Stem Cells and Society Flashcards
Stem cells are capable of what 3 things?
- Self renewal to make a copy of itself
- Potency to make a range of cell types
- Differentiate into a range of cell types
What are the 3 fates of stem cells?
- Self renewal
- Differentiation
- Death
Self renewal requires…
Growth factors to promote synthesis of macromolecules
Differentiation requires…
Loss of signals to drive cell down differentiation pathway
Cell death is mediated by…
- Death factors - promote apoptosis
- Survival factors - suppress apoptosis
Components for cell signalling process?
- Signal binds to membrane receptor
- Activation of intracellular effectors
- Activation of transcription factors in nucleus
- Change in gene transcription and state of cell
Chromatin structure
- Chromatin is wrapped around histones to form nucleosomes
- Allows condense packing of chromatin into chromosomes
What effect does acetylation of chromatin have?
How does it do this?
- Turns genes on
- Binds to histone tails and opens up chromatin to increase accessibility for transcription factors
Histone acetyl transferase…
Allows factors to load onto chromatin to increase transcription
Histone deacetylase…
Allows repressor proteins to load onto chromatin to decrease transcription
What effect does methylation of chromatin have?
How does it do this?
- Turns genes off
2 ways: - Direct methylation of CpG islands blocks binding of TFIID to TATA box of promotor to suppress transcription
- Recruits Histone deacetylase to condense chromatin and decrease transcription
Proximal control elements examples
- Promotor regions
- Transcriptional start sites
Distal control elements examples
Enhancer sequences
Introns in primary mRNA…
Are spliced out to form mature mRNA which is then 5’ capped and 3’ tailed
TFIID is transcription factor that binds to…
TATA box
miRNA and siRNA
- miRNA - blocks translation
- siRNA - degradation of mRNA
Ubiquitin tags proteins for…
Destruction by the proteosome
Cell cycle phases
- G1 phase - Cells increase in size and ribosome/RNA production
- S phase - Duplication of DNA content
- G2 Phase - DNA is checked for fidelity
- M phase - Mitosis
Cell cycle of Pluripotent stem cells is different because…
They cycle fast and don’t spend much time in G1
G1 checkpoint
- Is cell big enough
- Is environment favourable
- Is there DNA damage
G2 checkpoint
- Is all DNA replicated
- Is cell big enough
- Is environment favourable
Metaphase checkpoint
- Are all chromosomes aligned on spindle and attached
G0
State of dormancy not going through cell cycle
Cells enter G0 from…
G1
3 components of cell cycle activation
- Cyclin-dependent kinases (CDK)
- Cyclins
- CDK inhibitor proteins
Cyclin and CDK at start of G1
Cyclin D and CDK4/6
Cyclin and CDK at end of G1
Cyclin E and CDK 2
Cyclin and CDK in S phase
Cyclin A and CDK2
Cyclin and CDK in G2 phase
Cyclin A and CDK1
Cyclin and CDK in M phase
Cyclin B and CDK1
Most growth factors act by directly upregulating…
Cyclin D expression to drive cell through G1 for self renewal
G1 restriction point
Cell will either:
- Commit to division and proceed to S phase
- Exit cell cycle into G0
Molecular G1 restriction point
- Rb is bound to E2F
- Rb protein is phosphorylated by Cyclin D-CDK4 complex
- Rb protein is then hyperphosphorylated by Cyclin E-CDK2 complex
- Rb releases E2F
- E2F targets genes to activate S phase
2 families of CDK inhibitor proteins
- INK family - prevents binding of cyclin D to CDK4
- KIP family - binds to Cyclin E-CDK2 complex and inhibits activity
Apoptosis characteristics
- No loss of integrity
- Aggregation of chromatin at nuclear membrane
- Shrinking of cytoplasm and nuclear condensation
- Mitochondria release death signals
- No inflammation
Necrosis characteristics
- Loss of membrane integrity
- Swelling of cytoplasm and mitochondria
- Total cell lysis
- Disintegration of organelle
- Inflammation
Apoptosis activation signals
- Withdrawal of positive signals - adhesion to other cells
- Presence of negative signals - UV light, free radicals
Stages of apoptosis
- Death signal received and commitment to die - reversible
- Execution of cell - irreversible
- Dead cell phagocytosed
- Degradation of apoptotic bodies in phagocyte
Intrinsic pathway of Apoptosis
- Cytochrome C release from mitochondria
- Activates initiator caspase 9
- Activates effector caspase 3
- Triggers apoptosis
Extrinsic pathway of Apoptosis
- Death ligand bind to death receptors
- Activates initiator caspase 8
- Activates effector caspase 3
- Triggers apoptosis
Model of cell signalling
- Reception of signal
- Transduction of signal
- Cellular response
Signal transduction
Receiving information into a cell and acting to make choices on cell fate
5 crucial functions of signalling cascades
- Transduce signal into molecular form to stimulate response
- Relay signal from point of reception to point of action
- Amplify received signal
- Distribute signal to influence responses in parallel
- Each step open to modulation by other signals
2 mechanisms of signalling
Phosphorylation
- Signal taken in and protein is phosphorylated by a kinase
GTP-binding
- Signal taken in and G-portein activated by binding of GTP
Slow and Rapid acting signals
- Fast - directly alters cellular process
- Slow - indirectly alters cellular process by changing gene expression
Direct communication between cells
- Gap junctions
- Cell-cell recognition by surface molecules
Local communication between cells
- Paracrine signalling
- Synaptic signalling
- Hormonal signalling
2 clases of extracellular molecules in signalling
- Small hydrophobic molecules - pass through membrane and bind to intracellular receptors
- Large hydrophilic molecules - cannot pass through membrane and bind to membrane receptor
3 types of cell surface receptors
- Ion channel linked receptor
- G-protein linked receptor
- Enzyme linked receptor
3 types of enzyme linked receptors
- Receptor tyrosine kinase
- Cytokine receptors
- TGF-Beta receptors
Receptor Tyrosine Kinases
- Binding of ligand (soluble/membrane bound peptide hormones) allows tyrosine kinases to autophosphorylate
- This allows binding/activation of other proteins to form signalling complex
- Common component is G-protein Ras
Cytokine receptors
- Cytokines are small secreted proteins
- Control growth and differentiation of many tissues
- Signal to nucleus in direct pathway
TGF-Beta Receptors
- Used to keep pluripotent stem cells in an undifferentiated state
Binding of TGF-Beta to receptors causes phosphorylation of…
SMAD 2 and 3
Binding of BMP to receptors causes phosphorylation of…
SMAD 1, 5 and 8
Signals from TGF-Beta and BMP compete for…
SMAD 4 (coSMAD) to enter the nucleus
In human ES cells what does TGF-Beta and BMP cause respectively
- TGF-Beta - causes self-renewal
- BMP - causes differentiation
WNT OFF state
Beta-catenin is phosphorylated by CDK1 and then degraded by ubiquitination
WNT ON state
WNT binds to frizzled which prevents phosphorylation of Beta-catenin and it enters the nucleus to activate gene expression
Delta-Notch signalling
- Occurs between adjacent cells
- Cells carry both delta (ligand) and notch (receptor)
- Binding of delta to notch results in proteolytic cleavage of intracellular Notch domain that enters nucleus and turns on genes
- Leads to asymmetrical differentiation (1 cell becomes neuroblast, the rest become epidermis)
Cleavage
Cell division without increase in cell mass
Pattern formation
Laying down the spatio-temporal pattern in an embryo
Morphogenesis
Movement of cells into new positions
Differentiation
Cells become structurally and functionally different
Gastrulation
Produces the 3 germ layers:
- Ectoderm (outer layer) - skin, nervous system
- Mesoderm (middle layer) - heart, muscles, blood
- Endoderm (inner layer) - gut lining, liver
Epiblast cells migrate through the…
And emerge as…
- Primitive streak
- Emerge as the mesoderm and endoderm
4 main signalling centres in embryo
- Posterior Epiblast
- Anterior Visceral Endoderm
- Extra-embryonic Endoderm
- The Node
Formation of Primitive streak and notochord
- BMP4 has a gradient (decreases as you move towards Dorsal Visceral Endoderm)
- This results in restriction of Lefty1 to the DVE
- As embryo grows the DVE is pushed anteriorly which restricts WNT and TGF-Beta signals to the Posterior Epiblast
- WNT and TGF-Beta signals set up the primitive streak
- Node (where Nodal expression is greatest) is where notochord will start to form
What do BMP and Shh gradients specify?
The neural vs. non-neural ectoderm
In somitogenesis and axis elongation…
High FGF signalling at the posterior end
High FGF signalling at posterior end keeps cells in a proliferating state
In somitogenesis and axon elongation…
FGF and RA
FGF and RA give cells posterior identity
In somitogenesis and axon elongation…
Anti-WNTs
Anti-WNTs maintain anterior identities
In an ES cell what signals are needed to specify the primitive streak?
- Wnt
- Activin (TGF-Beta)
In an ES cell what signals are needed to specify the ectoderm?
BLOCK TGF-Beta and BMP signals
In an ES cell what signals are needed to specify ectodermal patterning?
FGF with no BMP added to drive neural differentiation
Where are ES cells derived from?
Inner cell mass (ICM) of blastocysts
Are ES cells transformed?
Do they have high or low telomerase activity?
- They are non-transformed so will live forever
- They have high telomerase activity
What is the significance of ES cells being non-transformed?
They have an indefinite proliferative potential
Are ES cells diploid or haploid?
Stable, diploid cells (46,XX or 46,XY)
What is the difference between mouse and human ES cells clonogenic capacities?
- Mouse ES cells have high clonogenic capacity
- Human ES cells have low clonogenic capacity
Are mouse ES cells naive or primed?
Do they express lineage markers?
- Naive
- Do not express lineage markers
Are mouse EpiS and human ES cells naive or primed?
Do they express lineage markers?
- Primed
- Express lineage markers
What surface markers are expressed on mouse ES cells?
- SSEA1
- SSEA3
- SSEA4
- TRA-1-60
- GCTM2
- Thy1
- MHC
- ALP
- SSEA1
- ALP
What surface markers are expressed on human ES cells?
- SSEA1
- SSEA3
- SSEA4
- TRA-1-60
- GCTM2
- Thy1
- MHC
- ALP
- SSEA3
- SSEA4
- TRA-1-60
- GCTM2
- Thy1
- MHC
- ALP
What gene is not expressed in mouse or human ES cells but is expressed in mouse EpiS cells?
Fgf5
How can pluripotent stem cells be characterised?
- Cell surface markers
- Genetic
- Epigenetic
- Gene expression
- Differentiation potential
- Single cell replanting ability
Adult or Tissue-specific stem cells function?
To build and repair tissue
Are adult stem cells pluripotent or multipoint?
Multipotent
Are adult stem cells easy or hard to isolate and grow in vitro?
Hard
Do adult stem cells have high or low telomerase?
They have NO telomerase
What is the significance of adult stem cells having no telomerase?
Telomeres will shorten until the cells senesce
What is the generic adult stem cel model?
After damage/physiological stress, Quiescent stem cell undergoes asymmetrical division to produce a copy of itself and a progenitor stem cell that goes on to expand
Are all adult stem cells multipotent?
No, some are unipotent e.g. epidermal basal cells only make keratinocytes
Are all adult stem cells quiescent?
No, some are continuously dividing e.g. intestinal crypt cells that continuously divide to give rise to more differentiated cells
Is the flow through adult stem cell hierarchies always unidirectional?
No, some can be bidirectional e.g. in the trachea damage to to the lining that causes loss of basal stem cells results in dedifferentiation of clara cells back into basal stem cells
Do all adult stem cells have a limited replicative capacity?
Yes
Stem cells in the Small Intestinal Crypt-Villus unit
- Continuous proliferation from the base
- Crypt Base Columnar (CBC) cells are the long term stem cells which give rise to cells of the intestine
- There is another type of stem cell that CBCs give rise to called +4LRC
- When tissue is damaged +4LRC can dedifferentiate into CBCs for repair
- Therefore have no professional G0 stem cells
How do the stem cells of the small intestinal crypt villus unit and blood differ?
- Stem cells in the small intestinal crypt villus can dedifferentiate to long term stem cells (have no professional G0 stem cells)
- Stem cells in the blood cannot dedifferentiate back into long term stem cells
Stem cells in the Mammary gland
- Long term stem cells (quiescent) give rise to 2 type of progenitor cells (one is unipotent and the other is bipotent)
- The bipotent progenitor cells give rise to 2 more types of progenitor cells however this is not unidirectional and there can be conversion between these 2 types of progenitor cells
Stem cells in Skeletal muscle
- Long term stem cells (quiescent) are the Satellite cells
- After damage satellite cells reenter cell cycle and undergo asymmetric division to form a copy of itself and a myoblast which goes on to repair damage
- Satellite cells are unipotent
What is a stem cell niche?
A local tissue microenvironment that hosts and influences the behaviours and characteristics of stem cells
What does the stem cell niche regulate?
What does removal of stem cells from the niche result in?
- Stem cell niche regulates self renewal
- Removal of stem cells from the niche results in cell differentiation
Niche concepts - Occupancy
Cell-cell adhesions between stem cells and niche keeps stem cells self renewing, if they move away these signals are lost and the cell begins to differentiate
Niche concepts - Fate
Signals from the niche regulate stem cell self-renewal (promote self-renewal and inhibit differentiation) and when you move away from niche these self-renewal signals are lost and differentiation occurs
Niche concepts - Asymmetric division
Physical organisation of niche polarises the stem cell to ensure asymmetric division (i.e stem cells makes a copy of itself and a cell which differentiates)
3 components of Mammalian niches and what do they do?
- Cell-cell interactions - stem cells and stromal support cells interact with each other through cell-surface receptors, gap junctions and soluble factors
- Cell-soluble factor interactions - autocrine and paracrine factors e.g FGF’s, BMPs etc
- Cell-ECM interactions - provides an anchor for stem cell to the niche and a polarity cue for stem cell mitosis
Hypertrophy
Increase in cell size
Hyperplasia
Increase in cell number
Metaplasia
Change in cell differentiation - replacing one mature cell type with another mature cell type
Dysplasia
Change in cell differentiation - replacing one mature cell types with another less mature cell type
What does IPS cells stand for and which cells are induced to produce them ?
- IPS= Induced pluripotent stem cells
- Adult stem cells are induced to become pluripotent , lots of signals and TF’s have to be replicated in order to induce pluripotency
What are 2 potential uses of IPS cells
- Disease remodelling
- Drug discovery
What 3 things have to be reset in order in order to reset the cell state (to produce IPS cells)?
- ) gene expression - somatic genes OFF, embryonic genes ON
- ) methylation (reset to totipotent configuration)
- ) chromatin - remodelled
Examples of some signals required to induce pluripotentcy in human cells
- Lentivirus + Oct4, Sox2, Nanog and Lin28
- Retrovirus + Oct4, Sox2, Klf4 and C-Myc
Some signals required to induce pluripotentcy in human cells also have additional factors , NAME 2
Additional factors used to help things – eg SV40Tm hTERT +c-myc these help the cells survive and grow
BUT: you are overexpressing factors to drive iPS formation How do you turn the expression off?
- Lentivirus will integrate BUT then be SILENCED.
- Alternative: use a non-integrating method
3 examples of non integrating methods of preventing over expression of factors to induce IPS cells
- Vector based approaches
- Protein based approaches
- Chemical based reprogramming
Delivery of reprogramming factors to make IPS cells
-VIRUS
- Efficient
- Can integrate instead of being silenced
- Retrovirus, only transduces dividing cells
- Lentivirus , can transduce dividing and non dividing cells
Delivery of reprogramming factors to make IPS cells
- Adenovirus
- Doesnt integrate
- Very inefficient
Delivery of reprogramming factors to make IPS cells
- Transposons
- Efficient
- Clones needed for checking of excision ad arrangement
Delivery of reprogramming factors to make IPS cells
- Episomal
- Does not use virus
- Very inefficient
Delivery of reprogramming factors to make IPS cells
- mRNA
- Does not integrate
- Very inefficient
Characteristics of IPS cells
- Appears any cells can be induced to become IPS cells
- IPS cells carry genotype of parent cells (ES don’t)
Why characteristics of IPS cells make them good for disease modelling ?
- Carry genotype , therefore we can capture a genotype forever
HOW are IPS cells used for disease modelling
Patients somatic cells compared against normal cells , can then be assessed
-is differentiation different ?
-Is phenotype affected ?
drug screens can be used , and drugs tested on normal cells to see if there is -ve impacts (drug testing)
Two forms of AMD
- 50% have WET amd (can be treated) - angiogenesis and subsequent haemorrhaging from them
- 90% have DRY amd (can’t be treated) - fatty deposits drusen on retina
LOSS OF RETINAL PIGMENT EPITHELIUM
Treatment procedure of AMD
1.) Neural retina folded back to reveal RPE layer
2.) RPE cells removed and transplanted
CENTRAL VISION RESTORED AT EXPENSE OF PERIPHERAL VISION
Produced human Retinal pigment epithelium which restored sight in rats, how was this tested ?
- Striped and head tracking to test visual acuity
3 issues with cultured adult RPE cells (from post mortem )
- Limited donor material
- Hard to grow in vitro
- Degenerate with time with in culture
Solution of failed attacthent of RPE cells to membrane
- Artificial membrane
- Artificial choriod
- Patch (coated with RPE cells)
In which disease do Lewy boys get deposited in the brain?
Parkinsons Disease
Treatments of Parkinson’s ?
- L-dopa (levodopa)
- Dopamine agonists
- MAO inhibitors
- COMT inhibitors
Thalamotry, Pallindotomy and Deep brain stimulation are surgical procedures to treat which disease ?
Parkinson’s
To make neurons you have to …
- Inhibit BMP and TGFB signalling
- To suppress mesodermal differentiation
Dopaminergic neurons anterior fates are determined by
- PAX6
- No morphogens or FGF and WNT inhibition= forebrain
Is a patent the right to use/perform the invention ?
NO, a patent is NOT the right to perform/use the invention
A patent doesn’t mean you have a monopoly of the market !
Transposable element
DNA sequence that can change its position within a genome
Transposase
Catalyses movement of transposon to another part of genome
AMD
Blurred vision or loss of central vision
RPE functions
Regeneration of bleached opsisns
Stem cell niche (3 things)
- Has a defined anatomical location
- Regulates self-renewal
- Removal from niche results in cellular differentiation
