Introduction to stem cells and cancer Flashcards
(104 cards)
1
Q
what are stem cells?
A
stem cells have the capacity to self-renew into more copies of itself, and can differentiate into more specialised cell types
2
Q
how do stem cells maintain their self-renewal state?
A
Stem cells undergoing self-renewal live in a close environment called a niche, which supports self-renewal
- Stem cell niche supports self-renewal
3
Q
what triggers stem cells to differentiate?
A
If stem cells are pushed out of the stem cell niche, they undergo differentiation
- This is triggered by signals distinct from the signals from the self-renewing niche
4
Q
what determines the behaviour of stem cells?
A
stem cells function based on cell signalling pathways:
- They interpret chemical gradients and perceive signals generated by neighbouring cells
- Signals determine the behaviour of cells
5
Q
what are progenitor cells/transit-amplifying cells?
A
these are cells that proliferate a limited number of cycles before differentiation
- their self-renewal capacity has a limited period
6
Q
what organism is a good model of early mammalian embryonic development?
A
Mouse embryo is a model of mammalian embryonic development
- Pregnancy cycle typically lasts 19-20 days until birth
- Amenable to genetic modification
7
Q
what is cell lineage?
A
Cell lineage: developmental history of a differentiated cell as traced back to the cell from which it arises
8
Q
what are the early stages of mammalian embryonic development in a mouse?
A
- Fertilisation of egg gives rise to zygote
- Between Embryonic stage 0 (E0) and 4.5 days, the zygote undergoes a series of transformations that involve proliferation
- 2 cells start dividing and their size reduces
- This generates a blastomere (cell type of the early embryo that is generated by zygote cleavage)
- a blastocyst is then formed
9
Q
what is a blastocyst?
A
Blastocyst: a spherical embryo (around 100 cells) that can be implanted into the uterus at E4.5
- blastocyst consists of 3 primary cell lineages
- formation of a blastocyst occurs between day 4.5 and 5 in mice (15 days in humans)
10
Q
what are the 3 primary cell lineages of the blastocyst?
A
- Trophectoderm = outer layer of the blastocyst which is the precursor of the placenta – supports the growth of embryo
Inner cell mass of the blastocyst gives rise to:
2. Epiblast = the founding tissue of the embryo proper that gives rise to foetal tissues (embryonic cell type)
3. Primitive endoderm = extraembryonic membranes/tissue that initially covers the epiblast surface and later gives rise to the yolk sac tissue
11
Q
what is the meaning of potency?
A
Potency: ability of a cell to differentiate into one or more cell types
12
Q
what is the meaning of totipotency?
A
Totipotency: ability of a cell to give rise to a fully functional organism (both embryonic and extraembryonic tissues)
- Occurs from zygote to 16-cell stage
13
Q
what is the meaning of pluripotency?
A
Pluripotency: ability of cell to develop into all embryonic cell types, including the germ cells, but cannot form extraembryonic cell types (trophectoderm and primitive endoderm)
- Seen at blastocyst stage (E4.5 – E9)
14
Q
what are the cell types of early embryonic cells?
A
early embryonic cells are pluripotent, meaning they can give rise to any cell type including the germ cells
- they are found in the post-implantation embryo
15
Q
what are the 2 hallmarks of pluripotency?
A
- expression of pluripotency transcription factors - descriptive hallmark
- teratocarcinoma formatinon - functional hallmark
16
Q
how is expression of pluripotency transcription factors a descriptive hallmark of pluripotency?
A
- Genes which are only found in pluripotent cells are pluripotency marker genes
- These markers are transcription factors which bind to DNA and activate gene regulatory programmes
- Inner cell mass contains the epiblast, where the pluripotent cells are (at E4.5)
- Via an expression analysis (in situ hybridisation) of the inner cell mass/epiblast, there is expression of a variety of genes encoding mRNA including Nanog, Oct4 and Sox2
- These form the major transcription factors which are the pluripotency markers
17
Q
which transcription factors are the major pluripotency markers?
A
Nanog
Oct4
Sox2
18
Q
how is teratocarcinoma formation a functional hallmark of pluripotency?
A
Pluripotent cells can be grafted onto the kidney of a host mouse and give rise to teratocarcinomas (type of tumours containing all cell types)
- Done by a teratocarcinoma assay
- Excite cells from early embryo and inject them into a kidney in adult mice, if the cells are pluripotent, they will give rise to large teratocarcinoma tumours
- Contains epidermal cells, gut epithelial cells, muscle cells
- Non pluripotent cells will form small growths of differentiated cell types
19
Q
what is gastrulation?
A
This is the onset of cell type specification and the loss of pluripotency (marks differentiation):
- occurs at E6 until E8.5
- Pluripotent cells organise as a single polarised epithelium stuck to a basement membrane with tight junctions
- At E8.5, the cells reorganise into 3 germ layers
20
Q
what are the 3 germ layers?
A
The germ layers are the first specialised precursors of different embryonic cell types:
- Outer layer = ectoderm
- Middle layer = mesoderm
- Innermost layer = endoderm
21
Q
what does the ectoderm give rise to?
A
Ectoderm gives rise to skin surface, neural structures (CNS and PNS) and neural crest
22
Q
what does the mesoderm give rise to?
A
Mesoderm gives rise to axial, paraxial, intermediate and lateral structures including blood, heart, muscle, kidney
23
Q
what does the endoderm give rise to?
A
Endoderm gives rise to the gut and internal visceral organs such as liver, pancreas, intestine
24
Q
what does gastrulation result in?
A
Gastrulation results in the formation of a fully formed embryo and its organisation into the anterior-posterior axis:
- The primitive streak (PS) is formed, which arises under influence of signalling pathways (WNT, BMP, FGF, Nodal) to define the posterior side
- When these pathways are blocked, the anterior of the embryo is formed
25
how does cell lineage specification during gastrulation occur in a regionalised manner?
- The location of a cell predicts its identity
- Specification occurs in response to distinct signals which activate lineage-specific transcription factors
26
what is the primitive streak organisation?
The organisation of the primitive streak is where cells from the epiblast undergo gastrulation to organise into the 3 germ layers
- this process is linked to EMT
- When epiblast moves towards the PS, there is a breakdown of the basement membrane, loss of cell-cell contacts, EMT and finally cell migration
- Once the cells have transitioned, they can differentiate into the 3 germ layers via gastrulation
27
what is an epithelium?
Epithelium: cells that line the surface of a structure, characterised by tight cell junctions and polarised morphology
28
what is epithelial-mesenchymal transition (EMT)?
Epithelial-Mesenchymal transition (EMT): a process where cells lose their epithelial characteristics, gain a less regular appearance and become migratory
29
what can occur once cells have organised into the 3 germ layers?
Once the cells have organised into the 3 germ layers and start to give rise to different cell types, they begin somitogenesis/axis elongation
30
what is somatogenesis/axis elongation?
- This involves elongation of cell body and construction of the trunk
- Embryo undergoes axis elongation, where the trunk buds via the production of somites
- This occurs from E8.5 to E13.5
31
what drives somatogenesis?
This is process is driven by progenitors called NMPs:
- Embryo acquires head, tail and other defining characteristics
- Niche of NMPs is within the posterior of the embryo
- As the embryo elongates and generates the trunk, the progenitors undergo self-renewal between E8.5 and E13.5 within their niche
When the NMPs are pushed out of their niche, they can differentiate into:
- Paraxial mesoderm/somites – future skeletal muscle, bone, cartilage, vertebrae
- Spinal cord and neurons
32
what type of potency do NMPs have?
they are bipotent, so can only give rise to 2 cell types:
- paraxial mesoderm/somites - future skeletal muscle, bone, cartilage, vertebrae
- spinal cord and neurons - CNS
33
how are the posterior NMPs defined?
WNT and FGF signalling activities are elevated in the NMP niche in the posterior of the embryo
- Expression of these mRNA are found in the tail of the embryo
34
what are the gene markers of NMPs?
T(BRACHYURY)-SOX2 co-expression:
- NMPs co-express 2 major transcription factors called Brachyury and Sox2
- Co-expression of these transcription factors maintains NMPs in self-renewal state (prevents differentiation)
- When forming paraxial mesoderm, SOX2 is downregulated, and Brachyury is only expressed
- When forming the spinal cord, Brachyury is downregulated and SOX2 is expressed
- This can be observed via immunofluorescence
35
what transcription factors are upregulated in NMP differentiation to paraxial mesoderm?
When forming paraxial mesoderm, SOX2 is downregulated, and Brachyury is only expressed
36
what transcription factors are upregulated in NMP differentiation to the CNS components?
When forming the spinal cord, Brachyury is downregulated and SOX2 is expressed
37
how can transcription factors be used in different cell types?
Transcription factors involved in embryonic development can be reused in different cell types
- E.g. sox2 is a pluripotency transcription factor which is later used to form spinal cord
38
how can defects in NMP transcription factors lead to disease?
Defects in NMP differentiation can lead to severe developmental abnormalities
- Defect in brachyury results in truncated embryo due to lack of mesodermal progenitors
- Defect in Wnt results in truncated embryo also
In humans, defects in NMPs results in issues in axial elongation, resulting in truncation problems:
- Spina bifida – spinal cord isn’t formed properly and neural tube stays open
- Currarino syndrome, sacral agenesis, spondylocostal dysostosis – all issues with spinal cord/vertebrae
39
what are neural stem cells (NSCs)?
NSCs are found specifically in CNS and are bipotent
- they can differentiate to neurons and glia
- they can maintain in self-renewal state
40
what signals and TFs maintain NSCs in their self-renewing state?
- Neural stem cells exist in niche maintained by EGF and FGF signals to self-renew
- SOX2 and RC2 are transcription factors which, when co-expressed, maintain the neural stem cells in their self-renewing state
41
what triggers NSCs to begin differentiation? which TFs are involved?
When the NSCs are removed from the niche due to downregulation of SOX2 and RC2, they can begin to differentiate into either:
- Neurons by TUJ1 TF
- glia (microglia, astrocytes, oligodendrocytes) by GFAP TF
42
which TF is involved in NSC differentiation to neurons?
TUJ1
43
which TF is involved in NSC differentiation to glia?
GFAP
44
what are haematopoietic stem cells (HSCs)?
HSCs are multipotent and give rise to all blood cell types
- HSCs arise in the aorta-gonad-mesonephros (AGM) region of the embryo, region where the aorta is near the kidney
- HSCs then migrate to the foetal liver
- At the bone marrow, they form their niche
- HSCs can reconstitute the entire haematopoietic system:
45
what assay can be used to functionally define HSCs and how they constitute the entire haematopoietic system?
- Sub-lethal radiation of mice results in immunocompromised mice lacking HSCs entirely
- The mice are then injected/transplanted with HSCs, resulting in the restoration of the entire haematopoietic system and their blood cells
46
why are embryos and stem cells hard to study?
- Small cell numbers
- In utero development
- Ethics
47
how can stem cells be studied?
Can isolate and capture stem cells in the petri dish to provide a solution
- This can be achieved by capturing pluripotent cells from the early embryo and expanding them in vitro to produce embryonic stem cells (ES cells)
48
why is in vitro modelling of embryonic development useful?
- In vitro modelling of embryonic development can lead to the production of clinically relevant cell populations
- Useful for disease modelling and cell replacement applications
49
how can pluripotent cells be captured in vitro?
1. Need to disassociate the pluripotent cells from the inner cell mass before implantation
2. The dissociated ICM is then plated on a layer of feeder cells
- Feeder cells are irradiated stromal cells, usually fibroblasts) derived from later embryos which support ES cell growth
- Feeder cells provide trophic signals to keep stem cells in pluripotent state
3. Once embryonic cells have divided a few times, they are disaggregated and re-plated
4. ES cells are then formed and can be observed using GFP
50
what are embryonic stem cells (ESCs)?
ESCs are pluripotent:
- ES cells in vitro express the main pluripotency markers (transcription factors) nanog, oct4 and sox2, just like their counter parts in the ICM in vivo
- No expression of genes indicative of differentiation
- A single cell can generate identical daughter cells (= stem cell)
- Self-renewal
- ES cells form teratocarcinomas when transplanted in permissive environments
51
how can feeder cells be replaced?
Critical trophic signals to maintain cells in self-renewing, undifferentiated state and can replace feeders:
- Feeder layers can be replaced by supplementing them with signals which block differentiation and promote self-renewal
- Mouse self-renewal signals: Leukaemia Inhibitory Factor (LIF), BMP
- Human self-renewal signals: FGF2, TGF-beta
- These signals maintain the ES cells in a self-renewing state
52
how can adult somatic cells be reprogrammed to a pluripotent stem cell state?
- Terminally differentiated somatic cells such as skin cells and fibroblasts are introduced to reprogramming pluripotency factors which are normally expressed in the early embryo, such as sox2, oct4
- Overexpression of these factors reverts adult cells from a terminally differentiated state to an early embryonic pluripotent state
- Cells require ability to develop to any cell type
- These cells are called induced pluripotent stem cells (iPSCs)
53
what is the advantage of iPSCs over ESCs?
iPSC production does not require the use of embryos, so there are reduced ethical concerns
54
how can ESCs be proved to be pluripotent?
- Mouse ES cells can be reintroduced to normal mouse embryos and continue normal development
- GFP-ES cells can be replaced into the epiblast pre-implantation, and then can be seen in the mice via green fluorescence expression of GFP
55
how can stem cell differentiation be engineered in vitro?
- Remove signals which promote self-renewing state, and add signals which favour differentiation of stem cells into specialised cell types
- Target cell type: defined by expression of the correct gene markers + functionality (does the cell type generated in vitro perform the function of its in vivo counterpart)
56
what are the 2 main approaches in triggering in vitro differentiation?
1. 3D
2. 2D
57
what is the process of 3D in vitro differentiation?
- Remove signals that keep cells in an undifferentiated state, e.g. BMP/LIF for mouse ES cells, FGF2/TGF-beta in human ES cell
- Grow in aggregates (= embryoid bodies or organoids) in presence or absence of signals
- Involves putting cells in close proximity so they can form 3D clusters
58
what are the ads and disads of 3D in vitro differentiation?
Advantage: recapitulates more accurately embryonic development
Disadvantage: difficult to observe/dissect role of individual signals
59
give an examples of 3D in vitro differentiation:
1. Cardiomyocytes from embryoid bodies start beating, indicating functionality
2. Cerebral organoid generate structures which resemble the early foetal brain
Gives rise to structures which have the correct gene markers in the correct location
60
what is the process of 2D in vitro differentiation?
- Plate a defined number of cells on the right substrate/extracellular matrix
- Use compounds that mimic the ECM in vivo
- Remove signals that keep cells in an undifferentiated state, e.g. BMP/LIF for mouse ES cells, FGF2/TGF-beta in human ES cell
- Grow in a defined medium with appropriate amounts of signals (FGF, WNT etc)
61
what are the ads and disads of 2D in vitro differentiation?
Advantage: more tractable system (e.g. for live imaging), easier to test the role of specific signals
Disadvantage: loss of cell interactions that may occur in vivo
62
give an example of 2D in vitro differentiation:
- GFP expression reflects T(brachyury) expression during ES differentiation
- T(brachyury) is an early mesodermal marker
63
what applications are there for in vitro cell engineering?
1. disease modelling
2. cell replacement
64
what is disease modelling via in vitro cell engineering?
- Reprogramming can enable taking skin cells from patients with mutations that give rise to diseases and reprogramme them to become iPSCs that resemble ES cells
- Using differentiation, we can then define what goes wrong and find the cause of the disease
65
what is an example of where in vitro cell engineering has been used to model disease?
microcephaly
- Neurodevelopmental disorder in which infants are born with an abnormally small brain
- Due to various autosomal recessive mutations
- Neurological defects, seizures
- Mouse mutants fail to recapitulate the condition, so cannot be used to model the disease
66
how was in vitro cell engineering used to model microcephaly?
- A skin biopsy of a patient who carries a mutation of the CDK5RAP2 gene (causes microcephaly) can be taken and reverted to an iPSC state by overexpression of pluripotency factors such as oct4, sox2, klf4, Myc
- The iPSCs could then be reprogrammed to form cerebral organoids
- Can then compare cerebral organoids from normal stem cells with the microcephaly mutant cerebral organoids
- Can examine expression of different factors in different tissues such as DCX in neurons, SOX2 in neural progenitors, DAPI in all cells
- Found that the microcephaly mutant organoids had fewer neural progenitors compared to the normal organoids
67
what is an example of where cell replacement therapy was used via in vitro cell engineering?
Parkinson’s
- Affects 1:500
- Symptoms: tremor, slow movement, rigidity, dementia, anxiety
- Progressive loss of dopaminergic neurons (mDA neurons) in the substantia nigra
- Can generate dopaminergic neurons from pluripotent stem cells and then transplant them into the patient
- hES-cell derived dopamine neurons
68
how was in vitro cell engineering used for cell replacement in Parkinson's disease?
Can generate dopaminergic neurons from pluripotent stem cells and then transplant them into the patient = hES-cell derived dopamine neurons
- Tyrosine hydroxylase (TH) is a marker of mDA neurons, as it is an enzyme involved in the synthesis of dopamine
- The in vitro-derived mDA neurons were transplanted into the brain of mice (model of Parkinson’s) and the TH marker was expressed, as well as other markers
- Indicated that the engineered mDA neurons had the correct expression in the correct place
- There was evidence of improved motor function of the mice – functionality
69
can we capture later (multipotent) stem cell/progenitor populations in vitro, such as NSCs?
Yes, can capture neural stem cells in differentiated state:
- Can plate the cells in presence of GFAP or TUJ1, removing the FGF2 and EGF
- The NS cells will then differentiate into glia and neurons respectively
70
what is mutipotency?
cell has restrictions on what cell types it can differentiate to
71
what is lineage specification?
Lineage specification starts in early embryonic development through gastrulation which involves organisation of pluripotent epithelial cells into a tri-layered structure forming the germ layers
72
what is growth and how does it occur?
Growth druves the rest of embryonic development frodrivesm mid-embryogenesis (after the body and trunk are formed)
growth occurs predominantly by cell proliferation in response to signals
73
what are the phases of the cell cycle?
- G1
- G0 (resting phase)
- S
- G2
- mitosis
74
how is the cell cycle regulated?
Cell cycle is controlled tightly by a variety of proteins and checkpoints:
- They operate in a calibrated manner to balance proliferation and homeostasis is maintained
- If this process is disturbed, it results in uncontrolled proliferation and cancer
75
what may cause uncontrolled proliferation?
Uncontrolled proliferation is caused by either/both:
- Activation of oncogenes (a gene capable of transforming a normal cell into a tumour cell)
- Inactivation of tumour suppressor genes (genes which restrict proliferation)
- Mutations in tumour suppressor genes leads to uncontrolled proliferation
76
what causes cancer?
genetic lesions may arise as a result of environmental insults:
- chemical e.g. smoking
- infections e.g. parasites
- radiation e.g. UV, ionising radiation
- viruses e.g. HPV, EBV, HBV
77
how are tumours heterogenous?
Cells within the same tumours often exhibit differences in terms of:
- Differentiation state
- Proliferation rate
- Migratory and invasive capacity – linked to metastatic behaviour
- Size
- Therapeutic response – some tumours are more refractory to chemotherapy than others
- Tumourigenicity
These all result in intra-tumour heterogeneity - there are a mix of cell types in tumours gives
78
what is the stochastic model of cancer?
- All tumour cells are equipotent and a proportion of them stochastically proliferate to fuel tumour growth
- Equipotent = the cells have the same capacity to generate other cell types within the tumour
- The other tumour cells differentiate
- This makes all cells within a tumour equally susceptible to chemotherapy, as they all have the capacity to generate a tumour
79
what is the limitation of the stochastic model of cancer?
- Tumours often tend to have the ability to recur after treatment
- Led to hypothesis that not all the cells are equally susceptible to anti-cancer treatments, and some of them have differential resistance to treatment
80
what is the cancer stem cell model?
- Only a small subset of tumour cells have the ability for long-term self-renewal - they act as stem cells
- It is these cells that give rise to committed progenitors with limited proliferative potential that eventually terminally differentiate
- In this model not all cells are equal
- There is a reservoir of self-renewing cancer stem cells which undergo commitment gradually
- There is then more differentiated derivatives of the CSCs
- It is the balance of the CSCs with the terminally differentiated cells that keeps the tumour surviving
81
what are the therapeutic implications of cancer stem cells (CSCs)?
- CSCs are typically dormant and have a slow cell cycle
- When the tumour is hit with drugs that target fast-proliferating cells, the dormant CSCs escape the toxic effects of the chemotherapy drug
- That renders the CSCs resistant to the drugs, and these may remain in tissues after treatment
- The CSCs can then drive tumour recurrence
- Eventually, the tumour can grow back with heterogeneity
82
what are the common features between CSCs and normal stem cells?
normal stem cells:
- self renewal for homeostasis
- differentiation - maintenance of organ functionality
- ability for functional reconstitution
CSCs:
- self-renewal for tumour growth
- differentiation - tumour heterogeneity selection advantage
- ability to initiate a tumour
both are regulated by the same signalling pathways e.g. WNT in colon cancer
- hyperactive WNT signalling is a driver for continuous proliferation and self-renewal of colon cancer
83
what are the 2 ways in which CSCs can be generated?
1. reprogramming somatic cells to a CSC state
2. reprogramming of a stem cell to acquire oncogenes
84
how may somatic cells be reprogrammed to a CSC state?
- Normal stem cell generates specialised derivatives
- At some point in normal development/ageing this differentiated derivative undergoes a somatic mutation
- This mutation reverts them to a stem cell/progenitor state that loses its ability to form specialised/differentiated daughter cells
- This reprogramming event gives rise to CSCs which hyper-proliferate, giving rise to tumours
85
how may stem cells be reprogrammed to acquire oncogenes?
- Oncogenic transformation of a pre-existing stem cell/progenitors
- This leads to hyperproliferation and skewed differentiation that gives rise to tumours
86
how can CSCs be captured in vitro?
In vitro potential: establishment of cell lines that can self-renew and differentiate
- Isolate the cancer cell lines that act as CSCs in a petri dish
87
How can a stem cell be proven to be cancerous?
In vivo potential: ability of CSCs to give rise to cancer following transplantation into animals
88
what is acute myeloid leukaemia? what stem cells are implicated in AML?
Acute myeloid leukaemia (AML): blood cancer affecting myeloid lineage
- HSC (haematopoietic stem cells) are CD34+ and CD38-
- These are markers of HSCs and are cell surface receptors
- HSCs have capacity to self-renew and generate multipotent progenitors which can differentiate to specialised blood cell types e.g. lymphocytes, red blood cells
89
what assay can be used to define acute myeloid leukaemia CSCs?
- CD34+ and CD38- AML stem cells give rise to leukaemia due to mutation
- The leukaemic stem cells can give rise to myeloid stem cells (cannot differentiate to whole haematopoietic system)
- If they are transplanted to immunocompromised mouse, then the mouse develops leukaemia
90
how do HSCs and leukaemia stem cells compare?
- There are parallels between the HSCs and the leukaemia stem cells e.g. they have the same CD34+CD38- cell surface markers
- But HSCs can generate entire range of blood cells, but leukaemic stem cells have restricted differentiation
91
what is glioblastoma?
Glioblastoma: most prevalent and lethal primary brain tumour
- Aggressive and invasive
- Treatment: surgical resection, radiation and chemotherapy
- Recurrent
- Average survival time = 12-18 months
- Only 25% patients survive more than a year
92
where do gliobastomas arise from?
GBMs are heterogenous and they arise from cells which resemble neural stem cells (bipotent so generate neurons and glia)
93
how can glioblastomas be captured and cultured, what TF's are involved
- Dissociate GBM tumour cells and plate them on laminin in presence of cytokines FGF2 and EGF - kept the culture in a self-renewing state
- Nestin/Sox2 were identified as undifferentiated, self-renewing TF markers (similar to RC2)
- If these signals are removed, GBM stem cells can differentiate to neurons, marked by Dcx, and glia, marked by GFAP
- This indicates that GBM stem cells are similar to NSCs and can be cultured in the same manner
- However, the differentiation potential of GBM stem cells varies depending on the tumour
this is a descriptive assay of glioblastomas
94
what is a functional assay of glioblastomas in vivo?
- Take isolated GBM stem cells from in vitro and inject into the brain of mice
- forms a GBM-like tumour within the brain
- Shows that the GBM stem cells in vitro have the same tumourigenic capacity as their in vivo counterparts
95
what drug was screened and found to stop glioblastoma development?
Scientists screened for drugs which stopped the development of GBM stem cells:
- If GBM stem cells are treated with indatraline, they die
- If foetal NSCs are treated with indatraline, the effects are less profound: there is some cell death but much less than with the GBMs
-Suggests that the drug is promising in treating glioblastoma as it selectively kills GBMs
96
what are the main approaches to studying cancer?
- Xenograft models – injecting tumour cells into animals
- Cancer cell lines – study tumour behaviour
- Genetically modified animals (overexpression/underexpression of oncogenes/tumour suppressor genes via mutations)
97
what are the limitations to methods in studying cancer?
- Failure to capture transition from a normal to a tumorigenic phenotype in a tractable manner and in a human context
- Lack of mechanistic insight into oncogenic transformation
98
how can human pluripotent cells be used to study cancer?
- Either: Can take established cancer stem cell lines from patients with tumours and reprogramme them back to pluripotent state
- Or: Can use human ESCs and introduce oncogenic mutations to them by overexpressing an oncogene or knockdown of a tumour suppressor gene
- In both of these methods we can then differentiate the cells and push them to generate a cell type that gives rise to cancer
- E.g. to generate a brain tumour, cells are pushed to generate NSCs
99
why is using pluripotent cells to study cancer advantageous?
Using pluripotent stem cells is advantageous, as we can examine it in the different stages of differentiation and see specifically when they start behaving in a cancerous manner e.g. overproliferation compared to a control
100
what is neuroblastoma?
Neuroblastoma: common tumour which arises in sympathetic ganglia in infants and young children
- 50% of patients die with neuroblastoma
- Originates in an embryonic cell type called the neural crest which generates peripheral neurons
101
what TF do neuroblastomas express?
- Aggressive neuroblastomas express high levels of the TF (transcription factor) MYCN
- Ectopic overexpression of MYCN in normal neural crest cells gives rise to neuroblastoma-like tumours
- MYCN is an oncogene
102
where do sympathetic ganglia arise from?
The progenitor of sympathetic ganglia is the neural crest, which is derived from the ectoderm:
- Neural crest gives rise to PNS
- Neural crest cells arise in response to WNT and BMP signals
- Neural crest cells are marked by Sox10 TF at E10.5 in mouse embryo
103
how can neural crest cells be generated?
Can generate neural crest cells in vitro using pluripotent stem cells by mimicking the in vivo developmental processes:
- can treat hESCs with WNT and BMP signals for 5 days
- This results in Sox10+ neural crest cells in the petri dish that resembles the in vivo counterparts
104
describe an assay that defines neuroblastoma cells which compare to normal hESCs:
- hESCs are treated with WNT, BMP and overexpression of MYCN oncogene (doxycycline is used on the cells which causes increased MYCN)
- The cells which were treated with doxycycline had an increased transcript level of MYCN expression
- Cell transformation and cell proliferation rates increased in the MYCN+ cells, indicating that the cells behave like neuroblastoma
- The untreated MYCN- controls did not show overproliferation
- When the MYCN+ cells were injected into mice brains, tumours formed which mimicked neuroblastoma
