BMS382 Stem Cell Biology Flashcards
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
What is a stem cell?
Cells that have the potential to generate specialised tissue (differentiation) as well as copies of themselves (Self-replication)
How are stem cells classified based on age of development?
Age of development
- Embryonic
- Adult
How are stem cells classified based on tissue of origin?
Tissue of origin
- Neural stem cells
- Hematopoietic
- Umbilical cord etc.
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)
Do stem cells divide quickly or slowly?
Stem cells divide very slowly until an external signal activates them to become transit amplifying cells
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
Give an example of stem cells being used is an allogenic therapy?
Embryonic stem cells, cord blood cells
Give an example of stem cells being used is an autologous therapy?
Auto-transplant from bone marrow or producing induced pluripotent stem cells
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
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
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
What is a teratoma?
A tumour formed of all three germ layers
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
What is an EC cell?
Embryonic carcinoma cell
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
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 fromin vitrocultures of mouse blastocysts. These cells are able to differentiate either in vitro or after inoculation into a mouse as a tumourin vivo.
Why are embryonic stem cells pluripotent?
Because they have the capacity to form cells of all three germ layers and the germ line
Where are embryonic stem cells found?
They come from the inner cell mast of the blastocyst
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
Why can’t human embryonic stem cells be used to produce chimeras or teratomas?
Due to ethical constraints
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
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.
What do feeder cells produce that is fundamental in maintaining mice ES cells?
LIF
BMP
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
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
When is LIF capable of regulating self renew and differentiation of mice ES cells?
In the presence of a serum
What is present in the serum that allows LIF to work?
BMPs
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
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
When and where was the first human embryonic stem cells isolated?
1998
- The first embryonic stem cells were isolated from IVF generated embryos
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
What signalling pathways are required for ES cell renewal in mice?
LIF and BMP
What signalling pathways are required for ES cell renewal in humans?
FGF and Activin
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
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
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
What are naive and primed ES cells?
The preimplantation ES cells are known as naïve ES cells whereas post are called primed ES cells
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
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
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
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
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
What three signalling pathways does LIF act through?
JAK/STAT3, PI3 kinase/AKT and MAP kinase
Give evidence for JAK/STAT activation in maintaining pluripotency
Activation of STAT3 has been found to be sufficient to maintain mouse ESC self-renewal
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
What regulated the JAK/STAT3 pathway
JAK/STAT3 pathway regulated by inhibitory proteins SOCS.
Give evidence for Socs role in regulating pluripotency
Socs2 knockdown ESCs retain pluripotency. This is shown by teratoma formation in nude mice
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
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
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
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
What pluripotent structures are present in the mouse embryo?
The inner cell mast (naive) and the epiblast (primed)
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
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
What are pluripotent factors?
They are all transcription factors with DNA binding domain
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
When was Oct4 discovered?
- Oct4 was defined in the 1990s and is a Pou transcription factor which were initially identified in the C. elegans
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
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
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
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
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
When was Nanog discovered?
- Discovered in the early 2000’s
- Was identified by two labs: Mitsui et al, 2003 and Chambers et al, 2003
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.
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
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
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
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
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
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
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
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
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
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
What is the problem with different ES cells lines?
- Not all pluripotent cell lines are equal in their capacity to differentiate into desired cell typesin 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
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
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
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.
What are thee advantages of embryoid bodies?
Cheap to produce
Can generate the three germ layers
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.
What can embryoid bodies shape and morphology cause?
The embryoid bodies shape and morphology can correlate to the cell lineage
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)
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
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
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
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
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.
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
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
What did Li et al (1998) do?
Generated pure neural precursors from ES cells by lineage selection
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
How can you further enrich a pure cell population?
Adding mitogens (encourages cell division) and will expand that particular cell type
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
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
What did D’Amour et al (2006) do?
Used developmental knowledge and directed differentiation to create pancreatic cell line and beta cell production
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
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
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
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
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
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
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
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
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
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
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
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
What is the future of 3D differentiation?
Organoids
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)
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
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
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.
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
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
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
What paper revolutionised IPS cells?
Takahashi K and Yamanaka S (2006)
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?
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
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
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
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
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
What was the problem with c-Myc as a pluripotent factor?
c-Myc is an oncogene
