Progenitor Cells from the Adult Human Inner Ear

Loss of inner ear hair cells leads to incurable balance and hearing disorders because these sensory cells do not effectively regenerate in humans. A potential starting point for therapy would be the stimulation of quiescent progenitor cells within the damaged inner ear. Inner ear progenitor/stem cells, which have been described in rodent inner ears, would be principal candidates for such an approach. Despite the identification of progenitor cell populations in the human fetal cochlea and in the adult human spiral ganglion, no proliferative cell populations with the capacity to generate hair cells have been reported in vestibular and cochlear tissues of adult humans. The present study aimed at filling this gap by isolating colony-forming progenitor cells from surgery- and autopsy-derived adult human temporal bones in order to generate inner ear cell types in vitro. Sphere-forming and mitogen-responding progenitor cells were isolated from vestibular and cochlear tissues. Clonal spheres grown from adult human utricle and cochlear duct were propagated for a limited number of generations. When differentiated in absence of mitogens, the utricle-derived spheres robustly gave rise to hair cell-like cells, as well as to cells expressing supporting cell-, neuron-, and glial markers, indicating that the adult human utricle harbors multipotent progenitor cells. Spheres derived from the adult human cochlear duct did not give rise to hair cell-like or neuronal cell types, which is an indication that human cochlear cells have limited proliferative potential but lack the ability to differentiate into major inner ear cell types. Anat Rec, 303:461–470, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Cellular distribution of myosin-V in the guinea pig cochlea

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.     

Role of osteopontin in the rodent inner ear as revealed by in situ hybridization

Osteopontin (OPN) is considered to be a non-collagenous bone matrix protein that is involved in the ossification process. However, OPN has recently been observed in ectopic sites such as the kidney and nervous tissue. In the present study, the expression of OPN mRNA was examined in the rat and mouse inner ear by nonradioisotopic in situ hybridization. Signals of OPN mRNA were observed in the marginal cells of the stria vascularis, spiral ganglion cells, vestibular sensory hair cells, and vestibular dark cells. OPN protein was detected only in the otoconia by immunohistochemistry. The presence of OPN mRNA in the cochlea and vestibular dark cells may indicate that OPN is involved in the regulation of ions in the inner ear fluid. Findings in the saccule and utricle suggest that OPN is one of the protein components of the rodent otoconia and that the vestibular sensory hair cells are involved in the production of otoconia.     

Tailchaser (Tlc): a new mouse mutation affecting hair bundle differentiation and hair cell survival

We have undertaken a phenotypic approach in the mouse to identifying molecules involved in inner ear function by N-ethyl-N-nitrosourea mutagenesis followed by screening for new dominant mutations affecting hearing or balance. The pathology and genetic mapping of the first of these new mutants, tailchaser (Tlc), is described here. Tlc/+ mutants display classic behavioural symptoms of a vestibular dysfunction, including head-shaking and circling. Behavioural testing of ageing mice revealed a gradual deterioration of both hearing and balance function, indicating that the pathology caused by the Tlc mutation is progressive, similar to many dominant nonsyndromic deafnesses in humans. Based on scanning electron microscopy (SEM) studies, Tlc clearly plays a developmental role in the hair cells of the cochlea since the stereocilia bundles fail to form the characteristic V-shape pattern around the time of birth. By young adult stages, Tlc/+ outer hair bundles are grossly disorganised although inner hair bundles appear relatively normal by SEM. Increased compound action potential thresholds revealed that the Tlc/+ cochlear hair cells were not functioning normally in young adults. Similar to inner hair cells, the hair bundles of the vestibular hair cells also do not appear grossly disordered. However, all types of hair cells in the Tlc/+ inner ear eventually degenerate, apparently regardless of the degree of organisation of their hair bundles. We have mapped the Tlc mutation to a 12 cM region of chromosome 2, between D2Mit164 and D2Mit423. Based on the mode of inheritance and map location, Tlc appears to be a novel mouse mutation affecting both hair cell survival and stereocilia bundle development.     

Growth factor regulation of the cell cycle in developing and mature inner ear sensory epithelia

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.     

Intratympanic injection of shRNA-expressing lentivirus causes gene silencing in the inner ear in chicken

The hair cells and their neural innervation in the avian inner ear can regenerate after injury. Identifying the genes involved in the regeneration and neuroplasticity of avian hair cell will enable us to experimentally induce new hair cell production and potentially harness this process for therapeutic replacement of hair cells in mammals and ultimately in humans suffering from sensorineural hearing loss. In this study, we developed a method for suppressing the expression level of genes in avian inner ear by intratympanic injection of shRNA-expressing lentivirus. The intratympanic injection approach is more convenient and presumably of less implication when compared with two existing methods, in which a nano-particles or gelfoam containing a recombinant virus is placed in the middle ear by surgery, or a recombinant virus is directly injected into the inner ear. Thus, we developed an easier method for identifying and characterizing molecules involved in the process of avian hair cell regeneration and re-innervation.     

Concise review: Inner ear stem cells--an oxymoron, but why?

Hearing loss, caused by irreversible loss of cochlear sensory hair cells, affects millions of patients worldwide. In this concise review, we examine the conundrum of inner ear stem cells, which obviously are present in the inner ear sensory epithelia of nonmammalian vertebrates, giving these ears the ability to functionally recover even from repetitive ototoxic insults. Despite the inability of the mammalian inner ear to regenerate lost hair cells, there is evidence for cells with regenerative capacity because stem cells can be isolated from vestibular sensory epithelia and from the neonatal cochlea. Challenges and recent progress toward identification of the intrinsic and extrinsic signaling pathways that could be used to re-establish stemness in the mammalian organ of Corti are discussed.     

Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery

Prior studies have shown that macrophages are recruited to sites of hair cell lesions in the avian inner ear in vitro (Warchol, 1997) and in vivo (Bhave et al., 1998). Although the avian ear has a high capacity for sensory regeneration (Oberholtzer & Corwin, 1997; Stone et al., 1998), the role of macrophages in the regenerative process is uncertain. The present study examined the possible influence of macrophages and immune cytokines on regenerative proliferation in the avian utricle, one of the sensory endorgans of the vestibular system. Utricles from post-hatch chicks were placed in organ culture and hair cell lesions were created by incubation in neomycin. The cultures were then maintained for an additional 24-48 hours in vitro, and some cultures were treated with dexamethasone, which inhibits macrophage activation and cytokine production. Following fixation, resident macrophages were identified by immunoreactivity to CD68. Labeled macrophages were present in all specimens and increased numbers of macrophages were observed following neomycin treatment. Regenerative proliferation in dexamethasone-treated specimens was reduced by about 50%, relative to untreated controls. Additional experiments showed that two macrophage secretory products-TGF-alpha and TNF-alpha-enhanced the proliferation of utricular supporting cells. The results are consistent with a role for macrophages in hair cell regeneration.     

Gene expression in the peripheral leukocytes and association analysis of PDLIM5 gene in schizophrenia

The terminal mitosis of hair cells (HCs) and supporting cells (SCs) in mammalian cochlea occurred during middle embryonic development. Most hearing loss results from the incapacity of the cochlear sensory epithelium to replace lost hear cells. Deafness due to hair cells loss is normally irreversible. The present study showed that cells acutely dissociated from the cochlea of young rat, cultured with EGF and FGF2, developed into otospheres that showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). The subcultured otospheres maintained for up to 10 passages. In addition, the cochlea sphere-derivatives contributed to a variety of cell types. They were found to differentiate to neuron, glia, hair cell and supporting cell phenotypes. The results suggest that the young rat inner ear cells have self-renewal capability and multipotent differentiation potential. This work raises the possibility that inner ear cells in the early post-natal rat have the character of pluripotent stem cells and might be a source for cell replacement therapy in the inner ear.     

The Meniere attack: An ischemia/reperfusion disorder of inner ear sensory tissues

We believe Meniere attacks arise as a chance association of endolymphatic hydrops and vascular risk factors for intracerebral ischemia. Hydrops acts as a variable Starling resistor upon the inner ear vasculature that is capable of inducing ischemic attacks only in people with reduced perfusion pressure in the ear. The unique characteristics of the attacks (loss of vestibular response and hearing acutely followed by a return to apparent normalcy over hours) are explained by the differential sensitivity of the inner ear tissues to transient ischemia, with the sensory tissues (dendrites, hair cells) vulnerable to hours-long ischemia/reperfusion injury, and the stria vulnerable to ischemia due to its high metabolic rate. Permanent hearing loss and vestibular damage after many attacks would result when small areas of irreversible sensory cell damage accumulate and become confluent.     

Developmental expression of monocarboxylate transporter in the gerbil inner ear.

The expression of H+-monocarboxylate cotransporters (MCTs) that facilitate cell uptake of lactate, pyruvate and other monocarboxylates was investigated in the adult and postnatally developing gerbil inner ear. In the mature cochlea, immunoreactive MCT1 was present in marginal cells of the stria vascularis and in type II, suprastrial and limbal fibrocytes. In the adult vestibular system, dark cells and a subpopulation of fibrocytes immediately underlying maculae and cristae stained strongly for MCT1. Satellite cells surrounding mature spiral and vestibular ganglia neurons also expressed MCT1. MCT1 immunoreactivity was present at birth in marginal and dark cells, at 8 days after birth in fibrocytes and at 12 days after birth in satellite cells, and coincided precisely with the developmental expression of Na,K-ATPase in these sites. The coexpression of MCT1 and Na,K-ATPase in these cell types points to MCT1 as an important source of energy to drive inner ear Na,K-ATPase activity.In the adult inner ear, MCT2 was detectable only in tectal cells of the cochlea and supporting cells of the crista ampullaris. Immunostaining was first observed at 16 days after birth in tectal and at 20 days after birth in supporting cells, and at the same time immunoreactive aquaporin 4 appeared in these cells. The coexpression of MCT2 and aquaporin 4 suggests a possible role for MCT2 in regulating transcellular water movement. Because MCT2 facilitates the transport of acidic intermediates, its biological significance also could relate to modulation of cell pH and volume.Maintenance of the inner ear's unique ion and fluid gradients is essential to normal hearing and balance and requires the expenditure of large amounts of energy. The cellular distribution of MCT1 and MCT2 points to their participation in generating these electrochemical gradients and their potential involvement in sensory deficits associated with various inner ear disorders.     

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development.

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.     

基于基底膜响应的圆窗激振性能评估偏差研究

目的 研究采用传统基底膜位移评价标准评估圆窗激振式人工中耳听力补偿性能的准确性,为圆窗激振式人工中耳的性能评估提供理论基础.方法 基于耳蜗几何结构的实验数据,建立耳蜗感声微观有限元模型,通过对比内听毛细胞、外听毛细胞、盖膜等部位位移响应的实验测量值,验证模型的可靠性.基于该模型,对比分析正向激振、圆窗激振下的基底膜位移...     

Basic helix-loop-helix gene Hes6 delineates the sensory hair cell lineage in the inner ear.

The basic helix-loop-helix (bHLH) gene Hes6 is known to promote neural differentiation in vitro. Here, we report the expression and functional studies of Hes6 in the inner ear. The expression of Hes6 appears to be parallel to that of Math1 (also known as Atoh1), a bHLH gene necessary and sufficient for hair cell differentiation. Hes6 is expressed initially in the presumptive hair cell precursors in the cochlea. Subsequently, the expression of Hes6 is restricted to morphologically differentiated hair cells. Similarly, the expression of Hes6 in the vestibule is in the hair cell lineage. Hes6 is dispensable for hair cell differentiation, and its expression in inner ear hair cells is abolished in the Math1-null animals. Furthermore, the introduction of Hes6 into the cochlea in vitro is not sufficient to promote sensory or neuronal differentiation. Therefore, Hes6 is downstream of Math1 and its expression in the inner ear delineates the sensory lineage. However, the role of Hes6 in the inner ear remains elusive.     

The mouse slalom mutant demonstrates a role for Jagged1 in neuroepithelial patterning in the organ of Corti

The Notch signalling pathway has recently been implicated in the development and patterning of the sensory epithelium in the cochlea, the organ of Corti. As part of an ongoing large-scale mutagenesis programme to identify new deaf or vestibular mouse mutants, we have identified a novel mouse mutant, slalom, which shows abnormalities in the patterning of hair cells in the organ of Corti and missing ampullae, structures that house the sensory epithelia of the semicircular canals. We show that the slalom mutant carries a mutation in the Jagged1 gene, implicating a new ligand in the signalling processes that pattern the inner ear neuro-epithelium.     

Riluzole rescues cochlear sensory cells from acoustic trauma in the guinea-pig

Acoustic trauma is the major cause of hearing loss in industrialised nations. We show in guinea-pigs that sound exposure (6 kHz, 120 dB sound pressure level for 30 min) leads to sensory cell death and subsequent permanent hearing loss. Ultrastructural analysis reveals that degeneration of the noise-damaged hair cells involved different mechanisms, including typical apoptosis, autolysis and, to a lesser extent, necrosis. Whatever the mechanisms, a common feature of noise damage to hair cells was mitochondrial alteration. Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is a neuroprotective agent that prevents apoptosis- and necrosis-induced cell death. Perfusion of riluzole into the cochlea via an osmotic minipump prevents mitochondrial damage and subsequent translocation of cytochrome c, DNA fragmentation, and hair cell degeneration. This was confirmed by functional tests showing a clear dose-dependent reduction (ED(50)=16.8 microM) of permanent hearing loss and complete protection at 100 microM. Although less efficient than intracochlear perfusion, intraperitoneal injection of riluzole rescues the cochlea within a therapeutic window of 24 h after acoustic trauma.These results show that riluzole is able to prevent and rescue the cochlea from acoustic trauma. It may thus be an interesting molecule for the treatment of inner ear injuries.     

脑缺血再灌注对大鼠耳蜗形态学及听觉的影响

目的：为探讨脑缺血再灌注对大鼠耳蜗形态学及听觉的影响。方法：采用闭塞大鼠肮脏了缺血模型,观察了脑缺血３０分钟及再灌注不同时间大鼠耳蜗结构和听阈的动态变化,形态学观察应用光镜、扫描电镜与透射电镜,听力测定采用４０ＨＺ听上关电位（４０ＨＺＡＥＲＰ）技术。结果：缺血３０分钟和再灌注２小时、２４小时、７２小时４０ＨＺＡＥＲＰ反应阈与缺血前比较显著提高（Ｐ〈０．０１）,随或注延长听阈逐渐降低。病理变化特点是     

The Meniere attack: An ischemia/reperfusion disorder of inner ear sensory tissues

We believe Meniere attacks arise as a chance association of endolymphatic hydrops and vascular risk factors for intracerebral ischemia. Hydrops acts as a variable Starling resistor upon the inner ear vasculature that is capable of inducing ischemic attacks only in people with reduced perfusion pressure in the ear. The unique characteristics of the attacks (loss of vestibular response and hearing acutely followed by a return to apparent normalcy over hours) are explained by the differential sensitivity of the inner ear tissues to transient ischemia, with the sensory tissues (dendrites, hair cells) vulnerable to hours-long ischemia/reperfusion injury, and the stria vulnerable to ischemia due to its high metabolic rate. Permanent hearing loss and vestibular damage after many attacks would result when small areas of irreversible sensory cell damage accumulate and become confluent.This theory is supported by the strong correlation of hydrops with Meniere attacks, the finding that autoregulation of cochlear blood flow is impaired in the hydropic ear, and studies demonstrating that symptoms and signs in people and in animal models vary with conditions that alter perfusion pressure in the inner ear. Induction of Meniere attacks in animal models requires both hydrops and a mechanism that reduces perfusion pressure, such as epinephrine injection or head dependency. There is a strong clinical association between Meniere attacks and disorders that increase the risk for cerebrovascular ischemia, such as migraine. The excitable tissues in the sensory structures have long been known to be more vulnerable to ischemia than the remaining aural tissues, and are now known to be vulnerable to excitotoxicity induced by ischemia/reperfusion. This correlates well with autopsy evidence of damage to dendrites and hair cells and with strial atrophy in late Meniere disease cases.If this hypothesis is confirmed, treatment of vascular risk factors may allow control of symptoms and result in a decreased need for ablative procedures in this disorder. If attacks are controlled, the previously inevitable progression to severe hearing loss may be preventable in some cases.