S317 Topic 7: Part 2: Stem cells and differentiation
Gut (stem cells)
Example of a niche, found at the base of the intestinal crypts. Could also use hair stem cells within the bulge region of the hair root. Play vital roles in replacing cells lost by natural turnover or injury.
Blood
Example of a tissue in which stem cells are continually dividing. Could also use the lymphoid system, intestinal epithelium or hair follicles.
Muscle
Example of a unipotent stem cell. Could also use skin or spermatogonial stem cells
Nuclear transfer
Method used to reprogramme differentiated cells to become pluripotent. AKA cloning Involves the transplantation of the nucleus from a cell of one individual into the enucleated egg cell (oocyte) of another individual. The dividing, pluirpotent cells are allowed to develop into a blastocyst, from which the inner cell mass is removed and cultured to provide ESCs. If the blastocyst is instead implanted into a host uterus, then an entire cloned organism can be generated.
MyoD
Myogenic transcription factor upregulated following muscle fibre damage.
TGF-beta (Transforming growth factor-β family)
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Can dictate either self-renewal or differentiation, depending on the activity of other signalling pathways. Activates Sox2 and Nanog Suppresses c-Myc
Wnt (Wingless-related integration site)
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Can dictate either self-renewal or differentiation, depending on the activity of other signalling pathways. Activates transcription factors c-Myc, Oct4 and Nanog via beta-catenin Beta-catenin suppressed by Klf4, Oct4. Suppresses Sox2
IGFs (Insulin-like growth factors)
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Helps to maintain ESC self-renewal. Activates transcription factors c-Myc, Klf4, Oct4 and Nanog
BMPs (Bone morphogenetic proteins)
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Triggers differentiation of ESCs Activates Oct4 and Sox2 Suppresses Nanog
Delta/Notch
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Triggers differentiation of ESCs Activates c-Myc and Sox2 Suppresses Klf4 and Nanog
ESCs (Embryonic stem cells)
Pluripotent stem cells derived from early embryos before formation of the tissue germ layers; capable of forming any cell of the body.
Myoblast
Proliferating muscle transit-amplifying cell.
Telomerase
Protective mechanism employed to lower intracellular levels of damaging ROS and efficiently remove toxic waste products. In this mechanism, the stem cell transcription factor Klf4 promotes _________________ (TERT) expression and prevents replicative senescence.
Export pumps
Protective mechanism employed to lower intracellular levels of damaging ROS and efficiently remove toxic waste products. In this mechanism, cells protect themselves against a build-up of toxic metabolites by using ATP-dependent ________________________ within the cell membrane to remove toxins from the cell. Stem cells have an increased number of ___________________ and are therefore better protected against toxins that could damage their cellular proteins and DNA. The way that stem cells use _______________________ to protect themselves from environmental stress is very similar to that of cancer cells. Current research is investigating whether some tumours arise from malfunctioning stem cells.
Force generating (complex)
Protein complex which becomes associated with the Par3/Par6/aPKC complex and captures astral microtubules emanating from the spindle pole and generates the microtubule-pulling forces responsible for the final orientation of the mitotic spindle. Differential cues instruct the position where the _______________________________________ attaches within the Par3/Par6/aPKC complex: either midway along or at each end. This ultimately decides whether the segregating determinants are distributed asymmetrically or symmetrically between daughter cells.
Klf4/Oct4/Sox2
Protein complex which regulates both passive and active demethylation of silenced target genes, re-activating transcription.
Par (Partitioning-defective proteins)
Proteins essential for asymmetric cell division and polarised cell growth.
Segregating determinants
Proteins that become selectively concentrated on one side of a stem cell prior to cell division. Depending upon the axis of division, the determinants can become segregated into just one of the daughter cells (asymmetric cell division) or can be equally divided between the daughter cells (symmetric division). Cues from the niche set up polarity of the stem cell, regulated by an evolutionarily conserved complex of three proteins - Par3, par6 and atypical protein kinase C (aPKC).
Tissue-specific (stem cells)
Stem cells which, in later embryonic development and postnatally, undergo further lineage restriction, such that they typically only contribute cells to the specific organs and tissues that they occupy. These processes of cell fate restriction are controlled by combinations of cell signalling, cell-cell contact, and epigenetic silencing of various 'stemness' genes that specify self-renewal
Gastrulation
The process in which the inner cell mass becomes organised into 3 distinct germ layers.
Potency
The range of commitment options available to a stem cell (of which there are four categories)
c-Myc (Myelocytomatosis viral oncogene homologue)
One of 5 transcription factors highly expressed in pluripotent embryonic stem cells, with subsets of these transcription factors being expressed in more specialised, multipotent stem cells. - Promotes cell cycle - Stimulates expression of growth-related genes - Prevents apoptosis Must be continually produced to maintain cell proliferation because it has a half-life of around 20 minutes. These same five transcription factors are expressed at much lower concentrations, or not at all, in most differentiated cells, suggesting roles for these proteins in stem cell self-renewal and inhibition of differentiation.
Nanog (homeodomain)
One of 5 transcription factors highly expressed in pluripotent embryonic stem cells, with subsets of these transcription factors being expressed in more specialised, multipotent stem cells. - When overexpressed, promotes entry into S phase and proliferation. - Blocks differentiation signals transduced by bone morphogenetic proteins (BMPs). - As ESCs commit to differentiation, __________ represses its own expression. - Enhances Oct4 expression. These same five transcription factors are expressed at much lower concentrations, or not at all, in most differentiated cells, suggesting roles for these proteins in stem cell self-renewal and inhibition of differentiation.
FGFs (Fibroblast growth factors)
One of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. Help to maintain ESC self-renewal. Activates transcription factors c-Myc, Sox2 and Nanog. Suppresses Oct4 Cause cells at the surface of the morula to activate the expression of the transcription factor Cdx2, essential for placenta formation.
HSCs (Haematopoietic stem cells)
Present in the bone marrow; generate all the cell types of the blood. Predisposed to apoptosis when subjected to cell stress.
Transit-amplifying (cells)
Proliferative stem-cell progeny fated for differentiation. Typically go through several rounds of division before differentiating into at least one specialised cell type
Antioxidants
Protective mechanism employed to lower intracellular levels of damaging ROS and efficiently remove toxic waste products. In this mechanism, stem cells possess efficient enzymatic and non-enzymatic mechanisms for detoxifying ROS. Eg, the stem cell transcription factor FOXO3 positively regulates expression of the __________________ enzyme manganese superoxide dismutase, thereby maintaining low levels of ROS in stem cells.
Smad2/3
Signalling molecule complex which binds to the Nanog transcription factor, forming a signalling unit that binds and activates several pluripotency genes in ESCs. Also binds to differentiation-specific genes that promote endodermal cell fates, but an as yet unknown inhibitor prevents transcription of these differentiation-promoting genes. As the ESC commits to differentiation, the amount of Nanog protein becomes lowered. Without Nanog, ______________ can no longer drive the transcription of pluripotency genes and, in combination with differentiation-specific transcription factors, is now competent to drive the transcription of endoderm cell fate genes
Clonogenic (stem cells)
Specified, but uncommitted, stem cells that are able to revert back to become a quiescent stem cell. Considered to be an intermediate stage between the stem cell and transit-amplifying cells.
Morula
Stage of the developing embyro, up to the 16-cell stage, just before the blastocyst stage. Contains totipotent stem cells capable of forming a complete individual, plus the trophectoderm
Super-enhancers
Stem cell transcription factors packed together into unusually large clusters on the enhancer regions of many cell identity-regulating genes in ESCs, including their own genes. Depending on the context of other transcription factors within this complex, these can either activate or repress the transcription of the targeted genes by the modification of histones
Oct4/Sox2
Stem cell transcription factors that, when complexed together, activate transcription. Singly they may act to repress gene transcription
Activin (and) Nodal
TGF-β family members which, via their low-level signalling, can maintain Human and mouse ESCs in a pluripotent state. Together they maintain the expression of Nanog. During the commitment to endodermal cell fates, the signalling via the TGF-β receptor actually increases, meaning the same signalling pathway is involved in both self-renewal and differentiation. This is due to the downstream signalling molecule complex of two Smads (Smad2/3)
Reprogramming
Technique which relies on the reactivation of silenced genes by the removal of inhibitory epigenetic methylation marks from cytosine or adenine DNA nucleotides. Demethylation may be passive or active. In passive demethylation, the transcription factor complex of Klf4/Oct4/Sox2 binds to specific DNA sequences and displaces the DNA methyltransferase, DNMT1, preventing excessive methylation that would halt transcription. In active demethylation, the same three transcription factors can bind a demethylase enzyme that removes methylation marks from DNA to restore transcription. Once genes have been demethylated, stem cell transcription factors also act, in the passive mode, to prevent key genes from becoming methylated again and thereby silenced.
Trophectoderm
The amniotic sac and the fetal portion of the placenta, formed by totipotent embryo-derived extra-embryonic cells.
TERT
The catalytic subunit of telomerase, an enzyme used to lengthen telomeres in self-renewing cells, including ESCs, germ cells, lymphocyte blood cells and many cancer cells
Inner cell mass
The cells which accumulate on one side of the blastocyst inner surface, comprising embryonic stem cells, which will go on to form the entire embryo. As development proceeds, this becomes organised into three distinct layers ('germ layers') that are committed towards more specialised fates. This process is called gastrulation and results in the formation of a gastrula.
Immortal strand (hypothesis)
The hypothesis of non-random mode chromosome segregation. Following initial symmetrical cell divisions, during which both DNA strands become radiolabelled, subsequent asymmetric divisions of a stem cell occur in a non-random way in which the same radiolabelled DNA strand of each DNA duplex is selectively and repeatedly segregated to the 'stem' daughter cell, division after division. One benefit of non-random segregation of DNA is that it could help protect the stem cell against DNA replication-induced errors that might occur as this template is being copied during mitosis, since the stem cell always retains a relatively 'pristine' copy of the genome.
aPKC (atypical protein kinase C)
The key component of the complex which regulates the polarity of stem cells. Phosphorylates at least two segregating determinants, called Numb and Miranda. Numb and Miranda are normally uniformly distributed within the cell but, following phosphorylation by ________, they can no longer bind to the Par3/Par6/________ complex. They are therefore displaced from the surface of the cell where the complex is located, forcing them instead to concentrate on the other side of the cell. __________ are divided into three groups (conventional, novel and ____________), according to the second messengers they use. Unlike other __________, ________ require neither calcium ions nor diacylglycerol (DAG) for their activity, but are instead activated by Par6.
(Cellular) differentiation
The process by which a less specialised cell becomes a more specialised cell type; for example, during the formation of specialised tissues and organs in the course of embryonic development. The fates that can be adopted by stem cell progeny depend primarily on intrinsic factors inherited from the mother stem cell and secondarily by the exact nature of extrinsic signals from the tissues they encounter as they move out of the stem cell niche. For example, differentiating daughters of neural stem cells will give rise to neurons, although the exact type of neuron is dependent upon the tissue environment.
Par3/Par6/aPKC
The three evolutionarily conserved proteins that make up a complex which regulates the polarity of a stem cell. Become associated with the inner cell membrane on only one side of the cell
Numb (and) Miranda
The two segregating determinants phosphorylated by aPKC which prevents them from binding to the Par3/Par6/aPKC complex, displacing them from the surface of the cell where the complex is located, forcing them to concentrate on the other side of the cell. The first of these is a repressor of Notch signalling, which, when activated, can commit a stem cell daughter to differentiation. Following asymmetric cell division of a stem cell, the daughter cell that is to remain a stem cell has a high concentration of _________ and therefore low Notch activity, while the daughter cell that commits to differentiate has less ________ and therefore high Notch activity.
Neural (stem cells)
These stem cells produce neurons and glia. They're used clinically for replacement cells after traumatic brain injury. Also for Parkinson's, Alzheimer's, multiple sclerosis and stroke.
Bone marrow
This HSC source produces blood cells, whose clinical use is to treat several blood disorders: anaemia, sickle-cell anaemia, leukaemia, non-Hodgkin lymphoma and immunodeficiencies.
Wnt3a/Activin
This signalling pathway works in opposition to the FGF/retinoic acid pathway, to promote mesendodermal fate and suppress ectodermal fate, by downregulating Sox2. FGF/retinoic acid in contrast downregulates Oct4 for the opposing differentiation fate.
Pax7 (and) Myf5
Two muscle specific transcription factors expressed by satellite cells.
Wnt (and) TGF-beta
Two of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. These two can promote both pluripotency/self-renewal or differentiation, depending upon the extrinsic signals experienced by the cell.
BMPs (and) Delta/Notch
Two of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. These two promote differentiation.
IGFs (and) FGFs
Two of the six main signalling pathways involved in maintaining the balance between ESC self-renewal and the commitment to differentiation. These two promote pluripotency and self-renewal.
Adult (stem cells)
Undifferentiated cells found in various tissues throughout the body that divide to generate cells that repair and replace cells in that tissue. In most tissues, they are quiescent or dividing slowly, but they can 'activate' to a more proliferative state when required.
Hippocampus
Along with the subventricular zone, the site of neural stem cells, responsible for the continual slow turnover of glia and neurons in the brain.
Yamanaka (factors)
A group of four transcription factors (Klf4, Oct4, Sox2 and c-Myc ) that are highly expressed in embryonic stem cells (ESCs) and whose over-expression in adult mouse fibroblasts was sufficient to generate ESC-like colonies from these cells. The resulting cells were described as induced pluripotent stem cells (iPSCs).
Cdx2
Activated by FGFs in the morula, this transcription factor causes the downregulation of Oct4 and Nanog in the outer cells of the morula, which disrupts the stem cell self-renewal loop and commits these cells to differentiate into extra-embryonic cells of the trophectoderm. The remaining cells in the interior of the morula do not receive sufficient FGF to activate _________ and, hence, they maintain their stem cell properties as pluripotent ESCs.
MSCs (Mesenchymal stem cells)
Mesoderm-derived multipotent stem cells found in bone marrow, connective tissue, adipose tissue and adult muscle that differentiate into these tissue specific cell types. In addition, given appropriate stimuli, ________ can differentiate into some ectodermal cell types including nervous system and skin cells. Do not produce blood cells. One in every 2000-4000 neurons in the brain are derived from _________ that migrate there from the bone marrow via the blood circulation. Transplanted bone marrow cells can also differentiate to form myocardial cells of the heart, liver hepatocytes and epithelial cells of the skin and gut. Bone marrow ________ are not restricted to the differentiated cell fates typical of their bone origin.
Cell fusion
Method used to reprogramme differentiated cells to become multipotent. Fusions of uninuclear diploid cells to form multinucleate cells (known as syncytia) occur naturally in skeletal and cardiac muscle, placenta and osteoclasts (cells that break down and remodel bones). Some cells can also be coerced to fuse in culture. Two distinct cell types are combined to form a single multinucleate cell and, if this artificially fused cell then proliferates, the two nuclei fuse together to give a cell with double the normal number of chromosomes (4n). By choosing appropriate culture conditions, the fused cell can be stimulated to undergo a reduction division back to the diploid state and differentiate into either of the starting cell types; so these fused cells are bi-potential.
Endoderm
Embryonic germ layer which will form cells of the gut and lungs, and cells lining the liver and pancreas.
Mesoderm
Embryonic germ layer which will form muscle and connective tissues including bone, cartilage, fat and blood.
Ectoderm
Embryonic germ layer which will form nervous system and skin.
(Transcription factor) transduction
Method used to reprogramme differentiated cells to become pluripotent. The technique by which iPSCs were first made. It involves using retroviruses to introduce four stem cell genes (klf4, oct4, sox2 and c-myc) into an adult diploid cell.
Muscle satellite (cells)
Example of a normally quiescent, non-dividing cell type that can be activated to a proliferative state following injury. In contrast, intestinal epithelial, skin and haematopoietic stem cells, are dividing all the time. When quiescent, these cells express Pax7 only. When activated by damage, they co-express Pax7 and MyoD. Following asymmetric division, one daughter downregulates MyoD and returns to quiescence, the other maintains MyoD and proliferates to form a cluster of myoblast cells. These eventually downregulate Pax7 expression and differentiate to produce new myonuclei that fuse with the parent muscle fibre.
Adipocyte (stem cells)
Example of multipotent stem cells. Could also use neural or mesenchymal stem cells.
ESCs
Example of pluripotent stem cells. They're used clinically for retinal stem cells (animal studies) and for pre-differentiation to cardiomyocytes, then used to repair myocardial infarction.
(Plant) meristem (cells)
Example of totipotent stem cells (in plants)
Wnt(3a)
Example signalling pathway (in its active form) involved in different differentiation fates depending upon its combination with other factors. -When repressed by FGF/Retinoic acid, it directs a cell's differentiation toward a neural ectodermal fate (inactive form). - When combined with activin (in its active form), it directs a cell's differentiation toward a mesendodermal fate.
Newt (limb regrowth)
Example species capable of de-differentiation. Following injury, epidermal cells migrate over the wound. Underlying cells de-differentiate to a multipotent state and generate transit-amplifying progeny that then redifferentiate into a variety of different cell types.
Klf4 (Kruppel-like factor 4)
One of 5 transcription factors highly expressed in pluripotent embryonic stem cells, with subsets of these transcription factors being expressed in more specialised, multipotent stem cells. - Binds the gene promoters of oct4, sox2 and c-myc - Activates its own transcription - Activates telomerase to prevent replicative senescence These same five transcription factors are expressed at much lower concentrations, or not at all, in most differentiated cells, suggesting roles for these proteins in stem cell self-renewal and inhibition of differentiation.
Sox2 (Sex-determining region Y box-2)
One of 5 transcription factors highly expressed in pluripotent embryonic stem cells, with subsets of these transcription factors being expressed in more specialised, multipotent stem cells. - Complexes with Oct4 - prevents neural differentiation These same five transcription factors are expressed at much lower concentrations, or not at all, in most differentiated cells, suggesting roles for these proteins in stem cell self-renewal and inhibition of differentiation.
Oct4 (Octamer binding transcription factor-4)
One of 5 transcription factors highly expressed in pluripotent embryonic stem cells, with subsets of these transcription factors being expressed in more specialised, multipotent stem cells. - Binds Sox2 protein to form a complex that binds the enhancers of several genes regulating self-renewal (including sox2, oct4 and nanog) - Represses Nanog expression These same five transcription factors are expressed at much lower concentrations, or not at all, in most differentiated cells, suggesting roles for these proteins in stem cell self-renewal and inhibition of differentiation. Inhibits commitment, allowing ESCs to progress in and out of the earlier specification stages of differentiation while still self-renewing.
Metabolism
Can be used by stem cells as a protective mechanism employed to lower intracellular levels of damaging ROS and efficiently remove toxic waste products. In this mechanism, rapidly proliferating cells switch their _________________, such that the initial ATP-generating steps in the glycolysis pathway are intensified, while the activity of the mitochondrial electron transport chain is decreased. This is less efficient in terms of ATP production but it has the benefit of producing far fewer ROS, making these cells less vulnerable to damage.
Pluripotent
Category of stem cell potency, characterised by: - Able to differentiate into all cell types of a particular organism, except the extra-embryonic lineages. - Embryonic stem cells (ESCs). - Several differentiated adult cell types (e.g. skin fibroblasts) have been reprogrammed to this state in culture to give induced ______________ stem cells (iPSCs).
Unipotent
Category of stem cell potency, characterised by: - Able to give rise to only one specialised cell type.
Multipotent
Category of stem cell potency, characterised by: - Capable of differentiating into diverse types of cells, but specific to the tissue they reside in.
Totipotent
Category of stem cell potency, characterised by: - The greatest plasticity - Derived from fertilised eggs up to the 16-cell stage - Able to form the entire organism - Able to differentiate into all the cells of the three germ layers, as well as embryo-derived extra-embryonic cells that go on to form the amniotic sac and the fetal portion of the placenta - Includes plant meristem cells, able to grow an entire plant from a cutting
Stem cells
Cells with the characteristic ability to divide asymmetrically. Most cells in mature multicellular organisms are fully differentiated, but small populations of these cells persist in the mature organism and support renewal and repair processes. Distinguishing features: - Undifferentiated - Able to divide asymmetrically - Able to protect themselves against metabolic stress - Able to protect themselves against damage to their proteins and DNA. Can either undergo symmetrical division to generate two daughter ___________________ (usually only seen in early development or after severe injury, when it is desirable to quickly increase the size of the ___________________ population.) or can undergo asymmetrical division to produce one daughter _________________ and one non-_________________ daughter (N).
Niche
Cellular microenvironment providing support and stimuli necessary to sustain self-renewal. The exact anatomy of the __________ in each tissue is different, but they all serve the same function: to maintain self-renewal and (in some cases) to regulate the balance between non-dividing (quiescent) and dividing (activated) states.
iPSCs (Induced pluripotent stem cells)
Created from fully differentiated cells by the re-expression of a core set of stem cell transcription factors, such as the Yamanaka factors: Klf4, Oct4, Sox2 and c-Myc.
Specified
Describes a cell that has received signals which result in it adopting a certain phenotype. ________________ cells need to commit before they can then differentiate. If it later receives additional or other signals, it may follow a different developmental pathway.
Committed
Describes cells that have engaged in a program leading to differentiation. For a stem cell, this means exit from self-renewal. The process towards differentiation in ESCs is initially reversible and can lead to several distinct differentiated cell fates. Cells first become specified and then become __________________. Although these cells are not fully differentiated, under normal circumstances their fate cannot be changed.
Umbilical cord (blood)
HSCs from this source, all produce blood cells. Their clinical use is the same as for circulation HSCs, but also includes immunodeficiencies and metabolic disorders. Advantage is that treatments don't require such a close immune compatibility match as is required for HSC transplants. Alternatively, the stem cells can be stored and used for autologous transplants later in life.
Circulating
HSCs from this source, all produce blood cells. They are used clinically for autologous transplantation in cancer patients after chemotherapy. Patients are given daily injections of growth factors to boost HSC production. Then blood is collected and stored. After chemotherapy (which destroys many of the HSCs), the HSC-rich blood is put back into the patient.
Epigenetic (changes)
Histone modifications, which underlie the priming of a cell towards a specific cell fate, to various genes that alter transcription. Create a cellular memory that restricts the cell and all of its progeny to specific lineages.
Blastocyst
Hollow ball of cells formed from zygote division. Has an accumulation of cells on one side of the inner surface called the inner cell mass, comprising embryonic stem cells, which will go on to form the entire embryo.
