Cerebral organoids: the passage from 2D to 3D

By Anastasia Alenova

Due to the complexity and cellular diversity of the human brain, disorders of the nervous system (NS) are challenging to study and to treat (Amin et al., 2018). Obtaining brain tissue for experimental studies presents ethical and practical challenges, and the rare biopsies only present a snapshot of a given disorder (StemCell Technologies; Arlotta et al., 2019). However, novel techniques promise to accelerate the investigation of NS disease mechanisms; the genomic revolution led to the identification of specific number and gene variants in various brain disorders and genome editing with CRISPR/Cas9 enabled investigation of gene-function relationships. Now, it is possible to bioengineer stem-cell derived self-organising 3D cell cultures, known as cerebral organoids, which can model complex brain structures (Amin et al., 2018).

Usually, monolayer 2D cultures of differentiated stem cells are used. Patient-derived somatic cells are converted to human induced pluripotent stem (hiPS) cells, which can differentiate into many cellular lineages. These cultures helped identify key mechanistic pathways, such as mitochondrial abnormalities in bipolar disorder, but lack distinguishing features of the human brain, such as signalling dynamics and important regulatory cell types like oligodendrocytes (Amin et al., 2018; StemCell Technologies, n.d.). 

The neurosphere assay was amongst the first 3D culture methods. It involved neural stem cells and progenitor cells being cultured in the absence of an adherent substrate. This led to single cells proliferating into small cell clusters grown in suspension. Neural aggregates are another type of 3D culture, where pluripotent stem cell aggregates representing early-stage embryo development could generate central NS-like progenitor cells. However, both neurospheres and 3D aggregates lack clear organisation. Another technique used is neural rosettes. Although it is a 2D culture system, it is believed to present the neural tube with cells surrounding a central lumen and undergoing mitosis at the luminal side (Lancaster et al., 2014). 

Cerebral organoids are heavily based on the culture method of cortical spheroids and are the most advanced 3D culture system. hiPS and human embryonic stem (ES) cells can be grown and differentiated into 3D cultures with ES cell feeders. The generated embryonic bodies are placed in 96-well U-bottom plates coated with non-adhesive compounds, such as neural induction media, and kept in suspension. They are cultured in conditions promoting self-organisation and self-patterning, and the obtained aggregates are embedded within Matrigel, composed of extracellular matrix proteins, which improves polarisation of neural progenitor sheets and promotes the growth of neuroepithelial buds. These buds then develop into layered brain regions (Arlotta et al., 2019; Lancaster et al., 2013). 

Lastly, the aggregates are transferred to a spinning bioreactor to promote improved oxygen and nutrient exchange. Over time, progenitor zones shrink as more neurons are produced. After 15-20 days, large continuous epithelia surrounded by fluid-filled cavities can already be observed and the resulting tissue can be maintained in vitro for long periods of time. However, organoids grow quickly beyond the limits of stationary diffusion of oxygen and nutrients, leading to a necrotic core (Arlotta et al., 2019; Lancaster et al., 2014). 

Organoids can be obtained via undirected differentiation, with cells grown in extracellular matrix in the absence of inductive cues, or directed differentiation, with exogenous signalling molecules applied in a specific combination or time. This guides the in vitro neurodevelopment specification and the formation of region-specific organoids (Amin et al., 2018; Arlotta et al., 2019). Although hard to master and analyse, 3D cultures allow the construction of complex arrangements of cells and extracellular matrices. Dynamic processes can also be modelled in organoids in a developmentally constrained and reproducible manner (Velasco et al., 2019).

As cerebral organoids mature, they display electrophysiological properties and network connectivity of neurons. Combining brain organoids with non-neuronal cell types, such as microglia, can model neuroimmunological interactions that may be protective in neuronal pathology. Multiple organoids can also be fused together to form assembloids, which model brain region interconnectivity (Amin et al., 2018).

Further work still needs to be conducted to advance functional maturation and assembly of neural circuits within organoids, as they lack sensory input and motor output. The system also lacks surrounding embryonic tissue, meninges and vasculature, amongst other elements (Arlotta et al., 2019). Advances in mouse transplantation may remedy this issue, with the emerging possibility of conducting functional studies and the integration of human neurons within living brain circuits (Amin et al., 2018). 

Cerebral organoids enable advanced study of brain disease, as they can model exposure to chemicals or viruses, with single-cell RNA sequencing allowing to investigate the transcriptional diversity in the human brain cell types and the underlying gene regulatory networks (Amin et al., 2018; Arlotta et al., 2019).

Neuropsychiatric disorders vary between individuals due to multiple genetic and psychosocial factors, and in the absence of diagnostic indicators rely heavily of behavioural and self-reported symptoms. With previous studies conducted on post-mortem tissue or on mice to research the environmental and genetic contributors, hiPS cell derived models are becoming essential for human neurological disease modelling (Amin et al., 2018; StemCell technologies). In cerebral organoids, effects of single gene mutations can be modelled using genetically modified stem cells or deriving hiPS cells from patients. Models of neurodevelopmental disorders, such as Zika virus-associated microcephaly, or neurodegeneration, such as Alzheimer’s Disease with APP gene overexpression leading to ß-amyloid plaque formation, can be developed (Amin et al., 2018). 

Cerebral organoids are a versatile and accessible tool, which can model relevant cellular interactions. However, they present certain limitations. As the organoids are extremely variable, reproducibility needs to be increased by potentially incorporating nanotechnology or bioprinted elements (Velasco et al., 2019). Also, the in vitro model lacks the typical patterning and environmental cues found in the intact embryo, and don’t grow past the equivalent of the prenatal brain, limiting its applicability in disease developing postnatally (StemCell Technologies, n.d.). Cerebral organoids still require refinement but may lead to better understanding of neurological disease and development of novel treatment to decrease disease morbidity and mortality. 

References: 

Amin, N. D. & Paşca, S. P. (2018) Building Models of Brain Disorders with Three-Dimensional Organoids. Neuron (Cambridge, Mass.). 100 (2), 389-405. Available from: https://search.datacite.org/works/10.1016/j.neuron.2018.10.007. Available from: doi: 10.1016/j.neuron.2018.10.007

Neural organoids, StemCell Technologies, viewed 14th November 2020, <https://www.stemcell.com/technical-resources/area-of-interest/organoid-research/neural-organoids/overview.html

Arlotta, P. & Paşca, S. P. (2019) Cell diversity in the human cerebral cortex: from the embryo to brain organoids. Current Opinion in Neurobiology. 56 194-198. Available from: https://search.datacite.org/works/10.1016/j.conb.2019.03.001. Available from: doi: 10.1016/j.conb.2019.03.001

Velasco, S., Kedaigle, A. J., Simmons, S. K., Nash, A., Rocha, M., Quadrato, G., Paulsen, B., Nguyen, L., Adiconis, X., Regev, A., Levin, J. Z. & Arlotta, P. (2019) Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature (London). 570 (7762), 523-527. Available from: https://search.datacite.org/works/10.1038/s41586-019-1289-x. Available from: doi: 10.1038/s41586-019-1289-x

Lancaster, M. A. & Knoblich, J. A. (2014) Generation of cerebral organoids from human pluripotent stem cells. Nature Protocols. 9 (10), 2329-2340. Available from: https://search.datacite.org/works/10.1038/nprot.2014.158. Available from: doi: 10.1038/nprot.2014.158

Lancaster, M. A., Renner, M., Martin, C., Wenzel, D., Bicknell, L. S., Hurles, M. E., Homfray, T., Penninger, J. M., Jackson, A. P. & Knoblich, J. A. (2013) Cerebral organoids model human brain development and microcephaly. Nature (London). 501 (7467), 373-379. Available from: https://search.datacite.org/works/10.1038/nature12517. Available from: doi: 10.1038/nature12517

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