Human communication and perception of the environment are mostly conveyed by information perceived through the ear and the eye. A number of different inherited conditions impair both sight and hearing. The most common of these inherited conditions is Usher syndrome a collection of severe autosomal recessive disorders. The sight loss is caused by gradual and progressive loss of photoreceptor cells and the hearing loss is caused by defects in the inner ear hair cells. Thus far there are no successful treatments for the retinal disorder and only a proportion of cases can benefit from cochlear implants. Here we use induced pluripotent stem (iPS) cells to model Usher2a retina and Usher1b inner ear diseases in vitro. The generation of a culture system to efficiently differentiate diseased sensory cells will aid the development of new treatments.
Usher2 patients account for almost half of all the Usher cases. We have generated a number of iPS cell lines from Usher2A patients and utilized our recently described 2D/3D protocol to differentiate these into retinal organoids. Immunohistochemistry and real time PCR analysis were used to assess the expression of cilial and Usher genes in controls and Usher2A retinal organoids. Transmission electron microscopy was used to evaluate photoreceptor ultrastructure. Photoreceptor degeneration was evaluated by Tunel staining.
Retinal organoid cultures generated photoreceptors containing synapses, connecting cilia, inner segments and outer segments. Usher proteins were present in iPSC-derived USH2a photoreceptor cells and electron microscopy did not demonstrate morphological abnormalities in cilia and outer segment formation. However, increased photoreceptor cell death was observed in Usher2a retinal organoids when compared to control organoids. We have now started to evaluate cellular metabolism pathways such ER stress, autophagy and oxidative stress to further understand the cause of degeneration in vitro.
Mutations in Usher1 genes cause the most severe form of Usher. Usher1b inner ear organoids containing hair cells were successfully generated and characterised using immunohistochemistry. A number of AAV capsids were tested in vitro to establish hair cells transduction efficiency.
These data suggest that Usher iPSC-derived retinal and inner ear organoids represent a potential tool to model disease and therefore enhance our understanding USH pathophysiologyand the mechanism of degeneration. Most importantly, diseased retinal organoids will aid the development of new treatments such as drug screening and gene therapy.