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Among the different layers of the cornea, the corneal endothelium, which is composed of corneal endothelial cells (CEC), plays a key role in the maintenance of cornea transparency. Based on the donor shortages and the limitations associated with transplantation, in this work we have developed collagen hydrogels with different patterned structures on the surface to provide topographies in ranges similar to the natural environment that CEC sense. This aimed at stimulating cells to maintain a typical CEC phenotype and provide alternatives to corneal transplantation. In this sense, we have elaborated curved collagen hydrogels (concave and convex) with three different topographies (50, 200 and 300 µm grooves), with the help of 3D printed mold and replicating the mold with the collagen hydrogel, onto which CEC were cultured in order to analyze its behavior. Flat hydrogels were used as controls. Cell morphology, cell circularity and gene expression of ATP1A1 and ZO-1 genes were analyzed after 3 and 6 days. Results showed an effect of the curvature and the topography compared to flat collagen hydrogels, showing higher expression of ZO-1 and ATP1A1 in curved non-patterned hydrogels at day 3. The patterned hydrogels did not have such a significant effect on gene expression compared to flat hydrogels, showing stronger effect of the curvature compared to the topography. The effect was opposite at day 6, showing higher gene expression at days 6 on the patterned hydrogels, especially for the ZO-1 gene. The gene expression results were in accordance with the cell morphology observed at the different time points, showing circularities closer to hexagon like morphology at shorter time points. Overall, this platform can serve as a system to culture cell under proper environment to further be able to transplant a CEC monolayer or to allow transplantation of thin collagen layers with cultured CEC.

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.