Application of stem cell-derived lens cells and new bioinformatics tools to investigate regulators of lens biology

Abstract

Cataract, caused by the opacification of the eye lens, is the most prevalent cause of blindness worldwide. Despite knowledge of growth factor pathways and transcription factors involved in lens biology, detailed mechanisms of normal lens and cataract development are yet to be fully identified. Understanding these signaling mechanism can provide opportunities to better understand cataract aetiology and, potentially, cataract treatment. The process of lens development is known to be regulated by multiple growth factor signaling pathways. Among them, FGF, PDGF, BMPs and TGFβ signaling play important regulatory roles in lens induction, proliferation and differentiation. These growth factors activate various intracellular kinases, including MAPK and AKT, that regulate lens growth, development and survival. However, many aspects of growth factor-initiated signaling circuitry in human lenses are yet to be explored, including crystallin protein expression. This is largely due to difficulties accessing human tissue, and species-specific differences between humans and animal models. To overcome these issues, this thesis describes a combined approach using stem cell-derived human lens epithelial cells (LECs) and micro-lenses (mi-ls), and signaling pathway predictions obtained via a recently developed algorithm (SPAGI). Discrete molecular hypotheses for FGF, PDGF, BMP and TGFβ signaling in lens cells, including expression of critical lens crystallin proteins, were generated using the SPAGI algorithm. Based on these predictions, inhibitor studies were initiated using the human LECs and mi-ls, with analysis methods including light microscopy, RNA-sequencing, mass spectrometry and histology. Chapter 1 presents a literature review that provides an overview of growth factors known to be involved in lens development, as well as key lens crystallin proteins and their role in normal and cataractous lenses. The experimental chapters are presented in manuscript format, and the author contributions for each chapter are included in chapter-specific ‘Acknowledgement’ sections presented immediately before the chapter-specific Refence sections. The figures and tables of each chapter are placed after the reference sections. In Chapter 2, the SPAGI algorithm and stem cell-derived LECs were used to investigate the role of FGF2 in regulating MAPK and AKT by applying inhibitors of MAP2K1 and AKT. In Chapter 3, the SPAGI algorithm was used to predict lens-specific, hierarchical relationships between FGF and PDGF signaling pathway members. Chapter 4 investigated the impact of BMP and TGFβ signaling on crystallin expression and development of light focusing in human mi-ls. The data show BMP and TGFβ signaling can both stimulate crystallin expression, and that inhibition or activation of these pathways decreases or increases mi-l focusing activity (respectively). Histology suggested both BMP and TGFβ pathways can regulate LEC differentiation to lens fiber cells, and candidate transcription factors were predicted that might mediate these effects. Lastly, Chapter 5 provides an overall discussion of how the research outcomes from the thesis have identified new and important questions related to lens and cataract biology for future research.

Date of Award	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Michael O'Connor (Supervisor)