Mestrado em Bioengenharia – Bioengenharia Molecular PDF

Mestrado em Bioengenharia – Bioengenharia Molecular 1º Semestre- 1ºAno-2024/2025 Engenharia Celular e Biologia de Células Estaminais Conteúdo Part 1 – stem cell fundaments........................................................................................... 2 Part 2 – therapeutic applications..................................................................................... 58 HSCs........................................................................................................................... 58 MSCs.......................................................................................................................... 65 Apoptotic MSCs for immune regulation - inhibit T cell proliferation................... 72 Neural Stem cells........................................................................................................ 74 Cellular engineering................................................................................................... 83 Cell engineering at a genetic level – gene engineering.......................................... 85 Cell bioprocessing.................................................................................................. 91 1 Note: analysis of an article 1. Complete read-through 2. Summarizing the article a. Conceptual body: theory behind the article b. Methods: which and why are they used; what other techniques could be used instead to obtain the same results c. Results: critical analysis of data, contextualized with the aims and methods used Part 1 – stem cell fundaments Nowadays, scientific magazines tend to overvalue the usage of new and novel technologies and methods, most of the time in detriment of the original scientific objective. This leads to conclusions that are far too grandiose and over-reaching for the data, controls and variables, that was in fact obtained. A thorough revision will thus gain great importance, guaranteeing that all conclusions are sufficiently backed up by data, if the statistical analysis has no calculation errors and makes use of a significant enough population, if the language used is universally understood and scientifically correct, etc. When one hears about pluripotent stem cells, those can be either induced or embryonic. These two differ in their origin, as the name suggests an induced pluripotent stem cell will be obtained by reprogramming an adult cell back to its pluripotent state, while an embryonic pluripotent stem cell will be obtained from an embryo. To note these can be obtained from the blastocyst, but also from the epiblast. A pluripotent stem cell is often defined as one capable of differentiating into a cell of any other lineage, except those that make up the extra-embryonic tissues, that is, the placenta. The name stem cell seems to be because all other cells stem from it, and it is, in its most literal sense, the stem father as well as the stem mother of all the countless generations of cells which later on the multicellular organism is composted, says Haeckel. Due to this definition, it becomes a necessity to define a tissue. In a certain way, one can think of a tissue as an association of cells organized in a given structure to fulfill one specific function or set of functions. It will thus have a cellular component, and one extracellular component, the extracellular matrix. In regards to the cellular component, it can include differentiated cells and their progenitors so that there can be renewal of the tissue, but it isn’t always as so. Not all tissues have the capacity to regenerate, or at least it’s not proven that all do. It is common belief that heart tissue, cartilage in adults, and neural tissue do not have the capacity to regenerate, even if recent knowledge suggests that there is some regeneration for neural tissue outside of the central nervous system. 2 One can consider that the renewal or turnover of the skin happens every 50 days, give or take. For the intestinal epithelium, with is villosities and microvillosities, has a much faster turnover of roughly 4 to 5 days. For blood, however, it is difficult to say a single number, since it has an incredible cellular diversity in circulation. There’s red blood cells and leucocytes, but within these there’s innumerous cell types belonging to the immune system, from NK cells, T and B lymphocytes, basophiles, monocytes, neutrophils, to other types that are still being characterized today. Some blood cells have a turnover of some hours, but others can take months, or even years as is the case for memory cells. The turnover of each tissue can vary from individual to individual, it will depend on their ontogenesis, the history of the organism’s formation from the egg that will generate all other cells. The liver will be different situation compared to the other three mentioned before. The cell turnover for this organ isn’t as fast, but it has the amazing capacity to recover from a large lesion or damage, restoring itself to the original volume quickly. What are the elements/factors responsible for the accelerated turnover in the other tissues, but are missing in the liver? Could it be active progenitor cells? It becomes necessary to distinguish a stem cell, a progenitor cell and a precursor cells, despite the fact these terms sometimes come up as synonyms in literature, especially older texts. 3 How is it, that all our body’s cells derive from such a small number of stem cells? Essentially, due to cellular division. A non-differentiated cell divides to give origin to a more differentiated cell that has less potential, and also another with the same characteristics as the original, that is, with the same differentiation potential. It is believed that it is the progenitor cells that are responsible for generating the majority of differentiated cells, due to a phenomenon called amplified transit, where there is more cell division. In fact, this amplified transit is present in the skin, blood and intestine. Semantically, progenitor could be used to describe stem cells and precursor cells, however, they have different differentiation potentials due to certain factors and checkpoints in the organism that lead to a change in that regard. All that to say, a stem cell will have more potential than a progenitor one, and this one more than a precursor. Going back to the characteristics of a tissue from a cellular standpoint. It has to be irrigated; therefore, it needs to have vasculature close by, which is always composed by endothelial cells no matter their type. This vasculature may also include muscle, smooth muscle cells, that are present in larger caliber vessels. For there to be extracellular matrix, there must be cells who produce it, such as fibroblasts. Despite the fact that fibroblasts are all considered simple and identical to each other, in truth there are certain molecular patterns on the inside of the cell itself that differ depending on their tissue of origin. A fibroblast from the heart will be slightly different from one found on the liver. Finally, in a tissue we have the cells of interest themselves, like cardiomyocytes in the heart, the many different types of neurons in the brain. The parenchyma will be comprised of the other cellular types that aren’t of interest in the particular study, the supporting cast one could say. For example, if one is studying the liver with an interest in the blood cells, that leaves out hepatocytes, fibroblasts, mesenchymal cells as the stroma or parenchyma of the liver. With this brief analysis, we can return to the goal at hand- the discussion of the article itself. 4 The goal was to develop a model that allows for a better recap of what goes on during the brain’s development, that is, how neurons mature and grow in vivo, and this is done through the manipulation of the microenvironment surrounding the developed organoids. A failure was identified, organoids of neural cells developed in an in vitro context could not mimic the properties and characteristics that are seen in vivo, in both architecture and overall complexity. They then identified the possible cause for this failure as a lack of stimulation from sensory inputs and signaling due to the absence of in vivo networks. It’s also worth mentioning that 2D cultures couldn’t replicate the complex networks that one can achieve by growing cells in 3D cultures, and that is why organoids were used. So, to start: 1- Identification of “problem”. Problem is a more formal definition, and may not always apply to the reason behind the research. In this situation, it does fit. Thus, the aim is to develop a model to help research a disease. 2- Strategy is to use organoids obtained in vitro as a model, and then test them in vivo. 3- Read out: the tests can also be incorporated in this part, but it mostly refers to the techniques used to assess the properties/functions of the strategy above, and as such answer the goal or problem. In this case we have a. MRI b. Immunostaining c. Electrophysiology d. Imaging e. Single cell RNA sequencing MRI and electrophysiology are compatible with a living individual, and imaging can be, but it depends on the type of imaging- on the type of tissue, the depth that needs to be reached. Immunostaining is a technique that makes use of antibodies coupled with a way to detect it, like a fluorophore. It may be necessary to permeabilize the membrane of the cell, if the target for the antibody is within the cell, but most of the time the target is a surface marker. However, finding a surface marker exclusive to a cell type is very hard. One such example is glycophorin A in humans or TER119 in mice for Red Blood Cells (RBC). Immunostaining is not so much based on the expression of a single protein, but more so on the context of expression for that cell. For example. A neural cell cannot express a marker associated with a lot of cells from the blood lineage (CD45). It also can’t express markers from oligodendrocytes, astrocytes, who are on the same environment, and they need to express 2 or 3 markers exclusive to the neuron lineage. Non expression is also called lineage negative. 5 The cell source considered is pluripotent stem cells. Specifically, they used induced pluripotent stem cells, that retain the genetic making of the individual, and as such retain any genetic mutation and therefore syndrome or disease encoded there, allowing the models to be more faithful in mimicking the specific conditions. They can, one reprogrammed, then be re-differentiated into practically any cell desired. The model used is newborn rats, and it is important that they are as young as possible due to the way the nervous system develops, as in, it doesn’t finish developing while in utero, therefore is still “pliant” enough after birth that it is possible to evaluate its development. Note: A definition of stem cells from the article - Stem cells reproduce themselves - Stem cells generate progeny destined to differentiate into functional cell types - Stem cells persist for a long time - Stem cell behavior is regulated by the immediate environment (the niche) Just what is, after all, a stem cell? We can define many “types” of stem cells: - Totipotent: o Can differentiate into any lineage o Differentiates into extra-embryonic lineages (EL) like the placenta - Pluripotent: o Differentiates into more than one cell lineage o Not differentiated (enough) o Capacity for infinite division o Cannot differentiate into EL - Multipotent From this short list it is possible to see a hierarchy of cell types based on their capacity to divide, but it’s not as straightforward as that, in the sense that the capacity to divide of a stem cell is not criteria enough to place it above or below another in terms of potency. Note: The Hayflick limit is the maximum amount of times a cell can divide before it accumulates too much damage and becomes senescent, that is, before it stops being able to divide itself. 6 Fun fact: the first iPSCs were generated from fibroblasts because of the dogma that they were somehow less differentiated and simpler, which in turn came about because scientists believed that their simple, “basic” morphology meant reprogramming this type of cells would be easier, since there were “less features to remove”. Fibroblasts are often part of the stromal cell support cast. The current goal is to immortalize stromal cells to make a layer of permanent “feeder” cells, a support layer to help the so called “noble” cells, the cells of interest like neural cells, or cardiomyocytes. Is infinite cell division an obligatory condition for a cell to be considered a stem cell? It’s not as clear cut. Multipotent stem cells have been shown to stop dividing after a while, they disappeared over generations of cell culture, but so far, the same hasn’t happened for pluripotent stem cells. However, the pluripotent stem cells don’t remain in the organism forever, they eventually differentiate into other cell lineages, losing their pluripotency. Where do they exist, then, and how was it proven they differentiate infinitely? Well, proving that they differentiate infinitely is done by expansion in vitro, in cell cultures, guaranteeing they maintain all characteristics for pluripotent stem cells in the process. In vivo, these cells only exist in the embryo, in the early stages of the pregnancy and disappear rapidly after. For a mouse, the gestation lasts for 20 days, counting the day 0,5 as the d.p.c (day post coitus) when the mice mate and there is a guarantee that the female is pregnant. The pluripotency of stem cells is only maintained in the mouse embryo until day 6,5. Multipotent stem cells are adult stem cells, they arise when the systems begin to form in the development process of new life. In truth, the formation of organ systems is the difference between the fetus and embryo stage, it is the organogenesis. Multipotent stem cells have a high division capacity and won’t be as differentiated as the other cells in the surrounding tissue. For example, HSC will differentiate into all blood cells, red or white. Note: The blood system is part of the hematopoietic system, which is the term that should be used. Blood is a mobile organ, comprised of red blood cells, erythrocytes, and white blood cells, leucocytes. These last ones are always involved in either acquired or innate immunity, and have a massive variety of cell types. Thus, multipotent stem cells have limited division capacity, but it is still broader than that of their neighboring cells. 7 There is, however, a stem cell that can only give rise to a single cell type, the ones that originate the germ cells. These will thus be called unipotent stem cells. Note: Soma, the somatic cells, are all cell types that are not the gamete, the reproductive cells. Those will be called germ cells, and there is one for male and one for female. Note: If pluripotent stem cells exist permanently only in vitro, are they actually real? Well, yes but not really. They are a good representation, in terms of genetics and behavior, of the ones that exist briefly in vivo, but they are a so-called artifact since their existence in vivo is ephemeral. A stem cell is capable of self-renewal, its division is capable of maintaining the pool of mother cells, that is, the division of a stem cell will originate a clone of itself, with the same characteristics, and another more differentiated cell. This is called asymmetrical cell division, and was thought to be the only possibility for stem cell division. However, it has been seen that in certain situations, some stem cells can divide symmetrically, giving rise to either two cells that are more differentiated, which will lead to the exhaustion of the stem cell pool, or 2 clones of itself and thus expanding the pool instead. This asymmetrical cell division is critical to the balance of stem cell self-renewal and differentiation. Much of what we know about this mechanism derives from experimentation in non-vertebrates, such as the Drosophila model, where asymmetric division predominate. We now know it may be induced by both intrinsic and extrinsic mechanisms. At the molecular level, multitudes of asymmetries, which may impact daughter cell behavior even if they may not determine the cell fate per se, have been described already. 8 The stem cell definition that we must have in consideration is the following, then: Stem cell: the self-replicating non-differentiated cell at the basis of a particular system endowed with high proliferation capacity. Thus, the key ideas here are: - Self-replication - Non differentiated - High proliferation capacity Planarias can regenerate a whole head and pharynx, a tube where food goes in and waste comes out, even from a single chunk of body. It seems to be because it is mostly constituted of pluripotent stem cells, with some slightly more differentiated ones in the mix, that allow it to do so. A blastema, which also shows up in amphibians like newts, salamanders, axolotls, is the little nub where the missing limb will grow back, and is a local gathering of cells in high proliferation. Every time we have a lesion, and not all lesions are the same nor trigger the same repair and regeneration systems, that was a lean cut like a lizard letting go of its tail to escape being trapped by a predator, the first thing the body does is closing the wound. This will prevent the loss of fluids, pathogen entry and body temperature changes. This is called epithelization, the formation of an epithelium to close the wound to the outside. There is a recruitment of cells to the so called blastema, the stem cells will reactivate and develop a specific genetic landscape to form a new limb that not only looks the same as the healthy old one, but also functions exactly the same. Stem cells seem to maintain affiliation and memory, they know what to form to create the exact same limb as before. Note: The paradigm of science is that the way organs are made during the body’s development can be used as a basis in assisting their regeneration later in life, remaking them in the eventuality of an injury. This functional repair is regeneration, the tissue formed is as good as new. It opposes mere repair, where tissue is replaced with a scar, fibrous tissue that doesn’t quite mimic the original function. For example, a lesion to the heart won’t be closed with cardiomyocytes, but with cartilage, scarring. While it doesn’t pass the impulses the same as the original cells, at least it doesn’t lead to necrosis of the apex of the heart due to the lack of oxygen that can’t diffuse through the open wound. 9 Some believe that the regeneration of a whole limb that appears in the newt is not possible in humans because they have a more immature immune system, however that has little credit so far. It is true that there seems to be an inverse relationship between the competence of the immune system and tissue regeneration. There is a delicate threshold, a balance point, where the pro-regenerative components of the immune response are maintained within the context of a more advanced immune system. During embryonic and postnatal development, the immune system regulates processes such as branching morphogenesis, ductal formation and angiogenesis. Similar functions are maintained in some adult tissues in order to maintain homeostasis. Injury or disease elicits an inflammatory response that can either promote functional restoration, regeneration, of a tissue, or a rapid healing response that may protect the organism at the expense of preserving structure and function. Inflammation often persists in wound healing and scar formation, ultimately impairing the normal function of the tissue. 10 It could be that newts build their limbs as blocks later in development than us humans. This is all regulated by a strict code of proteins and genetic expression- proteins is what differentiates each cell type, it’s how each cell communicates, how they exchange information in the form of molecules. Proteins are gathered in categories or families based on functions or other criteria, and how they interact with each other: - BMP: bone morphogenic protein - FGF: fibroblast growth factor - TGF-β: tumor growth factor - SH: sonic hedgehog - NOTCH: in notch signaling, part of the decision of symmetrical vs. asymmetrical division In the newt, a limb is grown as a block closer to the adult phase. Although it is not corroborated by the data, it’s believed that such makes it easier to reactivate the protein cascade that leads to the formation of limbs. Note: Pliotropy is the characteristic that some cytokines possess, the ability to be multifunctional, that is, to play different roles depending on the moment and what is required of it, leading to different reactions. The NOTCH family of proteins exhibit pliotropy. Regeneration on a tissue is associated with cells that have a higher turnover, while repair is associated with a lower cellular turnover. 11 Developmental biology addresses the evolution of an individual, from its formation towards maturity, passing by all stages. If the genome of all somatic cells is the same, why are there so many different cells? Differential gene expression, the way that a single genome can be expressed across different cells, is what leads to the diverse types of cells. Different cells will show different patterns of active genes, leading to the production of different protein patterns. There are many mechanisms that contribute to this: Differential gene transcription Selective RNA processing Selective RNA translation Processing/modification of proteins For example, methylation of DNA is when a moiety of methyl is added to the promoter of a certain gene in one of its cytosine residues, leading to the stabilization of the nucleosome. Transcription factors will avoid this promoter, and thus the gene will be inactivated. It is not a permanent alteration nor a decisive change in the DNA sequence itself, it is a reversible process where there are enzymes that will both promote the methylation and demethylation of the sequence. Translation factors are proteins that bind to specific zones in the genome called enhancers or repressors and change the expression of a certain gene, either increasing or suppressing its activity. They can even interact with other factors to add another layer of complexity to protein expression. Transcription factors act many a times on the crucial moments when deciding a cell’s fate. The most important transcription factors are often called master transcription factors. For example, the Yamanaka Factors, Oct4, cMyc, Sox2 and Klf4 were found to be overexpressed in pluripotent stem cells, but then their expression was lowered in more differentiated cells. Their overexpression was used to give rise to iPSC. Not all RNA produced in the nucleus is carried away from it, and thus not all RNA is processed and functional, leading to different protein expression in the long run for that cell. Alternative splicing in RNA also leads to the expression of different proteins, since certain genes that are considered exons in one splicing program may not be considered as such in another. Proteins themselves can also be modified after they are already produced to acquire different function or behavior. They can, for example, be glycosylated. RNA can be mobilized to different places in the cell due to information in their untranslated regions, UTR, that acts as signaling for molecular transport mechanisms. 12 Maternal genes, RNA, play an important role on the first divisions of the zygote, regulating the expression for the zygote until their own transcription factors can kick in and regulate all of the processes themselves. The concentration at which a protein exists will affect its function. Taking the example of morphogens, proteins important in the formation of tissues and the development of organs. Cells that produce it will secrete it to the environment around, meaning that the immediate surroundings of that cell will have the maximum concentration of morphogens. However, the factor diffuses throughout, establishing a gradient of concentration from its peak at the source, to an eventual null concentration far away. This is a way to decipher which types of cells occupy each zone relative to the source of morphogens. Depending on the way each different cell interacts and reacts with the factor, depending on the strength with which their receptors capture the signal, there will be different cell layers. This is called the French Flag Model. There are many different patterns that can be formed in terms of gradients, depending on how many morphogens are at play and how they diffuse not only in plain, but in space. Proteins often aren’t built in their functional state, they sit in the cell until a certain catalytic event that cleaves a specific domain happens due to a stimulus, allowing the protein to be activated and exert its function. The main stages in embryotic development are: Fertilization: formation of the egg 13 Segmentation/cleavage Gastrulation Organogenesis The one cell egg still needs support and help from maternal RNAs. IT than can go on to expressing its own, differentiating different cells and then tissues. This knowledge allows us to recapitulate it in vitro in order to assist in regenerative medicine. Cancer cells often show a dedifferentiated behavior, as they somehow manage to rollback their differentiation program. LOF means loss of function, it corresponds to an experiment where a certain gene is eliminated. Contrarily, GOF means gain of function and is when a certain gene has their activity increased. These are some of the manipulations one can do in a model’s genes. It allows us to see how this change in gene expression will translate in alterations of the in vivo activity of the model, like mice, C. elegans, zebrafish, drosophila, and others. These models can also undergo genetic screening tests. More recently there are human pluripotent stem cell models that allow for a broader scope of studies, in cell differentiation, manipulation of genes and high throughput models, where all the RNAs the cells produce are analyzed, together with all protein expressed. Xenopus, chicken, zebrafish, sea urchin, drosophila and mouse are the models most used in development biology, and each will have certain quirks that make them either more or less adequate for a certain study. Looking at embryos of many different vertebrates, one can see many similarities between them no matter whether they belong to a fish or a human. This could mean that the molecular mechanisms behind the embryonic development are rather conserved throughout. Indeed, this means one can be used as a rough model for the others. Sea Urchin o Advantages: ▪ Produces millions of eggs ▪ Fecundation and development occur outside of the body in the water, making it easier to manipulate o Disadvantages: ▪ Not very susceptible to mutations, and as such isn’t a very good genetic model 14 Frog (Xenopus laevis) o Advantages: ▪ Big cells, makes it easier to see and manipulate under a microscope. For example, it’s easy to do in citu hybridization where a small green fluorescence protein is coupled to a target to make it visible ▪ Easy to manipulate the embryos since the eggs are transparent o Disadvantages: ▪ Not very easy to manipulate at the genome level ▪ Complex genome that is not yet fully sequenced Fruit Fly (Drosophila) – also called the queen of genetic studies o Advantages: ▪ Small and easy to keep alive in a lab, not very costly too ▪ Fast development ▪ Small and fully sequenced genome o Disadvantages: ▪ Not a vertebrate ▪ Non transparent embryos Zebrafish o Advantages: ▪ Small and easy to keep alive in a lab ▪ Fast development ▪ Embryogenesis happens outside of the mother ▪ Transparent embryo allows for easy following of cell migration ▪ Easy to manipulate o Disadvantage: ▪ Relatively big genome Chicken o Advantages: ▪ Embryos galore ▪ Embryos develop outside of the mother ▪ Easy to manipulate embryos ▪ Amniote vertebrate, that means it has an amniotic sac ▪ Sequenced genome o Disadvantages: ▪ Hard to manipulate genome since KO technology is yet limited for this model 15 The embryo of a chicken can be monitored by cutting a small portion of the egg shell. Whenever we want to assess the evolution of the embryo, it is a matter of removing the already cut shell piece and put the egg under the microscope. When the data is gathered, we carefully put the shell piece back on and put the egg back in the incubator. Foreign cells can be grafted into a chick embryo, even if they belong to another animal entirely. A French scientist proved this by engrafting quail cells into a chick embryo that was already in the organ formation state, creating a chimeric being exhibiting both characteristics of a chic, but also of a quail like the wings of a quail on the overall body of a chic. This, however, could only happen once the chic is already on the organ development phase, earlier than that then there wouldn’t be a clear difference between what is chic, and what is quail. A chimera is defined a single organism that possesses two different genomes within itself. Using DNA and RNA probes specifically for certain markers, scientists were able to locate the precise location of where the quail gene possessing cells were within the chic. Note: techniques Western Blott is used for identifying DNA, Northern Blott for RNA and Western Blott for proteins. Mouse o Advantages: ▪ Mammal ▪ Relatively quick embryotic development ▪ Small size ▪ KO technology readily available o Disadvantage ▪ Very big genome ▪ Expensive maintenance Note: Rat is different from mouse Rat is a way bigger animal than a mouse, roughly 3 times larger which translates into different costs of maintenance and study. While the distinction is not clear in Portuguese since “rato” is rat and mouse is “ratinho”, it is a crucial difference. Rats aren’t used for genetic studies, but they can be used for behavior studies since they are larger and therefore easier to manipulate on the physical level. Mice bred in the lab are virtually clones of one another, they are inbred to reduce variables in statistic studies done. On other studies, it may be a disadvantage and that is when outbred mice belonging to other strains are brought in to diverse the pool. 16 Different strains of mouse will have different features, making them more suitable for different studies. For example, some strains deal better with infections, while others have a more permeable egg membranes which makes genetic manipulating easier. They live up to about 2 years, but at the 1 year of age a mouse is already considered old for this type of studies. When it comes to embryonic studies, it’s important to precisely control timings. How can one check if the female mouse did get pregnant after copulation? Lift the female mouse’s tail and check for a plug of mucus in its vagina. If it is there, then it is now day 0.5 dpc, and the female is pregnant. From 1 pregnancy, a female mouse can generate many embryos that will implant in one of the two horns of the mouse’s uterus. However, things can go wrong since mice are very sensitive to external stimuli, and the embryo can easily be reabsorbed and from what were initially 10 embryos, only 3 to 4 be born. Developmental biology studies are important in order to actually understand the molecular pathways needed for cells to develop and form tissues and organs, especially since some are very conserved throughout all kingdoms of life. Cell division, cell migration and cell death are examples of processes that exist in all life. The main steps of embryonic development are: Fertilization Segmentation/cleavage Gastrulation Organogenesis 17 Segmentation and gastrulation are the two most important steps for stem cell biology. Note: It’s important to look at an image and to try to figure out what techniques were used. For example, RNA probes can highlight specific tissues based on markers, making certain parts of the image darker. There’s not much size difference between zygote and morula, despite the fact that the morula is roughly 8 cells while the zygote is just 1 cell. This reinforces the idea that everything that happens is due to the elements already in the zygote. Fertilization starts the cytoplasmatic changes in the egg. An increase in the calcium ion concentration activates the cell division machinery, allowing for the arise of the next step, cleavage. Looking at a Xenopus egg, we can see a difference in color between the light exposed top side, and the water emerged bottom. Upon entry of the sperm, there is a rotation of the cytoplasm to the top, now being called gray crescent since its appearance changes. Cleavage is another name for cellular multiplication, the cells that result from the division are smaller in size and are called blastomeres. Blastomeres will be different depending on the plane of division, which is different depending on the organism. It depends on the orientation of the mitotic fuse within the cell, originating different polarities in division. This cell division is not dependent of one another, that is, one cell can divide while it’s “generational peer” won’t, so it’d have 3 cells instead of the expected 4 from the exponential growth. The mechanisms that dictate whether the fuse organizes one way or the another is not currently known. Especially in mammals, the planes of division lead to some heterogenicity in cell size, some cells can be smaller while others are larger. In humans, from fertilization to implantation it takes roughly a week. For mice, it is 4.5 days. Despite having wildly different gestation times, the first stages aren’t all that far apart, in terms of duration and processes that occur. 18 On the first few, 2~3, divisions we have 8ish cells that are equal in potency and can give rise to all cells in a full-grown organism. They are totipotent cells, but then cell fate starts to arise and that infinite potential is lost. An egg is about 60~100μm in diameter, which is much larger than the average size of a cell, 10μm. To note however that there are even smaller cells like the red blood cell, but also larger ones like macrophages at 30μm. Note: Hyperplasia happens in cardiomyocytes, it is when a cell grows in size due to the production of more protein, like sarcomeres, but it does not divide. Hyperplastic cell is one that can still decide. Blastomeres will compact to adhere better to one another and establish gap junctions. After the 2~3 divisions, the actin-myosin filaments pulse, and this physical stimulus kickstarts the reading and expression of the genes. From this moment on, as more and more cells arise and bond tightly together, communicating with each other, it is possible to see some distinct cell layers, those on the outside and those inside. These cell proximity points start leading to the upstart of cadherins that anchor cells tightly to each other. This organization also creates another polarity within the cells, the parts of the cell not connected to its neighbors are the polar parts and this leads to the upregulation of different genes and production of different proteins. Therefore, polarity and position are the 2 first events, mere morphological cues regarding space, and a mechanical stimulus are the key to starting differential gene expression and this different cell fates. This starts after the 8-cell state. Eventually, no cell is free of cell contact. The morula is the compacted egg of a hundred something cells. The blastocyst is the embryo stage after the morula state, when lineage specification events start happening. 19 The first event: the inner cells of the blastocytes have a different expression when compared to the external. A cavity in formed in the center with the cells around it organized in an epithelial-like layer. The cavity is filled with the fluid and pressure from cell division. The mass of cells pushed into the corner is the inner cell mass, and they are pluripotent. Those wall cells are called trophoblast or trophoectoderm. Note: Fluid from the extracellular matrix flows into the embryo and starts pushing the cells into one side. The second event: within the inner cell mass start acquiring different cell fates. Once again, cells within the inner cell mass will begin to distinguish themselves based on position. The cells acting as a barrier to the cavity become the hypoblast and the others, who retain their pluripotency, are the epiblast. Note: The HIPPO pathway is the king of mechanical transduction, it carries the stimuli into the nucleus. TAZ and YAP are important effector molecules in this pathway. 20 YAP is located within the cytoplasm of the cell. Upon activation of the HIPPO pathway on the cell’s outer layer, that cleaves the phosphors particle that inactivates YAP and prevents it from moving, YAP moves to the nucleus and activates differential gene expression. Note: Diapause happens in mice and some other species when the conditions aren’t favorable for pregnancy. It is a pause in development of the embryo where all processes are arrested until conditions are once again favorable. During the implantation of the embryo, the uterus walls are invaded by the cells of the embryo ectoderm. Gastrulation is a highly coordinated cell rearrange. The cells of the blastocyst are repositioned, establishing contact with new cells. They move the entire embryo and lead to the formation of 3 embryotic leaflets: ectoderm, mesoderm and endoderm. Once again, what distinguishes them at first is their special disposition. “Stem cells” is not a designation commonly attributed to these three germ layers. However, they are separated by division potential, have high proliferation and give rise to different lineages. So, they could be stem cells, but in truth they are progenitor cells, they give birth to all the different cells in our bodies. They have different signatures, produce different proteins, that can be identified within the embryo with, for example with antibodies. For example, T or Brachiury are identified in mesoderm. Mesoderm: blood, heart Ectoderm: skin, nervous system Endoderm: lungs, gut Note: When do stem cells cease to exist within the organism? Gastrulation, except for the primordial germ cells that remain for a bit longer. What about germ cells? In truth, there is a small group of cells kept apart before gastrulation that do not go through the process. The are the primordial germ cells. A mouse gastrulates at 7~7.5. Pluripotency ends here, except for the primordial germ cells that only stop being so at 13.5. This is the point where gonadal tissue that is required to receive those cells is ready, and thus the primordial germ cells migrate. 21 While mesoderm, ectoderm and endoderm cells no longer can give rise to germ cells, the primordial germ cells can still differentiate into mesoderm, ectoderm and endoderm cells. When gastrulation starts there is a redistribution with the formation of an obvious primitive streak with Hense’s nodes on top. From posterior to anterior, the cells differentiate and are pushed up. So, the cells on the posterior part differentiate after the anterior. At the same time somites are developing in pairs across the streak and they are progenitor cells. There is a movement similar to convection currents where cells more on the inside can move up and out. Cells differentiate first on the upper anterior part, and start to laminate into ectoderm, then endoderm and then the mesoderm will arise from a differentiation of the ectoderm. In multicellular organisms, cell behavior is tightly regulated to allow proper embryonic development and maintenance of adult tissue. A critical component in this control is the communication between cells via signaling pathways, as errors in intercellular communication can induce developmental defects or diseases such as cancer. It has become clear over the last years that signaling is not static but varies over time. Feedback mechanisms present in every signaling pathway lead to diverse dynamic phenotypes, such as transient activation, signal ramping or oscillations, occurring in a cell type and stage-dependent manner. In cells, such dynamics can exert various functions that allow organisms to develop in a robust and reproducible way. ERK, WNT and NOTCH signaling pathways are dynamic in several tissue types and organism, including the periodic segmentation of vertebrate embryos, and are often dysregulated in cancer. 22 How did we learn all the molecular pathways that mark the embryonic development in mice? Well, by research in vitro. For humans, it is not so easy because of ethical constraints. Even though nowadays we have the technology to keep human embryos for longer and even reprogram pluripotent stem cells to show characteristics of embryos, the concern of how far along we can take this embryo in research puts a stop to it. That begs the question, then, when do we call that blob of cells an embryo? Right after the sperm fertilizes the oocyte that was arrested on meiosis I and can now go ahead and finish meiosis II and then form an egg? Once we start seeing the physical characteristics of a vague human shape? There are many different definitions, especially when we compare to the time stamps of systems that mimic embryonic development in humans, and there are many different systems for different stages. In truth, there is no consensus and it varies from belief to belief. Some agree it’s with fertilization, others place it later since many a fertilized egg aborts itself before development and thus the pregnancy fails. The lack of in vivo assessment makes it hard to tell, and the different, clashing, intellectual definitions vs. emotional impact of losing said embryo turns the line murky. Typically, up to day 14 we are free to research in human embryos, which is the point where gastrulation happens where life starts according to some scientists. This is where the 3 germ layers arise. Morphological disposition greatly influences the characteristics of each layer. Depending on where they are, they will exhibit different potentials for differentiation. Mature cells will be the most important to our functions, as they are the ones that in fact act, but their sole destiny is to die, so are they even that interesting? On the flip side, cells with higher potential are much more interesting but they also don’t have any of the complex functions mature ones do. If we compare these two, a mature cell to an adult who has reproduced as much as it can, and an immature cell to a child, does that mean the adult’s sole purpose is to die? That would get us a more clear-cut answer, a resounding no. So, back to what differentiates the three germ layers, it is their molecular signatures. This is brought upon by their different gene expression and that is due to everything, everywhere, all at once. Yes, morphological distribution is a very big part, the cell adhesions they express due to the surfaces they contact - if they are in contact with a free surface or with other cells - how they break away from their neighbors to migrate and then establish different connections; all this leads to different genes activating and being silenced. But more than space, cell kinetics, how its cytoskeleton pulses and pulls in reaction to a mechanical stimulus, how it reacts to chemical signals outside itself, all of these events, while simple by themselves, will lead to the rise of different layers, and then each organ will arise from a specific layer. Like we mentioned before, from the ectoderm will come skin and nerves, endoderm guts and other inner organs, and from the mesoderm bone, heart and blood. 23 Thus, this can be the point where we can comprehend an individual will truly be formed. After gastrulation, neurulation follows. It is the formation of the notochord and the neural tube, which will finish at the end of this phase. Then, organogenesis happens and we are in the fetal phase. All this, to explain that we cannot work all the way through with a human embryo, so we often turn to the mouse embryo. While we have seen that a mouse embryo is rather similar to that of a human, it is also completely different in terms on intermediates. Even the way it implants into the uterine wall is different. As we can see on the image, the human is shaped more like a disk while the mouse is more like a cylinder. In both, there are enzymes to partly digest the uterine wall and allow implantation, but the way it happens is different. 24 The fetus arises at the 51st day in a human. Wouldn’t that be the point when mice and men would be totally different, since that’s when the phenotype starts to be obvious? Before that, wouldn’t the molecular signals also be different, however? Well, they aren’t all that different, it all boils down to gene expression and the proteins that form throughout, that start being different and different as time goes on. It is logical that it becomes increasingly apparent that the embryo that is growing is a human and not a mouse. It is important to know how to obtain a certain cell from a pluripotent stem cell. For example, to get a pancreatic cell we can first ponder: do we know of any stem cell in the pancreas? No, so we next ponder from each germ layer a pancreatic cell would need to arise from. As an inner organ, it would be from the endoderm. From there, it’s a matter of getting a pluripotent stem cell, forcing it to become a germ cell from the endoderm, and then giving it the correct stimuli for it to become a pancreatic cell. Stem cells have clonal growth, not unlike bacteria. This means that from a cell, a colony of identical clones is formed, with the potency of the original being maintained ideally. If a culture is good, we can’t even see the separation between each cell, they are almost syncytial. Poor culture, however, will show spaces between cells, with those in the periphery occasionally showing projections to the outside, indicating a more advanced degree of differentiation. This is when a decision has to be made. Do we trash the whole culture, those cells, or not? Cell culture is expensive, and stem cell culture doubly so. One has to buy correct media, get the correct factors, inhibitors for genes that would lead to differentiation, potentiators for the genes that maintain pluripotency. If the so-called damage to the culture, these cells that are no longer as undifferentiated desired, is contained to the border, we can disaggregate the colony with a gentle concoction. Trypsin should be avoided all-together for stem cells, as it is too aggressive and will hurt or even kill the cells. Instead, one should use a buffer with EDTA, which is a chelator for cations, such as calcium, sodium, magnesium. Calcium in specific is very important for cell-cell adhesion proteins such as cadherins, and integrins for cell- matrix. Inhibiting calcium won’t allow the cells to adhere that well to each other, and we can take them apart. Human cells don’t like to be alone and apart, however, since the communication they establish through gap junctions and molecule trades are essential to their survival. As such, these cells are passed in clusters. The molecular pathways, while essential to distinguish between a primed and naïve pluripotent stem cell, are to be seen in a broad view, focusing on a few key ones and an understanding of how a process itself leads to a difference in gene expression, and thus different behavior and different species. Human stem cells colonies are more irregular than that of mice. With experience, one can evaluate morphology, gene expression, molecular pathways, and still have only about 90% certainty that a cell is indeed a stem cell. We need a functional assay besides that, because a stem cell needs to look like a stem cell, but above all act like a stem cell. 25 A pluripotent stem cell exists only in the inner cell mass in the embryo, in the epiblast of the inner cell mass in the next step in development. They then differentiate into the 3 germ layers, so one assay would be to evaluate if a supposed pluripotent stem cell can in fact differentiate into the three. However, the main assay used is a rather obvious one in retrospect. If a pluripotent stem cell can only be derived naturally from the embryo, then it has to be able to be part of an embryo and behave like it. That is to say, the assay involves reintegrating our stem cell into a blastocyst and check if it will contribute to the differentiation of the other cells, if we can track the cells that arose from ours amidst the other “native” ones. So, we need to find a way to follow it without disturbing the forming tissue, and we need to check it was actually doing something and not just sitting there. This requires a discrimination marker that allows us to, even in way later stages, distinguish and pick out our cells. This means it needs to be stable, not suffer alterations and not be degraded. The first idea for this was DNA, and the original markers were introduced with the help of radiation induced mutations. Nowadays, GFP is a protein whose instructions are introduced in DNA and is easy to follow. For example, one introduced a small number of cells with a unique marker mutation into an organ niche, wanting to check if it differentiates into fibroblasts, cardiomyocytes or endothelium. Later on, we check and there are all 3 with our original markers, so from that small population of cells we got a lot, and from different lineages. Another form of giving rise to different cell types is induction and competence. This is related to the way cells respond to and/or produce certain chemical signals. Who won the Nobel Prize for Physiology and Medicine in 2007? Two groups did, Olivier Smithies and Marin J. Evans for embryonic stem cell identification, and Mario Capecchi for homologous recombination. Their research dates back to 1981 and is of great importance. The two topics are related because embryonic stem cells were proven to still be able to trigger homologous recombination that we typically associate with meiosis, but stem cells do it in a much lesser frequence and intensity. This still opened a window for genetic manipulation of stem cells, the first gain of function and loss of function studies for these cells that allow us to fix some genetic diseases we can detect early on, or make the cell show some different desired characteristic. Thus, any stem cell is the best vehicle to introduce a genetic change. Placing it on a pluripotent stem cell means any cell in a lineage derived from it will then express that genetic change. If we want it only one on specific cell type, we can place the mutation on a promoter region that we know is only active on that precise cell. Homologous recombination opened and started knockout technology, and scientists took it upon themselves to knock out one gene at a time to understand the purpose of each one. Back then, each new knockout gene was a paper all by itself, yet nowadays each mouse used in research has like 20 knockouts. 26 17 years later, in 1998, scientists isolated the first human embryonic stem cells. Still in 1981, scientists identified the first embryonic stem cells, these in mice, more specifically in the inner cell mass of the blastocyst where those cells were first identified. With the increasing cell number, cell polarity arises due to special distribution. These are accompanied by molecular changes, the expression of adhesion proteins like cadherins. All the mechanical stimuli translate into biochemical signals. Day 3.5 in mice and 5 in humans is when we have a blastocyst. It’s a polarized structure, and from its inner cell mass we can harvest the cells from which most of the embryo can be developed. These cells can be cultured in a lab, and they will grow undifferentiated. In labs, most of the cells used are tumor originated. If we obtain cells directly from a biopsy, we will have tedious lab work to isolate precious few cells. These, depending on their cell type, may not even divide in vitro, if they are terminally mature. Some primary cells can still divide a handful of times, and those could be called primary cel lines. However, most of the time, we use established cell lines, cell transformed into a tumor like state and can divide infinitely. In fact, most of these were isolated from certain tumors in an organ. Fortunately, some organs are exceedingly rare to get tumors on, like the heart. For those, there’s only one adult cardiomyocyte line for mice, and one immature cardiomyocyte line for humans, no more. In truth, since cardiomyocytes do not proliferate, cancers are harder to find. However, divisions are not the only way for a cancer to be formed, there are other ways to develop mutations that lead to cancer, like radiation, but there’s a catch here. Highly proliferative immature cells are way more sensitive to radiation and as such more susceptible to mutation. Once again, being a mature non proliferating cell saves the cardiomyocyte. Not to forget that some cells have the capacity to divide infinitely without a transformed karyotype. One of the awarded groups for stem cell studies placed blastocyst cells on a Petri dish and watched what grew. The other group did the same, but compared them to the other standards for pluripotency at the time, teratocarcinoma derived cells from male reproductive cells that could replicate infinitely and were noted to differentiate into other diverse cell lineages, albeit with a deficient and down right aborted, slow differentiation, into one or another lineage. 27 The scientists implanted the transformed teratocarcinoma cells into the blastocyst and noted that they become normal, characteristics wise. This thus led them to believe there would be a “normal”, healthy, cell counterpart that they could propagate in a lab. They did indeed try to do this, and they used the conditioned medium from the teratocarcinoma to promote growth. The problem is, each cell line has different needs, do they need gelatin, fibronectin, a hydrogel, specific extracellular matrix components, collagen, laminin? Do they require a cell monolayer of stromal cells for support? In fact, they did! They used a layer of mice embryonic fibroblasts, MEF, in this case the STO line. The cells cultured on top of MEF preferred it this way, they purposely migrated on top of the MEF layer, propagated infinitely and didn’t lose their potency. So, what is in the media that allows it? The blastocyst is isolated pre implantation when it still has the zona pellucida, before the so-called hatching of the embryo when it leaves said shell-like layer. They would then do microsurgery on it, removing the shell preventing it from growing. The inner cell mass will thus grow and grow and out-proliferate all other few cell types that exist in the culture. But they require a layer of support to propagate. This and the chemical signals in the conditioned teratocarcinoma medium, surface proteins and secreted molecules from the MEF layer, the paracrine effect, but also proteins secreted by the pluripotent stem cells themselves, the so-called autocrine effect, will allow the pluripotency to persevere. In the article, cells were isolated from the embryo and put to grow separately on monolayers of STO. When they had the karyotype analyzed after clonal growth, they still had that of a normal healthy cell and not a tumor. Note: in vitro fertilization for humans When a couple uses in vitro fertilization, all fecundated eggs must be checked for mutations, either those newly formed or already present, that will be eliminated or lead to the egg not being selected. These assays are done still on the totipotency state, on the first 2~3 divisions of the zygote when there are 4-ish cells with equal potency. The embryo truly will not miss it if a cell is removed in this state, and its genes can be analyzed. Then they asked what was the signal that makes the cell maintain its pluripotency. But before that, how would one even prove the cell is truly pluripotent? It needs to differentiate into more than one cell type in vitro, if not all 200 known cell types back then. However, they didn’t have the signals that guaranteed the cell didn’t differentiate into a certain cell type because it couldn’t, and not because it was lacking the right conditions. So, they instead went with the route to prove that the cell differentiates into at least one representative of each of the 3 germ layers, for example, a neuron cell for ectoderm, a cartilage cell for mesoderm, and an hepatocyte for endoderm. 28 This, in vitro. In vivo the result would’ve been absolute, but it is hard to do with the huge number of cells and huge lack of techniques and knowledge back then. In vitro is then the best proxy. Next, they needed to prove the indefinite propagation without differentiation unless wanted. That they did prove when they kept the culture alive and undifferentiated months upon months. Thus, we need to prove its functionality. For that, we return it to the blastocyst, essentially forming chimeric mice. The readout we were looking for is to be able to trace the cell down to somatic but also germ cells in the adult individual. So, how does one do that? Well, at the time they had experience with breeding animals to potentiate a certain wanted phenotype, as it has been done since time immemorial, so they used that knowledge. In particular, they used a cell from a mouse that had a black colored coat and injected it on a blastocyst of a mouse with a white coat. More specifically, the pluripotent stem cell removed from a black-coated mouse was implanted on the blastocyst of an albino female that was impregnated by an albino male, which would guarantee an albino offspring. This blastocyst was then placed in an albino surrogate mother who was hormonally induced into a state of pregnancy. To get some questions out of the way. Does the surrogate need to forcibly be an albino? Not really, but there’s always some little interferences from the mother that could be avoided by using a female much more similar to the one that gave origin to the host blastocyst. Why isn’t the blastocyst now with our study cell not reintroduced into its mother, but another mouse? When one removes the blastocyst from the female pregnant, it inevitably leads to an abortion, the mouse becomes unable to carry it. Not to mention, reintroducing the study object into a host that has already been tempered with would introduce new margins for mistake. Why does the surrogate mother need to be hormonally stimulated? Well, the surrogate was not pregnant, so its body was not primed to receive an embryo, hormonal stimulation is then needed to guarantee the body is ready to receive this blastocyst. In fact, it’s also what happens with women that use in vitro fertilization techniques. They are given hormonal therapies once the zygote to implant has been chosen to prep their body to adequately receive it. This assay is the gold standard, one of the best and most decisive one can do. However, it is costly in time, money and animal usage, so one would prefer cheaper assays for pluripotency. These won’t be as fail-proof, but they make do in a less restringing situation. 29 One example is the teratoma assay. We take a million of the cells we want to evaluate for pluripotency and inject them into the back flank of an immunodeficient mouse. These mice, NUDE or SCID for example, have no immune system, or little, to speak of, and will not develop a rejection reaction to these foreign cells. The nodes of cells will quickly become irrigated with the mouse’s vascularization and thus kept alive and thriving. They will start to grow and form a tissue mass, forming even bigger nodes. These nodes will be harvested and cultured for a while before being analyzed histologically with appropriate staining like H&E. More often than not, we get a variety of cell types coexisting in the nodes, like gut epithelium, muscle, epidermis, cartilage, cells across many germ layers. 30 Now we jump to 1998, where Thompson and crew proved they managed to isolate human pluripotent stem cells from a blastocyst. In this 17-year gap, many assays had since been developed to correctly evaluate, in vitro and in vivo, whether a cell was pluripotent or not. Pluripotent stem cells can be derived from an embryo or induced, so it is important to distinguish which we are talking about. The human pluripotent stem cells these scientists isolated were embryonic. They, too, were compared against the teratocarcinoma pluripotent stem cells, tumoral cells that had many mutations acquired and could not fully reacquire a normal karyotype. They knew that these cells were capable of multiplying and propagating overtime in vitro, and also were able to integrate into a blastocyst and give rise to any representative of the 3 germ layers, therefore they were pluripotent. This was thus the base for the evaluation of the human isolated supposed pluripotent stem cell. During the Bush Administration in the USA, the development of new stem cell lines was blocked when said research was funded by public money. This did not stop private researchers, who continued to develop new ones, but the information was a bit restricted nonetheless. During the isolation of a blastocyst, scientists will digest the zona pellucida with Acid Tyrode’s, then immune surgery will happen where rabbit antibodies against human RBC will be applied and then washed with human ES culture medium before a secondary antibody is applied against the primary ones. It is followed by a gentle mechanical trituration with a pipette, resulting in lysed trophectoderm cells, some intact ones, and the intact inner cell mass cells. These are isolated and left to grow on appropriate culture conditions for roughly a week to attach and proliferate. After roughly two weeks, half of the outgrowth will be disturbed and removed to pass onto a new container, while the other half will remain to grow. This important for the first few passages, after a while one can start adapting the cells to gentle enzymatic dissociation, but mechanical dissociation is preferable early on. 31 These protocols depend on the quality of the embryo itself, but also that of the culture conditions. The isolation of the ICM, and the experience of the operator, is essential in the second step. In the third, the outgrowth of the ICM isolate and the adherence to the support layer are the most important steps. The properties of these cell lines can change just by being poorly handled, so it is important to be as careful as possible. Some of these lines will thus have different potential, from 10 lines, maybe 2 or 3 will have the capacity to produce the cell type we desire, while the others won’t. The mechanisms that lead to this are still not fully known, so it is better to be safe and try to minimize mistakes. The composition of the media, the way they are isolated, the way they are passed have all been shown to lead to different characteristics, it has been shown that immune surgery can lead to changes, while mechanical disintegration can be tricky but lead to a higher quality inner cell mass isolate. How does this protocol stand up against what we do today? To be honest, there is not much difference, since stem cells seem to be a polemic topic. Research progresses very slowly; people fear what one can do by manipulating stem cells and therefore embryos. After the initial years, and the rise in interest that came with cloning, the hype died down and hasn’t progressed very much. We are still trying to figure out the fundamentals, what certain compound makes them have pluripotency, what signature makes them so special, what is the signals in their microenvironment that can modulate how they act, how do we identify a pluripotent stem cell with 100% certainty. Most of the stem cells in banks are induced pluripotent stem cells, 90% at least, to avoid controversy. Even in clinical usage, very many few people have actually received grafts of pluripotent derived cells, and those who did were in an experimental setting. It is thus very difficult to implement this technology in anything more than purely modeling, as it is expensive and still not fully established. Pluripotent stem cells need a support layer of embryonic fibroblasts, that we know from the moment of the isolation of mouse pluripotent stem cells. Thus, the quest for finding signals, transcription factors active in each stage of differentiation, especially if those just so happen to be master factors essential on the crossroads of differentiation, cytokines in the environment that conditioned the cell’s action. A cell has a road of differentiation ahead, and they wanted to find out how those transcription factors lock in their destination when connected to the correct gene. For example, PAX6 for B-cells is considered an important factor. If these factors did exist, then cells could be coached into a certain path of differentiation, representing each road to a certain cell type. If we find those signals, we can follow the step-by-step differentiation from a pluripotent stem cell all the way into a mature cell, therefore constituting a wonderful model of development. 32 So, we know a support layer is needed, and we also know that the teratocarcinoma medium was able to keep pluripotent stem cells thriving and not differentiating. So, what was in it that allowed pluripotency to remain? LIF, leukemia inhibitory factor, was one of the factors credited with such, but now we know it is not enough. The cell outlines start becoming more irregular, this means that those are beginning to differentiate. Although we are able to grow murine pluripotent cells without feeder layers, they can be brought back to the embryonic fibroblast layers to refresh their pluripotency and strengthen it due to the presence of specific factors like LIF. For humans, there is not clear factor that does it, we at best have a cocktail of cytokines. So, it is best to keep them growing in feeder layers, especially murine ones since human embryonic fibroblasts aren’t very ethical. Nowadays scientists are trying very hard to find alternatives to animal products due to ethical and economic constraints, especially the closer to clinic we are. Here we aren’t very close, but it is still important to. So, we have a putative line of pluripotent stem cells. No matter if it’s a human or mouse line, the assays are more or less the same with some key differences. If we want to identify it, we first phenotype the cell which is a description of the cell’s expression profile especially on the surface layer so as to not disturb or kill the cell. If it is not enough, however, we’ll have to investigate the cytoplasmatic proteins which involves permeabilizing the membrane to allow the passage of the antibodies used to identify them. 33 We use cell surface markers, like lectins and glycosylated proteins which are especially important in the identification of pluripotent stem cells. Using different antibodies against different glycosylated proteins may allow us to distinguish between naïve (closer to full pluripotency) and primed (closer to differentiation) pluripotent stem cell. Antibodies are used to stain the proteins, and these can carry a fluorochrome, or the fluorochrome can be carried by a secondary antibody that will amplify the signal. We then identify which cells have the fluorescent signal that discriminates the ones that carry the protein we wish to identify. We see two subpopulations that showed promise for pluripotency. With correct flow cytometry equipment, we can further separate these two for further in vitro testing. This, for surface markers. If the markers are in the nucleus, then the assay is terminal and they cannot be used for further testing other than identification of protein expression and similar end of line assays. 34 TRA-1-60 and TRA-1-81 are cell surface proteins characteristic of human pluripotent stem cells, and in mice though those have some difference. SSEA4 is a sugar surface marker for pluripotent stem cells exclusive to human cells. It can be quite useful to distinguish human cells from mice in the case of cross contamination. SSEA4 expressed in high quantities usually indicates a primed pluripotent stem cell, while lower expression indicates a naïve one. Oct-4A, Sox2 and Nanog are 3 transcription factors common in pluripotent stem cells. Some cells express one or another, but rarely if never the 3 at the same time. If a cell expresses the 3, then it is extremely likely we are looking at a pluripotent stem cell. These markers are phenotypical marker, they only grant us a good chance that a cell behaves like a pluripotent stem cell, but not the certainty that it functions and is in fact one. Another assay used to prove the pluripotency of a stem cell is the teratoma forming assay shown before. This one is more akin to a functional assay, but it is still not enough as it does not prove the cell can in fact differentiate into any of the 3 germ layers. By chance, it can in fact generate a representative of each of the 3 germ layers, but NOT of the germinative lineage. Before gastrulation, the germinative lineage destined cells will be kept apart from the rest, safely stored away in a membrane or another cavity derived from the epiblast. The remaining cells will rearrange themselves and lose their pluripotency, but the germinative bound cells will keep it for a bit longer. Once the somatic lineage bound cells form the gonadal tissue, the germinative cells will migrate to those and be kept there. We have pluripotent stem cells growing across a feeder layer. We want to evaluate if they keep their pluripotency, but we also want to see how they move forward towards differentiation. We separate different plates, ones without the LIF producing feeder layer that will inevitably start to differentiate, as it is the default mechanism when without the pluripotency maintaining factors. There are mainly 3 differentiation protocols. 35 One of them is the differentiation into embryoid bodies. Without LIF and feeder layers, the cells will start to cling to each other, aggregate due to the many molecules secreted in the autocrine and paracrine effects. If you take one of the aggregates in a specific point of time, and take them apart to analyze the different types of cells in them, we will check that there are differences between time points. The potential of cells at day 3 will be different than that of the cells at day 9 and so on. There is a loss of potential that is somewhat comparable to the development of an embryo in vivo. One of the first differentiated tissues to appear in an embryo is the heart, which happens at day 9 in mice, just a few hours after gastrulation. The heart, which is nothing more than a contractile tube at that point, will be the first one to form, alongside the blood progenitor cells. If these are isolated in culture, and given the necessary factors needed to differentiate into their respective destiny, they will do so in culture. These models allow us to understand development from a temporal standpoint and reinforced the usage of this system as a genetic manipulation tool to understand how different encoded instructions will influence the formation of specific structures. The next protocol uses a feeder layer, but it is not of embryonic cells, rather it is of stromal cells that gives out signals of differentiation into a desired cell type. It does not force the purchase of synthetic factors; it rather relies on the production of those molecules by the layer itself to induce differentiation into a cell type. The last protocol has more freedom. There is a more 2D culture, there is no feeder layer that could be considered a contamination upon our cells, but rather a strict protein composition enriched in the desired molecules required to nod the cells along a certain differentiation path such as neurons, if laminin is used for example. If these are used, monitoring is necessary to guarantee our readout is the desired. There are different markers to check this, we will know one for each of the germ layers. There are some promiscuous markers, that cross more than 1 germ layers. We do not wish to use those ones; we prefer markers that are contained to a single one. These are the three most used specific ones, Otx2 for ectoderm, brachyury for mesoderm and SOX17 for the endoderm. 36 Pluripotent stem cell is, in a way, an umbrella term that includes embryonic stem cells, iPSC and all pluripotent cells in different steps of differentiation. Besides the pluripotent stem cells, there are other cells in the blastocyst, like the extra embryonic tissues from the trophectoderm that will not be seen on this class, but do exist. Primitive germ cells migrate into the amniotic cavity between the epiblast and the primitive endoderm, and are kept away until organogenesis where they will migrate into the gonads. 37 When one needs to characterize stem cells, one may consider morphology, the physical aspect of the cell and its colonies, the surface markers expressed, the transcriptome of the cell, which is the total RNAs expressed in the cell, or even proteomics which are the proteins actually produced in the cell. While the transcriptome is a good indicator, it does not guarantee which functional proteins are in fact produced, since not all RNAs in the cell is translated into proteins and that will impact the cell type. Proteomics is, as such, the closest to a functional evaluation of the cell’s activity. So, what assay is used to evaluate transcriptomic? Northern Blott? It could be, but it is no longer used since it’s an old technique, it is very time consuming so it is only used when heavy evidence is required. Nowadays, we use PCR or real time PCR, where RNA is isolated and then a reverse transcriptase is used to obtain the DNA that originated it. Now, we need to check if that RNA does express itself into a protein. SDS-Page? Takes too long, and we lose the protein’s placement in the cell, so we’d want a technique that allows us to see the protein in the cell, in the organelle where it belongs. Immunofluorescence, where we use antibodies against a certain protein and then a fluorescent reporter that will give us a signal to locate. First, we fixate the cell. For that, we use a detergent to permeabilize the cell to allow access to the cell for the antibodies and other agents, and then crosslinkers to reticulate the proteins, allowing the cell to keep their integrity and make it a more accurate model. The colonial growth of stem cells requires a 2D substrate, it needs adhesion. We use MEFs, an adhesion substrate that also feeds the factors required for pluripotency. Once we know the cell well enough, one can swap out the feeder layer with adhesion proteins, but these require knowledge of the expression of protein signals that that cell requires. To evaluate and identify a stem cell, we have surface markers and others. Stage- specific embryonic antigen is a cell surface marker specific to each stage of embryonic development, and these will vary depending on species. Also, on the surface we have lectins and peptides. Sometimes the expression of 1 isn’t enough, we need co expression. SOX2, OCT4 and Nanog co-expression can basically guarantee that a cell has pluripotency. OCT4 specifically is like the orchestrator, it is the gene upstream of all the others. Besides evaluating the cell population, laminar flow cytometry allows us to check for certain surface markers and quantify the amount of each cell by correlation to signal strength. We can then sort cells according to the intensity of expression of that specific marker, thus trying to understand the difference it makes. SSEA-4 is a marker of primed pluripotent stem cells. 38 Vimentin is a protein characteristic of human embryonic stem cells. It is a component of the cytoskeleton. Trophectoderm markers are present in human embryonic stem cells only in the earlier moments and as such allows us to identify their stage of differentiation. LIFR is the receptor for LIF, a protein that is part of the IL-6 superfamily that is a pluripotency marker in mice. Gp130 is a chain of the LIFR, a part of it. That is all well and dandy, but in vitro assays are not enough. We thus need in vivo assays. We have the injection in NUDE mice to form a teratoma assay. Any mouse will do as long as it is immune deficient, and there are multiple available strains nowadays. Then, we allow the cells to form a teratoma, it is benign, but it can form a whole horrific variety of tissues, such as cartilage, even teeth. Then we have the gold standard, where we put the pluripotent stem cell back into its natural niche, the inner cell mass, the blastocyst. However, the epiblast is more organized and therefore less permissive to this engraftment, that’s why we use the blastocyst. This stem cell will be included in the blastocyst of the host, a different looking mouse. The litter, born from it, will show a different somatic phenotype that then parent that gave birth to it, but nothing is proven for the germ cells. For that, we breed the offspring with parent, or a mouse of the same lineage as the parent since all lab mice are inbred, and then we check the progeny. In accordance with Mendelian genetics, some of the children will inherit the contribution of the candidate pluripotent stem cell and thus prove it contributed to the germ cells. However, this isn’t the super gold standard, there is plenty room for mistake and variation. The true gold happens to simplify it, it is the chimeric aggregation, as it uses the cell’s aggregation tendency. It is easier for us to note the segregation between ICM and TE during the first differentiation event, since we have plenty of markers for it, we can thus use this timepoint. If 2 normal cells fuse, they get 4n ploidy, and this changes their expression, they can no longer form the embryonic cells. However, they still have the correct signals to form TE. Indeed, the 2n cells present will form the ICM, and the 4n stick together in the TE. We thus watched cell aggregation between 2n and 4n, and saw how this blastocyst formed will develop into a fully functional individual from the 2n cells, while the 4n cells will form the TE and extra embryonic tissues, allowing the 2n cells to commit to their thing. This assay is thus more potent in both its shorter time, and also because all the cells in the new being derive from the candidate stem cells. The fusion is done by different techniques, from electroporation to chemical reagents. 39 Differentiation can be undesirable. When the medium has certain molecules that lead our cells to differentiate, it becomes undesirable if the goal is to keep them undifferentiated. But other times, we want to force them to differentiate to prove pluripotency, or to give rise to a certain cell line. An interesting thing is to let the cells differentiate spontaneously. In mice, we remove the MEFs layer or the LIF in the medium, as well as any inhibitors used to suppress differentiation. Once these pluripotency maintaining factors are gone, cells naturally stick together and aggregate, beginning the differentiation events. We call it spontaneous, but in truth it only happens if there is culture serum available, and the different components it has that may lead the cell down one path or another. Each germ layer will have preferred factors, that will lead to the expression of different characteristic genes. The embryoid body is the 3D structure formed after gastrulation and is called that due to their morphological similarities to the embryo. It is one of the more common differentiation protocols. In 2012, John B. Gurdon and Shinya Yamanaka received the Nobel prize in Physiology and Medicine. Gurdon received his for his work during the 60s, for proposing the question answered by Yamanaka years later. This goes to prove that even before, scientists have always pondered how a cell becomes mature, does it mean it permanently loses information that was once coded in its genome? And if does, is it truly permanent or can one reverse it? Waddington defined a paradigm in cell differentiation with the epigenetic landscape. A cell, represented by a marble ball sliding down hills. When it reaches a crossroads, it will choose one path and never look back, never turn around. This, if following physics, means the ball would never roll upwards and, therefore, differentiation is nor reversible. However, scientists tried their all to prove him wrong. Yamanaka took a fully mature cell, a fibroblast, and tried to find a way to return to the earliest differentiation state they could, a pluripotent one. How would we approach this? Well, they didn’t know what a transcription factor really was, but they did know at the time that if you place the nucleus of a somatic cell in an embryo, that somatic cell in an embryo, that somatic cell would be reprogrammed to a less differentiated state. However, no embryos were used due to all the complications regarding their usage. They knew that a, what we now call, transcription factor, Myo-D, was what locked in a cell’s path to become a muscle cell, as it’s only expressed in the fully differentiated muscle cell and not in a myoblast, and removing Myo-D reverted it. Yamanaka thus hypothesized that there was a set of factors that were the key to pluripotency, and he did indeed find them. He did cDNA arrays to sequence the genes of the factors identified as possible key aspects in maintaining the stem cell identity. 40 He identified 24 of these factors, and started experimenting. First, he chose a model of an easy to obtain fully differentiated adult cell, and then put them into culture medium with all 24 at once. But how would we introduce them? And how would they introduce this DNA? Retroviruses! They are capable of delivering DNA to the nucleus and then introduce it randomly in the host cell’s genome. This process leads to unique insertions, since there is no guidance on where to introduce the genome. However, all the cells that come from that one will carry the same insertion in the same place. Retroviruses mostly need the cell to be dividing in order to introduce the DNA into the host cell, but other virus have no need for that. However, viruses are harmful. Even though they are modified to be incapable of vertical infection, that means, they cannot infect the progeny of an infected cell again, they are still extremely effective at doing what they do. The transfected cells produce virions, bubbles carrying the viral DNA that easily enter the cell membrane and integrate in another cell. These virions have a preference for certain cells, the so-called viral tropism. So, now we have a way to introduce the DNA, but how do we confirm that the cells actually got it? With a dual system! Besides the genes for the factors, they added genes for β-galactosidase and neomycin which is an antibiotic. They now needed a place to put the genes. They knew of a gene always expressed in pluripotent cells, and through knockout technology they checked it wasn’t, by itself, necessary for pluripotency: Fbx15. So, they inserted the cassette into the gene by homologous recombination. They used an embryonic fibroblast control for negative control, and an embryonic stem cell for positive. They argued that an embryonic fibroblast would be closer to obtain and easier to modify, so those were used for the first experiment, not skin fibroblasts. Comparing the controls to the candidate iPSC, we see similar growth rates. Seeing the RT-PCR done with the intent of evaluating the expression of the putative pluripotency genes, we check that the embryonic fibroblast controls do not express them, but out candidates did. Next, they start narrowing down the candidates by leaving some factors out and checking what happened, determining the minimum numbers of genes that allow the cell to keep the pluripotency. They did all the arrangements of combinations, and saw that with 2 factors no colony grew. With 10 factors they grew mostly ok. They narrowed it down to 4 factors, cMyc, Klf4, Oct4 and Sox2, that had the same effect as all the other 24 together. So, they evaluated expression and morphology. As we already know, next comes the differentiation, the functional in vivo assays. They used the teratoma assay to differentiate the cells into representatives of all 3 germ cells, and these not only had the morphology, but also the function, expressed proteins. 41 Again, as we know this is not the gold standard. They did in vitro assays, used an embryoid body differentiation protocol to check which cells of the embryoid body differentiate into representatives of the 3 germ layers. Then, they repeated all this with a later cell type, they used a skin fibroblast from a mouse tail tip, rather than MEFs. A year after, they tried to see if the same could be done for human cells, by interrogating if the same factors could be used to reprogram. More of the same followed. 2 years later, Science deemed it discovery of the year. But why 2 years later? Many scientists took Yamanaka’s protocol, verified it in labs across the world and started formulating new questions. For example, if a little guy has a mutation, and I take a cell from said little guy, fix the mutation, put it back into the blastocyst that will be little guy, will that mean that little guy would have the mutation fixed? This rationale was used for the treatment of sickle cell anemia, Parkison, and others, in mice. But, can this be translated into humans? That would mean making it 100% safe, with no risk of vertical infections from the retrovirus, no risk of mutations. Douglas Melton tried to use a shorter version of Yamanaka’s protocol, to dedifferentiate cells not into a fully pluripotent stem cell, but just into another line. His goal was to take adult exocrine cells from the pancreas, identify the transcription factors responsible for locking in that fate, remove them, and then add Ngn1+, Pdx, and others, to force them to undergo a path towards the insulin producing cells he wanted. Back to Waddington in the epigenetic landscape. He defended that every time the ball rolls down a path, the modification on that cell’s genome cannot be lifted, much like the ball can’t roll back up; he called those modifications that couldn’t be taken back epigenetic constraints. Nowadays, we know that epigenetic modifications are transient and reversible alterations to the DNA, through methylations and demethylations that change what is and what isn’t expressed. John Gurdon managed to clone a vertebrate back in the 60s by cloning a frog. On the matter of “is genome lost through differentiation”, he took an intestinal epithelium cell from an adult frog. Then, he checked to see if any information was lost in the nucleus. At the time, he knew that the oocyte has factors in its cytoplasm that guide the formation of the egg. He thus hypothesized that if he used the oocyte as an incubator for the intestine cell, to check if, by introducing the mature nucleus into the oocyte, that adult cell could be reprogrammed. First, he needed to remove the oocyte’s own nucleus, and he did so through UV radiation. Later on, he’d admit that the UV source and model frog chosen were essential points that set him apart from Biggs, who tried the same experiment and failed. Gurdon chose a species of frogs who are more permissible to manipulation, and UVs that can penetrate and kill the oocyte’s nucleus. 42 The oocyte with an intestine epithelium nucleus developed into a zygote. Some experiments worked and a full frog was born, in others it didn’t, and the embryo didn’t develop. In those, by excising a small number of cells and placing them in another blastocyst, they checked that those cells contributed to the embryo. Something similar was done with the cloning of Dolly. However, the usage of an oocyte, a germ cell, is still somewhat subjected to ethical constraints, so Yamanaka’s work offered a solution to avoid this issue. It was verified that the cytoplasm of an iPSC can act like an incubator for the reprogramming of a somatic cell. However, cloning is an absolute no go for humans. And even in animals it’s not a marvelous flawless thing. Dolly died of old age-related issues soon after birth, since the epigenetic markers from the adult somatic breast cell used to form it were not lifted. The three organ systems with the highest cellular turnover, and therefore regeneration, base themselves on stem cells and that is no coincidence. One of them, and the first to be thoroughly studied, is the blood system and its basis, the hematopoietic stem cell or HSC. HSC are therefore the paradigm for stem cells, they are the best studied by virtue of being the first identified and characterized. They also have a unique characteristic that sets them apart from all other cell lineages, they can all be told apart based solely on their cell surface markers. Leukocytes all express the pan-leukocyte antigen, or CD45. There’s a specific group of markers for each cell on the blood system, but the same can’t be said for any other system, for example, one can’t tell apart the many neural cells based on their surface markers. There is a hierarchy for cell differentiation from HSC, all the different cell types in our blood arise from a single cell population, HSC. 43 The numbers represented next to some of the blood cells represent their half life spam, which goes to show how little some cells live compared to others, despite having a common origin. From a single population of HSC, an entire blood system can be derived, and then transferred to a readily prepared vessel whose own hematopoietic system was depleted with radiation. The transplanted cells will be able to instill themselves and maintain a brand-new system. HSC cells are present in small amounts within the bone marrow, this is well known. Scientists verified this via a blood marrow transfer between twin brothers, in a time before immunosuppressors. One of the siblings was leukemic, and had received radiation therapy to fully destroy his original blood system. The transplant managed to install itself and generate a new blood system in the host, extending his life spam even if just for a little more. Nowadays, bone marrow transplants aren’t usually done by harvesting all of the cells in a chunk of bone marrow, this only still happens in the iliac crest. In truth, HSCs aren’t exclusive to the bone marrow, they have been shown to migrate in a small frequency, and as such show up

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