PROJECT SUMMARY Scaffolded stem cell transplantation has the potential to provide structured chemical and mechanical cues to guide cell growth into functional tissues for regenerative medicine. However, significant challenges have limited the clinical success of this approach. Cells implanted in large scaffolds often have limited viability due to hypoxic conditions in the host, while cells injected individually or in small clusters often suffer from poor retention at the site of injury. Hydrogels are commonly utilized as stem cell scaffold materials because they confer substantial flexibility in terms of chemical composition and ligand integration. However, hydrogels are also typically amorphous, limiting control over ligand presentation (important for adhesion and signaling), and lacking structural cues such as fibers that are present in biological ECM, which impacts mechanical strength. We have recently demonstrated that it is possible to generate stable, 1-nm-resolution functional patterns on amorphous polyacrylamide and polydimethylsiloxane surfaces, using sub-nm-thick films of highly ordered polydiacetylenes (PDAs) that are preassembled and covalently transferred to the hydrogel surface. Our approach potentially addresses both chemical and mechanical challenges associated with hydrogel stem cell scaffolds, enabling generation of cell-instructive hydrogel tapes that can be shaped to create 3D scaffolds. However, to be useful in clinical settings, this strategy will need to be validated with: (1) commonly used hydrogel stem cell scaffold materials, (2) hydrogel moduli matching the range commonly associated with tissues, and (3) films thin enough for adequate perfusion to prevent hypoxia and enable normal secretome interactions. Here, we develop a platform technology based on cell-instructive hydrogel tapes, benchmarking their chemical and mechanical properties, and their impacts on human mesenchymal stem cells (hMSCs). In Aim 1, we evaluate the hypothesis that PDA surface functionalization can improve chemical control over surfaces of hydrogels common in regenerative medicine, orienting and spatially clustering ligand presentation, to modify stem cell growth in a predictable fashion. We test this by culturing hMSCs on surfaces designed to maintain stemness or to induce specified differentiation behavior (angiogenesis, adipogenesis, chondrogenesis), benchmarking against common stochastic hydrogel modification strategies. In Aim 2, we evaluate the hypothesis that our PDA surface-functionalization approach can improve the mechanical and handling properties of thin, soft hydrogel films, enabling creation of cell-instructive hydrogel tapes. We generate and test impacts of paired tapes as cell sandwich scaffolds and 3D constructs that provide structured chemical surfaces and mechanical environments, while maximizing perfusion to and from cells. Overall, this proposal develops a modular surface functionalization strategy that can be easily integrated...