Endothelial Cells Offer a Better Option than Smooth Muscle Cells for the Treatment of Ischemic Limb Disease

Peripheral arterial disease (PAD) is estimated to affect at least 10 million people in the United States and over 200 million individuals worldwide. The clinical manifestations of PAD range from impaired mobility and pain with activity to chronic limb-threatening ischemia with risk of eventual tissue loss. Despite the significant global prevalence and debilitating consequences of the disease, non-option patients remain largely undertreated. Recognizing that ECs are well-known for their therapeutic role in enhancing perfusion and neovascularization in mouse models of critical limb ischemia, we hypothesize that a synergy of ECs with SMCs may achieve better recovery of perfusion and neovascularization than ECs or SMCs alone. In this study, we compared the therapeutic efficacy of hiPSC-ECs, hiPSC-SMCs, and hiPSC-ECs + hiPSC-SMCs for the treatment of critical limb disease using a NOD-SCID mouse model of hind limb ischemia (HLI). A total of 1 × 10⁶ hiPSC-ECs, 1 × 10⁶ hiPSC-SMCs, or 0.5 × 10⁶ hiPSC-ECs + 0.5 × 10⁶ hiPSC-SMCs were intramuscularly injected into the ischemic limb of the mouse on day 3 after HLI induction. Laser Doppler was recorded on day 0 before, days 3 (i.e., before cell administration) and 31 after HLI induction. Compared with only basal medium injection, hiPSC-ECs alone most effectively improved perfusion (63.8% after normalized non-ischemic limb, p < 0.001) and neovascularization (total vessel density = 180.9/magnification, p < 0.001) followed by co-administration of hiPSC-ECs + hiPSC-SMCs (perfusion = 59%, p = 0.008 and total vessel density = 170.5, p = 0.002) compared with basal medium injection (perfusion = 39.7% and total vessel density = 107.2), while hiPSC-SMCs administration achieved mild to moderate improvement (perfusion = 48.9% and total vessel density = 159.5, p = 0.01). When profiles of secreted growth factors or cytokines were determined using a protein array, 34 growth factors or cytokines involved in angiogenesis were highly enriched (at least >50% increase) in a hiPSC-ECs conditioned medium, including basic fibroblast growth factor and vascular endothelial growth factor, while there were 14 in hiPSC-SMCs conditioned medium. In conclusion, our results demonstrate that hiPSC-ECs alone most effectively improved perfusion and neovascularization, followed by co-administration of hiPSC-ECs + hiPSC-SMCs, which may be due to the abundance of growth factors and cytokines secreted by hiPSC-ECs. Future studies are warranted to evaluate the therapeutic efficacy of hiPSC-EC and hiPSC-SMC co-transplantation in aged animals with metabolic and cardiovascular comorbidities.