Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells.

UNLABELLED: The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A(WT/WT); C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. SIGNIFICANCE: Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs.

Stem cell therapy for the inner ear: recent advances and future directions.

In vertebrates, perception of sound, motion, and balance is mediated through mechanosensory hair cells located within the inner ear. In mammals, hair cells are only generated during a short period of embryonic development. As a result, loss of hair cells as a consequence of injury, disease, or genetic mutation, leads to permanent sensory deficits. At present, cochlear implantation is the only option for profound hearing loss. However, outcomes are still variable and even the best implant cannot provide the acuity of a biological ear. The recent emergence of stem cell technology has the potential to open new approaches for hair cell regeneration. The goal of this review is to summarize the current state of inner ear stem cell research from a viewpoint of its clinical application for inner ear disorders to illustrate how complementary studies have the potential to promote and refine stem cell therapies for inner ear diseases. The review initially discusses our current understanding of the genetic pathways that regulate hair cell formation from inner ear progenitors during normal development. Subsequent sections discuss the possible use of endogenous inner ear stem cells to induce repair as well as the initial studies aimed at transplanting stem cells into the ear.

Defining the cellular environment in the organ of Corti following extensive hair cell loss: a basis for future sensory cell replacement in the Cochlea.

BACKGROUND: Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss. METHODOLOGY/PRINCIPAL FINDINGS: Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca's, suggesting genetic background influences the rate of re-organisation. CONCLUSIONS/SIGNIFICANCE: The lack of dedifferentiation amongst supporting cells and their replacement by cells from the outer side of the organ of Corti are factors that may need to be considered in any attempt to promote endogenous hair cell regeneration. The variability of the cellular environment along an individual cochlea arising from patch-like generation of flat epithelium, and the possible variability between individuals resulting from genetic influences on the rate at which remodelling occurs may pose challenges to devising the appropriate regenerative therapy for a deaf patient.

Study on neural stem cell transplantation into natural rat cochlea via round window.

OBJECTIVE: The aim of the study is to investigate the survival of neural stem cells (NSCs) in normal rat cochlea and their potential effect on auditory function and cochlea structures via round window transplantation. METHODS: In comparison with the normal rats without any transplantation (group III), normal rat cochleae were transplanted with NSCs infected with adenovirus carrying green fluorescence protein (GFP) gene (group I) or the artificial perilymph (group II) via round windows. Auditory functions were monitored by thresholds of auditory brain stem responses (ABRs); the cochlea structures were examined by hematoxylin and eosin staining; survivals of implanted NSCs were determined by the expression of GFP; survivals of hair cells were accessed by whole mount preparation; and ultrastructures of hair cells were examined by scanning electron microscopy. RESULT: There were significant differences in the click-ABR thresholds in rats among all 3 groups neither at pretransplantation nor at posttransplantation; there were no significant differences in these values before and after transplantation in the same rats from each group. After transplantation, the cochlea structures were normal in both group I and group II. Grafted NSCs were visualized by the GFP expression in every turn of the cochlea in all animals of group I. There were no significant differences in the losses of outer hair cells (OHCs) among 3 groups. The inner hair cells and most OHCs were normal in every turns of cochleae of all groups. CONCLUSION: Neural stem cells survived in normal rat cochlea after transplantation via round window and showed no obvious effects on auditory functions and inner ear pathologic examination of the rat cochlea.

Methods for providing therapeutic agents to treat damaged spiral ganglion neurons.

Sensorineural hearing loss, characterized by damage to sensory hair cells and/or associated nerve fibers is a leading cause of hearing disorders throughout the world. To date, treatment options are limited and there is no cure for damaged inner ear cells. Because the inner ear is a tiny organ housed in bone deep within the skull, access to the inner ear is limited, making delivery of therapeutic agents difficult. In recent years scientists have investigated a number of growth factors that have the potential to regulate survival or recovery of auditory neurons. Coinciding with the focus on molecules that may restore function are efforts to develop novel delivery methods. Researchers have been investigating the use of mini osmotic pumps, viral vectors and stem cells as a means of providing direct application of growth factors to the inner ear. This review summarizes recent findings regarding the molecules that may be useful for restoring damaged spiral ganglion neurons, as well as the advantages and disadvantages of various delivery systems.

Strategies for replacing lost cochlear hair cells.

The auditory sensory epithelium is a mosaic composed of sensory (hair) cells and several types of non-sensory (supporting) cells. All these cells are highly differentiated in their structure and function. Mosaic epithelia (and other complex tissues) are generally formed by differentiation of distinct and specialized cell types from common progenitors. Most types of epithelial tissues maintain a population of undifferentiated (basal) cells which facilitate turnover (renewal) and repair, but this is not the case for the organ of Corti in the cochlea. Therefore, when cochlear hair cells are lost they cannot be replaced. Consequently, sensorineural hearing loss is permanent. In designing therapy for sensorineural deafness, the most important task is to find a way to generate new cochlear hair cells to replace lost cells.

Development of location-specific hair cell stereocilia in denervated embryonic ears.

The developmental mechanisms that allow physiological coding of acoustic pitch have remained unexplained. Cochlear hair cells that have different structures respond to different sound frequencies and synapse with neurons that project to different locations in the brain. How do these hair cells develop appropriate structures, and how are the connections between specific hair cells and the neurons that code for their pitch sensitivities matched? We have investigated one aspect of this by denervating embryonic chicken ears, before the time of hair cell production, and then transplanting them to the aneural chorioallantoic membrane of host embryos where they have continued to develop. We report that vestibular and auditory hair cell phenotypes differentiate appropriately and that correct gradients of hair cell structural phenotypes, as expressed in stereocilia bundles, develop in the cochleae of these denervated ears. Therefore, the normal development of gradients in hair cell stereocilia properties must be controlled by location-specific cues originating in the ear itself. Neuronally directed modification of target cell phenotypes is not required for the quite specific phenotype development represented by the stereocilia bundles of individual hair cells and the connectional matching in the numerous distinct peripheral information lines of the auditory system.