iPSC-derived organoids for organ-specific disease modeling

This article is based on a poster originally authored by Peter Hsueh, Zhen Qi, Spencer Chiang, An Ouyang, Zhan Guang Zuo, Shu Ge Guan, Lisa Chou, and Rosanna Zhang.

Organoids represent a promising drug development and disease modeling advancement to address traditionally high clinical drug failure rates. Despite rigorous preclinical testing, current organoid technologies face a series of limitations and often lack certain physiological cell types, standardized protocols, and translatable biological assays.¹

Regardless of these issues, organoids remain a gold standard model for preclinical drug development due to their translatable human genomic background, their potential for high throughput, and the capacity to mimic the three-dimensional architecture and function of their respective organs.²

Organoids as pre-clinical models have the potential to significantly improve drug development pipeline efficiency. This also reduces reliance on animal models, which are typically unable to accurately predict human responses. 

ACROBiosystems has developed novel hydrogel-free, human iPSC-derived, assay-ready cardiac and cerebral organoid systems, which are now commercially available.

Cerebral and cardiac organoids each contain several different cell types that can be verified using marker immunolabeling and RNA-seq. These cell types exhibit the functional characteristics of their respective modeled organs, which can be verified via MEA, patch clamp, and silicon probe recording.

The addition of alpha-synuclein pre-formed fibrils to the cerebral organoids created a Parkinson's disease model. The cardiac organoid was also utilized as a model to test a specific drug’s cardiac toxicity.

Both organoids were promising models for AAV screening for gene therapy developments.

ACROBiosystems’ advances in this area will help improve guidance for disease and toxicity modeling and enhance the potential of gene therapy development transitioning into clinical research.

Methods and materials

iPSC and organoid generation

Organoids were created from commercially acquired human iPSCs (Cat. No.: ACS-1007, ATCC). This was done using either a cerebral or cardiac organoid kit from ACROBiosystems (Cat. No. RIPO-BWM001K, RIPO-HWM002K, respectively), in line with the specific protocol outlined in the relevant data sheet.

Harvested iPSCs were initially seeded onto a 96-well ultra-low attachment plate. EB formation was carefully monitored, and cells were treated with the kit’s included media to trigger organoid formation and cell differentiation.

Assays were performed at the indicated time points using organoids. These organoids were fixed, permeabilized, and immunolabeled with the indicated cell markers for cell marker confirmation.

In the Parkinson’s disease model, at day 92, cerebral organoids were treated with 0.1 µM and 1 µM alpha-synuclein pre-formed fibrils (Cat. No. ALN-H5115) for a total of 12 days.

The cardiac toxicity model involved cardiac organoids being treated with different concentrations of ‘drug A’ (a confidential drug) on day 25.

AAV transduction saw cerebral organoids transduced at 101 days and cardiac organoids transduced at 11 days, each with 1011 vg of eGFP transgene bearing AAV5 WT as negative control. Alternatively, these were optimized AAV serotypes, including IVB-1 and IVB-2.

The entire range of organoids was incubated at 37 °C 5% CO₂, shaking these at 100 rpm until they were ready to be analyzed.

Results

Characterization of cerebral organoids: Cell marker verification

A Cerebral Organoid Differentiation kit was used to create iPSC-derived cerebral organoids (Figure 1A). These were then characterized at day 36 using immunolabeling for neural progenitor and immature neuronal cells.

Further characterization was performed at later stages of culturing for dopaminergic neurons and mature neurons (day 92), astrocytes (day 109), oligodendrocytes (day 119), and microglia (day 147) (Figure 1B-E).

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

Characterization of cerebral organoids: Transcriptomic and functional characterization

Bulk RNA-seq can be used to verify cerebral organoids’ neuronal subtypes and glial cell types (Figure 2A). MEA analysis (Figure 2B) and silicon probe recording (Figure 2C) can be used to verify cerebral organoids’ extracellular electrical activity.

Various neuronal groups were also revealed in early network activity using silicon probe recording (Figure 2D), while intracellular electrical activity was verified by patch clamp.

Figure 2. (A) Transcriptome profiling of cerebral organoids. Extracellular electrical activity by (B) MEA and (C) silicon probe recording. (D) Intracellular electrical activity by patch clamp. Image Credit: ACROBiosystems

Parkinson’s disease and AAV capsid screening modeling on cerebral organoids

It was observed that treating healthy 92-day-old cerebral organoids with alpha-synuclein pre-formed fibrils for a total of 12 days (Figure 3A, left) prompted dopaminergic neuron degeneration and disruption of other neurons’ dendrites in a concentration-dependent fashion (Figure 3A, right).

Cerebral organoids were grown for 101 days before being infected with AAV5-WT, IVB-1, and IVB-2. In this instance, the IVB-2 subtype showed comparatively higher infectiousness and transgene delivery toward cerebral organoids.

Figure 3. Cerebral organoids as models for (A) Parkinson’s disease and (B) AAV capsid screening. Image Credit: ACROBiosystems

Characterization of cardiac organoids – Cell marker verification

Human iPSC-derived cardiac organoids were generated using Cardiac Organoid Differentiation kits (Figure 4A). On day 12, they were characterized using immunolabeling to determine the presence of cardiomyocytes (Figure 4B) and mechanical beating (Figure 4C, beating not displayed).

Mechanical beating was visible following nine days of culturing in cardiac organoids. Calcium sensor imaging was also used to confirm the presence of calcium flux behavior on human heart organoids (Figure 4D).

From a morphological perspective, these cardiac organoids presented ventricular and atrial-like chambers in various regions, which were validated by the presence of ventricular and atrial cardiomyocyte markers (Figure 4E).

The presence of endothelial cells was also confirmed on the cardiac organoids (Figure 4F)

Figure 4. (A) Cardiac organoid differentiation steps. Immunofluorescence confirmation of (B) cTNT and (C) visualization of cardiac organoids on tissue culture plates. (D) Sequential images of calcium sensor imaging of cardiac organoids. (E) Presence of morphological and marker validated atrial (MLC2 A+ cells) and ventricular (MLC2V+ cells) chambers. (F) Vascularization was confirmed by CD31 positive endothelial cell immunolabeling. Image Credit: ACROBiosystems

Characterization of cardiac organoids: Functional characterization

The heart tissue was found to be electrically active, with MEA recordings used to probe this electrical activity. This investigation confirmed the consistent beating periods and amplitudes of the cardiac organoid model under investigation, and this was compared to three other cardiac models over periods of up to 6 hours (Figure 5A).

Silicon probe recordings can also measure extracellular electrical activity, a useful means of confirming physiological wave complex elements for heart activity (Figure 5B). A patch clamp was also used to verify intracellular electrical activity, enabling identification of a physiological cardiac waveform (Figure 5C).

Figure 5. (A) Electrical activity of the cardiac organoids were confirmed extracellularly by (A) MEA and (B) silicon probe recordings and intracellularly by (C) patch clamp recordings. The resemblance of a physiological waveform of cardiac beating was identified. Image Credit: ACROBiosystems

Cardiac toxicity and AAV capsid screening modeling on cardiac organoids

It was observed that treating healthy 25-day-old cardiac organoids with a suitable concentration of the confidential ‘drug A’ resulted in degeneration of both the cardiomyocytes and endothelial cells (Figure 6A).

Cardiac organoids grown for 11 days were infected with AAV5-WT, IVB-1, and IVB-2. The IVB-2 subtype was noted to demonstrate higher infectiousness and transgene delivery toward cardiac organoids, a phenomenon that was visualized using fluorescent intensity from GFP transgene expression.

Figure 6. (A) Cardiac toxicity model by Drug A treatment on cardiac organoids. (B) Cardiac organoids as models for AAV capsid screening. Image Credit: ACROBiosystems

Conclusion

High clinical failure rates are commonplace in pharmaceutical drug development, primarily due to efficacy issues and side effects. This issue is prevalent, even after rigorous preclinical testing, and can be attributed to the lack of translatable, relevant cell models.

Organoids are three-dimensional in vitro tissue models that can mimic their respective organs’ architecture and function. These models represent a promising advance in drug development and disease modeling due to their potential to help address the aforementioned issue of high clinical drug failure rates.

Current organoid technologies typically lack specific physiological cell types and translatable biological assays. Cerebral organoids bear several neuronal subtypes and all glial cell types and demonstrate functional activity.

Cardiac organoids bear all relevant cell types and exhibit mechanical beating, while also displaying functional activity.

The example presented here showed a Parkinson’s disease model created by adding alpha-synuclein pre-formed fibrils to the cerebral organoids, which then recapitulated the dopaminergic neuronal degeneration phenotype.

Cardiac organoids were observed to degenerate following the addition of a cardiotoxic drug. It was also possible to observe the cardiomyocyte and endothelial degeneration phenotype.

Both organoids were determined to be promising models for AAV screening for gene therapy developments, highlighting cerebral and cardiac organoids' excellent potential as preclinical models for drug toxicity, disease modeling, drug screening, and gene therapy.

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 Peter Hsueh, Zhen Qi, Spencer Chiang, An Ouyang, Zhan Guang Zuo, Shu Ge Guan, Lisa Chou, 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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