Dynamic ECM-Mimicking Biomaterials for Ischemia Treatment Peripheral artery disease (PAD) is the third most common cause of cardiovascular morbidity worldwide, present in 20% of the population over 65. If PAD is not treated, it can progress to critical limb ischemia, resulting in tissue necrosis and eventual limb amputation. Vasculogenesis, the process of de novo vessel formation from progenitor cells, may prove an effective therapeutic strategy. Vasculogenesis may be accomplished by delivering vascular progenitor cells derived from human induced pluripotent stem cells (hiPSCs-EPs), which have recently emerged as a promising, patient-specific therapy. However, the optimal conditions for iPSCs-EPs engraftment and anastomosis with the host vasculature are unclear, specifically, since the underlying molecular mechanisms that guide these cells' self-assembly into vascular networks are poorly understood. To overcome this hurdle, we propose to develop engineered vasculogenic hydrogels, presenting tunable cues at the cell-matrix interface, that can enhance the therapeutic vasculogenesis of iPSC-EPs for peripheral ischemia recovery and define the underlying mechanisms through which matrix properties control vasculogenesis. Previous work by us and others has shown that stable vascular network formation depends on both cell type and matrix properties such as stiffness and degradability. Highly degradable matrices such as collagen may support vasculogenesis initially, but long-term stability is challenging. Furthermore, these matrix properties are coupled and impact endothelial and perivascular cell sprouting at different time scales in neo-vascular network formation. Therefore, we hypothesize that temporal, in situ control over local matrix mechanics and degradability in synthetic matrices will synergistically regulate the vascular morphogenesis of hiPSC-EPs, lead to stable, mature vascular network formation and improve hind limb ischemia recovery. To test our hypothesis, we propose a hybrid interpenetrating hydrogel network (IPN) comprised of collagen and norbornene-modified hyaluronic acid (Coll/NorHA). This system has the advantage of combining the natural cues presented by collagen binding sites and fibrous architecture with the in situ dynamic tunability of synthetic NorHA. Our goal is to 1) elucidate the interplay between time-dependent matrix properties and mechanisms that govern vascular network development and 2) enhance therapeutic vasculogenesis for PAD. In Aim 1, we will modulate the elasticity in these hydrogels using in situ cross-linking reactions. We will study how stiffening at specific timepoints impacts the resulting vasculogenic response both in vitro and in vivo in a skin fold model. In a complementary approach to Aim 1, in Aim 2, we will isolate the effects of matrix degradability on iPSC-EPs vasculogenic potential using Coll/NorHA IPNs in which proteolytic susceptibility is tuned with matrix metalloprotease-degradable peptides. In Ai...