Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

JIALIN SHANG 0 JON CAFARO 0 RACHEL NEHMER 0 JENNIFER STONE 0 0 Department of Otolaryngology/Head and Neck Surgery, The Virginia Merrill Bloedel Hearing Research Center, University of Washington School of Medicine , Seattle, WA 98195-7923, USA In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro. - Mechanosensitive hair cells (HCs) are required for hearing. HCs are injured by ototoxic drugs, intense sound stimuli, and changes associated with aging. Mature mammals cannot replace auditory HCs, but mature birds can. Avian HC regeneration occurs via division of progenitor cells within the auditory epithelium (basilar papilla, BP; Corwin and Cotanche 1988; Ryals and Rubel 1988; Raphael 1992; Hashino and Salvi 1993; Stone and Cotanche 1994). These progenitor cells, called supporting cells (SCs), are quiescent unless HCs are injured (Corwin and Cotanche 1988; Ryals and Rubel 1988; Lippe et al. 1991; Warchol and Corwin 1996). Upon HC loss, SCs divide and progeny differentiate into HCs or SCs. Normal numbers of HCs return within 23 weeks (reviewed in Stone and Cotanche 2007). Restoration of normal structure and function, including cellular maturation and reinnervation, takes several weeks (reviewed in Bermingham-McDonogh and Rubel 2003). Several observations suggest that some HCs in the chicken BP may be regenerated by a process called direct transdifferentiation, which is the phenotypic conversion of SCs into HCs without cell division (reviewed in Morest and Cotanche 2004; Stone and Cotanche 2007). This process contributes to HC regeneration in cultured saccules of frogs and salamanders after aminoglycoside-induced injury (Baird et al. 1996, 2000; Taylor and Forge 2005). In chickens, some regenerated auditory HCs are unlabeled for a proliferation marker (3H-thymidine or bromodeoxyuridine (BrdU)) despite its continual perfusion into the inner ear after damage (Roberson et al. 1996, 2004). Cells with features of both HCs and SCs were seen in regenerating BPs, suggesting such cells were SCs converting into HCs (Adler et al. 1997; Cafaro et al. 2007). Also, attenuation of SC division did not prevent the reemergence of HCs in the BP (Adler and Raphael 1996). While this latter finding suggests HCs can be regenerated independent of SC division, inhibition of division in this study was incomplete. Therefore, despite substantial evidence for direct transdifferentiation in the chicken BP, further studies are warranted. Here, we describe an organotypic culture system for the chicken BP that has allowed us to examine the contribution of direct transdifferentiation to auditory HC regeneration after drug damage. Three studies have previously described patterns of auditory HC loss in control and drug-treated organ cultures (Oesterle et al. 1993; Frenz et al. 1998; Cheng et al. 2003). Oesterle et al. (1993) also showed that limited SC division occurs in organ cultures maintained without ototoxic drugs for 2 days. Other studies showed that substantial SC division occurs in organ cultures after drug damage (Navaratnam et al. 1996; Daudet et al. 2009) or HC ablation (Warchol and Corwin 1996), with some mitotic events leading to production of new HCs (Navaratnam et al. 1996). In the current study, we examined the time course, levels, and spatial patterns of SC division and HC regeneration in cultured chicken BPs treated with streptomycin. We found that high numbers of HCs regenerated after drug damage were formed by direct transdifferentiation, even when SC division was completely blocked. Fertile eggs of chickens (Gallus domesticus) of the White Leghorn variety were purchased from Hyline International (Graham, WA, USA) and maintained in a standard incubator until hatching. Hatchlings were housed in heated brooders with free access to water and food until the initiation of experiments, between posthatch days 5 and 10. All methods were approved by the University of Washingtons IACUC and conformed to AALAC standards. Preparation of uncultured tissue In some studies, comparisons were made between BPs that had been cultured and uncultured BPs that had been dissected from control or gentamicin-treated chickens and immediately fixed. For these experiments, chickens received a single subcutaneous injection of gentamicin (Sigma-Aldrich, St. Louis, MI, USA) at a dose of 250 mg/kg, administered on two consecutive days, and survived for various periods after gentamicin treatment. Chickens were euthanized by Nembutal overdose (100 mg/kg, intraperitoneal injection) and decapitation. The tympanic membrane and columella were dissected away, creating a hole into the inner ear. The head was then immersion-fixed in 4% paraformaldehyde for 1 to 1.5 h. Tissue was rinsed in phosphate-buffered saline (PBS). The cochlear duct was removed from the temporal bone, and the tegmentum vasculosum and tectorial membrane were mechanically removed from the cochlear duct. Chickens were euthanized by rapid decapitation. Following brief immersion in 70% ethanol, cochlear ducts were removed via a lateral approach through the middle ear and placed in ice-cold sterile Hanks buffered saline solution (HBSS; Sigma-Aldrich). The tegmentum vasculosum was dissected off the cochlear duct, and the remaining tissue of the cochlear duct was cultured free-floating in 500 l of media per well in 48-well plates. The remaining tissues (auditory end organs) were (...truncated)