BMS382 Stem Cell Biology Flashcards Preview

3rd year COPY > BMS382 Stem Cell Biology > Flashcards

Flashcards in BMS382 Stem Cell Biology Deck (310)
Loading flashcards...
1

Where did the term stem cell originate from?

Ernst Haeckel (1868)
- Tree of life with stem cells at the bottom (the stem of the tree)
- The first stem cell would be the fertilised zygote (pluripotent)
- Stammzelle – first term used

2

What is a stem cell?

Cells that have the potential to generate specialised tissue (differentiation) as well as copies of themselves (Self-replication)

3

How are stem cells classified based on age of development?

Age of development
- Embryonic
- Adult

4

How are stem cells classified based on tissue of origin?

Tissue of origin
- Neural stem cells
- Hematopoietic
- Umbilical cord etc.

5

How are stem cells classified based on their potential to produce different cell types?

- Totipotent: all cell types of the human body including trophoblasts (zygote)
- Pluripotent: derivatives from the three germ layers (embryonic stem cells)
- Multipotent: different cell types from a tissue or organ
- Unipotent: single cell type (e.g. muscle satellite cells)

6

Do stem cells divide quickly or slowly?

Stem cells divide very slowly until an external signal activates them to become transit amplifying cells

7

What are the ways that set cells can be used as a therapy?

- Stem cells are derived from a different donor and are expanded in the laboratory (allogenic)
- The stem cells to be transplanted are derived from the same patient (autologous)
- Recruitment of endogenous stem cells from the same tissue

8

Give an example of stem cells being used is an allogenic therapy?

Embryonic stem cells, cord blood cells

9

Give an example of stem cells being used is an autologous therapy?

Auto-transplant from bone marrow or producing induced pluripotent stem cells

10

What are some uses of stem cells?

- Excellent models to screen for new drugs
- Models to study genetic conditions
- Models for combining the former two (pharmacogenomics)
- Insight into fundamental biological problems

11

Why are developmental biology and stem cell biology closely linked?

However, there is a very complex map of development required to produce correct cell type

12

Why are cancer stem cells important when developing anti-caner therapies?

If drug does not target the cancer stem cell then the cancer will return after tumour shrinks as the stem cells will replenish the cells

13

What is a teratoma?

A tumour formed of all three germ layers

14

How did teratoma help in the understanding of pluripotent?

In the 1960’s, work showed that these complex tumours are produced by a single cell. They took a single cell from a tumour and transplanted into an animal and a tumour produced

15

What is an EC cell?

Embryonic carcinoma cell

16

How did the Brinster lab show that EC cells resembled pluripotent cells in 1970?

- They formed chimeras by taking an EC cell from a dark-skinned mouse strain and implanting it into the blastocyst of an albino mouse. It produces a chimera that has patches of pigmented skin.
- This initial EC cell incorporated into embryo and formed multiple cell types.
- This showed that despite the abnormalities present in the EC cells, they had the capacity to develop into multiple cell types both within a tumour and in an embryo

17

When were embryonic stem cells discovered?

Martin (1981)
- Isolated pluripotent cell line from early mouse embryos which form teratocarcinomas when injected into mice. This embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line.
- This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both.
Evans (1981)
- Established pluripotent cell lines in tissue culture which were isolated directly from in vitro cultures of mouse blastocysts. These cells are able to differentiate either in vitro or after inoculation into a mouse as a tumour in vivo. 

18

Why are embryonic stem cells pluripotent?

Because they have the capacity to form cells of all three germ layers and the germ line

19

Where are embryonic stem cells found?

They come from the inner cell mast of the blastocyst

20

What are the properties of embryonic stem cells?

- They are non-transformed cells (normal) but may acquire abnormalities if passage for a long time
- They are immortal – have an indefinite proliferative potential
- They have a stable diploid karyotype
- Clonogenic – will generate a colony of uniform cells from a single cell. This is more challenging in human stem cells than in mice

21

Why can't human embryonic stem cells be used to produce chimeras or teratomas?

Due to ethical constraints

22

How can you test if a cell population is pluripotent?

An ES cell injected into an adult isogenic host will form a teratoma.
Teratomas are non-invasive but can grow to a substantial size.
They produce different tissues

23

What are feeder cells?

A feeder layer is a carpet of fibroblasts that are put in a tissue culture dish and treated in a way to stop the fibroblasts from dividing. They produce factors which condition the media and produce an environment for the stem cells.

24

What do feeder cells produce that is fundamental in maintaining mice ES cells?

LIF
BMP

25

Outline the LIF pathway

LIF binds to the LIF receptor which interacts with the co-receptor Gp130. This activates the JAK pathway by activating the gp130 bound JAK through transphosphorylation. STAT3 is phosphorylated, inducing homodimerisation. STAT 3 then binds DNA and activates genes controlling self-renewal and pluripotency

26

How is self renew and differentiation maintained by LIF?

- LIF is capable of causing self-renewal and differentiation. There is a very carefully controlled balance that controls self-renewal
- If there is low LIF activation then the ERK pathway is activated which inhibits STAT3 and the cells will differentiate. If there are high amounts of LIF then JAK will be activated and cells will self-renew

27

When is LIF capable of regulating self renew and differentiation of mice ES cells?

In the presence of a serum

28

What is present in the serum that allows LIF to work?

BMPs

29

Give an experiment to show that BMPs are required for LIF action?

- They took ES cells and grow them in N2B27 media. N2b27 is serum free and is usually sued to drive neuronal differentiation. In this media, cells will express beta tubulin 1 and produce neurons. If added LIF, then neurons are still produced and no Oct4 (pluripotency factor) expression – doesn’t prevent differentiation.
- If add GDF6 (like BMPs) or BMP4 then it prevents neuronal differentiation and increases Oct4 expression.
- BMPs are more efficient then the serum but both result in LIF working

30

How do BMP and LIF work together to maintain mice ES cells?

Ying et al, 2003
- BMPs act through SMAD to block neuronal ectoderm differentiation
- At the same time LIF inhibits BMP from inducing mesoderm and endoderm
- If both molecules are present then all differentiation is blocked
- If only BMPs present then LIF does not block mesoderm and endoderm differentiation

31

When and where was the first human embryonic stem cells isolated?

1998
- The first embryonic stem cells were isolated from IVF generated embryos

32

What were the differences seen between mice and human embryonic stem cells?

- There are smaller raised colonies in mice ES whereas humans are nicely defined flat colonies.
- There are some different stem cell markers present. There were some similarities e.g. Stat3, Oct-Sox
- Human ES cells were not dependant on LIF. Instead they needed FGF signalling to remain pluripotent

33

What signalling pathways are required for ES cell renewal in mice?

LIF and BMP

34

What signalling pathways are required for ES cell renewal in humans?

FGF and Activin

35

How does the human epiblast differ from the mouse epiblast?

- Blastocyst stage is very similar between humans and mice
- Human blastocyst from the epiblast very early on where as mice take longer for the epiblast to form an is not a flat structure (tubular structure).
- This stage happens preimplantation in humans but after implantation in mice

36

When do mice ES cells appear the same as human ES cells?

Isolated cells from the later blastocyst stage in mice, ES cell lines look almost the same as human cell lines. Shows that the main difference is in timing of development

37

What is difference between the early and late embryonic stem cells in mice?

A preimplantation blastocyst has ES cells that are dependent on LIF that need GSK3 beta inhibition whereas a post plantation blastocyst is reliant on FGF and Activin signalling

38

What are naive and primed ES cells?

The preimplantation ES cells are known as naïve ES cells whereas post are called primed ES cells

39

Give a study which captured naive human stem cells?

Gafni et al, 2013
- Did a screening for different factors which induces the expression of Oct4 (a pluripotency gene). These factors made it possible to produce naïve human stem cells from ES cell lines
- The promotor that controls Oct4 has a distal and proximal promotor. One part of the promotor will work in naïve and the other in primed ES cells.
- They mutated each promotor and found that when one is missing, the expression of Oct4 is changed. This is a way of testing if the proximal or distal promotor is active. If primed cells then Oct4 is only activated from the proximal promotor as the distal is mutated and vice versa.
- These cells can produce chimeras in mice and incorporate into the tissue proving they are naive

40

Outline some differences between naive and primed stem cells

Naïve
- Mouse embryonic stem cells
- Easier to clone
- Dome shaped colonies
- Use of Oct4 distal enhancer
Primed
- Mouse epiblast cells
- Poor cloning
- Flat colonies
- Oct4 proximal enhancer

41

Why are naïve human pluripotent ES cells important?

- Naïve cells are important for the fundamental understating of pluripotency
- They are easier to genetically modify
- Could produce a chimeric animal using naïve human stem cells and produce an organs transplantation

42

How similar and different are ES cell lines?

Mouse ES cell lines are coming from inbred mice where the genome is pretty much the same whereas human embryos are all different

43

What applications might the differing human ES cell lines have?

Osafune et al, 2008
- There lines that are more capable of producing specific tissues
This is useful if you want cells for a specific thing e.g. insulin producing cells

44

What three signalling pathways does LIF act through?

JAK/STAT3, PI3 kinase/AKT and MAP kinase

45

Give evidence for JAK/STAT activation in maintaining pluripotency

Activation of STAT3 has been found to be sufficient to maintain mouse ESC self-renewal

46

What is the main JAK used in the LIF pathway

JAK1 is the main JAK involved in self renewal: Shown as JAK knockdowns ESCs require a higher concentration of LIF to maintain pluripotency than wild type cells

47

What regulated the JAK/STAT3 pathway

JAK/STAT3 pathway regulated by inhibitory proteins SOCS.

48

Give evidence for Socs role in regulating pluripotency

Socs2 knockdown ESCs retain pluripotency. This is shown by teratoma formation in nude mice

49

How does LIF act on PI3 kinase/AKT pathway?

LIF activates PI3Ks through phosphorylation of p85 which then activates AKT. AKT inhibits GSK3 beta. This in turn increases c-Myc which promote pluripotency

50

How does LIF act on MAPK pathway?

- LIF binding to its receptor activates PI3 kinase which activates Ras. This activates the MAPK pathway and inhibits self renewal
- This induces differentiation of mice ESCs by down regulating Nanog
- This is opposite to the other LIF activated pathways

51

How did Gafni et al, 2013 produce naive human stem cells?

- They identified polypeptide growth factors and small molecule inhibitors to produce naïve human stem cell medium (NHSM). This medium was able to produce human pluripotent stem cells which appeared in a naïve state

52

What defines pluripotency?

Pluripotency is not defined by a single molecule but instead a set of factors that are expressed together. It is the balance of these factors that define pluripotency

53

What pluripotent structures are present in the mouse embryo?

The inner cell mast (naive) and the epiblast (primed)

54

Describe a mouse embryo

- The morula forms the early blastocyst which initially has two cell types: the ectoderm (lineages that produce placenta) and the primitive endoderm
- The visceral endoderm, parietal endoderm and trophoectoderm are extraembryonic tissues and the epiblast is the embryo itself

55

What did Austin smith, 2005 discovery about defining pluripotency?

- There were tare key molecules important in this defining.
- These molecules are permanently fighting each other trying to drive differentiation in a specific way.
- Oct4 tries to drive mesodermal differentiation, Sox2 ectodermal differentiation and Nanog endodermal differentiation
- The balance of these three molecules therefore balance out and stop any differentiation but with the potential to differentiate in any direction if the balance changes – pluripotency

56

What are pluripotent factors?

They are all transcription factors with DNA binding domain

57

What are the three pluripotent factors?

- Nanog has a homeodomain which is like those described in the drosophila
- Oct4 has two DNA binding domains: Pou(Homeodomain) and Pou(Specific)
- Sox2 has the HMG binding domain and is important when interacting with other proteins

58

When was Oct4 discovered?

- Oct4 was defined in the 1990s and is a Pou transcription factor which were initially identified in the C. elegans

59

Give evidence for the role of Oct4

Nichols et al, 1998
- In vivo it has been shown that it is essential in maintaining the inner cell mast and without Oct4, the cells fail to acquire the potential to produce different lineages
- In vitro, ES cells leads to differentiation into trophoectoderm

60

What did Niwa et al, 2000 discover about the role of Oct4 on stem cell fate?

The relative levels of Oct4 influence ES cell fate
- Downregulation of Oct4 results in trophoectoderm whereas upregulation results in mesoderm
- The level of Oct4 expression therefore has to be carefully regulated

61

What did Niwa et al, 2005 discover about how Oct4 works?

- Oct4 does not act alone. The gene that is reciprocal to Oct4 is Cdx2. Cdx2 is not a pluripotency factor
- Oct4 has a repression loop with Cdx2. When Oct4 is downregulated, Cdx2 is upregulated resulting in trophoblast differentiation

62

What is the role of Sox2?

Pluripotency factor
- Has a very important role in the early epiblast as Sox2 deletion embryos fail to produce epiblast. Instead, trophoectoderm forms

63

Give evidence for the role of Sox2

Avillion et al, 2003
- Sox2 deletion, inner cell mast never forms
- In vivo, the epiblast is never formed and there is no Oct4 expression

64

When was Nanog discovered?

- Discovered in the early 2000’s
- Was identified by two labs: Mitsui et al, 2003 and Chambers et al, 2003

65

How did Mitsui et al, 2003 discover nanog?

- Used digital differential display to compare the expression of highly expressed genes in the embryonic cell population and identified a series of potential candidates
- used northern blotting and found that most were expressed in the ES cell population and found that Nanog was only expressed in ES cells suggesting it is involved in pluripotency.
- This was only an association.

66

How did Mitsui et al, 2003 show that nanog caused pluripotent and wasn't just an association?

- To test its role in pluripotency they investigated if it could act independently on LIF. If this gene could override the action of LIF or when LIF Is not present, then it is very important in pluripotency
- Transfected cells with Nanog and another mock gene. When LIf is present the cells did not differentiate. When LIF is not present, the cells with the mock gene differentiated whereas the Nanog cells did not.
- This shows that LIF is not required when Nanog is present

67

How did Chambers et al, 2003 discover nanog?

- Created a library of all the genes expressed by embryonic cells and transfected them into cells which did not have a LIF receptor
- They then looked for colonies that could self-renew despite no LIF activity. They identified an unknown sequence which turned out to be Nanog

68

Give evidence to Nanog being necessary for self renewal

- Without Nanog in vivo, embryos will have no epiblast and will form visceral/parietal endoderm
- Overexpression of Nanog removes the need for LIF
- Knockdown of Nanog in vitro loses ES cells pluripotency

69

Give an overview for the maintenance of pluripotency by the pluripotent factors

- In the early stages, Oct4 and Cdx2 compete between maintaining the inner cell mast and differentiating into the trophoectoderm. Cdx2 expressing cells become the trophoectoderm while the cells maintaining Oct4 become the inner cell mast
- Nanog keeps the cells in the epiblast stage and is counterbalanced by the gene GATA6 which is required to produce the primitive endoderm
- Sox2 is critical for the formation of the epiblastic tissue

70

How do the pluripotency factors work with each other?

- The pluripotency factors all drive expression of different tissues so the balance of them results in no differentiation and the cells remaining in a self-renewing state
- This is achieved by the factors mutually repressing each other. Oct4 drives mesoderm differentiation but represses trophoectoderm formation. Sox2 drives ectoderm but repressed trophoectoderm and mesoderm

71

Outline chambers et al 2007

- They created a cell line with a green and red fluorescent line that report on Nanog and Oct4 respectively and monitored the levels of these using antibodies
- They found that in a colony that has Oct4 expression there is also a valuable expression of green cells. This is very random and not in a pattern
- They separated the cells to see if the cells are different and differentiating or are they in a more intermediate state using FACS
- They separated the highly and low expressing Nanog cells
- After a week, there was a restoration in this and the positive group of cells produced a negative cell
- This shows that the cells haven’t fully differentiated and can return to an undifferentiated state
- They are transiently dynamic

72

Outline FACS sorting

FACS separates cells based on their fluorescence. The machine creates small droplets which contain a single cell and then is fired with a laser beam which will charge the cells to produce positive and negative cells and separates them apart based on their fluorescence

73

What is meant by stem cells being a heterogenous population?

Cells will go to a state that is more stable. A heterogeneous population may have subsets that are more likely to go into one state then the other

74

How genetic abnormalities lead to the discovery of genes involved in self renewal?

- Stem cells can either self-renew, differentiate or die. If cells re unable to differentiate or die then the cells ability to self-renew will be enhanced
- Cells cultured in vitro could develop genetic abnormalities and mutations that favour self-renewal would tend to be selected in culture. This could provide valuable insight into genes that control proliferation and self-renew

75

What did Andrews et al, 2007 discover about the location of proliferation genes?

Andrews et al, 2007
- The mutations that favour self renewal are not random - they do not occur everywhere. They seem to be found in certain chromosomes
- Found in chromosome 1, 12, 17 and X
- Further analysis showed that these chromosomes are often mutated
- Chromosome 4 is rarely mutated suggesting that it doesn’t contain genes that are required for expansion or that it has genes that are very critical for survival so mutation results in cell death

76

What is the problem with different ES cells lines?

- Not all pluripotent cell lines are equal in their capacity to differentiate into desired cell types in vitro. Genetic and epigenetic variations contribute to functional variability between cell lines and heterogeneity within clones
- These genetic variations could lead to a lineage bias

77

How can you force differentiation of stem cells?

Easy to push stem cells to differentiate by removing external signals that maintain pluripotency. Can also force differentiation by forcing the cells to aggregate

78

What are embryoid bodies?

Embryoid bodies can trigger gastrulation and division and form in balls of cells because they can’t attach (aggregates)
Can be used to drive differentiation

79

What did Derk ten Berge et al, 2008 find about the early stages of embryonic development using embryoid bodies?

- Wnt signalling mediates self-organisation and axon formation
- Axin2 gene is activated by Wnt signalling and is reported by LacZ so reports Wnt signalling
- After formation of the epiblast, start seeing Wnt signalling which then moves to the pole of the embryo at the moment of gastrulation - dependent upon Wnt signalling
- Embryoid bodies can have a polarised activity of Wnt based on the axin2 reporter.
- By changing the culture conditions, can modify the polarity using an activator or an inhibitor
- Wnt activity peaks around day4. If put Wnt in the media, segregation occurs at day 1. If add a wnt inhibitor then it is delayed.
- This shows that Intrinsically Wnt can be segregated and distinguish in the embryoid body.

80

What are thee advantages of embryoid bodies?

Cheap to produce
Can generate the three germ layers

81

What are thee disadvantages of embryoid bodies?

- Difficult to control aggregation in a reproducible way
- Number of days before they are collected
- Difficult to produce a specific lineage after the three germ layers but are good to produce mesoderm, ectoderm etc.

82

What can embryoid bodies shape and morphology cause?

The embryoid bodies shape and morphology can correlate to the cell lineage

83

Why are embryiod bodies described as a heterogenous population?

In any population, there will be a random selection of three types of cells
- There are cystic (produces endoderm)
- Bright cavity (most organised and can produce the three germ layers)
- Dark (not as organised but can produce three herm layers)

84

Outline the method to produce embryoid bodies efficiently

Must ensure that the population are Clonogenic (come from one cell)
- Use the hanging drop method where a lid of petri dish contains drops of media which contain one cell per drop. Then turn the dish upside down and the drops
will be hanging down due to the superficial tension. The cell inside of the media is then floating inside the drop and not attached to the surface
- Can use multiwall plates where cells will form an aggregate and not attach. There will be the same number of cells in each well and can then monitor the conditions in each well

85

What is the disadvantage of using the ganging drop method to generate embryoid bodies?

This method is okay for an initial start but not if want a particular cell type as need a given lineage – need directed differentiation

86

What are the important variables in directed differentiation?

- Starting point for directed differentiation could be embryoid bodies or plating cells into monolayers
- Culture them in the presence of growth factors. The type of growth factor, concentration, temporal controls and combination are all important variants when using growth factors to push a specific lineage
- The substrate that the cells are grown on are important when pushing cells in a specific way e.g. collagen or laminin

87

What is the problem with directed differentiation?

Even using best methods, it is unlikely that this will produce a pure sample of the desired lineage. Likely that other cells types will be produced. Therefore, needs to be purified

88

Give three methods to purify cells in directed differentiation?

- FACS – identify markers for intermediate progenitor state for desired lineage or marker of final cell type
- Can use density gradients as different cells have different buoyancies meaning cells will float at different levels. This is not widely used as not as accurate
- Can use a selectable marker. Can create a construct which will have an antibiotic resistance with the loci with the gene of interest. Only cells that have desired genes will survive antibiotic treatment.

89

What is the disadvantage of using antibiotic resistance to purify a sample?

Involves making a transgenic line so will limit what the cells can be used for e.g. can’t be used for treatments

90

How could you use specific growth factors to drive a mesodermal cell fate?

Can use specific growth factors to drive this differentiation e.g. block ectodermal formation by activating Wnt. Once this has occurred can add factors to induce anterior (Activin)/posterior areas (BMPs). Can then select for cell surface markers to enrich for a particular cell type

91

What did Li et al (1998) do?

Generated pure neural precursors from ES cells by lineage selection

92

How did Li et al (1998) generated pure neural precursors from ES cells?

- They inserted LacZ to report on Sox2 and give the cells the ability the survive in the present of G418 (antibiotic)
- Without the antibiotic, 43% of cells have LacZ. When use antibiotic, 90%
- This affected differentiation and resulted in a much purer population of cells

93

How can you further enrich a pure cell population?

Adding mitogens (encourages cell division) and will expand that particular cell type

94

What has directed differentiation been used to treat?

- The first cell therapy application treatment for diabetes
- Had a transplant of pancreatic islets from a donor. This was attempted with adult and foetal. Shows that the principle works

95

What gene expression is required for the generation of pancreatic islet cells from an embryo?

- First must create the patterning of the foregut using Fox2 and Sox17 expression
- Pdx1 specifies the pancreas
- Ngn3 marks the branching of the central pancreatic epithelium and Ins and Glc mark alpha and beta cell production

96

What did D’Amour et al (2006) do?

Used developmental knowledge and directed differentiation to create pancreatic cell line and beta cell production

97

What is C-peptide?

Insulin is produced as a pro-protein and must be cleaved to formed. The part of the insulin that is cleaved off is known as a C-peptide

98

How did D’Amour et al (2006) test if the production of the pancreatic cell line was successful?

- Identified transcripts using qPCR
- Then used western blotting to look for specific proteins: Sox17 marker of the endoderm etc. C-peptide was present showing that insulin cleavage is occurring

99

What was the problem with D’Amour et al (2006)?

This was not a very efficient protocol
- They were not glucose responsiveness – could not detect glucose and respond
- This was a good step forward but the cells were not mature enough

100

What did Kroon et al (2008) do?

- In vitro they produced the islet cells
- They then tested the functionality in vivo by transplanting into the mice and looked for hormone producing cells in the mice

101

How did Kroon et al (2008) test if the production of the pancreatic cell line was successful?

- Showed expression of the markers as well as C-peptide showing that mature insulin was present
- The beta cells have a specific type of granules and microscopy was used to show that these specific granules were present within the mouse
- Also tested functionality to show that the cells were acting in response to glucose. They used controls with adult islet cells. Give glucose to a fasting animal and measure C-peptide 30 minutes and 60 minutes after. If c-peptide increases, insulin is being released in response to glucose

102

How did Kroon et al (2008) show that the mice response was just to the transplanted cells?

They also tested for human insulin in the mice (human transplanted cells) showing it is in response to transplanted cells and not an endogenous response. Larger concentrations of glucose resulted in a larger production of insulin

103

How did Kroon et al (2008) show the effect of pancreatic cells in diabetic mice?

- Induce drugs that damage the endogenous pancreatic cells to produce a diabetic model and then transplant human islet cells
- 30-90 days after transplantation, blood glucose levels are the same as control
- They see that the effect of the cells in vivo. After removing the transplanted cells, the glucose levels increase showing that the transplanting cells were the reason for reduction in diabetic phenotype

104

What was the problem with Kroon et al (2008) diabetic cell replacement?

Problem is that 15% of the grafts developed tumours. Can try and stop this by purifying the cells in a specific way

105

What did Kelly et al, 2011 do?

- Identified a series of cell surface antigen markers are important as can separate the cells using magnetic beads which will have antibodies for these markers and bind the cells. This will purify the sample and reduce tumour formation when transplanted into animal models

106

What surface markers did Kelly et al, 2011 find?

CD142 which marks the pancreatic ectoderm – not fully differentiated but fated to produce desired lineage- and CD200 which is an endocrine marker and cells will be fully differentiated
- These markers are useful by association so it is important to validate them

107

How did Kelly et al, 2011 purify the pancreatic cell line using marker proteins?

- Combined the use of functional markers with these cell surface markers. The functional markers used were NKX6 which is a progenitor marker and CHGA which is a differentiated marker
- They separated the cells and then checked the factions for the expression of the other markers. They want the cells that are not fully differentiated (high in CD142 and NKX6).
- CD142 population contain a lot of NKX6 positive cells meaning they are the progenitor cells. These are the cells that they want to use to transplant

108

What did Kelly et al, 2011 find upon transplantation of purified pancreatic progenitor cells into diabetic mice?

- Non-sorted grafts resulted in 11/24 teratoma formation
- In the CD142 enriched grafts, almost none of them formed Teratomas. However, many of the cells failed to survive due to the rigorous selection process but if they did and were transplanted then they were successful

109

What is the future of 3D differentiation?

Organoids

110

What can organoids be used for?

- To model complex functions
- Many organs have been modelled like this such as the liver and brain (useful when modelling the Zika virus)

111

What is Waddington’s epigenetic landscape model?

- The idea that the way the cell develops is like a boulder rolling down a hill. It moves from a stable state to another stable state and uses the least energy demanding way to do this
- The idea of reprogramming challenges this as you’re asking the cells to go from a stable to an unstable state so to go backwards

112

What is the idea of direct conversion?

Direct conversion is the idea of going from a differentiated state to another differentiated state without going in the pluripotent stage e.g. pancreatic cells can be transformed into phagocytes in some conditions but this idea has not been concentrated on

113

What was the first evidence that cell do not lose genes only repress them?

John Gurdon
- Took cells from the intestine of albino frog and took the nucleus and places it into a cytoplasm of an green frogs egg. This lead to the egg differentiating and forming a complete embryo. This embryo was then placed into an adult green frog which gave birth to an albino offspring, showing that the genetic information came from the intestinal albino cell.

114

What was the first clone /

Wilmut et al, 1997 – Dolly
- Used a cell line from breast tissue of white faced sheep and transferred the nucleus into an oocyte from a black faced sheep. Lead to a white-faced offspring Dolly. Dolly could have offspring of her own. Dolly was a clone of the white-faced shape
- It is the environment in the egg that has allowed this reprogramming

115

What is a therapeutic clone?

Reprogramming patient’s cells for cell replacement therapy. These cells would be genetically identical to the patient and could be sued to treat them

116

What is a reproductive clone?

- If instead take the blastocyst and place into an organism to grow
- Banned everywhere in the world due to the ethical connotations

117

What paper revolutionised IPS cells?

Takahashi K and Yamanaka S (2006)

118

What did Takahashi K and Yamanaka S (2006) aim to find?

Asked how many genes controlled pluripotency, are there a few genes required or just one?

119

What system did Takahashi K and Yamanaka S (2006) use?

Created a new system where he could screen for pluripotency.
- He knew that Fbx-15 gene is highly expressed in ES cells but it is not important in pluripotency meaning it could be manipulated without damaging pluripotency.
- He knocked in Bgeo or G418 (neomycin selection gene) instead of Fbx-15. If this gene is active then neomycin resistance gene will therefore be active.
- Somatic cells can be killed with low levels of neomycin and if turn on this gene (only turned on in pluripotent ES cells) then they become resistant to this and the cells will survive

120

How did Takahashi K and Yamanaka S (2006) use their system to find factors needed to reprogram somatic cells?

- Used fibroblasts and with the G418/Fbx-15 system
- Used viral infection to add 24 genes (hypothesised to be involved in pluripotency) one by one. No cells survived meaning they hadn’t acquired ES cell properties.
- He then added them all together and this resulted in 22 clones and 5 of them looked very like ES cells.
- He then purified this further and found that they expressed pluripotent factors

121

What plutipotency factors did Takahashi K and Yamanaka S (2006) find were required to reporgamme somatic cels?

Oct4, Sox2, C-Myc and Klf4
- Nanog was dispensable

122

How did Takahashi K and Yamanaka S (2006) look to see if the IPS cells were the same as ES cells?

- Microarray analysis to see if the cells expressed the same genes as ES cells. Not quite the same but very similar
- Transplanted these cells into mice and can form Teratomas so can form all three germ layers

123

Did Takahashi K and Yamanaka S (2006) repeat using adult cells?

- Took adult fibroblasts from the adult tail and repeated this. Injected these cells into blastocyst and showed that the animal had these cells in all three germ layers – chimera

124

What was the problem with c-Myc as a pluripotent factor?

c-Myc is an oncogene

125

What factors did the Thomson lab use to reprogram cells?

Thomson lab therefore to remove oncogene C-Myc and use Oct4, Sox2, NANOG and Lin28 instead

126

What are the methods that can be used to reprogram cells?

Somatic cell nuclear transfer
- This is a rapid process that takes about 5 hours
Cell fusion with pluripotent stem cell
- Force the fusion between a somatic cell and the pluripotent stem cell but it can lead to the formation of a cell with twice the number of chromosomes so isn’t very useful for therapeutics
Transcription factors expression
- Slow process and not very efficient
Small molecule exposure
- Slow process
- Uses small molecules to control the epigenetics of genes to indirectly turn on transcription factors
- Often used in conjunction with transcription factors

127

What are the current methods of delivering the pluripotent transcription factor to the cells?

- Deliver the transcription factors by integrating virus.
- Non-integrating vectors such as mRNA or proteins
- Small molecules

128

What are the advantages and disadvantages of using integrating virus to deliver the pluripotent transcription factor to the cells?

The problem with integration of genes is that it can occur randomly anywhere in the genome which could affect gene expression.
We now know that there are regions of the genome which are called safe harbours which are being targeted for the genes to inserted as it won’t affect the function of the cell.
However, integration is always going to be a concern

129

What are the advantages and disadvantages of using non-integrating vectors to deliver the pluripotent transcription factor to the cells?

- Good because only need a high level of transcription factor expression for a short period of time.
- Can be a problem if need to maintain the levels. It can also lead to immune response and is expensive to manufacture.
- However, the advantages of these are that it does not affect the genome of the cells

130

What are the advantages and disadvantages of using small molecules to deliver the pluripotent transcription factor to the cells?

Small molecules can be used which involved no foreign genes and are cheap to make but drugs can affect unintended targets

131

Give an experiment into the functionality of IPS cells?

Hanna et al (2007)
- Treatment of sickle cell anaemia mouse model with iPS cells generated from autologous skin
- Sickle cell anaemia is caused by mutation in the haemoglobin affecting the carriage of oxygen. Allele so common as it provides resistance again malaria
- They used a mouse with human haemoglobin and human sickle cell anaemia mouse model. They reprogrammed skin cells from tip of the tail with the factors to produce IPS cells. They fixed the mutation using homologous recombination to correct the sickle cell anaemia mutation. Then transplanted the cells back into the mouse
- Haemoglobin A is the normal variant. In mutated animals, there is the 100% mutated version. Four weeks after transplantation, there is 60% normal haemoglobin present instead

132

What are the problems with hES cells?

- Genomic instability
- Continual supply of high quality embryos
- Potential for tumour formation
- Questions regarding functional differentiation
- Immune rejection
- Ethical issues

133

Are IPS cells and hES cells the same?

Exactly the same as ES cells but are very similar

134

What are the advantages of IPS cells?

- No requirement for administration of immunosuppressive drugs
- Opportunity to repair genetic defects by homologous recombination
- Opportunity to repeatedly differentiate IPS cells into desired cell type for continued therapy
- Less ethical issues

135

Do IPSC therapies require immunosuppression?

- The process of reprogramming could trigger changes in the antigen profile of cells
- If use ES cells from the same animal it can lead to tumours and If cross species then it leads to invasion of inflammatory cells like Cd4 and Cd3
- When they tested that differentiated IPCs they can still be rejected. This removed the idea IPS cells don’t result in immunosuppression

136

How was direct differentiation investigated?

Invetsgated direct differentiation
- Pushed fibroblasts into functional neuron. Found five factors that resulted in these changes

137

Where did information on adult stem cells come from?

Most information on adult stem cells came from investigations in animal regeneration

138

Can animals regenerate?

- Hydra can be cut into hundreds of pieces and each piece will regenerate into a new organism. Salamanders can regenerate limbs but humans and mammals have a very limited regenerative ability.
- There is a small ability e.g. wound healing, skin replacement.
- Some tissues regenerate very quickly e.g. blood while others are much slower are not at all e.g. muscle

139

Why does the blood need a stem cell?

- They have a limited proliferative ability and short life span
Evert day 10^11 red blood cells are lost and they are constantly being replenished.
- There therefore must be an uncommitted cell in the blood that can replenish all of these cell types and its own pool

140

What was the first evidence for the presence of adult stem cells?

- The research started around the second world war from research in ionising radiation due to the fear of nuclear warfare. Became clear that bone marrow is the most sensitive organ to the effects of ionising radiation.
- Irradiate mice with the ionising radiation and stopped production of blood cells leading to lethality. If, however, take bone borrow from a healthy mouse and transplant into the other mice then the mice would survive

141

What did Till and McCulloch 1961 do?

- Investigated these mice and found that these mice had nodules on their spleens
- Concluded that some of the cells from transplanted bone borrow migrate to the spleen and form colonies
- Think that each colony arose from a single cell as there is a linear relationship between the number of cells injected and the colonies found on the spleen.
- These single cells were termed colony forming units

142

What did Till and McCulloch 1961 find about the cells termed colony forming units?

- Found that the colonies formed by the colony forming units contained differentiated blood cells and new colony forming units. This was shown by taking these colonies from the spleen and transplanted into a second irradiated mice and see if the mice survive. They did survive and had nodules on their spleen
- This evidence shows that these cells can differentiate and self-renew. They are therefore stem cells
- These stem cells were later termed haematopoietic stem cells

143

Outline Haematopoiesis

Haematopoietic stem cells give rise to multipotent progenitors. This further advance into oligopotent progenitors. Examples of these are myeloid progenitors, which give rise to macrophage erythrocyte progenitors and granular macrophage progenitors, and lymphoid progenitors. These progenitors give rise to effector cells

144

What is the difference between a multipoint progenitor and a stem cell

The progenitor proliferative ability is limited whereas stem cells ability is life long

145

What is the difference between embryonic stem cells and haematopoietic stem cells?

Embryonic stem cells are pluripotent as they can give rise to all three germ layers whereas haematopoietic stem cells are multipotent as they can give rise to multiple cell types but these are tissue specific cells and not any cell type

146

What is meant by retroactive classification of stem cells?

To prove a cell is a stem cell, must prove it can differentiate into different lineages and self-renew. If force the cell to differentiate, it is no longer a stem cell. This means that there is always a retroactive classification

147

Why are cell surface markers useful when studying stem cells?

- Cell surface markers are used to generate antibodies to label cells with fluorescence and then use FACS to separate them.
- There is not a single marker that can define a stem cell population – need combination.
- This technique is very useful to understand how cells progress through lineage specification

148

What does the genes expression of cells tell us about stem cells?

Cells will express genes that are required for their function but not genes for other lineages. This tells us this that differentiation of the haematopoietic stem cells must involve upregulation of genes for one fate and suppression of genes for the other fates

149

What are the roles of transcription factors?

- Determine gene expression pathways
- Recruit coactivators and corepressors of transcription
- Often components of multiprotein complexes

150

Give examples of master regulators

Gata1
PU1

151

What must a master regulator do?

- To be called this it must be required for development into a specific lineage
- They also must supress other lineages

152

Give evidence for Gata1 being a master regulator

Must be required for development into a specific lineage
- Gata1 is a transcription factor that appears to be a master regulator for the erythrocyte cell fate from MEP progenitors. If delete gata1 from these progenitors there is no erythrocyte lineage development - it is essential. If introduce gata1 to GMP progenitors which don’t usually produce erythrocytes, the progenitors become MEP progenitors – reprograms the cell
They also must supress other lineages
- Gata1 downregulates myeloid markers and upregulated Erythroid markers

153

What is Transcription factor cross antagonism?

- PU1 binds to DNA and for it to bind and regulate transcription it must bind its coactivator c-Jun through its carboxyl terminal domain
- Gata1 outcompetes c-Jun through its zinc finger domain meaning that PU1 can no longer activate its target genes
- This also works the opposite way around: Gata1 binds to GATA on the DNA and activates target genes. PU1 has a transactivation domain and binds to GATA1 and prevent transcription of target genes

154

What ways can transcription factors act to regulate gene expression?

- Regulators negatively influence each other
- Autoregulation
- Positively and negatively regulate target genes

155

What has the understanding of transcription factor cross antagonism revealed about stem cell gene expression?

- This results in an undifferentiated cell with no net expression of target gene due to TF cross antagonism. An event then occurs that results in the upregulation of one transcription factor leading to outcompeting of the other transcription factor leading to a specific cell type.
- This is surprising because it was thought that stem cells will express stem cell markers and not TF for opposing lineages.

156

What is meant by multi-lineage priming?

Stem cells express transcription factors for various lineages which cross antagonise each other resulting in an undifferentiated cell

157

Why is multi-lineage priming needed?

So that cells can differentiate quickly

158

Give example for the need of multi-lineage priming

Upon a differentiation cue, cells can differentiate quickly into the specific lineages. The haematopoietic system must be able to be replaced upon damage and blood loss the stem cells are therefore ‘primed’ to do this

159

Are transcription factors the drivers of cell lineage?

It is still unknown whether transcription factors are the drivers of differentiation or whether it is stochastic drivers which push cells into a specific fate. It is possible that the transcription factors just respond to external cues but it is unclear how the initial choice is made

160

Give an extracellular cue involved in cell lineage

Cytokines are involved in regulation of cell fate e.g. EPO regulated erythropoiesis

161

What are the theories of how cytokines act on stem cells?

- Instructive role involves a progenitor receiving a signal that leads to the production of a single cell type
- Selective role involves the signal causing the stem cells do whatever the cell intrinsically is supposed to do. The cytokines then select for the cells that are needed and kills the unneeded cells

162

How was the way cytokines act on stem cells investigated?

Mossadegh-Keller et al, 2013
- M-CSF leads to an increase output of myeloid cells but they wanted to know whether it was selective or instructive
- They used PU1-GFP mouse model. PU1 is a marker for myeloid identity
- Time lapse video microscopy showed that cells can go from non-fluorescence to fluorescence suggesting that this cue is instructive. This is because there was no cell death
- This is however in the context of this specific in vitro experiment so may not prove that cytokines act instructively

163

Why is there thought to be stem cells in the hypothalamus?

The idea is that neurons can be generated in adulthood by this progenitor cell population anticipate the changing needs of the body e.g .puberty and pregnancy

164

How did Fu et al, 2017 show the present of a stem cell population in the hypothalamus?

-Targeted the FGF10 expressing cells at the early time point when its just been produced, let the cells develop and then asked what the cells gave rise to
- Found that the basal hypothalamus derives from these cells
- These FGF10 expressing cells could give rise to neurons from the anterior, tuberal and mammillary parts of the hypothalamus. This suggests that this cell population are multipotent
- The marker used for anterior neurons is islet 1 and the mammillary marker is Emx2

165

What are tanycytes?

- These cells have characteristic features: look like radial glial cells, basal extensions and their cell body at the third ventricle.
- Tissue specific lineage tracing showed that these cells are not only proliferative but are also neurogenic in adulthood and give rise to subsets of neurons in the arcuate nucleus.

166

What is meant by the epigenetic landscape?

- Cells don’t just roll down the hill – from pluripotent to differentiated but they stop on the hill in holes – mid points
- Every cell has different energy and the depressions in the hill metaphor represent the fates that are stable so the cell can adopt (attractants) whereas the high ground represents unstable states

167

Give a cell marker for undifferentiated stem cells

SSEA3

168

Are stem cell populations heterogeneous?

It is thought that there are different subsets of stem cells in the compartment which can all interact as they are all stem cells. When they leave the compartment, depending on what subset they are in, they are more likely to differentiate into a different state

169

Give evidence for stem cell populations containing subsets which show differentiation bias?

- SSEA3+ population expressed Oct4 and Nanog - stem cells. GATA6 (a TF which should not be expressed in stem cells but is turned on when differentiated into endoderm) was also expressed in some of these cells.
- To test if based towards endoderm, used GFP-GATA6. There is a small population where both GATA6 and SSEA3 are expressed.
- Created embryo bodies and used PCR to look at gene expression and found endoderm differentiation occurred

170

Can the subsets that the stem cells are in be manipulated?

- They addressed this using a MIXL1 reporter line. These cells could tend to make mesoderm rather than any other cell type
- Used E8 to grow MIXL1 cells – they disappeared. If activate Wnt signalling using GS3beta, the cells differentiate. LPA tends to inhibit differentiation and could produce a state where cells are trapped in the MIXL1 expressing state
- These SSEA3 MIXL1 cells are still stem cells are biased to produce mesoderm
- Balancing Wnt signalling, which drives them out of this compartment with LPA, which drives them back, traps them in this sub-state

171

What did Mossadegh-Keller et al, 2013 investigate?

Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival, expansion and differentiation of lineage-committed progenitors, but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. 
Therefore investigated the effect of cytokine M-CSF on HSC commitment

172

What is M-CSF?

Macrophage colony-stimulating factor (M-CSF, also called CSF1) is a myeloid cytokine released during infection and inflammation

173

How did Mossadegh-Keller et al, 2013 show that lineage-specific cytokines can act directly on HSCs?

- Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1+ cells 
- In Vivo, high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced PU.1-dependent myeloid differentiation
- Therefore, lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity.

174

What are RPE cells?

RPE are a type of supporting cells on the outer layer of the retina. They are linked between tight junctions. They have pigment to keep the eye from reflecting light so it cannot escape

175

What are ganglion cells?

Finally, there is the ganglion cells which are responsible for taking the information through the optic nerve through to the brain

176

What is the fovea?

The fovea is an area of the retina where there is a high density of photoreceptors

177

What is Age related macular degeneration (AMP)?

- This is the leading cause of blindness in the over 55s
- Leads to the degeneration of RPEs and their basement membrane
- Can be dry, degenerative, and wet, caused my neovascularisation of blood vessels which secrete a fluid
- It is polygenic – not caused by a single gene

178

What are the two types of AMP?

Dry
- It has slow progression and can be supplemented with vitamins and antioxidants
Wet
- Can be treated with anti-VEGF to prevent the formation of membranes but it does not always work
- Can also use surgery to graft a new RPE into the macular by rotating pedicled flaps. Does not lead to full vision

179

What is Stargardt’s Disease?

- Genetic and the most common cause of macular disease in children
- Most common is STGD1 mutation which is an autosomal recessive mutation in ABCA4

180

What is Retinitis pigmentosa?

Inherited condition that affects the photoreceptors

181

What kind of cell replacement occurs to treat retinal diseases?

RPE is happening in the clinic whereas photoreceptors are still experimental

182

What did MacLaren et al, 2006 do?

- Used retinal progenitors to transplant photoreceptors precursors to see of the cells are integrated
- They used GFP to measure the success. It was successfully integrated
- Asked if there was a particular time in development that these cells would be better at integrating

183

What did MacLaren et al, 2006 find?

- They found that the best results came from p3-p7 donor retina of animals. This is the time that the cells express the TF Nrl which is when the cells become post mitotic. If the cells are too mature they do not graft, and if not mature enough does not graft. If take cells before E11.5 (before Nrl expression) the cells do survive but it is not as affective. This was tested in a Wt mice
- Then used a mutated mice which were mutated in peripherin 2 and therefore blind. They then grafted the progenitors and differentiated and then measured the activity using the ganglion cells. There was activity showing that it was successful

184

How did Pearson et al, 2012 see if the cell replacement imported visual acuity ?

Asked if the mouse make sense if this information, can they actually see? Therefore, need to use behavioural tests
- Put in a water maze where there was either a blank screen or a screen with strips. Train when see stripes there is a platform to get out of the water. Therefore, need to see the right screen to be able to get out of the water. By chance there would be 50% but if could see would be about 70%
- Wild type mice had above 70% and blind had 50%. However, four out of the nine animals that were transplanted with the progenitors were above 70% and the others were in the middle, suggesting improvement

185

How did Gonzalez-Cordero et al, 2013 use stem cells in cell replacement therapy?

- Developed a method to develop retinas using mice ES cells
- They did they by combining the olfactory receptors with ECM and growing organoids that grow in a way that forms optic cups and the differentiation of photoreceptors
- They then allowed the photoreceptors to differentiate to different levels of maturity and transplanted into mice. Found that between day 26 and day 29 was peak efficiency in vitro and after D34 the cells did not work
- This lab is then looking at using human ES cells

186

Has cell replacement using human ES cells been investigated to treat AMD?

Ophthalmol et al, 2008
- Used foetal cells
- Ten patients with AMD or RP and found that seven improved in visual acuity
- However, there was a limited tissue source

187

Give examples of RPE cell replacement?

Haruta et al, 2004
- They did a functional test by implanting the cells into mice and doing a behavioural assay. After transplantation, the rats were able to follow stripes and the blind could not
Lu et al
- Looked at the long term safety of this transplantation. Ensure tumours did not develop and still working

188

What was the first clinical success of cell replacement therapy to treat AMD?

Schwartz et al, 2012

189

How did Schwartz et al, 2012 result in a treatment for AMD?

- They produced a cell line which can develop into RPE cells and showed in vitro that they have functionality. The cells were incubated with photoreceptors bio particles and fluorescently labelled. When at a low temperature or without particles there is no incorporation of the fluorescence and at high temperature it was incorporated. This is an indication that the cells from the ES cell line are working efficiently
- They then tested in an animal model by transplanting into mice and saw that there was a layer of fluorescently labelled RPE cells nine months after transplantation. There was no tumour formation
- There were two types of RPE cells produced and when transplanted into the back of the eye of patient then there was some functional recovery. Can see incorporation of photoreceptors in the retina

190

Give an experiment that used iPSC cells instead od ES cells in cell replacement

Mandai et al, 2017
- Developed two lines of iPSC cells from patients
- Only one line was used as the other was stopped due to several mutations present in the line. This created concerns
- Cells survived even without immunosuppression and there are no tumours to date. However, the neurovascular membrane (wet AMD) seemed to be forming again. This showed that the underlying problem of AMD was still there

191

What is the London project?

- They used human ES cells and developed a patch where the cells where grown on a membrane which could then be transplanted. If something went wrong then it would be easier to remove the whole membrane to remove the cells – improve safety
- Found that there was a gain in visual acuity and there was a safe follow up after 12 months

192

What are GMP?

- The manufacture of a reagent to be used in a clinical setting demands strict standards. These procedures are called the GMP
- Everything that is used needs to be proven to be pathogen free so not transferred into humans
- The way of writing the research protocol are also much stricter

193

What needs to be considered before a clinical reagent is used?

- Provenance
- Generation of the right cell line
- Purification
- Characterisation
- Quality control

194

What is a stem cell niche?

It is a local microenvironment that hosts and influences the behaviours or characteristics of stem cells

195

What was the fist adult stem cell niche discovered?

Bone marrow

196

How was the role of bone marrow as a stem cell niche first defined?

Schofield 1978 defined the niche as a HSCs need to reside in the bone marrow to retain their infinite potential. They have a defined anatomical location, they regulate self-renewal and removal from the niche results in differentiation

197

Why can we not study adult stem cells in vitro for a long period of time?

We cannot study adult SCs for prolonged periods in vitro but pluripotent stem cells can be kept in vitro indefinitely. Adult stem cells will stop self-renewing in vitro after a few weeks making experimentation difficult

198

What must be researched to allow the study of adult stem cells in vitro?

Comprehending the function of stem cell niches will enable designing improved treatments

199

Outline the drosophila testis stem cell niche

- At the apex of the embryo there is a small cluster of hub cells. These cells are in contact with germ line stem cells (which are also surrounded by somatic (cyst) stem cells).
- When this germ line stem cell divides, the daughter cell stays in contact with the hub cell and the other moves forwards and becomes a gonialblast.
- The somatic cells will exit the cell cycle and enclose the gonialblast

200

Outline the molecular pathways involved in the drosophila testis stem cell niche

- The hub cells in contact with germ line stem cells and cyst stem cells. These cells produce ligands like Dpp and Gbb which activate BMP through downstream Mad which prevents the expression of BAM inhibiting gene expression. This is not sufficient for cyst stem cell renewal.
- Also need JAK/STAT signalling which is activated by Upd which is also secreted by hub cells.
- Upd is also needed for maintenance of cyst stem cells. This is alone is sufficient for cyst stem cells

201

Outline the drosophila ovarian stem cell niche

The cap cells are the niche producing cells which are in contact with germ line stem cells. The stem cells will divide and one cell will remain in contact with Cap cells and the other will differentiate into cystoblast

202

Outline the molecular pathways involved in the drosophila ovarian stem cell niche

The Gbb and Dpp activation is sufficient for maintenance of these stem cells unlike in the male

203

Give an overview of how a stem cell niche results in differentiation and self renewal

Niche cell secretes short range singles that block the differentiation in the cell that is immediately adjacent to the niche cell. The other daughter cell is displaced further away and the short-range signals cannot reach it meaning its differentiation genes are upregulated and becomes a differentiated cell

204

How does one stem cell remain in contact with the hub cell while the other is displaced?

Orient their division
- The centrosome divides and the daughter centrosome will move to the opposite end of the cell
- Mitosis will then take place perpendicular to the hub cell so one daughter cell will be in contact and the other not
- Stem cells anchor to the hub cells through E-Cadherin (both germ line and cyst stem cells. Evidence I that over expression in cyst stem cells caused the out competition of germ line stem cells from the niche

205

How are Cell:cell interactions used in the stem cell niche?

Niches contain stromal support cells as well as stem cells which can interact with each other through: cell surface receptors, gap junctions and soluble factors

206

How are Cell:ECM interactions used in the stem cell niche?

- Cell:ECM interactions are important as they provide an anchor for stem cells to the niche
- They also proved a polarity cue so that cells know how to orient their mitotic spindle and achieve asymmetric division

207

How are Cell:soluble signal interactions used in the stem cell niche?

- Autocrine and paracrine factors
- Can act locally or diffuse throughout the niche
- Studies in flied and worms support that cells secrete factors that are required for maintaining stem cell identify
- Soluble factors include FGFs, BMPs and Wnts

208

What are the two types of bone tissue?

There are two types of bone tissue: cortical bone (dense) and trabecular bone (porous)

209

What separates the bone from the bone marrow?

The endosteum
- The endosteum are lined by bone forming cells (osteoblasts) and from the endosteum

210

What was originally thought to control the hematopoietic stem cell niche?

The endosteum
- Now in dispute due to technological advancements

211

What blood vessels are present in the hematopoietic stem cell niche?

There are different types of blood vessels: arteries which come through the cortical bone and branch into arterioles which transition into venous sinusoids to collect the blood and transport out of the bone marrow. These vessels have different diameters and differences in their basal lamina

212

How are hematopoietic stem cells distributed throughout the bone marrow?

The HCs cells are throughout the bone marrow but cluster around the sinusoids (80%), 10% by arterioles and only a small amount by the endosteum

213

What are the essential components in hematopoietic stem cell niche?

- Ablation experiments showed that essential components are vascular endothelial cells, periventricular stromal cells and megakaryocytes
- These essential components are in proximity or in contact with the HSCs but the accessory components are further away and secrete factors

214

What is CXCL12?

Important growth factor involved in retention in bone barrow expressed by vascular endothelial cells and periventricular stromal cells

215

How does CXCL12 act on hematopoietic stem cells?

Activates CXCR4 receptor which is expressed by HCSs

216

How has research into the stem cell niche resulted in treatment for multiple myeloma?

Autologous cell transplants for multiple myeloma
- For this to occur stem cells must be mobilised by granulocyte colony stimulating factor. The efficiency of this is very low
- Scientists were looking for another way: found a CXCR4 inhibitor AMD3100 which can mobilise stem cells which can be used for stem cell treatments
- This knowledge came from basic knowledge of the stem cell niche

217

How has research into the stem cell niche shed light on myeloproliferative disorders (blood cancers)?

- Research in paper focused on RA receptors (RARgamma) which bind RA
- Made RARgamma knockout mice and showed that there was perturbed haematopoiesis which was associated with myeloproliferative like disease
- Ageing RARgamma null mice have this disease
- Is this due to intrinsic stem cells or a consequence of the niche?
Not intrinsic to stem cells, they were going array due to a problem in the stem cell niche

218

How could targeting the stem cell niche lead to therapies?

- Enhancing the niche function could improve the efficiency of engraftment – allow more niches to stop competition between cells
- Modify the niche to allow different signals to alter the outcome of the stem cell differentiation

219

What is coronary heart disease?

- Coronary heart disease is the most common cause of death globally with WHO estimating 7.6 million/year
- Coronary arteries can form plaques in ulcerated areas. In vivo, the plaques would raise up and form a stenosis (blockage) in the artery. It is therefore difficult to treat and identify because there are still normal areas

220

What are the current treatments for atherosclerosis?

PCI- percutaneous coronary intervention
- This occurs by metal stents pushing plaque out of way to make the artery bigger. This Induces injury and triggers repair

221

What are drug eluting stents?

Metal stents now have drugs on surface, inhibiting cell proliferation. These are known as drug eluting stents. Drug eluting stents have revolutionised PCI. 2 common anti proliferative used in drug eluting stents are Taxol and rifampicin (sirolimus)

222

What is the problem with drug eluting stents as a treatment for atherosclerosis?

- If the metal cages were exposed to blood then no endothelium could grow over the stents resulting in a diminished efficacy.
How can we make sure we get the covering of cells over stent?
- Using pro healing alternative such as endothelial progenitor cells

223

What are Human trophoblast derived endovascular cells (HTEC)?

- Similar to cytotrophoblast cells
- Promote vessel growth
- Used as a treatment for atherosclerosis

224

How were HETCs used as a treatment for atherosclerosis?

Used to drug eluting stents are covered in cells to ensure repair
Incubated the stents in the cell and labelled with indium
- After 1 hour, there's quite a lot of indium left, but a lot falls off over the next few days.
- After 3 days, the cells are forming a nice monolayer. On the bare metal stent you can see gaps and dead cells. Stent coverage is greater with HTEC opposed to bare metal

225

Why can adult stem cells not be kept in culture for a long period of time?

Adult stem cells cannot be kept in culture for a long period of time as they cannot proliferate indefinitely. This is because when cells are cultured they are put in petri dishes which forced the cells to attach in a specific plane with a forced basal, apical polarity. Also, placing on a layer of matrix. It is therefore unsurprising that stem cells do not resemble the phenotypes in vivo

226

Why does 3D culture have an advantage over 2D culture of stem cells?

- Using 3D growth will give a more realistic result as cells can grow in all three dimensions and can spread and migrate. In 2D culture, the plastic dishes have a stiffness which is unlike vivo as the hardest tissue is bone which is still less stiff
- 2D cultures are experimentally quite tractable due to the good analytical tools. These tools are difficult to use in animal models. As the physiological relevance increases, the experimental tractability decreases. Organoids are therefore a good compromise between these two things

227

What is an organoid?

- A 3D structure resembling an organ
- This implies multiple organ specific cell types capable of recapitulating some specific function of the organ and are grouped together and spatially organised like an organ

228

Cells will spatially separate to their correct body plan. What are the two theories for how this occurs?

Differential adhesion hypothesis
- Different cell surface proteins so that cells of the same type will create more binds with each other
Spatially restricted lineage commitment
- The progenitor cells divide but the spatial constraints cause the progenitor cells to be pushed away from signals making them assume a different state

229

Outline the gut structure

Protrusions called villi line the lumen of the intestines and between the villi lie the crypt. The intestinal epithelium has a high turnover with the whole system regenerating in 5 days meaning stem cells are highly active

230

What cell types are present in the intestinal epithelium?

- Enterocytes - absorbance
- Paneth cells – secretes anti-microbial compounds
- Goblet cells – secretion of mucin
- Enteroendocrine cells – secrete various factors

231

What are paneth cells?

Paneth cells are the niche producing cells. The intestinal stem cells are sandwiched between the Paneth cells. They secrete factors which are important when keeping the stem cells in self renewing state

232

What is R-spondin?

R-spondin is also important for maintenance of intestinal stem cell

233

What did Sato et al, 2009 do?

Design a long-term culture of the intestinal system

234

What should long term cultures of intestinal stem cells have?

Long term cultures should have active Wnt signalling, EGF signalling, Noggin expression and overcome anoikis of isolated intestinal cells (commit to apoptosis if become detached – stops cancer)

235

What experimental approach did Sato et al, 2009 do?

Experimental approach
- Isolated of mouse intestinal crypts
- Crypt preparations were suspended in Matrigel – mixture of proteins enriched in laminin
- Optimised culture medium by titration of key signalling molecules

236

Was Sato et al, 2009 successful?

Produced crypt organoid
- Used FACS to isolate cells and found that the single cells can build crypt-villus like structures. These organoids are virtually indistinguishable to in vivo crypts. Contains differentiated cell types and Paneth cells and stem cells are located at the bottom of the crypt
- These gut organoids can be created from single Lgfr5+ stem cells and the organoids show polarisation and differentiation. These cells can be propagated for at least 1.5 years

237

What is cystic fibrosis?

A disease caused by mutations in CFTR which is a channel involved in the regulation of ions. When mutated there is incorrect exchange of ions and mucas builds up in carious organs

238

What potential applications are there of the development of a long term gut organoid?

Used to model cystic fibrosis (Dekkers et al, 2013)
- Common mutation is CFTR F508del
- Used organoids from WT and CF patients to see if gut organoids will allow testing of the response of various treatments
- Forskolin mediated organoid swelling
- In the wild type, the organoids swelling. In homozygous CFTR F508del mutants there was no swelling. Can use this to test different drugs to see if there is swelling in the presence of the drugs
- Found that some drugs did alleviate the symptoms. This allows for personalised medicine as different mutations will respond to different drugs

239

How have brain organoids been used to model disease?

IPS cells were used to generate brain organoids which have been used to generate microcephaly. The zika virus leads to microcephaly

240

What causes the symptoms of diabetes?

Due to vasculature – membrane thickens

241

Give an example of using organoids to study diabetes

Wimmer et al, 2019
- Used IPS cells
- Made small aggregates and induced mesoderm because mesoderm gives rise to the blood
- Specifically promote vascular lineage using BMP3, BEGF and FGF. Plate in matrigel and collagen and allow to culture
- First focus on the key features of diabetic microvasculopathy
- Basement membrane thickening in diabetic vessels
- Can recapitulate this phenotype if expose the organoids to high glucose
- Then used these organoids to test diabetic drugs

242

What are CFU-F cells?

- German pathologist Cohnheim proposed during the mid 19th century that cells involved in injury repair were derived from the bone marrow
- Friedenstein et al described them as adherent, fibroblastic like colonies from monolayer cultures of bone marrow, thymus and spleen. They were named CFU-F (colony forming units fibroblast

243

When were CFU-F cells names mesenchymal stem cells?

The name mesenchymal stem cell was given by Caplan in 1991. However, the identity of the stem cells was demonstrated in the late 1990s when transplants of clonal bone marrow MSCs produced bone in vivo

244

Why is it believed that some cells in the mesenchymal stem cell population should be names mesenchymal stromal cells?

- Found that the adherent cell population isolated from bone marrow are highly heterogeneous and consist of several subpopulations.
- Therefore, not all these cells fulfil the criteria to be considered stem cells as they have unlimited proliferative capacity and the ability to produce multiple lineages
- It has been proposed that they are called mesenchymal stromal cells, reserving the use of mesenchymal stem cells to those that meet the criteria
- However, MSC term is still used for both

245

What are the minimum criteria to define MSCs?

- Remain plastic adherent under standard culture conditions
- Express the markers CD105, CD73 and CD90
- Lack markers, CDC45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR
- Differentiate into osteoblasts, adipocytes and chondrocytes in vitro

246

Where are MSCs produced from?

MSCs in the head and neck area is derived from the neural crest. In the rest of the body, the MSCs are produced by the mesodermal somites with some contribution from the neural crest

247

Where are MSCs located in the adult?

- Initially isolated from the bone marrow
- Now been identified and purified from multiple tissues including adipose tissue, placenta, dental pulp, peripheral blood, synovial membrane, endometrium
- Evidence suggested that MSCs may be present virtually in any vascularised tissues throughout the whole body

248

Why is MSCs being located in the umbilical cord useful?

Stored in banks for years. Also has haematopoietic stem cells. They are not pluripotent like ES cells or IPS cells but can still be very useful for experimental purposes

249

What did Cristan et al, 2008 do?

Identified a perivascular origin for mesenchymal stem cells in multiple human organs - a niche

250

How did Cristan et al, 2008 isolate MSC cells?

Used immunofluorescence to label NG2+ and CD146. They then separated these cells using FACS and selected out the negative makers e.g. expressing CD56 – found in myogenic cells. For a positive selection they used CD146

251

How did Cristan et al, 2008 show that MSC were stem cells but not pluripotent?

- Grew the cells in vitro and found that they were able to expand in vitro and maintain the expression of CD146 and NG2 showing that they are quite stable. Then did functional assay by transplanting these cells into SCID mice (immunosuppressed – good host for cellular therapy)
- Found that the cells differentiated into muscle and bone. Shows that they are not pluripotent otherwise a teratoma will form

252

What are the clinical uses of MSCs?

- Cell replacement
- Healing and repair
- Immunomodulation
- Anti-cancer tools

253

How are MSCs used for cell replacement?

- MSCs have the potential for autologous transplants (personalised) so there is not concern about rejection
- Traditionally obtained from the bone marrow but fat tissue is increasingly becoming a source material

254

What tissue types are treated in cell replacement with MSCs?

- Mainly orthopaedics to replace bone and cartilage
- Can also replace muscle in a cardiac infarction. The data for these tests is inconclusive – unsure that MSCs cells different and graft well into the myocardium

255

What is meant by MSCs trophic and paracrine effect?

- MSCs secrete factors that help healing and repair. They produce cytokines, TGFbeta and prostaglandins, which attracts macrophages to the site of injury. This removes debris and kick-starts the repair response
- It also produces cytokines such as IL-2 which dampen the immune response that T cells and lymphocytes could trigger. These cells can make the injury bigger
- Therefore these two uses of MSCs are interesting in healing and repair research

256

What is the immunomodulation effect of MSCs?

The immunomodulation effect when dampens the immune system which is potentially important in acute injury. However, in chronic inflammation the effect of the MSCs may be negative

257

What allows MSCs to be used as an anti-cancer tool?

MSCs have an inherent tropism to tumour tissue. The nature of this interaction is not fully understood. Seems like the tumour produces enzymes such as SDF-1 that help tumour invasion which seem to target MSCs to the tumour. This could be used to an advantage in developing a therapy

258

What did Nakamizo et al, 2005 do?

Tried to explore MSCs as an anti-tumour agent delivery mechanism

259

How did Nakamizo et al, 2005 show that MSCs were localised to tumours?

- Induced gliomas in mice in the right frontal lobe by xenotransplantation of a U87 xenotransplantation. Then labelled hMSCs in red and injected into the carotid artery
- Within 7 days can see that the cells localised to the tumour
- In vitro tumour cells intermingle with MSCs
- Used other cell lines to ensure that the response wasn’t due to the cell line used. Found the same result
- This is unique to MSCs – not found when fibroblasts are injected

260

How did Nakamizo et al, 2005 investigate how tumour cells were attracting the hMSCs?

- Used trans well culture dish. With a Petri dish inside with a porous membrane. The outside well contained U87 (cancer cell line) conditioned media or control and the hMSCs in the well with the porous membrane
- The looked for the migration capacity of these cells. The percentage of migration
- U87 cancer cell line had a high migration percentage compared to fibroblasts. Medium alone or conditioned medium from fibroblasts did not promote MSC migration

261

How did Nakamizo et al, 2005 explore MSCs as an anti-tumour agent delivery mechanism?

- MSCs were genetically modified with adenovirus to secret IFN-beta
- INF-B triggers apoptosis in tumour cells
- Only the IFN-B secreted by MSC delivered intracranially increased the survival of the animals

262

What is TRAIL?

Tumour necrosis factor related apoptosis inducing ligand
- A cell surface protein which can trigger apoptosis in transformed cells but not in normal cells through the activation of TRAIL receptors

263

How did Loebinger et al, 2009 show that MSCs could be used to treat cancer cells?

- Used hMSCs that had been transduced with TRAIL-GFP antivirus that can be activated by doxycycline (dox)
- In vitro, the MSCs induce apoptosis in tumour cells in response to dox
- In vivo, reduced the growth of subcutaneous tumours. Can control the size of the tumour using TRAIL MSC cells
- Metastasis occurs in animals without TRAIL MSCs but half of these treated were free from tumours

264

What are ethics?

Moral principles that govern a person’s behaviour or the conducting of an activity

265

Why do stem cells present ethical issues now but not previously?

- Have been performing bone marrow transplants for years
- Organ transplantation is relatively uncontroversial
- IVF has been going on since 1978 giving families to many couples (5 million births)
- However, all of these are adult stem cells and do not have the potential to create life. These processes are for direct benefit of patients and not just research. However, embryonic stem cells are pluripotent and are derived from human embryos

266

What is the HFEA?

Licences research or treatment that involves the use of human embryos or the storage of eggs and sperm in the UK

267

Is there an international law on the use of human embryos stem cells?

- There is no overarching international law – no UN or EU law
- Decided by the individual state

268

Why is there believed to be a link between stem cells and ageing?

There is a tissue regeneration decline with age. Stem cells are responsible for tissue regeneration suggesting that ageing phenotypes are due to decline in stem cell numbers

269

Why is the link between ageing and stem cells difficult to prove?

- This theory is difficult to prove as it is hard to isolate some tissue type stem cells due to a lack of markers.
- These markers still do not prove that they are stem cells need to prove functionality
- There is a lack of adequate models

270

What type of stem cell can be easily isolated and used as a model?

Haematopoietic stem cells

271

What effects does ageing have on the blood?

- Decreased immunity
- Anaemia
- Increased evidence of bone marrow failure

272

How to haematopoietic stem cells change with age?

Seen that there is actually an increase in HSC number in the blood but they have skewed differentiation potential – bias to myeloid lineage and results in a decrease in red blood cells and lymph

273

What are the causes of stem cell ageing?

- Telomere shortening
- Energy metabolism and ROS
- DNA damage

274

How do the causes of stem cell ageing act?

They all contribute to cell senescence

275

What are telomeres?

Telomeres are repetitive DNA sequences at the end of chromosomes which maintain chromosomal integrity. They form a physical loop at the telomere

276

How does telomere shortening occur?

These telomeres are shortened with each round of cell division

277

How has telomere shortening been shown to be linked with stem cell ageing?

Young boy had bone marrow transplant from 60-year-old man. After a few years, there was a loss of bone marrow and upon investigation of the donor cells, the telomeres were much shorter than previously found

278

What are Reactive oxygen species?

Oxidative phosphorylation occurs in the mitochondria and the free radicle and reactive molecules derived from this process can damage the mitochondrial and nuclear DNA as well as proteins and lipids

279

What happens to ROS levels with age?

ROS levels increase in HSCs with age

280

Give evidence that ROS levels affect stem cell ageing

- Antioxidants increase the replicative potential of HSCs upon serial transplantation
- Conditional deletion of enzymes that regulate the expression of antioxidant enzymes in mice increased ROS levels and depleted HSCs and stem cells. These genes include FoxO1, FoxO3, FoxO4

281

What is the difference between spontaneous mutations and extrinsic mutations?

Random and spontaneous mutations occur during the cell cycle. Extrinsic mutations occur in response to environmental factors such as UV light

282

Give evidence that DNA damage is linked to ageing

- Despite the cells capability to repair these damages, some damaged DNA can persist and this is likely to be accumulated over time
- If the genes involved in DNA repair are deleted in mice then the DNA mutations persist overtime and the cells undergo premature replicate senescence and show early ageing phenotypes

283

How does DNA damage and telomere shortening lead to cell senescence?

DNA damage and telomere shortening activate tumour suppressor genes

284

Give evidence that senescence increases with age

- Senescence markers such as B-galactosidase increase with ageing in many tissues

285

What are the muscle stem cells?

Sateliite cells

286

What is meant by satellite cells normally being quiescent?

There is very negligible turnover in the muscle meaning satellite cells are usually quiescent. Satellite stem cells will only produce muscle upon damage

287

Outline the satellite stem cell niche

These stem cells are confined to the niche and found by the basal lamina. There is high ECM and the stem cells also receive signals from other cell types such as fibroblasts

288

How does the number of satellite cells change with age?

Aged muscles have reduced regenerative ability and reduced number of satellite cells. This is opposite to what occurs to HSCs

289

How is it been shown that the stem cell niche has a role in ageing?

Previously, it had been showed that the addition of serum from young mice then it can rejuvenate the satellite cell response

290

What did Chakkalakal et al, 2012 do?

Showed that the aged niche disrupts muscle stem cell quiescence

291

What transgenic mice did Chakkalakal et al, 2012 use to see if quiescence is disrupted in ageing?

TetO-H2B-GFP
- TetO is an inducible promotor.
- They did a pulse chase experiment that allows identifying of proliferating from non-proliferating cells.
- Cells will appear green upon activation of promotor. Then turn off promotor so that the cells will divide and the colour will be diluted out with each division.
- Can then find cells that aren’t dividing if the colour signal is as strong as it was to begin with – not been diluted as it is not dividing

292

How did Chakkalakal et al, 2012 show that quiescence is disrupted in ageing?

- Used the Tg mice to look at the population of satellite cells in the muscle based on their ability to retain GFP
- This lead to the identity of two populations, non-label retaining and label retaining (non dividing). The satellite pool is therefore heterogeneous
- They then compared these mice to aged counterparts. There is a more pronounced dilution in the aged cells suggesting that there is more division. They therefore concluded that aged satellite cells spend less time in quiescence

293

How did Chakkalakal et al, 2012 investigate if the loss of quiescence affect satellite cell function?

- In vivo transplantation of satellite cells from adult or aged mice into adult hosts
- Found that aged satellite cells are less able to regenerate muscle
- Aged cells lose the ability to self renew and gain differentiation markers

294

How did Chakkalakal et al, 2012 investigate what is causing the loss of quiescence?

- Investigated FGF family ligands as they are known to affect satellite cell proliferative activity
- Used qPCR and in situ hybridisation and found that there was lower FGF2 and Spry1 (downstream ligand) in aged mice

295

What model did Chakkalakal et al, 2012 come up with about how an ages niche affects quiescence?

Developed a model that said in young muscle there is a low niche FGF2 and high spry1 levels which act to maintain a quiescent state. In aged muscle, high FGF2 from the aged niche downregulates spry1 and contributes to sporadic cycling and cell loss

296

What is meant by tumours being heterogeneous?

- Some have a more differentiated phenotype than others
- Even though abnormal proliferation is a phenotype of a cancer as a whole, the cells within the tumour do not all proliferate

297

What two models can be used to explain the heterogeneity of tumours?

- Stochastic model
- Cancer stem cell model

298

Explain the stochastic model of tumours

- All tumour cells are equipotent
- Some cells proliferate and increase tumour growth while others differentiate. this occurs stochastically

299

Explain the cancer stem cell model of tumours

- Tumours are hierarchically organised like normal tissues and only certain cells contribute to long term tumour growth
- Progenitors have limited growth potential

300

How can we distinguish between the stochastic and cancer stem cell models?

- If the cancer stem cell model is correct then should be able to isolate the cells into populations. Only some of the cells should be able to develop into a tumour
- If the stochastic model is correct, should not be able to sort into populations and all cell as have the same chance of tumour development

301

How did Kleinsmith and Peirce 1964 show that there are cancer stem cells present in embryonal carcinoma?

They found that when one undifferentiated cell from an embryonal carcinoma is transplanted into an immunocompromised mouse a whole teratocarcinoma

302

Give evidence for the presence of cancer stem cells in other cancers

Acute myeloid leukaemia
- Increased number of myeloid cells
- It is possible to isolate cells within the malignant clone and see which cell can reinitiate the tumour upon transplantation in nude mice. Only some cells were capable of this and they are leukemic stem cells (LSCs)
Breast cancer
- Look for cells that initiate the cancer. Found CDC24- cells that were capable of initiating a breast cancer tumour

303

Give evidence that tumours are organised in a hierarchy like normal tissues

- Remove the non-tumour cells from tumour
- Using specific makers, identify different populations within the tumour. Some of the populations will be responsive for tumour proliferation (tumour propagating cells TPC), others will be progenitors and differentated
- They are organised with a hierarchy like normal tissues

304

How can we asses the tumour propagating potential of cells isolated from tumours?

- Inject all of the cells from tumour into a nude mice and will lead to a tumour – baseline
- If can isolate TPC and inject into nude mice it can increase the frequency of tumour formation. So less cells are required for formation of the tumour
- If used committed progenitor cells, upon first transplantation they act like propagating cells. Serial transplantation is therefore required to see the difference between the two as progenitor cells will eventually stop self-renewing and differentiate.
- Look at diagram

305

What is the problem faced with investigating the tumour propagating potential of cancer cells?

- Mice may not tolerate using human cells
- Tumour propagating cells may require a niche – injection may not work
- Tumorigenic potential must be tested under permissive conditions - Some TPCs do not proliferate in some environments but this does not mean that they are not the TPCs – might not have been tried in the permissive environment yet

306

How can cancer stem cells be used as a potential therapy for cancer?

- Therapies usually target proliferating cells. Stem cells can be quiescent though meaning that the tumour will come back as have not eliminated the cause of the cancer
- Instead target the cancer stem cells with the therapy to lose the stem cells and stop relapse. Could also cause the stem cells to differentiate, this would deplete the stem cell pool and remove the original cause

307

How did Al-Hajj et al, 2002 show that only some cells in the breast tumour are capable of tumour imitation

- Grown human breast cancer cells in immunocompromised mice and found that only a minority of breast cancer cells had the ability to form new tumours
- Used cell surface marker expression to distinguish the tumour initiating (tumorigenic) cells from non-tumorigenic cells
- Tumorigenic cells were identified to have the cell surface marker expression CD44+CD24-. Only 100 cells with this phenotype resulted in tumour formation but thousands of cells not in this lineage were unable to form tumours

308

Give a study into using brain organoids to study microcephaly?

Lancaster et al, 2013
- They took skin biopsy from a patient with microscopy and one without. They produced IPS cells and produced cerebral organoid
- In the control, a cerebral organoid was successfully produced and it reassembled a mini brain. They saw this using the markers DCX and SOX2
- In the organoid created from the patient with microcephaly, the ordered structure is lost and there are less cells present. This phenotype is seen in the disease. Therefore, have created a human model of the disease that cannot be done in other models

309

Give a study into using brain organoids to study the Zika virus?

Garcez et al, 2016
- Generated cerebral organoids from human IPS cells and added the zika virus
- Generated a cerebral organoid with the phenotype of microcephaly and cell death

310

Give an example of using organoids to develop a treatment for diseases?

Xu et al, 2016
- They stained the cells in the organoid with CAS3, a marker for dying cells. There were more dying cells in the organoid treated with the zika virus
- They then did a drug screening to find a drug that reversed this phenotype and they found a compound called emricasan that blocked increased cell death due to Zika virus infection