Embryonic stem cells Flashcards
(32 cards)
What are XEN?
Extra embryonic stem cells
What are TS?
Trophoblast stem cells
What are mES and hES?
Mouse embryonic stem cells
Human embryonic stem cells
What are EpiSC?
- Epiblast stem cells
- Derived just before gastrulation (post-implantation)
Examples of pluripotent stem cells?
XEN TS mES hES EpiSC
What is the trophoblast?
Cells forming the outer layer of a blastocyst (the trophoectoderm)
Where have XEN and TS cells been derived from?
Mouse NOT the human
What are 8 properties of ES cells?
1) Derived from the inner cell mass of blastocysts
2) Non transfomed
- But high telomerase acvtivity
3) Indefinite proliferative potential
- High amplification potential
4) Stable diploid karyotype
5) Clonogenic
- Whole population can be re-derived from a single cell
6) Pluripotent
- Make all 3 germ layers
7) Carry particular markers
- Surface markers
- Genes
8) Can be incorporated into chimeras
- ONLY MOUSE ES CELLS (not human)
Which stem cells are naive and what does this mean?
Mouse ES cells
- Don’t express lineage markers
- Only express SC genes
Which stem cells are primed and what does this mean?
Mouse EpiSC and human PSC
- Express lineage markers
- Start to switch on other genes, as well as having SC genes turned on
How can you characterise PSC?
1) Surface markers
- Proteins on the surface
2) Genetically normal
- 46 XX or 46XY
3) Epigeneticically normal?
- Methylation
- Impriniting
4) Gene expression
5) Functional tests
- What can they do?
- Differentiation
- Clonogenic assays
How are antibodies used to characterise cells?
- Cells carry different combinations of marker proteins on their surface
- Different antibodies recognise specific proteins on the surface
- Probe them
What analysis techniques are involved in picturing antibodies?
Immunohistochemistry (homogenate)
Immunofluorescence (in situ)
What controls the dose of gene expression?
- Bilateral expression of genes
OR
- Maternal imprints or paternal imprints of genes
How are pluripotent stem cells different from eachother?
- Different characteristics depending upon where cultured from (mouse/human) and the timings
- Characteristics are: gene expression, surface markers, epigenetic and functional (differentiation potential and single-cell re-plating ability)
How are human ES cells derived?
1) Take fertilized embryo
2) Form the blastocyt and ‘hatch’ outside of the zona pellucida
3) Remove the outer trophoectoderm layer
- By taking antibody which recognises the trophectoderm
4) Add compliment to the antibody to trigger cell lysis of the trophectoderm
5) Left with the inner cell mass and put into a medium
How can PSCs be geneticall characterised?
- Should be 46 XX or 46 XY
How can PSCs be epigenetically characterised?
1) Methylation
2) X-inactivation
- In the female lines one X is inactivated
3) Imprinting
Which gene is not expressed int the mouse or human ES cells but is expressed in mouse EpiS cells?
Fgf5
What are ‘gatekeeper’ genes of pluripotency’?
- Genes expressed by all ES cells (both naive and primed)
- Required to maintain chromatin in an ‘open’ state
- Oct 4
- Nanog
- Sox2
Can all ES cells (naive or primed) from teratomas?
Yes
- If cells are pluripotent, they should be able to form ALL three germ layers
What are the advantages of the embryoid body method of testing differentiation?
- 3D structure
What are the disadvantages of the embryoid body method of testing differentiation?
- Not reproducible
- Not directed
What are the advantages of the monolayer method of testing differentiation?
Differentiation is
- Reproducible and
- Directed
