Lecture 4 (The Self-Renewal of Pluripotent Stem Cells) - Lecture 5 (Induced Pluripotent Stem Cells) Flashcards
How is pluripotency regulated?
Pluripotency is regulated by a core regulatory circuit. Anti-mitogens such as as leukaemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) bind to receptors on the extracellular surface of the target cell and initiate the activation of various proteins. The binding of LIF activates STAT3, whilst the binding of BMP first activates Smad, which in turn activates the inhibitor of differentiation protein, Id. Both STAT3 and Id inhibit the progression of the pro-differentiation MAPK pathway, which is initiated by the binding of the mitogen fibroblast growth factor (FGF) to its constituent cell-surface receptor. If the MAPK pathway was not inhibited, it would progress to activate proteins, such as Gata6, which inhibit transcriptional factors essential to the maintenance of the pluripotent embryonic stem cell phenotype; for example, Oct-4, Sox-2 and Nanog.
Describe the action of epigenetic regulators Polycomb and Trithorax in the maintenance of the pluripotent phenotype.
Polycomb and Trithorax proteins, named after phenotype of mutations in the genes encoding them in Drosophila, are antagonistic epigenetic regulators.
Most Polycomb proteins are subunits of one of two classes of multi protein complexes: PRC1-type or PRC2. PRC2 complexes are thought to act initially by associating with specific repressors bound to their cognate DNA sequences early in embryogenesis, or ribonucleoprotein complexes containing long, non-coding RNAs. The PRC2 complexes contain histone deacetylases that inhibit transcription, in addition to an EZH2 domain, which contains a SET domain that is the enzymatically-active component of several histone methyltransferases. This SET domain in PRC2 complexes methylates histone H3 at lysine 27, generating di- and tri-methyl lysines. The PRC1 complex then binds the methylated nucleosomes through dimeric CBX subunits, each containing a methyl-lysine binding domain, otherwise known as a chromodomain, specific for methylated H3 lysine 27. Binding of the dimeric CBX to neighbouring nucleosomes is proposed to condense the chromatin into a structure that inhibits transcription of developmental-regulatory genes.
Trithorax proteins counteract the repressive mechanism of Polycomb proteins. Trithorax complexes contain a histone methyltransferase that trimethylates histone H3 lysine 4, a histone methylation associated with the promoters of actively transcribed genes. This histone modification creates a binding site for histone acetylase and chromatin-remodelling complexes that promote transcription, as well as TFIID, the general transcription factor that initiates preinitiation-complex assembly.
Overall, Polycomb proteins are responsible for cellular differentiation via transcriptional repression, whilst Trithorax proteins maintain the pluripotent phenotype.
Describe the role of the nucleosome remodelling complex in the maintenance of pluripotency and differentiation.
The nucleosome remodelling complex (NuRD) directly regulates the transcription of pluripotency genes, which means that it’s repressive activity is required for the differentiation of embryonic stem cells. NuRD mediates transcription heterogeneity in embryonic stem cell populations through the balancing of activation and silencing. Without the activity of NuRD, pluripotency genes are overexpressed and, conequently, the cell cannot differentiate.
Summarise stem cell energetics.
The stem cell equilibrium is bioenergetically and biosynthetically balanced through the proper regulation of the flux of pathways that metabolise glucose, glutamine and/or fatty acids. Mitochondria in stem cells are relatively inactive and stem cells rely heavily on aerobic glycolysis, whereas oxidative phosphorylation is associated with differentiation, as well as with impaired stem cell function. Fatty acid metabolism has recently been identified as a critical factor in the self-renewal of haematopoietic stem cells through the control it exerts over stem cell fate decisions.
How is genome integrity maintained in stem cells through redox homeostasis?
Genome caretaking describes the achievement of an optimal balance between maintaining sufficient numbers of embryonic stem cells and eliminating severely damaged stem cells. Low reactivity oxygen species levels maintain the self-renewal of stem cells by monitoring the redox homeostasis, which is well regulated by the antioxidant enzymatic defence systems and hypoxia niches, as well as through the actions of several key redox regulators, including:
- FoxO
- Nuclear factor erythroid-2-related factor 2 (Nrf2)
- Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE-1/Ref-1)
- Ataxia telangiectasia mutated (ATM)
High ROS levels cause abnormal differentiation, apoptosis or senescence of stem cells by the ROS-sensitive molecules, including hypoxia-inducible factors, p38 and p53.
Describe the process of cloning mice using somatic-cell nuclear transplantation from an olfactory neuron.
- The nucleus is removed from the mouse ovum
- The fluorescently-labelled (GFP) nucleus from the olfactory neuron is added to the ovum
- The ovum differentiates to the inner cell mass stage
- The embryonic stem cells are extracted
- The stem cells are added to a tetraploid blastocyst alongside host cells
- The GFP-labelled embryo develops into a mouse
Describe induced pluripotent stem cells.
Induced pluripotent stem cells are ordinary somatic cells which have been reprogrammed to resemble embryonic stem cells, through the administration of four key transcription factors, KLF4, SOX2, c-Myc and Oct-3/4, by a retroviral vector (between 1 and 2 cells in 10,000 are successfully reprogrammed).
In theory, iPS cells behave exactly like cloned embryonic stem cells, but without the associated cost and ethical concerns. iPS cells are similar to embryonic stem cells in terms of morphology, proliferation, surface antigens, gene expression, the epigenetic status of pluripotent cell-specific genes, and telomerase activity.
What are the problems associated with the use of induced pluripotent stem cells?
Whilst iPS cells are autologous, therefore eliminating any ethical concerns, the use of retroviral vectors for transfection increases the chance of tumourigenesis, as the provirus inserts randomly into the genome. iPS cells are also unsuitable for the treatment of genetic diseases, such as cystic fibrosis, and are not exactly identical to human embryonic stem cells, meaning that there is still a lack of knowledge with regard to their exact workings.
Describe the role of methylation in the induction of pluripotency.
The DNA hydroxylate Tet1 facilitates pluripotent stem cell induction by promoting Oct4 demethylation and deactivation. Oct4 ordinarily covalently modifies DNA by replacing the hydrogen H5 of cytosine nucleotides with a methyl group; this controls protein binding to target sites on DNA.
Describe the role of metabolism in haematopoietic stem cell differentiation.
Haematopoietic stem cells are located in the hypoxic environment of the bone marrow, meaning that they metabolise through the process of anaerobic glycolysis. However, a change in metabolism can be induced through the action of hypoxia-inducible factor 1α, which instigates a switch to mitochondrial oxidative phosphorylation. This efficient method of ATP production is required in order for the cell to enter the cell division program associated with differentiation. Panopolous (2012) showed that changes in cellular metabolism are critical for the reprogramming of somatic cells.
Describe the process of decellurisation.
Decellurisation is a technology used successfully in a variety of regenerative medicine applications. The technique works on all organs by simply running detergent through the blood vessels, in order to remove cells from their extracellular matrix. The vasculature is subsequently perfused with de-cell solution for over two hours, before ‘re-seeding’ with engineered stem cells, which can then develop into a fully functioning tissue.
State some of the values of human stem cell-based in vitro systems in predictive toxicology.
- Better understanding of the mechanism of action of toxicants
- Reduced animal usage
- Better physiological relevance: the ability to obtain data directly from human cells at various ages; the ability to obtain detailed dose-response data directly from human models
- Improved screening efficiency for potential toxicants: the ability to screen for re mediators and screen for toxicity of mixtures; the ability to screen for sensitive end-points that are conserved across species
- Better translation of findings: allows standardised metrics for comparing studies; allows benchmark criteria for linking in vitro to in vivo studies
Describe the types of in vivo and human cell-based in vitro systems for toxicity testing.
-Animal models in vivo
Non human, expensive, limited versatility for high throughout screening
-Immortalised human cell lines
Transformed cell lines, inexpensive, great versatility, poor relevance to human biology
-Human primary cultures
Limited supply, limited culturing lifespan, physiologically relevant
-Human embryonic stem cells
Embryonic origin, limited genetic diversity, unlimited supply, good versatility
-Human induced pluripotent stem cells
Unlimited supply, great genetic diversity, good versatility for high throughout screening
What is self-renewal?
Self-renewal describes the asymmetrical or symmetrical division of a stem cell in order to maintain potency and an undifferentiated state.
