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

Abstract

© 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