Define Stem Cells
A single cell that can replicate itself or differentiate into many cell types
What are the 2 types of Stem Cells and what can they differentiate into?
Embryonic Stem Cells - can differentiate into almost any cell type in the body except from placenta (pluripotent)
Adult Stem Cells - can differentiate into a subset of related cell types (multipotent)
What type of cell can differentiate into any cell including the placenta?
Totipotent
What part of embryonic stem cells can be removed and grown in culture?
Inner cell mass
How can you use embryonic stem cells to genetically engineer mammals (use mice as example)?
- extract mouse blastocysts
- genetically modify the cells and harvest and culture them
- insert back into another blastocyst
- transplant into a mother
- mother will have babies
- will be chimeric
- some cells will be modified and some will not
- can then selectively reproduce and create generations with the mutation wanted
Name one strategy for gene targeting in mice
- Start off with mouse blastocysts
- Harvest embryonic stem cells
- Create a targeting vector
- Need 2 arms of homologous DNA
- 1000 bases to match the dna wanted
- There is a resistant antibiotic section and a Negative selection bit - Introduce plasmid into embryonic stem cells
- Hope some stem cells will take up plasmid - To select for it - add puromycin
- Kills off any non transfected cells
- End up with population of pure embryonic stem cells
- Targeted construct is not incorporated into one of the stem cells.
- Homologous recombination occurs where we lose the negative selection marker - Take modified embryonic stem cell where one of the copies of the gene has the resistance introduced - interrupting gene function
- Introduce this into a blastocyst of a mouse
- Use coat colour to see how well this is working
- Modified cells being put back into a pseudo pregnant mother
- She will have a mosaic / chimeric mice
- The more pink she is, the more genes contributed and the more of the modified stem cells available - Mate one of the mice with a pink male mouse
- Creates knock out mice
knock out mice = both copies are mutated and can now target with CRSPR/Cas9
Name the 4 transcription factors that maintains the ES cell state
Myc
Oct4
Sox2
Klf4
What does transcription factor Nanog do?
Up regulate embyronic stem cell genes
down regulate the differentiation of genes
What does transcription factors Myc do?
Up regulate cell proliferation
Loosens the chromatin structure
What does activation of telomerase do?
Prevents cellular senescence
We lose parts of chromosomes without telomerase
What are Induced pluripotent stem cells (iPS)
Cells that reprogrammed back into pluripotent stem cells.
Where doe iPS’ come from?
Made from the patients own cells - typically skin cells or monocytes taken from blood
How do you create iPS cells?
- Isolate cells from patient (skin/monocytes)
- Introduce the 4 transcription factors
- Wait a few weeks
- Change culture conditions to stimulate cells to specialise the cells
How do you select iPS cells?
- Take fibroblast
- Introduce reporter construct that will allow to select for IPS cells
- Reporter has a promotor which is only active in embryonic stem cells
- Therefore is turned off in the fibroblast
- Will turn on once is specialised into the pluripotent cell - Add resistance marker for the antibiotic G418
- Wont be turned on in the fibroblast - Add the 4 transcription factors
- In vast majority of cells it wont work
- Cell will remain a fibroblast
- The reporter will not turn on
- The resistance gene is not expressed
- When the antibiotic is introduced, the cell will die - If it does work (rare)
- Promoter will become active
- The resistance gene is expressed
When the antibiotic is introduced, the cell will survive
Where do somatic stem cells reside in?
Blood Vessels
Teeth
Liver
Bone Marrow
What are the 2 well characterised adult stem cell types?
Stem Cells in the epithelia of small intestine
Haematopoietic stem cells
How does the gut lining renew itself?
Dividing cells push themselves up the crypt
- the transit amplifying cells moving up and still specialises
- is slothed off the top of the Villi
creates 4 types of differentiated cells
What 4 types of differentiated cells come from the stem cells in the crypt/ bottom of the Villi?
Absorptive Cell - takes up nutrients from gut lumen Goblet Cell - secrete protective coat of mucus Paneth Cell - Secrete proteins to kill bacteria Enteroendocrine Cell - Secrete serotonin and peptide hormones to regulate growth, proliferation and digestive activities of other cells
How do daughter stem cells know whether to stay as an embryonic stem cell or to proliferate?
Independent Choice model
What is the independent choice model?
Random
- When stem cell divided it could cause either:
○ 2 stem cells
○ 2 differentiated cells or 1 of each
What is the Asymmetric division model?
Some sort of localised determinant that marked the cell
- So when division of the stem ells occurred, they would be different
- One would have the determinant and one would not
- The one that did not have the determinant would differentiate
- The one that did have the determinant would stay as the stem cell
This would predict these outcomes
What experimental system was used to find out whether gut stem cells are multipotent?
Made a reporter construct which had a stem cell specific promoter
- Only active in the stem cells
- Will label stem cells green
- And produces the yellow CreERT2 enzyme
○ Enzyme is inactive until tamoxifen is added
- Activated form of CreERT2 recognises DNA sequences and causes recombination events to occur
○ Flip out a region of the dna sequence that is surrounded by recombinase sites (the blocks in pink)
- Another reporter construct is driven by ubiquitous promoter which is continuously turned on
- Expression of LacZ is blocked by blocking sequence
- Activated recombinase removes the blocking sequence allowing LacZ to be expressed
What keeps the stem cells in its place and maintains its stem cell character?
Paneth cells
How do Paneth cells create the stem cell niche?
There are Paneth cells surrounding the stem cells at the base of each crypt (in Villi in Gut cells).
These Paneth cells generate signals to activate pathways in stem cells.
Paneth cells release ligands that activate either the Wnt or Notch signalling pathway.
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STEM CELLS30
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MICROSCOPY46
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Cytoskleton43
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Cell Cycle, Cell Death and Tissue Homeostasis22
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Cell Cycle Regulation21
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Apoptosis18
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Methods in molecular cell biology24
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Embryology1
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Protein Targeting28
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Membrane Trafficking27
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Cell Signalling49
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Model Organisms21
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Plant Cell Biology17
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ECM & Cell Adhesion22
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Cell Adhesion21
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EM DISEASE18
