Abstract
Purpose :
A healthy corneal endothelial layer is imperative to maintain corneal transparency. Yet, corneal endothelial cells (CEC) have poor regenerative capability. Thus, any injury to this cell layer is irreversible and detrimental to sight. Corneal endothelial transplantation is performed as a therapy to replace affected endothelium, however, the scarcity of human corneal donor tissue, and the quality of aged cadaveric sources make the development of alternative approaches a much needed endeavor. The purpose of this study was to derive a CEC layer from human induced pluripotent stem cell (iPSC) using small molecule screening and a novel differentiation protocol
Methods :
Human iPSC were derived into CECs by a developmental stagewise approach. Initially, neural crest induction was achieved via a neural induction media for 10 days until neurospheres form. Neurospheres were dissociated and hiPSCs suspensions were seeded onto geltrex-coated dishes and gradually switched into a custom-designed CEC differentiation medium supplemented with inhibitors of TGFβ type I receptor kinase (ALK5), Glycogen synthase kinase 3 (GSK3), and Rho-associated kinase (ROCK). At various stages of the differentiation, RNA was extracted and quantitative PCR analysis (qPCR) performed for CEC gene profiles. After 40 days of differentiation, cells were fixed and immunofluorescence was performed to check for CEC phenotypic marker expression
Results :
qPCR analysis revelead that iPSC achieve a correct CEC gene expression profile with significant upregulation of ATP synthase subunit alpha 1 (ATPA1), Zonula Occludence 1 (ZO-1), and Paired Like Homeodomain 2 (PITX2) genes. Remarkably, cells treated with a novel ROCK inhibitor, GSK269962, achieved a robust corneal endothelium expression profile at an earlier differentiation time point when compared to those treated with Y-27632. Differentiated CECs presented the classical cobblestone morphology of human corneal endothelial cells and expressed ATPA1, ZO-1, as well as GJA1, and CDH1, confirming their CEC phenotype
Conclusions :
Our results demonstrate CEC differentiation of human iPSC cells in a stagewise manner. Our small molecule screen identified GSK269962 as a more potent and selective ROCK inhibitor for achieving a CEC genotypic and phenotypic profile. This project leads into a broader tissue-engineered program seeking full-thickness corneal graft constructs engineered from limbal stem cells, iPSC cells and biomaterial scaffolds
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.