8. ES and iPS cells Flashcards
where are ES cells derived from?
inner cell mass of blastocyst
what type of potency are ESC?
pluripotent
what can ESC give rise to?
all embryonic tissue and generate chimeric mice
what type of cells can proliferate indefinitely if kept in undifferentiated conditions?
ESC
what problem did the people that were trying to isolate SC that would not just exist transiently during development come across? and so what was a lot of effort put into?
- cells in culture started to differentiate
- a lot of effort was put into figuring out how to keep them pluripotent in culture
when did the first paper that was able to culture pluripotent mouse cells get published?
1981
what did this paper that managed to culture pluripotent mouse cells show?
> if you took cells from the inner cell mass of blastocysts, you could culture these for some time, and if injected into mice can form teratocarcinomas
they also showed that if you embryoid bodies could be formed from these cells are but back into blastocysts to generate mice
what are teratocarcinomas?
tumours that contain all three germ layers
in order to keep cells pluripotent in culture, cells were kept on what for a long time before better methods were established? and what is done now?
feeder cells
> now we know specific factors what can just be added to the stem cell media
what were ES cells initially called?
embryonic carcinoma cells
what did the next paper in 1982 establish?
the gold standard assays for pluripotent cells
> so that you could prove that the cells you have are really ES cells
what are the three golden assays required to show pluripotency?
in vitro differentiation (multiple lineages)
teratoma formation
chimeric mice
how are chimeric mice formed and describe their tissues?
> this is when the blastocyst of one mouse is injected with ESC of another mouse to form a chimeric mouse
every single tissue in the chimeric mouse is a mixture of the two mice it originated from, this is very evident in the skin and hair follicles
when were the first human ESC isolated and by who? and what did he show?
1998
James Thompson
he showed that they could differentiate into multiple different lineages in vitro
what did the discovery of ESC allow?
genetic modification and the formation of transgenic mice
> it was not possible to do this up until this point
what was done for the first time in 1989?
homologous recombination was used for gene targeting in ES cells
what was the nobel prize in 2007 for?
principle for introducing specific gene modifications in mice by the use of embryonic stem cells
if we understand more about why we are able to culture ESC for long period of time, what may this allow us to do?
be able to culture somatic stem cells in culture for longer
not all ESC are the same, what does this mean that we need to do?
use multiple ESC derived from different places (e.g. different animals) in order to validate out findings, and confirm findings are universal to all ESC and not species specific
what do feeder cells/culture serum provide to mice ESC? and what does it do?
Lif
this is a growth factor that maintains self-renewal
what happens when Lif is withdraw from the culture medium?
ESC start to differentiate
what does activation by LIF stimulate in cells? and what is this important for?
the activation of STAT3
this is very important to maintain self-renewal
what can we give to cells instead of LIF and why is this beneficial?
a chemical mimic instead of the entire proteins
> if we want to be able to maintain these cells in culture for regernate medicine purposes, giving them proteins derived from other cells risks cells contamination
> chemicals can be produced in a reactor with no potential contamination
name three TFs that are important in ESCs, and what is known about these in vivo?
- oct3/4
- nanog
- stat3
> these antagonise each other to mediate the progression of embryonic development
what does the blastocysts develop from?
the morula
what are the two components of the blastocyst?
trophectoderm and ICM
what is the trophectoderm?
the external part of the embryo which gives rise to the extra-embryonic tissues
what role Oct3/4 play in early development?
it promotes the development of the inner cell mass and antagonises the development of trophectoderm
what is very important to add to ESC in culture and why?
Oct3/4 as it blocks the formation of trophectoderm
what is the function of nanog in early development?
Nanog mediates the transition of ICM to epiblast and blocks the transition in primitive endoderm
what does nanog do in culture?
promotes self-renewal and blocks differentiation
when was John Gurdon’s paper published?
1962
what type of experiments did John Gurdon conduct? and what was he able to generate from this experiment?
nuclear transfer experiments
blastocysts and entire frog
what did John Gurdon notice that made the process more efficient?
if the nucleus was taken from the blastula as a pose to the developed tadpole
>the earlier in development the nucleus is harvested, the easier it was to drive it back to form blastula
what was john Gurdon able to obtain from his nuclear transfer experiments?
ESC
what was made possible by John Gurdon’s findings?
generating patients specific ESCs to model disease in a lab
what is the second way to generate ES like cells from adult cells? and what is generated from these experiments?
cell fusion
ESC-like tetraploids
how cell fusion used to generate ESC-like cells? and why was this useful?
fuse ESC with somatic cell
> this reduced the number of ESC lines that had to be used
why are nuclear transfer experiments and cells fusion no longer required?
now we can generate iPSCs
what does being able to generate iPSC allow us to do?
- it is possible to study patient specific tissues and test possible therapeutics
- CRISPR can be used to correct gene defects, these cells can be used to form tissue to put back into patients
in terms of agriculture, what did the making ESC from somatic cells allow?
the cloning of animals
> this is used a lot in agriculture
what paper was published in 2006?
Yamanaka’s paper on iPSCs from mouse embryonic fibroblast cultures by defined factors
>this paper was trying to develop a strategy that would start with somatic cells and revert them back to pluripotent cells
what reported system did the 2006 paper use?
they used a reporter system based on fbx15
what was expressed at this reporter locus in the 2006 paper?
beta-galactosidase
why was this locus picked by the 2006 paper?
it is expressed by pluripotent cells but is not super necessary for survival
what somatic cells did the 2006 paper start with?
mouse embryonic fibroblasts
what was the first thing the 2006 paper did?
selected 24 gene and put them into a cDNA library to transform cells
when the 2006 paper assayed their cells what did they do?
they cultured cells transformed with these 24 factors and waited for the generation of colonies that were beta-galactosidase positive i.e. an indication that they might be pluripotent
what was the first thing the 2006 paper did to prove beta-galactosidase positive cells might be pluripotent? and what was seen?
they compared these beta-galactosidase cells with ES cells and the cells of origin (MEF)
> cells treated with the 24 factors looked very similar to the gold standard ES cells
what did the 2006 paper then try and determine?
how many factors it required to induce beta-galactosidase positive cells
how the 2006 paper determine how many factors were required to induce pluripotency? and factors did this method narrow down to?
> they made libraries of 23 factors
if a colony is not obtained then you know that factor is important in establishing pluripotency
Oct3/4, c-Myc, Sox2 and Klf4
when the 2006 narrowed these factors down to three and two factors what was seen?
> minimal positive colonies were formed
>colonies were not formed
how did the 2006 paper prove they had generated iPSCs? (6)
- they looked at expression of TFs known to be expressed in ESC and not MEF
- these look at histone modifications for these genes and compared them to ESC and MEF
- they looked at DNA methylation
- they compared transcriptomes to ESC and MEF
- differentiation assay
- ability to generate chimeric mice
what was seen in the histone modifications of the transcription factors known to be expressed in ESC and not MEF? (2006 paper)
they looked similar to that of ESC
what was seen in the methylation of these genes? and what was interesting about this? (2006 paper)
> iPSCs methylation was somewhere inbetween ESC and MEF for Oct3/4 and nanog
the one gene where the methylation states were the same of ESC was that of Fdx15 (the reporter)
describe how the transcriptome of these iPSC faired against ESCs? (2006 paper)
the ES and the iPSC are a lot more similar than the MEF are to either
what was the result of the differentiation assay? (2006 paper)
they were able to produce cells of many different lineages in vivo
what was the limitation of this 2006 paper and why do people think they did this?
they only looked at the ability to form chimeric mice in terms of the ability to form a chimeric embryo
> it is suspected that they did this because they wanted their findings published first
when was the second paper that showed how to make iPSC published and how was it in relation to the first paper?
later that year
it was better - they used a different reporter and showed they were able to generate an adult chimeric mouse
what reporter did the second paper use and why?
Oct4
>its a gene that was known to be very important for these pluripotent cells
what did the second paper show about their reporters genes methylation? and what did this show?
little methylation in iPSC and ESC
high methylation in MEF
> this showed that these iPSCs were more ESC like than the ones obtained by screening with fbx15 expression
by choosing two difference reporter systems, how did the two papers achieve different levels of pluripotency?
> fbx15 is expressed by pluripotent cells but is not crucial for their existence
Oct4 is a critical factor for pluripotency
how are these four factors delivered to cells and why has this been chosen?
adenovirus
> factors are not introduced into the genome as this would lead to potential mutations
once the four factors have been introduced to cells, what happens after time?
> exogenous factors are silenced
>endogenous factors are re-activated and maintained
what increases generation of iPSC efficiency?
hand picking of colonies half way through the generation of iPSC increases efficiency of iPSC generation
> select for good looking colonies which makes harvest more efficient at the end
how many days does iPSC reprogramming take and why is this?
> several days
> this is because extensive chromatin modifications have to take place
what can be added to cells to increase the efficiency of reprogramming?
HDAC inhibitors
what is special bout NSC and reprogramming?
> they have more Sox2 and c-Myc than most other somatic cells
can be reprogrammed using only Oct4 and Klf4
when a somatic cells is obtained from iPSC, what can be done with this?
it can be reprogrammed again and again across many generations of mice
stem cells are easier to reprogram than differentiated cells, give an example of a very differentiated cell that is possible to reprogram
B cells are super differentiated cells
mice that are generated from iPSC are more susceptible to what, and why might be this the case?
tumours
> if the four factors are introduce by a retrovirus, this will integrate randomly into the cells genome and this may lead to mutations - factors may integrate into a specific loci
what type of genes are these frequency abnormalities between iPSC and ESC?
imprinted genes
what three types of factors are there still differences between iPSCs and ESCs?
chromatin, mRNA and miRNA
how do we determine what is a ‘good’ iPSC?
they generate chimeric mice
> we can also look at their epigenome to predict whether they will be able to make mice
describe the efficiency of the iPSC generation process
it is very inefficient
>need to start with a very high number of somatic cells and only very few of them will reprogram into iPSC, lots of time we don’t get colonies and the cells simply die
what is Transdifferentiation? and what is most likely to be happening when we reprogram cells?
when a cell becomes another cell without going through a less differentiated state
>the cells goes through a slightly less differentiated state before coming down into another ‘valley’
another paper was published to help us understand why the iPSC reprogramming process is so inefficient, what did it show?
once the four factors are put into the somatic cells, there are lots of possible events that can occur, which are all equally as likely to happen.
give an example of a stage in the reprogramming process where the somatic cells will it become an iPSC
when endogenous sox2 expression is high
what is the most prominent cause of blindness in the world? and how is it treated?
retinal disease
> it is difficult to treat. the disease mechanisms are poorly understood and so there are limited drugs available
what can be used to model disease and test therapeutics?
iPSCs
when was the first clinical application of iPSCs and what was it for?
2013
>a Japanese woman in her 70s, suffering from macular degeneration, was the first person to receive tissue (retinal pigment) derived from her own iPSCs
