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

Abstract

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.

EGFR signaling is required for regenerative proliferation in the cochlea: Conservation in birds and mammals

“…1I,J). Both the avian and the mammalian proliferation results are consistent with published findings (Daudet et al, 1998; Oesterle et al, 1993; Richardson et al, 1997; Shang et al, 2010). …”

“…1B, D) or in media containing the aminoglycoside antibiotic streptomycin (Fig. 1C, E), which kills auditory hair cells (Shang et al, 2010). Both sets of cultures then received the thymidine analogue EdU in media for another 24 hours (Fig.…”

Inhibition Of Notch Activity Promotes Nonmitotic Regeneration of Hair Cells in the Adult Mouse Utricles

“…Our observations are consistent with findings by Forge et al (1993) and Kawamoto et al (2009), even after months of recovery from aminoglycoside treatment in vivo . However, our findings are in stark contrast to the response of the mature avian utricular or auditory SE to in vitro hair cell loss, where hundreds of new hair cells differentiate within days of damage (Matsui et al, 2000; Shang et al, 2010; Warchol and Montcouquiol, 2010). …”

In vivo evidence for transdifferentiation of peripheral neurons

“…In the other two instances, when DRG number increased in a hemisegment, the DRG cells did not round up and retract their axons, suggesting that dedifferentiation did not occur prior to cell proliferation, as reported previously . Overall, our data support direct transdifferentiation of one cell type to another in the sensory system, as has been shown in the regenerating auditory system (Roberson et al, 2004;Shang et al, 2010).…”

“…transdifferentiate (Dupin et al, 2000;Dupin et al, 2003;Jee et al, 2010;Kanazawa et al, 2010;Lu et al, 2010;Rizvi et al, 2002;Roberson et al, 2004;Shang et al, 2010;Yin et al, 2010;Yoshii et al, 2007). Indeed, transdifferentiating cells have been found in the endocrine pancreas (Lu et al, 2010), the auditory system (Roberson et al, 2004;Shang et al, 2010), the retina (Yoshii et al, 2007), adipose tissue (Jee et al, 2010) and the sympathetic nerves (Kanazawa et al, 2010) in diverse vertebrate species such as amphibia (Yoshii et al, 2007), avia (Roberson et al, 2004;Shang et al, 2010), rodents (Kanazawa et al, 2010;Lu et al, 2010) and humans (Jee et al, 2010;Yin et al, 2010). The wide-ranging distribution of the evidence for transdifferentiation across tissues and organisms warrants a re-examination of our definition of differentiation to include the possibility of a plastically differentiated state.…”