Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.
Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.
1. Utricle Dissection and Culture
2. Adenovirus Infection of Supporting Cells
Important: Working with adenovirus requires Biosafety Level 2 (BSL2) procedures and certification. Check with your Institutional Biosafety Officer for guidance and training on BSL2 procedures.
3. Utricle Fixation, Otoconia Removal, and Immunochemistry
4. Representative Results
Utricles cultured using this method retain full complements of both hair cells and supporting cells (Figure 2). Hair cells in healthy cultures show round nuclear profiles surrounded by thin cytoplasmic regions that are myosin 7a-positive (Figure 2, top panel). Supporting cells (labeled with anti-Sox2) are smaller and more densely packed than hair cells (Figure 2, lower panel). Adenovirus infects 25-50% of the supporting cells in the utricle, and no hair cells are infected (Figure 2, Ad-GFP panels). It should be noted that the optimal working titer of each adenovirus must be determined empirically, since hair cell death is possible if the viral titer is too high. In addition, regions of mechanical damage (caused during the dissection) will take up large amounts of virus. These regions of mechanical damage are easily distinguishable by a continuous line of cells with very high levels of GFP expression and missing hair cells. This is in contrast to the scattered GFP expression in supporting cells of an undamaged culture (Figure 2).
Figure 1. Utricle Dissection. A: Auditory bulla (AB). B: The bulla is broken using two sturdy forceps. C: Bony cochlea (RW = round window, S = stapedial artery, C = cochlea, ST = stapes). D: A scalpel blade (SB) is used to cut off the apex of the cochlea apical to the VIIIth cranial nerve (8th N). ASC = anterior semicircular canal. E: The preparation is held firmly using a #3 forceps with one fork inserted into the anterior semicircular canal (ASC) and the other on the outermost bone (arrows). The membranous labyrinth is removed using forceps. F: With the membranous labyrinth removed, the basal turn of the osseous spiral lamina is visible near the hook region. A fine probe is inserted beneath this bony shelf to lift the shelf up and remove it (arrow). G: After removal of the saccule, utricle (U) is visible immediately adjacent to the stapes footplate (SF). H: Utricle (U) is removed using a #55 forceps (arrow). SF = stapes footplate. I: Utricle with otoconia (O) partially removed and sensory epithelium (SE) visible underneath. J: Otoconia (O) are removed from the utricle using a stream of media delivered by a syringe fitted with a 26-gauge needle. The shadow of the needle (S) is visible at the bottom of the image. K: Utricle with otoconia removed and sensory epithelium (SE) visible. L: Adenoviral infection: each utricle (U) is placed in an individual well of a mini-tray. Supporting cells are infected with adenovirus by pipetting virus into the well (P = Pipet tip).
Figure 2. Adenovirus-mediated infection of supporting cells. Utricles were infected with adenovirus driving expression of green fluorescent protein (Ad-GFP). Shown are confocal images of the hair cell layer and the supporting cell layer in the same area of one utricle. Hair cells are labeled with an antibody against Myosin 7a (magenta). Supporting cells are labeled with an antibody against Sox2 (red). Schematic shows the structure of the utricle sensory epithelium and the locations of the hair cells (HC) and supporting cells (SC). Locations of confocal (optical) sections shown in the upper (U) and lower (L) panels are indicated by dashed lines in the schematic. Upper panels: In confocal images taken at the level of the hair cell nuclei, Ad-GFP signal appears in the spaces between hair cells and does not overlap with the hair cell marker Myosin 7a. Lower panels: In confocal sections at the level of the supporting cell nuclei, Ad-GFP signal colocalizes with the supporting cell marker Sox-2. Ad-GFP infection results in GFP expression in supporting cells only, and no hair cells are infected.
Figure 3. Adenoviral infection does not result in death of hair cells or supporting cells. Utricles were infected with Ad-GFP at 4×108 PFU/ml. Utricles were labeled with antibodies against Myosin 7a (hair cell marker) and Sox2 (supporting cell marker). Hair cell and supporting cell counts were equal for control utricles and Ad-GFP infected utricles.
Sensory hair cells are susceptible to death caused by a variety of stresses, including aging, noise trauma, and exposure to ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. In mammals hair cell death results in permanent hearing loss and/or balance disturbance. In vitro model systems are critical tools for studies aimed at determining the cellular and molecular mechanisms underlying hair cell death, as well as those aimed at preventing or reversing hair cell death. Unlike cochlear hair cells from adult mammals, hair cells of the utricle survive well in culture. Utricle hair cells are sensitive to death from exposure to the same therapeutic drugs that are toxic to cochlear hair cells, and the cellular mechanisms underlying ototoxic hair cell death and survival are similar for both utricular and cochlear hair cells 12-18. Moreover, when data obtained in the utricle model system are tested in vivo, the utricle preparation has proven to be a reliable predictor of hair cell survival and death in the mature cochlea 12, 18-21. The mouse utricle preparation also allows us to examine the effects of specific proteins by utilizing utricles from transgenic and knockout animals.
The signals that mediate the survival and death of sensory hair cells under stress are poorly understood. However, emerging evidence suggests that other cell types in the inner ear may play important roles in determining whether hair cells under stress ultimately live or die 11, 22, 23. The utricle model system can be used to examine these critical cell-cell interactions in a mature mammalian sensory epithelium. Adenoviral infection provides a method of altering gene expression in supporting cells, thus facilitating studies of the role(s) of supporting cells in hair cell survival, death, phagocytosis, and regeneration.
The authors have nothing to disclose.
The authors are grateful to Dr. Shimon P. Francis for generating the confocal micrographs.
This work was supported by the Division of Intramural Research at the National Institute on Deafness and Other Communication Disorders. Additional support was provided by NIDCD 5R01 DC007613.
Reagent | Company | Catalog number |
Medium 199 | Gibco/Invitrogen | 12350 |
DMEM/F12 | Gibco/Invitrogen | 11320 |
Fine forceps (#55) | Fine Science Tools | 11255-20 |
Fine forceps (#5) | Fine Science Tools | 11252-30 |
Forceps (#3) | Fine Science Tools | 11231-30 |
Penicillin G | Sigma | P3032 |
Fetal bovine serum | Invitrogen | 10082-139 |
Nunc mini-tray | Fisher | 12-565-68 |
Adenovirus-GFP | Vector Biolabs | 1060* |
Cal-Ex decalcifier | Fisher | CS510-1D |
sodium borohydride | Sigma | 452882 |
Anti-Myosin 7a (polyclonal) | Proteus Biosciences | 25-6790 |
Anti-Myosin 7a (monoclonal) | Developmental Studies Hybridoma Bank | MYO7A 138-1 |
Anti-Calmodulin | Sigma | C 3545 |
Anti-Calbindin | Chemicon | AB1778 |
Fluoromount G | Southern Biotechnology Associates | 0100-01 |
Table 1. Reagents and tools used in utricle culture and adenovirus infection.
* Note: Ad-GFP is normally provided by Vector Biolabs at a stock titer of 1×1010 PFU/ml. We requested a custom amplification in order to provide the stock virus at a titer of 1.2×1011 PFU/ml.