lecture 3 - pluripotent stem cells I Flashcards
What are the 3 conditions required for the definition of a stem cell?
The ability to self-renew, be clonal (a single cell can generate more cells) and produce progeny with more restricted potential (differentiate)
How is embryonic stem cell pluripotency maintained?
Pluripotency is maintained during ES cell self-renewal by:
promotion of proliferation
suppression of differentiation
extrinsic signals such as growth factors and cytokines, as well as intrinsic factors, such as TFs
What are extrinsic factors maintaining pluripotency? How was the main factor discovered in mice?
LIF, STAT3, BMP2 and BMP4
Main factor produced by feeders was LIF - this was combined with serum and was able to maintain mESC in pluripotent state without feeders.
LIF is an extracellular cytokine
What is the LIF signalling pathway?
LIF activates STAT3, which is known to inhibit differentiation
activated forms of STAT3 can maintain pluripotency without LIF
LIF and BMP signalling pathways can counteract the pro-differentiation effects of FGF4 and stabilise the pluripotent epiblast
LIF signals through STAT3 to activate self-renewal
Klf4 are the Pluripotency-associated genes and are the target of LIF-STAT3
LIF represses non-neural differentiation
What can be used to replace serum and aid LIF in maintaining pluripotency?
BMP2/4 can be used and work together with LIF - involved in pathway that leads to activation of Smad4 and therefore Id (inhibitor of differentiation), leading to self-renewal
How does Wnt signalling lead to mESC renewal?
Wnt signals through GSK - when inhibited, phosphorylation of beta catenin is blocked, which accumulates and blocks TCF3, which forces self-renewal
What are 2i conditions?
Dual inhibition of MEK and GSK-3 are referred to as 2i conditions
mouse ESCs cultured in 2i conditions plus LIF is considered a ground state - stable naive pluripotent state
What are the three main TFs that control mESC pluripotency in the intrinsic pathways?
Oct-4, Sox-2 and Nanog - the master regulators
What is the role of Oct-4 in mESC?
Oct-4 can repress or activate transcription of genes
using tet-regulated expression systems
Cooperatively binds genomic sites, with Sox2, as heterodimers - these sites exhibit enhancer activity
Expressed at tightly controlled levels (with Sox2) across the entire population of ESCs, and are highly expressed in both native and primed Pluripotency states
Activates fgf4 (with Sox2), which is an autocrine factor that feeds back through the FGF4-ERK pathway to promote differentiation
What is the role of Sox2?
SRY-related high mobility-box protein-2
Implicated in regulation of transcription and chromatin
forms a complex and cooperates with Oct4
What is the role of Nanog and how is it identified?
Homeodomain-containing TF
it is identified as a factor whose expression is high in undifferentiated ESC, as well as a factor that can maintain mESC in an undifferentiated state in the absence of LIF
What are the ethical concerns around hESCs?
requires use of fertilised eggs - can only use spare IVF eggs as there are strict regulations in place
What are the difference between mESCs and hECSs?
hESCs are more difficult to culture, as they grow slower and do not like being separated into single cells
cannot be maintained by LIF - FGF-2 and activin A
hESC cells will die if dispersed into individual cells
