Browsing by Author "Dottori, Mirella"

Now showing 1 - 7 of 7
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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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    Generation of Neural Organoids from Human Embryonic Stem Cells Using the Rotary Cell Culture System: Effects of Microgravity on Neural Progenitor Cell Fate
    (Mary Ann Liebert, Inc., 2018-04) Mattei, Cristiana; Alshawaf, Abdullah; D’Abaco, Giovanna; Nayagam, Bryony; Dottori, Mirella
    Progress in aeronautics and spaceflight technologies requires in parallel further research on how microgravity may affect human tissue. To date, little is known about the effects of microgravity on human development. In this study we used the rotary cell culture system to investigate whether microgravity supports the generation and maintenance of neural organoids derived from human embryonic stem cells (hESCs) as a model of human brain development. Our results show that although neural organoids could be generated and maintained in microgravity conditions, there were changes in expression of rostral-caudal neural patterning genes and cortical markers compared to organoids generated in standard conditions. This phenomenon was also observed in hESC-derived cortical organoids exposed to microgravity for relatively shorter periods. These results are one of the first for analyzing human neurogenesis in a microgravity environment.
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    Generation of Vestibular Tissue-Like Organoids From Human Pluripotent Stem Cells Using the Rotary Cell Culture System
    (Frontiers, 2019-03) Mattei, Cristiana; Lim, Rebecca; Drury, Hannah; Nasr, Babak; Li, Zihui; Tadros, Melissa; D'Abaco, Giovanna; Stok, Kathyrn; Nayagam, Bryony; Dottori, Mirella
    Hair cells are specialized mechanosensitive cells responsible for mediating balance and hearing within the inner ear. In mammals, hair cells are limited in number and do not regenerate. Human pluripotent stem cells (hPSCs) provide a valuable source for deriving human hair cells to study their development and design therapies to treat and/or prevent their degeneration. In this study we used a dynamic 3D Rotary Cell Culture System (RCCS) for deriving inner ear organoids from hPSCs. We show RCCS-derived organoids recapitulate stages of inner ear development and give rise to an enriched population of hair cells displaying vestibular-like morphological and physiological phenotypes, which resemble developing human fetal inner ear hair cells as well as the presence of accessory otoconia-like structures. These results show that hPSC-derived organoids can generate complex inner ear structural features and be a resource to study inner ear development.
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    Graphene foam as a biocompatible scaffold for culturing human neurons
    (Royal Society Open Science, 2018-04) D'Abaco, Giovanna; Mattei, Cristiana; Nasr, Babak; Hudson, Emma; Alshawaf, Abdullah; Chana, Gursharan; Everall, Ian; Nayagam, Bryony; Dottori, Mirella; Skafidas, Efstratios
    In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation.
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    An In Vitro Model of Developmental Synaptogenesis Using Cocultures of Human Neural Progenitors and Cochlear Explants
    (Mary Ann Liebert Inc Publishers, 2013-03) Nayagam, Bryony; Edge, Albert; Needham, Karina; Hyakumura, Tomoko; Leung, Jessie; Nayagam, David; Dottori, Mirella
    In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be signifi- cantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of lo- cating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.This is a copy of an article published in the Stem Cells and Development Journal © 2013 [copyright Mary Ann Liebert, Inc.]; Stem Cells and Development is available online at: http://online.liebertpub.com.
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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.
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    Organotypic Cocultures of Human Pluripotent Stem Cell Derived- Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices
    (Hindawi, 2019-12) Hyakumura, Tomoko; McDougall, Stuart; Finch, Sue; Needham, Karina; Dottori, Mirella; Nayagam, Bryony
    Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.