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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
Q

by using a suicide gene, how can we prove we have marked all stem cells?

A

if we eliminate the stem cells from a tissue then we will lose more and more cells from a tissue over time

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26
Q

how many passages can primary cell last?

A

only survive one/a few passages

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27
Q

what are primary cells?

A

cells derived straight from a donor

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28
Q

which type of cells produce lots of variation?

A

primary cell lines

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29
Q

how are cell lines obtained?

A

through extensive culturing

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30
Q

which type of cells are more or less homogenous?

A

cell lines

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31
Q

what does the accumulation of mutations in cell lines allow them to do?

A

survive many passages

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32
Q

what is the problem with cell lines?

A

they are inherently different from the tissues that they initially came from

33
Q

describe two ways that BM can be obtained?

A
  1. crushing bone and filtering away the debris

2. cut the ends of tibias/femur and use syringe to flush out inside

34
Q

when BM is cultured name two cell types that are observed?

A

haematopoietic stem cells and BM stroma cells

35
Q

which cells seen in the BM adhere to tissue culture plastic?

A

BM stroma cells

36
Q

describe the appearance of HSC?

A

small and round cells

37
Q

describe the appearance of BM stroma cells?

A

large and fibroblast like

38
Q

what is a sphere formation assay?

A

cells are grown in 3D culture to assay their clonogenic growth potential

39
Q

in the sphere formation assay, what can only stem cells give rise?

A

serial spheres

40
Q

what are the pros of the sphere formation assay?

A

it can be used to gain an indication that there are stem cells in a poorly characterised tissue

41
Q

what are the cons of the sphere formation assay?

A

it is hard to control cells once in the sphere and so they may start to differentiate

42
Q

what are organoids?

A

a small simplified version of an organ produced in vitro in 3D, that shows realistic micro-anatomy

43
Q

what are the first organoid of?

A

mice intestine - with cells obtained from the crypt

44
Q

once cells were obtained from the crypt what was done to obtain organoids?

A

specific growth factors and media, and then allowed to organise themselves

45
Q

what is a colony formation assay?

A

an assay to see a single cells ability to grow into a colony (of at least 50 cells)

46
Q

why are cells seeded at a low density in the colony formation assay?

A

so that they are sufficiently far apart to not aggregate and so each colony will be derived from a single cell

47
Q

what does the size of the colony indicated in a colony formation assay?

A

information on the nature of the cell of origin i.e. stem cell, progenitor or differentiated post mitotic cell

48
Q

what does the number of large colonies indicate in a colony formation assay?

A

the number of stem cells in a tissue

49
Q

what does very few, small colonies indicate in a colony formation assay?

A

the tissue contained very few stem cells

50
Q

what can cerebral organoids be used to model?

A

human brain development and microcephaly

51
Q

what cells were used to make cerebral organoids?

A

iPSC

52
Q

what two things is the in vitro differentiation assay used to evaluate?

A
  1. the differentiation potential of a distinct cell population
  2. the role of genes/compounds in regulating differentiation
53
Q

what can mesenchymal stromal cells be differentiated into in vitro?

A

osteoblasts, chondrocytes and adipocytes

54
Q

what us time lapse microscopy?

A

the observation of live cells over a long period of time in vitro

55
Q

what can time lapse microscopy be used for?

A

to monitor differentiation and allow us to see how cells make lineage decisions

56
Q

what do in vitro assays allow us to do?

A

work with human cells

57
Q

what is the down side of in vitro assays?

A

they do not necessarily reflect what occurs in vivo

58
Q

what does a transplantation assay show?

A

the proliferative and multi-lineage potential of cells in vivo

59
Q

how can we determine the potency of a stem cells in vivo?

A

by adding them to a tissue that has its stem cells removed

60
Q

what is done to transplanted cells so that they can be recognised?

A

they are genetically marked, e.g. with a fluorescent protein, so that them and their progeny can be observed

61
Q

what are nude mice?

A

immunocompromised bald mice

62
Q

what do epidermal stem cells give rise to when transplanted into a nude mouse?

A

skin and the hair follicles

63
Q

what can reconstitute the bone marrow of a lethally irradiated mouse?

A

a single HSC

64
Q

what is graft-versus-host disease?

A

when an allogenic bone marrow/peripheral blood is transplanted into recipient and starts attacking recipient tissue

65
Q

give an example of grafts-versus-host disease?

A

when BM from a white mouse is transplanted into lethally irradiated black mouse

66
Q

what two common reported genes are used in lineage tracing?

A
  1. fluorescent probes

2. beta-galactosidase

67
Q

In lineage tracing, how do you ensure that the reporter gene is only expressed in certain cell types?

A

put CreER under the control of a tissue specific promoter

68
Q

what does lineage tracing rely on?

A

the availability of cell-population specific promoters

69
Q

what type of experiment allows us to follow lineage specification in physiological conditions?

A

lineage tracing

70
Q

how are confetti mice generated?

A

when Cre recombinase is activated there is a random recombination of a number of different fluorescent proteins

71
Q

why is it useful for different stem cells to be labelled in an infinite number of colours?

A

single stem cells in complex tissues can be followed

72
Q

what are transposons?

A

a sequence of DNA that can jump from one location to another

73
Q

what is transposases?

A

an enzyme that can recognise the end of transposon sequence and catalyses its move to another location

74
Q

in genetic bar coding, what is put upstream of transposase?

A

a tet-responsive promoter

75
Q

what activates the expression of transposase?

A

activated rtTA binding to tet-responsive promoter

76
Q

what needs to be administered in order to active rtTA in genetic barcoding?

A

doxycycline

77
Q

in genetic barcoding, what makes each cell individual?

A

the transposon can be inserted at any location in the genome

78
Q

how can the progeny of a specific cell be identified in genetic barcoding?

A

sequencing DNA to look for a specific ‘cell barcode’

79
Q

why must loci found over and over again in genetic barcoding be ignored?

A

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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