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3. stem cell proliferation and fate Flashcards

1
Q

what are the four phases of the cell cycle?

A

G1, S, G2 and M

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2
Q

what occurs in S phase?

A

DNA replication

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3
Q

what occurs in M phase?

A

mitosis

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4
Q

at what point of the cell cycle to cells exit and enter G0?

A

G1

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5
Q

what are the two types of G0?

A

post mitotic cells and quiescent cells

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6
Q

what type of cells are quiescent?

A

stem cells

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7
Q

what is quiescence?

A

the temporary persistence of G0 phase of the cell cycle

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8
Q

give an example of hyper-quiescent cells?

A

some mice HSC have been shown to only divide 5 times in their entire two years of life

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9
Q

given the right signal, what can stem cells in G0 do?

A

re-enter the cell cycle via G1

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10
Q

what phase of the cell cycle can mutations occur in?

A

S phase

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11
Q

what happens if too many DNA mutations occurs?

A

apoptosis

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12
Q

why is it beneficial for stem cells to remain quiescent?

A

they do not accumulate mutations

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13
Q

if a mutations occurs in stem cells how will this affect its progeny?

A

they will also have mutations

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14
Q

what two ways can proliferation in a tissue be measured?

A

using DNA labels or staining for cell cycle markers

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15
Q

what are the two types of cell cycle marker staining called?

A
  1. cyclin immunostaining

2. Ki67 staining

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16
Q

when is Ki67 expressed?

A

during all active stages of the cell cycle

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17
Q

when is Ki67 not expressed?

A

G0

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18
Q

when are cyclins expressed?

A

they are expressed at specific stages of the cell cycle

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19
Q

what method may used to look at the proliferative index of cancer? and what does a high index mean?

A

Ki67 immunohistochemistry

if there is a lot of staining this means the tumour is highly proliferative and malignant

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20
Q

what can Ki67 staining be used in conjunction with?

A

DNA labelling

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21
Q

is a cell has low Ki67 staining and low Hoechst staining, what stage of the cell cycle is it in?

A

G0

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22
Q

if a cell has high Hoechst staining and high Ki67, what stages of the cell cycle could it be in?

A

S, G2 or M

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23
Q

name two DNA dyes that bind to DNA?

A

DAPI and Hoechst

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24
Q

if a cell contains double as much DNA stain that another, what does this mean?

A

the cell has undergone S phase but has not yet undergone M phase

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25
if a cell has an intermediate amount of DNA what does this mean?
cell is in S phase replicating its DNA
26
what does coupling Ki67 staining with DNA staining allow you to do?
determine the percentage of cells in each stage of the cell cycle
27
what is BrdU?
an analogue of thymidine
28
how can cells that have incorporated BrdU label into their DNA be indented?
- antibodies specific for BrdU | - immunohistochemistry/flow cytometry
29
what does the label retaining assay look for?
quiescent cells
30
what does the pulse section of the label retaining assay involve?
BrdU label administered long enough to allow labelling of stem cells
31
what does the chase section of the label retaining assay involve?
allowing dividing cells to dilute their label
32
what are label retaining cells?
cells that have not divided enough to dilute their label
33
what can we suggest about these label retaining quiescent cells?
they are stem cells
34
what type of cells are most efficiently labelled in the pulse period?
the most highly proliferative
35
in addition to potential stem cells, what else might label retaining cells suggest?
location of stem cell niche
36
what are the 4 limitations of using BrdU in label retaining assay?
1. cells need to divide at the time of the pulse 2. BrdU is toxic and so injury to the tissue means we are not looking at homeostatic conditions 3. detection of brdU requires fixation and permeabilization (to allow antibodies into nucleus) - functional analysis cannot do done on dead cells 4. it is hard to distinguish intermediate levels of proliferation
37
what is an alternative method to BrdU labelling in label retaining assay?
tet-off-regulated H2BGFP
38
what problem does this alterative label retaining assay solve?
detection of label can be done on live cells and so functional assays can be performed
39
what happens during the pulse of tet-regulated H2BGFP system?
tTA is under the control of a ubiquitously expressed/tissue specific promoter, tTA binds tet-responsive sequence and drives expression of H2BGFP
40
why is this system cells tet-off?
when doxycycline is administered this bind tTA and prevents it from binding tet-responsive element
41
what happens in the chase period of this tet-off system?
doxycycline is administered and this prevents any further expression of H2BHFP
42
what happens to H2BGFP during the chase period?
it is diluted through cell division
43
what ensures that all cells are labelled with H2BGFP during the pulse period?
tetracycline inducible promoter expression is much larger than that of the endogenous H2B promoter
44
what are two advantages of using this tet-off system in the label retaining assay?
1. H2BGFP has very low toxicity | 2. dilution of H2BGFP can be followed by FACs analysis of live cells
45
what effect does inducing an injection/inflammation have on label retaining cells?
they rapidly divide in response and thus lose their label
46
what do label retaining HSC do better than non-label retaining HSC?
they transplant better and for more serial passages
47
what type of replicative potential do adult stem cells have?
they have a very high replicative potential | given the right signals they can proliferate
48
what is replicative potential?
the ability to undergo replication
49
what is a holoclone?
a colony forming stem cells that has a higher growth potential than a meroclone because it does not contain any differentiated cells
50
how does the replicative potential vary from stem cells to progenitors?
progenitors can only divide a certain number of times and so have a smaller replicative potential than stem cells that can self-renew
51
as a cell become more lineage committed what two abilities decrease?
1. the ability to self-renew | 2. their replicative potential
52
what is symmetric division?
when both daughter cells are the same
53
what is asymmetric division?
division that gives rise to a stem cell and a more differentiated cell
54
what can be used to see how cells divide and differentiate?
time lapse microscopy
55
why might what looks like an asymmetric division actually initially be a symmetric division?
stem cells may divide to produce two stem cells, one of which rapidly becomes more differentiated
56
what are the two mechanisms of asymmetric division?
1. environmental asymmetry (cell extrinsic) - once the cell have divided the two daughters are in slightly different environments, influencing them in different ways 2. divisional asymmetry (cell intrinsic) - different intrinsic factors inherited by the two cells
57
why might asymmetric division be a combination of these two factors?
- the environment of the mother stem cells may influence which factors are inherited by each daughter - cells are attracted to different environments due to slight difference in inherited factors
58
how is the human epidermis organised?
stem cells are at the basal layers of the epidermis. they can divide to produce more stem cells on the basal layer, or produce more differentiated cells that more up the layers of epidermis
59
what does looking at the orientation of spindles in the epidermis tell us about the divisions occurring in this tissue?
- when the spindle is perpendicular to the dermis this is asymmetrical division - when the spindle is parallel to the dermis this is symmetrical division - more asymmetric than symmetric division can be observed
60
what is apoptosis?
programmed cell death
61
what does a cell undergoing apoptosis look like?
it has a blebbing membrane
62
what is they hypothesis to why apoptosis occurs in tissues, using HSCs as an example
- apoptosis is a quick way of regulating how many cells we have in a tissue - one HSC might give rise to 5 progenitors, 1 undergoes apoptosis - when infections occurs and more immune cells are needed, the 5th progenitor does not undergo apoptosis
63
when can programmed cell death occur?
programmed cell death can occur any time during the differentiation process

SCRA (19 decks)

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