Stem cells Flashcards
1
Q
3 properties of stem cells …
A
- Self renewal
- Potency (can make a no. diff cell types)
- Differentiation
2
Q
3 extracellular signals that make the cell grow, divide survive or die
A
- Growth factors
- Death factors
- Survival factors
These cause a change in gene expression leading to self renewal diff. or death
3
Q
2 main types of EXTERNAL signals
A
-Soluble ligands for receptors
-Internally generated signals
external signals must be carried across the membrane to the nucleus (takes longer)
4
Q
What must be altered in order to ‘lock’ on/off gene expression
A
Chromatin
5
Q
Types of chromatin/gene modifications
A
- DNA methylation (of argines and lysines)
- Phosphorylation (serine)
- Histone acetylation (lysines)
6
Q
What are the 2 main pathways that DNA methylation blocks transcription
A
- DIRECT BLOCKING OF TFIID binding (TATA binding protein)
- Recruitment of histone deacteylases
7
Q
Method of transcription
A
- )chromatin extends out - EXPOSES ENHANCERS
- ) Mediator complex Binds-bridges gap between enhancers
- ) Promoter recruits transcription factors
- )RNA polymerase transcribes the gene
8
Q
Splicing
A
introns removed from pre mRNA to produce mRNA
9
Q
What causes degradation of mRNA ?
A
siRNA
10
Q
What causes blockage of transcription?
A
miRNA
11
Q
What destroys proteins
A
Proteasomes degrade ubiquitin tagged proteins
12
Q
Stages of the cell cycle ?
A
- G1
- S
- G2
- M
13
Q
Which stage of the cell cycle has the restriction point (R)?
A
G1
14
Q
What other phase can cells enter at restriction point
A
- Cell can enter G0 phase ‘quiescent state’ , stay here until needed
- CDKs and cyclins disappear
- can be temporary of lifelong
15
Q
What occurs at the restriction point in the G1 phase of the cell cycle ?
A
- somatic cells only
- key transition point
- integration of internal and external cues
- Cells either Commit to S PHASE or enter G0 phase
16
Q
What are the two checkpoints in the cell cycle ?
A
- G1 checkpoint
- G2 checkpoint
17
Q
What is checked at G1 checkpoint ?
A
- Is cell big enough
- Is environment favourable
- Is Dna damaged
18
Q
What is checked at G2 checkpoint ?
A
- Is all DNA replicated?
- Is cell big enough ?
- Is environment favourable
(checkpoint must be passed before entering M phase)
19
Q
What are the three principle components of the cell cycle ?
A
- Cyclin dependant kinases (CDK)
Amount stays the same throughout
-Cyclins
Amount fluctuates throughout the cell cycle
-CDK inhibitor proteins (INK4, KIP)
20
Q
Cyclin D …
A
Binds with CDK4/6
(G1)
- direct link between extracellular environment and cell cycle
- Growth factors act via up regulation of cyclin D
21
Q
Cyclin E …
A
Binds with CDK2
G1/S
22
Q
Cyclin A…
A
Binds with CDK2/1
S+G2
23
Q
Cyclin B
A
Binds with CDK1
G2/M
24
Q
Do ES cells have a restriction point ?
A
NO!
25
Process of restriction point progression
- PRB gets phosphorylated by Cyclin D+ CDK4
- Cyclin E and CDK2 hyperphosphorylate PRB
- E2F dissociates +binds to target genes = transcription/ progression
26
CDK inhibitors
INK4 family - prevents cell from entering S phase (binds to CDK4/6)
KIP family - binds to CDK
27
features of cancer cells
- Cannot arrest at G0
- loss of G1/S restriction control
- Unlimited replicative power
- NO longer responds to growth factors
28
Importance of cell death
- Digit formation
- Stopping immune response
- NS formation
29
Error in apoptosis can lead to disease...
Cancer,HIV,lupus
30
Too much apoptosis leads to ...
Tissue atrophy (loss of muscle mass/cells)
31
Too little apoptosis leads to...
Hyperplasia (Increase in organ size)
32
Characteristics of apoptosis
- PROGRAMMED cell death
- SHRINKS
- aggregation of chromatin at nuclear membrane
- mitochondria release death signals
33
Characteristics of Necrosis
- UNPROGRAMMED
- SWELL AND BURST
- disintegration of organelles
- NO vesicle formation
34
Withdrawal of +ve signals and presence of -ve signals leads to ...
apoptosis
35
Intrinsic pathway of apoptosis
DNA damage+p53 --> mitochondria/cytochrome C
--> INITIATOR CASPASE 9 ---> INITIATOR CASPASE 3
...cell death
36
Extrinsic pathway of apoptosis
Death ligands --> Death receptors -->
INITIATOR CASPASE 8 ---> INITIATOR CASPASE 3
...cell death
37
no signals to a cell...
leads to cell death
38
2 main types of signalling methods :
-Phosphorylation (kinase and phosphatase)
-GTP-binding protein (GTP binding = activation )
(GTP hydrolysis = inactivation)
The same signal can have many different effects!
39
3 ways of cell communication
- Direct cell-cell
- Local (synapses)
- Hormonal - long range
40
2 classes of extracellular signalling molecules ...
- Small hydrophobic molecules, pass through membrane, activate INTRACELLULAR receptors
- Hydrophilic Molecules , too large to cross membrane, rely on MEMBRANE RECEPTORS
41
2 examples of SMALL HYDROPHOBIC molecules
- Steroid hormones
| - Nitric Oxide
42
3 cell surface receptors
- Ion channels
- G protein coupled receptors
- Enzyme linked receptors
43
categories of enzyme linked receptors
- Receptor tyrosine kinases (RTKs), activate AKT pathway = acts as a survival signal that stimulates cell growth/prevents apoptosis
- Cytokine receptors , growth+diff , direct pathway
-TGF B receptors ,anti proliferative signals
mutations=cancer
44
Does TGF beta and BMP signalling compete ?
YES
- SMAD 2/3 (Phos. by TGF) competes for SMAD4 (co smad)
- SMAD 1/5/8(Phos. by BMP) competes for SMAD4 (co smad)
45
WNT in its OFF state=
ubiquitination , no signal
46
WNT in its ON state=
binds to frizzled, activated dishevelled (DVL)
| -B-catenin free to turn on target genes (displaces co-repressors)
47
KINASE INHIBITOR in WNT signalling leads to ....
ACTIVATION of pathway
48
Example of DIRECT cell communication
Notch signalling
- cleaved by gamma secretase (after delta has bound)
- cleaved domain activates gene transcription
49
Signalling = patterning of germ layer, what re the 4 main signalling centres ?
1. ) Post. Epiblast (bottom)
2. ) Anterior Visceral endoderm/AVE (left side)
3. )Extra Embryonic endoderm (top)
4. ) The Node (very bottom)
50
chick model .....Lefty 1 is
restricted to DVE
| also restricts nodal (which activates it)
51
chick model .....BMP4 gradient
(Induced in nodal epiblast)
| -Conc decreases as you go down to node (bottom)
52
As the embryo grows...
AVE+DVE are pushed anteriorly
53
Primitive streak is SET UP due to ?
WNT and TGFB signals which are/have to be restricted to posterior epiblast
-WNT induces TGFB
no signals=ectoderm instead of PS
54
The NOTOCHORD starts to form at the ?
NODE (highest point of NODAL expression)
55
chick model - Formation of Head/neural structures
due to :
-shh +BMP gradients
High BMP= non-neuronal ectoderm
Low BMP= neural crest cells
56
Gastrulation produces 3 Germ layers :
- Ectoderm (skin)
- Endoderm (gut, liver)
- Mesoderm (muscle, blood)
57
Primitive streak =
- thicker region on one side
| - Epiblast cells migrate through + produce mesoderm and endoderm
58
chick model- Somitogensis/axon formation is caused by ..
```
FGF + RA levels (give POSTERIOR identity)
Anti WNT (maintain ANTERIOR identities)
```
59
HIGH POSTERIOR FGF in somitogenisis results in ...
cell proliferation/axon elongation
60
In chick model - Hensons node forms...
Neural tube, somites , notochord
61
In chick model - Primitive streak forms
Mesoderm + endoderm
62
In an ES cell for primitive streak use...
WNT, Activin TGFB
63
In an ES cell for Ectoderm ...
BLOCK TGFB and BMP signals
64
In ES cells for Ectoderm patterning...
FGF with no BMP +drives neural differentiation
65
what types of cells cannot be gained from human embryos
XEN+TS
66
Which stem cells are derived from POST-implantations
Epiblast stem cells
67
Where are ES stem cells derived from ?
Inner cell mass of blastocysts
68
Properties of ES stem cells
- Non transformed survive forever
- pluripotent (make all 3 germ layers)
- stable diploid karyotype
- HUMAN embryonic stem cells have LOW clonegic capacity
- carry surface markers
69
Is incorporation of human ES cells into chimera plausible ?
NO, and neither is mouse ES cells into humans
70
Are Mouse ES cells Naive ?
yes
| -dont express lineage markers
71
Mouse EpiSC
- Primed
| - express lineage markers
72
Human PSC
primed
73
How are human ES cells derived
grown in IVF, trophectoderm removed via antibodies
| =Leaves inner mass cells
74
4 ways of characterising PSC's
- Surface markers
- Genetic
- epigenetic
- Gene expression
75
Functional tests for PSC's
- clonegenic assays
- 2d,3d differentiation
- teratoma
76
What can be used for analysis of surface markers
flow cytometry
77
mouse ES cell surface marker differences to human ES cells
- all negative APART FROM ALP
| - SSEA1 + (positive)
78
marker differentiation caused by
inhibiting TGFB and BMP
79
Epigenetic characterisation of PSC'S
- Methylation checking of genes (methylated=on)
(unmethylated-off)
-X inactivation Male XY=no inactivation
Female XX = 1 inactivated
-Imprinting
80
In epigenetic imprinting genes are exppressed ...
biallelically
OR
maternally or paternally
81
Physical test of ES cells
should be able to form
- embryos body (plating)
- organoid
- monolayer
82
Test for pluripotency
teratoma:
inject under skin
ALL 3 GERM LAYERS should form
83
what type of potency are adult stem cells ?
pluripotent
multipotent
unipotent
multipotent (some are unipotent)
84
are adult stem cells difficult to maintain in vitro and isolate ?
yes, they are very difficult to isolate and maintain in vitro
85
Do adult stem cells have telomerase ?
- yes
- no
NO, this means they cannot divide indefintely
86
DO adult stem cells fit the Hardwired hierarchy model?
| (that adult stem cells have a set limited fate)
NO
87
3 reasons why Adult stem cells don't fit the hardwired hierarchy model
- Not all are multi potent (epidermal basal cells=only kartocytes)
- Not all are quiescent (intestinal crypt constantly dividing)
- Not all have a unidirectional cell fate (clara cells in trachea= dedifferentiate into basal stem cell thats damaged)
88
whats the fastest self renewing tissue in a mammal ?
Small intestinal- crypt villus unit
| = crypt base columnar cells are long term stem cells
89
are there any GO professional stem cells in the intestinal crypt ?
NO
90
mammary gland- MaSC are multi potent and give rise to?
Myo-sc (unipotent)
| lum-sc (bipotent) interconvergance occurs
91
are Satellite cells (muscle stem cells) professional GO stem cells ?
YES, but are unipotent (reforms itself and myofibrils to replace damage)
92
What is a STEM CELL NICHE
A local tissue that hosts and influences the behaviour and characteristics of stem cells
- regulates cell diff
- regulates self renewal
- HAS DEFINED ANATOMICAL LOCATION
93
What does REMOVAL of stem cells FROM a niche results in ?
cellular differentiation (or death)
94
example of niche ... In c.elegans as cells move further away what happens
they lose niche signal + differentiate
95
Niche concepts :
- occupancy
- fate
- assymetric division
occupancy -cell cell adhesions between stem cell and niche keep it near self renewing signals
fate- signals from the niche regulate, self renewal, survival and maintenance
asymmetric divisions- niche can polarise/infulence half of a cell causing asymmetric division
96
Niches provide:
- physical support
- soluble factors
- cell-mediated interactions
To MAINTAIN and REGULATE stem cell functions
97
Cells in niches interact with each other through:
(i) cell-surface receptors
(ii) Gap junctions
(iii) Soluble factors
98
Examples of niche soluble factors are autocrine and paracrine factors which act locally and diffuse ... these factors include ?
FGF
WNT
BMPs
99
ECM/Cell adhesion molecules in niches have..
- Spatial cues
| - Mechanical inputs
100
What do epigenetic restrictions do ?
Prevent a cell from changing from one differentiated state to another
101
What does IPS cells stand for and which cells are induced to produce them ?
IPS= Induced pluripotent stem cells , adult cells are induced to become pluripotent , lots of signals and TF's have to be replicated in order to induce pluripotency
102
What are 2 potential uses of IPS cells
- Disease remodelling
| - drug discovery
103
What 3 things have to be reset in order in order to reset the cell state (to produce IPS cells)?
1. ) gene expression (somatic off, embryonic on)
2. ) methylation (reset to totipotent)
3. ) chromatin (remodelled)
first example-fibroblasts to muscle- CDNA for myod1
104
example of some signals required to induce pluripotentcy in human cells
- Lentivirus + Oct4, Sox2, Nanog and Lin28
| - Retrovirus + Oct4, Sox2, Klf4 and C-Myc
105
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
106
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
107
3 examples of non integrating methods of preventing over expression of factors to induce IPS cells
- vector based approaches
- Protein based approaches (TAT fusion)
- Chemical based reprogramming
108
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
109
Delivery of reprogramming factors to make IPS cells
| -Adenovirus + mRNA
- Doesnt integrate
| - Very inefficient
110
Delivery of reprogramming factors to make IPS cells
| -Transposons
- Efficient
| - clones needed for checking of excision ad arrangement
111
Delivery of reprogramming factors to make IPS cells
| -Episomal
- Does not use virus
| - very inefficient
112
Characteristics of IPS cells...
- Appears any cells can be induced to become IPS cells
| - IPS cells carry genotype of parent cells (ES don't)
113
Why characteristics of IPS cells make them good for disease modelling ?
-carry genotype , therefore we can capture a genotype forever
114
HOW are IPS cells used for disease modelling
patients somatic cells Compared against normal somatic 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)
115
Two forms of AMD
- WET amd (can be treated) -angiogensis +bleeding
- 90% DRY amd (can't be treated) - fatty deposits "drusen" between bruch's membrane and RPE
LOSS OF RETINAL PIGMENT EPITHELIUM in rear of retina
**RPE function = regeneration of bleached opsins**
costs 8.8 billion per year
116
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
117
produced human Retinal pigment epithelium which restored sight in rats, how was this tested ?
-Striped and head tracking to test visual acuity
118
3 issues with cultured adult RPE cells (from post mortem )
- limited donor material
- hard to grow in vitro
- degenerate with time with in culture
119
solution of failed attacthent of RPE cells to membrane
- Artificial membrane
- Artificial choriod
- Patch (coated with RPE cells)
120
In which disease do Lewy boys get deposited in the brain ?
Parkinsons
121
Treatments of parkinson's ?
- L-dopa (levodopa) *can cross BB barrier*,dopamine can't
- Dopamine agonists
- MAO inhibitors
- COMT inhibitors
122
Thalamotry, Pallindotomy and Deep brain stimulation are surgical procedures to treat which disease ?
Parkinsons
123
To make dopaminergic neurons from stem cells you have to ...
inhibit BMP and TGFB signalling
| suppress mesodermal diff.
124
specifying dopaminergic neurons
-pax6 (anterior fates)
-no morphogens or fgf+wnt inhibition= forebrain
-WNT FGF RA = Caudalize the brain
-midbrain produces - WNT1 FGF8
WNT signalling =patterning of brain
125
gradients of what define the fate of a neuroectodermal precursor on the D/V axis?
- Shh (high at bottom), BMP and WNT (High at top)
| - More Shh = more ventral fate
126
Hyperplasia...
Increase in no. of cells
reversible
127
Metaplasia...
change in cell differentiation (mature)
reversible
128
Dysplasia ...
change in cell differentiation (less mature)
reversible
129
Neoplasia (cancer) ...
new abnormal growth of tissue , IRRIVERSIBLE - autonomous
130
Usual fund size in life sciences?
£50 million -£300 million
131
proportion of fund used for overheads ?
1-2%
132
how is profit split ?
80% investors
| 20% venture fund managers
133
investment + return period ?
5-7 years investment
| 7-10 year return
134
target return from initial investment ?
3x
135
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 !
136
Which two people murdered people and sold them to doctors for medical research ?
William hare
| william burke
