User:Kinkreet/SCRAv2

Stem cells

Potency

The human gametes (ovum and sperm) are haploid cells, with 23 chromosomes in each. They merge into one during fertilization to become a diploid cell called the zygote, containing 44 autosomes and 2 allosome (sex chromosomes). This zygote is totipotent, because it is able to give rise to every cell of the human body as well as the extraembryonic tissues such as the placenta.

The zygote undergo embryonic cleavage (rapid division without significant growth); when it has undergone 5 divisions (to 32 cells), it has become a ball of cells collectively termed the morula. The cells at the outer edge of the morula binds tightly to each other through desmosomes and gap junctions. The cells of the morula secretes a viscous liquid, causing a central cavity to form, becoming a hallow ball of cells collectively known as the blastocyst. The cells on the edges of the blastocyst (trophectoderm) provides nutrients for the inner cells and will become the placenta and other extra-embryonic tissues, while a cluster of cells trapped on the interior of the blastocyst (inner cell mass, ICM) will give rise to the eventual embryo. The ICM is pluripotent because it is able to give rise to all three germ layers (thus all cells of the human body) but not the extraembryonic tissues, unlike the totipotent zygote. The blastocyst reorganizes from a single cell layer into a trilaminar gastrula, consisting of the ectoderm, mesoderm and endoderm. Each of these layers contains different types of multipotent stem cells, which contributes only to the tissue it resides in. These multipotent stem cells are able to divide asymmetrically, giving rise to progenitors/transit amplifying (TA) cells which divides rapidly and its progeny differentiate into terminal differentiated cells.

A stem cells is defined as cells which are able to give rise to multiple lineages while maintaining its own existence through self-renewal. A lineage is the genealogic pedigree of cells related through cell division; in order words, all cells in a lineage is derived from a common progenitor. Stem cells are able to give rise to many lineages by giving rise to different progenitor cells, each of which is committed to proliferate and differentiate to give rise to cells of their lineage. For example, the multipotent haematopoietic stem cells can give rise to common lymphoid progenitors which gives rise to lymphocytes; but it can also give rise to common myeloid progenitor which gives rise to the erythrocytes. The common lymphoid progenitors and lymphocytes are of the lymphoid lineage, and the common myeloid progenitor and erythrocytes are of the myeloid lineage; the haematopoietic stem cells is multipotent and able to give rise to both these lineages. On the grander scale, all cells of the lymphoid and myeloid lineages can also be said to be part of the haematopoietic lineage. Embryonic stem cells are derived from the inner cell mass (ICM) of the blastocyst and are pluripotent - able to give rise to the three germ layers and every lineage of the human body. Often a lineage can be defined by morphological and functional characteristic, gene regulation and expression of transcription factors, signalling molecules, non-coding RNAs (ncRNAs) and other epigenetic marks.^[1]

Embryonic stem cells

Embryonic stem cell were first isolated from mice in 1981^[2] and in humans in 1998^[3].

The embryonic stem cell niche is maintained by Nanog, LIF and Oct4.

Stemness

In stem cells, chromatin-remodelling genes that promotes a open conformation of DNA, such as histone acetyltransferases (HAT) is upregulated. DNA are mostly in the euchromatin conformation, and most genes, even those associated with differentiation, are transcribed. These genes are transcribed at low levels to allow the stem cell to respond rapidly to signals, such as those arising from injury; upregulating transcription is easier and faster than starting from scratch. However, the level of transcription of differentiation genes are kept low to allow the stem cells to maintain self-renewal and multipotent properties.

Generally, cell-cycle inhibitory genes (p16, p18, p21, p17...) are upregulated in stem cells, as this prevents stem cells from proliferating out of control. DNA repair genes are also upregulated to prevent DNA damage; this is important as its progeny will all inherit any mutations the stem cell accumulates.

Stem cell markers

Sox2

SRY (sex determining region Y)-box 2 (Sox2) is a member of the family of SRY-related HMG box transcription factors that is essential in maintaining the self-renewal and pluripotency of embryonic stem cells, as well as maintaining adult stem and progenitor cells in the adult. Sox2 is thought to be a tissue non-specific stem cell marker, found on the mouse in several epithelia, including stomach, cervix, anus, testes, lens, and multiple glands.

When EGFP is co-expressed with Sox2 in mice by knocking in the EGFP gene to be under the control of endogenous locus-regulatory region of the Sox2 gene, EGFP-positive cells are observed in the inner cell mass and in the extraembryonic ectoderm of blastocysts, in embryonic stem cell cultures, and in proliferating neural progenitor cells (NPCs) throughout the CNS throughout the lifetime of the mice, at the subventricular zone of the brain, neural retina, trachea, bronchiolar epithelium of the lungs, tongue, dermal papillae of the hair follicles, seminiferous and lens epithelium, glandular stomach, squamous epithelia lining the esophagus, forestomach, anus and cervix.^[4]^[5] The localization of the Sox2-GFP cells in these tissues are near or enriched in basal cells, the location of many stem cells. Consolidating the fact that Sox2⁺ cells are stem cells, when Sox2-GFP cells were analysed using the sphere formation assay, they were able to maintain their presence throughout many passages and was able to give rise to neurons, astrocytes and oligodendrocytes, exhibiting self-renewal and multipotency properties.^[4]

The self-renewal and multipotency of stem cells can be observed in vivo and analysed as it progresses using lineage tracing. Using Sox2-EGFP:ROSA26-lsl-EYFP cells, it was observed that a single Sox2⁺ is able to be maintained in tissues and give rise to differentiated progeny of different type. The single Sox2⁺ cell was able to be maintained at least 3 months in testes of mice, and 15-22 months in the glandular stomach of mice, as well as in other tissues, displaying its self-renewal properties.^[5] The stem cells of the stomach gives rise to mucus cells, parietal cells, enteroendocrine cells and chief cells; Sox2⁺ cells in the testes are able to reconstitute the testes and reinitiate spermatogenesis, displaying its multipotency.^[5]

Another properties of stem cells is their ability to proliferate and differentiate in order to regenerate damaged tissues. Ganciclovir (GCV) is released at a constant rate using an implanted osmotic pump system, into the flanks of Sox2-deltaTK mice. GCV is a pro-drug, which means it is not toxic as it is; GCV can be activated by phosphorylation by viral thymidine kinase (TK), which is present in the Sox2-deltaTK mice. Thus, GCV and deltaTK act in a suicide gene/pro-drug manner to ablate the Sox2⁺ stem cell populations in the mice. Mice treated with GCV showed signs of diseases such as severe inflammation and occasional edema, and most die after 2 weeks, probably due to the development of ulcers in the stomach and oral mucosa. As determined by histochemistry, Sox2⁺ stem cells near the basal membrane is ablated, but the Sox2^- cells in the stratified layers above remained unaffected, confirming the specificity of GCV for deltaTK-expressing Sox2⁺ cells. However, due to heterogeneity in the stem cell population, GCV would not have been able to ablate the entire Sox2⁺ stem cell population. Rescue of the mice by removal of the pump saw gradual recovery of the mouse in subsequent weeks. Recovery was complete after 3 months in the tongue, esophagus, forestomach and testes. This recovery is possible because residual Sox2⁺ cells are able to proliferate and differentiate in order to reconstitute the damaged cells.^[5]

If Sox2 is a stem cell marker, then it should only be present in stem cells and not in differentiated cells. Using Sox2 antibodies against different tissues, it was found that Sox2 is not expressed in committed cells, supporting the notion that Sox2 are uncommited stem cells.^[5] However, other evidence shows Sox2 is also observed in non-reconstitutive cells in the testis and also in transit amplifying cells. Thus, Sox2 must be used in conjunction with other markers to strictly mark for stem cells. Sox2 is not be the sole stem cell marker, as Sox2-GFP is not observed in the liver, kidney, heart, small intestine, colon, pancreas or bone marrow.^[5] Some tissues, such as the duodenum and submandibular gland, only express Sox2 transiently during fetal development, whilst most others continue into adulthood and constitute the adult tissues.^[5]

Nanog

Required for stem cell self-renewal

Oct3/4

Required for stem cell self-renewal

HoxB4

Required for stem cell self-renewal

Tissue-specific stem cell markers

Pluripotent and multipotent stem cell markers are gradually inactivated and tissue-specific markers gradually becomes activated. As a cell matures and differentiate, the chromatin remodelling leads to expression of tissue-specific genes and silencing of stemness genes. Tissue-specific stem cell markers, such as Bmi-1 (polycomb group protein), Sox9 (HMG box transcription factor), Tert (telomere subunit) and Lgr5 (GPCR), are absent in pluripotent stem cells.

Depletion of Zfx1 (zinc finger transcription factor 1) led to lack of self-renewal both in ESC and HSC.^[6] However, it cannot be the sole 'self-renewal' gene, as Zfx is also expressed in differentiated cells.

These tissue-specific genes require tissue-specific transcription factors, which activates themselves creating a positive feedback loop; knockdown or overexpression of these transcription factors can alter the lineage commitment of cells. The basic leucine zipper (bZIP) transcription factor E4BP4 (a.k.a NFIL3) is essential for natural killer cell and CD8α⁺ dendritic cell development in mice. Knockout mice developed B cells, T cells, NKT cells but lacks NK cells.^[7] The Ikaros gene encodes for a family of transcription factors that promotes the differentiation of pluripotential hematopoietic stem cell(s) into the lymphocyte pathways. A homozygous mutation in the Ikaros DNA-binding domain lead to an ablation of the lymphoid lineage, and all the progeny from the haematopoietic stem cell differentiated into the erythroid and myeloid lineages.^[8]

c-Myc

c-Myc promotes epidermal stem cell differentiation and proliferation along the epidermal and sebaceous lineages^[9]

Stem cell niche

The idea of a stem cell niche was first proposed by Schoefield in 1978.^[10] A stem cell niche is the in vivo microenvironment made up of a dynamic and complex network of cell-cell/matrix, hormonal, structural, physical, metabolic, neuronal and chemical signals which are required to maintain stem cells in quiescence.

The most widely studied stem cell niches are the invertebrate germ stem cell (GSC) niches and adult somatic stem cells niches. The most notable GSCs are the Drosophila ovary GSC, Drosophila testis GSC and the C. elegans gonad GSC; all these GSCs are maintained by associating with niche cells, which provides the signals to maintain the GSCs. Drosophila ovary GSCs associate with cap cells, Drosophila testis GSC associates with hub cells, and C. elegans gonad GSC associate with a single distal tip cell.

The Drosophila ovary GSC is housed in a larger structure called the germarium. The GSCs remain associated with cap cells through E-cadherins. Cap cells and escort cells (another niche cell which lines the sides of the germarium) expresses decapentaplegic (Dpp, theDrosophila counterpart to BMP); Dpp activates TGFβR signalling in GSC and block differentiation. A loss in Dpp would result in the cells differentiating and loses their ability to divide. This is what occurs when a GSC divides - division will usually occur on an axis perpendicular to the cap cells, and so one daughter cell will divide out of the niche, losing the Dpp signalling and begin differentiating into a cystoblast (CB); the other daughter will remain attached to the cap cells and stay a GSC. The CB will divide synchronously 4 times to give a cyst of 16 cystocytes (CC), one of which will further differentiate into an oocyte.^[11]

The presence of a stem cell niche is paramount if one wants to culture stem cells in vitro or use it for transplantation. For example, satellite cells (muscle stem/progenitor cells) will differentiate into myoblasts if cultured in vitro^[12] unless they are in contact with viable myofibers, which allows the stem cells to remain in quiescence even in the presence of mitogens.^[13]

It has also been shown that progeny of stem cells can migrate back into the niche and regain stemness; this is particularly important after injury in the stem cell population, where the progeny cells are able to replenish the lost cells.

Induced pluripotent stem cells (iPSCs)

Terminally differentiated cells can be induced back to being a stem cell using factors Sox2, Oct4, Klf4 and c-Myc^[14]

Tissue-specific stem cells

Different tissue-specific stem cells are distinguished from each by the type of niche cells, its position and its potency.

Gastrointestinal tract

The gastrointestinal tract includes the esophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (the colons), and there are resident stem cells in each of these organs. Stem cells in the gastrointestinal tracts are often the most active because the lumen of the GI tract is often under harsh conditions (such as the strong acidic conditions in the stomach) and so cells are lost at a higher rate than other tissues.

Intestines

The intestinal epithelium is the fastest turned-over tissue in mammals^[15]. The lumenal surface is subject to harsh proteolytic and a range of pHs (pH 6 in the duodenum to pH 7.4 in the terminal ileum, back to 5.7 in the caecum, increasing again to pH 6.7 in the rectum)^[16], and thus is damaged easily. Cells on the lumenal side is shed regularly and must be replaced by more cells; these cells are derived from intestinal stem (ISC) and progenitor cells.

The structure of the intestinal epithelium consists of microprojections called villi, which increases the surface area of the inside of the intestine, to ensure efficient nutrient absorption. The villi are most prominent in the small intestines, as the large intestine's function has moved away from absorbing nutrient to reabsorbing fluids. At the base and in between villi are small indentations termed crypts. Intestinal stem cells (ISCs) lie near the bottom of the crypt, and divides giving rise to progenitors which proliferate and differentiate, all the while migrating upwards towards the tip of the villi, evnetually reaching the top and shed. All cells in a single crypt originates from the same ISCs, and are thus clonal; villi are not clonal as cells in one villus can originate from multiple crypts.

Using Lgr5^[17]andBmi1^[18] as markers for intestinal stem cells, it was found in organoid cultures derived from crypt cells that the ISCs are located predominantly four cells above the base of the crypt, at the +4 position. The base of crypt (positions 0 to +3 are populated by Paneth cells, these cells are derived from the ISCs, but instead of migrating upwards, the progeny migrates downwards to reconstitute any lost Paneth cells.

The intestine is primarily made up of four types of cells - enterocytes, goblet cells, enteroendocrine cells and Paneth cells; ISCs constitute only a small population. Enterocytes are long column-shaped absorptive cells that is attached to the basal lamina; it forms a single-layered epithelium ensuring efficient transport of nutrients from the lumen into the blood stream, which arrives through the protal vein and into the liver to be filtered. Goblet cells secrets mucus and enteroendocrine cells secretes hormones such as ghrelin and leptin which regulates appetite. Paneth cells lies at the base of crypt and secretes lysozyme, TNF and anti-bacterial peptides to contribute to the immune system. Because the Paneth cells lies at the very bottom, they are landmark cells which helps in identifying ISCs.

ISCs are maintained inside their niche, when ISCs divides and a daughter leaves the niche, it is exposed to a range of signals which will guide their differentiation into different cell types.

The intestinal stem cell niche is primarily maintained by Wnt signalling. Overexpression of Wnt inhibitors (e.g. Dkk1) or the knockout of Wnt ligands (e.g. TCF4 and c-Myc) leads to the depletion of ISCs and the loss of crypts.^[19]Three signalling molecules - epidermal growth factor (EGF), noggin and R-spondin - alone are able to maintain Lgr5⁺ ISCs in culture; and thus these three signals are likely to be present in the native niche. Noggin inhibits BMP signalling, and thus prevent ISCs from differentiating; R-spondin activates Wnt; and EGF activates the KRAS signalling pathway.^[20] The niche itself can be established by the Lgr5⁺ ISCs autonomously, as evident by the fact that a single Lgr5⁺ ISC was able to create an organoid, with similar morphology to the native tissue and distribution of different cell types, without the need of a pre-existing niche.^[15]

As the daughter cells migrate up, Notch signalling ensures that secretory cells are distributed evenly and regularly in a layer dominated by enterocytes. Secretory cells displays and secretes high levels of the Notch ligand Delta onto the lipid membrane; the Delta ligand forms a gradient decreasing away from the secretory cells. Enterocytes display a larger proportion of the Notch receptor, which binds to the Notch ligand Delta and maintains its enterocyte fate. As the progeny of the ISCs migrate up, if they are near a secretory cell, it will encounter a high concentration of Delta and commit to the enterocyte fate; whereas if they are far away from a secretory cell, it will encounter a low level of Delta, and be committed to the secretory cell fate. In this way, secretory cells are spaced in regular intervals between enterocytes.

Eph/Ephrin signalling determines crypt formation. Eph is the ligand of Ephrin, and vice versa; Eph is expressed at high levels at the base of the crypt and maintains Paneth cells (which defines a crypt), and as you move up the crypt, the concentration of Ephrin increases. There are two types of Eph - EphB2 and EphB3 - and both are present in two chromosomal copies; only one copy of either is required for keeping the organization of Paneth cells.

Hedgehog signalling is observed at the approximate boundary between crypt and the villus, forming a gradient with the highest concentration at the villi and lowest near the crypt.

In humans, bone morphogenic protein (BMP) is a signal which induces differentiation. Thus unsurprisingly, BMP is expressed most highly on the top of the villi and lowest towards the base of the crypt. To prevent the ISCs from differenting and depleting the stem cell pool, BMP antagonist (such as noggin) exists at the base of the crypt to neutralize any BMP. (Note that BMP2/4 in germarium of female Drosophila melanogaster promotes blocks proliferation, showing cross-species difference of the same signalling molecule)

Other tissues

Similar to the intestinal crypts, the stomach is lined with gastric pits. Gastric epithelial stem cells^[21] have been located at the base of these gastric pits.

The esophagus is a tube made out of different layers - (from the lumen) mucosa, submucosa, adventia and muscularis propria. The mucosa layer of the esophagus is corrugated so as to allow for the expansion of the esophagus when food passes through the organ. The structure of the tissues in the esophagus resembles the interfollicular epidermis, however there are no dormant stem cells in the basal layer and the esophagus maintains itself by continuous proliferation, even in the absence of injury.

Epidermis

The epidermis is the outermost layer of cells of an organism; it lies above the (from the out in) dermis, a layer of subcutaneous fat, and a thin layer of muscle (responsible for facial expressions). The structure of the epidermis consists of a basal lamina above which stratified layer of squamous cells lie. There are three major types of stem cells in the epidermis - interfollicular epithelial (IFE) stem cells, which is part of the basal/germinal layer; and bulge stem cells and sebaceous gland stem cells which resides in the hair follicle at the bulge and sebaceous glands, respectively. During homeostasis, the three stem cell populations maintain its respective tissue; but after injury, one stem cell population is able to reconstitute the others.

Interfollicular epithelial (IFE) stem cells

The interfollicular epidermis is made up of 4-5 layers of cells. The innermost layer (closest to the dermis) is the basal/germinal layer (stratum germinativum), it is not flat but made up of undulating ridges and valleys, and contains the population of IFE stem cells. IFE stem cells divide and is pushed laterally to the side and replaces any cells that has migrated up into the upper layers. Cells are kept attached to the basal laminae using hemidesmosomes. Proliferating cells then migrate up through the spinous layer (stratum spinosum) and into the granular layer (stratum granulosum), where they lose their nuclei and their cytoplasm becomes granular. Cells migrate further and become part of the cornified layer (stratum corneum), made up of 10 to 30 layers of polyhedral, anucleated, merged corneocytes. As the cells migrate up, they become flatter, cells on the cornified layer are dead is has become a mesh of keratin fibers with lipids.

Hair follicle and the dermal papilla (DP)

Even in the same organism, there are different types of hair follicles, with heterogeneity represented as having different thickness, length and shape of the hair shaft^[22]. The dermal papilla (DP) cells are Sox2⁺hair follicle stem cells, evident by its ability to generate whole and complete hair follicles of all types, including the bulge and sebaceous gland stem cell populations. DP cells resides near the peak of the ridges on the basal/germinal layer of the epidermis. Primary DP cell cultures can be cultured (regenerative potential only lasts a few passages) and transplanted into individuals with alopecia (hair loss) to restore hair growth.^[22]

Different types of hair follicles are developed at different stages during postnatal development. Reinoic-acid-binding protein 1 (CRABP1), CD133 (a.k.a. PROM1) and alkaline phosphatase are expressed on all DP cells and can be used as a marker to isolate DP cells.^[23]^[24] Type-specific DP can also be isolated because all DP cells express Sox2 at different times, identifying Sox2⁺ cells at different developmental stages allows for the isolation of different types of DP cells.^[25]

During embryonic development, the formation of hair follicles begin with a thickening of the embryonic epidermis, into a structure called placode containing Sox2⁺ cells; the placode also prevents neighbouring cells from becoming placodes. A group of fibroblasts condenses into the dermal condensate underneath the placode. The placode and condensate reciprocally signal to each other, mainly through Wnt signalling, and causes cells of the placode to proliferate down to the dermis, creating a pit, forming the overall shape of the hair follicle. A group of epidermal cells envelops the dermal condensate which becomes the dermal papilla (DP)^[22]. The DP signals to neighbouring epithelial cells to proliferate and differentiate into different cell lineages that constitute the inner root shaft (IRS). The inner root shaft is the base structure of a hair follicle, and is made up of layers (IRS cuticle, Huxley layer, and the Henle layer) of cells, each with their own progenitor cell. The medulla, cortex and the hair cuticle layers form the hair shaft, the most central part of a hair follicle.^[22] Wnt an Hedgehog (downstream of Wnt^[26]) signalling is required throughout the development.

Throughout the life of a mammal, a hair follicle would have gone through many cycles of growth and regression. It cycles through three phases of telogen (resting; days to months), anagen (growth) and catagen (regression). The hair follicle is only one of few tissues (such as the mammary gland) which is able to undergo cycles of growth and regression; this is mediated by stem cells called dermal papilla (DP) cells.

During catagen, the epithelial cells at the base of the follicle undergo apoptosis which leads to the hair being shed; the dermal papilla migrates upwards next to the bulge and remains there throughout telogen until anagen. On the onset of anagen, the DP migrates or is pushed down towards the base of follicle and initiate hair growth. The new hair will push the old hair out of the follicle. The longer the anagen phase, the long the hair; the serine protease Corin and Sox2 is upregulated during anagen^[27]^[28], and so can be used to isolate DP cells at anagen. Sox2⁺ DP cells are also able to differentiate into skin-derived progenitor cells (SKPs), and consequently be able to replenish neurons, glia, smooth muscle cells and adipocytes.^[29]

Fgf7 and Fgf10are expressed by DP cells to promote proliferation of neighbouring epithelial cells^[30], Wnt is required for the proliferation of DP cells themselves.^[31]

Mammary gland

The development of the mammary gland begins at the early fetus stage, and originates from the ectoderm and mesoderm layers. A layer of ectodermal cells on the ventral (towards the abdomen) of the embryo thickens to form the mammary band, which differentiates sequentially into the mammary streak, mammary line, mammary crest, mammary hillock, and at about 3 months post-fertilization, into the mammary bud. The developed mammary bud remain unchanged until about 5 mpf, at which point the mammary bud differentiates into primary sprout(s). The primary sprout(s) will eventually become openings on the teat to which milk is expelled from. These openings are termed galactophores, and its number per teat or nipple can differ greatly between mammals. The deeper layer of the bud epithelium differentiate further into 10-25 (in humans) secondary buds, which eventually form the lactiferous ducts and cavities where milk is produced and transported to the nipple to be expelled. The secondary buds lengthens, forming solid cords of epithelial cells that grows into the mesenchymal cell populations and reaches the subcutaneous tissues below. These solid 'cords' of cells will become canalized (hollowed) shortly before birth. After birth, the cells at the end of the secondary sprouts will continue to branch to form new cords; this growth is isometric, occurring at a similar rate to body growth.

Prior to puberty, the anterior pituitary glands of females releases follicle-stimulating hormones (FSH) and lutenizing hormone (LH) in a cyclic pattern. FSH and LH causes the ovaries to release the female sex steroid hormones estrogen (estradiol) and progestins (progesterone), respectively; both contribute to the growth of the mammary gland during puberty. Estrogen encourages cells at the terminal buds to proliferate in order for the ducts to elongate and branch; progesterone develops these ducts and enlarge them.

The mammary develops significantly during puberty, but the majority of growth occurs during pregnancy. There is a fat pad in every mammary gland; during pregnancy, significantly elevated levels of estrogen and progesterone causes fat pad to be gradually replaced by ducts, blood vessels, connective tissues, and alveoli. Furthermore, estrogen and progesterone continues to signal for elongation, branching and enlargement of ducts. At the same time, somatotropin and prolactin is synthesized in the anterior pituitary gland, which encourage more nutrients to be used for milk production at the cavities at the ends of the ducts.

After birth, the levels of sex steroid hormones decreases; secretory cells undergo apoptosis, leading to reduced milk production, and when apoptosis occurs at a greater rate than generation, the mammary will regress and revert back to pre-pregnancy state. It is one of a few tissues that are able to undergo cycles of growth, functional differentiation and regression.

Stem cells are postulated to be central to the growth of the mammary glands after regression; and indeed mammary stem cells have been identified which can regenereate a complete and functional mammary gland.^[32] Although the existence of mammary stem cells is well-established, the location of the mammary stem cells is highly disputed. Some suggests that the luminal epithelial and myoepithelial cells at the sides of the terminal bud are the stem cells, they'd proliferate laterally to give rise to the body cells and cap cells; others suggests that the cap cells at the end of the terminal end bud are the stem cells.^[33]

Bone Marrow

The bone marrow lies inside the bone and is primarily made up of haematopoietic, mesenchymal, and endothelial cells; other cell populations such as osteomacs (resident macrophages adjacent to osteoblasts), T cells, adiopocytes, fibroblasts and CD146⁺ osteoprogenitors also exists. It is lined by a calcified bones maintained by osteoblasts and osteoclasts.

The haematopoietic stem cell population maintains T cells and macrophages, whereas nestin⁺ mesenchymal stem cells (MSCs) gives rise to osteocytes (in bones), chondrocytes (in cartilage), myocytes (in muscles), fibroblasts (in skin, tendon and ligaments), astrocytes (in the central nervous system), stromal cells (in the bone marrow) and adipocytes (in fat).

Haematopoietic stem cells

Haematopoietic stem cells remains in the yolk sac for up to 3 weeks post-gestation, after which it gradually migrate to the newly-formed liver. 7 weeks post-gestation the HSC can also leave the yolk sac and faetal liver into the bone marrow, this can occur upto ~40 weeks post-gestation, often even after birth. HSCs migration occurs in clusters, where HSCs migrate towards the blood, pulling neighbouring cells along with it; only when it forms a cluster will it be released into the bloodstream.

The HSC population is maintained by the niche, which residues in the stroma and numerous factors produced by niche cells, primarily mesenchymal stem cells^[34]. HSC have the characteristics of not having that many characteristics - it lacks differential gene expression as this is associated with terminally differentiated cells. BMI1 remodels chromatin to silence many genes to allow LT-HSCs to maintain its potency. The Dicer complex involved in RNA interference generates the microRNA Mir-125a, which also silence apoptotic genes. PTEN is also required for HSC maintenance.

CXCL12 is a chemokine produced primarily by osteoblasts and bone marrow stromal cells, and CXCL4, a 7TM receptor, is it ligand. Normally, HSCs are retained in the bone marrow through binding of CXCL12 by CXCL4 expressed on the surface of HSCs. The sympathetic nervous system (SNS) is involved in its regulation - a stimulated SNS releases norepinephrine (NE), which suppresses osteoblasts so they do not produce CXCL12, and the HSCs are no longer retained.

For HSC to maintain and/or regenerate tissues, it must be able to come out of quiescence and differentiate. MLL (Myeloid/Lymphoid, or Mixed-Lineage, Leukemia) is a histone methyltransferase that activate gene transcription, including c-Myc which, in mammals, promotes proliferation and differentiation. p21 expression is also required for HSC proliferation.

Haematopoietic stem cells are unique amongst other stem cells because it has the ability to migrate out and then return to its niche, while still maintaining its stem cell properties. At any given time, a few HSC will migrate out and into the circulation. a process similar to the HSC migration during development. Once in the vasculature, it will remain there for ~2 minutes, before rolling on the inner wall of the endothelium, adhering to to the vessel wall with interactions mediated by VCAMs and selectins. It binds strongly to the chemokine molecule (CXCL12) and begin migrating through the blood vessel wall and back to the stroma, where the niche cells are.

This mobility of HSC makes HSC-based therapies more convenient. Obtaining HSC requires a simple administration of granulocyte colony-stimulating factor (G-CSF) which mobilizes the HSCs into circulation, and we can simply collect the HSCs in the blood. To administer HSCs, for example to replenish a HSC population after chemo/radiotherapy, or to repair damaged tissues, one just have to inject HSCs into the blood, and the HSCs will automatically home to the bone marrow and other CXCL12-expressing cells.

Hepatic stem cells

The liver is made up of many lobes. In each lobe, the portal vein brings in blood from the intestine for the liver to filter, the hepatic artery (HA) brings in oxygenated blood from the heart and the vena cava transport away deoxygenated blood to the heart. Blood from the portal vein passes through a narrow sinusoid (very small and loose vessel) to the vena cava, and through this journey will exchange its nutrients into hepatocytes, which will metabolize the nutrients and release the modified products back into blood. Bile can also be produced from this metabolism is released into the bile duct which feeds back into the duodenum where it aids the digestion of fats. The HA, BD and PV lie in close proximity and is known as the triad; it is believed that many triad feed into a common vena cava, and this system makes up a lobule, with each lobule having one common vena cava.

The turnover for hepatocytes is ~200-300 days, and is normally maintained through simple proliferation of existing hepatocytes. After local injury, such as by hepatotomy, HGF, IL6, TNFα and EGF encourages these hepatocytes to proliferate to regenerate the liver; while blood bile levels and other regulators ensure the liver do not regrow. After chemical injury, normally not-present progenitor cells (e.g. oval cells from the canal of Hering) appear to replenish the tissue. Over 75% of the liver may be surgically removed and the whole liver will be completely regenerated in a matter of months.

Other stem cells

Neural stem cells are present throughout the central nervous system during development but remains only in the subventricular zone (SVZ) and subgranular zone (SGZ) during the adult, where they remain largely quiescent.

Bronchioalveolar stem cells (BASC) are found at junctions between the bronchioles and alveoli. It was able to repair the bronchioles and alveoli after damage, able to self-renew, and is able to differentiate into all cell types in the bronchioles and alveoli as determined by a clonal differentiation assay.^[35]

Lin^-c-kit⁺ cells have been found to be self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells, and are thus likely to be cardiac stem cell.^[36] When these Lin^-c-kit⁺ cells were injected into infarcted rat myocardium, the progeny of these cells differentiate into blood vessels and myocytes. Isl1⁺ (a LIM-homeodomain TF) in rat, mouse and human myocardium are shown to be able to develop into mature cardiac cells which display myocytic markers in the absence of cell fusion, intact Ca²⁺-cycling, and the generation of action potentials, and contributes to the right ventricle, both atria, the outflow trac and regions of the left ventricle.^[37]Thus, isl1⁺ cells may be cardiogenic progenitors.

Obtaining stem cells

Haematopoietic stem cells

Haematopoietic stem cells are able to leave its niche and into the circulation before homing back into the bone marrow again. We can thus simply collect the HSCs from the blood. This process occurs naturally but at a low rate, and thus not many HSCs can be harvested. To counteract this problem, administration of granulocyte colony-stimulating factor (G-CSF) mobilizes more HSCs into circulation, giving more HSCs to be harvested.

Dnmt3a is a methyltransferase which methylates multipotency genes to promote differentiation of HSCs; Dnmt3a^- HSCs can be obtained from transgenic organisms to give HSCs which do not differentiate even in culture.

Somatic cell nuclear transfer (SCNT)

Somatic cell nuclear transfer (SCNT) is a technique first introduced in the 1950s, where the genetic material of an individual is extracted and injected into the emptied nucleus of an egg cell. The resulting cell will be like a zygote, and may develop into a blastula. The cells in the blastula closely resembles ES cells.^[38] These cells can be used as ES cells to regenerate tissues and organs, as well as be used to clone an individual, as seen in Dolly the Sheep.^[39] Originally, SCNT was only successfully performed in mice, but now also in primates.^[40]

Induced pluripotent stem cells (iPSCs)

On a separate note, DP cells can be more readily reprogrammed into iPS cells than most other cell types.^[41]

Laboratory applications

Transgenics

Mammalian ESCs can be transfected with a viral vector, which encourages homologous recombination to occur at specific sites of the ESC genome, replacing the gene of interest and thus knocking this gene down in the ESC. The embryonic stem cell will subsequently give rise to all cells of the organism, meaning all cells of this organism will have the gene of interest knocked out. Which gene is knocked down is determined by the recombination sequence which must be considered when designing the viral vector sequence. Transgenics have been achieved in mice^[42]^[43] and humans^[44]

Martin Evans, Oliver Smithies and Mario Capecchi shared the 2007 Nobel Prize in Physiology or Medicine for creating a transgenic mice.

Chimera

Chimeras can be generated by extracting the ICM from the blastula of the organism displaying a certain desired phenotype, and inject it into the ICM of the same or different species of a different phenotype. The ICM of both will incorporate into the embryo, creating a chimera with patches of cells of one phenotype, and patches of cell of a different phenotype.

Aging

Regeneration

Regeneration is the ability to recreate lost or damaged tissues, organs and limbs. It has high clinical relevance as it allows the regeneration of tissues, for third-degree burns victims, organs for those with organ failure, and of limbs for amputeees.

The control of regeneration is also important, as tissues or organs that overregenerates will lead to disease, such as cancers, or osteoarthritis when bones regenerates too much and grind against each other.

Mechanism

The most common way of regeneration is re-entering cells into the cell cycle and through mitosis, where they are prevented from apoptosing and proliferates; this is known as compensatory hyperplasia. It is a process most attributed to the liver where up to 75% of the liver can be removed and regenerated within a few weeks because the hepatocytes undergo hyperplasia. The human pancreas and blood vessels, and the newt cardiac muscle, can also regenerate in this way.

Another way of regeneration is through proliferation of stem cells/dedifferentiated cells, and the subsequent differentiation of its progeny.

The mechanism of regeneration of some organs such as bird beaks, nipple, testis, ovary, spleen and gills are not yet elucidated.

Methods

Identifying factors involved in regeneration

In humans

Regeneration in humans is effective during the first third of gestation, where wounds heal without scarring. In the post-natal human, regeneration of most part of the body is ineffective and induces scarring.

Stem cells, aging and regeneration

The three topics are closely related. Regeneration are often facilitated by stem cells, regenerative properties of mammals decrease with age, up to the point of failure.

Model organisms

Model organisms are commonly used in research because they are more convenient than studying humans. Ethical restrictions on animal is looser, animal studies are usually cheaper than human studies, the lifespan of an animal is shorter and so process such as development and aging can be studied in a much shorter time frame than with humans. However, as with any model organisms, they are merely models, and there will be aspects between these model organisms and humans, differences that are often fundamental. BMP in humans promotes differentiation whereas inDrosophila it inhibits differentiation; although both have haematopoietic lineages, an immune system with complement system, the blood cells in humans have differences with that of the zebrafish.

Mouse

The mouse model is a good model because it is a mammalian model and so is relatively close to humans; techniques for generating transgenics are readily available. It has a short lifespan (4-5 weeks) and so any developmental/regeneration process can be studied in a short period of time.

Zebrafish

Zebrafish is widely used as a model organism in stem cell research because its embryo is transparent, allowing scientists to track individual cells (including stem cells and cancer cells) and their progeny during embryogenesis or metastasis in real time. A transgenic zebrafish which remains transparent throughout adulthood (casper) have also been developed by crossing nacre^-/- and roy^-/- fish, and allows lineage tracing experiments.

Zebrafish are highly prolific (gives many progeny) which develops quickly. Due to their small size, transcient (a few days) knockdown of genes using morpholinos injctions requires little morpholinos, and drugs can simply be added to the fish water and the fish will absorb the drug. Care must be taken when interpreting results based on morpholino treatments, as the morpholino itself may induce a response from the cells; and the use of a control (e.g. rescue using mRNA, scrambled sequence of morpholino etc) is paramount.^[45]

The zebrafish genome is sequenced and has been characterization using genetic screens^[46], molecular screens^[47] and chemical screens.^[48]^[49]

Although it is a fish, it has an immune system, a complement system and others which resembles humans. Even though at a low incidence, zebrafish are capable of forming every form of tumours.

Using zebrafish as a model organisms has its disadvantages too. The lifespan of the fish is ~90 days, which is relatively long; thus results cannot be obtained quickly. It is difficult to obtain transgenic fish because many genes are duplicated, and both genes must be knocked-out before the fish display the desired phenotype. Currently, there are few markers and antibodies designed to work with zebrafish, and so tracking stem cells might not be feasible.

Signals

Stem cells are often found at hypoxic (low oxygen) locations, such as deep inside tissues or near veins. It is thought that low oxygen levels induces hypoxia-inducible factors (HIFs) which acts to prevent stem cells from differentiating.^[50]

Signalling pathways

Notch, JAK-STAT, DPP/BMP, Hedgehog, Wnt, angioprotein-1 (ANG1) and OPN are some signalling pathways involved in stem cell maintenance and/or differentiation. These signalling pathways often crosstalk with each other, taking extrinsic signals, such as cytokines, hormones, growth factors, cell-cell/matrix adhesion, physical forces and other chemicals or reactive species, and converting these signals to affect transcription, ultimately deciding cell fate.

Wnt

Wnt have different effects on stem cells at different spatial and/or temporal coordinates; and the levels of Wnt is also important. Wnt can act to prevent proliferation of stem cells, because high levels of Wnt is found in stem cell niches, as well as aged myosatellite cells; both cell types do not proliferate.

Wnt also maintains genomic stability in stem cells. β-catenin complexes with Klf4 to induce the transcription of telomerase reverse transcriptase TERT, which is a component of the telomerase that elongates telomeres. Telomerase is a ribonucleoprotein which addsDNA repeats of T T A G G G to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. Telomere shortening is associated with genome instability because it does not protect the end sequences of a chromosome, where 100-200 nucleotides can be lost after each replication event. The length of telomeres in β-catenin-deficient mice is shorter than those of mice with an activated β-catenin.^[51].

Enhanced β-catenin can enhance telomere lengths and prevent and even reverse age-related degradation.^[52]

There are three different Wnt pathways - canonical pathway mediated by β-catenin, the non-canonical pathway and the Ca²⁺ pathway.

BMP

Bone morphogenetic protein (BMP) is a member of the TGFβ superfamily of proteins. In mammals it encourages differentiation whereas inDrosophila it inhibits differentiation.

Integrated pathways

Noggin is a protein that binds to TGFβ signalling ligands such as BMP to prevent its action. Noggin also lead to the expression of Lef1, which can complex with β-catenin to form a transcription factor complex that drives expression of multipotency genes. Thus, noggin both prevents differentiation and maintains multipotency; noggin is found around the base of the intestinal crypt, antagonizing BMP and promoting Wnt, to maintain intestinal stem cells (ISCs).

Cell cycle

A major difference between stem cells and its progeny is that stem cells tend to lie outside the cell cycle and divides only rarely; in mice, some haematopoietic stem cells divide only 5 times throughout the lifetime of the organism. Stem cells tend to be quiescent (temporarily persists in G₀ phase) and do not have a specialized morphology (small cytoplasm and little mRNA transcripts); this should be distinguished from terminally differentiated cells, which are also in G₀, but this is permanent and terminally differentiated cells are not able to re-enter the cell cycle and divide (cancer is the exception)

If the stem cells proliferate too much, the excess cells are trimmed by apoptosis. Apoptosing cells first shrinkage, have its chromatin condensed, moving on to membrane blebbling, nuclear collapse, continued blebbing, apoptotic body formation and the lysis of apoptosis bodies.

Epigenetics

Epigenetics is the study of heritable changes in gene expression or cellular phenotype which are not caused by mutations. The genome of stem cells is exactly the same as that of terminally differentiated cells, but they have different transcriptome and proteome. These difference arise from covalent modifications, such as methylation, acetylation, phosphorylation and ubiquitination, to the genome and its associated proteins, modifications which does not alter the primary sequence.

DNA modifications

Modifications to DNA tend to be more stable compared to proteins, such as histones. The two most common modifications to DNA are acetylation and methylation. Methylation occurs after replication to allow the cell to know which is the newly synthesized strand, so any DNA repair systems can correctly distinguish which strand is the correct one. Methylation can only occur on the cytosine bases that are located 5' to a guanosine in a CpG dinucleotide; because the methyl group is relatively small, a strand of methylated DNA is able to be packaged tightly, into a conformation called heterochromatin. Because it is so tightly packaged, transcription factors and enhancer proteins cannot associate with the DNA sequence due to steric hindrance, and thus transcription is generally repressed. On the other hand, acetylated DNA pack more loosely, due to the larger (compared to methyl) acetyl group, in a conformation termed euchromatin. Transcription factors and enhancer proteins are able to bind and activate transciption more easily, and so euchromatin are usually associated with active transcription. However, many DNA sequences show bivalent marks, meaning it has both methylated and acetylated sequences close to each other; thus it is likely that the expression level of the associated gene is often dependent on the ratio between the various modifications.

A high level of euchromatin is associated with young, stem or tumour cells, because in these cells many genes are not repressed, as these cells age, become more specialized, or loses its potency, their differentiation will lead to more repression of genes, and hence a larger proportion of DNA is in the heterochromatin state in aged, terminally differentiated and/or non-tumour cells.

The DNA methyltransferases Dnmt3a and Dnmt3b is required for stem cells to differentiate. Dnmt3a and Dnmt3b methylates SC-specific genes involved in maintaining multipotency, such as Runx1 and Gata3. The ablation of Dnmt3a and Dnmt3b leads to a lack of differentiation of HSC, even after injury using a pulse of 5-fluoro-uracil to kill all proliferating cells.^[53]

The state of DNA methylation can be determined by bisulfite sequencing, where DNA is treated with bisulfite that converts all unmethylated cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. After bisulfite treatment, the mutated DNA is sequenced and the difference between the treated and untreated DNA sequences will give us single-nucleotide-resolution data on the methylation state of each nucleotide.

DNA methylation patterns are hereditary, so daughter cells will also have the same/similar patterns of modifications.

Chromatin Immunoprecipitation (ChIP)

Some epigenetic marks are mediate by protein-DNA interactions; an example is histones interacting with DNA indicates nucleosome-bound DNA sequences. To identify these protein-DNA interactions, a technique called chromatin immunoprecipitation is used. First, the DNA and protein are cross-linked to each other using formaldehyde; the cell is lysed and the DNA is then cut at random loci to give DNA fragments, some of which will be associated with our protein of interests and others will not. All fragments then passes through an affinity column where the beads are coated with antibodies against our protein of interest (against histones to identify nucleosome associated DNA sequences, against lamin to identify S/MAR sites). The protein of interest, along with the DNA sequence that is bound to it, will remain attached to the column while other protein-DNA will flow through. Any remaining protein-DNA is eluted. To obtain the DNA sequences, we must break the crosslinks and digests the protein using proteases, leaving only the DNA sequences which are then analysed quantitatively using q-PCR, microarrays (ChIP-chip) and/or are sequenced (ChIP-seq).^[54]^[55]

To analyse using q-PCR, one must have a test sequence in mind. Primers are designed and made specific for this test sequence; the DNA sequences obtained are then mixed into a solution with PCR primers, free fluorescently-labelled nucleotides and buffers, and put through repeated PCR cycles in a thermal cycler. Any DNA matching the test sequence will be amplified and have free nucleotides incorporated into it. Everytime a nucleotide is incorporated, a fluorescent signal will be emitted, and these signals are collected which are proportional to the level of DNA in that eluent. q-PCR is the most quantitative but is limited to a few test sequences.

In ChIP-chip, the obtained DNA sequences are fluorescently-tagged and allow to hybridize with a microarray chip with known sequences printed in a regular matrix. If the test DNA sequences match a particular sequence on the chip, it will hybridize and the fluorescence signal there will be high. Using ChIP-chip is easy but gives a low resolution read out, depending on the size of the chip and the length of the nucleotides.

ChIP-seq sequences every DNA fragment obtained and map it onto the genome. The sequences where most overlaps of the fragment occurs are the loci where the protein of interest is most likely to bind. ChIP-seq can be quantitative given a large enough number of sequences.

ChIP is limited by the availability and strength of antibodies against the protein of interest.

Non-coding RNAs (ncRNAs)

The microRNA miR-150 targets the transcription factor MYB to guides megakaryocyte-erythrocyte progenitors into the megakaryocytic lineage during differentiation.^[56]

Therapy

Stem cell-based therapies

The generation of patient-specific embryonic-like stem cells allows for the regeneration of any organs or tissue of the patient. These patient-specific pluripotent stem cells can be dervied from iPSCs. Potentially totipotent cells can be derived from somatic cell nuclear transfer (SCNT).

Cancer

Tumor cells often exists, and is maintained, within a different niche than normal healthy cells. An altered niche can lead to tumorigenesis, but the reciprocal might also be true, where the tumor produce factors that alter the niche to make it more accessible for more tumor cells. These altered niches increase proliferation, mutation rates, metastatic potential, encouraging the formation of cancers.

For example, CXCL12 may be expressed by tissues and creates a gradient where cells that expresses the CXCL12 ligand, chemokine receptor CXCR4, can home to. Normally, cells expresses a high level of CXCL12 so as to saturates its own CXCR4, as to prevent metastasis. Breast cancer cells stop expressing CXCL12 and thus is encouraged to metastasize elsewhere.^[57]^[58]^[59]

Methods

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^ Hoffmeyer, K.; Raggioli, A.; Rudloff, S.; Anton, R.; Hierholzer, A.; Del Valle, I.; Hein, K.; Vogt, R.; Kemler, R. (2012). "Wnt/ -Catenin Signaling Regulates Telomerase in Stem Cells and Cancer Cells". Science. 336 (6088): 1549–54. doi:10.1126/science.1218370. PMID 22723415.
^ Jaskelioff, Mariela; Muller, Florian L.; Paik, Ji-Hye; Thomas, Emily; Jiang, Shan; Adams, Andrew C.; Sahin, Ergun; Kost-Alimova, Maria; Protopopov, Alexei (2010). "Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice". Nature. 469 (7328): 102–6. doi:10.1038/nature09603. PMC 3057569. PMID 21113150.
^ Challen, GA; Sun, D; Jeong, M; Luo, M; Jelinek, J; Berg, JS; Bock, C; Vasanthakumar, A; Gu, H (2011). "Dnmt3a is essential for hematopoietic stem cell differentiation". Nature Genetics. 44 (1): 23–31. doi:10.1038/ng.1009. PMC 3637952. PMID 22138693.
^ Carey, M. F.; Peterson, C. L.; Smale, S. T. (2009). "Chromatin Immunoprecipitation (ChIP)". Cold Spring Harbor Protocols. 2009 (9): pdb.prot5279. doi:10.1101/pdb.prot5279. PMID 20147264.
^ Hawkins, R. David; Hon, Gary C.; Ren, Bing (2010). "Next-generation genomics: An integrative approach". Nature Reviews Genetics. 11 (7): 476–486. doi:10.1038/nrg2795. PMC 3321268. PMID 20531367.
^ Lu, Jun; Guo, Shangqin; Ebert, Benjamin L.; Zhang, Hao; Peng, Xiao; Bosco, Jocelyn; Pretz, Jennifer; Schlanger, Rita; Wang, Judy Y. (2008). "MicroRNA-Mediated Control of Cell Fate in Megakaryocyte-Erythrocyte Progenitors". Developmental Cell. 14 (6): 843–53. doi:10.1016/j.devcel.2008.03.012. PMC 2688789. PMID 18539114.
^ Mirisola, Valentina; Zuccarino, Ambra; Bachmeier, Beatrice E.; Sormani, Maria Pia; Falter, Judith; Nerlich, Andreas; Pfeffer, Ulrich (2009). "CXCL12/SDF1 expression by breast cancers is an independent prognostic marker of disease-free and overall survival". European Journal of Cancer. 45 (14): 2579–2587. doi:10.1016/j.ejca.2009.06.026. PMID 19646861.
^ Wendt, M. K.; Cooper, A. N.; Dwinell, M. B. (2008). "Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells". Oncogene. 27 (10): 1461–1471. doi:10.1038/sj.onc.1210751. PMID 17724466.
^ Teicher, B. A.; Fricker, S. P. (2010). "CXCL12 (SDF-1)/CXCR4 pathway in cancer". Clinical Cancer Research : An Official Journal of the American Association for Cancer Research. 16 (11): 2927–2931. doi:10.1158/1078-0432.CCR-09-2329. PMID 20484021.

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