Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One... Show moreNumerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application. Show less
Beelen, E.S.A. van; Valk, W.H. van der; Groot, J.C.M.J. de; Hensen, E.F.; Locher, H.; Benthem, P.P.G. van 2020
Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph... Show moreMelanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+/K+-ATPase alpha 1 by week 14. Furthermore, we investigated the expression of the migration-related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders. Show less
In recent years, there has been an increased interest in stem cells for the purpose of regenerative medicine to deliver a wide range of therapies to treat many diseases. However, two-dimensional... Show moreIn recent years, there has been an increased interest in stem cells for the purpose of regenerative medicine to deliver a wide range of therapies to treat many diseases. However, two-dimensional cultures of stem cells are of limited use when studying the mechanism of pathogenesis of diseases and the feasibility of a treatment. Therefore, research is focusing on the strengths of stem cells in the three-dimensional (3D) structures mimicking organs, that is, organoids, or organ-on-chip, for modeling human biology and disease. As 3D technology advances, it is necessary to know which signals stem cells need to multiply and differentiate into complex structures. This holds especially true for the complex 3D structure of the inner ear. Recent work suggests that although other factors play a role, the extracellular matrix (ECM), including its topography, is crucial to mimic a stem cell niche in vitro and to drive stem cells toward the formation of the tissue of interest. Technological developments have led to the investigation of biomaterials that closely resemble the native ECM. In the fast forward moving research of organoids and organs-on-chip, the inner ear has hardly received attention. This review aims to provide an overview, by describing the general context in which cells, matrix and morphogens cooperate in order to build a tissue, to facilitate research in 3D inner ear technology. Anat Rec, 303:408-426, 2020. (c) 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Show less
