The process begins when the notochord induces the formation of the central nervous system (CNS) by signaling the ectoderm germ layer above it to form the thick and flat neural plate. The neural plate folds in upon itself to form the neural tube, which will later differentiate into the spinal cord and the brain, eventually forming the central nervous system. Computer simulations found that cell wedging and differential proliferation are sufficient for mammalian neurulation.
Different portions of the neural tube form by two different
processes, called primary and secondary neurulation, in different
species.
In primary neurulation, the neural plate creases inward until the edges come in contact and fuse.
In secondary neurulation, the tube forms by hollowing out of the interior of a solid precursor.
Primary neurulation
Cross section of a vertebrate embryo in the neurula stage
Primary neural induction
The concept of induction originated in work by Pandor in 1817. The first experiments proving induction were attributed by Viktor Hamburger to independent discoveries of both Hans Spemann of Germany in 1901 and Warren Lewis of the USA in 1904. It was Hans Spemann
who first popularized the term “primary neural induction” in reference
to the first differentiation of ectoderm into neural tissue during
neurulation. It was called "primary" because it was thought to be the first
induction event in embryogenesis. The Nobel prize-winning experiment was
done by his student Hilda Mangold. Ectoderm from the region of the dorsal lip of the blastopore of a
developing salamander embryo was transplanted into another embryo and
this "organizer" tissue “induced” the formation of a full secondary axis
changing surrounding tissue in the original embryo from ectodermal to
neural tissue. The tissue from the donor embryo was therefore referred
to as the inducer because it induced the change. While the organizer is the dorsal lip of the blastopore, this is not
one set of cells, but rather is a constantly changing group of cells
that migrate over the dorsal lip of the blastopore by forming apically
constricted bottle cells. At any given time during gastrulation there will be different cells that make up the organizer.
Subsequent work on inducers by scientists over the 20th century
demonstrated that not only could the dorsal lip of the blastopore act as
an inducer but so could a huge number of other seemingly unrelated
items. This began when boiled ectoderm was found to still be able to
induce by Johannes Holtfreter. Items as diverse as low pH, cyclic AMP, even floor dust could act as inducers leading to considerable consternation. Even tissue which could not induce when living could induce when boiled.[14] Other items such as lard, wax, banana peels and coagulated frog’s blood did not induce. The hunt for a chemically based inducer molecule was taken up by
developmental molecular biologists and a vast literature of items shown
to have inducer abilities continued to grow.
More recently, the inducer molecule has been attributed to genes,
and in 1995, there was a call for all the genes involved in primary
neural induction and all their interactions to be catalogued, in an
effort to determine “the molecular nature of Spemann’s organizer”. Several other proteins and growth factors have also been invoked as inducers, including soluble growth factors such as bone morphogenetic protein and a requirement for “inhibitory signals” such as noggin and follistatin.
Even before the term induction was popularized, several authors, beginning with Hans Driesch in 1894, suggested that primary neural induction might be mechanical in nature. A
mechanochemical-based model for primary neural induction was proposed
in 1985 by G.W. Brodland and R. Gordon. An actual physical wave of contraction has been shown to originate
from the precise location of the Spemann organizer which then traverses
the presumptive neural epithelium and a full working model of how primary neural inductions was proposed in 2006. There has long been a general reluctance in the field to consider the
possibility that primary neural induction might be initiated by
mechanical effects. A full explanation for primary neural induction remains yet to be found.
Shape change
As neurulation proceeds after induction, the cells of the neural plate become high-columnar and can be identified through microscopy as different from the surrounding presumptive epithelial ectoderm (epiblastic endoderm
in amniotes). The cells move laterally and away from the central axis
and change into a truncated pyramid shape. This pyramid shape is
achieved through tubulin and actin
in the apical portion of the cell which constricts as they move. The
variation in cell shapes is partially determined by the location of the
nucleus within the cell, causing bulging in areas of the cells forcing
the height and shape of the cell to change. This process is known as apical constriction. The result is a flattening of the differentiating neural plate which is
particularly obvious in salamanders when the previously round gastrula
becomes a rounded ball with a flat top.
Folding
The process of the flat neural plate folding into the cylindrical neural tube is termed primary neurulation.
As a result of the cellular shape changes, the neural plate forms the
medial hinge point (MHP). The expanding epidermis puts pressure on the
MHP and causes the neural plate to fold resulting in neural folds and the creation of the neural groove. The neural folds form dorsolateral hinge points
(DLHP) and pressure on this hinge cause the neural folds to meet and
fuse at the midline. The fusion requires the regulation of cell adhesion
molecules. The neural plate switches from E-cadherin expression to
N-cadherin and N-CAM expression to recognize each other as the same
tissue and close the tube. This change in expression stops the binding
of the neural tube to the epidermis.
The notochord plays an integral role in the development of the
neural tube. Prior to neurulation, during the migration of epiblastic
endoderm cells towards the hypoblastic endoderm, the notochordal process
opens into an arch termed the notochordal plate and attaches
overlying neuroepithelium of the neural plate. The notochordal plate
then serves as an anchor for the neural plate and pushes the two edges
of the plate upwards while keeping the middle section anchored. Some of
the notochodral cells become incorporated into the center section neural
plate to later form the floor plate of the neural tube. The notochord
plate separates and forms the solid notochord.
The folding of the neural tube to form an actual tube does not
occur all at once. Instead, it begins approximately at the level of the
fourth somite at Carnegie stage 9 (around embryonic day 20 in humans). The lateral edges of the neural plate touch in the midline and join together. This continues both cranially (toward the head) and caudally (toward the tail). The openings that are formed at the cranial and caudal regions are termed the cranial and caudal neuropores. In human embryos, the cranial neuropore closes approximately on day 24 and the caudal neuropore on day 28. Failure of the cranial (superior) and caudal (inferior) neuropore closure results in conditions called anencephaly and spina bifida,
respectively. Additionally, failure of the neural tube to close
throughout the length of the body results in a condition called rachischisis.
Patterning
Transverse section of the neural tube showing the floor plate and roof plate
According to the French Flag model
where stages of development are directed by gene product gradients,
several genes are considered important for inducing patterns in the open
neural plate, especially for the development of neurogenic placodes. These placodes first become evident histologically in the open neural plate. After sonic hedgehog
(SHH) signalling from the notochord induces its formation, the floor
plate of the incipient neural tube also secretes SHH. After closure,
the neural tube forms a basal or floor plate and a roof or alar plate in response to the combined effects of SHH and factors including BMP4
secreted by the roof plate. The basal plate forms most of the ventral
portion of the nervous system, including the motor portion of the spinal
cord and brain stem; the alar plate forms the dorsal portions, devoted
mostly to sensory processing.
The dorsal epidermis expresses BMP4 and BMP7. The roof plate of the neural tube responds to those signals by expressing more BMP4 and other transforming growth factor beta
(TGF-β) signals to form a dorsal/ventral gradient among the neural
tube. The notochord expresses SHH. The floor plate responds to SHH by
producing its own SHH and forming a gradient. These gradients allow for
the differential expression of transcription factors.
Complexities of the model
Neural tube closure
is not entirely understood. Closure of the neural tube varies by
species. In mammals, closure occurs by meeting at multiple points which
then close up and down. In birds, neural tube closure begins at one
point of the midbrain and moves anteriorly and posteriorly.
Secondary neurulation
Primary
neurulation develops into secondary neurulation when the caudal
neuropore undergoes final closure. The cavity of the spinal cord extends
into the neural cord. In secondary neurulation, the neural ectoderm and some cells from the
endoderm form the medullary cord. The medullary cord condenses,
separates and then forms cavities. These cavities then merge to form a single tube. Secondary neurulation
occurs in the posterior section of most animals but it is better
expressed in birds. Tubes from both primary and secondary neurulation
eventually connect at around the sixth week of development.
In humans, the mechanisms of secondary neurulation plays an
important role given its impact on the proper formation of the human
posterior spinal cord. Errors at any point in the process can yield
problems. For example, retained medullary cord occurs due to a partial
or complete arrest of secondary neurulation that creates a
non-functional portion on the vestigial end.
Paraxial mesoderm surrounding the notochord at the sides will develop into the somites (future muscles, bones, and contributes to the formation of limbs of the vertebrate ).
Masses of tissue called the neural crest
that are located at the very edges of the lateral plates of the folding
neural tube separate from the neural tube and migrate to become a
variety of different but important cells.
Neural crest cells will migrate through the embryo and will give
rise to several cell populations, including pigment cells and the cells
of the peripheral nervous system.
Failure of neurulation, especially failure of closure of the neural tube are among the most common and disabling birth defects in humans, occurring in roughly 1 in every 500 live births. Failure of the rostral end of the neural tube to close results in anencephaly, or lack of brain development, and is most often fatal. Failure of the caudal end of the neural tube to close causes a condition known as spina bifida, in which the spinal cord fails to close.
Gastrulation occurs when a blastula, made up of one layer, folds inward and enlarges to create a gastrula. This diagram is color-coded: ectoderm, blue; endoderm, green; blastocoel (the yolk sac), yellow; and archenteron (the primary gut), purple.
Gastrulation is the stage in the early embryonic development of most animals, during which the blastula (a single-layered hollow sphere of cells), or in mammals, the blastocyst, is reorganized into a two-layered or three-layered embryo known as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body (e.g. dorsal–ventral, anterior–posterior), and internalized one or more cell types, including the prospective gut.
Gastrula layers
In triploblastic organisms, the gastrula is trilaminar (three-layered). These three germ layers are the ectoderm (outer layer), mesoderm (middle layer), and endoderm (inner layer). In diploblastic organisms, such as Cnidaria and Ctenophora, the gastrula has only ectoderm and endoderm. The two layers are also sometimes referred to as the hypoblast and epiblast. Sponges do not go through the gastrula stage.
Gastrulation takes place after cleavage and the formation of the blastula, or blastocyst. Gastrulation is followed by organogenesis, when individual organs develop within the newly formed germ layers. Each layer gives rise to specific tissues and organs in the developing embryo.
Following gastrulation, cells in the body are either organized into sheets of connected cells (as in epithelia), or as a mesh of isolated cells, such as mesenchyme.
Basic cell movements
Although
gastrulation patterns exhibit enormous variation throughout the animal
kingdom, they are unified by the five basic types of cell movements that
occur during gastrulation:
The terms "gastrula" and "gastrulation" were coined by Ernst Haeckel, in his 1872 work "Biology of Calcareous Sponges". Gastrula (literally, "little belly") is a neo-Latin diminutive based on the Ancient Greek γαστήρgastḗr ("a belly").
Importance
Lewis Wolpert,
pioneering developmental biologist in the field, has been credited for
noting that "It is not birth, marriage, or death, but gastrulation which
is truly the most important time in your life."
Model systems
Gastrulation
is highly variable across the animal kingdom but has underlying
similarities. Gastrulation has been studied in many animals, but some
models have been used for longer than others. Furthermore, it is easier
to study development in animals that develop outside the mother. Model organisms whose gastrulation is understood in the greatest detail include the mollusc, sea urchin, frog, and chicken. A human model system is the gastruloid.
Protostomes versus deuterostomes
The distinction betweenprotostomes and deuterostomes is based on the direction in which the mouth (stoma) develops in relation to the blastopore.
Protostome derives from the Greek word protostoma meaning "first mouth"
(πρῶτος + στόμα) whereas Deuterostome's etymology is "second mouth"
from the words second and mouth (δεύτερος + στόμα).
The major distinctions between deuterostomes and protostomes are found in embryonic development:
Mouth/anus
In protostome development, the first opening in development, the blastopore, becomes the animal's mouth.
In deuterostome development, the blastopore becomes the animal's anus.
Sea urchins exhibit highly stereotyped cleavage patterns and cell fates. Maternally deposited mRNAs establish the organizing center of the sea urchin embryo. Canonical Wnt and Delta-Notch signaling progressively segregate progressive endoderm and mesoderm.
The first cells to internalize are the primary mesenchyme cells (PMCs), which have a skeletogenic fate, which ingress during the blastula stage. Gastrulation – internalization of the prospective endoderm and non-skeletogenic mesoderm – begins shortly thereafter with invagination and other cell rearrangements the vegetal pole, which contribute approximately 30% to the final archenteron length. The gut's final length depends on cell rearrangements within the archenteron.
The sperm contributes one of the two mitotic asters needed to complete first cleavage. The sperm can enter anywhere in the animal half of the egg but its exact point of entry will break the egg's radial symmetry by organizing the cytoskeleton. Prior to first cleavage, the egg's cortex rotates relative to the internal cytoplasm by the coordinated action of microtubules,
in a process known as cortical rotation. This displacement brings
maternally loaded determinants of cell fate from the equatorial
cytoplasm and vegetal cortex into contact, and together these
determinants set up the organizer. Thus, the area on the vegetal side opposite the sperm entry point will become the organizer. Hilde Mangold, working in the lab of Hans Spemann, demonstrated that this special "organizer" of the embryo is necessary and sufficient to induce gastrulation.
The dorsal lip of the blastopore is the mechanical driver of gastrulation, and the first sign of invagination seen in the frog.
Germ layer differentiation
Specification of endoderm depends on rearrangement of maternally deposited determinants, leading to nuclearization of Beta-catenin. Mesoderm is induced by signaling from the presumptive endoderm to cells that would otherwise become ectoderm.
Cell signaling
In the frog, Xenopus, one of the signals is retinoic acid (RA). RA signaling in this organism can affect the formation of the endoderm
and depending on the timing of the signaling, it can determine the fate
whether its pancreatic, intestinal, or respiratory. Other signals such
as Wnt and BMP also play a role in respiratory fate of the Xenopus by activating cell lineage tracers.
In preparation for gastrulation, the embryo must become asymmetric along both the proximal-distal axis and the anteroposterior axis.
The proximal-distal axis is formed when the cells of the embryo form
the "egg cylinder", which consists of the extraembryonic tissues, which
give rise to structures like the placenta, at the proximal end and the epiblast at the distal end. Many signaling pathways contribute to this reorganization, including BMP, FGF, nodal, and Wnt. Visceral endoderm surrounds the epiblast. The distal visceral endoderm (DVE) migrates to the anterior portion of the embryo, forming the anterior visceral endoderm (AVE). This breaks anterior-posterior symmetry and is regulated by nodal signaling.
The primitive streak is formed at the beginning of gastrulation and is found at the junction between the extraembryonic tissue and the epiblast on the posterior side of the embryo and the site of ingression. Formation of the primitive streak is reliant upon nodal signaling in the Koller's sickle within the cells contributing to the primitive streak and BMP4 signaling from the extraembryonic tissue.Furthermore, Cer1 and Lefty1 restrict the primitive streak to the appropriate location by antagonizing nodal signaling. The region defined as the primitive streak continues to grow towards the distal tip.
During the early stages of development, the primitive streak is the structure that will establish bilateral symmetry, determine the site of gastrulation and initiate germ layer formation. To form the streak, reptiles, birds and mammals arrange mesenchymal
cells along the prospective midline, establishing the first embryonic
axis, as well as the place where cells will ingress and migrate during
the process of gastrulation and germ layer formation. The primitive streak extends through this midline and creates the antero-posterior body axis, becoming the first symmetry-breaking event in the embryo, and marks the beginning of gastrulation. This process involves the ingression of mesoderm and endoderm progenitors and their migration to their ultimate position, where they will differentiate into the three germ layers. The localization of the cell adhesion and signaling molecule beta-catenin is critical to the proper formation of the organizer region that is responsible for initiating gastrulation.
Cell internalization
In order for the cells to move from the epithelium of the epiblast through the primitive streak to form a new layer, the cells must undergo an epithelial to mesenchymal transition (EMT) to lose their epithelial characteristics, such as cell–cell adhesion. FGF signaling is necessary for proper EMT. FGFR1 is needed for the up regulation of SNAI1, which down regulates E-cadherin, causing a loss of cell adhesion. Following the EMT, the cells ingress through the primitive streak and spread out to form a new layer of cells or join existing layers. FGF8 is implicated in the process of this dispersal from the primitive streak.
Cell signaling driving gastrulation
During gastrulation, the cells are differentiated into the ectoderm or mesendoderm, which then separates into the mesoderm and endoderm. The endoderm and mesoderm form due to the nodal signaling. Nodal signaling uses ligands that are part of TGFβ family. These ligands will signal transmembrane serine/threonine kinase receptors, and this will then phosphorylate Smad2 and Smad3. This protein will then attach itself to Smad4 and relocate to the nucleus where the mesendoderm genes will begin to be transcribed. The Wnt pathway along with β-catenin plays a key role in nodal signaling and endoderm formation. Fibroblast growth factors (FGF), canonical Wnt pathway, bone morphogenetic protein (BMP), and retinoic acid (RA) are all important in the formation and development of the endoderm. FGF are important in producing the homeobox
gene which regulates early anatomical development. BMP signaling plays a
role in the liver and promotes hepatic fate. RA signaling also induce
homeobox genes such as Hoxb1 and Hoxa5. In mice, if there is a lack in
RA signaling the mouse will not develop lungs. RA signaling also has multiple uses in organ formation of the pharyngeal arches, the foregut, and hindgut.
Gastrulation in vitro
There have been a number of attempts to understand the processes of gastrulation using in vitro techniques in parallel and complementary to studies in embryos, usually though the use of 2D and 3D cell (Embryonic organoids) culture techniquesusing embryonic stem cells (ESCs) or induced pluripotent stem cells
(iPSCs). These are associated with number of clear advantages in using
tissue-culture based protocols, some of which include reducing the cost
of associated in vivo work (thereby reducing, replacing and refining the use of animals in experiments; the 3Rs), being able to accurately apply agonists/antagonists in spatially and temporally specific manner which may be technically difficult to perform during Gastrulation.
However, it is important to relate the observations in culture to the
processes occurring in the embryo for context.
To illustrate this, the guided differentiation of mouse ESCs has resulted in generating primitive streak–like cells that display many of the characteristics of epiblast cells that traverse through the primitive streak (e.g. transient brachyury up regulation and the cellular changes associated with an epithelial to mesenchymal transition), and human ESCs cultured on micro patterns, treated with BMP4, can generate spatial differentiation pattern similar to the arrangement of the germ layers in the human embryo. Finally, using 3D embryoid body- and organoid-based techniques, small aggregates of mouse ESCs (Embryonic Organoids, or Gastruloids)
are able to show a number of processes of early mammalian embryo
development such as symmetry-breaking, polarisation of gene expression,
gastrulation-like movements, axial elongation and the generation of all
three embryonic axes (anteroposterior, dorsoventral and left-right
axes).
In vitro fertilization occurs in a laboratory. The process of in vitro
fertilization is when mature eggs are removed from the ovaries and are
placed in a cultured medium where they are fertilized by sperm. In the
culture the embryo will form. 14 days after fertilization the primitive streak forms. The formation
of the primitive streak has been known to some countries as "human
individuality". This means that the embryo is now a being itself, it is its own entity.
The countries that believe this have created a 14-day rule in which it
is illegal to study or experiment on a human embryo after the 14-day
period in vitro. Research has been conducted on the first 14 days of an embryo, but no known studies have been done after the 14 days. With the rule in place, mice embryos are used understand the
development after 14 days; however, there are differences in the
development between mice and humans.
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.
Instructive growth factors regulating fate decisions in embryonic NCSCs
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
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.
A cerebral organoid can be formed by inducing ectoderm cells to differentiate into a 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.
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 assays 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/ electroporation
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.
There are many potential applications for cerebral organoid use, such as cell fate potential, cell replacement therapy, 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 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, as well as neurons in
general, 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 the 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 as a therapeutic treatment. They can fuse with host vasculature 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 methods to cerebral organoids. After 2015, significant effort has gone into fabricating microscale
devices to generate reproducible cerebral organoids at high-throughput.
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
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
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 isoform CYP3A5.
Microcephaly
In one case, a cerebral organoid grown from a patient with microcephaly
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 gene. The disease is difficult to reproduce in mouse models because mice lack the developmental stages for an enlarged cerebral cortex
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, SOX2, MYC 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 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 tangles, 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 disorders
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 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 that produced a larger amount of GABAergic
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 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, 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 is 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 seem 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 glioblastomas
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
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 counterparts 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 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
neurodevelopmental timeline, such as synaptogenesis, 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.
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. 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.
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.
Additionally, the "humanization" of animal models has been raised as a
topic of concern in transplantation of human stem cell 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.
Scope–severity grid from Bostrom's paper "Existential Risk Prevention as Global Priority"
Risks of astronomical suffering, also called suffering risks or s-risks, are risks involving much more suffering than all that has occurred on Earth so far. They are sometimes categorized as a subclass of existential risks.
According to some scholars, s-risks warrant serious consideration
as they are not extremely unlikely and can arise from unforeseen
scenarios. Although they may appear speculative, factors such as
technological advancement, power dynamics, and historical precedents
indicate that advanced technology could inadvertently result in
substantial suffering. Thus, s-risks are considered to be a morally
urgent matter, despite the possibility of technological benefits.
Sources of possible s-risks include embodied artificial intelligence and superintelligence, as well as space colonization, which could potentially lead to "constant and catastrophic wars" and an immense increase in wild animal suffering
by introducing wild animals, who "generally lead short, miserable lives
full of sometimes the most brutal suffering", to other planets, either
intentionally or inadvertently.
Types of S-risk
Artificial intelligence
Artificial intelligence
is central to s-risk discussions because it may eventually enable
powerful actors to control vast technological systems. In a worst-case
scenario, AI could be used to create systems of perpetual suffering,
such as a totalitarian regime expanding across space. Additionally, s-risks might arise incidentally, such as through
AI-driven simulations of conscious beings experiencing suffering, or
from economic activities that disregard the well-being of nonhuman or
digital minds. Steven Umbrello, an AI ethics researcher, has warned that biological computing may make system design more prone to s-risks.
Space colonization
Space colonization
could increase suffering by introducing wild animals to new
environments, leading to ecological imbalances. In unfamiliar habitats,
animals may struggle to survive, facing hunger, disease, and predation.
These challenges, combined with unstable ecosystems, could cause
population crashes or explosions, resulting in widespread suffering.
Additionally, the lack of natural predators or proper biodiversity on
colonized planets could worsen the situation, mirroring Earth’s
ecological problems on a larger scale. This raises ethical concerns
about the unintended consequences of space colonization, as it could
propagate immense animal suffering in new, unstable ecosystems. Phil
Torres argues that space colonization poses significant "suffering
risks", where expansion into space will lead to the creation of diverse
species and civilizations with conflicting interests. These differences,
combined with advanced weaponry and the vast distances between
civilizations, would result in catastrophic and unresolvable conflicts.
Strategies like a "cosmic Leviathan" to impose order or deterrence
policies are unlikely to succeed due to physical limitations in space
and the destructive power of future technologies. Thus, Torres concludes
that space colonization could create immense suffering and should be
delayed or avoided altogether.
Magnus Vinding's "astronomical atrocity problem" questions
whether vast amounts of happiness can justify extreme suffering from
space colonization. He highlights moral concerns such as diminishing
returns on positive goods, the potentially incomparable weight of severe
suffering, and the priority of preventing misery. He argues that if
colonization is inevitable, it should be led by agents deeply committed
to minimizing harm.
Genetic engineering
David Pearce has argued that genetic engineering is a potential s-risk. Pearce argues that while technological mastery over the pleasure-pain axis and solving the hard problem of consciousness could lead to the potential eradication of suffering,
it could also potentially increase the level of contrast in the hedonic
range that sentient beings could experience. He argues that these
technologies might make it feasible to create "hyperpain" or "dolorium"
that experience levels of suffering beyond the human range.
Excessive criminal punishment
S-risk
scenarios may arise from excessive criminal punishment, with precedents
in both historical and in modern penal systems. These risks escalate in
situations such as warfare or terrorism, especially when advanced
technology is involved, as conflicts can amplify destructive tendencies
like sadism, tribalism, and retributivism.
War often intensifies these dynamics, with the possibility of
catastrophic threats being used to force concessions. Agential s-risks
are further aggravated by malevolent traits in powerful individuals,
such as narcissism or psychopathy. This is exemplified by totalitarian
dictators like Hitler and Stalin, whose actions in the 20th century inflicted widespread suffering.
Exotic risks
According to David Pearce, there are other potential s-risks that are more exotic, such as those posed by the many-worlds interpretation of quantum mechanics.
Mitigation strategies
To
mitigate s-risks, efforts focus on researching and understanding the
factors that exacerbate them, particularly in emerging technologies and
social structures. Targeted strategies include promoting safe AI design,
ensuring cooperation among AI developers, and modeling future
civilizations to anticipate risks. Broad strategies may advocate for
moral norms against large-scale suffering and stable political
institutions. According to Anthony DiGiovanni, prioritizing s-risk
reduction is essential, as it may be more manageable than other
long-term challenges, while avoiding catastrophic outcomes could be
easier than achieving an entirely utopian future.
Induced amnesia
Induced amnesia has been proposed as a way to mitigate s-risks in locked-in conscious AI and certain AI-adjacent biological systems like brain organoids.
Cosmic rescue missions
David
Pearce's concept of "cosmic rescue missions" proposes the idea of
sending probes to alleviate potential suffering in extraterrestrial
environments. These missions aim to identify and mitigate suffering
among hypothetical extraterrestrial life forms, ensuring that if life
exists elsewhere, it is treated ethically. However, challenges include the lack of confirmed extraterrestrial
life, uncertainty about their consciousness, and public support
concerns, with environmentalists advocating for non-interference and
others focusing on resource extraction.
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