Stem Cells 1

Question 1

What is differentiation ?

Answer 1

Specialisation of a cell: when the fate of a cell is sealed.
The key decision of a stem cell is to keep cycling or to differentiate

Question 2

How is the fate of a cell decided ?

Answer 2

The cell of a fate is decided through its ability to receive and respond to signals.
It is determined by receptors, transcription factors and epigenetics.
The gene expression patterns control the futur of a cell

Question 3

Which technique is used to investigate gene expression pattern ?

Answer 3

DNA microarray “heat” map.

There is a Colour contrast illustrating how the gene expression changes.
If the genes appears in red they are induced.
If green, they are repressed

Question 4

How epigenetics is involved in fate decision ?

Answer 4

There is a state where the fate of a cell is already decided.
It can’t response to new signals.

If a promoter is modified by epigenetic mechanisms it could results in its inaccessibility.

Question 5

What is the G0 state ?

Answer 5

A special property of adult stem cells. (It’s not the same as being post-mytotic)
It’s a quiescence state. The cells are just waiting for signals to go back into the cycle and cycle again.
It’s a pause state, they are waiting for activation

Question 6

What are the key characteristics of stem cells ?

Answer 6

They can divide, they divide by mitosis to give daughter cells from a mother cell.

It can self-renew
That means that from mitosis, it will produces two daughters cells and at least one of them will stay as a stem cell.
For the genome, this is complicated
And at least one of these daughter can differentiate.

So 1 of the cell needs to keep its genome completely unchanged, the other might have to differentiate (= change its gene expression patter)

Question 7

What means the potency of a stem cell ?

Answer 7

It defines how many cell type it produces

Stem cells differ in their potency: the number of cell types that they can produce.

Question 8

What is a totipotent stem cell ?

Answer 8

It can produces a whole organism including extraembryonic tissue, they remain for 4-5 days.

Question 9

What is an Embryonic stem cell (ES) ?

Answer 9

Pluripotent cells, they can produces all the cell of an organism but excluding extraembryonic tissue (eg: placenta)

They comes from the ICM from the blastocyst
The blastocyst is a set of cell that allow implantation into the womb associated with the inner cell mass (ICM).

They can self renew indefinitely in culture and are pluripotent (= they can differentiate into all 3 germ layer)

Question 10

What is an induced pluripotent stem cell (IPS/IPSC) ?

Answer 10

Pluripotent stem cells

Question 11

What are adult tissue specific stem cells ?

Answer 11

They have a reduced potency, they are multipotent
They can produces cells specific to one particular region and can only give differentiate cell from one tissue eg: brain-neuro stem cells

The genome becomes more and more restricted.
Certain genes are switched off.

Question 12

What are the three germ layers and what is their fate ?

Answer 12

After fertilisation, a zygote is produces, then it becomes a blastocyst and then a gastrula.
During the gastrula state, the 3 germ layer begin to form.

Ectoderm, the external layer of the gastrula will form skin cell of epidermis, neurons of brain and pigment cells.

Mesoderm, the middle layer, will give cardiac muscle, skeletal muscle cells, tubule cells of the kidney, red blood cells

Endoderm, the internal layer, will give lung cells (alveolar cells), thyroid cells, pancreatic cells.

Question 13

How do you check pluripotency ?

Answer 13

The cell has to be able to differentiate into all the cell of the 3 layers.

Question 14

Why are pluripotent cells useful ?

Answer 14

As tools:
understand development
Investigate the molecular and cell biology of disease
Test drugs and other therapeutics on

As therapies:
To repair damaged/diseases tissues
To replace diseased/dying cell
To support diseased cell

Question 15

What are the three main transcription factors of pluripotency ?

Answer 15

Oct4
Nanog
Sox2

They are expressed in ES cell and are essential for their behaviour. Just a few TF can control enough genes to make the genome pluripotent.
These 3TF occupied promoters of + 353 genes and miRNAs in human ES cell lines.

Question 16

How is ES cell pluripotency maintained ?

Answer 16

TF such as Tgfbeta and Wnt Activate genes that maintain pluripotency and repress genes involved in differentiation. It is often the physical state of the chromatin that gonna define the potency of a cell.

Question 17

What are the difference between IPSCs and ES cells ?

Answer 17

IPSC -> engineered cells by a Japanese group are post mytotic cell to become like ES cells ie pluripotent. It is a reprogrammation of differentiate cells into undifferentiated cells. It is achieve using a small cocktail of TF.

IPSC need to pass a serie of tests
They need to looked like ES cells, divided and self-renewed, express the same proteins

The key difference is that they expressed many ES cell genes but not all of them

Question 18

What are the different test used to prove pluripotency of IPSC ?

Answer 18

The teratoma test :
see what kind of tumour the injection of the presumed IPSC cells can induce to a nude mice (without immune system). If they induces tumors that contained cells from all 3 germ layers, the cells are pluripotent.

Embryoid bodies:
Withdrawing factors keeping culture cells ES, that will induce spontaneous differentiation. The cells will stop sticking to the plate and aggregate. It results in the formation of embryoid bodies. They mimic some embryonic behaviour but in a disordered manner. (If do, the cells are pluripotent)

Epigenetics:
Eg: pluripotency genes demethylated

The goal is to see if IPSCs can developed an embryo. A labelling IPSCs with GFP is usually done.

Question 19

How are IPSCs made ?

Answer 19

With different factors.
They are delivered using viruses but many problems: TF (used to induces redifferentiation) need to be silenced for pluripotency to developed, there is a risk of re expression

C-MYC part of the cocktail, it’s a TF that controls a powerful oncogene.

More recently, plasmids, microRNA, recombinant proteins and small molecules.

Question 20

What is NANOG ?

Answer 20

It is a useful marker of pluripotency.
If the cell expresses NANOG (linked to GFP), the cell has been reprogrammed and is pluripotent.

The process of reprogrammation is extremely inefficient

Question 21

Other problems with IPSCs ?

Answer 21

They often remember a little bit about where they came from (cell memory).
The chromatin of IPSCs are not identical to the one of ES.
Some leads to tumors

Question 22

Epigenetics and gene expression ?

Answer 22

Unmethylated genes, with euchromatic histone patterns likely to be express.
Unmethylated genes, with heterochromatic histone patterns likely to be silenced.
Methylated gens have heterochromatic hi stones and ARE silenced.

Question 23

Example of cell memory

Answer 23

IPSC derived from blood made blood more frequently than IPSC derived from fibroblast

Depending on which signal in which order and the state of your SC, you might get quite different fate.
It’s a really precise process, you always get a mix population of cell.

Question 24

What are the key signals in midbrain development

Answer 24

Wnt, FGF8, Shh

If you want to make dopanergic neurons, using these factors you can make them in a dish.

Question 25

What is direct conversion

Answer 25

A tissue specific cell directly concerts into a related tissue specific cell or into a cell type of another germ layer

Question 26

What are the application of IPSC ?

Answer 26

IPSC from patient could be used to model diseases, test treatments, grow and engineer replacement cell.

Question 27

IPSC into neurons ?

Answer 27

1. Take embryoid bodies formed from IPSC
2. Treat with a Shh and RA (= retinoic acid) agonist, grow on laminin
3. The cell differentiate into motor neurons (using combination of TF)

IPSC -> could be used to understand Parkinson and Alzheimer’s disease or Rett syndrome.

In vitro modelling of diseasing -> how does the disease causing mutation affect cell survival ?

Interaction with others cells

Response to stresses on therapies