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Epigenetic Regulation and Differentiation. In this class, we will talk about epigenetics and its mechanisms, the epigenetics of cell differentiation, cell reprogramming and stem cells, and global epigenetic modifications that incorporate three types of changes. Some affect the germline and trophoblast, others genomic imprinting, and others X chromosome dosage compensation. The path of differentiation requires the loss of potentiality. Differentiation is a gradual process involving several cell generations. Possible fates are gradually restricted, as indicated by Waddington's epigenetic landscape, where the zygote is represented by this higher ball that will descend on its developmental path and make differentiation decisions, like the first one here, left, right, which will then determine the progress towards a different fate that will again require a decision towards one side or the other. At each step, the possible fates of the cell are restricted, and reprogramming to recover potentiality is very difficult and, in nature, irreversible. Only from a methodological in vitro point of view can we recover the capacity by reprogramming cells towards more potent stages than they are currently in. Here, then, in terms of potential, we have a cascade of decreasing potential from the zygote towards the functional differentiation of neuronal cells, already differentiated neuroendocrine cells, and where in embryogenesis these cells are found. Epigenetics involves variations in chromatin and not in the DNA sequence of a cell. Changes in the capacity or organization of DNA then modify gene expression. Waddington's epigenetic landscape is a metaphor for how gene expression regulation modulates development. Waddington asks us to imagine a ball corresponding to the zygote rolling downhill towards the viewer. There is a first alternative to roll to the right or to the left. Along the subsequent paths, a second similar alternative appears. The marbles will show the grooves on the slope and will stop at the lowest points. These points represent cell fates, i.e., tissue types. Thus, the initial zygote will, as its cells duplicate and begin to determine different decisions, undergo changes at the epigenetic level, as the genome will remain intact throughout embryonic development. Each time a cell makes a differentiation decision, it will have or undergo a new change in its epigenetic profile. During development, external or environmental factors, such as, for example, exposure to a morphogen, alter gene expression, and this leads to specific changes in chromatin structure. These changes are persistent and heritable to cellular progeny during the mitosis of daughter cells that underwent the epigenetic change. These changes can, however, be reversible under very particular conditions, such as cell reprogramming. Let us then see what these epigenetic modifications that occur during development consist of, at what levels they occur, and what effects they produce on gene expression. The selective expression of genes that will give rise to the protein pattern determining the identity of the differentiated cell is controlled by chromatin, which acts as a filter for the use of genomic information. Chromatin acts at various scales of size, from the topological organization of chromatin, where different colors represent different topological domains determined by the association of different proteins, to chromatin accessibility, where the triangles show regions of DNA depleted of chromatin, of nucleosomes, and therefore accessible to interaction with the proteins indicated in blue and red. Here we see in gray the more assembled nucleosomes. Then, the modifications, of which are shown here as colored circles representing each of the different covalent modifications that can be performed on the terminal tails of histones. And finally, DNA methylation at cytosines, which we saw previously. Chromatin is then decorated by modifications and binding of different factors that are specific to the function of the sequence of the transcriptional state. In this case, several of the most important factors are listed. In the case of, for example, H3, H2, H4, they represent the specific modified histones, and the modification is indicated by the type of residue, for example, lysine four, position four in the protein, and in this case, for example, a methylation or a dimethylation or a trimethylation, meaning three distinct modifications on histone H3 at the same residue. And as well as methylation, there are acetylations, ubiquitinations, and phosphorylations. We also have specific proteins that mark, like CTCF and cohesin, the boundary of different topological domains; they act as a barrier to the expansion of the chromatin state at the chromatin level. Here we also have RNA polymerase, whose association also marks a particular chromatin state, and here DNA methylation as a specific chemical change. The type of modifications, the set of modifications that a DNA region has, will determine whether the gene included within the region is active or repressed. And note that we know today that in active genes, enhancers are associated with this type of intensely indicated modifications and a low level of DNA methylation, as well as an absence of this type of modifications. This is slightly different for the promoter and the gene body, where there will be another type of modifications that identify active genes. In regions that are chromatin insulators, we see these that I showed you previously. Now, when a gene is repressed, note that the type of modifications of its chromatin are totally different, with all the activating chromatin marks disappearing and now maintaining other, very different types of modifications that maintain the repressed state of the chromatin. Thus, there is a greater increase in methylation at all levels of the gene. Purely heterochromatic regions, note that they also have an intense density of these types of repressive modifications. Therefore, epigenetic modifications in development occur at several levels: chromatin packaging, nucleosome positioning, histone modifications, and DNA methylation, and they produce different effects on gene expression, activation, and repression of varying intensities. We therefore know in what different epigenetic states a gene can be. However, how is it possible for epigenetic patterns to be modified over time during development? How is it possible for epigenetic profiles to be established in response to induction at a specific moment of development, for example, with a morphogen, and then maintained as a fixed memory in a specific cell lineage? The epigenome changes during development in response to inductive signals that activate chromatin-modifying proteins. Various modifying proteins alter the chromatin state in response to signals. Among them, we distinguish enzymes called writers and enzymes called erasers. Among the writers that modify histones are histone acetyltransferases and histone deacetylases. We also have enzymes that add methyl groups to histones, histone methyltransferases or demethylases. Similarly, we have writer enzymes for writing DNA methylation, known as DNA methyltransferases, and eraser enzymes for methylation, known as TET enzymes. And then we have other enzymes for each of the types of modifications we have seen on histones, such as phosphorylations and ubiquitinations. Once modifications are established due to the modification of the activity of these enzymes in response to a signal, they are inherited and maintained through cell divisions, even when the initiating signal is no longer present, ensuring the memory of the epigenetic identity of the cell lineage. Let's look in more detail at the changes that occur specifically at the chromatin level. Here we have a diagram summarizing the possible modifications of the histone octamer that forms the nucleosome in the terminal tail of the histones. Primarily, I would like you to observe the two types of histone-modifying proteins we saw, which are writer enzymes that add modifications to histones and eraser enzymes that remove these modifications. Furthermore, the diagram shows another class of proteins, readers. These reader proteins, some of which are nucleosome modifiers, bind to the histone modifications produced by the writers and are also capable of altering adjacent nucleosomes, thus propagating the chromatin pattern along the chromosome until blocking signals are encountered that separate chromatin domains. Nucleosome remodeling proteins, which are included in this complex of reader proteins, can be of these types. For example, this is a reduced list, and they have histone-binding domains such as bromodomain, chromodomain, etc. Nucleosome modification and remodeling work together to increase DNA accessibility, and in other cases, to completely silence DNA regions. Once an epigenetic signature triggered by developmental signals has been established, it must be maintained to ensure lineage identity. What allows cells to maintain the epigenetic configuration of their own lineage? The answer is, chromatin patterns are inherited by daughter cells, making epigenetic memory possible. Once chromatin has been remodeled in response to a developmental signal, the established epigenetic pattern must be inherited by daughter cells to maintain cell lineage identity. This is possible because when the cell proliferates, not only the DNA sequence is replicated, but also the chromatin state in which it is packaged. Let's observe a replication fork with two newly synthesized strands, where the old histones have dark colors and the new ones have lighter colors. As the replication fork passes with the two newly synthesized strands and the parental double helix opens with the old nucleosomes, the old H3 and H4 tetramers remain attached to one of the daughter duplexes randomly and will never be released from the DNA into the free histone pool. On the contrary, the H2A-H2B dimers are released into the local environment, becoming available for new nucleosome assembly in the vicinity of the fork. Recycled and new histones will associate to restore half of the missing nucleosomes in each strand. The distributive inheritance of old histones during chromosome duplication provides a mechanism for the propagation of the parental histone modification pattern. Through this mechanism, old modified histones will tend to rebind to daughter chromosomes in a position close to their previous position on the parental chromosome. Old histones have an equal probability of binding to either of the two daughter chromosomes. This localized inheritance of modified histones ensures that a subset of modified histones is located in similar positions on each daughter chromatid. Let's observe what happens with the acetylation of histones H3 and H4, indicated by the blue arrows. Acetylation itself recruits a histone acetyltransferase through the binding of a bromodomain to the modified residue. That is, histone modifications are used to recruit enzymes that perform the same modifications on neighboring nucleosomes, facilitating the propagation of the modification to both daughter chromosomes. While this is an example of an activating modification, both activating and repressive chromatin structures occur. Let's look at an example of the inheritance of repressive chromatin complexes. Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are widely used repressors to epigenetically silence DNA regions throughout development. The PRC2 complex is recruited by sequence-specific DNA-binding proteins, such as the FORC complex, which recognizes Polycomb response elements (PREs) in the DNA. PRC2 contains a histone methyltransferase that trimethylates lysine 27 on the H3 histone tails. This methylation leads to the recruitment of the PRC1 complex, which is believed to condense chromatin or lead to the placement of a nucleosome at or near the transcription start site. PREs are often found near promoters. A classic example of Polycomb repression occurs at the level of Hox genes early in the anteroposterior axis development. Like chromatin patterns, DNA methylation patterns are also inherited by daughter cells. Methylcytosines are generated from a reaction catalyzed by a DNA methyltransferase from a cytosine. This modification is reversible by the action of TET enzymes or 10-translocation, which catalyze the oxidation of 5-methylcytosine in various steps to cytosine. There are two types of DNA methyltransferases. Type 3 can methylate any cytosine in response to an appropriate signal, such as a developmental signal, thus being a de novo chromatin remodeler. Meanwhile, Type 1 DNA methyltransferases only methylate cytosines in CpG dinucleotides of the DNA of newly synthesized cells during DNA replication. Therefore, they are responsible for maintaining the methylation level and transmitting it to daughter cells. Here we have the de novo action on any DNA sequence by DNMT3A or 3B, and conversely, the maintenance action by Type 1, which places methyl groups on the DNA in newly synthesized strands. Finally, it is important to mention that although epigenetic changes occur at different levels of DNA compaction in chromatin, there is extensive interaction between these levels. As an example, we see the interaction between DNA methylation and nucleosome modification. The methyl-CpG-binding protein 2 (MECP2) recognizes DNA methylation and is capable of recruiting histone deacetylases, which remove acetyl groups, or it can also recruit histone methyltransferases that add methyl groups. Both of these modifications promote nucleosome stability and strong DNA packaging, thus collaboratively repressing gene expression. That is, DNA methylation causes gene silencing by itself, but MECP2 binding recruits nucleosome remodeling complexes that are capable of increasing silencing and achieving complete silencing of genes in that region. As we mentioned, DNA methylation regulates gene transcription. The addition of a methyl group (CH3) to cytosines in the promoter decreases the binding capacity of transcription factors and polymerase, similar to what occurs in enhancer regions that regulate gene expression. Mammalian gene promoters have continuous clusters of CG bases, forming what are called CG islands. Here, C refers to C on one strand and G on the complementary strand. This is an example of a gene denoted as having a CpG island. This is the promoter. This would be an enhancer region. You see stretches rich in C and CG dinucleotides here on one strand and on the other, and in this case, it is available for the association of Egr1, the transcription factor that would be promoting the transcription of this gene by RNA polymerase II in this scheme. However, when these red dots, representing methyl groups covalently attached to cytosine, are added, the association of this factor is inhibited, and therefore it cannot promote gene transcription, and the gene is inactive. These conformations called CG islands are found in one-third of mammalian gene promoters, meaning they are highly regulated in this way. Not all are used, but many are. And this is something that does not exist, it is not as common in other animal groups, for example, Drosophila does not have it, and it is used as a silencing strategy that often operates long-term and is also heritable. This is an example of the change in globins from the embryonic to the fetal stage. In this case, at six weeks, you can see that on this chromosome, there are two genes, one after the other, for globins, epsilon-embryonic and gamma, which is silenced at six weeks because its promoter is methylated in the DNA. However, a few weeks later, around six weeks, a specific remodeling of that site occurs, which removes the methyl groups from the cytosines of the gamma-globin promoter, while methyl groups are introduced into the epsilon-globin promoter. Therefore, a shift occurs towards the expression of gamma-globin. This will also happen later with beta-globin. So, while the absence of methylation allows gene transcription, it should also be remembered that it is not sufficient for transcription to occur, but it is necessary. There are four mechanisms that allow a cell to inherit its information epigenetically. In the two above, part A, epigenetic mechanisms are shown that are said to act in cis because the molecule inducing the change acts on itself. Here, on one hand, we have DNA methylation; a non-methylated region can be methylated, and that methylated region can be inherited by daughter cells through the action of DNA methyltransferases that work when DNA replicates, generating a daughter strand with the same methylation characteristics as the parental strands. This same process now occurs at the level of histone modifications, which can go from active chromatin states to inactive chromatin states, which are also replicated during DNA replication and transmitted to daughter cells. Epigenetic regulation also occurs in ways that do not involve direct changes associated with DNA. In this case, these are mechanisms that act in trans, meaning the inducing molecule, for example, acts on another molecule, in this case, DNA. Here we have a cell that does not synthesize a protein and begins to synthesize protein A in response to a signal. And since this protein is a transcription factor for its own gene, a positive feedback loop is produced, and these types of positive feedback loops can be transmitted to daughter cells as long as factor A is in the cytoplasm of the mother cell and can pass to the daughter cells. Something different that we have not seen is the epigenetic propagation of a cellular state via proteins. In this case, normal proteins can change their conformation, misfolding and producing protein aggregates or misfolded proteins like prions. These misfolded proteins propagate the misfolding of proteins of the same type, and this propagation is passed from a daughter cell to its mother cells. Excuse me, from mother cells to daughter cells. Cells can combine these mechanisms to ensure that gene expression patterns are maintained and inherited accurately and reliably over a period of up to 100 years or sometimes more in our own case. Having thus seen the general mechanisms governing epigenetic regulation, let us now look in more detail at the effect of epigenetics on cell differentiation itself. In development, epigenetic changes that lead to the activation and maintenance of differentiation programs are induced in response to extracellular signals. These signals can consist of ligands, a gradient, a concentration of certain ligands, a combination of several of them, or communication with neighboring cells or the extracellular matrix. And this will lead to interaction in the recipient cell and signal transduction, which at some point will lead to a modification in the combination of transcription factors and chromatin, or also hormones that can enter the cell directly and activate some transcription factor intracellularly. This will then cause new gene transcription, transcription factors to bind to DNA, and specific genes to be turned on and off in response to these signals. And these patterns will have to be maintained in subsequent generations by the chromatin transmission mechanisms we saw previously: positive feedback, DNA methylation, and changes in chromatin state. That is, epigenetic changes in differentiation produce activation that leads to gene silencing and gene activation maintained by these mechanisms. Let's look at an example of transcription factor activation by an epigenetic mechanism that we saw last week in group discussion one, referring to this gene that encodes MyoD, which is a master transcription factor for muscle differentiation. Here we have a group of cells in the somite, specifically the region known as the dermomyotome, which will be the precursors of skeletal muscle cells. And these cells, as you can see, are located in a place where they are exposed to various signals from different surrounding structures. In this case, what we are seeing is a chicken embryo of two to four days, a cross-section at the trunk level. This shows the neural tube, the notochord, the epidermis, the somites, and the lateral plates on both sides. So, these cells are exposed, as you can see, to the action of a gradient of Wnt ligands, Wnt1 and Wnt3a, which is stimulated by the expression of BMP4 and occurs from the dorsal portion of the neural tube. They are also exposed to the expression of Sonic Hedgehog or Shh, which is secreted by the notochord, and also the basal plate of the neural tube. On the other hand, BMP4 and FGF8 are secreted from the lateral plate, and the combined action of Wnt here also manages to trigger the expression of Pax3 transcription factors, which are the most important activators of MyoD expression. Here we have MyoD, which we will now recall what it does and the importance of its epigenetic regulation, and how it also controls the epigenetics of the genes it regulates later. This is the region we are observing in particular, and I want you to see the same longitudinal section, but by scanning microscopy, where you can see the actual structures like the notochord, the neural tube, the somites, the epidermis. And how you can visualize the individual cells that are occupying these structures and that do not yet have clear differentiation. The progenitor cells of the somites we just saw will transform into myoblasts, where the expression of MyoD begins. After MyoD initiates a cascade of downstream regulatory events, the cells will then begin to form multinucleated microtubules characteristic of skeletal cells, and then mature muscle fibers, fully differentiated, will form. This would then be the stage where the expression of genes that will irreversibly lead the cell to its muscular fate appears. This is known as determination; it depends on extracellular signals, as we saw in the previous diagram, precisely located in time and space and at an adequate concentration to be interpreted by the somitic cells, which are competent because they have already had a history of chromatin remodeling that allows them, for example, to express Pax3, which will be a competence factor at that moment. And the expression of genes like MRFs causes Pax3 to then activate the expression of MyoD, which in turn, remember, after being translated, enters the nucleus and again activates the transcription of its own gene, creating a self-regulatory loop that maintains its expression from then on in the differentiated cell. The activation of Myf5 and MRF4 also occurs, which are also factors of myoblasts, where other transcription factors are expressed in the vicinity. At this moment, the expression of these factors does cause the synthesis of proteins specific to the myotube, and which, mainly with myogenin, will finally achieve the maturation of these cells into a differentiated muscle cell. You will recall from discussion one that the expression of MyoD, which was off before these signals arose around the dermomyotome, will be triggered by the removal of epigenetic repressors that were blocking the regulatory regions of the gene. Thus, we go from MyoD chromatin, which was repressed, to active chromatin with the characteristics we recently saw, due to these signals. And the other interesting thing is that MyoD, being a transcription factor itself, is capable of binding to the DNA of all these genes, or several of these genes involved in differentiation, and thus also changing their chromatin state, making them epigenetically activated and therefore transcribing all of them, including others. This will allow the muscle fiber cell to acquire that muscle-specific gene pattern. Recently, the roadmap of the human epigenome was deciphered, and this was carried out by a consortium of many research centers that worked on determining the epigenetic patterns of humans at the level of many organs and tissues, different stages of embryonic development, and the first steps of development with their different cell types. This figure shows the different analyses performed on each of these cells, involving the transcriptome, i.e., the determination of all transcriptions of histones, how accessible chromatin is throughout the genome. And we also have analyses of chromatin structure in the nucleus and DNA methylation analysis, among others. That is, for each cell, in each place and at each time, all these aspects of its chromatin situation were determined. This was done for around 111 different regions. One of the results obtained is that if we, for example, compare all or several of the analyzed cells, considering only the distribution and quantity of this specific modification of H3K4me1, and we compare one cell with another, we manage to order the cells based on their common ancestors. For example, this entire group of green cells are T cells from different subclasses, and you can see that they all come from a common ancestor that can be identified by this type of histone modification present in differentiated cells. Look in more detail here; different colors mark different lineages, cells derived from the same precursor cell that can be perfectly ordered based on just one type of histone modification. Another interesting finding is that by observing epigenetics, regulatory modules determined by the epigenome can be deduced. If you look, for example, at this entire group of genes in all these different tissues, they correspond to genes that are essential for maintenance in practically all pluripotent stem cells. They share some of them, but not all. Then we have specific genes, for example, that are related to neural crest development, to the development of specific structures that are associated with specific tissues, which are epigenetic modifications that allow the expression of gene groups that define specific tissues. Let's focus now on the possibility of cell reprogramming and what the concept of stem cells and their potential applications implies. The diversity of cell types that a stem cell can generate in vivo defines its natural potency. During development, we start from a totipotent cell to a successive loss of potentiality, going from pluripotent, multipotent to a progenitor, a precursor, and a differentiated cell. This is a particular example of neuron differentiation, but it is the same that occurs in other tissues. If you observe the diagram, it indicates which cells have these levels of potentiality during development: zygote, embryonic cells, stemosomes, multipotent stem cells, progenitors, etc. And at the corresponding moments of development: the zygote, the blastocyst, the inner cell mass, and then the embryo and different regions of the tissue in question. Totipotent, then, is a cell capable of producing both embryonic and extraembryonic tissues and all cell types. It occurs in a zygote of four to eight cells in mammals, and then the potentiality of embryonic cells begins to decline. Pluripotent is the cell capable of producing all cells of the embryo, but not extraembryonic ones. In a pluripotent cell, the cells are not yet determined. And here we have the inner cell mass and extraembryonic cells as examples of pluripotent cells. Multipotent. These are cells capable of generating cell types with more restricted specificity for the tissue in which they reside. These cells are then determined for a particular tissue but can give rise to different cell types within that tissue. We are talking here about adult stem cells from adult tissues. A precursor is designated in a less specific way to any ancestral cell type. As cell populations within each germ layer expand and differentiate, resident stem cells are maintained within these developing tissues. These stem cells are multipotent and function to generate cell types with restricted specificity for the tissue in which they reside. From the embryonic to the adult intestine, small intestine, or neural tube to the adult brain, multipotent stem cells play critical roles in fueling organogenesis in the embryo and regeneration in adult tissues. Adult stem cells have the capacity for self-renewal but have a finite number of generations to do so. Adult stem cells have the capacity to divide symmetrically to produce self-renewal and can also divide asymmetrically to produce a cell one step further in its determination pathway, called a determined cell. A committed stem cell produces one or very few cell types to a progenitor cell that is of transient amplification, which can proliferate for several divisions and then commits to differentiating into a particular cell type. Having reviewed these concepts, let's look at Waddington's epigenetic landscape from the perspective of developmental potential and cell epigenetic states at each stage of development. Violet indicates the cell with the highest potential, totipotent potential or zygote, which has global DNA demethylation. A little further down the hill, we find pluripotent cells whose identity we have already mentioned. From an epigenetic point of view, they have active X chromosomes, global repression of differentiation caused by proteins of the Polycomb complex group, and also low methylation of their promoters. These are conditions that allow pluripotency. As cells decrease their proliferative potential and their differentiation potential, they move further down, becoming multipotent cells, including adult stem cells, or reprogrammed cells, which we are not discussing now. These cells already have X chromosome inactivation, repression of lineage-specific cells active by Polycomb proteins, and now have hypermethylated promoters. Finally, when the cell achieves its terminal differentiation state, it has a single cell type. In this case, X chromosome navigation continues, but there is derepression of lineage genes that were silenced by Polycomb complexes, and promoter hypermethylation is maintained. Thus, this is a more modern view of the specific chromatin situation at different developmental stages. Embryonic stem cells can be obtained from blastocyst cells, from the inner cell mass, or from the primordial germ cells in the fetus, and can be cultured in a controlled culture system to maintain their undifferentiated state and prevent their differentiation or proliferation. This has been developed in the last two decades, and today we have systems for culturing stem cells or pluripotent stem cells in laboratories. The mechanisms of stem cell regulation are diverse. This diagram shows an overview of these systems. It shows general external characteristics and internal molecular mechanisms that influence the quiescent state, the proliferative state, or differentiation. In the case of stem cells, there are adult stem cells in adult tissues that ensure the cell number is restored in case of injury or cell death in tissues exposed to erosion or natural cell death, such as skin or mucosal tissues. The basic functions of these cells are therefore self-renewal and differentiation. Now, how is it regulated that these cells remain quiescent until they are needed to meet needs, either in morphogenesis or in adult tissue? This regulation is highly influenced by the microenvironment surrounding the stem cell and has been termed the stem cell niche. The evidence we have implies that all tissues have a unique stem cell niche, even though there are many differences in the type of niche depending on the cell type. In principle, it involves extracellular mechanisms that lead to changes in cellular behavior. Extracellular mechanisms involve physical influences, including adhesion factors between cells that support the niche architecture, different types of cell-cell or cell-extracellular matrix adhesion, as well as cell density in the niche, which can also be altered by mechanical forces and by chemical regulation, such as the proximity and exposure to certain molecules, for example, signals from other surrounding cells that signal to maintain either the quiescent state or trigger proliferation. What is a stem cell? A stem cell is a cell that has the capacity to generate cells with different phenotypes, meaning more than one cell type. Normally, they are cells that are quite undifferentiated and, in response to a stimulus, can generate a cell that is determined towards a cell type and can become a differentiated cell that is different from itself, the origin cell. It has been shown that stem cells have the capacity to divide symmetrically, generating a cell identical to itself. Therefore, of the same potentiality state, this is called self-renewal, but they also have the capacity to divide asymmetrically, generating a cell different from themselves through asymmetric division. This different cell, called a determined stem cell, in this case a multipotent stem cell, is also capable of advancing one step further in determination, to the point where at some point they form what are called progenitor cells, called transient amplification cells, which are capable of proliferating intensely for a brief period of time. And then, in this case, these cells will be able to differentiate towards specific fates that will depend on the potentiality of the original stem cell. Stem cells can be produced by induced reprogramming and are called induced pluripotent stem cells (iPSCs). These cells are produced by isolating skin fibroblasts, for example. It is now known that other tissues can also be used, but they are differentiated adult cells to which reprogramming factors such as Oct4 and the other three factors are forcibly incorporated. These reprogramming factors, naturally expressed in stem cells, are capable of making the cell regain pluripotency and be capable of self-renewal and, in response to appropriate inductive signals, give rise to various cell types. In principle, this has many applications, some of which are underway and quite advanced, while others are not yet. But with these cells, taken from adult cells and their potentiality restored, we can not only correct genes that are, for example, faulty, to generate cells that can refill a tissue damaged by transplantation into the patient's own organism, avoiding rejection because these are the patient's own cells. I mean immune rejection. They can also be used to derive them into specific tissue organs and use them as disease models to discover drugs or compounds that may be useful for treatment. It is possible to induce the differentiation of stem cells into different specific cell lineages. This is achieved by mimicking the steps of mammalian epiblast development, using a set of paracrine signals, which are what cells normally receive to reach final differentiation. These signals must be added sequentially, in a well-ordered manner, and at adequate concentrations, mimicking what these cells are naturally exposed to when reaching differentiation. Thus, the addition of specific growth factors in the ordered sequence they receive can determine the acquisition of the three germ layers of a normal embryo. At the same time, organoids or rudimentary organs can be produced from the in vitro differentiation of stem cells. This is done with various strategies that also use specific growth factors, conditioned media that promote the morphogenesis of specific tissue types, such as organs. In most cases, these are isolated induced cells or also adult pluripotent cells specified towards one of the three germ lines, which then differentiate more specifically into defined fates, such as a type of organ. And often, a three-dimensional matrix is also required to allow the generation of an architecture similar to that of an organ. In this sense, these stimuli with these different morphogenic factors will lead the cell to express specific tissue genes, but fundamentally to acquire an architecture that depends on cell-cell interactions for the construction of layers and tissues that often resemble essential characteristics of tissues that normally originate with a much lower level of complexity. This image schematically shows the production of cerebral organoids by adding to the medium, a medium that is also very special, designed for stem cells so that they do not differentiate. Basic fibroblast growth factor is added, a medium specifically for neuronal induction. Note that there is a clear protocol in terms of timing, as well as the specific concentrations and compositions of each medium. And then an artificial extracellular matrix, which provides architectural support and allows interactions and the deposition of a specific extracellular matrix that will be a fundamental mediator of the generation of three-dimensional structures of these organs. This can also be done using bioreactors and, well, and more morphogens, in this case, retinoic acid. And here you can see microscopic images of the structures generated at each of these stages. In this micrograph, you see a section of a cerebral organoid stained with three antibodies conjugated to different fluorophores that recognize the specific proteins indicated here as SOX2, TH, and nuclear DNA in blue. And you can see how the different neurons and neuronal progenitors are located in very different positions and fold and interact in quite different structures. There will be other cells that do not express these markers, so they cannot yet be categorized as neurons or neuronal progenitors, but are likely other types of cells, such as support cells. In the following micrographs, immunohistochemistry, a radial glial cell marked with an antibody against vimentin is shown, which is undergoing division and shows morphological characteristics with a long apical process, an apical membrane, and a long basal process, reminiscent of a cell from the ventricular lumen type.