Generation of human pluripotent stem cell-derived micro-lenses to investigate lens development and lens toxicity

Abstract

Cataract is an opacification of the eye's lens. It is the leading cause of blindness and causes life-impacting vision loss for millions of people worldwide. Currently the only treatment is surgical implantation of an intra-ocular lens. Due to the number of annual surgeries this approach is expensive and can result in a range of sightaffecting complications. Difficulties arise in accessing vision-restoring surgery for people with cataract living in developing countries due to inadequate access to cataract surgery. Greater understanding of cataract molecular mechanisms is required for development of anti-cataract drugs that may be easier to access than surgery. Several cataract risk factors have been postulated; however, their specific molecular mechanisms are poorly identified. This is due largely to a lack of functional human lens tissue available for studying cataract. Previous investigations of cataract have used animal models to study lens development and cataract; however, there are significant differences between humans and animal lenses including protein expression and cell membrane composition. Human pluripotent stem (PS) cells are a potential source of human lens cells and an elegant 3-stage protocol for generating lens cells and rudimentary lens tissue was published in 2010. Several groups have attempted to refine this protocol with varying success. Nevertheless, all of these approaches suffer from three main issues: i) production of heterogeneous cultures of human lens and non-lens cells; ii) poorly- or uncontrolled-production/loss of rudimentary lens tissue; and iii) limited or no evidence that the stem cell-derived lens tissues have focusing ability (a defining property of the lens). Very recent work in our laboratory overcame the first of these issues, allowing simple and large-scale production of purified, ROR1-positive human lens epithelial cells (LECs) from heterogeneous cell cultures generated from PS cells. This ROR1+ LEC purification protocol provided a starting point for this thesis to investigate conditions for generating light-focusing human lens tissue in vitro. The studies presented here resulted in a method to produce tens-of-thousands of uniform, transparent and light-focusing human PS cell-derived lens organoids, termed micro-lenses. These micro-lenses appear able to model a clinically-relevant cataract associated with exposure to the cystic fibrosis drug Vx-770. These functional human micro-lenses represent a useful tool for drug discovery and toxicity. In addition to the successes of producing the world-first human PS cell-derived micro-lenses, there were several lessons learned during this project. Repeated failures of the cells to survive and proliferate on RGDS-chitosan film negated some of the first approaches at human lens regeneration (i.e., attempts at producing a chitosan-based explant pair system); these difficulties required consideration of an alternate approach. A wider understanding of lens biology gave rise to the idea of trying to mimic non-human lens developmental in the lab, inspired by teleost lens development. This LEC aggregation method was made possible by thinking laterally and using simple, available, materials to replicate embryonic aspects of teleost lens development. This method ultimately proved successful in producing human PS cellderived micro-lenses. Furthermore, the rapid assessment lesson learned by experiencing some unworkable situations in the course of the project were then applied during the hydrogel 3D growth environment trials. Repeatable aggregate loss in some hydrogels confirmed that the agarose-based gels best supported the developing micro-lenses and facilitated progression of investigation of micro-lenses derived from enriched ROR1 cells. Additionally, this project was not without its share of failures. Reflection upon these challenges and ascertaining the reasons for (and ways to move beyond) them have changed my approach to managing difficulties and improved my analytical abilities. These skills will be applied to my future undertakings.

Date of Award	2018
Original language	English