Research team
Expertise
Development and characterization of a 3D neurospheroid model for ischemic stroke research. In this context, I gained experience in: (1) 2D and 3D cell culture (incl. stem cells, neural cells and primary monocytes, microglia(-progenitors)) (2) differentiation of induced pluripotent stem cells to neural stem cells, and further differentiation into neurons and astrocytes, (3) development of reporter cell lines, by means of lentiviral vector transduction, (4) in vitro luminescent assays to monitor neurospheroid viability and growth, (5) execution of hypoxia experiments on 2D and 3D cell cultures, (6) flow cytometry of dissociated neurospheroids, (7) immunocytochemical stainings and microscopic imaging of neurospheroids in semi-high throughput, (8) ELISA to detect cytokines produced by neurospheroids.
The study of alpha-synuclein pathology and related neuroinflammation in a human brain-like context: a human neurospheroid approach.
Abstract
Synucleinopathies, encompassing diseases like Parkinson's disease and dementia with Lewy bodies, are a group of neurodegenerative disorders characterized by the formation of alpha-synuclein (αSyn) aggregates that are able to propagate in a prion-like manner between cells of the nervous system. However, the exact role of αSyn pathology in the disease progression of these synucleinopathies remains to be elucidated. Moreover, microglia have been pointed to as a major player in synucleinopathy pathophysiology, but how these cells affect αSyn pathology remains unclear. Current in vitro models are limited in their ability to replicate human responses to pathological αSyn with sufficient fidelity. Recently developed (microglia-containing) brain organoids represent a promising new tool to study αSyn pathology in a human-brain like environment. In this study, we will use human brain organoids or 'neurospheroids' (NSPHs) to enhance our understanding of αSyn pathology, focusing on propagation and associated pathophysiological pathways. By using NSPHs with and without microglia (annotated as tri- and bipartite NSPHs), we aim to determine the role of microglia and neuroinflammation in these processes. To this end, pre-formed αSyn fibrils will be added to NSPHs. Staining of NSPHs for pathological αSyn, by phosphorylated αSyn antibody and thioflavin, at different timepoints allows to monitor internalization, accumulation and propagation of αSyn pathology. Altered pathophysiological pathways will be determined by RNA-sequencing and validated at the protein level by means of immunocytochemistry (ICC). In summary, this project will help to identify major alterations associated with αSyn pathology and clarify the role of microglia in a human brain-like context.Researcher(s)
- Promoter: Van Breedam Elise
Research team(s)
Project type(s)
- Research Project
Development of isogenic human embryonic stem cell-derived 3D neuro-immune cell culture models: pre-clinical evaluation of interleukin 13 for microglia and macrophage immunomodulation under stroke-like pathology.
Abstract
Development of three-dimensional (3D) in vitro cell culture models for human neuro-immunological research is currently a hot topic in medical cell biology research. Although multiple protocols have been described for generating human 3D brain organoids starting from pluripotent stem cells, current models display several limitations, including the lack of extracellular matrix (ECM), the absence of multiple types of immune cells and a functional blood-brain-barrier (BBB). With this project we aim to develop and optimize a new method for generating 3D neuro-immune cell culture models to study and modulate human neuro-inflammatory responses. For this, isogenic 3D cell cultures comprising human embryonic stem cell (hESC)-derived neurons, astrocytes and microglia will be established on decellularized mouse brain sections in order to provide growth and organizational support by original brain ECM proteins. In addition, hESC-derived astrocytes and endothelial cells will be used to create a BBB model for physical separation of hESC-derived macrophages. Further inclusion of genetic engineering strategies, to allow for real time bioluminescence imaging and (live cell) confocal microscopy, will be applied to ensure profound validation and high throughput screening applications. Once established, we will use this technology to further extend our research efforts to optimize therapeutic strategies based on interleukin (IL)13-mediated immunomodulation, following hypoxic and hypoglycemic stress (i.e. stroke-like conditions). Once validated, we believe that implementation of the proposed 3D brain organoid technology by academia and/or pharmaceutical industry will not only have great impact on the reliability of pre-clinical drug screening, and consequently on the medical and social investments associated with patient care, but also will find application in advanced human toxicology research.Researcher(s)
- Promoter: Ponsaerts Peter
- Fellow: Van Breedam Elise
Research team(s)
Project website
Project type(s)
- Research Project