Pluripotent Stem Cells Flashcards
(42 cards)
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
