Sabapathy, S; (2015) Optimisation of methods for the differentiation of human embryonic stem cells into retinal progenitor cells. Doctoral thesis , UCL (University College London).

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Abstract

Many ocular diseases result in visual impairment often leading to blindness with disease progression, one such example is Retinitis pigmentosa (RP). The underlying cause for blindness is often due to the degeneration of photoreceptors and supporting cells in the retina. Photoreceptors are responsible for the conversion of light signals into electrical impulses which are processed by the brain to form a visual image. Unlike other cells, photoreceptors cannot be regenerated by the body. For this reason current treatment for many ocular diseases aims to preserve what little vision patients hold. The only way such diseases could be cured is by regenerating photoreceptors and transplanting them into patients to replace lost cells and function. The differentiation of retinal cells from human embryonic stem cells (hESCs) is a difficult process. Current strategies produce low efficiencies of retinal cells which could not supply cells in a clinical environment. It is evident that the key lies within the tight control of the microenvironment of these cells at various stages of the differentiation process. In this study various optimisation avenues were investigated in order to increase the yield of retinal cells. By investigating the impact of various dissociation reagents and enzymes it was found that, the dissociation of cells using TrypLE Express significantly increased retinal gene expression. Using this reagent resulted in over a 100 fold increase in expression of photoreceptor precursor marker Nrl in comparison to the mechanical dissection with Collagenase IV (the control). In this investigation it was also found that the dissociation of cells during the process, in order to reduce cell densities is highly detrimental. The investigations were successful in finding some changes to the published protocols that optimised the differentiation process of pluripotent Shef3 hESCs into retinal cells.

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