User:Kinkreet/MCBII/Stem cell

During the very early stages of development, all cells have the potential to give rise to all cell types of an organism - they are totipotent. In mammals, there are about 200 different cell types. But only after a few rounds of cell division, the cells differentiate and loses its totipotency. In a human 8-cell morula, all cells expresses CDX2 and OCT3/4; but in a 16-cell morula, CDX2 expression is down-regulated in the innermost cells, whereas OCT3/4 remains expressed throughout the morula. After about 10 rounds of cell division, the embryo has become a hollow the morula becomes a blastocyst. The outermost layer of the blastula is made up of cells that expresses CDX2, this outer layer (trophoblast) will become the placenta by adhering to the endometrium. Inside the blastula, there is a cluster of cells which expresses OCT3/4 that are the pluripotent stem cells, which can give rise to all the lineages of the organism.

Characteristics of Stem Cells
Stem cells, by definition, are cells capable of self-renewal - the ability to replicate itself indefinitely - and pluripotency - the ability to give rise to different cell types.

Self-renewal
When stem cell divides, they can divide symmetrically, giving rise to two cells with roughly equal concentrations of proteins and factors, producing two stem cells from one. But it can also divide asymmetrically, maintaining one stem cell while giving rise to another cell, which will eventually lose its stem cell properties and differentiate.

Stem cells themselves rarely divide, but the divided cells (transit amplifying cells) can leave the stem cell niche and divide and differentiate rapidly to give many cells, and many cell types. TA cells are therefore easier to detect.

Stem cells has the property of self-renewal, which is parallel to that of cancer cells, which means many cancer cells originate from the transformation of normal stem cells. Normally, cancer cells needs several (two) hits to its DNA to cause cancer, because the other strand will still carry the normal function; for a normal cell, this would mean it will proliferate and metastasise, for a cancer stem cell, however, it will mean it can proliferate indefinitely and differentiate into different cell types at the same time, and so is better adapted to very part of the body.

Stem cells are cells which can perpetuate themselves by self-renewal and differentiate some of the cells to generate mature cells. The ratio of stem cells to mature cells in each tissue is small and so it is hard to isolate them from the rest of the tissue for study. Haematopoietic stem cells (HSCs) have been isolated from mice and humans and is responsible for generating and regenerating cells of the blood-forming and immune (haematolymphoid) systems, because of this, it is widely used for therapy.

It is later found that bone marrow and HSCs can give rise to non-haematopoietic tissues which means they have higher potency than first thought.

Oncogenesis
Persistence of stem cells for its whole lifetime, through self-renewal, is fundamental, and there are similarities between the mechanism of self-renewal for stem cells and cancer cells. So by understanding the mechanism behind stem cell renewal, will also help us know possible malfunction in pathways that lead to unregulated self-renewal.

The ability to self-renew is different for different stem cells in the haematopoietic system. Multipotent

progenitors constitute 0.05% of mouse bone-marrow cells, and can be divided into three different populations: longterm self-renewing HSCs, short-term self-renewing HSCs, and multipotent progenitors without detectable self-renewal potential. The long-term HSCs mature into short-term HSCs and then multipotent progenitors, losing its self-renewal ability progressively, but also becoming more mitotically active. Normally, the long-term HSCs will only divide in response to a need for blood cells.

Mice which has been lethally irradiated can still reconstitute itself with long-term HSCs, but can last no longer than 8 weeks using only short-term HSCs and multipotent progenitors.

The mechanism for regulation is still unclear, as growth factors which induces proliferation do not prevent differentiation, as it is observed in long-term HSCs.

There are evidence that shows that mechanism for oncogenesis of cancer cells, such as the Wnt, Shh and Notch pathways, when regulated forms the normal self-renewal mechanism for stem cells; the genes which expresses the proteins involved in these pathways are called oncogenes, because when overexpressed, they cause cancer; one such gene is the bcl-2, which when overexpressed, prevent apoptosis. The activation of the Notch pathway using the ligand Jagged-1 leads to an increased number of primitive progenitor activity, suggesting the Notch pathway promotes HSC self-renewal or at least to maintain its multipotentiality.

Human HSC (CD34+Lin-CD38-) shows increased self-renewal when stimulated with Shh and other growth factors.

The Wnt signalling pathway is show to regulate both self-renewal and oncogenesis in different organs and in different organisms. Wnt proteins are intercellular molecules. Wnt proteins are expressed in the both marrow, and when activated b-catenin, a downstream activator of the Wnt signalling pathway, is added to long-term HSCs, the number of transplantable HSCs, with the same phenotype and function, increased. When Axin-1 is expressed using recombination of the AXIN1 gene, it inhibits Wnt signalling and leads to inhibition of HSC proliferation, increased deaths of HSCs in vitro, and reduced reconstitution of the haematopoietic system in vivo. Human keratinocytes with a higher proliferative potential have higher levels of b-catenin than those with low proliferative capacity. Retroviral transduction of activated b-catenin in epidermal stem cells showed an increased capability for self-renewal but decreased capability for differentiation.

Apart from HSC, the Wnt signalling pathways also regulate the self-renewal of epidermal and gut stem cells.

Leukaemogenesis
Stem cells are more likely than other cells to become cancerous, this is because its self-renewal pathway is already activated, and so fewer mutations are required to maintain or deregulate this self-renewal; furthermore, because of self-renewal, a stem cell will persist longer than other mature cells, meaning there are more opportunities for mutations to accumulate in a single cell.

Target for mutation
The target for mutations for most cancers are unknown, however, certain types of cancer has shown to arise almost exclusively from the accumulation of mutations in HSCs. Cells that can initiate human acute myeloid leukaemia (AML) in NOD/SCID (non-obese diabetic/severe combined immunodeficiency) in mice has a CD34+CD38- phenotype, similar to that of human HSCs.

Restricted progenitor and differentiated cells may also be the target for transformation. Using the hMRP-8 promoter, it is possible to target the expression of transgenes only in restricted myeloid progenitors.

Natural
Create a whole organism from a few cells during development. Replenishing and repairing lost and dead cells; most notably blood and gut cells. It has been used to replenish blood cell populations in patients following radiation therapy. However, even embryonic stem cells still have markers such as MHC class I (albeit usually in very low concentrations) on their cell surface which can cause transplant rejections.

Crypt stem cells
The cells that line the inner walls of the gut contain villi, which increases the surface area for efficient absorption. At the base of the villi are crypts, which contains slowly dividing stem cells (>24 hours cycle time) that self-renew as well as producing progenitors (>12 hours cycle time) which migrates upwards that replaces cells which are shed near the tip of the villi. The time it takes for cells to move from the bottom of the crypt to being shed at the top of the villus is about 3-5 days.

The primary signalling pathway involved in crypt formation is Wnt, as the down-regulation of Wnt using Dkk-1 (an inhibitor of Wnt signals) led to the loss of crypts; and the up-regulation of Wnt using R-spondin-1 lead to the hyperproliferation of crypts. Wnt is involved in causing the proliferation of progenitors and thus its absence will lead to terminal differentiation. Furthermore, the down-regulation of TIF4 lead to the absence of the progenitor section of the crypt, although the differentiated cells were unaffected.

At the crypt base, Wnt, hedgehog and maybe other signals induces bone morphogenetic protein 4 (BMP4) to be expressed in the villus core ('upwards' of the crypt base). The BMP4 in turn inhibit Wnt and hedgehog signalling further up the villus, and so the cells will not proliferate, and so preventing them from turning into a crypt. Patients with BMP4 mutations may develop Juvenile Polyposis Syndrome (JPS), characterized by predisposition to hamartomatous polyps (growths, like tumours found in organs as a result of faulty development) in the gastrointestinal (GI) tract. This is due to ectopic crypts forming further up the villus, because BMP4 signal is blocked. At the crypt base, Wnt keeps gut stem cells dividing and induces Notch expression. Notch, in turn, along with hedgehog, induces differentiation where when one cell expressing a signal would inhibit nearby cells to have the same cell fate. As a result, the villus is lined with absorptive cells, with regularly-spaced secretory cells. Cells at the crypt base expresses EphB and those up the crypt expresses ephrin. Ephrin and EphB repulse each other, and so this signalling keeps the cells fo the crypt base at the crypt base, and prevent those further up the villus from migrating down.

Bulge stem cells
Bulge stem cells are multipotent epidermal stem cells in the bulge region of hair follicles. They are capable of forming all cells of the hair lineage, as well as sabaceous gland cells and interfollicular epidermis. Very similar to crypt cells, Wnt is essential for follicle formation: the absence of Lef1 reduced the number of hair follicles whereas increasing β-catenin levels increased the number of follicles. The up-regulation of Lef1 allows de novo follicle formation. Here, the gene that TCF binds include multiple keratin genes, which is the primary protein in hair. Wnt signalling is only observed for development along the hair lineage, and thus Wnt signalling is thought to commit bulge stem cells to the hair lineage, in place of sebaceous or epidermal lineages.

Neural stem cells
Neural stem cells are known to make new nerve cells (neurogenesis), originally only in the embryonic and perinatal stages in mammals, but now known to occur throughout the life of that organism, in at least two places - the subgranular zone (SGZ) in the dentate gyrus of the hippocampus (of the limbic system), where new dentate granule cells are generated; and the subventricular zone (SVZ) of the lateral ventricles (fluid-filled space which acts as padding to the brain), where new neurons are generated, originally from radial-like cells, the progenitor cells, neuroblast, immature neruons and finally neurons. The new neurons then migrate through the rostral migratory stream (RMS) to the olfactory bulb to become interneurons. Major questions arises from this model, such as:
 * How do the new neurons integrate into the existing circuitry?
 * Can you recruit stem cells to damaged tissues for repair, such as from the bone marrow?

In 2005, Buffo found that after injury, glia cells are recruited to the site and promotes proliferation but no neurogenesis. Buffo found that these cells expressed Olig2, which suppresses neurogenesis. So the model states that we actively stop new neurons being produced using Olig2, therefore, by inhibiting Olig2, we, in theory, can induce regeneration.

Heart stem cells
The heart, along with nervous system cells, pancreas cells, regenerate slowly after injury. If heart stem cells exists, then it would mean we can activate them and encourage them to regenerate damaged tissues. Previously, the patient's skeletal muscles was biopsied and expanded in vitro, but this can cause arrhythmias if the myoblast don't couple with the heart.

There are 14 genes that develop the heart cells. Ieda transfected fibroblast with each of these 14 genes to see if they develop into cardiomyocyte-like cells. It was found that mouse postnatal or dermal fibroblasts can be directly reprogrammed into differentiated cardiomyocyte-like cells using Gata4, Mef2c, and Tbx5. The resulting cells showed characteristics of spontaneous Ca2+ flux, electrical activity and beating. This transdifferentiation process does not involve the fibroblast first becoming a iPSC.

Muscle stem cells
Muscle stem cells (satellite cells) exist between the muscle fibre and basal lamina, and are usually rested at G0. It serves to make more muscle fibres during old age and/or inflammation after injury.

TNF from inflammation activates p38 MAPK signalling which causes the satellite cells to proliferate and block differentiation. These newly divided cells transiently differentiate the regenerate the lost tissues.

Research
Stem cells are used as tools in looking for drug targets, toxicity testing, studying early development and disease. It can also be used in therapy to regenerate cells and repair tissues, such as implanting bone marrow for leukemia and after chemotherapy to regenerate all the blood cells; nerve stem cells for treating Parkinsons and Alzhiemer's disease; heart muscle for heart disease; and pancreatic islet cells for diabetes.

Stem cell-based therapies and regenerative medicine
As mentioned already, neurons, pancreas and heart cells do not regenerate readily after injury. In the USA, one million non-fatal infarction are reported every year. Infarction is caused by cardiomyocyte cell death, and this induces inflammation, fibroblast accumulation, and fibrosis (scar forming). The scar tissue may interfere with the contraction. The drugs currently available are not ideal, as they are either inefficient or produce too many undesirable side effects.

The rate of regeneration varies between different organisms. In teleost fishes and urodeles, there are a high to moderate level of regeneration; in zebrafish and newts, de novo cardiogenesis is observed; in rodents and humans, fibrosis is observed with little or no cardiomyocyte regeneration.

In mice, if an E14 (embryonic day 14, with conception marking the beginning of embryonic day 1) is intentionally damaged and grown in a serum-free culture, the heart regenerates and no inflammation or scarring is observed. However, just 4 days later, at E18, the damaged heart is scarred with connective tissues, and do not regenerate. Humans cannot regenerate much other than the finger tips. (on an unrelated note, the regenerative properties of salamander is not stem cell based)

Implanting stem cells into the tissues does not always induce permanent improvements - the stem cells may, through paracrine signalling, make neighbouring cells exhibit stem cell properties, but it does not cause the transdifferentiation of these cells to stem cells. In 2005, Laugwitz identified isl1+(LIM-homeodomain transcription factor, islet-1) cardiac progenitors in the myocardium of postnatal mouse, rat and humans. It is found to give rise to the right ventricle, both atria, the outflow tract and regions of the left ventricle.

Activating resident stem cells are always better than implanting new cells because it eliminates the problem of rejection. In 2011, Smart found adult progenitor cells in the heart tissue and it is characterized by the expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide that can generate new vessels and reverse differentiation.

Research into how mechanical forces can affect stem cells have been ongoing. It is found that cyclic stretch and compression stopped the migratory and spreading of stem cells, and induced their division.

Transplantation of artificial tissue
The idea is to generate the tissue in vitro using a scaffold layered with cells and stem cells which grow on it. After the cells have established substantial mass, the scaffold can be digested. This method can be used on most cells of the body, and can be moulded into the desired shape, depending on the shape of the scaffold.

This can be done using cells from the recipient and so no problem of rejection. The scaffold can also be from a donor, for example the trachea, if it is to remain as part of the tissue. Cells and MHC antigens are removed from the donor tissue before the recipient cells grows on it.

A human test has been done where a lung from a tissue bank is decellularized using very vigorous detergent, but leaving the alveoli structure in place. This scaffold is seeded with A549 human epithelial carcinoma cells and human endothelial cells from cord blood progenitors. The A549 adhered well to the alveolar surfaces, while the endothelial cells adhered well to the vasculature.

Levels of Differentiation of Stem Cells
There are three broad types of stem cells, defined by their potency, or the number of cell types it can differentiate into. Embryonic Stem (ES) cells have the most potency, followed by induced pluripotent stem cells(iPS/iPSCs), and embryonic/adult tissue specific stem cells. The test for potency is to see if the cell can generate all three germ layers - ectoderm (will form epidermal layer of skin), mesoderm (will form muscle, bone, kidneys, blood, gonads and connective tissues), and endoderm (will form the lining of the gut, the liver and the lungs. They tried to find the genes responsible for the pluripotency of stem cells, or genes which stops the cell from differentiating, thus allowing stem cells the ability to self-renew. Boyer screened some of the genes from developmental biology and found only 3 transcription factor genes scattered across the genome which are responsible for these properties; they are OCT4, SOX2, and NANOG. These proteins are involved in chromatin remodelling, by keeping DNA physically open at genes that encourage pluripotency and discourages differentiation; and/or involved in self-renewal, for example using signal transducer and activator of transcription 3 (STAT3). Stem cell chromatin is less compact that in differentiated cells, shown by its faster turn-over rate (faster time of recovery in fluorescence recovery studies), meaning proteins, such as histones, binding to chromatin is a more dynamic process (both on and off) than in differentiated cells. One factor is the nucleosome assembly factor HirA; ES cells lack this factor and histones do not bind tightly, thus are more dynamic. In contrast, ES cells which expresses HirA differentiates. thus stem cells are more sensitive to signals and transcription factors than differentiating and differentiated cells.

Homeobox
The homeobox is a DNA sequence found within genes that are involved in the regulation of patterns of anatomical development (morphogenesis) in animals, fungi and plants, such as the Hox gene clusters. It encodes the homeodomain, which has 60 conserved amino acids. The homeodomain binds with some specificity to the promoter region of genes, forming complexes with other transcription factors, such SOX2, which together increases its specificity. Homeodomain-containing proteins are involved in promoting many homeodomain genes that determine cell fate in many organisms; two such proteins are OCT4/POU5F1 and NANOG. As OCT4 and NANOG are also transcription factors for their own genes, there is a positive feedback loop.

Disruption of OCT4 results in the inappropriate differentiation of ES and ICM cells to trophectoderm cells; and disruption of NANOG results in the differentiation to extra-embryonic endoderm. It was also found that overexpression of OCT results in a similar phenotype to that of knock-out NANOG.

Hox genes
Hox genes are homeobox-containing genes which encodes for proteins that determine the identity of cells along the anterio-posterior axis

Embryonic Stem Cells
ES cells are cells taken from the inner cell mass (ICM) of the blastocyst. They are taken out 4-5 days after fertization, when the blastocyst has reached a cell count of about ~100. They can propagate in culture

ES cells cultured on a bed of stromal fetal cells treated with conductive factors such as bone morphogenic protein 4 (BNP-4) and sonic hedgehog, tend to differentiate into neural lineages. ES cells treated with retinoic acid (RA) and sonic hedgehog agonist, grown as free-floating cells, differentiated into motoneurons. By experimenting with different treatment conditions, one can push ES cells into differentiating into lineages which usually heal slowly following injury or from degenerative diseases, such as nervous system cells, pancreas and heart.

Because obtaining the ICM will destroy the embryo, ethical issues have been raised against ES cell research.

Induced Pluripotent Stem Cells (iPS/iPSCs)
It was already known that differentiated cells can be reprogrammed to stem cells by transferring the nuclear contents from that cell into oocytes, or by fusing it with an ES cell. Unfertilized eggs and ES stem cells contain factors which gives it its toti- or pluripotency. Suspected factors include Oct3/4, Sox2 and Nanog, and the upregulation of Stat3, E-Ras, c-myc, Klf4, and β-catenin.

Takahashi and Yamanaka introduced four factors - Oct3/4, Sox2, c-Myc, and Klf4 (note NANOG was dispensable) - under ES cell conditions, into mouse embryonic or adult ﬁbroblasts. The resulting cells exhibited self-renewal nad pluripotent properties, in morphology, in gene expression (most ES genes are expressed) and surface markers. Fluorescently-labelled iPS cells were able to contribute to mouse embryonic development when injected into the blastocyst, and thus can act as stem cells in vivo; when injected into nude mice in a teratoma test, it formed a tumour which was not rejected and had all three germ layers. In vitro, they formed embryoid bodies, clusters of cells in which there is a dividing centre, and an outer layer with differentiated cells. The chromatin in iPS are demethylated, just like ES cells, meaning it has the chromatin dynamics observed in ES cells.

This method of obtaining stem cells for therapy resolves two major issues:
 * Ethical - Because obtaining the cells to which the iPS are induced from do not involve human embryos, the issues arising from destroying embryos are not valid here
 * Clinial - Because we can induce stem cells from the patient's own cells, the problem of transplant rejection is eliminated.

However, it raises issues of its own:
 * Because it has self-renewal properties, it can potentially become a cancer stem cell, such as in the teratoma test
 * The induction of the four factors into the cells require transfection, often using viruses. It is also possible not to use viruses, but this method is less efficient.
 * It is thought that the iPS still retain some memory, but it is unclear how the type of cell iPS is derived from can affect its behaviour.
 * iPS-derived mice are more likely to die of cancer than ES-derived mice.
 * iPS cells are not exactly like ES cells, for example the histone modifications and transcription is slightly different.

Regulation
Because stem cell is so important, it must be regulated so the right proportions of dividing cells differentiate while the others self-renew, ensure the stem cell pool is not depleted. Mechanisms must also be in place to ensure they do not divide too much, otherwise it may turn in cancer stem cell.

Stem cell niche
Signals which helps the stem cell remain a stem cell are provided by localized and specialized supporting cells (usually stromal cells), other stem cells, components of the extracellular matrix etc. Collectively, they are known as the stem cell niche.

The niche can provide a wide range of conditions in which the stem cell can be maintained. It provides a physical anchor for which the stem cell can grow on, e.g. DE-cadherin in Drosophila testes, or N-cadherin in bone marrow hematopoietic (HSC) niche bind homophilicly. The niche provides regulating factors of stem cell differentiation and proliferation, such as Wnt, Shh, BMPs, Notch, FGFs etc. It also has signals which determine the symmetry of division.

The extracellular matrix and its physical forces can affect stem cell fate; the mitotic spindle can also affect the fate of the divided cells. If the spindle is twisted or have deposition of proteins, then the division may not be symmetrical, giving rise to one stem cell and one transit amplifying (TA) cell. Stem cells can be imaged in culture, but few are transparent enough to be imaged in vivo. Because stem cells can only be maintained within the stem cell niche, to study stem cells accurately, one must be able to image it in vivo, within its stem cell niche. In this paper, Sheng and Matunis used spinning-disk confocal or 2-photon microscopy that enabled us to image stem cells within intact testes for up to 12 hours - about half of the GSC cell cycle. The niche is here to provide signals but it may also serve to rejuvenate the stem cell. Stem cells without rejuvenation will age and die eventually. Older mice's satellite cells tend to produce more fibrous cells than muscle cells; this is known as fibrosis, and causes excessive connective tissue in an organ or tissue. This process is known to be caused, at least partly, by canonical Wnt signaling pathway, and can be suppressed by Wnt inhibitors. It is thought that components in the serum of the old mice binds to Frizzled, the receptor for Wnt, and induces Wnt signalling. In 2007, Brock, operated on a young and old mice, fusing their circulation together (parabiosis). The results showed that the serum of the old mice aged the progenitors of the young mice, but the same effect was also observed - the serum of the young mice made the progenitor of the old mice behave like young cells.

Storage niche
Keeps stem cells at G0, and only enter it into the cell cycle when injured.

Symmetrical division niche
Where one stem cell divides into two, with one staying in the niche, and the other moving out of the niche to differentiate.

Asymmetrical division niche
Where signals from outside the niche polarize the stem cell and so it divides asymmtrically.

In Drosophila testes
In the Drosophila testes, there is a cluster of non-dividing stromal cells which provides the stem cell niche for germline and somatic stem cells. Hub cells express 'unpaired' which causes stem cells adjacent to the hub cells to exhibit Jak-STAT signaling, preventing their differentiation. The stem cells may divide and move away from the hub cells, at which point they can differentiate to form sperm.

The hub cells bind to GSCs via cadherin junctions, it is this junction which determines the orientation of the mitotic spindle. And it is the orientation of the mitotic spindle which determines where the spatial coordinates of the daughter cell after division. Sheng and Matunis used the technique described above to track stem cell division and found that most germline stem cells (GSCs) divide with their spindle orientated perpendicular to the hub cells, and thus one of the daughter cells are displaced from the niche and go on to differentiate. Therefore, they concluded the typical division gives rise to one renewed stem cells and the other a daughter cell. However, they also found in wild type young testes, some GSCs swiveled after division so the daughter cell remained in contact with the hub cells, and thus in these divisions, one GSC gave rise to two GSCs. In contrast, there were also observations where the GSC divides and both of the resulting cells lose contact with the hub cells and go on to differentiate. Sheng and Matunis suggests that these three process - asymmetric division, symmetric renewal, and symmetric differentiation - occur simultaneously in vivo, and are used to balance the output of cells during steady state.

They also observed instances where differentiated cells (transit-amplifying stem cell daughters) move back into the niche and regain its stem cell properties, used when the stem cell population around the hub cell is depleted, and thus is used in regeneration. This breaks the status quo where long-lived, slow cycling cell divides to give daughter cells, while itself remained attached to the hub, and remains like so for the whole lifetime of the organism. This has only been demonstrated in Drosophila, and not yet in mammals.

Hub cell equivalents
Cap cells in Drosophila ovaries, distal tip cells in C. elegans gonad, osteoblast in M. musculus bone marrow and endothelial cells in M. musculus brain are all equivalent niche cells in other organism