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Intestinal crypt

Intestinal sub-epithelial myofibroblasts are SMA, desmin cells that secretes HGF, KGF, TGFβ and regulates epithelial cell's differentiation. Interstitial cells of Cajal are SMA, desmin, c-Kit and CD45 cells in proximity of neurons, to transmit electrical potential that regulates movement of the gastrointestinal tract.

Previously, mesenchymal cells (which are normally the niche cells) are thought to be required to maintain primary intestinal epithelial culture. However, in these cell cultures, Lgr5 ISCs are not observed.

Note that Lgr are also found in multipotent stem cells of the pylorus, and that Lgr cells and Sox2 cells do not overlap, at least not at the levels observable by immunohistochemistry. Using Ki67 as a proliferation marker, it was shown that about half of the Sox2 cells proliferate in homeostatic conditions,showing heterogeneity within the adult stem cell population.

Hair follicle

The cells near the dermis expresses markers such as keratin 5/14. Different markers are also expressed - keratin 1/10, involucrin, cornifin, and transglutaminase-1 are xpressed in the spinous layer of the epidermis; loricrin, filaggrin, and keratohyalin are expressed in granular layers of the epidermis.

The heterogeneity can be attributed to the location in the body. Follicle morphogenesis comes in waves, guard follicles are generated around E14.5 and have long straight shafts, awl and auchene follicles are generated around E16.5 and have long, straight or singly-kinked shafts, shafts on the back of mice are generated around E18.5 and is shaped like a zigzag (with two kinks).

Only a low level of β-catenin is required for hair follicle fomation from sebaceous glands and interfollicular epidermis, whereas a high level of β-catenin is required for new follicles to form from preexisting follicles; this can be a mechanism in which hair follicle formation is controlled which has a large leeway.

The mouse dermis taken from sites of hair formation can induce follicles in any epithelium when transplanted, whereas dermis taken from sites of non-hair formation cannot induce follicles.

fibroblasts from the head and face derive from the neural crest, dorsal and ventral trunk fibroblasts are derived from the dermomyotome of somitic and lateral plate, respectively.

Mice are born nude, and hair begin to grow only after about 10 days. The first two cycles are synchronized, after which they become desynchronized. Thus, it is advisable to study young mice, as they are all synchronized and expresses the same factors.

HSC

Haematopoietic and mesenchymal stem cells are known to regulate the immune response.

Canonical Wnt signalling is thought not to be involved in HSC regulation as KO of α- β- and γ-catenin produced no apparent phenotype. However, different experiments show different effects of Wnt signalling. Some shows that APC, part of the destruction complex destroying intracellular β-catenin levels, is required for HSC survival; while GSK3β, also part of the destruction complex, when inhibited promotes progenitors to expand while retaining HSCs. Dkk1 (binds to LRP6 and inhibit Wnt) expression impairs HSC function. These contrasting results probably means that different Wnt levels leads to different outcomes, and Wnt signalling in the HSC niche needs to be within optimal level.

Other factors that contribute the niche, and the associated receptor on the HSC includes VCAM1/α4β1 integrin, SCF/KIT, THPO/MPL, ANGPT1/TIE2, HoxB4

In vitro, osteoblasts produce haematopoietic growth factors that support HSC.

Calcium ion contribute to the niche and binds to CASR on the HSC for signalling, and is generated from osteoclasts eating the bone.

Parathyroid hormone (PTH) is a regulator which encourages bone resorption to harvest the calcium stored in bones; however, when given as a periodic dose, actually increases bone mass by increasing the number of osteoblasts. PTH binds to PTH/parathyroid hormone-related protein receptor (PPR or PTHR1) on bone cellsand expands the osteoblast compartment and HSC pool via activation of the Wnt and Notch signalling. However, immunofluorescence of bone sections shows the HSC are located near blood vessels and not next to osteoblasts, thus, there is a debate as to whether osteoblasts are the niche cells which interact with HSC.

HSC homing is mediated by the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt) mice is a line which exhibit aberrant nerve conduction, dysregulated adrenergic tone (lack of stimulation from adrenaline via the sympathetic nervous system), dysregulated osteoblast function as well as dysregulated bone CXCL12; in these mice, virtually none of the HSPCs observed to migrate out from the bone marrow, even after stimulation with granulocyte colony-stimulating factor (G-CSF) or fucoidan.

The mechanism is as follows: the sympathetic nervous system stimulation leads to a release of norepinephrine (NE), which suppresses G-CSF-induced osteoblast and they become flatter and have a shorter protrusion. CXCL12 is downregulated and this means the HSPC mobilize and exit the bone marrow. Ablation of NE signals using drugs or genetic engineering leads to lack of mobilization, adminstration of a β₂adrenergic agonist further increase mobility in both wildtype and NE-deficient mice.

adipocytes (inhibits HSC, and transplantation into marrow with no fat allows faster engraftment)

HSC proliferate after injury by BrdU treatment, IFNγ treatment, LPS treatment and 5-fluoro-uracil treatment, but goes back into quiescence after.

MSC are purified from the bone marrow using the fact that MSCs are adherent to plastic culture dishes, whereas HSCs are not. Macrophages are also adherent and so must be separated from MSCs.

Neural stem cells

Neural stem cells differentiate into early neuroepithelial stem cells which transitions into radial glia which forms astrocytes. Some astrocytes remain as stem cells and produces new neurons that get integrated into the network; this differentiation occurs in a defined temporal sequence.

Lung stem cells

The exchange of gases from the air in the lungs and the blood is mediated through structures called alveoli. There are three types of cells in the alveoli - alveolar cell type I, II and alveolar macrophages. Alveolar cell type II makes up 3% of the alveolar surface, they are usually found in clusters and secretes surfactants, with the major component being dipalmitoylphosphatidylcholine (DPPC). Surfactants are agents which lowers the surface tension between different phases, such as that between gases and liquids, allowing the alveolar wall to be more permeable to gas exchanges. Alveolar cell type I makes up the rest of the alveolar surface, and contributes to turning over surfactants via pinocytotic vesicles. Alveolar macrophages are not part of the alveolar wall but found on the wall and helps fight off pathogens. Activity of the alveolar macrophage is relatively high, because they are located at one of the major boundaries between the body and the outside world.

The bronchioles are tubular structures next to the alveoli that connects the alveoli with the bronchi, it is the last branching structure before the alveoli and dissipates the air pressure to prevent physical damage. There are different types of cells in the bronchioles - cuboidal epithelial cells, ciliated cells, clara cells (which secretes glycosaminoglycans surfactants to protect the bronchiole lining) and neuroendocrine cells (which senses hypoxia through O₂-activated K channels and regulates growth and regeneration)

Liver stem cell

The liver is made up of many lobes and is composed of hepatocytes, bile duct epithelium, Stellate cells, Kupffer cells, vascular epithelium, fibroblasts and leukocytes.

Pancreatic stem cell

The pancreas can be viewed as two organs into one. Exocrine pancreas produces digestive enzymes to release into the intestine, while the endocrine pancreas contains islet cells that produce insulin, glucagon and somatostatin, required for maintaining glucose level. In type I diabetes, autoimmune responses eliminates beta cells, and so no insulin is produced, meaning the body no longer stores glucose and so leads to high blood sugar levels.

Epigenetics

DNA modifications

lysine 4 residue on histone 3 (H3K4)

A high-resolution map for the methylation of 20 histone lysine and arginine methylations have been established.

The Trithorax group (TrxG) proteins activate genes, often by methyltransferase reactions. Opposes PcG

Polycomb group (PcG) proteins silence genes, probably using methyltransferase reactions, but can also involve noncoding RNAs and the RNAi machinery.

Histone acetyltransferase (HAT) and histone deacetylase (HDAC); HAT opposes HDAC

Niche and signalling

Adhesion is generally required and is mediated by β-catenin, cadherins and integrins.

Signalling pathways

Wnt

• Canonical - beta catenin are usually at cell-cell junctions and adhesion junctions, excess beta-catenin are degraded.
• The following genes have been reported to be direct targets of Wnt signaling pathway: Drosophila Ultrabithorax (Ubx), the Xenopushomeobox genes siamois, twinned, and engrailed-2, the Xenopus genes encoding nodal-related factor Xnr-3, fibronectin, metalloproteinase-7 (matrilysin), PPARdelta (a member of the nuclear receptor superfamily), c-Jun and Fra-1 (two components of the AP-1 transcription complex), E-cadherin, connexin-43 (a gap junction subunit), cyclin D1 (a major regulator of cell cycle progression), the transcription factor T (brachyury), micropthalmia transcription factor (MITF, a key gene regulating pigment cell formation), the oncogeneWISP-1, the anhidrotic ectodermal dysplasia (EDA) gene (which causes the X-linked disorder anhidrotic ectodermal dysplasia), thepolycystic disease-1 gene, the proto-oncogene c-myc, mouse ovo (involved in hair formation), immunoglobulin transcription factor-2 (ITF-2), the ectodermal dysplasia gene (EDA), the polycystic kidney disease-1 gene (PKD1); and genes for Vascular Endothelial Growth Factor, Survivin, Interleukin-8, Nr-CAM, Chemotaxin-2, the transcription factor BRN-2, and PML.

Hedgehog

• Cascade still not well-understood
• Gli family are both activator or repressive and can often depend on other transcription factors

Notch

• What gene promotes the expression of NICD

The Notch receptor Notch1 is expressed in all epidermal layers, but the Notch ligand, Delta-like 1, is highly expressed only on stem cells in the basal layer which instructs other cells to differentiate as well as aiding stem cells to stick to each other.

TGF-beta

Activins, Nodal, TGF-β1,2,3, BMP 2, 3, 4, 7 activates TGF beta siganlling. Follistatin, cerberus, Noggin, Chordin ihibits TGF-beta ignalling.

Noggin leads to expression of Lef1; Wnt signalling stabilizes beta-catenin. The beta-catenin-Left1 complex (transcrition factor) then promotes the expression of P-cadherin and the down-regulation of E-cadherin, leading to the migration of hair follicle promordial cells to form a new hair bud.

JAK-STAT

In the Drosophila midgut, after injury the progeny enterocytes produce cytokines (Upd, Upd2, Upd3) which activates the JAK-STAT pathway leading to proliferation and progenitor differentiation.

Interactions

The differentiation of the epidermis into hair follicles require Wnt, Notch and Hedgehog signalling. First active canonical Wnt signalling, then Notch signalling is used to drive differentiation at the same time as Hedgehog signalling, which drives proliferation.

β-catenin signalling is activated in intestinal tumorigensis. It integrates with the Notch pathway, more specifically with the Notch effector Hes1, and causes the Notch pathway to be activated downstream of canonical Wnt.

Glossary of terms

Asymmetric division - Generation of distinct fates in progeny from a single mitosis. Oriented division may position daughter cells in different microenvironments or intrinsic determinants may be segregated into only one daughter. Observed in some but not all stem cells and can occur in other types of progenitor cell.
Cancer cell of origin - Precancerous cell that gives rise to a cancer stem cell. May be a mutated stem cell, or a committed progenitor that has acquired self-renewal capacity through mutation.
Cancer-initiating cell - General term that encompasses both cancer cell of origin and cancer stem cell.
Cancer stem cell - Self-renewing cell responsible for sustaining a cancer and for producing differentiated progeny that form the bulk of the cancer. Cancer stem cells identified in leukaemias and certain solid tumours are critical therapeutic targets.
Cell replacement therapy - Reconstitution of tissue by functional incorporation of transplanted stem-cell progeny. Distinct from ‘bystander’ trophic, anti-inflammatory or immunomodulatory effects of introduced cells.
Clone - the progeny of a single cell. All cells in a clone are genetically identical.
Clonal analysis - Where a single cell is isolated and its progeny studied. Essential for formal demonstration of self-renewal (by exhibiting persistence on the plate) and potency of stem cells (by observing the different cell types it can differentiate into).
Commitment - Engaging in a programme leading to differentiation. For a stem cell, this means exit from self-renewal.
Embryonic stem cell - Pluripotent stem-cell lines derived from early embryos before formation of the tissue germ layers.
Founder/ancestor/precursor cell - General terms for cell without selfrenewal ability that contributes to tissue formation. In some cases they generate tissue stem cells.
Immortal strand - The hypothesis of selective retention of parental DNA strands during asymmetric self-renewal. Potential mechanism to protect stem cells from the mutations associated with replication.
In vitro stem cell - Self-renewal ex vivo in cells that do not overtly behave as stem cells in vivo. Occurs due to liberation from inductive commitment signals or by creation of a synthetic stem-cell state.
Label-retaining cell - Candidate for adult tissue stem cell because of slow division rate and/or immortal strand retention. Interpret with caution.
Lineage - the genealogic pedigree of cells related through cell division. I.e. cells which are derived from the same progenitor cell or cell population.
Lineage commitment - the restriction of only being able to give rise to a set of differentiated cells.
Lineage priming - Promiscuous expression in stem cells of genes associated with differentiation programmes.
Long-term reconstitution - Lifelong renewal of tissue by transplanted cells. The definitive assay for haematopoietic, epidermal and spermatogonial stem cells. Transplantation assay may not be appropriate for all tissues.
Niche - Cellular microenvironment providing support and stimuli necessary to sustain self-renewal.
Plasticity - Unproven notion that tissue stem cells may broaden potency in response to physiological demands or insults; or the ability for stem cells of one lineage to be reprogrammed into a different lineage.
Potency - The range of commitment options available to a cell.

Totipotent - cells which have the ability to differentiate into any cells of the adult body and of cells of the extraembryonic membrane (e.g. placenta). The only totipotent cells are the fertilized egg and cells resulting from the first 2 or 3 mitosis events; plant meristem cells are also totipotent.

Pluripotent - Able to form all the body’s cell lineages, including germ cells, and some or even all extraembryonic cell types. Example: embryonic stem cells.

Multipotent - Can form multiple lineages that constitute an entire tissue or tissues. Example: haematopoietic stem cells.

Oligopotent - Able to form two or more lineages within a tissue. Example: a neural stem cell that can create a subset of neurons in the brain.

Unipotent - Forms a single lineage. Example: spermatogonial stem cells.

Differentiated cell - Usually a post-mitotic cell with a specified function.

Progenitor cell - Generic term for any dividing cell with the capacity to differentiate. Includes putative stem cells in which self-renewal has not yet been demonstrated. Proliferative stem-cell progeny fated for differentiation. Initially may not be committed and may retain self-renewal. Same as transit amplifying cell. Usually called progenitors when they have a marker associated with it.
Regenerative medicine - Reconstruction of diseased or injured tissue by activation of endogenous cells or by cell transplantation
Replicative potential - ability to undergo mitosis. Stem cells have very high replicative potentials, but most often they do not use it.
Reprogramming - Increase in potency. Occurs naturally in regenerative organisms (dedifferentiation). Induced experimentally in mammalian cells by nuclear transfer, cell fusion, genetic manipulation or in vitro culture.
Self-renewal - Cycles of division that repeatedly generate at least one daughter equivalent to the mother cell with latent capacity for differentiation. This is the defining property of stem cells. The ability to self-renew tends to decrease over time, apart from embryonic stem cells.
Stem cell - A multi- or pluripotent cell that self-renews and can continuously produce clonal populations of unaltered daughters.
Stem-cell homeostasis - Persistence of tissue stem-cell pool throughout life. Requires balancing symmetric self-renewal with differentiative divisions at the population level, or sustained asymmetric self-renewal.
Stemness - Unproven notion that different stem cells are regulated by common genes and mechanisms.
Tissue stem cell - Derived from, or resident in, a fetal or adult tissue, with potency limited to cells of that tissue. These cells sustain turnover and repair throughout life in some tissues.
Transit Amplifying cell - Proliferative stem-cell progeny fated for differentiation. Initially may not be committed and may retain self-renewal. Same as progenitor cell. Usually called transit amplifying cells when they have no markers associated with it.

Model organisms

Zebrafish

In zebrafish, haematopoiesis occurs in the kidney marrow (also observed in clonal ginbuna crucian carp (Carassius auratus langsdorfii), and the haematocytes have differences compared to that of human's. For example, erythrocyes in zebrafish have a nucleus, the platelets are slightly larger, neutrophils have 2 lobes instead of 3 etc. Although the zebrafish contain no osteoblasts, there are similar signals which maintains the haematopoietic niche. Transplantation of whole kidney marrow in irradiated adult zebrafish confirms that kidney marrow is the source of all haematopoietic lineages; the technique has been well characterized.

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