PROJECT SUMMARY The mortality rates for patients with critical limb ischemia (CLI) are 15-40%, depending on the treatment. Moreover, the quality of life for surviving patients remains severely reduced. Limb revascularization focused on limb rescue is critical to saving lives and life quality. Cell therapy strategies currently under investigation inject a variety of progenitor cells into ischemic tissue with varying levels of success. However, no one has examined the ability of angiogenic tip-specific endothelial cells (ECs) to promote revascularization in an ischemic limb. We hypothesize that angiogenic ECs that contain tip ECs will more robustly revascularize ischemic limbs compared with nonangiogenic ECs. The proposed studies will use highly expandable pluripotent stem cells as the starting cell source and derive tip and non-tip cell-containing ECs from human induced pluripotent stem (iPS) cells. We will then compare the ability of tip ECs versus non-tip ECs to rescue the muscle in a hind-limb ischemic mouse model. For delivery, we employ spatially nanopatterned collagen biomaterial scaffolds to enhance cell survival after transplantation into the ischemic limb. Lastly, we will incorporate placental growth factor (PIGF) mRNA delivery from aligned nanopatterned collagen biomaterials to support the release of PlGF protein for maintaining the angiogenic phenotype of tip ECs, and/or directing in vivo the differentiation of stalk-to-tip ECs. We expect these studies to show that angiogenic tip-specific ECs, not only integrate into and support current vasculature in the ischemic limb, but also initiate the growth of new blood vessels, faster re-establishment of tissue perfusion, and reproducibly rescue the ischemic limb.