Zingaro, Simona;
(2023)
Exploring the microenvironment for 3D inner ear organoid and otic neuron differentiation from mouse embryonic stem cells.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Hearing loss is one of the most common forms of sensory impairment in humans, affecting more than 5% of the world’s population. To date, our understanding of the mechanisms of hearing loss and the generation of therapies have been hampered by the lack of a human in vitro model. Previous work showed that it is possible to generate inner ear organoids (IEOs) containing functional hair cells from mouse (mPSCs) or human pluripotent stem cells (hPSCs) through a three-dimensional (3D) culture system; however, their translation into an in vitro model for drug screening or developmental modelling is limited by low differentiation yield, lack of reproducibility and standardisation of the differentiation protocol. This study aims to explore microenvironments for IEOs and otic neuron (ONs) differentiation from mESCs that could ultimately be combined with engineering devices for translational research. IEOs containing hair cells, supporting cells and neurons were generated with a Atoh1/nGFP mouse embryonic stem cell (mESC) line. The microenvironment was explored by culturing mESC aggregates in microwells or in 3D MatrigelTM, collagen or a combination of both. Results show that microwells allow the control of mESC aggregates size but affect otic differentiation. On the other hand, culture of aggregates in gel domes revealed that these can sustain formation of IEOs and also support the formation of neural-like structures in the surrounding matrix, in particular when embedded in collagen combined with MatrigelTM. Additional characterisation by immunostaining, electrophysiology and single nuclei RNA sequencing confirmed the presence of a population of functional neurons, often observed in clusters, formed after culture in 3D Collagen supplemented with MatrigelTM. This thesis provides a step forward the characterisation of microenvironment suitable for development of IEOs and functional presumptive ONs. This model would allow future works directed on understanding ONs development and for investigating the mechanisms underlying sensory neural hearing loss.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Exploring the microenvironment for 3D inner ear organoid and otic neuron differentiation from mouse embryonic stem cells |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > The Ear Institute |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10163104 |
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