Dr Marcelo Rivolta: Research Themes
Human auditory stem cells and the development of a cell-based therapy for deafness
Deafness and the lack of conventional treatment. More than 3 million adults in the UK have a bilateral hearing impairment that is moderate to profound (larger than 45 dB HL). The numbers rise to around 10 million if we include sufferers of mild impairments. This is important, since severity increases rapidly after 50 years of age in what is known as presbyacusis. To compound the problem, congenital deafness affects 1 in 1000 children. Almost 90% of people affected suffer a sensorineural deficit, which involves loss of the two first cells in the auditory pathway: the sensory hair cells and their associated auditory neurons.
In mammals, the progenitor cells that give origin to the sensory cell types are only produced during foetal and early postnatal stages. Therefore, damaged cells are not replaced, making hearing loss irreversible. There is no medical treatment for deafness although, in patients that maintain a suitable nerve, the sensory function of the inner ear can be partially restored by a cochlear implant (CI). While the CI can functionally replace damaged hair cells, there is virtually no treatment to compensate for the loss of auditory neurons. The social and economic implications of hearing impairment are enormous, as is the size of the population affected.
The need for a new therapy: Stem cell opportunity
Current developments in stem cell technology could offer new hopes for the treatment of deafness. One therapeutic approach could be to trigger sensory regeneration from existing cells or to replace lost cells by transplantation of exogenous, in vitro-maintained stem populations with the potential to produce hair cells and neurons.
Isolation and identification of human Foetal Auditory Stem Cells (hFASCs)
Since the auditory sensory cells are only generated during development, we initially try to identify a population of stem cells in the human foetal cochlea (1) and then developed a protocol that allowed their isolation and expansion in vitro (2). By culturing cells from sensory epithelia from 9 to 11 weeks-old foetuses in a serum-free media supplemented with defined growth factors, we expanded a population that expressed stem cell markers such as NESTIN, SOX2, OCT4 and REX1 and have the ability to differentiate into functional sensory neurons and hair cell-like cells. These cells are an excellent system to study human ear differentiation and allowed us to define a media to sustain stem cell growth as well as conditions to induce the differentiation into neurons and hair cells.
Generation of otic phenotypes from human embryonic stem cells
We have also established a protocol to coerce hESCs into auditory phenotypes using FGF3 and 10, the ligands involved in ear induction during development in mice (3). We started differentiation of hESCs by plating them without feeders in a serum-free, chemically defined media supplemented with human FGF3 and FGF10. This treatment triggered differentiation of otic placode cells, as determined by the combined expression of PAX8, SOX2 and FOXG1. After FGF treatment, 18.3%±0.8 cells were double labelled, high expressing for PAX8 and SOX2 while 18%±2 of the total cells were double labelled for PAX8 and FOXG1. These cells were also positive for the otic markers PAX2, NESTIN and GATA3
Two different types of colonies with different lineage potential are obtained after induction
After FGF3 and FGF10 induction, two morphologically distinct types of otic colonies were obtained and, given their morphological appearance we have operatively named them hESC-derived otic epithelial progenitor (hESC-OEPs) and hESC-derived otic neural progenitor (hESC-ONPs). To test the differentiation potential of these cells they were transferred to either a ‘hair cell’ or to a ‘neuralizing’ culture condition, previously described by using hFASCs (2). When hESC-derived OEP colonies were cultured under ‘hair cell’ conditions for 2-4 weeks, they differentiated into hair cell-like cells, expressing markers such as ATOH1, BRN3C and MYO7A. On the other hand, when ONPs were cultured in ‘neuralizing’ conditions they became elongated displaying the typical bipolar morphology of inner ear neurons and expressing TuJ1, NF200 and other neuronal markers. Differentiating hair cell-like cells and neurons also expressed typical currents and functional markers. The electrophysiological profile of both differentiated cell types was remarkably similar to those obtained from hFASCs.
The potential therapeutic application of the ONPs was studied by transplanting them into a gerbil model of neuropathic deafness (see figure below). Transplanted ONPs grafted into the modiolus, extended projections and differentiated into TuJ1+ neurons. Histological analysis after 10 weeks post transplantation (PT) showed that cells expressed 3A10 neurofilament-associated antigen and NKAalpha3, a marker of type I neurons and afferent fibers in the inner ear. Significantly, projections from the transplanted cells that reached the organ of Corti were targeting the hair cells, and fibers positive for NKAalpha3 and GluA2 were observed next to the basal pole of the inner hair cells suggesting the presence of synaptic connections. Moreover, fibers from the transplanted cells also stained for synaptophysin were found in the cochlear nucleus, suggesting synaptic connections with the central auditory path. Hearing function was studied at 1-2 week intervals. Control animals showed no sign of functional recovery throughout the experiment. However in the transplanted animals, there was a detectable improvement in the ABR thresholds starting approximately four weeks PT. The mean auditory threshold shift, calculated as the difference between the thresholds at 10 weeks PT versus the one before onset of deafness, was of 53±1.7 dB in the control animals compared to 28.6±3.6 dB in the transplanted cohort (p 0.0002).
Work in my lab is supported by Action on Hearing Loss, Deafness Research UK, the Medical Research Council and collaborations with Cochlear and Pfizer.
- Chen, W; Cacciabue-Rivolta, D; Moore, HD and Rivolta, MN.
The human fetal cochlea can be a source for auditory progenitors/stem cells isolation.
Hearing Res 233: 23-29, 2007.
- Chen, W; Johnson, SL; Marcotti, W; Andrews, PW; Moore, HD and Rivolta, MN.
Human Fetal Auditory Stem Cells (hFASCs) can be expanded in vitro and differentiate into functional auditory neurons and hair cell-like cells.
Stem Cells, 27:1196-1204, 2009.
- Chen, W; Jongkamonwiwat, N; Abbas, L; Jacob Eshtan, S; Johnson, SL; Kuhn, S; Thurlow, JK; Andrews, PW; Marcotti, W; Moore, HD and Rivolta, MN.
Restoration of auditory evoked responses by human ES cell-derived cochlear progenitors.
Nature, 2012 DOI 10.1038/nature11415.