Hair cells are the sensory cells of the inner ear, and transduce physical sound waves into neural signals to be deciphered by higher brain regions. Unfortunately, losing one’s hearing (to varying degrees) is a fact of aging, since we are born with all the hair cells that we will ever have. These cells do not regenerate like many other cells in the body, but are subject to a constant onslaught of damage via environmental noise or infection, not to mention a slew of known genetic mutations responsible for hearing impairment. Therefore, it is obvious why hair cell replacement strategies are a hot topic in hearing research—the very topic of a recent paper published by Zhengqing Hu and Jeffery Corwin in PNAS. This paper succeeds in generating new hair cells from avian embryonic vestibular tissue, and provides evidence that these new cells have functional transduction channels. It marks an important first step in understanding how hair cells might one day be propagated in culture and forced to properly differentiate.

Why start with birds? Unlike mammals, birds can regenerate hair cells throughout their lives and even following multiple rounds of deafening. Furthermore, for unknown reasons, vestibular tissue (in both birds and mammals) is more likely to spontaneously regenerate hair cells than cochlear epithelium. So, embryonic avian vestibular tissue was a logical starting place for the development of an in vitro hair cell differentiation model. Their methods were relatively simple: dissect away an interior portion of the E14 chick utricle, dissociate the cells in 6 or 7 passages, start a new cell culture in suspension, and then observe the morphology of the resultant spheres.

Following the dissociation of the vestibular epithelium, the hair cells and supporting cells began to lose their epithelial junctions, with a concomitant reduction in E- and N- cadherin expression (proteins important for making cell junctions). They transitioned into a mesenchymal phenotype, and began to grow as solitary cells rather than as epithelia. After that transition, cells could be dissociated and allowed to thrive in a suspension, which prevented them from adhering to substrates as opposed to each other. Within a few days, the cells began to aggregate and form hollow spheres. As early as day six after suspension, some of the cell spheres started differentiating into hair cells. This was concluded based on the expression of the hair cell markers myosin VIIa, calretinin, parvalbumin 3, and otoferlin. Two days after this initial expression, actin-filled stereocilia could be seen projecting from 3-5% of cells in the sphere (the same cells which were positive for hair cell markers). Four days later, about 15-20% of the sphere’s cells had become hair cells. It is interesting to note that the hair cells’ stereocilia always projected away from the sphere’s lumen, into in the surrounding media. Other cells comprising the sphere were positive for the supporting cell marker SCA, suggesting that the spheres were capable of producing diverse cell types mimicking the auditory epithelium.

However, it is important to prove that these new hair cells are functionally, as well as morphologically, well-developed. This can be tested with a dye called FM1-43, which enters and stains an active hair cell through open mechanosensory transduction channels. When Corwin’s group incubated spheres (passaged 15 or 19 times) with FM1-43 for 10 seconds, only the hair cells in the spheres were labeled with the dye. This suggests that the resultant hair cells from this group’s cell culture method have developed functional mechanosensory transduction channels.

In addition to providing an in vitro population of hair cells which could be useful for drug testing or studies on development, this work has important implications for the replacement of human hair cells. Corwin’s group was able to generate new hair cells from a homogenous line of cells which had been frozen, thawed, and exponentially expanded. The cells survived for months in culture, and did not require implantation into the inner ear before differentiating into functional hair cells. The next step might be to implant these new hair cells into vestibular or auditory avian epithelium in order to observe whether they are capable of integrating into the cellular matrix, and ultimately, within the auditory or vestibular circuit.

1. What might prevent the same culture methods from being used with human hair cells?
2. Are there explanations for why FM1-43 entered hair cells, other than through functional mechanosensory transduction channels? How might this be tested?
3. Why might birds and reptiles have retained the ability the regenerate hair cells?
4. Why might mammalian vestibular hair cells retain the ability to regenerate while cochlear hair cells have not?
5. Integrating new hair cells into the mature epithelium might prove to be daunting in humans. What might improve the feasibility of this approach?