This article is based on a poster originally authored by Malika Bsibsi, Catalina Gomez Puerto, Lieke Geerts, Matteo Zanella, Stefan Kostense, and Marijn Vlaming, presented at SfN 2024.
The chronic neurodegenerative disease Alzheimer’s disease (AD) generally begins at around 60 years old, progressively impairing language and cognition.
A central symptom of AD is accumulating plaques containing β-amyloid causing synaptic failure, leading to neuronal death.
Thanks to the advent of induced pluripotent stem cells (iPSCs), the reproduction and study of the mechanisms underpinning AD’s pathology and β-amyloid plaque-dependent degeneration have been made possible in recent years.
The study presented here aims to demonstrate a robust AD in vitro model for drug development, by treating iPSC-derived glutamatergic neurons with commercially available β-amyloid aggregates.
Methods
ioGlutamatergic Neurons from bit.bio were derived from human iPSCs. Opti-ox technology was used for iPSC precision reprogramming.
Pre-differentiated neurons were thawed and cultured for nine days to facilitate full maturation. Cells were then exposed to 5, 10, 20, or 40 μM β-amyloid aggregates for 72 hours before assessing cell toxicity.
This was evaluated via two approaches:
Immunohistochemistry
Cells were fixed on day 12 before immunocytochemistry for DAPI and βIII-tubulin. The Yokogawa CV8000 was used for high-content imaging, while PE Columbus 2.9.1 was employed to generate an algorithm for quantification, focusing on neuritic structures, nuclei size, and signal intensity (DAPI).
Meso scale discovery
This assay was completed following the supplier’s manual and used to measure neurofilament light chain (NfL) release in the supernatant, as a translational marker of neurodegeneration.
Figure 1. Schematic representation of experimental setup of assays performed to evaluate the impact of the exposure of ioGlutamatergic neurons to β-amyloid aggregates. (A) Workflow and experimental timelines (B) High content imaging and analysis was performed using the Yokogawa CV8000 and an algorithm that was developed in-house using PE Columbus 2.9.1 for quantification. Image Credit: Charles River Laboratories
Results
Toxicity assessment by high content analysis
Figure 2. Exposure of ioGlutamatergic neurons to β-amyloid aggregates impaired neurite structure and the number of healthy nuclei. (A) Representative images of ioGlutamatergic neurons exposed for 72 hours to different concentrations (0-40 μM) of β-amyloid aggregates and stained for βIII-tubulin (red) and DAPI (blue). (B) Examples of nuclei segmentation in ioGlutamatergic neurons not treated or treated with β-amyloid aggregates. Green dots: healthy nuclei; red dots: unhealthy, condensed nuclei. High content imaging was performed using the Yokogawa CV8000. Image Credit: Charles River Laboratories
Figure 3. Exposure of ioGlutamatergic neurons to β-amyloid increased nuclei intensity, decreased nuclear area, and reduced the number of healthy nuclei. (A) Quantification of healthy versus condensed (unhealthy) nuclei, (B) DAPI intensity and nuclei area (μm2), and (C) neurite area (μm2) in untreated or 40 μM β-amyloid-treated ioGlutamatergic neurons. Bars represent the mean values of six wells (n=6) with error bars indicating standard deviation. Image Credit: Charles River Laboratories
Toxicity assessment by MSD analysis of neurofilament light chain (Nf-L)
Figure 4. Exposure of ioGlutamatergic neurons to β-amyloid aggregates induced Nf-L release. Quantification of total Nf-L release by MSD in untreated or 40 μM β-amyloid-treated ioGlutamatergic neurons. Light blue bars depict Nf-L release as measured by MSD analysis in supernatants. Dark blue bars depict Nf-L release normalized to cell number (nuclei count quantified by high content analysis). Bars represent the mean values of six wells (n=6) with error bars indicating standard deviation. Image Credit: Charles River Laboratories
Conclusion
Charles River Laboratories have successfully developed a robust AD in vitro model that treated iPSC-derived glutamatergic neurons with commercially available β-amyloid aggregates.
This approach led to a quantifiable reduction of neuronal viability in line with patient pathology.
Neurons exposed to β-amyloid for 72 hours show toxicity compared to the vehicle control and untreated cells. This was confirmed by reduced DAPI-positive healthy nuclei and the destruction of neurite structures (stained for β-III tubulin).
These effects occurred in a β-amyloid concentration-dependent fashion, confirmed by quantifying 40 μM β-amyloid-treated samples via high-content analysis.
Neurodegeneration was also confirmed by a higher release of NfL in 40 μM β-amyloid aggregate-treated neurons.
These preliminary results support the model’s validity and robustness, demonstrating its potential for future disease-relevant applications, including compound screening for effective AD treatments.
Acknowledgments
Produced from materials originally authored by Malika Bsibsi, Catalina Gomez Puerto, Lieke Geerts, Matteo Zanella, Stefan Kostense, and Marijn Vlaming from Charles River, Leiden, Netherlands.
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