Lecture 10: induction of pluripotency and a cell's identity

Question 1

transcription factors can be thought of a s

Answer 1

• defining a cell type

- SOX2, NANAG, OCT4 + others define an ES cell

Question 2

metaphor for cellular differentiation:

Answer 2

• Waddington
• differentiation is like a ball rolling down a hill
• Valleys = epigenetic restriction
• cells can’t just change easily form one ‘state’ to another
• can change state with correct factors

Question 3

two ways or resetting the cell state:

Answer 3

Cloning

Inducing pluripotency

Question 4

Resetting the cell state Cloning:

Answer 4

• a cell nucleus is manipulated to ‘reset’ its state. What has to be reset?
• Gene expression: somatic genes turned off, embryonic genes turned on
• Methylation: reset back to a ‘totipotent’ configuration
• chromatin: Remodelled

Question 5

Resetting the cell state: Inducing Pluripotency

Answer 5

• there must be factors in oocytes that are resetting the state
• a set of sequential chromatin & gene expression changes

Question 6

You are ____ factors to drive iPS formation

Answer 6

OVEREXPRESSING

• how do u turn the expression off?
• lintivirus will integrate but then be silenced
• Alternative: use a non-integrating method

Question 7

non-integrating delivery of reprogramming factors: 3 types

Answer 7

• Vector-based approach
• Protein based approaches
• Chemicals based reprogramming

Question 8

Vector-based approach

Answer 8

• transient transfection
• adenovirus
• floxed vectors
• transposon
• mRNA

Question 9

protein based approaches:

Answer 9

TAT-fusion

Question 10

chemicals used reprogramming:

Answer 10

VPA, 5-AzaC

Question 11

delivery of reprogramming factors RETROVIRUS delivery

Answer 11

can only transduce DIVIDING cells

Question 12

delivery of reprogramming delivery: LENTIVIRUS delivery

Answer 12

can transduce both DIVIDING and NON-DIVIDING cells

Question 13

in reprogramming factors using virus’ what does Cre do

Answer 13

recombinase recognises LOX sites. Excises the intervening sequences BUT leaves ONE lox site behind

Question 14

a Transposable element (TE or transposon) =

Answer 14

• DNA sequence that can change its position within a genome

- can create and remove mutations

Question 15

Transposase:

Answer 15

enzyme that binds to the end of a transposon: catalyses the movement of the transposon to another part of the genome..

by:
- cut and paste
- a replicative transposition

Question 16

Delivery of reprogramming factors: Episomal:

Answer 16

-Episomal replication: some vectors can replicate EXTRA_CHROMOSOMAL i.e remains outside the chromosomes. If conc limiting can be diluted by cell division

Question 17

delivery of reprogramming factors: mRNA

Answer 17

Exogenous mRNAs can trigger an ancient anti-viral response – which causes degredation. You need to use drugs to supress this degradation + 5’-protected mRNA

Question 18

how do you identify bone fide iPS colonies?

Answer 18

• Morphology
• protein expression
• gene expression
• differentiation
• epigenetics

Question 19

what other cells have iPS cells been derived from?

Answer 19

epidermis, neuron, blood, beta-call.

In reality, it appears any cell can be programmed to an iPS cells

Question 20

iPS compared to ES

Answer 20

• Both pluripotent BUT iPS unlike ES carry the genotype of the parent cells.
• This means you have captured a particular genotype potentially forever (remember iPS cell can grow indefinitely).
• Therefore, you could potentially make iPS cells from different genotypes (including people with diseases)

Question 21

what can we use induced pluripotent cells for?

Answer 21

• pluripotent stem cells grow forever
• compare diseased iPS cells with normal iPS cells & COMPARE
• is differentiation affected? (is the disease caused by the cells not forming properly)
• is the phenotype affected? (is it caused by the cells not functioning properly)
• DRUG SCREEN

Question 22

what diseases fibroblasts have iPS cells been derived from?

Answer 22

-Duchene muscular dystrophy
-PArkinsons
-ADA-SCID
-type 1 diabetes
Gaucheries disease
-Lesch-Nyhan syndrome

Question 23

transdifferentiation: Direct reprogramming

Answer 23

• can we change any cell into ant other cell???
• lots have been competed so far
• predict TF needed
• approx. 2000 TF
• approx 20 TF expressed in a cell type
• -could 3-5 TF work??