Pluripotent Stem Cells Flashcards
What is special about stem cells?
They retain pluripotency
What gives a cell its identity?
Stable genetic profiles that are less likely to be modified by extracellular signals
What allows a cell to maintain its identity?
Gene regulatory network gives robustness, which can withstand pertubations and return to its stable state
Where are mESCs derived from?
Inner cell mass of blastocysts
How are mESCs initially cultured?
On MEF ‘feeders’ in serum-containing media
What are MEF “feeders”?
Cells used to support the growth
MEF cells secrete growth factors into the medium, which help maintain the pluripotency of ES cells.
MEF cells provide a cellular matrix for ES cells to grow on.
MEF cells are often mitotically inactivated so that they remain viable but cannot replicate and overgrow the ES cell culture.
What factor was found to be the key component provided by MEF ‘feeders’?
LIF
What is serum-containing media?
Fetal bovine serum (FBS), which is a common supplement added to cell culture media to provide essential growth factors and nutrients for cell proliferation and survival
How do we know ESCs are truly pluripotent?
Because when injected into tetraploid embryo an alive fetus was produced
Shows ESC have the potential to form ALL fetal cell lineages
What pathways does LIF activate?
STAT3 and ERK pathways
Explain the STAT3 pathway
LIF binds LIFR, which is coupled to co-receptor gp130
Activation of JAK = phosphorylates STAT3 on specific tyrosine residue
Activated STAT3 allows it to dimerize and translocate to nucleus
Inhibits differentiation
Explain the ERK pathway
LIF binds LIFR, which is coupled to co-receptor gp130
Ras is activated by converting GDP to GTP
Ras phosphorylates Raf
Which phosphorylates MEK
Which phosphorylates ERK
Promoting differentiation
What effect do the two pathways LIF activates have on differentiation?
STAT3 inhibitis differentiation
ERK promotes differentiation
What happens when ERK activation is supressed in mESCs?
mESCs require less LIF to self-renew
How does STAT3 inhibit differentiation in mESCs?
LIF binds LIFR, which is coupled to co-receptor gp130
Activation of JAK, which then phosphorylates STAT3 on speciifc Tyr residue
Activated STAT3 dimerizes and translocates to nucleus
How does ERK promote differentiation in mESCs?
LIF binds LIFR, which is coupled to co-receptor gp130
Ras activated by conversion of GDP to GTP
Phosphorylates Raf > MEK > ERK
What do mESC need to be maintained?
Shown that they can be maintained without serum
With LIF + BMP4
What is LIF needed for to maintain mESC?
Leukaemia inhibtory factor (LIF)
To induce STAT3 = inhibitor of differentiation
Why is BMP4 needed to maintain mESC?
Bone morphogenetic protein (BMP)
To induce inhibitor-of-differentiation proteins
How do STAT3 and ERK regulate gene expression?***
What factors do pluripotent cell express?
Transcription factors = Sox2, Oct4, Klf4 and Nanog
(Nanog is not needed for maintenance of pluripotency; only initiation)
What is the effect of deleting pluripotency TFs?
Phenotype would change, causing stem cells to stop being pluripotent and start differentiating
What is the effect of overexpressing pluripotency TFs?
Causes more resistnace to differentiation = more robustnuss
But this depends on WHICH factors
Which pluripotency TFs are NOT targets of LIF pathway?
Sox2, Oct4 & Nanog
Which pluripotency TFs are targets of LIF pathway?
Klf4 indirectly by LIF
What is the role of Nanog in pluripotency?
It is important when MAKING pluripotent cells
But NOT needed to MAINTAIN pluripotency
What is the “ground state” hypothesis?
The ability to grow mESC without serum because pluripotency is the default
So only need to inhibit differentiation signals to allows pluripotency to occur
Inhibit ERK and GSK3 signalling
What conditions create ground state pluripotency?
2i = 2 inhibitors
Inhibit ERK and GSK3 signalling
Define ground state pluripotency
Pluripotency does not need to be promoted
Cell require input to exit from it = default
How are ground state cells different to those not grown under 2i?
More homogeneous = high level of Nanog
How are hESC different to mESC?
Don’t respond to LIF
Die in 2i = no ground state
What do hESC need to self-renew?
Activin and FGF
How does FGF act on hESC compared to mESC?
FGF causes hESC to self-renew
FGF activates ERK in mESC = promoting differentiation
hESC react very differently to conditions than mESC, what does this tell us?
The way hESC and mESC maintain their identitiy is different!!!
How does LIF work in mice vs humans?
In mouse embryos, LIFR = not required for early embryo development
hESC are LIF indepencent
Why are LIFR not required for early mouse embryo development?***
LIF receptors are not required for early mouse embryo development because while LIF plays a crucial role in uterine receptivity for implantation
Studies have shown that mouse embryos lacking LIF or its receptor can still develop normally to the blastocyst stage, indicating that LIF signaling is not essential for the early stages of embryonic development before implantation.
Is there are specific function of LIF signalling in mice that is not required in humans?***
YES hESC are LIF-independent
LIF plays a crucial role in maintaining the pluripotency and self-renewal of mESCs
What is the epiblast?
A tissue of the post-implantation embryo that generates the embryo proper
At implantation, the embryo is composed of the epiblast, a pluripotent derivative of the inner cell mass
What is the difference between mESCs and EpiSCs derivation?
mESC obtained from mouse embryo BEFORE impantation in uterus
EpiSCs derived from epiblast, which is tissue of post-implanted embryo
What do naive and primed states mean for ESC?
These are pluripotency states
In mESC naive state goes before primed state then to differentiation
High Nanog = naive
Low Nanog = primed to differentiaate
How is the naive mESC phenotype stabilized?
2i conditions
What factors influence ESC phenotype?
Culture conditions = signalling
Developmental stage
Species differences
Heterogeneous, fluid state
