L3 ES Cells and Self-Renewal II Flashcards
What is the role of Oct4?
It is crucial to maintaining pluripotency. Relative levels of Oct3/4 influence ES cell fate.
What happens when there’s no Oct4/too much/not enough?
With no Oct4, the embryo fails to produce the lineages found in gastrulation and produces extra embryonic trophoectoderm.
With too much Oct4, extra embryonic trophoectoderm is produced.
Not enough Oct4 produces only trophoectoderm.
Describe the interaction between Oct4 and Cdx2.
Oct4 acts as a counterbalance to the gene Cdx2 which drives differentiation of the trophoblast. There is a reciprocal repression loop between the two genes.
- A decrease in Oct3/4 leads to an increase in Cdx2.
- Forced expression of Cdx2 leads to tropholast.
- Forced repression of Oct3/4 also leads to trophoblast.
Oct3/4 and Cdx2 appear to bind in a complex that inhibits their individual transcriptional activity.
What is the role of Sox2?
Sox2 is required in the lineage leading to epiblast formation and in their absence trophoectoderm is formed.
How did Mitsui et al (2003) identify Nanog?
They screened ES cells to identify the genes that were being highly expressed in them. They then checked the degree of expression of these molecules using a Northern Blot.
They then put Nanog cDNA into a plasmid vector, expressed it in ES cells and grew them with or without LIF. It was discovered that, even though the cells without LIF are supposed to lose their capacity to be pluripotent, Nanog was capable of making the cells grow and maintain undifferentiation.
How did Chambers et al (2003) identify Nanog?
They put various genes into cells with their receptor for LIF mutated. They then took the cells that appeared to be pluripotent even without LIF and identified Nanog to be in one of those cells.
Describe the effect of Nanog mutations.
- In vivo -/- mutations lead to no formation of pluripotent ectoderm. Visceral and parietal ectoderm form instead.
- Upregulation in vivo overcomes the cell’s requirements for LIF.
- In vitro -/- (ES cells and the ICM) mutations lose their pluripotency and causes the cells to differentiate into extra embryonic endoderm.
What genes help make mesoderm, ectoderm, endoderm and hypoblast?
Mesoderm = Oct4
Definitive Ectoderm = Sox2
Definitive Endoderm = Nanog
Hypoblast = Tbx3
What are heterogenous populations and why are they important?
This is where you have more than one cell type. These cell types are distinguishable by the different cell markers. They can be sorted by FACS (fluorescent activated cell sorting).
Heterogeneity is important because cells from different states may be ‘primed’ for particular lineages.
How did Chambers et al (2007) discover the concept of heterogeneity?
They used ES cell lines with GFP downstream of Nanog enhancers in order to monitor transcription factors. They then used FACS (fluorescent activated cell sorting) to determine the cells that have activated GFP.
They determined that in any given culture, even if there is a perfect population of ES cells, there will always be a fraction of +ve and -ve cells.
They also showed that over time, when you culture +ve and -ve cells together, the +ve population start to make -ve cells and vice versa. Each population is able to generate the other.
What way did Chambers et al (2007) suggest Nanog expression was working?
They suggested that Nanog may act as a ‘rheostat’ providing variable resistance to differentiation. This means Nanog expression has a gradient of control, not an ‘on-off’ switch.
Describe how you get a Nanog -ve differentiated cell.
Within an ES cell population, you have +ve cells and transient -ve cells that are interchangeable (they can go back to being the other type). When you commit to a cell fate, the -ve Nanog ES cell becomes a -ve Nanog differentiated cell.
What are the three paths a cell can take?
1) Self-renewal.
2) Differentiation.
3) Die.
What happens to cells in vitro and how can this help us?
Cells cultured in vitro tend to develop genetic abnormalities. The majority of these mutations favour self-renewal.
This could provide an insight into the genes that control self-renewal and proliferation.
