L2 ES Cells and Self-Renewal I Flashcards
What are teratomas?
A tumour made up of multiple different tissues that originate in the gonad.
Describe the history of teratomas and ES cells.
- Stevens (1954) showed 129 strains of mice were capable of producing teratomas spontaneously.
- Kleinsmith and Pierce (60s) showed that teratomas can be produced from a single cell and named them embryonic carcinoma cells (ECCs). He did this by taking a cell from a teratoma in one mouse and injecting it into another.
- Brinster (1974) showed that ECCs have a very high amount of pluripotency in vivo. If you inject an ECC into a blastocyst, it will become part of the organism.
- Finally, Evans and Martin (1981) isolated mouse embryonic stem cells.
What are feeder cells?
These are a carpet of fibroblasts that have been taken from the embryo and treated chemically/with radiation to stop them proliferating (they have been inactivated).
What happens to mouse ES cells when injected into:
a) Blastocyst from another mouse.
b) An adult mouse.
c) No injection (they are left to divide).
a) Chimeras are formed.
b) Tumours are formed.
c) Embryoid bodies are formed.
Name 10 properties of mouse ES cells.
1) They are derived from the inner cell mass of the blastocyst.
2) Non-transformed and fairly stable (normal).
3) Indefinite proliferative potential with high amplification capacity.
4) Stable diploid karyotype.
5) Clonogenic (they can grow from a single cell).
6) Pluripotent.
7) Can be incorporated into chimeras.
8) Can do germline transmission in chimeras. (Where the injected stem cells will incorporate into the germline so the genes from that cell will be passed on to the offspring).
9) Permissive to genetic manipulation (e.g. transgenics).
10) When injected into an adult isogenic (very similar genotype) host, they will form teratomas.
What is LIF and what does it do?
Leukaemia Inhibiting Factor.
LIF is responsible for maintaining the self-renewal and proliferation of the ES cells.
How are LIF and BMP working together to keep ES cells pluripotent?
- BMP via Smads induce Id genes that stop the cell from following a neural lineage.
- At the same time, LIF/Stat3 are inhibiting BMPs from inducing mesoderm and ectoderm.
What are the three main differences between mES and hES cells?
1) Mouse ES cells form elevated colonies that form a dome and grow outwards. hES cells form flat, defined colonies.
2) Different surface markers.
3) Mice depend on LIF and 2i (ERK1/2 inhibitor PD0325901, GSK3 beta inhibitor CHIR99021). Humans depend on FGF2 and Activin.
What are the two stages of pluripotency and how did scientists discover them?
1) A naive or ground-state. These are the early cells, also called inner cell mass-like.
2) A primed state. These are the later cells, also called epiblastic-like.
These states were discovered when Brons et al (2007) derived ES cells from the epiblast not the ICM. These cells were shown to be dependant on FGF and Activin like hES cells.
How were naive hES cells discovered?
In 2013, a cell line was created that was resistant to an antibiotic and had an Oct4 GFP reporter line. The cells that became pluripotent not only expressed high amounts of GFP, but they were resistant to the antibiotic.
This cell line was then exposed to a range of different factors to see what would induce naive pluripotency.
Regulation of distal and proximal enhancers of Oct 4 was a way to distinguish between naive and primed cells .
Why are naive hES cells important?
- Fundamental for understanding pluripotency.
- Easier to modify genetically (more efficient homologous recombination).
- Have provided confirmation of human-mouse chimerism because they can integrate well into the ICM.
