Tissue Engineering for Ocular Surface Reconstruction: Differentiation of human pluripotent stem cells towards corneal epithelium

Corneal epithelium, the outermost layer of the transparent and avascular cornea, is renewed by tissue-specific stem cells, termed limbal epithelial stem cells (LESC). These stem cells are located in specialized niches of the limbus, a narrow transition zone between the cornea and sclera, which also serves as a physical barrier between the clear avascular cornea and the vascularized conjunctiva. Extensive trauma to the limbus, or LESC dysfunction as a result of certain chronic inflammatory diseases may lead to limbal stem cell deficiency (LSCD), characterized by the spread of the vascularized conjunctival tissue over the damaged ocular surface. This vision-threatening condition varies in its severity, but commonly causes severe symptoms and is difficult to treat. Conventional corneal transplantation is not a feasible treatment option, as it only replaces the central corneal epithelium, and not the damaged limbus. Therefore, various strategies aimed at replacing damaged LESCs have been explored. Limbal transplantation, with or without ex vivo expansion of LESCs, has shown great promise. However, patients suffering from LSCD in both eyes are common and donor corneal tissue is scarce. Alternative approaches for ocular surface reconstruction are therefore needed. Human pluripotent stem cells (hPSC) are capable of virtually unlimited selfrenewal and can differentiate into any cell type, including LESCs and corneal epithelial cells. To date, only a few studies have demonstrated successful differentiation of corneal epithelial cells from hPSCs, and these methods rely on the use of chemically undefined and xenogeneic culture components. Such factors are subject to biologic variation and pathogen transmission, and thereby require a thorough quality control to ensure the safety of transplantable cell populations. Nevertheless, hPSC-derived LESCs could provide an alternative cell source for cell-based therapy of severe ocular surface disorders. In addition, hPSC-derived LESCs could be applied to studying corneal development and modelling corneal tissue as a testing platform for in vitro drug development. This dissertation aimed to investigate differentiation of LESCs and corneal epithelial cells from hPSCs under conditions that would allow for a smooth transition to clinical applications. A directed and efficient two-stage differentiation method was developed, generating LESC-like cells capable of self-renewal and terminal differentiation towards mature corneal epithelial cells. Differentiation was carried out in close to chemically-defined and xeno-free conditions, and several modifications could be explored further. Although there was a certain degree of variation in differentiation efficiency between the studied cell lines, the method was consistent and reproducible overall. Thorough characterization of hPSC-derived cells is crucial to confirm their authenticity. In this dissertation, several key characteristics of LESCs were examined. It was demonstrated that hPSC-derived LESCs possess the appropriate cell morphology, as well as gene and protein expression profiles. They were also able to self-renew and proliferate in culture, and terminally differentiate towards mature corneal epithelial cells. Moreover, hPSC-derived LESCs were similar to native ocular surface epithelial cells, as verified using relative mass spectrometrybased proteomics. Cell transplantation to the ocular surface would require a transparent, mechanically durable, yet elastic carrier biomaterial. Currently, human amniotic membrane is the gold-standard material commonly used to transplant donor LESCs, despite its limitations. The final aim of this dissertation was to evaluate the suitability of a bioengineered collagen matrix for the culture and transplantation of hPSC-derived LESCs. The fabrication of this biomaterial is standardized, it is suitable for clinical use, and has shown promise in earlier biocompatibility studies using an animal model. In this study, the bioengineered collagen matrix supported the attachment and growth of hPSC-derived LESCs in vitro, and could therefore be applied to clinical use in the future. In conclusion, this dissertation as a whole describes a novel tissue engineering approach to ocular surface reconstruction. These results have contributed to increasing the knowledge of hPSC differentiation towards LESCs, their characteristics, and potential for use in cell-based therapy.