A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

Background Failure of the corneal endothelium cell (CEC) monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form CEC monolayer. We hypothesize that by using dental pulp stem cells (DPSC) that share an embryological origin with CEC, 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 (iPSC). Methods In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem cells and, then, into CEC. Results Initially, we compared the efficiency of direct differentiation of DPSC with the differentiation of iPSC to express NCSC related genes in adhesion culture, showing significantly higher levels of early stage marker AP2 for the DPSC compared to iPSC. To provide better environment for NCSC gene expression, suspension method was performed, which induced the formation of neurospheres. The results showed neurosphere formation after few days, obtaining the peak of NCSC marker expression after 4 days, showing overexpression of AP2, p75 and CHD7 markers, confirming the formation of NCSC like cells. Furthermore, pluripotent markers Oct4, Nanog and Sox2 were as well upregulated in suspension culture. Neurospheres were then directly cultured in CEC conditioned medium for the second differentiation, showing the conversion of DPSC into polygonal like cells expressing higher levels of ZO-1, ATP1A1, COL4A2 and COL8A1 markers, providing proof of the successful conversion into CEC. Conclusions Therefore, our findings demonstrate that patient-derived dental pulp stem cells represent an autologous cell source for corneal endothelial regeneration that avoids actual transplantation limitations as well as reprogramming techniques.