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SCRA > 2. how do we work with stem cells? > Flashcards

2. how do we work with stem cells? Flashcards

1
Q

name a marker for adult stem cells and progenitor cells

A

sox2

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2
Q

what is one way of looking for cells that express this marker?

A

putting GFP under the control of the sox2 promoter in transgenic mice

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3
Q

if a cells expresses this marker is it defiantly a stem cell?

A

no, we may have also marked progenitors, functional analysis must be carried out

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4
Q

what is lineage tracing?

A

identifying the progeny of a single cell

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5
Q

why can sox2 not be used as a marker of lineage tracing?

A

it may not be present in the progeny

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6
Q

in lineage tracing, what is put under the control of the sox2 promoter?

A

Cre recombinase

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7
Q

what sequences of DNA does Cre recombinase recognise?

A

Flox sequences

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8
Q

what is Cre recombinase fused to?

A

ER - the portion of the oestrogen receptor that binds the ligand

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9
Q

what is tamoxifen?

A

an analogue of oestrogen

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10
Q

what happens when tamoxifen binds ER?

A

tamoxifen displaces heat shock proteins bound to ER in cytoplasm
CreER migrates into the nucleus

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11
Q

why is tamoxifen ideal?

A

it does not interfere with androgynous ER

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12
Q

what sort of promoter is YFP placed under?

A

one that is ubiquitously expressed

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13
Q

give an example of the promoter YFP may be placed under?

A

collagen

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14
Q

what is floxed upstream of the YFP but before its promoter?

A

a stop codon

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15
Q

when CreER is expressed and localised to the nucleus, what occurs?

A

Cre recombinase removed the floxed stop codon and YFP is expressed

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16
Q

why do we need to do control with this CreER system?

A

to ensure that oestrogen does not give us any false positives

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17
Q

what type of cells will be genetically labelled at the time of the tamoxifen pulse?

A

all cells expressing Sox2

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18
Q

why are the progeny of these cells labelled?

A

a genetic recombination event has occurred, this is not reversible

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19
Q

how do we know if we have targeted the stem cells?

A

look at 18 months
if we targeted a progenitor we will no longer see any labelled cells
if we targeted a stem cell then we will still see labelled stem cell and progeny

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20
Q

what does sox2 deletion lead to?

A

short and long term tissue failure

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21
Q

if the tissue fails when all Sox2+ cells are killed what does this mean?

A

Sox2 is a marker for stem cells

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22
Q

when a cell expresses thymidine kinase what is it sensitive to?

A

ganciclovir

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23
Q

where is thymidine kinase inserted in the genome?

A

at the Sox2 locus for one allele

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24
Q

what happens when ganciclovir is administer?

A

all cells expressing Sox2, and thus thymidine kinase, will die

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25
by using a suicide gene, how can we prove we have marked all stem cells?
if we eliminate the stem cells from a tissue then we will lose more and more cells from a tissue over time
26
how many passages can primary cell last?
only survive one/a few passages
27
what are primary cells?
cells derived straight from a donor
28
which type of cells produce lots of variation?
primary cell lines
29
how are cell lines obtained?
through extensive culturing
30
which type of cells are more or less homogenous?
cell lines
31
what does the accumulation of mutations in cell lines allow them to do?
survive many passages
32
what is the problem with cell lines?
they are inherently different from the tissues that they initially came from
33
describe two ways that BM can be obtained?
1. crushing bone and filtering away the debris | 2. cut the ends of tibias/femur and use syringe to flush out inside
34
when BM is cultured name two cell types that are observed?
haematopoietic stem cells and BM stroma cells
35
which cells seen in the BM adhere to tissue culture plastic?
BM stroma cells
36
describe the appearance of HSC?
small and round cells
37
describe the appearance of BM stroma cells?
large and fibroblast like
38
what is a sphere formation assay?
cells are grown in 3D culture to assay their clonogenic growth potential
39
in the sphere formation assay, what can only stem cells give rise?
serial spheres
40
what are the pros of the sphere formation assay?
it can be used to gain an indication that there are stem cells in a poorly characterised tissue
41
what are the cons of the sphere formation assay?
it is hard to control cells once in the sphere and so they may start to differentiate
42
what are organoids?
a small simplified version of an organ produced in vitro in 3D, that shows realistic micro-anatomy
43
what are the first organoid of?
mice intestine - with cells obtained from the crypt
44
once cells were obtained from the crypt what was done to obtain organoids?
specific growth factors and media, and then allowed to organise themselves
45
what is a colony formation assay?
an assay to see a single cells ability to grow into a colony (of at least 50 cells)
46
why are cells seeded at a low density in the colony formation assay?
so that they are sufficiently far apart to not aggregate and so each colony will be derived from a single cell
47
what does the size of the colony indicated in a colony formation assay?
information on the nature of the cell of origin i.e. stem cell, progenitor or differentiated post mitotic cell
48
what does the number of large colonies indicate in a colony formation assay?
the number of stem cells in a tissue
49
what does very few, small colonies indicate in a colony formation assay?
the tissue contained very few stem cells
50
what can cerebral organoids be used to model?
human brain development and microcephaly
51
what cells were used to make cerebral organoids?
iPSC
52
what two things is the in vitro differentiation assay used to evaluate?
1. the differentiation potential of a distinct cell population 2. the role of genes/compounds in regulating differentiation
53
what can mesenchymal stromal cells be differentiated into in vitro?
osteoblasts, chondrocytes and adipocytes
54
what us time lapse microscopy?
the observation of live cells over a long period of time in vitro
55
what can time lapse microscopy be used for?
to monitor differentiation and allow us to see how cells make lineage decisions
56
what do in vitro assays allow us to do?
work with human cells
57
what is the down side of in vitro assays?
they do not necessarily reflect what occurs in vivo
58
what does a transplantation assay show?
the proliferative and multi-lineage potential of cells in vivo
59
how can we determine the potency of a stem cells in vivo?
by adding them to a tissue that has its stem cells removed
60
what is done to transplanted cells so that they can be recognised?
they are genetically marked, e.g. with a fluorescent protein, so that them and their progeny can be observed
61
what are nude mice?
immunocompromised bald mice
62
what do epidermal stem cells give rise to when transplanted into a nude mouse?
skin and the hair follicles
63
what can reconstitute the bone marrow of a lethally irradiated mouse?
a single HSC
64
what is graft-versus-host disease?
when an allogenic bone marrow/peripheral blood is transplanted into recipient and starts attacking recipient tissue
65
give an example of grafts-versus-host disease?
when BM from a white mouse is transplanted into lethally irradiated black mouse
66
what two common reported genes are used in lineage tracing?
1. fluorescent probes | 2. beta-galactosidase
67
In lineage tracing, how do you ensure that the reporter gene is only expressed in certain cell types?
put CreER under the control of a tissue specific promoter
68
what does lineage tracing rely on?
the availability of cell-population specific promoters
69
what type of experiment allows us to follow lineage specification in physiological conditions?
lineage tracing
70
how are confetti mice generated?
when Cre recombinase is activated there is a random recombination of a number of different fluorescent proteins
71
why is it useful for different stem cells to be labelled in an infinite number of colours?
single stem cells in complex tissues can be followed
72
what are transposons?
a sequence of DNA that can jump from one location to another
73
what is transposases?
an enzyme that can recognise the end of transposon sequence and catalyses its move to another location
74
in genetic bar coding, what is put upstream of transposase?
a tet-responsive promoter
75
what activates the expression of transposase?
activated rtTA binding to tet-responsive promoter
76
what needs to be administered in order to active rtTA in genetic barcoding?
doxycycline
77
in genetic barcoding, what makes each cell individual?
the transposon can be inserted at any location in the genome
78
how can the progeny of a specific cell be identified in genetic barcoding?
sequencing DNA to look for a specific 'cell barcode'
79
why must loci found over and over again in genetic barcoding be ignored?
transposons may insert at insert at one loci more than others, meaning that this barcode will not be specific to a certain cell

SCRA (19 decks)

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