Publicação
Harnessing human iPSC-derived 3D brain models to study the innate immune response
| Resumo: | Neuroinflammation is a protective response to central nervous system (CNS) injuries that, when prolonged, may contribute to CNS pathologies. Microglia, the CNS-resident innate immune cells, undergo morphological, transcriptomic and functional changes upon injury, acquiring a proinflammatory profile and increasing proliferation. Recombinant adeno associated virus (rAAV)-mediated gene therapy offers therapeutic options for previously untreatable neurological disorders. However, some patients experienced unexpected immune reactions after the delivery of rAAV-based gene therapy, highlighting the translational gap and the need for innate immune competent CNS preclinical models. Three dimensional (3D) CNS models incorporating microglia provide a better mimicry of tissue architecture and cell-cell interactions. However, most models combine full differentiated microglia and neurons, limiting the evaluation of interactions occurring during differentiation. This project aimed to develop a human induced pluripotent stem cell (hiPSC)-derived neurospheroid model through the 3D co-differentiation of neural and erythromyeloid progenitor cells in stirred tank bioreactors, optimizing medium composition to promote microglial differentiation. The evaluation of the cellular composition of the neurospheroids allowed the detection of functional neurons, astrocytes and microglia at day 28. To characterize the morphofunctional status of microglia, a morphometric analysis workflow was developed. Microglia were characterized and clustered based on a toolbox of morphometric parameters fitting the specific morphotypes observed in the neurospheroids, which lacked typically ramified microglia and mostly exhibit immature characteristics. Pro- and anti-inflammatory challenge demonstrated that microglia were functional, responding as expected regarding gene expression, and exhibited different, cell line-dependent, morphological changes, highlighting the need for customized morphometric workflows. Overall, this work culminated with the development of a human preclinical CNS model incorporating functional microglia, along with the development of a bioanalytical imaging tool to better assess the morphofunctional status of microglia. These will help to unravel the complex and dynamic nature of innate immunity in the CNS and reduce the translational gap. |
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| Autores principais: | Delgado, Maria Margarida dos Santos |
| Assunto: | neuroinflamação microglia hiPSCs neuroesferóides morfologia Teses de mestrado - 2024 |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Neuroinflammation is a protective response to central nervous system (CNS) injuries that, when prolonged, may contribute to CNS pathologies. Microglia, the CNS-resident innate immune cells, undergo morphological, transcriptomic and functional changes upon injury, acquiring a proinflammatory profile and increasing proliferation. Recombinant adeno associated virus (rAAV)-mediated gene therapy offers therapeutic options for previously untreatable neurological disorders. However, some patients experienced unexpected immune reactions after the delivery of rAAV-based gene therapy, highlighting the translational gap and the need for innate immune competent CNS preclinical models. Three dimensional (3D) CNS models incorporating microglia provide a better mimicry of tissue architecture and cell-cell interactions. However, most models combine full differentiated microglia and neurons, limiting the evaluation of interactions occurring during differentiation. This project aimed to develop a human induced pluripotent stem cell (hiPSC)-derived neurospheroid model through the 3D co-differentiation of neural and erythromyeloid progenitor cells in stirred tank bioreactors, optimizing medium composition to promote microglial differentiation. The evaluation of the cellular composition of the neurospheroids allowed the detection of functional neurons, astrocytes and microglia at day 28. To characterize the morphofunctional status of microglia, a morphometric analysis workflow was developed. Microglia were characterized and clustered based on a toolbox of morphometric parameters fitting the specific morphotypes observed in the neurospheroids, which lacked typically ramified microglia and mostly exhibit immature characteristics. Pro- and anti-inflammatory challenge demonstrated that microglia were functional, responding as expected regarding gene expression, and exhibited different, cell line-dependent, morphological changes, highlighting the need for customized morphometric workflows. Overall, this work culminated with the development of a human preclinical CNS model incorporating functional microglia, along with the development of a bioanalytical imaging tool to better assess the morphofunctional status of microglia. These will help to unravel the complex and dynamic nature of innate immunity in the CNS and reduce the translational gap. |
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