homogenization of the aqueous solution of gelatin. We prepared a mixture of induced ECs and MCs (1.0 x106 ECs+1.0 x106 MCs / mouse) with or without GS. We injected them into the ischemic thigh muscles just after induction of ischemia. We compared control group (medium), vehicle group (GS + medium) and treatment group (hiPSC-derived ECs and MCs) with or without GS. Measurements of the limb blood flow were performed on 0 (post), 3, 7 and 14 days after surgery using laser Doppler perfusion imaging (LDPI).
Results: VE-cadherin+ CD31+-ECs were 96.3±2.5% (n=3) and PDGFRβ+-MCs were 56.7±11.2% (n=3) according to the flow cytometry analysis, respectively. LDPI index (ischemic/normal blood flow ratio) revealed that limb blood flow in treatment group with GS was significantly higher than the other groups at 7 and 14 days after treatment (blood flow ratio at 14 days after treatment: control group vs. vehicle group vs. treatment group without GS vs. treatment group with GS, 0.56±0.09 vs. 0.55±0.03 vs. 0.64±0.05 vs. 0.82±0.07; P<0.001, n=6). Immunohistochemical analysis indicated that α-smooth muscle actin-positive vessel density was significantly higher in treatment group with GS (P<0.0001, n=6). Gene expression analysis by qPCR indicated that VEGFA mRNA expression levels were significantly higher in treatment group with GS (P<0.005).
Conclusion: Transplantation of hiPSC-derived ECs and MCs with GS increased the blood flow of hindlimb ischemia in a murine model. Further pre-clinical and clinical studies are required for the standardization of this method.