Using cerebral organoids as neuroinflammation models

This article is based on a poster originally authored by Zhen Qi, Zhanguang Zuo, Shuge Guan, Juan Quintana, Idil Arioz and Rosanna Zhang.

High clinical failure rates are common in drug development targeting neurological disorders, including Parkinson’s disease (PD) and Alzheimer’s disease (AD), even after rigorous preclinical testing. This is primarily due to efficacy issues and side effects and is often attributed to a lack of relevant and translatable cell models.

Organoids represent a highly promising advance in the field of drug development and disease modeling, with the potential to help address high clinical drug failure rates.

This article introduces a novel, hydrogel-free, assay-ready, human iPSC-derived cerebral organoid system from ACROBiosystems designed to exhibit the functional characteristics of the brain. This commercially available system includes several cell types, all verified by marker immunolabeling and RNA-seq.

The company’s cerebral organoids function as essential platforms in AD modeling with the small molecule Aftin-4. They are also suitable for modeling PD using alpha-synuclein (α-syn) pre-formed fibrils (PFFs). Both of these approaches have been assessed by evaluating immune response to quantify neuroinflammation. 

Methods and materials

Organoids were generated using a Cerebral Organoid kit from ACROBiosystems (RIPO-HWM002K) according to the data sheet’s protocol. The results section lists the organoids used for assays, in line with their indicated time points.

Cell marker confirmation was achieved by fixing, permeabilizing, and immunolabeling whole organoids with the specified cell markers on the specified days. Aftin-4 and alpha-synuclein pre-formed fibril treatments were each performed on culture day 86 and day 92, respectively, adding these directly to the organoid culture medium at the specified concentrations and durations.

Results

Characterization of cerebral organoids: Cell marker verification

Production of human iPSC-derived cerebral organoids was achieved using ACROBiosystems’ Cerebral Organoid Differentiation Kit (Figure 1A). These organoids were characterized at key stages via immunolabeling (Figure 1B-E):

• Neural progenitor and immature neuronal cells (day 36)
• Dopaminergic neurons and mature neurons (day 92)
• Astrocytes (day 109)
• Oligodendrocytes (day 119)
• Microglia (day 147)

Figure 1. (A) Cerebral organoid differentiation steps. Immunofluorescence confirmation of (B) NESTIN and TUJ1, (C) MAP2 and TH, (D) GFAP, (E) OLIG2, and (F) IBA1 positive cells at different stages of organoid maturation. Image Credit: ACROBiosystems

Characterization of cerebral organoids: Transcriptomics and electrophysiological characterization

Using bulk RNA-seq (Figure 2A), several cerebral organoids’ neuronal subtypes and glial cell types can be verified. It is also important to note that cerebral organoids demonstrated extracellular electrical activity, which can be verified by MEA (Figure 2B and Figure 2C) and silicon probe readings (Figure 2D).

In the example presented here, silicon probe recording highlighted several neuronal groups participating in early network activity (Figure 2E). A patch clamp was also used to verify intracellular activity.

Figure 2. (A) Transcriptome profiling of cerebral organoids. Extracellular electrical activity was measured by MEA and silicon probe recordings. (B) Microscopic image of MEA on an 82-day old cerebral organoid along with its (C) MEA recording. (E) Intracellular electrical activity was measured by patch clamp recordings. Image Credit: ACROBiosystems

Modeling Alzheimer’s disease with cerebral organoids

Aftin-4 (an Aβ-42 peptide inducer) was used to chemically induce an AD model using cerebral organoids. IL-6 and CCL2 mRNA levels were observed to significantly increase on induction for 2 to 5 days. This can be reduced using LY450139 (Eli Lilly Semagacestat) (Figure 3).

Figure 3. (A) Healthy hiPSC-derived cerebral organoids treated with Aftin-4. (B) Morphological rescuing of the organoid morphology with LY450139/Semagacestat. (C) Aftin-4 induced Aβ-42 production, without changing total A β-peptide level. Aftin-4 increased IL-6 and CCL2 mRNA levels significantly, which was rescued by LY450139. There was no significant changes seen in Aβ Precursor Protein (APP) and BACE1 levels upon Aftin-4 induction. Image Credit: ACROBiosystems

Modeling Parkinson’s disease with cerebral organoids

In the example presented here, a non-chemical, non-genetic PD model was created through the addition of α-syn PFFs. This was done for 12 days on healthy 92-day-old cerebral organoids.

It was noted that α-syn PFFs prompted the degeneration of dopaminergic neurons, as well as disrupting the dendrites of other neurons in a concentration-dependent manner (Figure 4).

Figure 4. (A) Healthy hiPSC-derived cerebral organoids treated with α-Syn PFFs. (B) PFFs, formed in vitro, have seeding activity and can induce neurodegenerative pathologies by recruiting soluble pathological proteins. (C) α-Syn PFFs damage dopaminergic neuron network (TH+ cells) in addition to overall causing neuronal damage. (D) α-Syn increased IL-6 and CCL2 mRNA levels. No significant changes were seen in other cytokine levels. Image Credit: ACROBiosystems

Conclusion

The examples presented here highlighted iPSC-derived cerebral organoids’ capacity to show marker expression of astrocytes, oligodendrocytes, neuronal subtypes, microglia, and endothelial cells organized in a 3D manner. This approach enables a more accurate representation of human brain characteristics, including functional electrophysiological activity.

Preclinical models of both AD and PD were developed, with these models recapitulating the disease-associated immunological response marked by IL-6 and CCL2 increase.

The AD model used a drug undergoing clinical trials to alleviate immune responses triggered by Aftin-4, while the organoid-based PD model highlighted damage to the dopaminergic network alongside the immunological response. This was found to be reflective of PD-associated neurodegeneration.

Cerebral organoids represent useful preclinical models due to their ability to replicate disease-associated immune responses and phenotypes. These innovative models have the potential to offer valuable insights when used in therapy development.

References and further reading

1. Kim, J., Koo, B.-K. and Knoblich, J.A. (2020). Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology, (online) 21(10), pp.571–584. https://doi.org/10.1038/s41580-020-0259-3.
2. Zhao, Z. (2022). Organoids. Nature Reviews Methods Primers, (online) 2(1), pp.1–21. https://doi.org/10.1038/s43586-022-00174-y.

Acknowledgments

Produced from materials originally authored by Zhen Qi, Zhanguang Zuo, Shuge Guan, Juan Quintana, Idil Arioz, and Rosanna Zhang from ACROBiosystems.

About ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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