Research Elise Van Breedam

Abstract

Stroke has a dramatic, lasting influence on the lives of millions of patients worldwide. Despite decades of research, none of the candidate neuroprotective drugs have made it to an effective therapy to date. This is partially due to the lack of appropriate model systems that recapitulate the human ischemic responses. Fortunately, induced pluripotent stem cell (iPSC)-technology has provided a new entry point for generating relevant human-based in vitro brain models, such as neurospheroids. Capitalizing on the successful research efforts of my host group to generate human iPSC-derived tripartite neurospheroids that contain mature neurons, astrocytes and microglia, we hypothesize that such models will help unravel biologically relevant cellular and molecular events in the context of stroke pathology. To investigate this, we will emulate stroke-like conditions in these model systems by means of oxygen and glucose deprivation and subsequently characterize the functional and molecular changes using a combined imaging and multi-omics approach. Furthermore, we will refine the current tripartite model, by allowing the infiltration of peripheral immune cells (i.e., monocytes and neutrophils) to mimic the in vivo response even more comprehensively. This way, we intend to unravel the neuroinflammatory cascade that follows ischemic stroke and identify new pathways and associated markers that may help protect or repair the neurological damage.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: De Vos Winnok
• Co-promoter: Van Breedam Elise
• Fellow: Van Calster Siebe

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Synucleinopathies, including Parkinson's disease, are neurodegenerative disorders characterized by the formation of alpha-synuclein (?Syn) aggregates that propagate prion-like between nervous system cells. The exact role of ?Syn pathology in disease progression remains unclear. Moreover, how microglia precisely affect ?Syn pathology remains to be elucidated. Current in vitro models are limited in their ability to faithfully replicate human responses to pathological ?Syn. In this study, we will use human neurospheroids (NSPHs) to enhance our understanding of ?Syn pathology, more specifically the pathophysiological pathways associated with ?Syn accumulation. By using NSPHs with and without microglia, we aim to clarify the role of microglia and neuroinflammation in general in ?Syn accumulation/propagation and downstream cellular responses. Hereto, pre-formed ?Syn fibrils will be added to NSPHs and ?Syn accumulation/propagation will be monitored over time by staining NSPHs for pathological ?Syn. Next, pathways elicited with ?Syn accumulation will be determined at the transcriptome and proteome level and further characterized at the cellular and functional level, by means of immunocytochemistry and functional assays (e.g. electrophysiology), respectively. In summary, this project will help to identify pathophysiological mechanisms associated with ?Syn pathology possibly leading to neuronal dysfunction or loss, and clarify the role of microglia, in a human brain-like context.

Researcher(s)

• Promoter: Ponsaerts Peter
• Fellow: Van Breedam Elise

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

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)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

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)

• Laboratory for Experimental Hematology (LEH)

Project website

Project type(s)

• Research Project