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Title
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Development, characterization and application of luminescent human iPSC-derived neurospheroids in ischemic stroke research
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Author
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Abstract
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| Although stroke is one of the world’s leading causes of death and disability, and more than a thousand candidate neuroprotective drugs have been proposed based on extensive in vitro and animal-based research, an effective neuroprotective/restorative therapy for ischemic stroke patients is still missing. Especially the high attrition rate of neuroprotective compounds in clinical studies should make us question the ability of in vitro stroke models currently used for ischemic stroke research to recapitulate human ischemic responses with sufficient fidelity. The ischemic stroke field would greatly benefit from the implementation a physiologically relevant human in vitro stroke model. The development and application of human iPSC-derived 3D neurospheroid models represent an appropriate approach to fulfill this need. In a first part, a luminescent human iPSC-derived neurospheroid model enabling the real-time read-out of neural viability after ischemia-like conditions was developed and characterized. By depriving neurospheroids from oxygen and glucose, the ability of the applied bioluminescent system to detect neurotoxicity was demonstrated. Moreover, differences in behaviour after oxygen-glucose deprivation between different ages of neurospheroids were observed, whereby 1-week-old but not 4-week-old neurospheroids displayed spontaneous recovery. This underscores the need for more mature neurospheroids in in vitro stroke research that more faithfully recapitulate the in vivo adult situation. Furthermore, evaluation of the pan-caspase inhibitor Z-VAD-FMK in the established model demonstrated its inability to increase overall neural survival in neurospheroids in contrast to a 2D culture of the same hiPSC-derived neural stem cells, where neuroprotection was observed. This exemplifies how the increased complexity of spheroid models can result in a different outcome when testing neuroprotective compounds. In a second part, the foundation was laid to further increase the complexity and predictivity of the developed human neurospheroid model by generating more mature, multicellular neurospheroids. In a first set of explorative experiments, culture conditions were optimized in order to obtain neurospheroids with increased maturity and the presence of astrocytes. Hereby, increasing the culture time of the neurospheroids markedly increased neuronal maturity as well as the spontaneous development of astrocytes. It was also noted that culture of neurospheroids in selected differentiation media did not give rise to astrocytes for the evaluated time points despite their ability to display faster differentiation and maturation. In a second set of explorative experiments, the integration of hematopoietic progenitors cells into neurospheroids was explored for the future creation of an immune-competent, microglia-enriched neurospheroid model for ischemic stroke. Comparison of different time points for the addition of hematopoietic progenitor cells to neurospheroids, showed higher integration efficiency when added to pre-established neurospheroids. |
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Language
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English
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Publication
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Antwerp
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University of Antwerp, Faculty of Medicine and Health Sciences
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2022
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Volume/pages
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xv, 162 p.
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Note
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Ponsaerts, Peter [Supervisor]
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Full text (open access)
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