טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLaura Serror
SubjectStudy of Corneal Pathophysiology and Therapy Using
Induced Pluripotent Stem Cells
DepartmentDepartment of Medicine
Supervisors Professor Shalom-Feuerste Ruby
Full Professor Aberdam Daniel
Full Thesis textFull thesis text - English Version


Abstract

Corneal diseases such as limbal stem cell deficiency (LSCD), are affecting millions of individuals. The disease mechanisms are largely unknown and as specific drugs do not exist, patients undergo corneal grafting. The difficulty in developing treatment stems from the lack of physiologically relevant models. Human induced pluripotent stem cells (iPSCs) hold promises for regenerative medicine as they may provide an alternative source for therapy, and moreover allow disease modelling and drug design. The objective of the project was to test the hypothesis that iPSCs-derived corneal epithelial cells (DCEC) can provide a model to study corneal pathophysiology and the design of therapies. To this end we established a well-characterized model of corneal differentiation system from iPSCs. Importantly, iPSCs differentiation recapitulated corneal epithelial embryogenesis in vivo. To evaluate the therapeutic potential of iPSCs DCEC, we established a rabbit model of LSCD and we established a method to produce transplantable corneal-reconstituted tissues from human limbal epithelial cells. Next, our method allowed us to design a unique cellular model that recapitulated major embryonic defects related to a syndrome called ectodermal dysplasia (EEC). Fibroblasts from healthy donors and EEC patients carrying mutations in the DNA binding domain of p63 were reprogrammed into iPSCs. EEC-iPSCs from both patients showed early ectodermal commitment but failed to differentiate into corneal epithelial progenitors. APR-246, a compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSCs for p63-related disorders and paves the way for future therapy of EEC. Finally our new model of corneal differentiation was used to study the molecular circuitry by which Pax6, the master regulator of the eye is regulated.  It was found that miR-450b-5p can bind to the 3’UTR of Pax6, suggesting that it may be a direct repressor of Pax6. Expression profile of miR-450b and Pax6 revealed a reciprocal expression in iPSCs differentiation, during lens development and in adult corneal epithelial compartments. Additionally, bone morphogenic protein 4 (BMP4), which is an essential morphogen for eye embryogenesis, upregulated miR-450b and repressed Pax6, during corneal commitment of iPSCs and lens morphogenesis. Altogether, these data suggests a new mechanism of Pax6 regulation by BMP4-mediated miRNA that may have significance for corneal/lens development, homeostasis and diseases. Finally, this study shows that iPSCs are valuable for modeling the mechanisms underlying eye morphogenesis, pathophysiology, drug development, and regenerative medicine.