A cerebral organoid, or brain organoid, describes an artificially grown, ''
in vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
,'' miniature organ resembling the
brain
A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a v ...
. Cerebral
organoid
An organoid is a miniaturized and simplified version of an Organ (anatomy), organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few Cell (biology), cells from a Tissue (biology), tissue, ...
s are created by culturing
pluripotent stem cells Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
in a three-dimensional rotational
bioreactor
A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical reaction, chemical process is carried out which involves organisms or biochemistry, ...
, and they develop over a course of months.
The brain is an extremely complex system of heterogeneous tissues and consists of a diverse array of
neuron
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
s. This complexity has made studying the brain and understanding how it works a difficult task in neuroscience, especially when it comes to neurodegenerative diseases. The purpose of creating an ''in vitro'' neurological model is to study these diseases in a more simple and variable space. This 3D model is free of many potential ''
in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' limitations. The varying physiology between human and other mammalian models limits the scope of study in neurological disorders. Cerebral organoids are synthesized tissues that contain several types of nerve cells and have anatomical features that recapitulate regions of the cortex observed in brains.
Cerebral organoids are most similar to layers of neurons called the
cortex
Cortex or cortical may refer to:
Biology
* Cortex (anatomy), the outermost layer of an organ
** Cerebral cortex, the outer layer of the vertebrate cerebrum, part of which is the ''forebrain''
*** Motor cortex, the regions of the cerebral cortex i ...
and
choroid plexus
The choroid plexus, or plica choroidea, is a plexus of cells that arises from the tela choroidea in each of the ventricles of the brain. Regions of the choroid plexus produce and secrete most of the cerebrospinal fluid (CSF) of the central nerv ...
. In some cases, structures similar to the
retina
The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then ...
,
meninges
In anatomy, the meninges (, ''singular:'' meninx ( or ), ) are the three membranes that envelop the brain and spinal cord. In mammals, the meninges are the dura mater, the arachnoid mater, and the pia mater. Cerebrospinal fluid is located in th ...
and
hippocampus
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, a ...
can form.
Stem cell
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
s 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 since.
Similar techniques are used on stem cells used to grow cerebral organoids.
Model development
Using human
pluripotent stem cells Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
to create ''in vitro'' cerebral organoids allows researchers to summarize current developmental mechanisms for human neural tissue as well as study the roots of
human neurological diseases. Cerebral 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 simple 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, leading to an understanding of how regions of the brain work. An ''in vitro'' human brain model would allow for the next wave in an understanding of the human brain.
Culturing methods
To make an organoid
An organoid is a miniaturized and simplified version of an Organ (anatomy), organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few Cell (biology), cells from a Tissue (biology), tissue, ...
, an embryoid (tissue that has some embryo
An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...
nic features) grown from natural stem cell
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
s is used. Embryos have three layers: endoderm
Endoderm is the innermost of the three primary germ layers in the very early embryo. The other two layers are the ectoderm (outside layer) and mesoderm (middle layer). Cells migrating inward along the archenteron form the inner layer of the gast ...
, mesoderm
The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.Langman's Medical E ...
and ectoderm
The ectoderm is one of the three primary germ layers formed in early embryonic development. It is the outermost layer, and is superficial to the mesoderm (the middle layer) and endoderm (the innermost layer). It emerges and originates from t ...
. Each turns into various body parts. The nervous system
In biology, the nervous system is the highly complex part of an animal that coordinates its actions and sensory information by transmitting signals to and from different parts of its body. The nervous system detects environmental changes th ...
grows from the ectoderm (which also contributes dental enamel
Tooth enamel is one of the four major tissues that make up the tooth in humans and many other animals, including some species of fish. It makes up the normally visible part of the tooth, covering the crown. The other major tissues are dentin, ...
and the epidermis
The epidermis is the outermost of the three layers that comprise the skin, the inner layers being the dermis and hypodermis. The epidermis layer provides a barrier to infection from environmental pathogens and regulates the amount of water rele ...
). Ectodermal cells were placed into gel droplets and floated in a nutrient broth in a rotating bioreactor
A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical reaction, chemical process is carried out which involves organisms or biochemistry, ...
, which supported cell growth without forming by the container. After ten days the organoid developed neuron
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
s. After 30 days it displayed regions similar to parts of brains. Lacking a blood supply
The blood circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. It includes the cardiovascular system, or vascular system, tha ...
, cerebral organoids reach about 4 mm across and can last a year or more. The general procedure can be broken down into 5 steps. First human pluripotent stem cells are cultured. They are then allowed to cultivate into an embryoid body
Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells.
EBs are differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers.
Background
The pluripotent cell type ...
. Next the cell culture is induced to form a neuroectoderm
Neuroectoderm (or neural ectoderm or neural tube epithelium) consists of cells derived from ectoderm. Formation of the neuroectoderm is first step in the development of the nervous system. The neuroectoderm receives bone morphogenetic protein-inh ...
. The neuroectoderm
Neuroectoderm (or neural ectoderm or neural tube epithelium) consists of cells derived from ectoderm. Formation of the neuroectoderm is first step in the development of the nervous system. The neuroectoderm receives bone morphogenetic protein-inh ...
is then grown in a matrigel
Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular ...
droplet. The matrigel provides nutrients and the neuroectoderm
Neuroectoderm (or neural ectoderm or neural tube epithelium) consists of cells derived from ectoderm. Formation of the neuroectoderm is first step in the development of the nervous system. The neuroectoderm receives bone morphogenetic protein-inh ...
starts to proliferate and grow. It's important to note that, 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. Normally, SMAD inhibition is used in usual cerebral organoid culturing processes; recent studies show that inhibition of this process generates microglia in cerebral organoids. It's important to note that the lack of vasculature limits the size the organoid can grow. This has been the major limitation in organoid development; recently, however, new methods using a spinning bioreactor
A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical reaction, chemical process is carried out which involves organisms or biochemistry, ...
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.
This was the original method outlined by Madeline Lancaster
Madeline Lancaster is an American developmental biologist studying neurological development and diseases of the brain. Lancaster is a group leader at the Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge, UK.
Education ...
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 i
this video
by Dr George M. Church
George McDonald Church (born August 28, 1954) is an American geneticist, molecular engineer, chemist, and a serial entrepreneur who is widely regarded as the "Founding Father of Genomics", and a pioneer in personal genomics and synthetic bio ...
.
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
Oct-4 (octamer-binding transcription factor 4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. I ...
and NANOG. These two markers are diminished during the course of development for the organoid. Neural identity markers that note successful neural induction, SOX1
SOX1 is a gene that encodes a transcription factor with a HMG-box (high mobility group) DNA-binding domain and functions primarily in neurogenesis. SOX1, SOX2 and SOX3, members of the SOX gene family (specifically the SOXB1 group), contain tra ...
and PAX6
Paired box protein Pax-6, also known as aniridia type II protein (AN2) or oculorhombin, is a protein that in humans is encoded by the ''PAX6'' gene.
Function
PAX6 is a member of the Pax gene family which is responsible for carrying the geneti ...
, 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
Forkhead box protein G1 is a protein that in humans is encoded by the ''FOXG1'' gene.
Function
This gene belongs to the forkhead family of transcription factors that is characterized by a distinct forkhead domain. The complete function of thi ...
and SIX3
Homeobox protein SIX3 is a protein that in humans is encoded by the ''SIX3'' gene.
Function
The SIX homeobox 3 (SIX3) gene is crucial in embryonic development by providing necessary instructions for the formation of the forebrain and eye deve ...
are highly expressed throughout organoid development. However, hindbrain markers EGR2 and ISL1
Insulin gene enhancer protein ISL-1 is a protein that in humans is encoded by the ''ISL1'' gene.
Function
This gene encodes a transcription factor containing two N-terminal LIM domains and one C-terminal homeodomain. The encoded protein play ...
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
The cerebral cortex, also known as the cerebral mantle, is the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals. The cerebral cortex mostly consists of the six-layered neocortex, with just 10% consisting of ...
and occipital lobe
The occipital lobe is one of the four major lobes of the cerebral cortex in the brain of mammals. The name derives from its position at the back of the head, from the Latin ''ob'', "behind", and ''caput'', "head".
The occipital lobe is the vi ...
have been tested. Many regions that have forebrain marker FOXG1
Forkhead box protein G1 is a protein that in humans is encoded by the ''FOXG1'' gene.
Function
This gene belongs to the forkhead family of transcription factors that is characterized by a distinct forkhead domain. The complete function of thi ...
, 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
LIM domain transcription factor LMO4 is a protein that in humans is encoded by the ''LMO4'' gene.
LIM domain only 4 is a cysteine-rich, two LIM domain-containing protein that may play a role as a transcriptional regulator or possibly an oncogene. ...
with the first representing cerebral cortex and the two after representing the occipital lobe. Genetic marker A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be ...
s 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
T-box, brain, 1 is a transcription factor protein important in vertebrate embryo development. It is encoded by the ''TBR1'' gene. This gene is also known by several other names: ''T-Brain 1'', ''TBR-1'', ''TES-56'', and ''MGC141978''. TBR1 is ...
is present in the preplate, the precursor to the cortical plate, and is present, along with MAP2
Microtubule-associated protein 2 is a protein in humans that is encoded by the ''MAP2'' gene.
Function
This gene encodes a protein that belongs to the microtubule-associated protein family. The proteins of this family were originally isolated s ...
, 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
Reelin, encoded by the ''RELN'' gene, is a large secreted extracellular matrix glycoprotein that helps regulate processes of neuronal migration and positioning in the developing brain by controlling cell–cell interactions. Besides this import ...
+ 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
T-box, brain, 1 is a transcription factor protein important in vertebrate embryo development. It is encoded by the ''TBR1'' gene. This gene is also known by several other names: ''T-Brain 1'', ''TBR-1'', ''TES-56'', and ''MGC141978''. TBR1 is ...
exhibiting preplate cells. Late-born neurons with markers SATB2
SATB is an initialism that describes the scoring of compositions for choirs, and also choirs (or consorts) of instruments. The initials are for the voice types: S for soprano, A for alto, T for tenor and B for bass.
Choral music
Four-part harmo ...
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 GFP may refer to:
Organisations
* Gaelic Football Provence, a French Gaelic Athletic Association club
* Geheime Feldpolizei, the German secret military police during the Second World War
* French Group for the Study of Polymers and their Applicat ...
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
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
and inhibited through tetrodotoxin
Tetrodotoxin (TTX) is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish; several of these species carry the toxin. Although tetrodotoxin was discovered ...
.
Interactions with environment
In ''DishBrain'', grown human brain cells were integrated into digital systems to play a simulated ''Pong
''Pong'' is a table tennis–themed twitch arcade sports video game, featuring simple two-dimensional graphics, manufactured by Atari and originally released in 1972. It was one of the earliest arcade video games; it was created by Allan Alcor ...
'' via electrophysiological stimulation and recording. The cells "showed significantly improved performance in ''Pong''" when embodied in a virtual game-world.
;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.
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. Transcriptome analysis has been used as an assay to examine the pathology of cerebral organoids derived from individual patients. Additionally, TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis.
Method
TUNEL is a method for dete ...
s have been used in studies as an evaluative marker of apoptosis in cerebral organoids. Other assays used to analyze cerebral organoids include the following:
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 depends 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 cerebral organoid cells. If, however, the genetic modification is made via “genetic cargo” insertion (such as through Adeno-associated virus
Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus ''Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in di ...
/ electroporation
Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...
methods) then it has been found that the genetic modification becomes less present with each round of cell division in cerebral organoids.
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.
Applications
There are many potential applications for cerebral organoid use, such as cell fate potential, cell replacement therapy
Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition. , the only established therapy using stem cells is hematopoietic stem cell transplantation. This usually takes the form of a bone marrow, bone-marrow transpla ...
, and cell-type specific genome assays. Cerebral organoids also 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. Further potential applications for cerebral organoids include:
Tissue morphogenesis
Tissue morphogenesis with respect to cerebral organoids covers how neural organs form in vertebrate
Vertebrates () comprise all animal taxa within the subphylum Vertebrata () ( chordates with backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the phylum Chordata, ...
s. 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
Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular dire ...
. Neural glial cells cover a wide variety of neural cells, some of which move around the neurons. The factors that govern their movements, as well as neurons in general, can be studied using cerebral organoids.
Clonal lineage tracing
Clonal lineage tracing is part of fate mapping
Fate mapping is a method used in developmental biology to study the embryonic origin of various adult tissues and structures. The "fate" of each cell or group of cells is mapped onto the embryo, showing which parts of the embryo will develop into ...
, 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 could 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
A neurodegenerative disease is caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic ...
as a therapeutic treatment. They can fuse with host vasculature
The blood circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. It includes the cardiovascular system, or vascular system, tha ...
and be immunologically silent. In some cases, the genomes of these cerebral organoids would first have to be edited. Recent studies have been able to achieve successful transplantation and integration of cerebral organoids into mouse brains; development of cell differentiation and vascularity was also observed after transplantation. Cerebral organoids might serve as the basis for transplantation and rebuilding in the human brain due to the similarity in structure.
Drug testing
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 drug high-throughput screening
High-throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology, materials science and chemistry. Using robotics, data processing/control software, liquid handlin ...
methods to cerebral organoids.
Developmental biology
Organoids can be used for the study of brain development, for example identifying and investigating genetic switches that have a significant impact on it. This can be used for the prevention and treatment of specific diseases (see below) but also for other purposes such as insights into the genetic factors of recent brain evolution (or the origin of humans and evolved
Evolution is change in the heritable characteristics of biological populations over successive generations. These characteristics are the expressions of genes, which are passed on from parent to offspring during reproduction. Variation t ...
difference to other apes), human enhancement
Human enhancement (HE) can be described as the natural, artificial, or technological alteration of the human body in order to enhance physical or mental capabilities.
Technologies
Existing technologies
Three forms of human enhancement curre ...
and improving intelligence, identifying detrimental exposome
An environmental factor, ecological factor or eco factor is any factor, abiotic or biotic, that influences living organisms. Abiotic factors include ambient temperature, amount of sunlight, and pH of the water soil in which an organism lives. Bi ...
impacts (and protection thereof), or improving brain healthspans.
Disease study
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 individual patients. Additionally, the development of vascularized cerebral organoids could be used for investigating stroke therapy in the future.
Zika Virus
Zika virus
''Zika virus'' (ZIKV; pronounced or ) is a member of the virus family ''Flaviviridae''. It is spread by daytime-active ''Aedes'' mosquitoes, such as '' A. aegypti'' and '' A. albopictus''. Its name comes from the Ziika Forest of Uganda, whe ...
has been shown to have teratogenic effects, causing defects in fetal neurological development. 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. Increased apoptosis was also found in cerebral organoids infected with Zika virus. Another study found that neural progenitor cell (NPC) populations were greatly reduced in these samples. The two methods by which NPC populations were reduced were increased cell death and reduced cell proliferation. TLR3
Toll-like receptor 3 (TLR3) also known as CD283 (cluster of differentiation 283) is a protein that in humans is encoded by the ''TLR3'' gene. TLR3 is a member of the toll-like receptor family of pattern recognition receptors of the innate immune s ...
receptor upregulation was identified in these infected organoids. Inhibition of this TLR3 receptor was shown to partially halt some of the Zika induced effects. 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
Cocaine has also been shown to have teratogenic effects on fetal development. Cerebral organoids have been used to study which enzyme isoforms are necessary for fetal neurological defects caused by cocaine use during pregnancy. One of these enzymes was determined to be cytochrome P450
Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
isoform CYP3A5
Cytochrome P450 3A5 is a protein that in humans is encoded by the ''CYP3A5'' gene.
Tissue distribution
''CYP3A5'' encodes a member of the cytochrome P450 superfamily of enzymes. Like most of the cytochrome P450, the CYP3A5 is expressed in the ...
.
Microcephaly
In one case, a cerebral organoid grown from a patient with microcephaly
Microcephaly (from New Latin ''microcephalia'', from Ancient Greek μικρός ''mikrós'' "small" and κεφαλή ''kephalé'' "head") is a medical condition involving a smaller-than-normal head. Microcephaly may be present at birth or it ...
demonstrated related symptoms and revealed that apparently, the cause is overly rapid development, followed by slower brain growth. Microencephaly is a developmental condition in which the brain remains undersized, producing an undersized head and debilitation. Microcephaly is not suitable for mouse models, which do not replicate the condition. The primary form of the disease is thought to be caused by a homozygous mutation in the microcephalin
Microcephalin (MCPH1) is a gene that is expressed during fetal brain development. Certain mutations in ''MCPH1'', when homozygous, cause primary microcephaly—a severely diminished brain. Hence, it has been assumed that variants have a role in ...
gene. The disease is difficult to reproduce in mouse models because mice lack the developmental stages for an enlarged cerebral cortex
The cerebral cortex, also known as the cerebral mantle, is the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals. The cerebral cortex mostly consists of the six-layered neocortex, with just 10% consisting of ...
that humans have. Naturally, a disease which affects this development would be impossible to show in a model which does not have it to begin with. To use cerebral organoids to model a human's microcephaly, one group of researchers has taken patient skin fibroblasts and reprogrammed them using four well known reprogramming factors. These include OCT4
Oct-4 (octamer-binding transcription factor 4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. I ...
, SOX2, MYC
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' (MYC), ''l-myc'' ( MYCL), and ''n-myc'' (MYCN). ''c-myc'' (also sometimes refe ...
and KLF4. The reprogrammed sample was able to be cloned into induced pluripotent stem cells. The cells were cultured into a cerebral organoid following a process described in the cerebral organoid creation section below. The organoid that resulted had decreased numbers of neural progenitor cells and smaller tissues. Additionally, the patient-derived tissues displayed fewer and less frequent neuroepithelial tissues made of progenitors, decreased radial glial stem cells, and increased neurons. These results suggest that the underlying mechanism of microcephaly is caused by cells prematurely differentiating into neurons leaving a deficit of radial glial cells.
Alzheimer's disease
Alzheimer's disease
Alzheimer's disease (AD) is a neurodegeneration, neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in short-term me ...
pathology has also been modeled with cerebral organoids. Affected individual’s pluripotent stem cells were used to generate brain organoids and then compared to control models, synthesised from healthy individuals. It was found that in the affected models, structures similar to that of plaques caused by amyloid beta proteins and neurofibrillary tangle
Neurofibrillary tangles (NFTs) are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary biomarker of Alzheimer's disease. Their presence is also found in numerous other diseases known as tauopathies. Little is kn ...
s, that cause the disease's symptoms were observed. Previous attempts to model this so accurately have been unsuccessful, with drugs being developed on the basis of efficacy in pre-clinical murine models demonstrating no effect in human trials.
Autism spectrum diseases
Cerebral organoids can also be used to study autism spectrum disorders. In one 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. Autism
The autism spectrum, often referred to as just autism or in the context of a professional diagnosis autism spectrum disorder (ASD) or autism spectrum condition (ASC), is a neurodevelopmental condition (or conditions) characterized by difficulti ...
has also been studied through the comparison of healthy versus affected synthesised brain organoids. Observation of the two models showed the overexpression of a transcription factor FOXG1
Forkhead box protein G1 is a protein that in humans is encoded by the ''FOXG1'' gene.
Function
This gene belongs to the forkhead family of transcription factors that is characterized by a distinct forkhead domain. The complete function of thi ...
that produced a larger amount of GABAergic
In molecular biology and physiology, something is GABAergic or GABAnergic if it pertains to or affects the neurotransmitter GABA. For example, a synapse is GABAergic if it uses GABA as its neurotransmitter, and a GABAergic neuron produces GABA. A ...
inhibitory neurons in the affected models. The significance of this use of brain organoids is that it has added great support for the excitatory/inhibitory imbalance hypothesis which if proven true could help identify targets for drugs so that the condition could be treated.
The field of epigenetics
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
and how DNA methylation
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts t ...
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, so cerebral organoids have been proposed as an alternate method of studying the potential effect that epigenetic mechanisms may have on the development of autism. This use of the cerebral organoid model to examine ASD and epigenetic patterns might provide insight in regards to epigenetic developmental timelines. However, it's important to note that the conditions in which cerebral organoids are cultured in might affect gene expression, and consequentially affect observations made using this model. 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.
Preterm hypoxia/ischemia
Preterm hypoxic injury remain difficult to study because of limited availability of human fetal brain tissues and inadequate animal models to study human corticogenesis. Cerebral organoid can be used to model prenatal pathophysiology and to compare the susceptibility of the different neural cell types to hypoxia during corticogenesis. Intermediate progenitors seems to be particularly affected, due to the unfolded protein response pathway. It has also been observed that hypoxia resulted in apoptosis in cerebral organoids, with outer radial glia and neuroblasts/immature neurons being particularly affected.
Glioblastomas
Traditional means of studying glioblastoma
Glioblastoma, previously known as glioblastoma multiforme (GBM), is one of the most aggressive types of cancer that begin within the brain. Initially, signs and symptoms of glioblastoma are nonspecific. They may include headaches, personality ch ...
s come with limitations. One example of such limitations would be the limited sample availability. Because of these challenges that come with using a more traditional approach, cerebral organoids have been used as an alternative means to model the development of brain cancer. In one study, cerebral organoids were simulated to reflect tumor-like qualities using CRISPR CAS-9. Increased cell division was observed in these genetically altered models. Cerebral organoids were also used in mice models to study tumorigenesis
Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...
and invasiveness. At the same time, the growth of brain cancers is influenced by environmental factors which are not yet replicable in cerebral organoid models. Cerebral organoids have been shown to provide insight into dysregulation of genes responsible for tumor development.
Multiple Sclerosis
Multiple sclerosis is an auto-immune inflammatory disorder affecting the central nervous system. Environmental and genetic factors contribute to the development of multiple sclerosis, however, the etiology of this condition is unknown. Induced pluripotent stem cells from healthy human controls, as well as from patients with multiple sclerosis were grown into cerebral organoids creating an innovative human model of this disease.
Limitations
Cerebral organoids are preferred over their 3D cell culture counter parts because they can better reflect the structure of the human brain, and because, to a certain extent, they can reflect fetal neocortex development over an extended period of time. While cerebral organoids have a lot of potential, their culturing and development comes with limitations and areas for improvement. For example, it takes several months to create one cerebral organoid, and the methods used to analyze them are also time consuming. Additionally, cerebral organoids do not have structures typical of a human brain, such as a blood brain barrier. This limits the types of diseases that can be studied. Other limitations include:
Necrotic centers
Until recently, the central part of organoids have been found to be necrotic
Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...
due to oxygen as well as nutrients being unable to reach that innermost area. This imposes limitations to cerebral organoids' physiological applicability. Because of this lack of oxygen and nutrients, neural progenitor cells are limited in their growth. However, recent findings suggest that, in the process of culturing a cerebral organoid, a necrotic center could be avoided 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. Future steps to streamline organoid culturing include analyzing more than one sample at a time.
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
Synaptogenesis is the formation of synapses between neurons in the nervous system. Although it occurs throughout a healthy person's lifespan, an explosion of synapse formation occurs during early brain development, known as exuberant synaptogenes ...
, is not yet suggested in cerebral organoid models. Since cerebral organoids are reflective of what happens during fetal neurodevelopment, there has been concern over how late onset diseases manifest in them. Future improvements include developing a way to recapitulate neurodegenerative diseases in cerebral organoids.
Ethics
Sentient organoids
Ethical concerns have been raised with using cerebral organoids as a model for disease due to the potential of them experiencing sensations such as pain or having the ability to develop a consciousness
Consciousness, at its simplest, is sentience and awareness of internal and external existence. However, the lack of definitions has led to millennia of analyses, explanations and debates by philosophers, theologians, linguisticians, and scien ...
. Currently it is unlikely given the simplicity of synthesised models compared to the complexity of a human brain, however models have been shown to respond to light-based stimulation, so present models do have some scope of responding to some stimuli at present. If such sensations could be proven to be present in any of the models, then the ethics of their use would be questionable.
Guidelines and legislation
Steps are being taken towards resolving the grey area such as a 2018 symposium at Oxford University where experts in the field, philosophers and lawyers met to try to clear up the ethical concerns with the new technology. Similarly, projects such as Brainstorm from Case Western University aim to observe the progress of the field by monitoring labs working with brain organoids to try to begin the ‘building of a philosophical framework’ that future guidelines and legislation could be built upon.
Humanized animals
Additionally, the "humanization" of animal models has been raised as a topic of concern in transplantation of human SC derived organoids into other animal models.
For example, potential future concerns of this type were described when human brain tissue organoids were transplanted into baby rats, appearing to be highly functional, to mature and to integrate with the rat brain. Such models can be used to model human brain development and, as demonstrated, to investigate diseases (and their potential therapies) but could be controversial
Controversy is a state of prolonged public dispute or debate, usually concerning a matter of conflicting opinion or point of view. The word was coined from the Latin ''controversia'', as a composite of ''controversus'' – "turned in an opposite ...
.
See also
* Neural tissue engineering
* Evolution of the brain#Genetic factors of recent evolution
References
{{reflist
Developmental neuroscience
Stem cells
Central nervous system
Synthetic biology