Derivation of cochlear cells from pathological or isogenic human iPSCs for modeling hereditary hearing loss

Alström Syndrome (AS) is a human autosomal recessive genetic disorder characterized by numerous clinical symptoms including deafness. AS is caused by mutations in the ALMS1 gene encoding for ALMS1 protein expressed at the basal body and implicated in ciliogenesis, cell cycle and proliferation (Jagger et al., 2011; Zulato et al., 2011 & Shenje et al., 2014). We are interesting in understanding the unknown mechanisms involving this protein in the genetic deafness of AS patients. To develop a model as closer as possible to the human pathology, we are using human induced pluripotent stem cells (hiPSCs) generated from fibroblasts of healthy and AS patients. Using a stepwise protocol, we demonstrated that healthy hiPSCs (waiting for isogenic hiPSCs) can generate a population of cells with gene and protein expression patterns consistent with the ones of otic progenitor cells (OSCs). At this differentiation stage, we observed some proliferation and apoptotic defects between healthy and AS cells. When human OSCs are co-cultured with mouse feeder cells, they are able to differentiate into hair cells (HCs). We successfully differentiated AS hiPSCs generated from AS patients into HCs. We are currently confirming gene expression pattern and testing HCs functionality.  To exclude patient linked epigenetics and differentiation defects, we are correcting the genomic mutation in the AS hiPSCs to generate isogenic hiPSCs using the CRIPSR/Cas9 system. Thanks to the isogenic hiPSCs we will be able to confirm that these defects are well due to the ALMS1 mutation.Derivation of cochlear cells from pathological or isogenic human iPSCs for modeling hereditary hearing los

Unravelling Cemip expression and functions in the auditory portion of the inner ear.

The inner ear is a complex organ composed of the vestibular system – which is the balancing system – and the cochlea – which is the earing system. The cochlea is a coiled shape organ composed of three main structures: the spiral ligament sitting on top of the stria vascularis, the organ of Corti with sensory hair cells and supporting cells and the spiral ganglion composed of neurons and glial cells. After an auditory stimulus, the sound wave progresses in the scala media filled with endolymph and induces a stimulation of sensory hair cells. These cells then transmit the information to the spiral ganglion neurons connected to them. Of course, the correct ionic homeostasis of endolymph is required for a good sound wave transmission. This homeostatic function is assured by the stria vascularis and the spiral ligament. The alteration of one of the structures mentioned before induces deafness. Currently, numerous genes have been associated to this kind of hearing loss. In the present work, we focus our attention Cemip – also known as KIAA1199 – that has been associated to human hereditary neurosensory deafness. Indeed, three missense mutations consisting in non-synonymous amino acid changes (R187L, R187H and H783Y) have been associated to this form of deafness. Therefore we would like to understand the role of Cemip in the cochlea. For that we have analysed Cemip mRNA pattern of expression by in situ hybridization at different developmental stages on cochlear sections. It seems Cemip mRNA is not present in the auditory portion of the inner ear at early embryonic stage 14 (E14) while it is largely present at E17 in the spiral ganglion, in supporting cells of the organ of Corti and in the spiral ligament. This expression is maintained post-nattily until P7. At P21 the expression is restricted to the spiral lamina - an osseous structure surrounding the spiral ganglion. Our on going work is aimed at revealing the biological role of Cemip in the cochlea in conditional knock-out mice

Pluripotent stem cell-derived cochlear cells: a challenge in constant progress

Hearing loss is a common affection mainly resulting from irreversible loss of the sensory hair cells of the cochlea; therefore, developing therapies to replace missing hair cells is essential. Understanding the mechanisms that drive their formation will not only help to unravel the molecular basis of deafness, but also give a roadmap for recapitulating hair cells development from cultured pluripotent stem cells. In this review, we provide an overview of the molecular mechanisms involved in hair cell production from both human and mouse embryonic stem cells. We then provide insights how this knowledge has been applied to differentiate induced pluripotent stem cells into otic progenitors and hair cells. Finally, we discuss the current limitations for properly obtaining functional hair cell in a Petri dish, as well as the difficulties that have to be overcome prior to consider stem cell therapy as a potential treatment for hearing loss

Matrice extracellulaire et fonction auditive : Rôle de l'acide hyaluronique

Hyaluronic acid (HA), a non-sulfated glycosaminoglycan, is part of the ground substance, a gel-like structure that bathes all the ECM components. HA has been involved both in tissue biomechanics, as it awards visco-elastic properties, and in cell signaling, through cell-ECM receptors. Thus, HA takes part in several biological processes, including morphogenesis and inflammation. Multiple studies demonstrated the importance of ECM during inner ear development and in hearing and some HA-related genes have been associated with hearing impairments. As such, the gene Cemip (or KIAA1199), encoding a Hyaluronidase, has been reported to be mutated in deaf patients. We therefore aim at characterizing the role of HA in hearing function. By combining specific immunostainings, RNAscope and qRT-PCR assays, we first explored the spatio-temporal distribution of cochlear HA as well as the expression profile of enzymes responsible for its synthesis and degradation. In addition, we generated a mutant mouse model in which Cemip gene has been invalidated. We took advantage of this model to investigate the impact of HA accumulation on cochlear morphogenesis and hearing function, by performing morphological analyses and Auditory Brainstem Response (ABR) recordings. We found that HA is highly enriched in the basilar membrane (BM), for which visco-elastic properties are instrumental in sound wave decomposition, frequency discrimination and mechanical sound wave conversion. The main enzymes involved in HA metabolism are present at embryonic and postnatal stages in the cochlear duct and in the spiral ganglion. Despite an accumulation of HA in the BM region below hair cells, the global morphology of Cemip-deficient cochleae is preserved, suggesting that Cemip has no prominent role in cochlear development. However, we evidenced that hearing function of Cemip KO mice is slightly impaired, as ABR recordings revealed an increase in peak 1 amplitude at some frequencies. Although unexpected, this result suggests that either more sensory cells are stimulated by sound, or that spiral ganglion neurons are over-activated compared to control mice. Altogether, our data suggest that HA might be instrumental in cochlear biomechanics, by awarding visco-elastic properties to the BM. Cemip loss has no critical impact on cochlear development and hearing, although an overstimulation of the cochlear nerve has been observed. We are currently investigating further this phenotype to identify the cause of this neuronal over-stimulation and we particularly focus on BM morphology and cochlear perineuronal nets. In the future, we also plan to examine whether Cemip KO mice are more prone to noise-induced hearing loss due to neuron excitotoxicity, for example

Gene expression pattern of NEDD4-1/2 in the developing inner ear

Protein ubiquitination is an important post-translational modification that is involved in multiple cellular processes such as protein degradation, endocytosis, sorting, trafficking and turnover. A recently identified mutation in human NEDD4L gene, which encodes the E3 ubiquitin-ligase NEDD4-2, has been shown to lead to several abnormalities in patients such as cerebral atrophy, optic atrophy, and hearing impairment. This observation, combined with the fact that NEDD4-2 knockout mice are deaf lead us to investigate the roles of NEDD4-1/2 in the development of the inner ear. The first part of the project focuses on establishing the expression pattern of Nedd4-1/2 mRNAs and proteins in the inner ear by in situ hybridization and immunohistofluorescence, respectively. We show that both NEDD4-1 and NEDD4-2 are dynamically expressed in the embryonic cochlea, with transcripts present in the mechanosensory hair cells, the greater epithelium ridge, spiral ganglion neurons and the stria vascularis. We notice that while NEDD4-1 expression remains in these structures at postnatal stages, NEDD4-2 expression is significantly reduced. This NEDD4-2 expression profile suggest a role of NEDD4-2 in cochlear cell proliferation, differentiation and patterning. Functional and histological studies are ongoing on NEDD4-2 knockout animals in order to decipher its role on hearing and cochlear development.Functions of NEDD4-2 in the auditory portion of the mouse inner ea