Applications of Lgr5-Positive Cochlear Progenitors (LCPs) to the Study of Hair Cell Differentiation

The mouse cochlea contains approximately 15,000 hair cells. Its dimensions and location, and the small number of hair cells, make mechanistic, developmental and cellular replacement studies difficult. We recently published a protocol to expand and differentiate murine neonatal cochlear progenitor cells into 3D organoids that recapitulate developmental pathways and can generate large numbers of hair cells with intact stereociliary bundles, molecular markers of the native cells and mechanotransduction channel activity, as indicated by FM1-43 uptake. Here, we elaborate on the method and application of these Lgr5-positive cochlear progenitors, termed LCPs, to the study of inner ear development and differentiation. We demonstrate the use of these cells for testing several drug candidates, gene silencing and overexpression, as well as genomic modification using CRISPR/Cas9. We thus establish LCPs as a valuable in vitro tool for the analysis of progenitor cell manipulation and hair cell differentiation

The potential of induced pluripotent stem cells for spiral ganglion neuron replacement

© 2014 Dr. Niliksha GunewardenePublications included in thesis:Gunewardene, N., Dottori, M. & Nayagam, B. A. (2012). The convergence of cochlear implantation with induced pluripotent stem cell therapy. Stem Cells Reviews and Reports, 8(3), 741-754. DOI: 10.1007/s12015-011-9320-0Needham, K., Hyakumura, T., Gunewardene, N., Dottori, M. & Nayagam, B. A. (2014). Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells. Stem Cell Research, 12(1), 241-249. DOI: 10.1016/j.scr.2013.10.011In mammals, overexposure to loud noise, ototoxic medication or even ageing can incur irreversible damage to the sensory hair cells and spiral ganglion neurons (SGNs) resulting in sensorineural hearing loss (SNHL). Currently, the cochlear implant is the only available treatment for SNHL, but its functionality is dependent on a healthy complement of SGNs. Therefore in cases of severe SNHL, where the numbers of SGNs are significantly depleted, the efficacy of this neural prosthesis may be compromised. Using stem cells to replace damaged SGNs is an emerging therapeutic strategy for deafness. Whilst previous studies have explored the potential for several stem cell types, particularly human embryonic stem cells (hESCs) to replace SGNs, it will eventually be important that transplanted cells are from an autologous source. This thesis therefore aims to explore the potential of human induced pluripotent stem cells (hiPSCs) for SGN replacement. These cells offer the option of transplanting SGNs generated from a patient’s own cells to potentially restore hearing function and/or improve the efficiency of the cochlear implant. To investigate the potential of hiPSCs, it is first necessary to assess their potential to differentiate into a SGN lineage. In the first study of the thesis, an established neural induction protocol was used to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC, H9) towards a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyse the expression of key markers involved in SGN development, at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain and otic placodal markers (PAX7 and PAX2), pro-neurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160) and sensory SGN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC-and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Whilst all cell lines analysed produced neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared to the hESC controls. Thereby, suggesting that hiPSCs have a more variable differentiation potential compared to the hESCs. The functionality of the hiPSC-derived neurons was next assessed using patch clamp electrophysiology and in vitro co-culture assays. It was found that the cells were capable of firing action potentials in response to depolarisation and exhibited a phasic profile of activity, thus indicating that the neurons were physiologically active. Following co-culture of cochlear explants or denervated explants with hiPSC- and hESC-derived neurons, their neural processes were observed to make direct contact and form extensive synaptic connections with inner and outer hair cells in vitro. However the hiPSC-derived neurons were observed to innervate fewer hair cells, compared to hESC-derived neurons. Preliminary data also suggests that hiPSC-derived neurons are able to survive and maintain a neural phenotype two weeks post-transplantation in the mammalian cochlea. Overall, this thesis demonstrates that hiPSCs are capable of differentiating into functional neurosensory-like progenitors and innervating developing hair cells in vitro. However the differentiation and innervation potentials of hiPSC-derived neurons were observed to be less consistent, compared to hESC-derived neurons. While it is important that these variabilities are minimised prior to the clinical translation of this treatment, the use of hiPSCs for SGN replacement in the deaf cochlea holds potential

The Convergence of Cochlear Implantation with Induced Pluripotent Stem Cell Therapy

According to 2010 estimates from The National Institute on Deafness and other Communication Disorders, approximately 17% (36 million) American adults have reported some degree of hearing loss. Currently, the only clinical treatment available for those with severe-to-profound hearing loss is a cochlear implant, which is designed to electrically stimulate the auditory nerve in the absence of hair cells. Whilst the cochlear implant has been revolutionary in terms of providing hearing to the severe-to-profoundly deaf, there are variations in cochlear implant performance which may be related to the degree of degeneration of auditory neurons following hearing loss. Hence, numerous experimental studies have focused on enhancing the efficacy of cochlear implants by using neurotrophins to preserve the auditory neurons, and more recently, attempting to replace these dying cells with new neurons derived from stem cells. As a result, several groups are now investigating the potential for both embryonic and adult stem cells to replace the degenerating sensory elements in the deaf cochlea. Recent advances in our knowledge of stem cells and the development of induced pluripotency by Takahashi and Yamanaka in 2006, have opened a new realm of science focused on the use of induced pluripotent stem (iPS) cells for therapeutic purposes. This review will provide a broad overview of the potential benefits and challenges of using iPS cells in combination with a cochlear implant for the treatment of hearing loss, including differentiation of iPS cells into an auditory neural lineage and clinically relevant transplantation approaches

Innervation of Cochlear Hair Cells by Human Induced Pluripotent Stem Cell-Derived Neurons In Vitro

Induced pluripotent stem cells (iPSCs) may serve as an autologous source of replacement neurons in the injured cochlea, if they can be successfully differentiated and reconnected with residual elements in the damaged auditory system. Here, we explored the potential of hiPSC-derived neurons to innervate early postnatal hair cells, using established in vitro assays. We compared two hiPSC lines against a well-characterized hESC line. After ten days' coculture in vitro, hiPSC-derived neural processes contacted inner and outer hair cells in whole cochlear explant cultures. Neural processes from hiPSC-derived neurons also made contact with hair cells in denervated sensory epithelia explants and expressed synapsin at these points of contact. Interestingly, hiPSC-derived neurons cocultured with hair cells at an early stage of differentiation formed synapses with a higher number of hair cells, compared to hiPSC-derived neurons cocultured at a later stage of differentiation. Notable differences in the innervation potentials of the hiPSC-derived neurons were also observed and variations existed between the hiPSC lines in their innervation efficiencies. Collectively, these data illustrate the promise of hiPSCs for auditory neuron replacement and highlight the need to develop methods to mitigate variabilities observed amongst hiPSC lines, in order to achieve reliable clinical improvements for patients

Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons

Directing Human Induced Pluripotent Stem Cells into a Neurosensory Lineage for Auditory Neuron Replacement

Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESCderived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages

Viral-mediated transduction of auditory neurons with opsins for optical and hybrid activation

Abstract: Optical stimulation is a paradigm-shifting approach to modulating neural activity that has the potential to overcome the issue of current spread that occurs with electrical stimulation by providing focused stimuli. But optical stimulation either requires high power infrared light or genetic modification of neurons to make them responsive to lower power visible light. This work examines optical activation of auditory neurons following optogenetic modification via AAV injection in two species (mouse and guinea pig). An Anc80 viral vector was used to express the channelrhodopsin variant ChR2-H134R fused to a fluorescent reporter gene under the control of the human synapsin-1 promoter. The AAV was administered directly to the cochlea (n = 33) or posterior semi-circular canal of C57BL/6 mice (n = 4) or to guinea pig cochleae (n = 6). Light (488 nm), electrical stimuli or the combination of these (hybrid stimulation) was delivered to the cochlea via a laser-coupled optical fibre and co-located platinum wire. Activation thresholds, spread of activation and stimulus interactions were obtained from multi-unit recordings from the central nucleus of the inferior colliculus of injected mice, as well as ChR2-H134R transgenic mice (n = 4). Expression of ChR2-H134R was examined by histology. In the mouse, transduction of auditory neurons by the Anc80 viral vector was most successful when injected at a neonatal age with up to 89% of neurons transduced. Auditory neuron transductions were not successful in guinea pigs. Inferior colliculus responses to optical stimuli were detected in a cochleotopic manner in all mice with ChR2-H134R expression. There was a significant correlation between lower activation thresholds in mice and higher proportions of transduced neurons. There was no difference in spread of activation between optical stimulation and electrical stimulation provided by the light/electrical delivery system used here (optical fibre with bonded 25 µm platinum/iridium wire). Hybrid stimulation, comprised of sub-threshold optical stimulation to ‘prime’ or raise the excitability of the neurons, lowered the threshold for electrical activation in most cases, but the impact on excitation width was more variable compared to transgenic mice. This study demonstrates the impact of opsin expression levels and expression pattern on optical and hybrid stimulation when considering optical or hybrid stimulation techniques for neuromodulation