Suzanne de Bruijn

300 Chapter 6 BOX 1 - A limitation: Tissue availability to study vision and hearing diseases For both multi-omics approaches and single cell approaches, it is crucial that the optimal tissue of interest is selected for investigation. However, tissue availability imposes a major limitation to study both retinal and inner ear diseases. Over the years, different animal models have been employed to study sensory diseases, which led to the successful validation of several candidate disease genes (for example EYS -associated RD 105 and TRRAP -associated HL 106 in zebrafish). The use of animal models is time and money consuming, and raises several ethical concerns. Due to the evolutionary distance, and potential differences in physiology, findings in animal models are not always easily translatable to the patient situation. For example, disease genes are not always conserved among species (e.g. EYS 105 ), or exert the same function (e.g. DFNA5 107 ) Patient-derived induced pluripotent stem cells (iPSC) provide a valuable alternative. Several protocols are available that allow the differentiation of iPSCs into cells that optimally mimic the sensory (precursor) cells of the eye and ear. 108-110 Many of the omics and single cell technologies mentioned in sections 1 and 2 have been successfully applied in stem cell-derived models and crucial insights were gained regarding disease mechanisms. Novel splice defects have been identified using 2D photoreceptor precursor cells 83 , and enhanced splice and ciliation defects were previously observed in 3D retinal organoids compared to fibroblast cells. 111 Additionally, these cell models have proven value for in vitro validation of personalized therapies: an AON treatment in 3D retinal organoid and RPE cells derived from a patient with pathogenic CEP290 variants rescued the ciliary defects. 112 Although efficient protocols are available to produce cochlear hair cells or retinal photoreceptors, not all cellular specializations or cell types are equally represented or generated in the differentiated cell models. In most retinal organoid models, only limited development of photoreceptor outer segment discs is described, and neuronal cell types often display a fast degeneration. 108 Also, the development of retinal organoids is significantly slower than that of the human native retina. 113 Direct interactions of photoreceptor cells with the retinal pigment epithelial (RPE) layer are absent, which are essential when studying multifactorial diseases that affect the multiple cell layers. The latter maybe can be resolved by generating multi-layer tissue models in a retina-on-a-chip platform. 114 The established differentiation protocols for inner ear organoids still have some important limitations as well. Current iPSC-derived inner ear models only represent a small subset of the diverse cell populations in the inner ear and the main focus has been on the generation of sensory cell types. 110 However, many of the HL-associated genes are expressed in non-sensory or mesenchymal cell populations and therefore more attention should be paid to a broader set of cell types to study pathogenesis for defects of these genes as well. To illustrate, an important example is the investigation of SLC26A4- associated pathogenic mechanisms). As described in chapter 5 , the genetic defect of the CEVA haplotype found in the majority of monoallelic patients is still unknown. A logical next step would be to interrogate the RNA for aberrant expression levels of SLC26A4 or splice alterations, which would require a suitable cell model with detectable expression levels. SLC26A4 is not expressed in readily available cell types (e.g. blood and fibroblasts) and also not induced in iPSCs or otic progenitor cells. 115 Fortunately, a differentiation protocol aimed to specifically generate sulcus-like cells that do express SLC26A4 has been described. 11,115 The sulcus-like cells generated by this protocol are less complex than the multi- cellular organoid 3D systems. However, they can still potentially help to overcome the current deadlock in transcript analysis to unravel SLC26A4 pathogenicity.