Inner ear hair cells produced in vitro by a mesenchymal-to-epithelial transition

Abstract

Sensory hair cell loss is a major contributor to disabling hearing and balance deficits that affect >250 million people worldwide. Sound exposures, infections, drug toxicity, genetic disorders, and aging all can cause hair cell loss and lead to permanent sensory deficits. Progress toward treatments for these deficits has been limited, in part because hair cells have only been obtainable via microdissection of the anatomically complex internal ear. Attempts to produce hair cells in vitro have resulted in reports of some success but have required transplantation into embryonic ears or coculturing with other tissues. Here, we show that avian inner ear cells can be cultured and passaged for months, frozen, and expanded to large numbers without other tissues. At any point from passage 6 up to at least passage 23, these cultures can be fully dissociated and then aggregated in suspension to induce a mesenchymal-to-epithelial transition that reliably yields new polarized sensory epithelia. Those epithelia develop numerous hair cells that are crowned by hair bundles, composed of a single kinocilium and an asymmetric array of stereocilia. These hair cells exhibit rapid permeance to FM1-43, a dye that passes through open mechanotransducing channels. Because a vial of frozen cells can now provide the capacity to produce bona fide hair cells completely in vitro, these discoveries should open new avenues of research that may ultimately contribute to better treatments for hearing loss and other inner ear disorders.balance ͉ culture ͉ hearing ͉ regeneration ͉ stem cell

Sensory Epithelial Cells Acquire Features of Prosensory Cells Via Epithelial to Mesenchymal Transition

“…Color images available online at www.liebertonline.com/scd 1818 ZHANG AND HU cells are able to undergo an EMT when they are cultured on 2D substrates. These results are similar to those of chick utricular cells [10]. However, N-cadherin, a mammalian mesenchymal marker, was observed in utricular sensory epithelial cells derived from chick embryos.…”

“…Pure sensory epithelial sheets were harvested from 3-month-old Swiss Webster female mice using previously described methods [10]. The pure sensory epithelial sheets were cut into 1-2 mm 2 pieces that were then transferred into a new 15 mL centrifuge tube.…”

“…First, cell number was evaluated with a hemocytometer at each passage and the doubling time was calculated using the following formula: td = (t1-t0)/log2(C1/C0). C1 = cell number at time point t1, C0 = cell number at time point t0, td = doubling time for cell population [10].…”

“…Indeed, when epithelial cells from pancreatic islet are cultured on 2-dimensional (2D) substrates in vitro, they de-differentiate into mesenchymal-like cells that can be expanded to a large number for use in regeneration study [8,9]. In our previous efforts for inner ear regeneration, utricle epithelial cells from avian embryos are found to undergo an EMT to proliferate and subsequently become sensory epithelial cells through a reverse EMT, mesenchymal-to-epithelial transition (MET) [10]. Since avian inner ear sensory epithelium is known to be able to regenerate, it might not be surprising to observe that these cells are able to proliferate via EMT.…”

Can neurosphere production help restore inner ear transduction?

“…For example, stem cells isolated from mammalian inner ear tissues such as the stria vascularis, the auditory organ (4,5), the greater epithelia ridge of the cochlea (6), and the inner ear organs of balance (4, 7) appear to possess the potential to proliferate and generate hair cell-like cells under certain conditions or after transplantation. Cells with bona fide hair bundles have been generated in vitro from cell lines derived from embryonic chick ears (8). Stem cells derived from sources outside the inner ear such as embryonic stem cells (9), bone marrow mesenchymal stem cells (10), and adult mouse olfactory precursor cells (11) are also able to differentiate into cells that express hair cell markers.…”