Browsing by Author "Crombie, Duncan"

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    Directing human induced pluripotent stem cells into a neurosensory lineage for auditory neuron replacement
    (Mary Ann Liebert, 2014) Gunewardene, Niliksha; Van Bergen, Nicole; Crombie, Duncan; Needham, Karina; Dottori, Mirella; Nayagam, Bryony
    Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (AN) 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) towards 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 hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), pro-neurosensory 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. Whilst 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 to the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs towards a neurosensory lineage, but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.
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    Innervation of Cochlear Hair Cells by Human Induced Pluripotent Stem Cell-Derived Neurons In Vitro
    (Hindawi Publishing Corporation, 2016) Gunewardene, Niliksha; Crombie, Duncan; Dottori, Mirella; Nayagam, Bryony A.
    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 hiPSCderived 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.