Prosensory signals: from discovery to application using inner ear organoids

Our senses of hearing and balance depend on specialized 'hair' cells located in the sensory organs of the inner ear. The hair cells are only produced during the embryonic development of the inner ear, and their disappearance in adult life is the leading cause of hearing loss, in particular in the ageing population. The treatments for hearing loss are limited to hearing aids or cochlear implants, and there is considerable need for new and more efficient therapies. The most promising venues in terms of cellular therapies rely on i) the re-activation of developmental signals to induce hair cell regeneration and ii) the use of stem cell systems such as 3D inner ear "organoids" to model diseases and test new therapeutic approaches.
In our previous MRC-funded research, we have uncovered new genes and signalling pathways that are crucial for "prosensory specification", the developmental process leading to the formation of hair cell progenitors. In this project, we will test the function of these candidates by artificially modifying their activity in the embryonic chicken inner ear. We will also use single-cell DNA sequencing technologies to gain new insights into the gene expression profile of mammalian prosensory cells, thereby helping us to pinpoint the most critical signals controlling prosensory specification. Finally, we will use inner ear organoids derived from mouse embryonic stem cells to investigate the mechanisms of prosensory specification in vitro. The results of this project will help to uncover the developmental genes and cell-to-cell communication pathways that could be manipulated to trigger hair cell regeneration in the damaged inner ear. They will also contribute to the improvement of current methods for the generation of hair cells from embryonic stem cells, which are essential from a basic and clinical research perspectives. In the future, this work could open up new venues for more efficient therapies for hearing loss in humans.

In the UK alone, one in six people has a hearing disability, rising to one in two for over 70 year-old. The main cause of sensorineural hearing loss is the loss of auditory hair cells residing in the inner ear. A potential therapy for hearing loss could be to induce in the damaged auditory organ the regeneration of new hair cells - a process that is occurring spontaneously in non-mammalian vertebrates. However, the current strategies relying on the manipulation of the molecular signals controlling hair cell differentiation alone are relatively inefficient. An alternative approach could be to re-activate the developmental signals that drive the formation of the 'prosensory' cells, which are the progenitors for both hair cells and their associated supporting cells. However, this requires a better understanding of the molecular mechanisms of prosensory specification.
In our previous MRC-funded project, we have uncovered a new role for the Wnt signalling pathway as a dose-dependent regulator of prosensory specification. We also started RNA-Seq and functional screenings aimed at identifying new genes participating to this process. Here, we will pursue this research with new methods to maximize our chances of identifying key regulators of prosensory specification. We will use Crispr/Cas9 mediated gene-knockdown for testing the function of candidate factors, such as the Tcf/Lef effectors of the Wnt pathway, in the embryonic chicken inner ear. We will use single-cell RNA-Seq to characterize mouse prosensory cells and bioinformatics methods to pinpoint the key genes associated to prosensory specification. Finally, we will translate our findings to 3D inner ear organoids, using a mouse stem cell line carrying a fluorescent reporter of Sox2, a prosensory marker. The outcomes of this research could be exploited in the future to induce hair cell regeneration by means of gene therapies or to improve existing protocols for the generation of hair cells in 3D organoids.