Neuronal Growth and Health – Assay Development

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Being able to investigate neuronal differentiation and development is key to developing in vitro models of neurological disease and degeneration. During this article we will showcase Charnwood Discovery’s capabilities in the CNS therapeutic area.  

Challenges

Neurological conditions contribute significantly to global disease burden, with prevalence set to rise alongside our ageing populations. These diseases account for 9 million deaths a year globally plus the additional financial burden of care for those with these diseases. For drug discovery purposes within this therapeutic area, it is vital to have access to physiologically relevant models amenable to bioassay development. This allows for the investigation of potential leads and for the discarding of inappropriate compounds in disease-appropriate models. However, this approach can be challenging as neuronal cells are non-proliferative and very difficult to handle.

Neurons can be obtained as primary cells or through the differentiation of inducible Pluripotent Stem Cells (iPSC’s) into neurons across multiple weeks. iPSC differentiation is a useful option when developing assays for compound screening, as it can provide a stock of proliferative progenitor cells from which to continually produce differentiated neuronal cells. 

What We Did...

Differentiation of iPSC Derived Neural Stem Cells into Neurons

iPSC-derived neural stem cells were differentiated into neurons across 28 days in culture. After seeding at day 0, cells were cultured in differentiation medium for 14 days, before the addition of Ara-C (a compound toxic to dividing cells), to remove any remaining undifferentiated cells (Ara-C removed on day 19). Neurons were then further matured in differentiation medium until at least day 28 prior to using for drug discovery work.

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Figure 1. iPSC-derived neural stem cells differentiated over 28 days, stained with the neuronal-specific marker β-iii-Tubulin (green) and Hoechst (blue).

Characterization of neurons was achieved through flow cytometry and high content imaging, staining for mature neuronal markers (such as Microtubule associated protein 2 – MAP2) and neuronal lineage specific markers (such as the vesicular glutamate transporter vGLUT1 for glutamatergic neurons, or transcription factor FOXA2 for dopaminergic neurons).

Figure 2. 28-day differentiated neurons. Left image labelled with MAP2 and vGLUT1; Right image with β-iii-Tubulin and FOXA2.

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Figure 3. Lineage map with individual expression markers of different neuronal cell types. (lineage map taken from Abcam website)

Investigating Neuronal Disorder /Disease using High Content Imaging and Immunoblotting

Pathological deposits of hyperphosphorylated Tau are a hallmark of neurodegenerative tauopathies, including Alzheimer’s disease. Here we use high content imaging to track phosphorylation levels of Tau in neurons treated with different concentrations of a kinase inhibitor. This powerful technique can provide an insight into how compounds interact with targets at the subcellular level within a cell-based, disease-relevant assay. 

Figure 4. Treatment of iPSC derived neuronal cells with a kinase inhibitor changes the phosphorylation landscape of the cells. Representative images of differentiated neurons stained with P-Tau (green), Tau (red) and Hoechst (blue), dosed with a kinase inhibitor. 

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Figure 5. Kinase inhibition resulted in a dose-dependent decrease in Phospho-Tau expression from the iPSC derived neurons.

As an orthogonal assay for tracking phospho-protein levels, we perform immunoblotting using the capillary-based Bio-techne JESS system. Targets can be multiplexed or probed by RePlex™ (the equivalent of stripping and re-probing), allowing comparison of Phosphoprotein to total protein levels.

Here we show the reduction in levels of a phosphorylated protein across a compound dose response with probe 1, normalized to the total levels of the protein with probe 2 after stripping of probe 1.

Figure 6. Pseudo-blot of neuronal lysates probed for phospho- and total levels of a target protein. Graph shows phospho-signal over background normalized to total protein.

Tracking Differentiation by Live Cell Imaging

At Charnwood Discovery, we can monitor the differentiation process in real time using the IncuCyte SX5 live cell imaging system, tracking metrics such as neurite length, branch points and cell body area.

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Figure 7. Images and associated graphs of neurons captured on the IncuCyte SX5, displayed with and without overlayed analysis mask – neurites in pink and cell bodies in yellow. 

Differentiation of Neurons in 3D Culture

Another exciting area in the drive for physiological relevance is the ability to grow cells as 3-dimensional systems (3D). 

At Charnwood Discovery, we can do this either in ultra-low attachment plates or in the CelVivo ClinoStar rotating incubator / bioreactor. In the CelVivo, cells can be grown as spheroids or attached to electrospun magnetic scaffold material for easy manipulation.

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Figure 8. Z-slices of iPSC-derived neural stem cells, differentiated for 2-weeks on a 400 µM scaffold. Cells can be seen growing around the darker strands of the scaffolds. Samples imaged on the ImageXpress® Confocal HT.ai High-Content Imaging System using a 20X water immersion objective and a 50 µm split disk. A. Cells stained with with β-iii-tubulin (green), vGlut1 (red) and Hoechst (blue). B. Cells stained with with β-iii-tubulin (green), vGlut1 (red) and Hoechst (blue). C. Cells stained with with MAP2 (green), vGlut1 (red) and Hoechst (blue).  

Summary

At Charnwood Discovery we have successfully developed a suite of assays to screen compounds against neuronal cells differentiated in-house from iPSC-derived neural stem cells. 

Here we show only mono-culture work, however we have the capability to culture two or three cell types (astrocytes, microglial cells) together with neurons to recapitulate the complex 3-dimensional architecture of the brain in a more physiological environment.

By using high content imaging, flow cytometry, immunoblotting and kinetic imaging, we are able to study the functions of both normal and disease cells and the effects of compounds on these cell types.