Stem Cells and Society Flashcards
(141 cards)
1
Q
Stem cells are capable of what 3 things?
A
- Self renewal to make a copy of itself
- Potency to make a range of cell types
- Differentiate into a range of cell types
2
Q
What are the 3 fates of stem cells?
A
- Self renewal
- Differentiation
- Death
3
Q
Self renewal requires…
A
Growth factors to promote synthesis of macromolecules
4
Q
Differentiation requires…
A
Loss of signals to drive cell down differentiation pathway
5
Q
Cell death is mediated by…
A
- Death factors - promote apoptosis
- Survival factors - suppress apoptosis
6
Q
Components for cell signalling process?
A
- Signal binds to membrane receptor
- Activation of intracellular effectors
- Activation of transcription factors in nucleus
- Change in gene transcription and state of cell
7
Q
Chromatin structure
A
- Chromatin is wrapped around histones to form nucleosomes
- Allows condense packing of chromatin into chromosomes
8
Q
What effect does acetylation of chromatin have?
How does it do this?
A
- Turns genes on
- Binds to histone tails and opens up chromatin to increase accessibility for transcription factors
9
Q
Histone acetyl transferase…
A
Allows factors to load onto chromatin to increase transcription
10
Q
Histone deacetylase…
A
Allows repressor proteins to load onto chromatin to decrease transcription
11
Q
What effect does methylation of chromatin have?
How does it do this?
A
- 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
12
Q
Proximal control elements examples
A
- Promotor regions
- Transcriptional start sites
13
Q
Distal control elements examples
A
Enhancer sequences
14
Q
Introns in primary mRNA…
A
Are spliced out to form mature mRNA which is then 5’ capped and 3’ tailed
15
Q
TFIID is transcription factor that binds to…
A
TATA box
16
Q
miRNA and siRNA
A
- miRNA - blocks translation
- siRNA - degradation of mRNA
17
Q
Ubiquitin tags proteins for…
A
Destruction by the proteosome
18
Q
Cell cycle phases
A
- 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
19
Q
Cell cycle of Pluripotent stem cells is different because…
A
They cycle fast and don’t spend much time in G1
20
Q
G1 checkpoint
A
- Is cell big enough
- Is environment favourable
- Is there DNA damage
21
Q
G2 checkpoint
A
- Is all DNA replicated
- Is cell big enough
- Is environment favourable
22
Q
Metaphase checkpoint
A
- Are all chromosomes aligned on spindle and attached
23
Q
G0
A
State of dormancy not going through cell cycle
24
Q
Cells enter G0 from…
A
G1
25
3 components of cell cycle activation
- Cyclin-dependent kinases (CDK)
- Cyclins
- CDK inhibitor proteins
26
Cyclin and CDK at start of G1
Cyclin D and CDK4/6
27
Cyclin and CDK at end of G1
Cyclin E and CDK 2
28
Cyclin and CDK in S phase
Cyclin A and CDK2
29
Cyclin and CDK in G2 phase
Cyclin A and CDK1
30
Cyclin and CDK in M phase
Cyclin B and CDK1
31
Most growth factors act by directly upregulating...
Cyclin D expression to drive cell through G1 for self renewal
32
G1 restriction point
Cell will either:
- Commit to division and proceed to S phase
- Exit cell cycle into G0
33
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
34
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
35
Apoptosis characteristics
- No loss of integrity
- Aggregation of chromatin at nuclear membrane
- Shrinking of cytoplasm and nuclear condensation
- Mitochondria release death signals
- No inflammation
36
Necrosis characteristics
- Loss of membrane integrity
- Swelling of cytoplasm and mitochondria
- Total cell lysis
- Disintegration of organelle
- Inflammation
37
Apoptosis activation signals
- Withdrawal of positive signals - adhesion to other cells
| - Presence of negative signals - UV light, free radicals
38
Stages of apoptosis
- Death signal received and commitment to die - reversible
- Execution of cell - irreversible
- Dead cell phagocytosed
- Degradation of apoptotic bodies in phagocyte
39
Intrinsic pathway of Apoptosis
- Cytochrome C release from mitochondria
- Activates initiator caspase 9
- Activates effector caspase 3
- Triggers apoptosis
40
Extrinsic pathway of Apoptosis
- Death ligand bind to death receptors
- Activates initiator caspase 8
- Activates effector caspase 3
- Triggers apoptosis
41
Model of cell signalling
- Reception of signal
- Transduction of signal
- Cellular response
42
Signal transduction
Receiving information into a cell and acting to make choices on cell fate
43
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
44
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
45
Slow and Rapid acting signals
- Fast - directly alters cellular process
| - Slow - indirectly alters cellular process by changing gene expression
46
Direct communication between cells
- Gap junctions
| - Cell-cell recognition by surface molecules
47
Local communication between cells
- Paracrine signalling
- Synaptic signalling
- Hormonal signalling
48
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
49
3 types of cell surface receptors
- Ion channel linked receptor
- G-protein linked receptor
- Enzyme linked receptor
50
3 types of enzyme linked receptors
- Receptor tyrosine kinase
- Cytokine receptors
- TGF-Beta receptors
51
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
52
Cytokine receptors
- Cytokines are small secreted proteins
- Control growth and differentiation of many tissues
- Signal to nucleus in direct pathway
53
TGF-Beta Receptors
- Used to keep pluripotent stem cells in an undifferentiated state
54
Binding of TGF-Beta to receptors causes phosphorylation of...
SMAD 2 and 3
55
Binding of BMP to receptors causes phosphorylation of...
SMAD 1, 5 and 8
56
Signals from TGF-Beta and BMP compete for...
SMAD 4 (coSMAD) to enter the nucleus
57
In human ES cells what does TGF-Beta and BMP cause respectively
- TGF-Beta - causes self-renewal
| - BMP - causes differentiation
58
WNT OFF state
Beta-catenin is phosphorylated by CDK1 and then degraded by ubiquitination
59
WNT ON state
WNT binds to frizzled which prevents phosphorylation of Beta-catenin and it enters the nucleus to activate gene expression
60
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)
61
Cleavage
Cell division without increase in cell mass
62
Pattern formation
Laying down the spatio-temporal pattern in an embryo
63
Morphogenesis
Movement of cells into new positions
64
Differentiation
Cells become structurally and functionally different
65
Gastrulation
Produces the 3 germ layers:
- Ectoderm (outer layer) - skin, nervous system
- Mesoderm (middle layer) - heart, muscles, blood
- Endoderm (inner layer) - gut lining, liver
66
Epiblast cells migrate through the...
| And emerge as...
- Primitive streak
| - Emerge as the mesoderm and endoderm
67
4 main signalling centres in embryo
- Posterior Epiblast
- Anterior Visceral Endoderm
- Extra-embryonic Endoderm
- The Node
68
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
69
What do BMP and Shh gradients specify?
The neural vs. non-neural ectoderm
70
In somitogenesis and axis elongation...
| High FGF signalling at the posterior end
High FGF signalling at posterior end keeps cells in a proliferating state
71
In somitogenesis and axon elongation...
| FGF and RA
FGF and RA give cells posterior identity
72
In somitogenesis and axon elongation...
| Anti-WNTs
Anti-WNTs maintain anterior identities
73
In an ES cell what signals are needed to specify the primitive streak?
- Wnt
| - Activin (TGF-Beta)
74
In an ES cell what signals are needed to specify the ectoderm?
BLOCK TGF-Beta and BMP signals
75
In an ES cell what signals are needed to specify ectodermal patterning?
FGF with no BMP added to drive neural differentiation
76
Where are ES cells derived from?
Inner cell mass (ICM) of blastocysts
77
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
78
What is the significance of ES cells being non-transformed?
They have an indefinite proliferative potential
79
Are ES cells diploid or haploid?
Stable, diploid cells (46,XX or 46,XY)
80
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
81
Are mouse ES cells naive or primed?
| Do they express lineage markers?
- Naive
| - Do not express lineage markers
82
Are mouse EpiS and human ES cells naive or primed?
| Do they express lineage markers?
- Primed
| - Express lineage markers
83
What surface markers are expressed on mouse ES cells?
- SSEA1
- SSEA3
- SSEA4
- TRA-1-60
- GCTM2
- Thy1
- MHC
- ALP
- SSEA1
| - ALP
84
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
85
What gene is not expressed in mouse or human ES cells but is expressed in mouse EpiS cells?
Fgf5
86
How can pluripotent stem cells be characterised?
- Cell surface markers
- Genetic
- Epigenetic
- Gene expression
- Differentiation potential
- Single cell replanting ability
87
Adult or Tissue-specific stem cells function?
To build and repair tissue
88
Are adult stem cells pluripotent or multipoint?
Multipotent
89
Are adult stem cells easy or hard to isolate and grow in vitro?
Hard
90
Do adult stem cells have high or low telomerase?
They have NO telomerase
91
What is the significance of adult stem cells having no telomerase?
Telomeres will shorten until the cells senesce
92
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
93
Are all adult stem cells multipotent?
No, some are unipotent e.g. epidermal basal cells only make keratinocytes
94
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
95
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
96
Do all adult stem cells have a limited replicative capacity?
Yes
97
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
98
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
99
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
100
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
101
What is a stem cell niche?
A local tissue microenvironment that hosts and influences the behaviours and characteristics of stem cells
102
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
103
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
104
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
105
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)
106
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
107
Hypertrophy
Increase in cell size
108
Hyperplasia
Increase in cell number
109
Metaplasia
Change in cell differentiation - replacing one mature cell type with another mature cell type
110
Dysplasia
Change in cell differentiation - replacing one mature cell types with another less mature cell type
111
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
112
What are 2 potential uses of IPS cells
- Disease remodelling
| - Drug discovery
113
What 3 things have to be reset in order in order to reset the cell state (to produce IPS cells)?
1. ) gene expression - somatic genes OFF, embryonic genes ON
2. ) methylation (reset to totipotent configuration)
3. ) chromatin - remodelled
114
Examples of some signals required to induce pluripotentcy in human cells
- Lentivirus + Oct4, Sox2, Nanog and Lin28
| - Retrovirus + Oct4, Sox2, Klf4 and C-Myc
115
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
116
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
117
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
118
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
119
Delivery of reprogramming factors to make IPS cells
| - Adenovirus
- Doesnt integrate
| - Very inefficient
120
Delivery of reprogramming factors to make IPS cells
| - Transposons
- Efficient
| - Clones needed for checking of excision ad arrangement
121
Delivery of reprogramming factors to make IPS cells
| - Episomal
- Does not use virus
| - Very inefficient
122
Delivery of reprogramming factors to make IPS cells
| - mRNA
- Does not integrate
| - Very inefficient
123
Characteristics of IPS cells
- Appears any cells can be induced to become IPS cells
| - IPS cells carry genotype of parent cells (ES don't)
124
Why characteristics of IPS cells make them good for disease modelling ?
- Carry genotype , therefore we can capture a genotype forever
125
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)
126
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
127
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
128
Produced human Retinal pigment epithelium which restored sight in rats, how was this tested ?
- Striped and head tracking to test visual acuity
129
3 issues with cultured adult RPE cells (from post mortem )
- Limited donor material
- Hard to grow in vitro
- Degenerate with time with in culture
130
Solution of failed attacthent of RPE cells to membrane
- Artificial membrane
- Artificial choriod
- Patch (coated with RPE cells)
131
In which disease do Lewy boys get deposited in the brain?
Parkinsons Disease
132
Treatments of Parkinson's ?
- L-dopa (levodopa)
- Dopamine agonists
- MAO inhibitors
- COMT inhibitors
133
Thalamotry, Pallindotomy and Deep brain stimulation are surgical procedures to treat which disease ?
Parkinson's
134
To make neurons you have to ...
- Inhibit BMP and TGFB signalling
| - To suppress mesodermal differentiation
135
Dopaminergic neurons anterior fates are determined by
- PAX6
| - No morphogens or FGF and WNT inhibition= forebrain
136
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 !
137
Transposable element
DNA sequence that can change its position within a genome
138
Transposase
Catalyses movement of transposon to another part of genome
139
AMD
Blurred vision or loss of central vision
140
RPE functions
Regeneration of bleached opsisns
141
Stem cell niche (3 things)
- Has a defined anatomical location
- Regulates self-renewal
- Removal from niche results in cellular differentiation
