Proliferation and fate

Question 1

What is quiescence?

Answer 1

Quiescence - temporary persistence in the G0 phase of the cell cycle. Most stem cells are quiescent especially when compared to their direct progeny. Note: differentiated cells are not quiescent; they are post mitotic and cannot proliferate anymore.

Question 2

What are the two kinds of G0 phase?

Answer 2

There two kinds of G0

• quiescent - the one stem cells use
• post mitotic state - cells that are in this stage are out of the cell cycle and are never going back.

Question 3

How can we check how much proliferation we have?

Answer 3

To answer these questions we can check for markers such as Ki67 and BrdU

Question 4

What is BrdU?

Answer 4

BrdU is an analogue of thymidine and gets incorporated in stems that are in S phase. So when we use BrdU we are specifically labelling cells that are duplicating their DNA.

Question 5

How can we see which cells are in particular phases?

Answer 5

We can see which cells are in particular phases. We can use a dye such as DAPI or Hoechst to label the cells (because they are fixed the dye will go in and not be pumped out).. Then we can use flow cytometry on the cell suspension and get the plot. The more dye there is in the cell the more DNA is present.

Question 6

What chemicals can also be used to characterise cells based on the stage of the cell cycle they are in?

Answer 6

We can also use different cyclins as markers as they are expressed at different stages of the cell cycle

Question 7

What is a FUCCI reporter system?

Answer 7

FUCCI reporter system - fluorescent, ubiquitination based cell cycle indicator

Question 8

What phase does Cdt1 indicate?

Answer 8

Cdt1-KuOrange in G1

Question 9

What phase does Geminin indicate?

Answer 9

Geminin-GFP in S, G2 and M

Question 10

Describe a label retaining cell assay

Answer 10

1. Short term administration of the lable highlights all cells
2. Afterwards dividing cells dilute the lable between daughters.
3. Non-dividing cells retain the lable and are detected as label retaining cells

Question 11

What happens during the chase phase and why is it important?

Answer 11

during the chase phase proliferation happens. The more a cell proliferates the less dye remains in the cell → cells that don’t proliferate much (such as stem cells) remain strongly labeled.

Question 12

What are some limitations of BrdU?

Answer 12

• BrdU
  
  • you need to fix cells to use BrdU so you cannot perform functional assays afterwards and can’t really tell if the cell is the stem cell
  • is slightly toxic so some cells die which the activates stem cells
  • you need to have dividing cells so you need to perform the staining at a young age of the mice. If the stem cell s in quiescence then they won’t be labeled

Question 13

How does the Tet-regulated H2BGFP system work?

Answer 13

The Tet gene expression system functions when a recombinant tetracycline-controlled transcription factor (either tTA for Tet-Off or rtTA for Tet-On) binds to theTet-op promoter, subsequently driving the expression or controlling the inhibition of the target gene (see figure below).

Gene expression is regulated by the presence or absence of tetracycline or one of its derivatives such as doxycycline. Tetracycline binds directly to the transcription factors.

Tet-Off system: doxi binds the tTa and takes it away from the promoter turning the system off.

Tet-On system: doxi binds the rtTa and allows it to bind to the promoter turning the sysetm on.

Question 14

Which cell will remain bright in the Tet-regulated H2BGFP system?

Answer 14

only the cells that do not proliferate remain bright

Question 15

Which cells are marked in an ubiquitous system?

Answer 15

• in the example above we use an ubiquitous system which allows us to monitor cells everywhere in the mouse which might be nuce as we can then see the dilution of fluorescence in different tissues
• we can also use tissues specific systems to only monitor the events in particular tissues

Question 16

What are some benefits of the Tet system?

Answer 16

• H2BGFP has no or very low toxicity
• H2BGFP dilution can be followed by FACS analysis on live cells

Question 17

What are the two possible symmetries of division?

Answer 17

symmetric divisions (you get two identical cells) and asymmetric divisions (you get two different cells)

Question 18

What two kinds of asymettric cell division can you get?

Answer 18

environmental asymmetry or divisional asymmetry

Question 19

Where can apoptosis as one of the cell fates be seen?

Answer 19

can be seen in quick changes in the output of cells for example when we need to produce a lot of immune cells then we can do it for example at the expense of red blood cells and the red blood cell progenitors that would normally go down the red blood cell lineage just die so we can have more resources for new lymphocytes

Question 20

What is the marker for apoptottic cells?

Answer 20

the marker for apoptotic cells is caspase 3

Question 21

Why do we tend to look at apoptosis in situ?

Answer 21

look at appoptosis in situ cuz flow cytometry can induce apoptosis and then you have falsly positive results

Question 22

What is the default behaviour of hematopoetic cells in the absence of CSFs?

Answer 22

the default behaviour of hematopoetic cells in the absence of CSFs (colony stimulating factors) is death by apoptosis and the control of cell survival plays a central part in regulating the numbers of blood cells

Question 23

What is the reasoning behind so many blood cells going through apoptosis?

Answer 23

• The amount of apoptosis in the vertebrate hematopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. In fact, most neutrophils produced in the bone marrow die there without ever functioning
• the reasoning behind this behaviour is to presumably maintain a reserve of cells that could be mobilised to fight infection if that was needed. Compared to the life of the organism the life of the cell is cheap