Neural organoid
A neural, or brain organoid, describes an artificially grown, in vitro, tissue resembling parts of the human brain. Neural organoids are created by culturing pluripotent stem cells into a three-dimensional culture that can be maintained for years. The brain is an extremely complex system of heterogeneous tissues and consists of a diverse array of neurons and glial cells. This complexity has made studying the brain and understanding how it works a difficult task in neuroscience, especially when it comes to neurodevelopmental and neurodegenerative diseases. The purpose of creating an in vitro neurological model is to study these diseases in a more defined setting. This 3D model is free of many potential in vivo limitations. The varying physiology between human and other mammalian models limits the scope of animal studies in neurological disorders. Neural organoids contain several types of nerve cells and have anatomical features that recapitulate regions of the nervous system. Some neural organoids are most similar to neurons of the cortex. In some cases, the retina, spinal cord, thalamus and hippocampus. Other neural organoids are unguided and contain a diversity of neural and non-neural cells. Stem cells have the potential to grow into many different types of tissues, and their fate is dependent on many factors. Below is an image showing some of the chemical factors that can lead stem cells to differentiate into various neural tissues; a more in-depth table of generating specific organoid identity has been published. Similar techniques are used on stem cells used to grow cerebral organoids.
Model development
Using human pluripotent stem cells to create in vitro neural organoids allows researchers to analyze current developmental mechanisms for human neural tissue as well as study the roots of human neurological diseases. Neural organoids are an investigative tool used to understand how disease pathology works. These organoids can be used in experiments that current in vitro methods are too simplistic for, while also being more applicable to humans than rodent or other mammalian models might be. Historically, major breakthroughs in how the brain works have resulted from studying injury or disorder in human brain function. An in vitro human brain model permits the next wave in our understanding of the human nervous system.Culturing methods
An embryoid body cultivated from pluripotent stem cells is used to make an organoid. Embryoid bodies are composed of three layers: endoderm, mesoderm and ectoderm, which has the potential to be differentiated into different types of tissue.A cerebral organoid can be formed by inducing ectoderm cells to differentiate into cerebral organoids. The general procedure can be broken down into 5 steps. First human pluripotent stem cells are cultured. They are then cultivated into an embryoid 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. 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. SMAD inhibition may be used in usual cerebral organoid culturing processes to generate microglia in cerebral organoids. The lack of vasculature limits the size the organoid can grow. This has been the major limitation in organoid development. The use of a spinning bioreactor may improve the availability of nutrients to cells inside the organoid to improve 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.
Components
Differentiation
It has been shown that cerebral organoids grown using the spinning bioreactor 3D culture method differentiate into various neural tissue types, such as the optic cup, hippocampus, ventral parts of the teleencephelon and dorsal cortex. Furthermore, it was shown that human brain organoids could intrinsically develop integrated light-sensitive optic cups.The neural stem/progenitor cells are unique because they are able to self-renew and are multipotent. This means they can generate neurons and glial cells which are the two main components of neural systems. The fate of these cells is controlled by several factors that affect the differentiation process. The spatial location and temporal attributes of neural progenitor cells can influence if the cells form neurons or glial cells. Further differentiation is then controlled by extracellular conditions and cell signaling. The exact conditions and stimuli necessary to differentiate neural progenitor cells into specific neural tissues such as hippocampal tissue, optic nerve, cerebral cortex, etc. are unknown. It is believed that cerebral organoids can be used to study the developmental mechanisms of these processes.
Gene expression
To test if the neural progenitor cells and stem cells are differentiating into specific neural tissues, several gene markers can be tested. Two markers that are present during pluripotent stages are OCT4 and NANOG. These two markers are diminished during the course of development for the organoid. Neural identity markers that note successful neural induction, SOX1 and PAX6, are upregulated during organoid development. These changes in expression support the case for self-guided differentiation of cerebral organoids. Markers for forebrain and hindbrain can also be tested. Forebrain markers FOXG1 and SIX3 are highly expressed throughout organoid development. However, hindbrain markers EGR2 and ISL1 show early presence but a decrease in the later stages. This imbalance towards forebrain development is similar to the developmental expansion of forebrain tissue in human brain development. To test if organoids develop even further into regional specification, gene markers for cerebral cortex and occipital lobe have been tested. Many regions that have forebrain marker FOXG1, labeling them as regions with cerebral cortical morphology, were also positive for marker EMX1 which indicates dorsal cortical identity. These specific regions can be even further specified by markers AUTS2, TSHZ2, and LMO4 with the first representing cerebral cortex and the two after representing the occipital lobe. Genetic markers for the hippocampus, ventral forebrain, and choroid plexus are also present in cerebral organoids, however, the overall structures of these regions have not yet been formed.Organization
Cerebral organoids also possess functional cerebral cortical neurons. These neurons must form on the radially organized cortical plate. The marker TBR1 is present in the preplate, the precursor to the cortical plate, and is present, along with MAP2, a neuronal marker, in 30-day-old cerebral organoids. These markers are indicative of a basal neural layer similar to a preplate. These cells are also apically adjacent to a neutral zone and are reelin+ positive, which indicates the presence of Cajal-Retzius cells. The Cajal-Retzius cells are important to the generation of cortical plate architecture. The cortical plate is usually generated inside-out such that later-born neurons migrate to the top superficial layers. This organization is also present in cerebral organoids based on genetic marker testing. Neurons that are early born have marker CTIP2 and are located adjacent to the TBR1 exhibiting preplate cells. Late-born neurons with markers SATB2 and BRN2 are located in a superficial layer, further away from the preplate than the early born neurons suggesting cortical plate layer formation. Additionally, after 75 days of formation, cerebral organoids show a rudimentary marginal zone, a cell-poor region. The formation of layered cortical plate is very basic in cerebral organoids and suggests the organoid lacks the cues and factors to induce formation of layer II-VI organization. The cerebral organoid neurons can, however, form axons as shown by GFP staining. GFP labeled axons have been shown to have complex branching and growth cone formation. Additionally, calcium dye imaging has shown cerebral organoids to have Ca2+ oscillations and spontaneous calcium surges in individual cells. The calcium signaling can be enhanced through glutamate and inhibited through tetrodotoxin.Interactions with environment
In DishBrain, grown human brain cells were integrated into digital systems to play a simulated Pong via electrophysiological stimulation and recording. The cells "showed significantly improved performance in Pong" when embodied in a virtual game-world. In the 2020s, significant changes in how these electrophysiological systems are made and interact with brain organoids could lead to better stimulation and recording data across the organoind in 3D.;Interactions with surrounding tissues
It is not fully understood how individual localized tissues formed by stem cells are able to coordinate with surrounding tissues to develop into a whole organ. It has been shown however that most tissue differentiation requires interactions with surrounding tissues and depends on diffusible induction factors to either inhibit or encourage various differentiation and physical localization. Cerebral organoid differentiation is somewhat localized. The previously mentioned markers for forebrain and hindbrain are physically localized, appearing in clusters. This suggests that local stimuli are released once one or more cells differentiate into a specific type as opposed to a random pathway throughout the tissue. The markers for subspecification of cortical lobes, prefrontal cortex and occipital lobe, are also physically localized. However, the hippocampus and ventral forebrain cells are not physically localized and are randomly located through the cerebral organoid. Cerebral organoids lack blood vessels and are limited in size by nutrient uptake in the innermost cells. Spinning bioreactors and advanced 3D scaffolding techniques are able to increase organoid size, though the integration of in vitro nutrient delivery systems is likely to spark the next major leap in cerebral organoid development.