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Disease Study

Cerebral organoids can be used to study the crucial early stages of brain development, test drugs and, because they can be made from living cells, study diseases of individual patients.

Teratogens

Cerebral organoids have been used to study the effects of certain teratogens on fetal neurological development.

Zika Virus

Cerebral organoids have been used in studies in order to understand the process by which Zika virus affects the fetal brain and, in some cases, causes microcephaly.Cerebral organoids infected with the Zika virus have been found to be smaller in size than their uninfected counterparts, which is reflective of fetal microcephaly. Additionally, lumen size was found to be increased in organoids infected with Zika virus.The results found from studying cerebral organoids infected with Zika virus at different stages of maturation suggest that early exposure in developing fetuses can cause greater likelihood of Zika virus-associated neurological birth defects.

Cocaine

Potential enzyme versions responsible for fetal neurological developmental defects caused by cocaine use during pregnancy have also been identified using cerebral organoids. Cerebral organoids were used to determine the enzyme necessary for development of abnormalities associated with cocaine use. This enzyme was determined to be cytochrome P450 isoform CYP3A5 .

Autism

Cerebral organoids can also be used to study autism spectrum disorders. In a recent study, cerebral organoids were cultured from cells derived from macrocephaly ASD patients. These cerebral organoids were found to reflect characteristics typical of the ASD-related macrocephaly phenotype found in the patients. By cultivating cerebral organoids from ASD patients with macrocephaly, connections could be made between certain gene mutations and phenotypic expression. The field of epigenetics and how DNA methylation might influence development of ASD has also been of interest in recent years. The traditional method of studying post-mortem neural samples from individuals with ASD poses many challenges; cerebral organoids have been proposed  as an alternate method of studying the effect of epigenetic mechanisms on the development of autism. This model might provide insight in regards to developmental timelines. However, the conditions in which the organoid is cultured in might affect gene expression. Additionally, there is concern over the variability in cerebral organoids cultured from the same sample. Further research into the extent and accuracy by which cerebral organoids recapitulate epigenetic patterns found in primary samples is also needed.

Other Applications

A list of potential applications for cerebral organoids is highlighted below. Potential applications include:

Tissue morphogenesis

Cerebral organoids can be used to study tissue morphogenesis, which, as it relates to cerebral organoids, illuminates how neural organs form in vertebrates. Cerebral organoids can serve as in vitro tools to study the formation, modulate it, and further understand the mechanisms controlling it.

Migration assays

Cerebral organoids can help to study cell migration. Neural glial cells cover a wide variety of neural cells, some of which move around the neurons. The factors that govern their movements can be studied using cerebral organoids.

Clonal lineage tracing

Clonal lineage tracing is part of fate mapping, where the lineage of differentiated tissues is traced to the pluripotent progenitors. The local stimuli released and mechanism of differentiation can be studied using cerebral organoids as a model. Genetic modifications in cerebral organoids might one day serve as a means to accomplish lineage tracing.

Transplantation

Cerebral organoids can be used to grow specific brain regions and transplant them into regions of neurodegeneration as a therapeutic treatment. In some cases, the genomes of these cerebral organoids would first have to be edited. The "humanization" of animal models has been raised as a topic of concern in transplantation of human SC derived organoids into other animal models.

Cross species developmental timing

Cerebral organoids provide a unique insight into the timing of development of neural tissues and can be used as a tool to study the differences across species.

Drug high-throughput screening

Cerebral organoids can be used as simple models of complex brain tissues to study the effects of drugs and to screen them for initial safety and efficacy. Testing new drugs for neurological diseases could also result from this method of applying drugs high-throughput screening methods to cerebral organoids.

Cell replacement therapy

Cerebral organoids can be used as a simple model to show how cell replacement therapy would work on brain tissues.

Implantation

In addition to being used as tools to study disease pathology and treatments, future application of cerebral organoids include direct implantation into a human host. The organoid can fuse with host tissue in areas of neurodegeneration, being incorporated with the host vasculature and be immunologically silent.

Stroke Therapy

In the future, cerebral organoids with vascularity might provide insight into stroke therapy.

Cell-type specific genome assays

Culturing Methods

The general procedure used to culture a cerebral organoid can be broken down into 5 steps. First, human pluripotent stem cells are cultured. They are then allowed to cultivate into an embryoid

Figure: This flow chart outlines the basic steps to create a cerebral organoid. The process takes a span of months and the size of the organoid is limited to the availability of nutrients.

body. Next, the cell culture is induced to form a neuroectoderm. The neuroectoderm is then grown in a matrigel droplet. The matrigel provides nutrients and the neuroectoderm starts to proliferate and grow. However, the lack of vasculature limits the size the organoid can grow. This has been the major limitation in organoid development, however new methods using a spinning bioreactor

have allowed an increase in the availability of nutrients to cells inside the organoid. This last step has been the key breakthrough in organoid development. Spinning bioreactors have been used increasingly in cell culture and tissue growth applications. The reactor is able to deliver faster cell doubling times, increased cell expansion and increased extra-cellular matrix components when compared to statically cultured cells. Some developmental markers include the fact that, after ten days the organoid developed neurons, and after 30 days, it displayed regions similar to parts of brains. Lacking a blood supply, cerebral organoids reach about 4 mm across and can last a year or more. Additionally, while these cells are self organizing, replication of specific brain regions in cerebral organoid counterparts is achieved by the addition of extracellular signals to the organoid environment during different stages of development; these signals were found to create change in cell differentiation patterns, thus leading to recapitulation of the desired brain region. While SMAD inhibition is used in usual  cerebral organoid culturing processes,  inhibition of this has been shown to generate microglia in cerebral organoids. This adjustment to culturing methods would allow for research into diseases such as ASD.

This was the original method outlined by Madeline Lancaster and has since been developed and refined. Newer methods allow development of cerebrovascular organoids, and micro pumps to provide circulation through them are being developed, as explained in this video by Dr George M. Church.

Assays

Cerebral organoids have the potential to function as a model with which disease and gene expression might be studied. However, diagnostic tools are needed to evaluate cerebral organoid tissue and create organoids modeling the disease or state of development in question.

Genetic Modifications

Cerebral organoids can be used to study gene expression via genetic modifications. The degree to which these genetic modifications are present in the entire organoid depends on what stage of development the cerebral organoid is in when these genetic modifications are made; the earlier these modifications are made, such as when the cerebral organoid is in the single cell stage, the more likely these modifications will affect a greater portion of the cells in the cerebral organoid.

The degree to which these genetic modifications are present within the cerebral organoid also depend on the process by which these genetic modifications are made. If the genetic information is administered into one cerebral organoid cell's genome via machinery, then the genetic modification will remain present in cells resulting from replication. Crispr/Cas 9 is a method by which this long-lasting genetic modification can be made. A system involving use of transposons has also been suggested as a means to generate long-lasting genetic modifications; however, the extent to which transposons might interact with a cell genome might differs on a cell to cell basis, which would create variable expressivity between cells.

If, however, the genetic modification is made via “genetic cargo” insertion (such as through Adeno-associated virus/ electroporation methods) then it has been found that the genetic modification becomes less present with each round of cell division.

Computational Methods

Use of computational methods have been called for as a means to help improve the cerebral organoid cultivation process; development of computational methods has also been called for in order to provide necessary detailed renderings of different components of the cerebral organoid (such as cell connectivity) that current methods are unable to provide. Programming designed to model detailed cerebral organoid morphology does not yet exist.

Transcriptome analysis

Transcriptome analysis has also been suggested as a potential assay to examine the pathology of cerebral organoids derived from individual patients.

Apoptotic Markers

TUNEL assays have been used in studies as an evaluative marker of apoptosis in cerebral organoids.

Limitations

Necrotic Centers

Until recently, the central part of organoids have also been found to be necrotic due to oxygen as well as nutrient being unable to reach that innermost area.This imposes limitations to cerebral organoids' physiological applicability. However, recent findings suggest that, in the process of culturing a cerebral organoid, a necrotic center could be avoided later on by using fluidic devices to increase the organoid's exposure to media.

Reliability in Generation

The structure of cerebral organoids across different cultures has been found to be variable; a standardization procedure to ensure uniformity has yet to become common practice. Future steps in revising cerebral organoid production would include creating methods to ensure standardization of cerebral organoid generation. One such step proposed involves regulating the composition and thickness of the gel in which cerebral organoids are cultured in; this might contribute to greater reliability in cerebral organoid production. Additionally, variability in generation of cerebral organoids is introduced due to differences in stem cells used. These differences can arise from different manufacturing methods or host differences. Increased metabolic stress has also been found within organoids. This metabolic stress has been found to restrict organoid specificity.

Maturity

At the moment, the development of mature synapses in cerebral organoids is limited because of the media used. Additionally, while some electrophysiological properties have been shown to develop in cerebral organoids, cultivation of separate and  distinct organoid regions has been shown to limit the maturation of these electrophysiological properties. Modeling of electrophysiological  neurodevelopmental processes typical of development later in the neurodeveopmental timeline, such as synaptogenesis, is not yet suggested in cerebral organoid models.