Treatment strategies for peripheral artery disease (PAD) remain limited due to the many challenges of surgical intervention. Stem cell therapy offer a promising option, but barriers exist to their clinical translatability. Among these barriers include lifestyle choices like nicotine use that may influence the efficacy of stem cell therapy. The objective of this study aims to examine the efficacy of iPSC-EC therapy for treatment of PAD in mice with prior chronic exposure to nicotine. We hypothesize that chronic nicotine exposure negatively affects the therapeutic potential of implanted iPSC-ECs in a murine model of PAD. NSG mice were implanted with an osmotic pump for controlled exposed to nicotine or saline (control) for 28 days. Plasma cotinine was measured to confirm nicotine exposure. After 28 days of nicotine or saline exposure, the mice underwent induction of hindlimb ischemia and before receiving an injection of 10 6 iPSC-ECs. A second dose of cells were injected to the ischemic limb on day 7 post-surgery. All mice were monitored for blood perfusion recovery using laser Doppler spectroscopy imaging. Survival of the transplanted cells in vivo were tracked by bioluminescence imaging. The mice receiving iPSC-ECs were also imaged for cell survival using bioluminescence imaging. At day 14 post-surgery, mice were euthanized, and the gastrocnemius muscle and thigh muscle were harvested for histological analysis. The results show that the average cotinine concentration in sera was ~200 ng/mL, which is within physiological range for a tobacco user. Blood perfusion recovery at day 14 showed trends of nicotine treated groups have hindered perfusion recovery, compared to those treated with saline. Importantly, nicotine exposure trended with poorer perfusion recovery after cell therapy. Bioluminescent imaging showed iPSC-ECs persisted in vivo for all groups with a gradual decline in cell numbers over time. In conclusion, nicotine exposure was associated with poorer blood perfusion recovery after iPSC-EC therapy, compared to control saline exposure, suggesting that chronic nicotine exposure may have adverse effects on the efficacy of iPSC-EC therapy for treatment of PAD.

Dysfunctional endothelial cells (EC) can restrict flow in the extremities, leading to limb ischemia. We aim to improving revascularization of ischemic limbs using cell therapy, augmented with spatially patterned scaffolds that promote survival. The purpose was to evaluate the effect spatially patterned scaffolds on endothelial cell survival. The biophysical parameters of collagen scaffolds (alignment, crosslinking, and fibril diameter) were characterized with atomic force microscopy and degradation assays. Cytoskeletal arrangement of primary human coronary artery ECs and human induced pluripotent stem cell (iPSC)-derived ECs on collagen scaffolds was evaluated by F-actin staining. To evaluate the effect of mechanical properties on cell viability in vitro, Live/Dead and MTS assays were used. Cell seeded scaffolds were implanted into a mouse hindlimb ischemia model, and cell survival was tracked by bioluminescent imaging. Both primary EC and iPSC-ECs reorganized their F-actin cytoskeleton corresponding to the alignment of the fibrils, irrespective of crosslinking degree, stiffness or fibril diameter. When cells were grown in hypoxic mimicking ischemia however, viability was improved using aligned scaffolds with 200 nm fibril diameter. Degradation assays showed that scaffolds with higher crosslinking had significantly more durability. When cell-seeded scaffolds were implanted to the ischemic limb, the aligned scaffolds with 200nm fibril diameter had the highest survival, confirming in vitro studies. Aligned collagen scaffolds with 200nm fibril diameter and high cross-linking degree led to improved viability of primary and iPSC-ECs in a mouse model of PAD.