In vitro stem cells

Question 1

What are pluripotent stem cells?

Answer 1

Can give rise to any cell types of an embryo- including germ cells

Question 2

What are multipotent cells?

Answer 2

Can only give rise to a small subset of cells

Question 3

What are unipotent cells?

Answer 3

Can only give rise to one type of cell.

Question 4

What does self renew mean?

Answer 4

The make another copy of themselves. Stem cells live in a niche where they sit and divide to give rise an identical cells and a differentiated cell

Question 5

What are some pluripotent cell markers in mice ES cells?

Answer 5

Nanog, Oct4, Sox2. They are TFs

Question 6

What is gastrulation?

Answer 6

The formation of the germ layers, requires differentiation and organisation
The germ layers are the first founders of the cells which will form the embryo proper
Stem cell potency is diminished over time as the embryo develops

Question 7

Why is it hard to capture stem cells in vitro?

Answer 7

They are very hard to study because of the small number and in-utero developments. Also some ethical implications

Question 8

How to maintain stem cells in vitro?

Answer 8

Need to replicate the stem cell niche by adding the factors which the support cells secrete

Question 9

What are some of these factors in mice?

Answer 9

Leukaemia inhibitory factor (LIF), BMP agonists

Question 10

What are some niche factors in humans?

Answer 10

FGF2 and TGFBeta

Question 11

How to isolate ES cells?

Answer 11

Take cells from 4 day embryo in mice- dissociate the ICM and plate this on a dish with feeder cells which produce trophic factors

Question 12

How to make IPS cells?

Answer 12

remove skin cells and de-differentiate them back to their pluripotent state. If you over express pluripotent factors- cell will return to the pluripotient state and can differentiate into all cell types in the body

Question 13

How do we know we have captured Pluripotent cells?

Answer 13

Expression of pluripotent factors
They can differentiate into any type of cell
No expression of genes which are indicative of differentiation
A single cell can generate an identical daughter cell
ES cells form teratocarcinomas when transplanted into permissive environments
When transplanted into a mouse they will contribute to normal development- can see using GFP

Question 14

What are the two types of cell a neural stem cell will differentiate into?

Answer 14

Radial glia, neural cell. If you block differentiation you can culture neural stem cells in.

Question 15

How to plate neural stem cells

Answer 15

From foetal brain and dissociate and plate them on laminin in the presence of cytokines, FGF and EGF- They will self-renew and produce neural cell markers e.g. RC2. They do not express factors associated with differentiated neurons or glial cells e.g. GFAP

Question 16

How to initiate differentiation

Answer 16

Remove self-renewal markers. To get the right cells- we need to make sure that the cells are expressing the right markers.
Grow cells on aggregates in the presence or absence of differentiation signals. By plating them on organoids or embryonic bodies- this is a 3D approach and more accurately recpitulates embryonic environment, but it is more difficult to see the effect of individual signals

Question 17

How to make 2D differentiation model

Answer 17

Plate a defined number of cells on the right substrate
Remove undifferentiation markers
Grow in a medium with appropriate amounts of signals
This is a more tractable system and you can more easily determine the role of specific signals. However there is a loss of cell interaction which may occur in vivo

Question 18

What are the main applications for in vitro stem cells

Answer 18

Most commonly disease modelling
Take patients with a genetic disease and turn cells into IPS cells. Induce them into the affected cell type and test drugs on them. Allows us to study diseases which are hard to model

Question 19

What is familial dysautonomia

Answer 19

genetic disorder which affetcs the development and survival of neurons which control involuntary actions such as digestions breathing etc

Question 20

When do the symptoms of familial dysautonomia begin to show

Answer 20

Infancy, symptoms include lack of muscle tone, feeding difficulties, poor growth, lack of tears- later on difficulty walking, spinal curvature etc

Question 21

What is familial dysautonomia caused by

Answer 21

mutations in IKBKAP gene. Trying to model this disease by using the IPS technique and tested Kinetin treatment and this causes an increase in in vitro neurons- could be used in the future to treat FD