Epithelium

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https://en.wikipedia.org/wiki/Epithelium

Epithelium

Types of epithelium Epithelium or epithelial tissue is a thin, continuous, protective layer of cells with little extracellular matrix. An example is the epidermis, the outermost layer of the skin. Epithelial (mesothelial) tissues line the outer surfaces of many internal organs, the corresponding inner surfaces of body cavities, and the inner surfaces of blood vessels. Epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. These tissues also lack blood or lymph supply. The tissue is supplied by nerves.

There are three principal shapes of epithelial cell: squamous (scaly), columnar, and cuboidal. These can be arranged in a singular layer of cells as simple epithelium, either simple squamous, simple columnar, or simple cuboidal, or in layers of two or more cells deep as stratified (layered), or compound, either squamous, columnar or cuboidal. In some tissues, a layer of columnar cells may appear to be stratified due to the placement of the nuclei. This sort of tissue is called pseudostratified. All glands are made up of epithelial cells. Functions of epithelial cells include diffusion, filtration, secretion, selective absorption, germination, and transcellular transport. Compound epithelium has protective functions.

Epithelial layers contain no blood vessels (avascular), so they must receive nourishment via diffusion of substances from the underlying connective tissue, through the basement membrane. Cell junctions are especially abundant in epithelial tissues.

Classification

Simple epithelium

Simple epithelium is a single layer of cells with every cell in direct contact with the basement membrane that separates it from the underlying connective tissue. In general, it is found where absorption and filtration occur. The thinness of the epithelial barrier facilitates these processes.

In general, epithelial tissues are classified by the number of their layers and by the shape and function of the cells. The basic cell types are squamous, cuboidal, and columnar, classed by their shape.

Type	Description
Squamous	Squamous cells have the appearance of thin, flat plates that can look polygonal when viewed from above. Their name comes from squāma, Latin for "scale" – as on fish or snake skin. The cells fit closely together in tissues, providing a smooth, low-friction surface over which fluids can move easily. The shape of the nucleus usually corresponds to the cell form and helps to identify the type of epithelium. Squamous cells tend to have horizontally flattened, nearly oval-shaped nuclei because of the thin, flattened form of the cell. Squamous epithelium is found lining surfaces such as skin or alveoli in the lung, enabling simple passive diffusion as also found in the alveolar epithelium in the lungs. Specialized squamous epithelium also forms the lining of cavities such as in blood vessels (as endothelium), in the pericardium (as mesothelium), and in other body cavities.
Cuboidal	Cuboidal epithelial cells have a cube-like shape and appear square in cross-section. The cell nucleus is large, spherical and is in the center of the cell. Cuboidal epithelium is commonly found in secretive tissue such as the exocrine glands, or in absorptive tissue such as the pancreas, the lining of the kidney tubules as well as in the ducts of the glands. The germinal epithelium that covers the female ovary, and the germinal epithelium that lines the walls of the seminferous tubules in the testes are also of the cuboidal type. Cuboidal cells provide protection and may be active in pumping material in or out of the lumen, or passive depending on their location and specialisation. Simple cuboidal epithelium commonly differentiates to form the secretory and duct portions of glands. Stratified cuboidal epithelium protects areas such as the ducts of sweat glands, mammary glands, and salivary glands.
Columnar	Columnar epithelial cells are elongated and column-shaped and have a height of at least four times their width. Their nuclei are elongated and are usually located near the base of the cells. Columnar epithelium forms the lining of the stomach and intestines. The cells here may possess microvilli for maximizing the surface area for absorption, and these microvilli may form a brush border. Other cells may be ciliated to move mucus in the function of mucociliary clearance. Other ciliated cells are found in the fallopian tubes, the uterus and central canal of the spinal cord. Some columnar cells are specialized for sensory reception such as in the nose, ears and the taste buds. Hair cells in the inner ears have stereocilia which are similar to microvilli. Goblet cells are modified columnar cells and are found between the columnar epithelial cells of the duodenum. They secrete mucus, which acts as a lubricant. Single-layered non-ciliated columnar epithelium tends to indicate an absorptive function. Stratified columnar epithelium is rare but is found in lobar ducts in the salivary glands, the eye, the pharynx, and sex organs. This consists of a layer of cells resting on at least one other layer of epithelial cells, which can be squamous, cuboidal, or columnar.
Pseudostratified	These are simple columnar epithelial cells whose nuclei appear at different heights, giving the misleading (hence "pseudo") impression that the epithelium is stratified when the cells are viewed in cross section. Ciliated pseudostratified epithelial cells have cilia. Cilia are capable of energy-dependent pulsatile beating in a certain direction through interaction of cytoskeletal microtubules and connecting structural proteins and enzymes. In the respiratory tract, the wafting effect produced causes mucus secreted locally by the goblet cells (to lubricate and to trap pathogens and particles) to flow in that direction (typically out of the body). Ciliated epithelium is found in the airways (nose, bronchi), but is also found in the uterus and fallopian tubes, where the cilia propel the ovum to the uterus.

Summary showing different epithelial cells/tissues and their characteristics.

By layer, epithelium is classed as either simple epithelium, only one cell thick (unilayered), or stratified epithelium having two or more cells in thickness, or multi-layered – as stratified squamous epithelium, stratified cuboidal epithelium, and stratified columnar epithelium, and both types of layering can be made up of any of the cell shapes. However, when taller simple columnar epithelial cells are viewed in cross section showing several nuclei appearing at different heights, they can be confused with stratified epithelia. This kind of epithelium is therefore described as pseudostratified columnar epithelium.

Transitional epithelium has cells that can change from squamous to cuboidal, depending on the amount of tension on the epithelium.

Stratified epithelium

Stratified or compound epithelium differs from simple epithelium in that it is multilayered. It is therefore found where body linings have to withstand mechanical or chemical insult such that layers can be abraded and lost without exposing subepithelial layers. Cells flatten as the layers become more apical, though in their most basal layers, the cells can be squamous, cuboidal, or columnar.

Stratified epithelia (of columnar, cuboidal, or squamous type) can have the following specializations:

Specialization	Description
Keratinized	In this particular case, the most apical layers (exterior) of cells are dead and lose their nucleus and cytoplasm, instead contain a tough, resistant protein called keratin. This specialization makes the epithelium somewhat water-resistant, so is found in the mammalian skin. The lining of the oesophagus is an example of a non-keratinized or "moist" stratified epithelium.
Parakeratinized	In this case, the most apical layers of cells are filled with keratin, but they still retain their nuclei. These nuclei are pyknotic, meaning that they are highly condensed. Parakeratinized epithelium is sometimes found in the oral mucosa and in the upper regions of the oesophagus.
Transitional	Transitional epithelia are found in tissues that stretch, and it can appear to be stratified cuboidal when the tissue is relaxed, or stratified squamous when the organ is distended and the tissue stretches. It is sometimes called urothelium since it is almost exclusively found in the bladder, ureters and urethra.

Structure

Epithelial tissue cells can adopt shapes of varying complexity from polyhedral to scutoidal to punakoidal. They are tightly packed and form a continuous sheet with almost no intercellular spaces. All epithelia is usually separated from underlying tissues by an extracellular fibrous basement membrane. The lining of the mouth, lung alveoli and kidney tubules are all made of epithelial tissue. The lining of the blood and lymphatic vessels are of a specialised form of epithelium called endothelium.

Location

Normal histology of the breast, with luminal epithelial cells annotated near bottom right.

See also: Table of epithelia of human organs

Epithelium lines both the outside (skin) and the inside cavities and lumina of bodies. The outermost layer of human skin is composed of dead stratified squamous, keratinized epithelial cells.

Tissues that line the inside of the mouth, the esophagus, the vagina, and part of the rectum are composed of nonkeratinized stratified squamous epithelium. Other surfaces that separate body cavities from the outside environment are lined by simple squamous, columnar, or pseudostratified epithelial cells. Other epithelial cells line the insides of the lungs, the gastrointestinal tract, the reproductive and urinary tracts, and make up the exocrine and endocrine glands. The outer surface of the cornea is covered with fast-growing, easily regenerated epithelial cells. A specialised form of epithelium, endothelium, forms the inner lining of blood vessels and the heart, and is known as vascular endothelium, and lining lymphatic vessels as lymphatic endothelium. Another type, mesothelium, forms the walls of the pericardium, pleurae, and peritoneum.

In arthropods, the integument, or external "skin", consists of a single layer of epithelial ectoderm from which arises the cuticle, an outer covering of chitin, the rigidity of which varies as per its chemical composition.

Basement membrane

The basal surface of epithelial tissue rests on a basement membrane and the free/apical surface faces body fluid or outside. The basement membrane acts as a scaffolding on which epithelium can grow and regenerate after injuries. Epithelial tissue has a nerve supply, but no blood supply and must be nourished by substances diffusing from the blood vessels in the underlying tissue. The basement membrane acts as a selectively permeable membrane that determines which substances will be able to enter the epithelium.

The basal lamina is made up of laminin (glycoproteins) secreted by epithelial cells. The reticular lamina beneath the basal lamina is made up of collagen proteins secreted by connective tissue.

Cell junctions

Cell junctions are especially abundant in epithelial tissues. They consist of protein complexes and provide contact between neighbouring cells, between a cell and the extracellular matrix, or they build up the paracellular barrier of epithelia and control the paracellular transport.

Cell junctions are the contact points between plasma membrane and tissue cells. There are mainly 5 different types of cell junctions: tight junctions, adherens junctions, desmosomes, hemidesmosomes, and gap junctions. Tight junctions are a pair of trans-membrane protein fused on outer plasma membrane. Adherens junctions are a plaque (protein layer on the inside plasma membrane) which attaches both cells' microfilaments. Desmosomes attach to the microfilaments of cytoskeleton made up of keratin protein. Hemidesmosomes resemble desmosomes on a section. They are made up of the integrin (a transmembrane protein) instead of cadherin. They attach the epithelial cell to the basement membrane. Gap junctions connect the cytoplasm of two cells and are made up of proteins called connexins (six of which come together to make a connexion).

Development

Epithelial tissues are derived from all of the embryological germ layers:

• from ectoderm (e.g., the epidermis);
• from endoderm (e.g., the lining of the gastrointestinal tract);
• from mesoderm (e.g., the inner linings of body cavities).

However, pathologists do not consider endothelium and mesothelium (both derived from mesoderm) to be true epithelium. This is because such tissues present very different pathology. For that reason, pathologists label cancers in endothelium and mesothelium sarcomas, whereas true epithelial cancers are called carcinomas. Additionally, the filaments that support these mesoderm-derived tissues are very distinct. Outside of the field of pathology, it is generally accepted that the epithelium arises from all three germ layers.

Cell turnover

Epithelia turn over at some of the fastest rates in the body. For epithelial layers to maintain constant cell numbers essential to their functions, the number of cells that divide must match those that die. They do this mechanically. If there are too few of the cells, the stretch that they experience rapidly activates cell division. Alternatively, when too many cells accumulate, crowding triggers their death by activation epithelial cell extrusion. Here, cells fated for elimination are seamlessly squeezed out by contracting a band of actin and myosin around and below the cell, preventing any gaps from forming that could disrupt their barriers. Failure to do so can result in aggressive tumors and their invasion by aberrant basal cell extrusion.

Functions

Forms of secretion in glandular tissue

Different characteristics of glands of the body

Epithelial tissues have as their primary functions:

1. to protect the tissues that lie beneath from radiation, desiccation, toxins, invasion by pathogens, and physical trauma
2. the regulation and exchange of chemicals between the underlying tissues and a body cavity
3. the secretion of hormones into the circulatory system, as well as the secretion of sweat, mucus, enzymes, and other products that are delivered by ducts
4. to provide sensation
5. Absorb water and digested food in the lining of digestive canal.

Glandular tissue

Glandular tissue is the type of epithelium that forms the glands from the infolding of epithelium and subsequent growth in the underlying connective tissue. They may be specialized columnar or cuboidal tissues consisting of goblet cells, which secrete mucus. There are two major classifications of glands: endocrine glands and exocrine glands:

• Endocrine glands secrete their product into the extracellular space where it is rapidly taken up by the circulatory system.
• Exocrine glands secrete their products into a duct that then delivers the product to the lumen of an organ or onto the free surface of the epithelium. Their secretions include tears, saliva, oil (sebum), enzyme, digestive juices, sweat, etc.

Sensing the extracellular environment

Some epithelial cells are ciliated, especially in respiratory epithelium, and they commonly exist as a sheet of polarised cells forming a tube or tubule with cilia projecting into the lumen." Primary cilia on epithelial cells provide chemosensation, thermoception, and mechanosensation of the extracellular environment by playing "a sensory role mediating specific signalling cues, including soluble factors in the external cell environment, a secretory role in which a soluble protein is released to have an effect downstream of the fluid flow, and mediation of fluid flow if the cilia are motile.

Host immune response

Epithelial cells express many genes that encode immune mediators and proteins involved in cell-cell communication with hematopoietic immune cells. The resulting immune functions of these non-hematopoietic, structural cells contribute to the mammalian immune system ("structural immunity"). Relevant aspects of the epithelial cell response to infections are encoded in the epigenome of these cells, which enables a rapid response to immunological challenges.

Clinical significance

Epithelial cell infected with Chlamydia pneumoniae

The slide shows at (1) an epithelial cell infected by Chlamydia pneumoniae; their inclusion bodies shown at (3); an uninfected cell shown at (2) and (4) showing the difference between an infected cell nucleus and an uninfected cell nucleus.

Epithelium grown in culture can be identified by examining its morphological characteristics. Epithelial cells tend to cluster together, and have a "characteristic tight pavement-like appearance". But this is not always the case, such as when the cells are derived from a tumor. In these cases, it is often necessary to use certain biochemical markers to make a positive identification. The intermediate filament proteins in the cytokeratin group are almost exclusively found in epithelial cells, so they are often used for this purpose.

Cancers originating from the epithelium are classified as carcinomas. In contrast, sarcomas develop in connective tissue.

When epithelial cells or tissues are damaged from cystic fibrosis, sweat glands are also damaged, causing a frosty coating of the skin.

Etymology and pronunciation

The word epithelium uses the Greek roots ἐπί (epi), "on" or "upon", and θηλή (thēlē), "nipple". Epithelium is so called because the name was originally used to describe the translucent covering of small "nipples" of tissue on the lip. The word has both mass and count senses; the plural form is epithelia.

Neurulation

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Neurulation  

Neurulation refers to the folding process in vertebrate embryos, which includes the transformation of the neural plate into the neural tube. The embryo at this stage is termed the neurula.

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.

Early brain development

The anterior portion of the neural tube forms the three main parts of the brain: the forebrain (prosencephalon), midbrain (mesencephalon), and the hindbrain (rhombencephalon). These structures initially appear just after neural tube closure as bulges called brain vesicles in a pattern specified by anterior-posterior patterning genes, including Hox genes, other transcription factors such as Emx, Otx, and Pax genes, and secreted signaling factors such as fibroblast growth factors (FGFs) and Wnts. These brain vesicles further divide into subregions. The prosencephalon gives rise to the telencephalon and diencephalon, and the rhombencephalon generates the metencephalon and myelencephalon. The hindbrain, which is the evolutionarily most ancient part of the chordate brain, also divides into different segments called rhombomeres. The rhombomeres generate many of the most essential neural circuits needed for life, including those that control respiration and heart rate, and produce most of the cranial nerves. Neural crest cells form ganglia above each rhombomere. The early neural tube is primarily composed of the germinal neuroepithelium, later called the ventricular zone, which contains primary neural stem cells called radial glial cells and serves as the main source of neurons produced during brain development through the process of neurogenesis.

Non-neural ectoderm tissue

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 ).

Neural crest cells

Main article: Neural crest

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.

Neural tube defects

Main article: Neural tube defect

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

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Gastrulation 

 

Gastrulation
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.

• The ectoderm gives rise to epidermis, the nervous system, and to the neural crest in vertebrates.
• The endoderm gives rise to the epithelium of the digestive system and respiratory system, and organs associated with the digestive system, such as the liver and pancreas.
• The mesoderm gives rise to many cell types such as muscle, bone, and connective tissue. In vertebrates, mesoderm derivatives include the notochord, the heart, blood and blood vessels, the cartilage of the ribs and vertebrae, and the dermis.

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:

1. Invagination
2. Involution
3. Ingression
4. Delamination
5. Epiboly

Etymology

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 γαστήρ gastḗr ("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 between protostomes 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.
• Cleavage
  • Protostomes have what is known as spiral cleavage which is determinate, meaning that the fate of the cells is determined as they are formed.
  • Deuterostomes have what is known as radial cleavage that is indeterminate.

Sea urchins

Further information: Sea urchin § Development

Sea urchins have been important model organisms in developmental biology since the 19th century. Their gastrulation is often considered the archetype for invertebrate deuterostomes.

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.

Amphibians

The frog genus Xenopus has been used as a model organism for the study of gastrulation.

Symmetry breaking

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.

Amniotes

Overview

In amniotes (reptiles, birds and mammals), gastrulation involves the creation of the blastopore, an opening into the archenteron. Note that the blastopore is not an opening into the blastocoel, the space within the blastula, but represents a new inpocketing that pushes the existing surfaces of the blastula together. In amniotes, gastrulation occurs in the following sequence: (1) the embryo becomes asymmetric; (2) the primitive streak forms; (3) cells from the epiblast at the primitive streak undergo an epithelial to mesenchymal transition and ingress at the primitive streak to form the germ layers.

Symmetry breaking

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.

Epithelial–mesenchymal transition – loss of cell adhesion leads to constriction and extrusion of newly formed mesenchymal cell.

Germ layer determination

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 techniques using 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.

Cerebral organoid

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Cerebral_organoid 

A flask containing human cerebral organoids

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.

An embryoid body cultivated from pluripotent stem cells is used to make an organoid. Embryoid bodies are composed of three layers: endoderm, mesoderm and ectoderm, which has the potential to be differentiated into different types of tissue.

A cerebral organoid can be formed by inducing ectoderm cells to differentiate into 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.

Applications

See also: Aging brain, Neurobiological effects of physical exercise, Wetware computer, Metabolome, and Neurogenomics

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.

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 difference to other apes), human enhancement and improving intelligence, identifying detrimental exposome impacts (and protection thereof), or improving brain health spans.

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 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.

Ethics

See also: Neuroethics and Wetware computer § Ethical and philosophical implications

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. 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.

Humanized animals

See also: Humanzee and Human intelligence § Non-human intelligence

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.