Trends in stem cell biology paper discussions
Lecture 2. Epigenetics and pluripotency in mouse embryonic stem cells.
Prolonged Mek1/2 suppression impairs the developmental potential of
embryonic stem cells. Choi et al., Nature. 2017 548(7666)
Common theme: how can pluripotency be tested; how can pluripotency be captured in
vitro and how does it compare to in vivo?
→ What do we actually need to keep cell pluripotent in vitro (dish)?
What could be the reason for all these differences in this traditional medium which
people have been using the last 20 years. These substances which people add to the
medium directly talks to the pluripotency network and stabilize this pluripotency
network.
→ but it does not stop the reason why these cells want to differentiate. They want to
differentiate mainly because these cells themselves excrete FGF4 and induce amoxicillin
→ and therefore want to differentiate.
LIF + BMP4 they overwrite FGF signalling and stop differentiation in vivo. (But the
differentiation cues are still there). → one of the main problems. They want to
differentiate but they cannot because other substances in the media stopping them for
differentiation and remain pluripotent.
Another research: don’t want to overwrite the signals but just stop differentiation. So,
he blocked FGF4 signalling pathway with the inhibitor. By blocking phosphorylation
Erk.
→ also, here direct interaction with pluripotency network via nucleus.
Okay, we now don’t use serum anymore. That good.
These cells are one of the two very unique stages where there is almost no DNA
methylation!!! In ES cells, when they cultured them in serum, DNA methylation was
actually very high. So, they do not look at inner cell mass is we culture them in serum.
2i ESCs are much closer to early embryo than classical serum ESCs in term of DNA
methylation.
→ naïve state pluripotency.
Reduced DNA methylation in 2i medium. (2i = 2 inhibitors)
,Paper is about the concerns. DNA methylation is important for DNA integrity. =
gnomically instable without. Another problem is imprinting phenomenon where a gene
in the early embryo is only expressed from the maternal allele or the paternal allele
(normally it is equally expressed). It has been shown that imprinting is regulated by
DNA methylation.
→ DNA methylation is key for
imprinting → imprinting is key
for pluripotency.
→ 2i ESCs lack DNAN
methylation.
→ How stable are 2i ESCs?
They isolated inner cell mass cells. Culture in traditional medium (serum + Lif) vs new
serum (2i + Lif).
1. Looked at DNA methylation → imprinting region.
DNA methylation levels should be 0.5 (1 allele methylated, the other allele not)
→ Indeed, in serum mostly 0.5, in 2i down to 0 (-> methylation lost,
higher, biallelic expression)
→ Does loss of imprinting affect pluripotency?
- At the end you can look at the colour of the coat and then look what
was the contribution of my ES cells. If it is black = only receiver cells. If it
is brown = whole mouse is derived from the ES cells. Or in between.
→ 2i cells lost their capacity of pluripotency and do not contribute to the
development anymore after a while.
, → 2i ESCs are unstable and loose pluripotency
Can this be fixed?
We have two inhibitors which he adds to the medium. Adding Chir is doing not much so
is safe to use (will not influence imprinting). Inhibitor PD of the medium does causes
defective DNA methylation of the imprinting region.
Lecture 3. Epigenetics and pluripotency in mouse embryonic stem cells.
Main technique they use is ATAC-seq for open chromatin regulatory regions. We know
genes are expressed under certain control promotor. But also, enhancers and
repressors can enhance or repress gene expression.
→ ATAC-seq. → assay for chromatin accessibility → identify open chromatin
regions → TF motif prediction.
If the chromatin regions are open, the transposase can get near the DNA and cut the
DNA. TN5 can cut the open chromatin regions (peaks in results). And amplify open
chromatin regions. TF motif prediction can be made.
Pioneer transcription factor = the first TF to be used. They are able to open the
chromatin and recruit other factors to bind to the DNA.
In the paper they also used reporter assays: check whether the cells are at certain state.
You introduce jamanaka factors into the cells. Check how the chromatin has changed
and how gene expression has changed. What was the mechanism. DNA methylation was
not important in this process.
→ they use FACS sorting to sort cells
based on the marker gene expression.
Therefore, they can separate potential
pluripotent stem cells and cells that are
failed in reprogramming during the
complete process.
Lecture 2. Epigenetics and pluripotency in mouse embryonic stem cells.
Prolonged Mek1/2 suppression impairs the developmental potential of
embryonic stem cells. Choi et al., Nature. 2017 548(7666)
Common theme: how can pluripotency be tested; how can pluripotency be captured in
vitro and how does it compare to in vivo?
→ What do we actually need to keep cell pluripotent in vitro (dish)?
What could be the reason for all these differences in this traditional medium which
people have been using the last 20 years. These substances which people add to the
medium directly talks to the pluripotency network and stabilize this pluripotency
network.
→ but it does not stop the reason why these cells want to differentiate. They want to
differentiate mainly because these cells themselves excrete FGF4 and induce amoxicillin
→ and therefore want to differentiate.
LIF + BMP4 they overwrite FGF signalling and stop differentiation in vivo. (But the
differentiation cues are still there). → one of the main problems. They want to
differentiate but they cannot because other substances in the media stopping them for
differentiation and remain pluripotent.
Another research: don’t want to overwrite the signals but just stop differentiation. So,
he blocked FGF4 signalling pathway with the inhibitor. By blocking phosphorylation
Erk.
→ also, here direct interaction with pluripotency network via nucleus.
Okay, we now don’t use serum anymore. That good.
These cells are one of the two very unique stages where there is almost no DNA
methylation!!! In ES cells, when they cultured them in serum, DNA methylation was
actually very high. So, they do not look at inner cell mass is we culture them in serum.
2i ESCs are much closer to early embryo than classical serum ESCs in term of DNA
methylation.
→ naïve state pluripotency.
Reduced DNA methylation in 2i medium. (2i = 2 inhibitors)
,Paper is about the concerns. DNA methylation is important for DNA integrity. =
gnomically instable without. Another problem is imprinting phenomenon where a gene
in the early embryo is only expressed from the maternal allele or the paternal allele
(normally it is equally expressed). It has been shown that imprinting is regulated by
DNA methylation.
→ DNA methylation is key for
imprinting → imprinting is key
for pluripotency.
→ 2i ESCs lack DNAN
methylation.
→ How stable are 2i ESCs?
They isolated inner cell mass cells. Culture in traditional medium (serum + Lif) vs new
serum (2i + Lif).
1. Looked at DNA methylation → imprinting region.
DNA methylation levels should be 0.5 (1 allele methylated, the other allele not)
→ Indeed, in serum mostly 0.5, in 2i down to 0 (-> methylation lost,
higher, biallelic expression)
→ Does loss of imprinting affect pluripotency?
- At the end you can look at the colour of the coat and then look what
was the contribution of my ES cells. If it is black = only receiver cells. If it
is brown = whole mouse is derived from the ES cells. Or in between.
→ 2i cells lost their capacity of pluripotency and do not contribute to the
development anymore after a while.
, → 2i ESCs are unstable and loose pluripotency
Can this be fixed?
We have two inhibitors which he adds to the medium. Adding Chir is doing not much so
is safe to use (will not influence imprinting). Inhibitor PD of the medium does causes
defective DNA methylation of the imprinting region.
Lecture 3. Epigenetics and pluripotency in mouse embryonic stem cells.
Main technique they use is ATAC-seq for open chromatin regulatory regions. We know
genes are expressed under certain control promotor. But also, enhancers and
repressors can enhance or repress gene expression.
→ ATAC-seq. → assay for chromatin accessibility → identify open chromatin
regions → TF motif prediction.
If the chromatin regions are open, the transposase can get near the DNA and cut the
DNA. TN5 can cut the open chromatin regions (peaks in results). And amplify open
chromatin regions. TF motif prediction can be made.
Pioneer transcription factor = the first TF to be used. They are able to open the
chromatin and recruit other factors to bind to the DNA.
In the paper they also used reporter assays: check whether the cells are at certain state.
You introduce jamanaka factors into the cells. Check how the chromatin has changed
and how gene expression has changed. What was the mechanism. DNA methylation was
not important in this process.
→ they use FACS sorting to sort cells
based on the marker gene expression.
Therefore, they can separate potential
pluripotent stem cells and cells that are
failed in reprogramming during the
complete process.
