The inner ear and the in vitro system.

The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.     

Embryogenesis of the inner ear. II. The late differentiation of the mammalian crista ampullaris in vivo and in vitro

The embryonic development of the crista ampullaris of the CBA/CBA mouse was followed both in organ culture of explanted inner ears of the 16th gestational day and in vivo from the 16th gestational day until the 21st day, an age corresponding to birth. Cytodifferentiation of the sensory epithelium of the crista ampullaris occurs during this period. At partus, there is a rather mature crista with well developed hair cells and 1-2 layers of supporting cells. Innervation and differentiation into type I and type II hair cells have started prior to partus but occur mainly post partum. The in vitro development followed that of the in vivo but with a slight delay, especially concerning the later stages of the in vitro development. At the time corresponding to partus, differentiation of hair cells is almost identical in the two groups but innervation is delayed in the in vitro group of inner ears. Because of the very high reproducibility/stability in vitro and morphologic maturation of both hair cells and gross structure of the crista ampullaris, organ culture of the 16th gestational day inner ear explant is a suitable model in the study of the late embryonic development under normal and pathological conditions.     

The inner ear and the in vitro system

Summary The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.Supported by grants from Karolinska Institutet, The Swedish Medical Research Council (grant no 12X-720) and The Swedish Society of Medical Sciences     

Regenerative Medizin in der Therapie der Innenohrschwerhörigkeit

Regenerative medicine offers the prospect of causal treatment of sensorineural hearing loss. In humans, the loss of sensory hair cells is irreversible and results in chronic hearing loss. Other vertebrates, particularly birds, have the capability to spontaneously regenerate lost sensory hair cells and restore hearing. In the bird model, regeneration of hair cells is based on the proliferation of supporting cells. In mammals, supporting cells have lost their proliferative capacity and are terminally differentiated. To gain an understanding about regeneration of hair cells in mammals, cell division of supporting cells has to be controlled. Gene disruption of the cell cycle inhibitor p27(Kip1) allows supporting cell proliferation in the organ of Corti in vivo. Furthermore, in vitro studies indicate that newly generated cells may differentiate into hair cells after p27(Kip1) disruption. Other current methods to induce hair cell regeneration include the gene transfer of Math1 and transplantation of stem cells to the inner ear.     

Expression of calretinin by fetal otocyst cells after transplantation into damaged rat utricle explants

Severe damage by acoustic overstimulation or ototoxins induces inner ear hair cell loss, resulting in permanent hearing loss and balance disorders because hair cell regeneration scarcely occurs in the inner ear sensory organs of mammals. In this study, to evaluate the possibilities of cell transplantation therapy for damaged inner ear sensory organs, dissociated cell cultures of fetal otocyst cells (FOCs) were established from embryonic day 12.5 (E12.5) rat inner ears, and transplanted into gentamicin-treated explants of vestibular sensory epithelia. Two weeks after transplantation, immunohistochemical analysis demonstrated that some of the grafted FOCs survived within the vestibular sensory epithelia and expressed epitopes of calretinin. one of the hair cell marker proteins. These findings indicate that FOCs have the potential to migrate into damaged vestibular epithelia and differentiate into hair cell immunophenotypes. Cell transplantation therapy may be available for functional regeneration in inner ear diseases.     

Strategies to regenerate hair cells: identification of progenitors and critical genes

Deafness commonly results from a lesion of the sensory cells and/or of the neurons of the auditory part of the inner ear. There are currently no treatments designed to halt or reverse the progression of hearing loss. A key goal in developing therapy for sensorineural deafness is the identification of strategies to replace lost hair cells. In amphibians and birds, a spontaneous post-injury regeneration of all inner ear sensory hair cells occurs. In contrast, in the mammalian cochlea, hair cells are only produced during embryogenesis. Many studies have been carried out in order to demonstrate the persistence of endogenous progenitors. The present review is first focused on the occurrence of spontaneous supernumerary hair cells and on nestin positive precursors found in the organ of Corti. A second approach to regenerating hair cells would be to find genes essential for their differentiation. This review will also focus on critical genes for embryonic hair cell formation such as the cell cycle related proteins, the Atoh1 gene and the Notch signaling pathway. Understanding mechanisms that underlie hair cell production is an essential prerequisite to defining therapeutic strategies to regenerate hair cells in the mature inner ear.     

Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear

The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER−/− mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.     

Supernumerary outer hair cells arise external to the last row of sensory cells in the organ of corti

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells.     

Transgene expression in neonatal mouse inner ear explants mediated by first and advanced generation adenovirus vectors

The mouse serves as a valuable model for treatment leading to the prevention and therapy of inner ear disease. Transgenic correction of genetic inner ear disease in mice may help develop treatment for human genetic inner ear disease. In mutations involving hair cells (HCs) or supporting cells (SCs), it is necessary to insert the wild-type transgenes directly into these cells. We used inner ear explants to characterize the transgenic expression using adenovirus-mediated reporter genes (bacterial lacZ). The variable parameters were the age of the explants (P1-P5), the type of vector (first and advanced generation adenovirus) and the genotype of the mouse (wild-type versus shaker-2 mutant). Transduction of cochlear HCs was detected at P1 and in some of the P3 cochleae. Low efficiency transduction of SCs was observed in P1 explants, but the efficiency increased with age and reached high levels at P5. The pattern of transduction was similar regardless of the genotype and the type of vector used. The data demonstrate that differentiating HCs and SCs in mouse explants can be transduced by adenovirus vectors, suggesting that cultures of mouse ears are a valuable model for developing inner ear gene therapy protocols.     

Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo

Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy. In bullfrogs, GTTR uptake was prominent in mature hair cells, but not in immature hair cells. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla. GTTR was associated with the hair bundle; and, in guinea pigs and mice, somatic GTTR fluorescence was initially diffuse before punctate (endosomal) fluorescence could be observed. A baso-apical gradient of intracellular GTTR uptake in guinea pig cochleae could only be detected at early time points (<3h). In 21-28 day mice, cochlear GTTR uptake was greatly reduced compared to guinea pigs, 6-day-old mice, or mice treated with ethacrynic acid. In mice, GTTR was also rapidly taken up, and retained, in the kidney, dorsal root and trigeminal ganglia. In linguinal and vibrissal tissues rapid GTTR uptake cleared over a period of several days. The preferential uptake of GTTR by mature saccular, and proximal hair cells resembles the pattern of aminoglycoside-induced hair cell death in bullfrogs and chicks. Differences in the degree of GTTR uptake in hair cells of different species suggests variation in serum levels, clearance rates from serum, and/or the developmental and functional integrity of the blood-labyrinth barrier. GTTR uptake by hair cells in vivo suggests that GTTR has potential to elucidate aminoglycoside transport mechanisms into the inner ear, and as a bio-tracer for in vivo pharmacokinetic studies.     

Characterization of supporting cell phenotype in the avian inner ear: implications for sensory regeneration

The avian inner ear possesses a remarkable capacity for the regeneration of sensory receptors after acoustic trauma or ototoxicity. Most replacement hair cells are created by renewed cell division within the sensory epithelium, although some new hair cells may also arise through nonmitotic mechanisms. Current data indicate that epithelial supporting cells play an essential role in regeneration, by serving as progenitor cells. In order to become progenitors, however, supporting cells may need to undergo partial dedifferentiation. In this review, I describe molecules that are expressed by supporting cells in the avian ear. Although a number of these molecules are likely to be critical to the maintenance of the supporting cell phenotype, we presently know very little about phenotypic changes in supporting cells during the early phase of regeneration.     

Determinants of ganglion-receptor cell interaction during development of the inner ear. A heterochronic ganglia study

It has been suggested that inner ear sensory receptors produce attractant fields that guide neurite outgrowth from statoacoustic ganglion (SAG) neurons to appropriate target sites within the developing labyrinth. This experiment tested the temporal limitations of SAG neurons in their ability to respond to these attractant fields. Statoacoustic ganglia were excised from 12, 13, 14 and 15 gestation day (GD) mouse embryos. This temporal series of SAG was implanted into aganglionic 12 GD otocysts. All cultures were grown for 7 days in vitro, then fixed and processed for nerve fiber staining. Specimens were evaluated for the presence of neurites associated with the inner ear sensory receptors that developed within the otic explants. All of the implanted heterochronic ganglia (i.e. 13, 14 or 15 GD) as well as the homochronic (i.e. 12 GD) ganglion controls extended neurites to sensory epithelium of both vestibular and auditory character. Neurites made contact with the base of hair cells in all of the sensory structures. These findings demonstrate that SAG neurons are capable of extending processes in response to otic attractant fields for an extended period during the embryonic development of this ganglion. This observation supports the hypothesis that the onset and duration of receptor generated attractant fields may act as a controlling factor in establishing patterns of innervation within the developing inner ear.     

Nestin expression in the developing rat cochlea sensory epithelia

An intermediate filament (IF), nestin, is used as an immature cell marker because nestin occurs in neural progenitors during early development. Recent cell culture studies have indicated that proliferating otic progenitor cells express nestin in vitro. However, localization of nestin in the developing inner ear has not yet been clarified. In this study, the ontogenetical expression of nestin epitopes in the rat cochlea was examined immunohistochemically. Sensory epithelial cells in the rat Corti organ (e.g. hair cells and support cells) transiently demonstrated immunoreactivity for nestin during the late embryonic period. After birth, nestin expression in the sensory epithelia disappeared gradually. The findings of this study indicate that the expression of nestin epitopes in the developing cochlea is linked with the plasticities of sensory epithelial cells, such as proliferation or differentiation.     

Neurogene Stammzelltransplantation in die Kochlea

Background

Stem cell therapy is especially interesting for inner ear related diseases, since the hair cells are very sensitive and do not regenerate. Hair cell loss is therefore irreversible and is accompanied by hearing loss. In the last few years, different research groups have transplanted stem cells into the inner ear with promising results. In the presented study, our aim was to gain insight into how neuronal stem cells behave when they are transplanted, both in vitro and in vivo, into a damaged inner ear.

Methods

Neuronal stem cells from E9.5 day old mouse embryos were collected and infected with an adenoviral vector encoding green fluorescent protein (GFP). GFP+ cells were then transplanted into a damaged organ of Corti in vitro or into a damaged mouse inner ear in vivo.

Results

We were able to detect GFP+ cells close to the organ of Corti in vitro and in the organ of Corti in vivo. The GFP+ cells do not seem to be randomly distributed in either the in vitro or in vivo situation. Most interestingly, GFP+ cells could be detected close to places where hair cells had been lost in vivo.

Conclusion

Neuronal stem cells are interesting candidates to replace lost hair cells. However, a great deal of research is still needed before they can enter clinical trials.     

Low-dose gamma irradiation effects on labyrinthine development in vitro

The 13th and 16th gestational day inner ear anlagen, respectively, were exposed after explantation to an organ culture system to low-dose gamma irradiation with a 2 Gy single dose. The explants were thereafter cultured in vitro for 8 vs. 5 days to an age corresponding to birth in vivo. The explants were analysed with regard to gross morphology and at the light and electron microscopic levels. The 13th gestational day inner ear anlage showed malformations of the gross shape. The gross morphology of the 16th gestational day inner ear explant was unaffected. Irradiated specimens showed a delayed development in general as compared with controls. A defective cytodifferentiation of hair cells was observed at the ultrastructural level. Sensory hair fusion occurred, the sensory hair rootlets were poorly developed as also was the cuticle. Nerve terminals were not identified. The observations in the present in vitro study are in agreement with corresponding earlier published in vivo investigations. The organ culture model can thus be used for irradiation induced selective effects on labyrinthine development.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.     

Transplantation of neural stem cells into explants of rat inner ear

Damage and loss of hair cells in the inner ear is the most frequent cause of hearing loss and balance disorders. Mammalian hair cells do not regenerate in the conventional ways. To regenerate the hair cell in the mammalian inner ear we transplanted neural stem cells into explants of rat inner ear. The stem cells integrated successfully into the sensory epithelium of the vestibular organs, but not into the organ of Corti. This method is useful to investigate efficient ways to transplant stem cells into the inner ear.