Microenvironment based optimization of retinal induction using CRISPR-CAS9 reporter pluripotent stem cells as an expandable source of retinal progenitors and photoreceptors.

A primary objective of this work was to develop the technical capability to generate large pools of reproducible stem ell derived retinal tissues that could potentially be used for transplantation purposes. A limitation to the use of stem cell derived retinal tissues has historically been the low rates of conversion of stem cells into retinal tissues and reproducibility that raises questions about safety. We began this project by making generating retinal cell type specific reporters using CRISPR-Cas9 gene-editing. The tools we developed allow us to monitor cells as they transition from a stem cell into a retinal neuron. Using these tools we began a series of experiments to optimize retinal cell growth using high content imaging of large numbers of stem cell derived organoids. Through these experiments we were able to optimize conditions ranging from the microenvironment the cells grow in to growth factor regulation. We successfully achieved our goal of producing reliable and robust retinal tissues. To verify the authenticity of these tissues we used a variety of next generation sequencing techniques to quantify gene expression, microRNA expression and the chromatin landscape of developing cells. Using these tools we have identified a number of biomarkers that we believe will aid in identifying transplant capable retinal tissues from retinas derived from any stem cell source.