Periodic Reporting for period 1 - MAL-ZOO (The malaria zoo: dissecting cerebral malaria in three in vitro primate blood-brain barrier models)
Berichtszeitraum: 2023-04-01 bis 2025-03-31
Malaria is a parasitic disease that continues to affect hundreds of millions of people every year, causing over 600,000 deaths, mostly among children in sub-Saharan Afrihttps://ec.europa.eu/research/participants/grants-app/reporting/VAADIN/themes/sygma/icons/ico6-save.pngca. Although the numbers of infections and deaths has strongly decreased in recent decades, progress in further reducing malaria has stalled. Therefore, advances in our understanding of how the disease develops is essential to finding new strategies to reduce the high mortality.
The most severe complication, and the major cause of deaths, particularly in children, is cerebral malaria (CM), with a mortality rate of 15 – 25%. During CM, parasites accumulate in the brain’s blood vessels, where they can damage the blood-brain barrier. This can lead to brain swelling, coma and death. However, the exact mechanism is not entirely understood.
Interestingly, CM can occur in certain macaque species, but not in zoonotic malaria (spreading from macaques to humans). This suggests that host-specific factors define the outcome of the disease.
A big limitation in studying human CM, is that animal models do not reflect the extent of parasite accumulation and brain swelling seen in patients. In contrast, studies on human patients is limited to non-invasive and post-mortem inspections. To overcome these barriers, tissue engineering is undergoing enormous advances by creating complex tissue models from human cells grown in culture. These in vitro models can replicate key physiological processes, including those involved in CM.
A powerful tool for building such models is the use of induced pluripotent stem cells (iPSC). iPSCs can differentiate into all cell types, including those forming our blood vessels. Freshly differentiated cells can self-organize into tissue-like structures and often mimic the behavior of real human tissue. iPSCs can also be derived from specific donors, and therefore allow host-specific disease modelling, using cells of humans or animals like macaques.
This project uses iPSCs to study CM by addressing the following objectives:
1. Develop an iPSC-method to generate human brain blood vessel models for in vitro studies of CM.
2. Reproduce key features of CM in the model, such as parasite accumulation and vessel damage, to better understand how infection leads to brain pathology.
3. Adapt the methods to macaque iPSCs to explore species-specific differences during infection. This will help identify mechanisms that either contribute to or protect against CM.
The most severe complication, and the major cause of deaths, particularly in children, is cerebral malaria (CM), with a mortality rate of 15 – 25%. During CM, parasites accumulate in the brain’s blood vessels, where they can damage the blood-brain barrier. This can lead to brain swelling, coma and death. However, the exact mechanism is not entirely understood.
Interestingly, CM can occur in certain macaque species, but not in zoonotic malaria (spreading from macaques to humans). This suggests that host-specific factors define the outcome of the disease.
A big limitation in studying human CM, is that animal models do not reflect the extent of parasite accumulation and brain swelling seen in patients. In contrast, studies on human patients is limited to non-invasive and post-mortem inspections. To overcome these barriers, tissue engineering is undergoing enormous advances by creating complex tissue models from human cells grown in culture. These in vitro models can replicate key physiological processes, including those involved in CM.
A powerful tool for building such models is the use of induced pluripotent stem cells (iPSC). iPSCs can differentiate into all cell types, including those forming our blood vessels. Freshly differentiated cells can self-organize into tissue-like structures and often mimic the behavior of real human tissue. iPSCs can also be derived from specific donors, and therefore allow host-specific disease modelling, using cells of humans or animals like macaques.
This project uses iPSCs to study CM by addressing the following objectives:
1. Develop an iPSC-method to generate human brain blood vessel models for in vitro studies of CM.
2. Reproduce key features of CM in the model, such as parasite accumulation and vessel damage, to better understand how infection leads to brain pathology.
3. Adapt the methods to macaque iPSCs to explore species-specific differences during infection. This will help identify mechanisms that either contribute to or protect against CM.
A prerequisite for this project is the successful development of a brain blood vessel model. Using iPSC technologies, published differentiation methods were integrated into a microfluidic setup that generates perfusable blood vessels with highly elevated barrier function, one of the key features of the brain’s vasculature. While applying this state-of the-art differentiation approach, concerns arose in the iPSC community about the cellular identity of this protocol. Therefore, genetic tools were used to generate an iPSC line in which the gene regulation is controlled by the induction of so-call endothelial transcription factors. This led to the development of an improved iPSC-differentiation approach that produces blood vessel models with the correct cellular identity. Strikingly, these vessels still show elevated barrier function and are suitable for in vitro malaria infections.
During infection, the new blood vessel model shows increased parasite accumulation. In addition, when exposed to malaria-released toxins, disruption of the vessel barrier can be observed in a temporal and dose-dependent manner. Through a comparative analysis of the transcriptional response, meaning the cell’s gene expression response to the malaria toxins, we identified biological processes that are either up- or downregulated. We observed that the newly generated cell type shows responses linked to cellular architecture and contact points. These results highlight that we have engineered a blood vessel model that supports malaria infection, reflects key features of CM disease, and allows us to identify the cellular changes that are at least partly responsible for observed vascular dysfunction in patients.
The project originally aimed to develop a matching model using macaque iPSCs to study host-specific differences in brain vascular pathology. While the initial protocol showed promising results in macaque cells, efforts shifted toward improving the human iPSC model, given its greater medical relevance. Our findings reveal that endothelial transcription factors are essential for generating functional blood vessel infection models and provide a pipeline that can be adapted for other iPSC sources, including from different species.
During infection, the new blood vessel model shows increased parasite accumulation. In addition, when exposed to malaria-released toxins, disruption of the vessel barrier can be observed in a temporal and dose-dependent manner. Through a comparative analysis of the transcriptional response, meaning the cell’s gene expression response to the malaria toxins, we identified biological processes that are either up- or downregulated. We observed that the newly generated cell type shows responses linked to cellular architecture and contact points. These results highlight that we have engineered a blood vessel model that supports malaria infection, reflects key features of CM disease, and allows us to identify the cellular changes that are at least partly responsible for observed vascular dysfunction in patients.
The project originally aimed to develop a matching model using macaque iPSCs to study host-specific differences in brain vascular pathology. While the initial protocol showed promising results in macaque cells, efforts shifted toward improving the human iPSC model, given its greater medical relevance. Our findings reveal that endothelial transcription factors are essential for generating functional blood vessel infection models and provide a pipeline that can be adapted for other iPSC sources, including from different species.
One of the key outcomes of this project is the integration of endothelial transcription factor-based regulation into iPSC differentiation. We generated what we believe to be the first stable iPSC line expressing three transcription factors that produce blood vessels with improved barrier function. This has broad relevance, not only for malaria research but also for studying various brain vascular diseases.
There is already strong interest from collaborators to apply our model in areas like brain trafficking and blood-brain barrier studies. However, each application beyond malaria will require validation of disease-specific features, for example, confirming the presence of relevant transport proteins.
Our model also has strong potential for larger-scale applications, such as pharmaceutical screening. We are currently in discussion with biotech and pharma partners and are working closely with our institution on intellectual property and patent matters.
We are among the few groups worldwide using iPSC technology in malaria research. iPSCs are uniquely suited for this, as they can be differentiated into many tissue types. However, the technical complexity remains a barrier for many researchers in the malaria field. Our study provides a clear roadmap for how to combine iPSC-based models with infection biology, opening new possibilities to study how malaria affects different tissues. Continued integration of iPSC models into infection research will be essential to advance the field.
There is already strong interest from collaborators to apply our model in areas like brain trafficking and blood-brain barrier studies. However, each application beyond malaria will require validation of disease-specific features, for example, confirming the presence of relevant transport proteins.
Our model also has strong potential for larger-scale applications, such as pharmaceutical screening. We are currently in discussion with biotech and pharma partners and are working closely with our institution on intellectual property and patent matters.
We are among the few groups worldwide using iPSC technology in malaria research. iPSCs are uniquely suited for this, as they can be differentiated into many tissue types. However, the technical complexity remains a barrier for many researchers in the malaria field. Our study provides a clear roadmap for how to combine iPSC-based models with infection biology, opening new possibilities to study how malaria affects different tissues. Continued integration of iPSC models into infection research will be essential to advance the field.