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Summary Trends In Stem Cell Biology NWI-BM073
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Samenvatting Trends in StemCell Biology
Contents
Epigenetics and pluripotency in mouse embryonic stem cells................................................................2
Key questions paper 1:......................................................................................................................6
Reprogramming to induced pluripotency...............................................................................................9
Key questions paper 2:....................................................................................................................13
Hematopoeitic stem cells.....................................................................................................................16
Key questions paper 3:....................................................................................................................21
Stem cells in neurobiology....................................................................................................................23
Key questions paper 4:....................................................................................................................30
Epidermal stem cells in treatment........................................................................................................33
Key questions paper 5:....................................................................................................................39
,Epigenetics and pluripotency in mouse embryonic stem
cells
What are stem cells? Can differentiate into other cells and can divide to produce more of the same
type of stem cells (cell renewal). 2 broad types of stem cells:
- Embryonic stem cells, which are isolated from the inner cell mass of blastocysts. They are
pluripotent and can grow an entire new organism.
- Adult stem cells, later in development which can still divide in every kind of kidney cell for
instance but are not pluripotent cannot form entire new organism.
There are different forms of cell potency during development. Totipotency is at the basis of the whole
cascade can be from trophectoderm/trophoblast and inner cell mass. The trophectoderm cannot
form the embryo anymore but forms the placenta..
During formation of an organism cells lose potency and can become pluripotent: cannot form
placenta anymore but can still differentiate into all the germ layers. After that they become
multipotent, oligopotent or unipotent. Pluripotent cells are the most important for research.
DNA does not change the stem cells, pluripotency / toti potency is not connected to DNA or genome
changes. They are influenced by epigenetics (environment determines behaviour).
The pluripotent state is a rather dynamic state. Pluripotent cells are most important for research (are
at the hill of epigenetics). Pluripotent state from which ecto-, endo- and mesoderm can form. There is
some early and late pluripotent states in which the cells develop towards a lineage. ESC from
blastocyst embryo and grown in plate = early pluripotent state cannot become placenta anymore
but can become epiblasts these can become Ecto- endo and mesoderm.
,You can also culture cells from a little bit later time such as epiblast =late pluripotent state these
could also not form placenta anymore but could become ecto- endo and mesoderm.
Why do we call these cells pluripotency? Capability to differentiate into ecto-, endo- and mesoderm
and be able to do self-renewal. How can you test / prove this?
- Grow a mouse out of the cell: Inject cell into embryo repopulating inner cell mass
mouse grows out of this. Not allowed in humans.
- Inject teratomas (cancer tumours) under skin of mouse show that they can form
endoderm, mesoderm and ectoderm. Could potentially do this with human cells inside
mouse.
- Also just in a dish with grow factors and looking at the cells if they grow into different
lineages.
- Gene markers. (however not absolute proof) (Oct4, Nanog, Sox2, SSEA, etc) YAMAKA factors.
Conclusion: Embryonic stem cells, like some other in vitro culture cells, are pluripotent.
Application of embryonic stem cells (ES) / pluripotent stem cells: ES cells are the only ones that will
form the complete body! You can use them in any quantity and can grow them into any type you
want. This is not the case for adult stem cells.
ES cells / pluripotent cells can be used for:
- Regenerative medicine (they can grow into large quantities and differentiate)
- Generation of KO mouse
- Disease model (because differentiate into desired specialisation)
- Cytotoxicity tests (especially during pregnancy for the foetus)
ES cells for therapies: replace lost cells during disease. Dangers: graft rejection (not when the stem
cells comes from the patient itself), graft versus host, teratocarcinoma. ES cells are very useful for
regenerative medicine and to study embryonic development.
Molecular mechanisms to maintain pluripotency:
You need to stabilize pluripotent stem cells in a dish otherwise they will differentiate. This can be
done with 2 different things:
- Serum + feeders
- Serum + LIF
- 2i (+LIF) = 2 kinase inhibitors
The pluripotency network is stable and acts to self-induce its own expression and other pluripotency
genes by binding the promoter. SOX2, Oct4 and Nanog are highly expressed in pluripotent stem cells.
These make sure the cells stay in their pluripotent state by 1. Regulating themselves, binding to its
,own promotor inducing itself. 2. promoting expression of the other two. 3. Performing feedback loop
within them. This network makes sure that self-induce own expression happens. They also repress
the differentiation linked genes. Few master regulators that can control the cell type from
differentiating. Cells can only differentiate when this network is inhibited.
Master regulators: are also associated as enhancers. They regulate everything such as expression of
different cell types as promotors but also as enhancers. If you modulate levels of these proteins you
change differentiating.
STAT3 is signalled by adding LIF and the main active component in serum is BMP4 which signals
SMAD 1/5/8. these directly talk to the pluripotency network. If you don’t add this the cells will
differentiate. If you add this cross talk to master regulaters OCT4, SOX2 and Nanog to not
differentiate and maintain pluripotency.
DNA is packaged in histones. Histomodifications are important. These chains of histones can be
modified. These are also critical for embryonic development. ES cells have specific histomodicications
contributing to the pluripotency. Embryonic stem cells have both the histomodifications for
enhancing and repression / silencing. The ES cells can very quickly turn on or off these modifications
depending in which cells they differentiate. So turn and turn off genes depending on which cells they
differentiate into.
These heterochromatin structures are still quite loose in ES stem cells. In differentiated cells they are
more fixed and dense. is a hypothesis. Could be needed for cells to differentiate.
paper
How do you culture and maintain pluripotency in ES cells?
LIF and serum with STAT3 from LIF and Bmp4 from serum cross talks to pluripotency network. Does
not affect self-renewal but overrides the FGF4 signalling. So they want to differentiate but they cant.
Instead of serum and LIF the 2 kinase inhibitors were used (2i and LIF).
2i does top the wanting of the cells to differentiate by inhibiting the FGF4 signalling pathway.
Cells grown in 2i they are demethylated, while cells grown in serum they are more methylated so
2i cells might be good for multiple reasons. However, DNA methylation is important for genome
integrity for genomic imprinting. about 100 genes that are expressed from only the paternal or
maternal alleles essential for embryonic development. If this does not happen the embryo dies.
Paper asks:
2i induces demethylation of the genome: what is the effect?
Method:
,Take blastocyst grow inner cell mass to ES stem cells and maintain them pluripotent (on serum+
LIF or 2i + Lif) split them into two kind of media. look at DNA methylation, gene expression,
H2AX deposition, developmental potential and genomic stability.
DNA methylation should be 0,5 (half mother half father allele). 0 methylation = both alleles are
active, 100 methylation = both alleles are inactive. In 2i culture cells the methylation is lost (almost 0).
meaning whole imprinting mechanism is lost imprinting is essential for embryonic
development.
Assay this:
- transfer cells into blastocyst of mouse do they grow into mouse? Difference between 2i
serum or without 2i serum.
- transfer cells into blastocyst of mouse Look at coat color in chimera. If the color is
changed the injected stem cells contribute a lot to the final mouse, if there is not enough
change in color the stem cells did not contribute to the final mouse and probably died.
Difference between 2i serum or without 2i serum.
- Teratomas using 2i ESCs and normal ESCs some 2i lines could form teratomas but not very
efficient.
- Long time culture in 2i results in genomic abnormalities that can be shown in a karyotype.
2i ESCs are unstable and loose pluripotency. Can this be fixed?
They found that PD causes defective imprinting PD inhibits MAPK and causes demethylation. So
another kinase inhibitor that does not cause demethylation should be used.
A2i ESCs using an Src inhibitor maintains imprints.
However, clear differences between humans and mouse.
Questions from the lecture + answers:
1. Why do researchers like to culture mESCs in media containing 2i+ LIF?
To prevent the cells from differentiating and keep them in their pluripotent state. LIF and serum with
STAT3 from LIF and Bmp4 from serum cross talks to pluripotency network. Does not affect self-
renewal but overrides the FGF4 signalling. So they want to differentiate but they cant.
Instead of serum and LIF the 2 kinase inhibitors were used (2i and LIF).
2i does top the wanting of the cells to differentiate by inhibiting the FGF4 signalling pathway.
2. What is imprinting and why is it important?
, Imprinting: inactivating either the allele from the maternal or the paternal side by methylating the
DNA. DNA methylation should be 0,5 (half mother half father allele). 0 methylation = both alleles are
active, 100 methylation = both alleles are inactive. In 2i culture cells the methylation is lost (almost 0).
meaning whole imprinting mechanism is lost imprinting is essential for embryonic
development.
3. What is the reason that the authors perform blastocyst injections with cultured mESCs?
To create chimeras
4. How do the researchers show that developmental potential is compromised after long time
culture of mESCs in 2i?
They did multiple assays:
- transfer cells into blastocyst of mouse do they grow into mouse? Difference between 2i
serum or without 2i serum.
- transfer cells into blastocyst of mouse Look at coat color in chimera. If the color is
changed the injected stem cells contribute a lot to the final mouse, if there is not enough
change in color the stem cells did not contribute to the final mouse and probably died.
Difference between 2i serum or without 2i serum.
- Teratomas using 2i ESCs and normal ESCs some 2i lines could form teratomas but not very
efficient.
- Long time culture in 2i results in genomic abnormalities that can be shown in a karyotype.
Key questions paper 1:
Paper 1: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem
cells. Choi et al., Nature. 2017 548(7666):219-223. PMID: 28746311
- Why do researchers like to culture mESCs in media containing 2i + LIF?
To prevent them from differentiating and keeping them in their pluripotent state.
- What is imprinting and why is it important?
imprinting: inactivating either the allele from the maternal or the paternal side by methylating the
DNA.
DNA methylation should be 0,5 (half mother half father allele). 0 methylation = both alleles are
active, 100 methylation = both alleles are inactive. In 2i culture cells the methylation is lost (almost 0).
meaning whole imprinting mechanism is lost imprinting is essential for embryonic
development.
- What is the reason that the authors perform blastocyst injections with cultured mESCs?
To create chimera’s
- How do the researchers show that developmental potential is compromised after long time
culture of mESCs in 2i?
They did this by performing 4n blastocyst injections. Culture them in both conditions how many
pups are there created.
- What was the hypothesis of the authors?
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