Introduction of pluripotency Flashcards
How are cells arranged in a ‘state space’?
1) Give each gene a numerical value
2) Each different SET of genes will have a different numerical value
3) Arranged in an abstract space called a ‘state space’ depending on the SET of genes and their numerical values
What defines the state of a cell?
The combination of genes in the cell
In the ‘state space’, why would you not find cells represented at each position?
- Some combinations of genes are not compatible to be a cell
- Only STABLE configurations make cells and are shown in the state space
Ultimately, what defines a cell type?
The transcription factors present
Are the probabilities of changing between states equal? Why?
NO
- Difficulty due to how many genes need to be turned on or off
- If the two states are far away from each other on the state space
- More genes have to be turned on/off
- Harder to switch between the two states
What does the stability of a cell state depend upon?
- Where the state is positioned on the ‘state space’
(Think of the state space having hills and troughs:
- Hills are unstable
- Troughs are stable, as hard to roll around)
What is the metaphor for differentiation?
- It is like rolling a ball down a hill with valleys
- Only certain pathways can be taken
- When enter one valley (ie. switch certain genes on/off) it becomes much harder to switch to a different pathways
INEVITABILITY of differentiation
What is transdifferentiation? Is it difficult?
- Switching between different unipotent differentiated cells
(ie, jumping between valleys) - Some are more difficult than others
- Some can be overcome with the right factors
What must be reset in order to reset the cell state?
Doesn’t occur naturally
1) Gene expression
- Somatic genes turned off
- Embyronic genes turned on
2) Methylation
- Reset back to ‘totipotent’ configuration
3) Chromatin
- Remodelled
- Different patterns in different states
What POTENTIALLY be used to reset any cellular state and why?
Transcription factors as they are said to define a cell state
When fibroblasts are transfected with MyoD1 transcription factor what happens? How?
- Fibroblasts converted into muscle precursors
- MyoD1 turns on target genes
- Proteins coded for by target genes may switch on other genes
- This may form a cascade and switch the cell state as the genes expressed override the existing gene regulatory netowrk
How induce pluripotency in mouse or human?
Transfect somatic cells with transcripption factors that are known to be expressed in a pluripotent cell
Why does the induction of pluripotency have a low efficiency?
- As the genes which are expressed in a cell are changed, the cells may be able to respond to new signals
- These signals may cause the cell to change to a different state which is ‘off course’ of the original state that was the original target
What are the additional factors which must be added to make iPS cells?
Factors which help the cells to survive and divide such as:
- c-mvc
- hTERT
- SV40T
What are the problems with using viral transduction to create iPS cells?
1) Viruses insert into the genome and could cause a mutation
2) Viruses may not be silenced and could remain active in the cell, causing overexpression of genes which keep the cell in the stem cell stage, can’t differentiatte
What are 3 other ways of creating iPS cells which don’t involve integration?
1) Vector-based approaches
2) Protein based approaches
3) Chemical based reprogramming
Examples of vector based approaches?
- Transient transfection
- mRNA
- Adenovirus
- Transposons
What is the difference between integration by retroviruses and lentiviruses?
R - can only transduce dividing cells
L- can transduce both dividing and non-dividing cells
Why is delivery by adenoviruses inefficient?
Doesn’t integrate
How does transistent transfection work?
- Integrate into the DNA but have attached recognition sites
- Can put recomibinase into the cells, which recognises the sites and loops of the integrated DNA
What is the disadvantage of transistent transfection?
- Leaves a mark (not complete excision)
- Risk of mutagenesis
What is a transposon?
- DNA sequence that can change its position within a genome
- Can create and remove mutations
What are the advantages of using transposons to deliver reprogramming factors?
They can excise in a ‘scar free’ way
How is mRNA used to deliver reprogramming factors?
- mRNA transcribed in vitro
- Capped and polyA tail
- Transfect cells
Why are drugs needed to be prescribed with mRNA transfection?
- Exogenous mRNA triggers anti-viral response
- Drugs stop interferons (immune response) and prevent degredation of the mRNA
What are the advantages of using mRNA to deliver reprogramming material?
- No viral components
- No genomic integration
What are the disadvantages of using mRNA to deliver reprogramming material?
- Multiple rounds of transfection requires
- Lower levels of expression than when integrated
How do you identify that you have iPS cells?
1) Morphology
2) Protein and gene expression
3) Differentiation
- Makes teratomas
4) Epigenetics
- Methylation at specific loci
How are iPS cells different to the ES cells and how is this an important advantage?
- They carry the genotype of the parent cells
- Capture a genotype
What are the advantages of iPS cells?
- You can capture a particular genotype potentially forever (iPS cells can grow indefinitely)
1) Can capture disease genotypes for studying
2) Capture the genotype of a particular human population for studying
3) Drug discovery
Describe the process of iPS cells in drug discovery
1) Capture normal genotype and diseased genotype in vitro
2) Compare the differentiation and phenotype in vitro
3) Drug screen with the effect on target known or unknown
4) Does the drug relieve the the cell phenotype?
5) What is the effect of the drug on the normal phenotype?
What diseased fibroblast have iPS cells been derived from?
- DMD
- Parkinson’s
- Type 1 diabetes
What genes are put into cells to make iPS celld?
OCT4 NANNOG SOX2 KLF4 C-MYC
Which viruses are used to tranduce cells, which integrate into the host genome?
Lentivirus
Retrovirus
