Marcelo N. Rivolta

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear

The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8–E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5–10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5–13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling. J. Comp. Neurol. 442:378–391, 2002.

Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca2+ dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca2+ sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca2+ dependence of immature spiking cells. However, the nature of the Ca2+ sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca2+ dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca2+ dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca2+ sensitivity of vesicle fusion at hair cell synapses.

Auditory hair cell precursors immortalized from the mammalian inner ear.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the α9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

GATA3 IS DOWNREGULATED DURING HAIR CELL DIFFERENTIATION IN THE MOUSE COCHLEA

GATA3 is a transcription factor expressed in the inner ear during the early stages of development. A monoclonal antibody revealed that it is expressed in spiral ganglion cells and in all cells of the developing auditory sensory epithelium in the mouse before the hair cells differentiate at embryonic days 14–16. Expression decreases selectively in the hair cells as they differentiate progressively from the base to the apex of the developing organ of Corti. GATA3 subsequently decreases in the supporting cells and cannot be detected by immunofluorescence in any cell of the adult sensory epithelium. It is not expressed in the vestibular sensory epithelia or surrounding tissues from embryonic day 14. We suggest that GATA3 could act as a repressor of critical genes involved in cell differentiation in the organ of Corti, enabling a progressive formation of the adult cellular pattern.

Human Fetal Auditory Stem Cells Can Be Expanded In Vitro and Differentiate Into Functional Auditory Neurons and Hair Cell‐Like Cells

In the quest to develop the tools necessary for a cell‐based therapy for deafness, a critical step is to identify a suitable stem cell population. Moreover, the lack of a self‐renovating model system for the study of cell fate determination in the human cochlea has impaired our understanding of the molecular events involved in normal human auditory development. We describe here the identification and isolation of a population of SOX2+OCT4+ human auditory stem cells from 9‐week‐old to 11‐week‐old fetal cochleae (hFASCs). These cells underwent long‐term expansion in vitro and retained their capacity to differentiate into sensory hair cells and neurons, whose functional and electrophysiological properties closely resembled their in vivo counterparts during development. hFASCs, and the differentiating protocols defined here, could be used to study developing human cochlear neurons and hair cells, as models for drug screening and toxicity and may facilitate the development of cell‐based therapies for deafness. STEM Cells 2009;27:1196–1204

Calcium signalling mediated by the α9 acetylcholine receptor in a cochlear cell line from the Immortomouse

1 We have investigated the characteristics of the α9 acetylcholine receptor (α9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC‐2 developed from the organ of Corti of the transgenic H‐2Kb‐tsA58 mouse (the Immortomouse) using both calcium imaging and whole‐cell recording. 2 Ratiometric measurements of fura‐2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 μM ACh. The calcium increase was seen in 66 % of the cells grown at 39 °C in differentiated conditions. A smaller fraction (34 %) of cells grown at 33 °C in proliferative conditions responded. 3 Caffeine (10 mM) elevated cell calcium. In the absence of caffeine, the majority of imaged cells responded only once to ACh. A small proportion (< 2 % of the total) responded with an increase in intracellular calcium to multiple ACh presentations. Pretreatment with caffeine inhibited all calcium responses to ACh. 4 In whole‐cell tight‐seal recordings 10 μM ACh activated an inward, non‐selective cation current. The reversal potential of the ACh‐activated inward current was dependent on the extracellular calcium concentration with an estimated PCa/PNa of 80 for the α9 receptor at physiological calcium levels. 5 The data indicate that ACh activates a calcium‐permeable channel α9AChR in UB/OC‐2 cells and that the channel has a significantly higher calcium permeability than other AChRs. The results indicate that the α9AChR may be able to elevate intracellular calcium levels in hair cells both directly and via store release.

Generation of Inner Ear Cell Types From Embryonic Stem Cells

The senses of hearing and balance are mediated by hair cells located in the cochlea and in the vestibular organs of the vertebrate inner ear. Loss of hair cells and other cell types of the inner ear results in hearing and balance disorders that substantially diminish the quality of life. The irreversibility of hearing loss in mammals is caused by the inability of the cochlea to replace lost hair cells. No drugs are available that stimulate inner ear cell regeneration. We describe here protocols to generate inner ear progenitor cells from murine ES cells and to differentiate these progenitors into hair cells and potentially into other inner ear cell types. In addition, we provide a modification of the protocol describing culture conditions in which human ES cells express a similar set of inner ear markers. Inner ear progenitor cells, generated from ES cells, may be used for the development of cell replacement therapy for the diseased inner ear, for high-throughput drug screening, and for the study of inner ear development.

Genomic Analysis of the Function of the Transcription Factor gata3 during Development of the Mammalian Inner Ear

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKBβ, a dramatic increase in Akt1/PKBα protein and relocation of Akt1/PKBα from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27kip1, a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome.