Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic ...interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.
Abstract Losing either type of cochlear sensory hair cells leads to hearing impairment. Inner hair cells act as primary mechanoelectrical transducers, while outer hair cells enhance sound-induced ...vibrations within the organ of Corti. Established inner ear damage models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 h, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained until three days post-treatment, after which deterioration in structure and number was observed, culminating in a complete hair cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.
The avian hearing organ is the basilar papilla that, in sharp contrast to the mammalian cochlea, can regenerate sensory hair cells and thereby recover from deafness within weeks. The mechanisms that ...trigger, sustain and terminate the regenerative response in vivo are largely unknown. Here, we profile the changes in gene expression in the chicken basilar papilla after aminoglycoside antibiotic-induced hair cell loss using RNA-sequencing. We identified changes in gene expression of a group of immune-related genes and confirmed with single-cell RNA-sequencing that these changes occur in supporting cells. In situ hybridization was used to further validate these findings. We determined that the JAK/STAT signaling pathway is essential for upregulation of the damage-response genes in supporting cells during the second day after induction of hair cell loss. Four days after ototoxic damage, we identified newly regenerated, nascent auditory hair cells that express genes linked to termination of the JAK/STAT signaling response. The robust, transient expression of immune-related genes in supporting cells suggests a potential functional involvement of JAK/STAT signaling in sensory hair cell regeneration.
Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. ...Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells’ bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4–5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.
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•New auditory hair cells appear 5 days after hair cell loss•New synapses appear near the basement membrane at the tips of hair cell projections•Functional recovery starts 11 days after hair cell loss and is complete after 4 weeks•Cytomorphological maturation time course aligns with recovery of hearing thresholds
Sato et al. provide a timetable of the cytomorphological maturation and reinnervation of newly regenerated avian auditory hair cells, leading to functional recovery of hearing thresholds. Synapse reformation happens via an unusual mechanism at the tips of hair cell projections that reach toward afferent neurites near the basement membrane.
In sharp contrast to the adult mammalian cochlea, which lacks regenerative ability, the mature avian cochlea, or basilar papilla (BP) is capable of complete recovery from hearing loss after damage. ...Avian sensory hair cell regeneration relies on rousing quiescent supporting cells to proliferate or transdifferentiate after hair cell death. Unlike mammalian cochlear supporting cells, which have clearly defined subtypes, avian BP supporting cells are deceptively indistinguishable and molecular markers have yet to be identified. Despite the importance of supporting cells as the putative stem cells in avian regeneration, it is unknown whether all supporting cells possess equal capability to give rise to a hair cell or if a specialized subpopulation exists. In this perspective, we reinvigorate the concept of a stem cell in the BP, and form comparisons to other regenerating tissues that show cell-cycle reentry after damage. Special emphasis is given to the structure of the BP and how anatomy informs both the potential, intrinsic heterogeneity of the supporting cell layer as well as the choice between mitotic and nonmitotic regenerative strategies.
The organ of Corti harbors highly specialized sensory hair cells and surrounding supporting cells that are essential for the sense of hearing. Here, we report a single cell gene expression data ...analysis and visualization strategy that allows for the construction of a quantitative spatial map of the neonatal organ of Corti along its major anatomical axes. The map displays gene expression levels of 192 genes for all organ of Corti cell types ordered along the apex-to-base axis of the cochlea. Statistical interrogation of cell-type-specific gene expression patterns along the longitudinal gradient revealed features of apical supporting cells indicative of a propensity for proliferative hair cell regeneration. This includes reduced expression of Notch effectors, receptivity for canonical Wnt signaling, and prominent expression of early cell-cycle genes. Cochlear hair cells displayed expression gradients of genes indicative of cellular differentiation and the establishment of the tonotopic axis.
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•Unbiased clustering of single organ of Corti cells based on gene expression data•Clusters correspond to nine major medial-to-lateral aligned organ of Corti cell types•Spatial reconstruction of apex-to-base localization of each organ of Corti cell•Statistical analysis reveals changes in gene expression along the tonotopic axis
Waldhaus et al. reconstruct a spatial 2D map of the organ of Corti from single cell gene expression profiles. Statistical interrogation of cell-type-specific gene expression along the map reveals expression gradients and provides a tool to integrate functional data with complex gene expression patterns.
The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in ...pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.
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•Skin organoids can be generated from mPSCs under defined conditions•Skin organoids are composed of self-assembled epidermal and dermal layers•Skin organoids produce hair follicles, sebaceous glands, and adipocytes•Hair follicle induction from skin organoids mimics normal hair folliculogenesis
Lee at el. describe a defined in vitro 3D culture system that generates skin organoids from mouse pluripotent stem cells. The skin organoids contain self-organized skin layers and skin appendages, including hair follicles, sebaceous glands, and adipocytes.
Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale ...shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.
In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are ...known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.
Single cell trajectory analysis is a computational approach that orders cells along a pseudotime axis. This temporal modeling approach allows the characterization of transitional processes such as ...lineage development, response to insult, and tissue regeneration. The concept can also be applied to resolve spatial organization of cells within the originating tissue. Known as temporal and spatial transcriptomics, respectively, these methods belong to the most powerful analytical techniques for quantitative gene expression data currently available. Here, we discuss three different approaches: principal component analysis, the ‘Monocle’ algorithm, and self-organizing maps. We use a previously published qRT-PCR dataset of single neuroblast cells isolated from the developing mouse inner ear to highlight the basic features of the three methods and their individual limitations, as well as the distinct advantages that make them useful for research on the inner ear. The complex developmental morphogenesis of the inner ear and its specific challenges such as the paucity of cells as well as important open questions such as sensory hair cell regeneration render this organ a prime target for single cell trajectory analysis strategies.
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