SCGI W1-5 Flashcards
1
Q
days 0-6
A
oocyte release
12-24 hrs fertilisation - zygote formed
pronuclei fusion
30 hrs the 2-cell stage
3 days the morula
4 days advanced morula
4.5-6 days the blastocyst
potential to for dizygotic twins - 2 oocytes released and fertilised
2
Q
Describe stages of development : blastocysts
A
4.5-6 days
Outer cell mass pumps fluid in forming the blastocyst
3
Q
what is Oct4
A
The POU-domain transcription factor Oct4
- maintaining pluripotency in stem cells
- tightly regulated transcription factor associated with a number of target genes implicated in pluripotency maintenance.
- regulatory elements in target genes are in close vicinity of Sox2 binding sites
key factor in the transcriptional framework of self-renewing stem cells
4
Q
Implication of Oct4 on a knockout blasotcyst
A
In the knockout blastocyst, if Oct4 isn’t expressed, there will not be a functional inner cell mass, and therefore, there will not be an epiblast or hypoblast stage. The epiblast is a single layer embryo, with the hypoblast surrounding it, and the placenta surrounding both.
5
Q
What is fate of cells in the outer cell mass ?
A
The outer cells of the blastocyst form the placenta as trophoblasts
The outer cell mass pumps in fluid, forming the blastocyst cavity
By 6 days, the outer cells must differentiate into different cell types, such as fibrous structures, to implant in the uterine structure.
6
Q
what do the synctiotrophoblast cells do
A
fuse together to form a single layer without intercellular boundaries. The cells directly below the syncytiotrophoblast form the cytotrophoblast, which consists of an irregular layer of ovoid, mono-nucleated cells.
7
Q
what is the fate of the inner cell mass
A
Day 9
Becomes the epiblast and hypoblast.
8
Q
What is the fate of the cells in the hypoblast ?
A
- Form part of the inner cell mass
- Become part of the inner layer of the developing embryo
9
Q
Gastrulation regulators
A
cell migration and specification = controlled by FGF8 - synthesised by the primitive streak cells
FGF8 controls cell movement by downregualting E-cadherin
FGF8 controls specification into the mesoderm by regulating BRACHYURY (T) expression
10
Q
primitive streak formation and gastrulation
A
day 15
the primitive streak and primitive node appear
the initiation of gastrulation
epiblast cells migrate to the primitive streak and slip below
- cells migrate btw the epiblast and the hypoblast - form the definitive mesoderm
- cells displace the cells of the hypoblast - form the definitive endoderm
the remaining cells form the ectoderm
11
Q
What is the fate of cells in the epiblast?
A
Epiblast cells form the embryonic disk, which gives rise to the embryonic structures such as the amniotic cavity, embryonic disk, and eventually the fetus.
12
Q
What is significance of primitive node and singalling molecules
A
- Releases signaling molecules that determine body axis and cell fate.
- NODAL, a member of the TGF-B family, maintains the primitive streak and allows signals to be passed out to other cells.
- The primitive node upregulates developmental genes, including BMP4, which stimulates the formation of skin.
- Chordin and Noggin block the action of BMP4 and stimulate the production of neural structures and cranial features.
13
Q
What is significance of primitive streak
A
- Indicates gastrulation
- Controlled by FGF8 as cells of the epiblast migrate and slip beneath it
- Significant for the formation of the three germ layers.
14
Q
Describe process of gastrulation
A
- Formation of the primitive streak from thickened epiblast cells
- Migration of epiblast cells towards the primitive streak and slipping beneath it
- Formation of the notochord - a rod-like structure that runs along the body axis and acts as a scaffold for the development of the nervous system
- Controlled by FGF8 signaling molecule
- Results in the formation of three germ layers: ectoderm, mesoderm and endoderm.
15
Q
Describe the importance of the ectoderm, mesoderm and ectoderm
A
- Ectoderm: Forms skin, hair and nervous system * Mesoderm: Forms muscles, bones and connective tissue
*Endoderm: Forms GIT and respiratory system
and urinary tract .
16
Q
Explain the importance of signalling molecules in embryonic development
A
- Directing cell differentiation, migration and growth.
These molecules include: NODAL, BMP4, Chordin, Noggin, FGF8
They regulate gene expression and help to establish the axes of the body and the formation of tissues and organs.
17
Q
HOX transcription factor
A
Regulates gene expression during embryonic development
Controls the formation of the axial skeleton and segmentation of the body
Controls the differentiation of embryonic cells into specific tissue types and organs
Acts as a “master regulator” in early development and is crucial for proper formation and function of the body.
18
Q
GSC gene
A
needed for cranial development
goosecoid (HOX TF) can stimulate the production of cerberus
19
Q
Formation of the neural plate
A
day 19
the presence of the notochord and prechordal mesoderm leads to the thickening of the overlying ectoderm, resulting in the formation of the neural plate.
20
Q
formation of the somites
A
day 20
as the neural plate lengthens its lateral edges elevate to form neural folds and the depressed midregion forms the neural groove
21
Q
Formation of the neural tube
A
day 22
neural folds approach each other in the midline where they fuse
fusion begins in the cervical region (fifth somite) and proceeds cranially and caudally
22
Q
Formation and closure of neuropores
A
day 23 - formation
the cephalic and caudal ends of the neural tube communicate with the amniotic cavity by way of the anterior (cranial) and posterior (caudal) neuropores
days 25-28 - closure
23
Q
what are the differentiation singals of the neural tube cell
A
sonic hedgehog (SHH) - patterns the ventral neural tube (F = floor plate cells)
bone morphogenic proteins (BMPs) - pattern the dorsal neural tube (R = roof plate cells)
chordin, noggin - block the action f BMP4 stimulate formation of NS and cranial structures
24
Q
What does the neural tube form
A
- cells on the ventral side of the blastula secrete proteins such as BMP4 - inducing the ectoderm above to become the epidermis
- noggin and chordin physically bind to BMP4 molecules in the extracellular space preventing BMP4 from binding to receptors on ectoderm cells causes the ectoderm cells to follow their intrinsic pathway forming the neural folds and the brain and spinal cord
blocks the action of BMP4 - causing the default pathway to be formed
25
How does sonic hedgehog differentiate and develop the spinal cord
The notochord releases SHH, which communicates with and patterns the ventral neural tube, leading to the differentiation and development of the spinal cord.
26
differentiation of mesoderm
day 20-21
1st phase = periodisation/segmentation
- segmented blocks of somites appear progressively from the anterior (carinal) end of the animal
mesoderm cells "epithelise" and become fibroblast like - these somite cells form donut shapes
27
lateral folding
dermatome --> dermis
sclerotome --> muscle
myotome --> tendon, cartilage, bone (vertebrae-ribs)
SHH, noggin -> sclerotome -> PAX1 -> vertebrae formation
BMP4, WNT, NT-3 -> PAX3 -> dermis
BMP, WNT -> MYF5, MyoD -> muscle
28
somite differentiation
dermatome --> dermis
sclerotome --> muscle
myotome --> tendon, cartilage, bone (vertebrae-ribs)
SHH, noggin -> sclerotome -> PAX1 -> vertebrae formation
BMP4, WNT, NT-3 -> PAX3 -> dermis
BMP, WNT -> MYF5, MyoD -> muscle
29
how does bone morphogenic protein differentiate and develop the spinal cord
- Expressed. by roof cells
- Pattern the dorsal neural tube
Bone morphogenic protein (BMP) plays a role in the differentiation and development of the spinal cord. BMP signals direct the differentiation of surrounding cells into specific spinal cord cell types, influencing the formation and patterning of the spinal cord.
30
Importance of notochord in differentiate and develop of spinal cord
: The notochord is a flexible rod-like structure found in the embryonic stage of all chordates, including vertebrates. It acts as a structural support and a signaling center
- either side of notochord are somites, the notochord communicates with floor plates od neural groove, the roof plates form the neural tube
- notochord releases SHH which communicates and patterns the ventral tube
31
Formation of notochord
-days 17-18
notocord formation occurs ina cranial to caudal sequence
important site of signal secretion for NS development
remains to become part of the invertebrate discs in the adult
32
Migration of neural crest cells
migrate to the ectoderm
migrate to the melanocytes and become the mesoderm the cells specify into:
- connective tissue and bonterm-22es of the face and skull/dermis in face and neck/odontoblasts
- cranial nerve ganglia/sympathetic ganglia/dorsal root ganglia/adrenal medulla/glial cells
33
How does developmental spinal bifida arise
Failure of proper neural tube closure: This results in an opening in the spinal column, exposing the spinal cord and surrounding tissues to the amniotic fluid.
Genetic and environmental factors: It is believed to be a complex interaction between genetic and environmental factors such as maternal nutrition and exposure to toxins.
34
What is developmental spinal bifida
type of congenital birth defect that affects the proper development of the spinal cord and surrounding tissues. It results in the formation of a spinal cyst or an incomplete closure of the spinal column.
35
What is neuralation?
days 15-18
prenotochordal cells invaginating in the primitive node move forward cranially in the midline until they reach the prechordal plate
formation of neural tube
36
stages of neurulation
1. Ectoderm overlying the notochord and
somite regions becomes neural
ectoderm.
2. Tissue begins to fold, with the neural
plate in the middle and the neural folds
on the ends.
3. The folds fuse to give the neural tube.
4. The cells at the tips of the folds migrate away and become neural crest cells.
5. The remainder of the ectoderm (non-
neural) becomes epithelial and
forms the epidermis.
6. During neurula, the body elongates
7. Anterior-posterior axis is particularly
obvious.
37
How do embryonic and adult stem cells differ?
both exhibit self-renewal and the capacity to differentiate
ES cells are derived from early embryos but don't represent a cell the normally exists - have the potential to give rise to every cell type in the body
adult stem cells arise in the foetus and serve to maintain organs throughout life - have the potential to give rise to one to several different cell types depending on the organ from which they derive
38
Why is cell replacement necessary for tissues?
Cell replacement is necessary for tissues due to normal turnover and also disease or traumatic damage that may require new cells.
39
How do embryonic stem cells change during development?
The proportion of total cells that are stem cells decreases during development.
From stage 1-8, the stem cells are totipotent. Then, the morula is reached, and the cells change to a blastocyst, which contains inner mass cells that are pluripotent. They can go through gastrulation to form the embryo proper.
40
What is self-renewal, and how does it relate to stem cells?
Self-renewal is the ability to undergo symmetrical division without differentiation. It is a characteristic of stem cells.
41
What are the essential features of a stem cell
self-renewal and differentiation potential.
2 ways of dividing
- asymmetrically - maintenance - stem cells associated with tissues
- symmetrically - expansion - transient stem cells involved in development
quiescence
42
Embryonic stem cells: ICM isolation
isolate the inner cell mass (ICM) cells from IVF
feeders provide factors that maintain embryonic stem cell growth
- human ESCs cultured on foreskin fibroblasts that release the GFs and cytokines to maintain the stem cell niche
43
Embryonic stem cells: in vitro
differentiation triggered whey grown in suspension, embryoid body formation
different cells obtained spontaneously
specific growth factors can be used to direct the differentiation of ES cells into specific cells
44
How do embryonic and adult stem cells differ?
Embryonic stem cells (ES cells) are derived from early embryos and have the potential to give rise to every cell in the body except for embryonic tissues such as placenta, while adult stem cells arise in the fetus and serve to maintain organs, and can give rise to one to several different cell types depending on the organ from which they are derived.
45
What are the different potentials of stem cells?
Stem cells can have different potentials, including totipotent, pluripotent, and multipotent.
46
What is a totipotent stem cell?
A totipotent stem cell is the most versatile stem cell
when the sperm and egg fuse the cell formed is totipotent - it has the potential to give rise to any cell in the human body
1 cell can give rise to an entire functional organism
post fertilisation the zygote divides in synchrony - each cell is totipotent up to the 8 cell stage
47
What is a pluripotent stem cell?
can give rise to all tissue types
they cannot give rise to the entire organism - don't give rise to the extra-embryonic tissues
day 4 of development - the embryo forms 2 layers
- trophectoderm which forms the placenta
- inner cell mass which forms the embryo proper - all the tissues and organs of the developing human body
48
What is a multipotent stem cell?
less plastic and more differentiated
give rise to a limited range of cells within a tissue type
daughter cells become progenitors of cell lines e.g. blood/skin/nerve cells
become 1 of several cell types within a give organ
bi-potent - self renewal and 2 cell types
uni-potent - self renewal and 1 cell type
49
What is the difference between bi-potent and uni-potent stem cells?
Bi-potent stem cells can self-renew into two cell types, while uni-potent stem cells can only self-renew into one cell type.
50
How do embryonic stem cells change during development?
The proportion of total cells that are stem cells decreases during development. From stage 1-8, the stem cells are totipotent. Then, the morula is reached, and the cells change to a blastocyst, which contains inner mass cells that are pluripotent. They can go through gastrulation to form the embryo proper.
51
What triggers differentiation in stem cells?
Changing growth factors and media surrounding stem cells trigger differentiation.
52
Why is spontaneous cell differentiation bad when trying to maintain pluripotency?
Spontaneous cell differentiation can lead to loss of pluripotency in stem cells.
53
What is required for using stem cells therapeutically?
Stem cells must be seeded on human foreskin fibroblasts for therapeutic use.
54
What are transcription factors?
Transcription factors are proteins that bind to specific DNA sequences, controlling the rate of transcription of genetic information from DNA to RNA.
55
ESC- Oct3/4 and what is its function?
Oct3/4 is a POU-domain transcription factor that maintains pluripotency in different types of stem cells. It is a tightly regulated transcription factor and is associated with a number of target genes implicated in pluripotency maintenance.
56
ESC- What is Sox2 and what is its function?
Sox2 is a transcription factor involved in embryonic development and determination of cell fate. It is necessary for embryonal development and to prevent ES cell differentiation. Many ES cell pluripotency-associated genes are coregulated by SOX2 and Oct4.
57
ESC- What is Nanog and what is its function?
Nanog is a unique homeobox transcription factor involved in self-renewal of undifferentiated embryonic stem cells.
- contains a homeobox domain
downstream effectors of signals of LIF and BMP
elevated levels excludes inclusion of LIF and feeder layer
works with other key factors including Oct4 and Sox2
58
What is LIF and what is its function?
LIF is a cytokine from the interleukin-6 family that is essential for maintaining pluripotency in vitro in the presence of serum. Binding of LIF to a heterodimeric receptor comprising of LIF-receptor and gp130 on the cell membrane results in activation of Jak/stat signal transduction pathway. Activated Stat3 maintains pluripotency.
59
How do mouse ES cells and human ES cells differ in terms of growth and differentiation?
Mouse ES cells grow adherently and require LIF for maintaining pluripotency. Human ES cells are grown in conditioned medium with bFGF and MEF-CM, and if bFGF is removed, the outside of the cell becomes jagged, and the colony differentiates.
60
How are stem cells identified?
appearance
- through analysis of the binding of labelled antibodies specific to cell surface proteins (not enough on its own)
function
- assays e.g in vitro self-renewal and differentiation
- serial transplantation into mice
identification of stem cells = an iterative process of subdivision based on appearance followed by functional testing by the most rigorous means possible
61
How are stem cells identified?
Through analysis of binding of labelled antibodies specific to cell surface proteins and a variety of assays may be employed including in vitro self-renewal and differentiation, but the gold standard test is serial transplantation into mice.
62
What must be provided to prevent spontaneous differentiation in stem cells?
a niche
63
What pushes stem cells down specific pathways?
Specific cytokines or growth factors.
64
What are some factors that must be considered when conducting a UV irradiation experiment?
Dosage, exposure time, and background reading.
65
What information about the cell line used must be provided in an experiment?
The specific cell line used, if it lies on a feeder layer, culture media, and culture parameters such as temperature and CO2.
66
How is gene expression studied in ES cells?
RNA is made into stable CDNA and checked for integrity on an agarose gel. Techniques such as RTPCR, Western blotting, and RNA sequencing can be used to identify controls.
67
Intestinal crypts as a stem cell niche
non-dividing differentiated Paneth cells at the bottom of the crypts
stem cells in the bottom of the crypts (cycle time = 24hrs)
rapidly dividing cells (cycle time = 12hrs)
non-dividing differentiated cells
epithelial cell migration from birth at the bottom of the crypt to loss at the top of the villus (transit time = 3-5days)
68
intestinal crypts: differentiated cell types
absorptive cell
goblet cell
paneth cell
enteroendocrine cell
69
intestinal crypts: lineage tracing
lineage tracing shows that the stemm cells in the crypt produce all the cells in the villus
mark cells using a reporter e.g. LacZ
marker can be activated in stem cells and expression remains in the daughter cells - daughter cells from one stem cell will all be marked
70
mechanism to generate cellular diversity
asymmetric division: sister cells are born different
symmetric division: sister cells become different as a result of influences acting on them after their birth
71
What techniques can be used to extract and measure protein in an experiment?
SDS page is used to extract protein and measure concentration, and the amount to be loaded onto the SDS-PAGE must also be determined.
72
What is the turnover rate of the intestinal epithelium in adults?
The entire epithelium turns over entirely every 3-5 days.
73
Where do slowly dividing stem cells reside in the intestine?
At the bottom of crypt.
74
What type of cells make up the crypt at the base of villi?
Absorptive brush border cells and mucus secreting goblet cells.
75
What is the importance of the balance between self renewal and differentiation in stem cells?
It is important for tissue maintenance, as too many stem cells can compromise the function of the tissue, and not enough can exhaust the niche.
76
How do stem cells choose their fate?
They can choose between self renewal and differentiation through asymmetrically localized determinants and signals from neighboring cells.
77
What are the 4 main types of signal transduction pathways?
paracrine signalling
juxtacrine signalling
endocrine signalling
autocrine signalling
78
What is juxtracrine signalling: notch pathway
A type of signaling in which the signal remains bound to the cell membrane of the signaling cell, meaning only cells in direct contact with the signaling cell can be activated.
- notch is a cell surface protein with extracellular and intracellular domain
notch binds either delta or serrate - membrane proteins with an extracellular domain and a minimal intracellular domain
notch contains 36 EGF-like repeats
binding to notch requires EGF repeats 11 and 12
79
juxtacrine singalling: notch cleaving
notch engagement causes 2 sequential proteolytic cleavage events
which liberate the notch intracellular domain (ICD)
1st cleavage in the extracellular domain by TACE
2nd cleavage in the transmembrane domain by y-secretase
80
juxtacrine signalling: notch transcriptional activation
notch ICD traffics to the nucleus form the cell membrane
in the nucleus the ICD binds to the transcription factor CSL
unstimulated cells CSL binds a co-repressor NCoR and represses transcription
ICD-binding to CSL displaces co-repressors and recruits co-activators to activate transcription
81
What genes are turned on by notch signaling?
HES genes.
82
What are HES gene products?
transcription repressors that are basic helix-loop-helix (bHLH) transcription factors with an N-terminal bHLH domain. They bind as homo- or heterodimers with other HES proteins and recognize the E-box sequence. The basic amino acids at the N-terminus of the first helix bind to DNA.
83
juxtacrine signalling: recruitment of co-repressors
C-terminal WRPW motif recruits the co-repressor Groucho
HES genes are transcriptional repressors
84
juxtacrine signalling: transcriptional repression
notch activation ultimately represses transcription
notch signalling controls gut cell diversification and helps maintain the stem-cell blocks
85
juxtacrine signalling: notch and specialisation
mediates competitive cell interactions that limit specialisation
notch mediates lateral inhibition
delta on 1 cell binds notch on neighbouring cells activating the notch pathway and repressing delta
cells compete forming mutually repressive loop
1 cell wins and maintains delta activating the notch pathway in its neighbours and blocking their differentiation
singularised cell fates in an equivalence feild
can be biased/influenced by
- asymmetrically segregated products
- differential delta expression
- post-translational modification of the notch receptor
86
juxtacrine signalling: notch and gut cell diversification
notch used at 2 stages
1. paneth cells next to ISCs in the crypts express notch ligand delta
- activates notch pathway in ISCs in contact with the paneth cells
- preserves the stem cell state
- when a daughter cell divides 1 daughter cell loses contact with the paneth cell and differentiates
2. lateral inhibition via notch
- used to space differentiated structures in an initially equivalent field of cells
87
paracrine signaling
allows signal gradients (morphogen gradients)
secreted molecule (morphogen) diffuses from its site of synthesis (source) to its site of degradation (sink)
a concentration gradient forms from the source to the sink
if cells respond differently according to the level of the morphogen different cell fates can be achieved across the gradient
88
paracrine signalling: Wnts
Wnts are morphogens that maintain the gut stem-cell compartment
Wnt signalling pathway
- activation of wingless signalling pathway occurs by inhibition of an inhibitor (in this case GSK3)
- a DNA-binding TF (TCF) is converted from a transcriptional repressor to an activator by changes in protein associations
- activation is by relief of repression
Wnt can control proliferation
Wnt is secrete by Paneth cells at the base of the crypt
89
lateral inhibition
Lateral inhibition is a process used to space differentiated structures in an initially equivalent field of cells by controlling the appearance of specialised cells from a field of initially equivalent cells.
Notch mediates lateral inhibition by activating in equivalently spaced cells, resulting in selective activation of Notch, and the cells become specialised and acquire specific fate as a result of Notch activation.
90
paracrine signalling: Wnt signalling cascade
signal - ligand: Wnt
receptor: frizzleds
transducers: beta-catenin
targets: genes and cytoskeleton
91
What are the essential elements of any signalling cascade?
The essential elements of any signalling cascade include signal (ligand), receptor, transducer, and targets.
92
paracrine signalling: properties of the Wnt proteins and their receptors?
proteins : secreted proteins of 350-400 amino acids
act over 1-5 cell diameters
gradients of action
multiple Wnts
Receptors : receptor is composed of frizzled and co-receptor LRP
frizzled:
- the core receptor
- a seven-pass transmembrane protein
- multiple frizzled family members (10 human)
LRP:
- a co-receptor for wingless
- a single-pass transmembrane protein
93
paracrine singalling: properties of the transducer
beta-catenin/armadillo is a cytoplasmic-nuclear signalling mediator
- also in cell adherens junctions
- stability is controlled by phosphorylation by GSK3
- phosphorylated beta-catenin is targeted for proteosome degradation by a destruction complex inducing APC/Axin
94
paracrine signalling: control of beta-catenin stability
axin forms a scaffold of a destruction complex
beta-catenin is phosphorylated by casein kinase 1alpha at ser45. GSK3 then phosphorylates Thr41, Ser37 and Ser33
phosphorylated beta-catenin is bound by the ubiquitin ligase beta-TRCP ubiquitination of beta-catenin targets it for proteosome-dependent degradation
- APC regulates the transition from phosphorylation to ubiquitination
95
paracrine signalling: beta catenin effect on target genes
TCF identified as a binding partner for beta-catenin
DNA binding protein that contains an HMG (high mobility group) domain
in the absence of Wnt signalling TCF binds a co-repressor (groucho) and represses transcription
binding of beta-catenin to TCF switched it to a transcriptional activator
96
paracrine signalling: TCF switch froma repressor to an activator
in the absence of Wnt signalling TCF binds a co-repressor (Groucho) and represses transcription
beta-catenin displaces Groucho from TCF by binding an additional, low-affinity site that overlaps the Groucho-binding site
beta-catenin/TCF activates transcription
97
paracrine signalling: mutations
TCF mutants lack villi and show no proliferation in crypts
conditional mutants in beta-catenin impair ISC proliferation
98
paracrine signalling: cancer
dysregulation of Wnt signalling leads to cancer
aberrant Wnt signals in APC mutants drives hyper proliferation
CRC/FAP - mutant APC
99
What are the different types of adult stem cells?
hematopoietic stem cells (HSCs)
intestinal stem cells
mesenchymal stem cells (MSCs)
neural stem cells
100
What are the different types of adult stem cells?
Hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, hepatic stem cells, and testicular cells.
101
HSCs come from
1st HSCs are generated by blood islands during embryo development
1st HSCs that stay with you for life are generated in the aorta-gonad-mesonephros (AGM) region at the ventral wall of the dorsal aorta
102
HSCs formation
at the ventral wall of the dorsal aorta a specialised type of endothelium undergoes a transition from an adherent cell type into a non-adherent type - EHT (endothelial to haematpoietic transition)
after this emergence of stem cell progenitor cells further expansion of the haematopoietic system occurs in the foetal liver then it move to the bone marrow
103
HSCs short term vs long term
in mice
- both ST and LT HSCs can reconstitute all blood lineages in an irradiated mouse - they are equally pluripotent
- serial transplantation only possible using LT HSCs
104
HSCs self renewal vs differentiation
LT-HSCs are able to fully self renew and to maintain/even expand the pool of LT-HSCs in the long term
ST-HSCs aren't able to as differentiation outcompetes self-renewal
105
HSCs proliferation
high proliferation occyrs at progenitor stage
106
HSCs stem cell niche
the bone marrow
quiescent cells located on the outside
middle area = stem cells that are active
relationship btw where they are and how they are proliferating = due to signals from the surroundings
bone marrow contains many cell types - each interacting with their environment by uptake and excretion of soluble factors
what signals and/or cells that help regulate HSC quiescence, retention, proliferation, expansion is not clear yet
- likely to be perivascular cells/endothelial cells/macrophages/nerves/O2 levels
107
MSCs are
stromal (support) cells
provide support and structural scaffold to the parenchymal cells of tissues (which provide the main function)
108
MSCs are multipotent bone marrow stromal cells
identification of colony-forming units fibroblasts (CFU-F)
form spindle shape adherent cells in culture
act as a feeder layer for HSCs in vitro
tri-lineage potential following in vitro transplantation
multi-potent stromal cells = MSCs
109
MSCs location
perivascular
110
What are ISCs
crypts and villi - forming a large SA
ISCs located at the bottom of the crypt
regulated by BMP signalling - determines differentiation
They divide continuously throughout life and produce the cells lining the surface of the small and large intestines. They reside near the base of the stem cell niche, called the crypts of Lieberkuhn.
111
What are neural stem cells and what is the difference between symmetrical and asymmetrical division?
They exist in the adult brain and can generate new neurons. They can divide in a symmetrical or asymmetrical manner, with symmetrical division leading to the expansion of the stem cell pool and asymmetrical division leading to the generation of differentiated cells.
112
What are olfactory adult stem cells and what is their unique characteristic?
They are harvested from the human olfactory mucosa cells and can develop into many different cell types, similar to embryonic stem cells.
113
What are hepatic stem cells and where are they located?
They reside in the liver and can regenerate the organ upon loss of a substantial part of it.
114
What are mammary stem cells?
Mammary stem cells provide the source of cells for growth of the mammary gland and can give rise to both the luminal and myoepithelial cell types of the gland.
115
Where are mesenchymal stem cells located in the body?
Mesenchymal stem cells have a perivascular location around the blood vessel, and their exclusive markers for in vivo identification and isolation are unclear.
116
What is the Haematopoietic hierarchy and how does it change as it goes down?
The Haematopoietic hierarchy is a series of stages in the development of blood cells. As it goes down, self-renewal decreases and differentiation increases.
117
What is EHT and where does it occur?
EHT is a transition from an adherent cell type into a round non-adherent type, which occurs at the ventral wall of the dorsal aorta.
118
What are multipotent bone marrow stromal cells and what is their function?
Multipotent bone marrow stromal cells are identified as colony-forming units fibroblasts (CFU-F) and form spindle-shaped adherent cells in culture. They act as a feeder layer for HSC in vitro.
119
What is cloning and what does it allow for?
scientific technique to create genetically identical organisms
production of 1+ individual plants/animals (whole/in part) that are genetically identical
120
What are the three types of cloning?
embryo cloning
reproductive cloning
therapeutic cloning
121
What is embryo cloning and how is it done?
produce monozygotic twins/triplets
- 1 egg fertilised that divides into 2 cells which separate
in vitro
- allow the embryo to divide in vitro
- remove 1+ cells and encourage to develop into embryo - twins/triplets have identical DNA
122
What is reproductive cloning and why is it controversial?
produce a duplicate of existing animal
used to clone sheep + other mammals
produce several genetic defects
medical ethicists - immoral procedure to be done of humans as it is unsafe and unethical
123
What is reproductive cloning and why is it controversial?
Reproductive cloning is the production of a duplicate of an existing animal, but it can produce several genetic defects. It is considered immoral and unsafe to be done on humans by medical ethicist
124
Therapeutic cloning : SCNT in animal
hold egg in place
remove the nucleus from the egg
isolate the nucleus from a somatic cell
inject nucleus into egg beneath protective egg covering
allow nucleus and egg to fuse
allow embryo to grow to blastocyst stage in vitro
125
therapeutic cloning: SNCT in animals
enucleate the eggs produced by scottish blackface ewes
- treat the ewes with gondotropin-releasing hormone (GnRH) which causes them to produce oocytes ready to be fertilised
- the oocytes are arrested at metaphase of the 2nd mitotic division (MII)
- micropipette into the egg over the polar body and suck out the polar body and the haploid pronucleus within the egg
126
therapeutic cloning: dolly the sheep - fusion
fuse each enucleated egg with a diploid cell growing in culture
- cells from the mammary gland of an adult finn dorset ewe (white faced) are grown in tissue culture
- 5 days before usethe nutrient level in the culture is reduced so that the cells stop dividing and enter G0 of the cell cycle
- donor cells and enucleated recipient cells are placed together in culture
- the cultures are exposed to pulses of electricity
-- cause the respective plasma membranes to fuse
-- stimulate resulting cell to begin mitosis (mimicking the stimulus of fertilisation)
127
therapeutic cloning: dolly the sheep - growth
culture the cells until they have grown into a morula or even into a blastocyst (6 days)
transfer several of these into the uterus of each scottish blackface ewe (previously treated withGnRH to prepare them for implantation)
wait
the result = 1 ewe gave birth to dolly
128
are cloned embryos identical to fertilised embryos
most cloned embryos that are derived from SCNT die during gestation
those that survive birth and grow to adulthood experience respiratory problems and have developmental defects associated with an enlarged placenta/have metabolic disorders/diabetes/neurological disorders
genome wide expression analysis indicates that approx5% of the genome (up to 50% of the imprinted genes) are abnormally expressed in tissues of newborn SCNT derived mice
the epigenetic inheritance may be the principle biological barrier to normal development of cloned animals - primarily influence normal placental development at early stages
129
What is the difference between nuclear and mitochondrial genes in cloning?
Nuclear genes will remain the same in cloning, but mitochondrial DNA would be different. In the case of Dolly the sheep, her nuclear genome came from the Finn Dorset ewe, but her mitochondria came from the cytoplasm of the Scottish blackface ewe.
130
what is therapeutic cloning
removing nucleus of egg cell replace with nucleus of somatic cell stimulate cell division
somatic cell nuclear transferase (SCNT)
biomedical cloning
research cloning
regenerative medicine
nuclear transplantation therapy (NTT)
131
stem cell research gives way for
drug development and toxicity tests
experiments to study development and gene control
cultured pluripotent stem cells -> tissues for therapy -> bone marrow/nerve cells/heart muscle cells/pancreatic islet cells
132
animal cloning advantages
cloned animals are genetically identical - all have the good characteristics of their parent
transgenic animals can be cloned so producing a small herd that produces specific human protein in their milk
endangered animals can be cloned so preserving their genes for future
rapidly improve the quality of herds
133
animal cloning disadvantages
cloned animals are all genetically identical - may have unknown genetic disease or susceptibility to disease
quality of life of cloned animals may be poor - smaller groups, isolation, controlled conditions, shorter life expectancy
clones are genetically identical and so could be problems of genetic drift and evolutionary bottlenecks
success rate is low for some types of cloning
134
What is reprogramming and how did it emerge as a concept?
Reprogramming is the process of reverting differentiated cells back to pluripotent stem cells. It emerged as a concept as a result of the fight against aging and the consequences associated with it. The idea is based on development studies as it is the reversion of the differentiation process.
135
What is the difference between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)?
ESCs are cells that are made in the lab taken from the blastocyst, whereas iPSCs are derived from somatic cells that have been reprogrammed to become pluripotent.
136
What are the four transcription factors used in reprogramming somatic cells to iPSCs?
The four transcription factors used in reprogramming somatic cells to iPSCs are Oct4, Sox2, Klf4, and C-Myc. These transcription factors are involved in regulating the pluripotency of embryonic stem cells.
137
What is the difference between using skin fibroblasts and blood cells for reprogramming?
Reprogramming using blood cells is less invasive than using skin fibroblasts. Urine cells are not invasive at all to create iPSCs. Reprogramming using neurons or disease cells can provide insights into disease mechanisms and allow for disease modeling.
138
What is the goal of therapeutic cloning by SCNT in humans?
The goal of therapeutic cloning by SCNT (somatic cell nuclear transfer) in humans is to use the embryo as a source for ES cells and to use ES cells to generate an organ that carries genetic markers of the patient or to correct genetic errors in ESCs at the blastula stage.
139
SCNT therapeutic cloning
embryo cloning - nuclear transplantation of patients own cells to make an oocyte and stem cells from which immune-compatible cells can be derived for transplants
(stem cells generation require the destruction of an embryo)
3 goals of therapeutic cloning by SCNT in humans:
- use embryo as source for ES cells
- use ES cells to generate an organ
- correct genetic error in the ESC in blastula stage
very little done with ESC clinically to date
140
What is the role of transcription factors Oct4 and Sox2 in maintaining the self-renewal and pluripotency of ESCs?
Oct4 and Sox2 are required for the formation of the ICM (inner cell mass) in the blastocyst stage during embryogenesis. Their expression levels need to be tightly regulated in order to maintain self-renewal and pluripotency of ESCs. They bind to DNA in adjacent sites and co-stimulate their binding and synergistically activate transcription of target genes.
141
What is the core set of transcription factors that maintain the ES cell state?
The core set of transcription factors that maintain the ES cell state includes Oct4, Sox2, and Nanog. These factors regulate pluripotency and self-renewal of embryonic stem cells.
142
What are some challenges in reprogramming?
Reprogramming is inefficient and can involve oncogenes in the reprogramming cocktail. There is a need for better ways to reprogram cells, and there are ongoing efforts to decode the molecular mechanisms of reprogramming.
143
How can reprogramming be used for disease modeling?
Reprogramming can be used to create iPSCs from cells from patients with specific diseases, allowing for the study of disease mechanisms and potential treatments.
144
What are some advantages of reprogramming?
A: Reprogramming can replace damaged tissue, is self-renewing, can help understand how stem cells develop into healthy and diseased cells, and has no immune rejection.
145
What are induced pluripotent stem cells (iPSCs)?
iPSCs are cells that have been reprogrammed from somatic cells (such as skin cells or blood cells) by adding specific transcription factors, including Oct4, Klf, Sox2, and C-Myc, to make them pluripotent and able to produce any cell type in the body.
146
What are some potential applications of iPSCs?
iPSCs have the potential to replace damaged tissues, help researchers understand how stem cells develop into healthy and diseased cells, and serve as a tool for drug screening and disease modeling.
147
IPSCs: TF Oct4
homeodomain TF encoded for by the Pou5f1 gene
required for formation of the ICM in the blastocyst stage during early embryogenesis and defines the identity of ESCs
its expression levels must be tightly regulated in order to maintain self-renewal and pluripotency of ECSs
148
IPSCs: TF Sox2
SOX family TFs
required for ICM and epiblast development during embryogenesis
ESCs Sox2 and Oct4 bind in adjacent sites co-stimulating its binding and synergistically activate transcription of its target genes such as Fgf4
Oct4 and Sox2 together with the shared target nanog co-occupy a large set of genes involved in the regulation of pluripotency and form super-enhancer domains at key genes which determine ESC identity
149
IPSCs: TF Klf4
zinc finger-containing TF
associated with inhibition of cell proliferation by controlling the expression of cell-cycle regulatory genes
either activates/represses the transcription of genes e.g. Cdh1 and p53 that must be regulated in order for reprogramming to occur
150
What is the role of C-Myc in pluripotency?
C-Myc is a proto-oncogene that is needed for the regulation of cell cycle, self-renewal, and pluripotency of ESCs. Its expression is crucial during the first few days of reprogramming since it upregulates genes in DNA replication and cell division. It is dispensable for the reprogramming cocktail.
151
What are some challenges associated with reprogramming somatic cells into iPSCs?
Reprogramming is inefficient, and the use of retroviruses to deliver reprogramming genes inside the cell can be problematic. Oncogenes in the reprogramming cocktail can cause concerns about tumorigenicity.
152
What are the three types of stem cells?
Embryonic
Tissue-specific (adult)
Induced pluripotent
153
How can iPS cells be created?
Isolate cells from a patient (e.g. skin or fibroblast)
Treat cells with reprogramming factors
Create pluripotent stem cells
Change culture conditions to stimulate cells to differentiate into a variety of cell types
154
What are the potential uses of adult stem cells?
Used to repopulate a damaged tissue with stroma of tissue or to modify chronic inflammatory response.
MSC therapy
155
How can stem cell rejection be avoided?
Use iPS cells derived from the adult themselves instead of hES cells to avoid rejection.
156
How does MSC therapy work?
Injected MSC can go to sites of injury and modify the disease process
The MSC may differentiate into stromal cell when in the organ or can produce factors to give orders to the surrounding cells.
MSC can also produce immunosuppressive factors or produce factors that are presented on the endothelial to pull in cells such as T-reg cells.
157
What are the advantages and disadvantages of hepatocytes for stem cell therapy?
Advantages: suitable for many enzyme deficiency states, metabolic diseases, coagulation disorders, and liver failure
Disadvantages: donor organ shortages and limitations in transplanted cell engraftment and proliferation
158
What are the advantages and disadvantages of hemopoietic cells for stem cell therapy?
Advantages: non-limited plasticity to the tissue they derived from
Disadvantages: procedure for obtaining involves bone marrow aspirate
159
What are the advantages and disadvantages of immune cells for stem cell therapy?
Advantages: easy to isolate and expand for autologous therapy
Disadvantages: only used in HCC and tend to form inflammatory storms
160
What are the advantages and disadvantages of EPCs for stem cell therapy?
Advantages: appear anti-fibrotic and pro-regenerative for autologous therapy
Disadvantages: isolation process is complicated and clinical use is unclear
161
what is the zygote
pronuclei
- male and female haploid genome, approach each other and the nuclear membranes break down
- chromosomal pairing, DNA replicates, 1st mitotic division
spermatozoa contributes the centriole
oocyte contributes the mitochondria
zona pellucida
- mechanical protection of egg involved in the fertilisation process
adhesion of sperm to the egg initiates the acrosome reaction - causes the release of enzymes that break down cortical granules to block polyspermy
162
describe compaction and formation of the inner/outer cell mass
8 cell stage
inner cells segregate from the outer cells
inner cell mass --> embryo
outer cell mass --> trophoblast --> placenta
163
blastocyst implantation
day 12
trophoblasts differentiate into
- cytrophoblasts: an irregular layer of ovoid, mononucleated cells directly below the syncytiotrophoblast
- syncytiotrophoblast: an external layer with no intracellular boundries, the cells form cord infiltrating the endometrium, derived from the fusion of cytotrophoblast cells through apoptosis of uterine stromal cells spaces are created through which the blastocyst penetrates further into the endometrium
164
stem cells differ by ...
origin: embryonic/adult
different potentials: totipotent/pluripotent/multipotent
165
what is pluripotency
the ability to give rise to differentiated cell types derived from all three primary germ layers of the embryo: endoderm, mesoderm and ectoderm
166
Stem cells vs tumour cells
stem cell
- the RB/E2F and p53 pathways are INACTIVE in stem cells
- Cdk1 is the major kinase regulating all the phases
differentiated cell
- cells proliferate under the strict control of multiple Cdk/cyclin complexes and their inhibitors
tumour cell
- inhibitory pathways are inactivated leading to hyper activation of the Cdk/cyclin complexes
167
juxtacrine signalling: notch and gut cell diversification
notch used at 2 stages
1. paneth cells next to ISCs in the crypts express notch ligand delta
- activates notch pathway in ISCs in contact with the paneth cells
- preserves the stem cell state
- when a daughter cell divides 1 daughter cell loses contact with the paneth cell and differentiates
2. lateral inhibition via notch
- used to space differentiated structures in an initially equivalent field of cells
168
paracrine signalling: Wnt signalling cascade
signal - ligand: Wnt
receptor: frizzleds
transducers: beta-catenin
targets: genes and cytoskeleton
169
paracrine signalling: Wnt signalling cascade
signal - ligand: Wnt
receptor: frizzleds
transducers: beta-catenin
targets: genes and cytoskeleton
170
paracrine signalling: TCF switch froma repressor to an activator
in the absence of Wnt signalling TCF binds a co-repressor (Groucho) and represses transcription
beta-catenin displaces Groucho from TCF by binding an additional, low-affinity site that overlaps the Groucho-binding site
beta-catenin/TCF activates transcription
171
paracrine signalling: target genes of Wnt signalling
cell cycle regulators: C-myc/Cyclin D1
tissue specific genes
tissue remodeling proteins: MMPs/ephrin receptors and ligands/adhesion molecules/cyclin D1
angiogenesis: VEGF
172
HSCs self renewal vs differentiation
173
HSCs proliferation
high proliferation occyrs at progenitor stage
174
MSCs location
perivascular
175
Embryo cloning: embryo splitting
artificially splitting a single embryo at a very early stage of development
done at n early stage - less than 8 cells - can only make a few clones
both nuclear and mitochondrial genes would be identical
176
therapeutic cloning: dolly the sheep - fusion
fuse each enucleated egg with a diploid cell growing in culture
- cells from the mammary gland of an adult finn dorset ewe (white faced) are grown in tissue culture
- 5 days before usethe nutrient level in the culture is reduced so that the cells stop dividing and enter G0 of the cell cycle
- donor cells and enucleated recipient cells are placed together in culture
- the cultures are exposed to pulses of electricity
-- cause the respective plasma membranes to fuse
-- stimulate resulting cell to begin mitosis (mimicking the stimulus of fertilisation)
177
stem cell research gives way for
drug development and toxicity tests
experiments to study development and gene control
cultured pluripotent stem cells -> tissues for therapy -> bone marrow/nerve cells/heart muscle cells/pancreatic islet cells
178
animal cloning disadvantages
cloned animals are all genetically identical - may have unknown genetic disease or susceptibility to disease
quality of life of cloned animals may be poor - smaller groups, isolation, controlled conditions, shorter life expectancy
clones are genetically identical and so could be problems of genetic drift and evolutionary bottlenecks
success rate is low for some types of cloning
179
Mammalian nuclear transfer
reproductive cloning
use of electrofusion/sendi virus to fuse cells of 8/16 cell embryos into enucleated eggs of sheep -> obtained 2 healthy clones
180
Imporrance of DNA repair
1 unrepaired DNA DSB is sufficient to kill a cell
defects in DNA repair pathways strongly contribute to tumourigenesis e.g. promote chromosomal translocations
DNA damage can be induced by exposure to agents such as ionising radiation/x-rays/chemotherapeutic drugs
181
sensing DNA DSBs
2 protein complees
Ku70/Ku80 heterodimer:
- essential for repairing DSBs by NHEJ
- NHEJ repairs 80-85% all DSBs throughout the cell cycle
- required to activate the PI3 kinase-like kinase (PIKK) DNA-PK
Mre11/Rad50/Nbs1 (MRN) complex:
- essential for repairing DSBs by HR
- HR repairs 15-20% of DSBs in late S/G2-phase of the cell cycle
- required to activate the PIKKs/ATM/ATR
182
Ku-dependent recognition of DNA DSBs
Ku70/80 is essential for NHEJ and DNA-PK activation
NHEJ is involved in repairing 80-85% of all DSBs irrespective of cell cycle phase
DNA-P activation is a stepwise process that requires Ku70/80 binding DNA ends, synapsis of the Ku-bound DNA ends by PAXX and the recruitment of DNA-PKcs
183
the MRN complex
2xMre11, 2xRad50, 1-1.5xNbs1 = MRN complex
majors sensor of DNA DSBs
essential for HR and ATM/ATR activation
MRN-dependent pathways repair 15-20% of all DSBs - particularly those associated with heterochromatin
recruitment and maintenance of the MRN complex at DSBs is dependent upon DNA binding and binding phosphorylated and/or PARylated proteins on the chromatin
ATM activation requires NBS1 binding and the autophosphorylation of ATM homodimers in trans
184
assaying the recruitment of DNA damage response proteins: a practical approach
grow cells on slides
-/+IR or laser stripe
fix cells in 3.6% PFA
permeabilise cells 0.1% TX-100
incubate with primary Ab (anti-Rad51)
wash
incubate with secondary Ab couples to fluorophor
wash
incubate DAPI to stain DNA, put on coverslip, visualise by fluorescence microscopy
185
Factors influencing the decision between Ku-dependent and MRN-dependent DSB recognition and repair
previously thought that Ku and MRN complex could not occupy the same DSB - high resolution microscopy indicates that this is not the case
induce DSBs in S phase - don't induce Ku fosi - cell cycle phase can influence if Ku can recognise DSBs
Ku foci recognise DSBs in S phase if you inhibit ATM - ATM plays a role in actively suppressing Ku-dependent recognition of DSBs in certain phases of the cell cycle to prevent inappropriate repair
inhibition of ATM unrepaired DSBs - close proximity to heterochromatin - ATM involved in detecting/repairing DSBs associated within regions of heterochromatin
NHEJ-dependent pathways are inefficient at recognising and repairing complex DNA lesions induced by high LET radiation
186
regulation of CDK activity
G1 -> G1 Cdks on
S -> G1 Cdks off/S Cdks on
G2 -> S Cdks off/G2 Cdks on
M -> G2 Cdks off/mitotic Cdks on
mitotic exit -> all Cdks off
cyclin levels are controlled transcriptionally and by targeted protein degradation
the activity of Cdks can also be controlled by the presence of CKIs e.g. p21
187
the cell cycle
the series of biochemical structural events involving the growth, replication, and division of eukaryotic cells
188
cell cycle checkpoints
a point in the eukaryotic cell cycle where progress through the cycle can be halted until conditions are suitable for the cell to proceed to the next stage
checkpoints prevent the propagation of deleterious genetic errors e.g. DNA damage/mis-segregated chromosomes
189
G1/S checkpoint
prevents entry into S-phase and replication of damaged DNA
target for inhibition: G1-phase Cdk activity
controlled by ATM/p53-dependent DDR after IR
190
DNA damage activated G1/S-phase checkpoint: practical approach FACS
cells grown in culture
-/+IR and BrdU
wash
trypsinize and fix cells 70% ethanol
wash
incubate in 1.5M HCl
wash
incubate with primary Ab (anti-BrdU)
wash
incubate with secondary Ab coupled to fluorophor
wash
incubate with propidium iodide and RNAse
FACS
191
DNA damage activated G1/S-phase checkpoint: practical approach SDS-page
cells grown in culture
-/+IR
cell lysis
SDS-page and electrotransfer to nitrocellulose membrane
ponceau S stain membrane to check transfer
wash
incubate with anit-protein X primary Ab
wash
incubate with secondary Ab coupled to HRP
wash
incubate with ECL and expose to light sensitive film
192
intra-S-phase checkpoint
prevents replication of damaged DNA
both elongation and late origin firing are inhibited
target for inhibition: replication associated Cdk activity (Cdk2/Cdk7)
controlled by ATM/ATR-dependent DDR pathway after IR
193
DNA damage-activated S-phase checkpoint: practical approach - DNA replication suppression
addition of radiolabelled thymidine-C^14 (24hrs)
wash off excess
IR
thymidine-H^3 (30mins)
isolate the DNA - measure the ratio of the thymidine C^14 to H^3 incorporated - H^3 only incorporated after DNA damage
cells lacking S-phase checkpoint damage induced inhibition of DNA synthesis doesn't occur
194
DNA damage-activated S-phase checkpoint: practical approach - DNA fibre analysis
incubation of cells with thymidine analogue CldU - incorporates into the DNA (20mins)
wash off excess
IR
incubate cells with IdU (20mins)
take cells and lyse down a glass slide - DNA spreading produces DNA fibres
use fluorescently coupled Abs that recognise CldU IdU
see the ongoing fork - 2 colours
stalled fork - 1st colour
new origin - 2nd colour
DNA DSB - suppression of new origin firing/slows elongation of ongoing forks
195
G2/M-phase checkpoint
prevents entry into mitosis with DNA damage
target for inhibition: G2/M-phase Cdk activity e.g. CyclinB/Cdc2
controlled by ATM/ATR-dependent DDR after IR
ATR activation requires resection of DSB
196
G2/M-phase checkpoint
prevents entry into mitosis with DNA damage
target for inhibition: G2/M-phase Cdk activity e.g. CyclinB/Cdc2
controlled by ATM/ATR-dependent DDR after IR
ATR activation requires resection of DSB
197
DNA damage-activated G2/M-phase checkpoint: practical approach
cells in culture
-/+ IR
trypsinize cells
fix cells 70% ethanol
permeabilise cells 0.1% TX-100
incubate with primary Ab (anti-phospho-H3 Serine-10)
wash
incubate with secondary Ab coupled to fluorophor
wash
incubate with propidium iodide and RNAse
FACS
198
assaying the DSB repair capacity of a cell
grow cells on slides
-/+ IR
fix cells in 3.6% PFA
permeabilise cells 0.1% TX-100
incubate with primary Ab (anti-Rad51)
wash
incubate with secondary Ab coupled to fluorophor
wash
incubate DAPI to stain DNA, put on coverslip, visualise by fluorescence microscopy
or use gammaH2AX
199
non-homologous DNA end joining (NHEJ)
DNA DSB repair
doesn't require a homologous template, can occur at any phase of the cell cycle
accurate, although small deletions can occur at sites of the DSB during repair
essential for cell viability
required for immune system development/maturation
defects in NHEJ are associated with RS-SCID and/or severe growth deficiency
200
NHEJ pathway
DSB Ku70/Ku80 heterodimer binds
ends of the DNA held together by PAXX
recruit DNA-PKcs - serine, threonine PK phosphorylated proteins including Artemis
Artemis trims DNA
PNKP enzymes make sure ends are correct for ligation
XRCC4 and XLF form filaments along the DNA
LigVI ligates the DSB
201
NHEJ assaying repair: practical - western blotting
cells in culture
-/+ IR
cell lysis
SDS-page and electrotransfer to nitrocellulose membrane
poncaeu S stain membrane to check transfer
wash
incubate with anti-protein X primary Ab
wash
incubate with secondary Ab couple to HRP
wash
incubate with ECL and expose to light sensitive film
202
NHEJ assaying repair: practical - IP-kinase assay
cells in culture
-/+ IR
cell lysis
add anti-DNA-PKcs Ab
add protein A/G beads
isolate protien-Ab-bead complex by centrifugation
wash
add substrate dsDNA and 32P-labelled ATP
incubate at 37 degrees
SDS-page
stain gel, dry and expose to radioactivity sensitive film
203
NHEJ assaying repair: practical - immunofluorescence
grow cells expressing GFP-tagged protein in BrdU
UV laser
allow time to recover
visualise in real-time protein relocalisation
204
NHEJ assaying repair: practical - FACS
cells in culture
RFP-plasmid/Sce-GFP-plasmid/I-Scel-plasmid (may not go into the same cell
trypsinize cells
fix cells in 70% ethanol
FACS
RFP is used to control for transfection efficiency
NHEJ is quantified as the ratio of RFP:GFP
205
alternative DNA end joining (Alt-EJ)
DSB
in absence of core NHEJ machinery - recognised by MRN complex and CtlP
uncontrolled resection of DSB - by Exo1
DNA synapse formation - PolQ
microhomology annealing
polymerases carry out DNA synthesis
Fen1 removes the flaps
potential loss of >4nt
206
Alt-EJ assaying repair: practical - DNA reporter assay
Alt-EJ results in GFP expression
207
homologous recombination (HR)
DNA DSB repair
requires a homologous template - only occurs when the genome has been replicated (late S/G2-phase)
high degree of accuracy - low frequency of mistakes
essential for cell viability
defects in HRR are associated with hereditary breast cancer and neurodegenerative disorders e.g. A-T/A-TLD/NBS/FA
208
HR pathway
DSB
recognised by the MRN complex
MRN complex binds to CltP - activation of endonuclease activity
creates nicks in the DNA - resect away from the DSB
53BP1 complex functions to limit resection and HR
RPA binds to resected DNA
BRCA2-BRCA1-POUB2 complex removes RPA and load on Rad51
Rad51 forms a nucleofilament along the ssDNA
promotes invasion of the non-damaged DNA
invasion allows DNA polymerase to uses this as a template - DNA synthesis across the DSB - forms a double holiday junction
resolved by either
- topoisomerase/helicase complex resulting in non-crossover
- structure-specific nuclease complex which depending on the direction of cutting results in cross over/non-cross over
209
assaying HR: practical - immunofluorescence
to assess the ability of HR proteins to relocalise to DSBs
grow cells on slides
-/+ IR
fix cells in 3.6% PFA
permeabilise cells 0.1% TX-100
incubate with primary Ab (anti-Rad51)
wash
incubate with secondary Ab coupled to fluorophor
wash
incubate DAPI to stain DNA, put on coverslip, visualise by fluorescence microscopy
210
assaying HR: practical - assess frequency of sister chromatid exchanges
label the different sister chromatids
look at the ability of the cell to form crossovers
211
assaying HR: practical - FACS based HR reporter assay
cells in culture
add RFP-plasmid, Sce-GFP-plasmid, I-Scel-plasmid - may not end up in the same cells
trypsinize cells
fix cells in 70% ethanol
FACS
RFP is used to control for transfection efficiency. HR is quantified as the ratio of RFP:GFP
212
basic DNA replication
G1-phase : prepares the cell for replication
S-phase : DNA synthesis
G2-phase : preparation for mitosis
M-phase : mitosis
213
DNA replication in human cells
3 billion bases per cell division
30000-50000 initiations per genome
214
3 stages of replication
initiation - loading
elongation - copying
termination - removing
215
terminology of DNA synthesis
template strands
lagging strand template
leading strand template
daughter strands
lagging strand
leading strand
replication fork
origin
sister chromatids
216
replication proteins
DNA helicase - separates DNA double helix
RNA primase - DNA template-directed, synthesises RNA primer
DNA polymerase - DNA template-directed, synthesises DNA
DNA ligase - seals DNA ends together
DNA topoisomerase - relaxes topological strain/supercoiling in DNA
217
replication initiation
not all origins of replication will be fired
early/mid/late synthesis
G1 replication proteins loaded onto the DNA
S Cdks phosphorylate the proteins + further protein recruitment, then firing of the origin and bi directional replication
218
replication fork progression
RNA primase generates the RNA primer
leading strand = 1 primer
lagging strand = multiple primers
MCM, GINS + CDC45: replicative helicase
Pol alpha: RNA primase
219
replication termination
TOPII - DNA topoisomerase II
TOPII cuts DNA and resolves the structures
separation of the 2 sister chromatids
loss of proteins via post translational modifications
220
replication stress
DNA damage during DNA replication
when the replication fork slows down
replication forks that aren't moving properly can be converted into DSBs
221
replication stress: intracellular obstacles
DNA secondary structures
repetitive DNA
RNA nucleotide incorporation
transcription
spontaneous DNA damage
222
replication stress: extracellular obstacles
UV damage
other carcinogens e.g. cigarette smoke
chemotherapies
223
measuring replication stress
DNA fibre method
CidU 20mins
wash
IdU 20mins
run cells down a slide
Ab against CldU - red
Ab against IdU - green
stalled fork = only 1st Ab present
224
results of replication forks slowing down
mitosis after incomplete replication
- super coiled resolved
- under replicating DNA
- anaphase - lagging chromosomes or anaphase bridge
fulty mitosis causes
- chromosomal instability
- cell death by mitotic catastrophe
225
results of replication forks stalling
caused by DNA damage on template -> DNA polymerase unable to continue -> replication stalls
stalled fork -> replication fork collapse, loss of replication proteins -> nuclease processing into 1-ended DSB -> collapsed fork
SSB -> replication fork collapse, loss of replication proteins -> SSB transformed into 1-ended DSB -> collapsed fork
DSB
- prevent proper functioning of chromosome, especially in mitosis
- highly toxic - lager amounts are sensed by the cell and activate apoptosis
- lower numbers of DSBs are highly mutagenic
226
importance of replication stress
cancer cells have some level of replication stress compared to normal cells
activation of some oncogenes can induce replication stress
oncogenes of the GF signalling pathways inc. Cdk activity and accelerate S-phase entry and replication initiation
inc initiation causes replication stress by
- too much initiation
- nucleotide depletion
- replication fork slowing
- DNA damage on template
227
replication stressin stem cells
mechanisms of replication stress can occur in stem cells when they are induced to grow/growing fast
- stem cell reprogramming/iPS cells
- fast cell cycles in embryonic stem cells/pre-implantation embryos
228
cell response to replication stress - checkpoint
ATM
- signals gamma-H2AX
- CHK2 which causes p53 activation and apoptosis/DNA repair/CDK1, 2 inhibition and cell cycle arrest
ATR
- signals gamma-H2AX
- CHK1 which causes DNA repair: HR/CDK1, 2 inhibition and cell cycle arrest
229
cell response to replication stress - HR
function of the RAD51 recombinase
1. Rad51 forms protein-DNA filaments on 3' ss overhangs
2. the Rad51 filament searches for homologous sequences and invades them
3. this results in a displacement loop (D-loop) structure containing 1 holiday junction (HJ)
4. the 3' end in the D-loop structure can be used by the replication machinery to establish replication + copy genetic information
230
visualising replication stress
increased damage
- damage foci: 53BP1/gamma-H2AX/RAD51
fibre analysis
- DNA structures/replication speeds
