This Multi-PI project combines the efforts of two research groups with different areas of expertise to address the long-term goal of developing bioengineered corneal stroma and endothelial tissues to provide therapy for individuals with corneal blindness. These tissues will be bioengineered from adult stem cells, which can be obtained from the individuals to be treated as autologous or from allogeneic cell storage since the stem cells are immunosuppressive. Over the past 5 years we have demonstrated that organization of these cells into tissues can be guided by scaffolds constructed of native extracellular matrix proteins, fabricated using a biomimetic, surface-induced assembly process. The Du Lab at the University of Pittsburgh will obtain stem cells from limbal stroma of donated human corneas. Their extensive work with these corneal stromal stem cells (CSSC) shows that they differentiate to stromal keratocytes and to corneal endothelial cells, tissues responsible for most corneal opacity. We have demonstrated that CSSC can be obtained from biopsy samples, presenting the opportunity to generate patient-specific, autologous bioengineered tissues. The Feinberg Lab at Carnegie Melon University has developed novel approach of assembling native extracellular matrix proteins to produce tissue-like scaffolding with defined 3-D architecture. Aim 1 will build on our previous work, which has showed that we can engineer spatial and biochemical cues provided by the scaffolding to generate stroma-like tissue from CSSCs that can be stacked to form multilamellar 3-D tissue similar to that of the corneal stroma. Bioengineered stroma produced in the proposed experiments will be subjected to biomechanical loading to simulate cornea development and further improve mechanical and optical properties. Function of the bioengineered stroma in lamellar keratoplasty will be evaluated in an in vivo rabbit model. Aim 2 will build on our work differentiating CSSC into endothelial cells and growing these on engineered basement membrane protein scaffolds to form polygonal monolayers that express genes typical of corneal endothelium. Previously, we demonstrated the ability to create the equivalent of Descemet's membrane to bioengineer an entire sheet of endothelium suitable for lamellar keratoplasty, but we also developed an alternative approach to engineer small patches of endothelium and deliver these via simple injection. Bioengineered endothelium produced in the proposed studies will use our newly developed “shrink-wrapping” technology to create microscale patches of corneal endothelium that can be injected into the anterior chamber for injury-free engraftment. Functionality of the constructs will be demonstrated in rabbit models in vivo, focused on boosting cell-density to improve pump function as an alternative to lamellar keratoplasty for endothelial disease. This project will build on the innovative experimental approaches we developed during the first 5 years of this ...