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SCRA > 7. bone marrow haematopoietic and mesenchymal stem cells > Flashcards

7. bone marrow haematopoietic and mesenchymal stem cells Flashcards

1
Q

when does haematopoiesis occur in development and how does it occur?

A

it occurs very early on in development and occurs in waves

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2
Q

where are HSC initially produced in development?

A

yolk sac - this is outside the embryo

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3
Q

can HSC from yolk sac engraft adult mice?

A

no - they are therefore not equivalent to adult HCs

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4
Q

where and how are the first definitive intra-embryonic HSC are generated? and can these cells engraft recipients?

A

the floor of the dorsal aorta
>epithelial cells line aorta
>some of these cells changes and become haematopoietic
>HSC shed into aorta and go into circulation
>these cells can engraft recipients and reconstitute haematosis

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5
Q

why is the first definitive intra-embryonic HSC generation hard to see?

A

it happens very early on in development

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6
Q

where does haematopoiesis occur during embryonic development?

A

in the liver

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7
Q

how do foetal liver HSC compare to adult HSC?

A

they are much more proliferative but can still engraft adult mice and so are equivalent to adult HSC

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8
Q

later on there is a switch from the foetal liver to what, and what is known about this?

A

to the bone marrow – not much is known about this switch

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9
Q

when does this switch to the BM occur?

A

as soon as their is a bone cavity for HSC to colonies

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10
Q

by the time the baby is born where is haematopoiesis taking place?

A

entirely in the bone marrow

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11
Q

why are zebrafish good for studying embryonic development?

A

they are are transparent and so it is easy to see cells that are fluorescent

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12
Q

what can be used in order to observe biological systems in vivo at high resolution?

A

intravital microscopy

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13
Q

intravital microscopy was used to observe what happening in zebrafish?

A

it was used to see HSC being born in the embryo

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14
Q

what was seen to happen in zebrafish before cells leave the epithelium of the aorta?

A

the cells either side close in on it to maintain the vessel

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15
Q

what was seen happening in the aorta of zebrafish that we do not think occurs in mice and humans? comment on this

A

cells do not bud into the aorta, instead they bud out through the mesenchyme
>we have never been able to observe this in mice in vivo and so we cant say this doesn’t happen in mice
>what we have observed from sections may be artefacts
>t may genuinely just be differences between organisms

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16
Q

what things are similar between zebrafish and mice HSC and the aorta?

A

> endothelial cells that become haematopoietic
both come from the same place in the aorta
similar molecular signals

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17
Q

what can we not say about these cells viewed in the intravital microscopy of zebrafish?

A

whether they are HSC or HC
>until we obtain them and transplant them we cannot say they are stem cells
>reprogramming of endothelial might be just to haematopoietic cells and only some are then further reprogrammed to stem cells

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18
Q

what has been shown to be important when these HC bud from the aorta?

A

circulation (stress due to plasma and blood circulating the aorta) - endothelial cells will only bud if they sense this
>production of HC is less efficient if they don’t sense this pressure

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19
Q

if we understand the mechanism for that is going on here where endothelial cells become haematopoietic, what can this be used for?

A

we can use this in the lab to generate HSCs

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20
Q

what different lineages can HSC become?

A

lymphoid lineages and myeloid lineages

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21
Q

what is challenged fairly regularly in the literature? and why is this?

A

HSC differentiation hierarchy

>differentiation can occur in this way, but there are also other ways that it can occur

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22
Q

give some example of how this haematopoiesis hierarchy has been challenged?

A

there may be some MMP that only give rise to one cell type = common myeloid progenitors may not exist, they may already be primed to go down certain lineages

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23
Q

flow cytometry with lots of markers identifies CMPs, this population of cells is very homogenous, unlike CLP and GMP that cluster close together, transcriptome analysis revealed how many clusters in CMP?

A

19 sub populations

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24
Q

classically define CMP are highly heterogeneous, what do individual CMP yield? and where does a true common progenitor between these cells types lie?

A

most individual CMPs yield either erythrocytes or myeloid cells
>in the MMP pool

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25
Q

name three proteins involved in HSC self-renewal, and what function do they have?

A

HoxB4 - high levels to maintain SR

Gfi1 - negative regulator of HSC proliferation

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26
Q

what happens when Gfi1 is KO in HSC?

A

they will proliferate more

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27
Q

what transcription factors Leads to HSC proliferation and subsequent differentiation? and what does it regulate?

A

c-Myc

>it regulates the expression of a number of adhesion molecules

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28
Q

what are the two hypothesis regarding c-Myc and changes in adhesion molecule expression

A
  1. HSC upregulate c-Myc and this changes how they interact with environment, they move away from the niche and differentiate
  2. stem cell proliferate and progeny may be pushed out the niche and this results in c-Myc upregulation
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29
Q

name three proteins that are involved in quiescence and what else are they involve in?

A

p21
p27
p16
preventing ageing

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30
Q

what happens when p21 is KO in mice?

A

they rapidly lose HSC due to their fast proliferation and exhaustion

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31
Q

what happens when p27 is KO in mice?

A

this does not affect HSC but they have an expanded pool of progenitors

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32
Q

what happens when p16 is KO in mice?

A

reduced number of HSC in young mice, but rescues HSC ageing in old mice

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33
Q

name three chromatin modifiers important in maintaining HSC

A

Bmi-1
Mll
Dicer
PTEN

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34
Q

what is Bmi1 and what happens when it is KO?

A

Is a polycomb gene necessary for maintenance of LT-HSCs

>when KO HSC are rapidly lost

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35
Q

describe Mll KO mice?

A

> no phenotype observed

>this BM cannot reconstitute mice - this is very stressful on cells

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36
Q

name another stress that can be used to test HSC? and what might these stresses show?

A

> chemotherapy, this kills all proliferating cells, pushing HSCs to proliferate in response
inflammatory signals to mimic infection, this is known to activate HSC
they may bring out a phenotype that is otherwise not visible

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37
Q

what does being able to KO Mll and having no obvious phenotype indicate?

A

the haematopoietic system is very robust

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38
Q

what is for keeping HSC persistent in vivo?

A

Dicer

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39
Q

what does Dicer do?

A

processes miRNA

40
Q

name a particular miRNA that control HSC apopotosis and what does it do?

A

mir-125a

this down regulates pro-apoptotic genes such as Bak1

41
Q

what is indispensable for HSC maintenance but not for leukaemia maintenance?

A

PTEN

42
Q

why is it useful in therapeutics to identify molecule mechanism that cancerous cells and non-cancer cells vary on?

A

this makes cancerous cells easier to target over non cancerous cells

43
Q

give an example of a protein needed by leukaemia and no by HSC

A

CD44
>when KO HSC can still engraft
>leukaemia cells less good at engrafting

44
Q

what did GWTA on HSPCs reveal? and what does this mean?

A

a densely interconnected network of coding and non-coding genes
>we need to be able to understand this in order to be able to manipulate it
>if we affect one gene and have an unexpected outcome this may be bc we have effected the balance of other genes and signalling

45
Q

describe a competitive BM transplant

A 
>BM from a donor and competitor mouse 
>donor will either be WT or KO 
>BM mixed 1:1 
>added to lethally irradiated host 
>peripheral blood checked every 4 weeks for 4 months
46
Q

what is expressed on all HC cells and what two forms does it come in? which mice have each form?

A

CD45
CD45.1 and CD45.2
the test mice are CD45.2 and the competitor and host mice are CD45.1

47
Q

what is the aim of the competitive transplant?

A

we want to see how well the KO cells are doing compared to control cells

48
Q

what happens when both donor and competitor as WT?

A

the fitness of the BM is the same

>they each reconstitute about 50%

49
Q

what might be see in a competitive transplant?

A

KO HSC might have better or worse fitness than WT

50
Q

how can the two types of cells be identified in competitive BM transplant?

A

using antibodies for CD45.1 and CD45.2

51
Q

what happens with the KO is much between or worse than the competitor?

A

the ratio may be changes from 1:1 to something like 10:1 for example

52
Q

what is the relationship between the immune system and HSC?

A

HSC give rise to immune cells and immune cells are part of many stem cell niches

53
Q

what is immune privilege and where can it be found?

A

these are sites where immune responses do not take place

>lots of niches are sites of immune privilege

54
Q

describe sites of immune privilege

A

lots of T reg cells that dampen down the immune response

55
Q

how do we know the HSC niche is a site of immune privilege?

A

BM from one mouse transplanted to another will be rejected. if we just take HSC, they will engraft and give rise to progeny, which once out the niche will be attacked. after a number of weeks, HSC are still seen in BM. they can be use to engraft the first mouse to show they are still HSC.

56
Q

what does the serial BM transplant test?

A

the self renewal capacity of HSC

57
Q

describe the serial BM transplant

A

donor mice BM out into WT primary recipient, wait 4 mounts, take the BM out and put it into secondary recipients. we wait for months and put this into tertiary recipients.

58
Q

what can be seen if the initial BM transplanted is WT?

A

survival of tertiary recipients

59
Q

if the initial BM that was transplanted in the primary mouse was from a transgenic mouse, what might be seen?

A

the mice may not survive as the BM did not engraft sufficiently for survival

60
Q

what can serial transplants highlight?

A

subtle phenotypes

61
Q

what is DKK1?

A

an inhibitor of Wnt signalling

>it interacts with frizzles receptor and competes with Wnt

62
Q

what happens when DKK1 is expressed on osteoblasts?

A

mice have no phenotype

>when BM used for serial transplant tertiary recipient do not survive as self renewal of HSC is affected

63
Q

what affect does dampening down Wnt signalling have on HSC? but beta catenin KO have no phenotype, why is this?

A

it reduces their self-renewal

>other signalling pathways can compensate for it

64
Q

what affect does GSK3 inhibitors have on HSC?

A

increase HSC ability to repopulated transplanted recipients by expanding progenitors and retaining HSC

65
Q

what is important for ability of stem cells to repopulate transplant recipients?

A

having a good level of Wnt signalling

66
Q

what does GSK3 inhibiting do to Wnt signalling? and what affect does this have on engraftment?

A

it increases Wnt signalling as beta-catenin is stabilised

>GSK3 inhibitions results in better engraftment

67
Q

how do osteoblast support HSC colony formation in vitro?

A

by producing haematopoietic growth factors

68
Q

what treatment expands to osteoblast compartment? and how does this affect HSC?

A

Parathyroid hormone treatment (PTH) acts as a bone forming agent
>bones with more active osteoblasts have lots more HSC

69
Q

what signalling regulates osteoblast number and HSC number?

A

BMP

70
Q

what happens when osteoblasts are ablated?

A

this severally alters BM haematopoiesis

the BM empties

71
Q

how can osteoblasts be ablated from mice?

A

thymidine kinase expressed in osteoblast cells. these cells are susceptible to ganciclovir treatment.

72
Q

when ganciclovir is given to thymidine kinase mice BM empties, what happens when ganciclovir treatment is stopped? and what does this suggest?

A

BM will repopulate

>this suggests that the ganciclovir left some very primitive HSC as the BM can repopulate

73
Q

what has been used for in vivo imaging of mouse HSC microenvironment?

A

intravital microscopy of the skull of mice

74
Q

we don’t have very good promoters for the haematopoietic system in general, even worse for HSC. how is this problem over come?

A

> isolate HSC from donor mice using flow cytometry and cell sorting
label cells ex vivo
inject them intravenously and look for them in recipient

75
Q

what did imaging of HSC homing to the BM after 5 hours show for non-radiated and irradiated mice?

A

> in non-irradiated mice you can see the vessel very well

>in irradiated mice radiation has affected vessels, dye used to stain vessels as leaked out

76
Q

if we irradiate the recipients we have engraftment whereas if we do not irradiate the recipients then we do not have engraftment . where are HSC in irradiated mice observed?

A

fairly close to osteoblasts

77
Q

there is a tight relationship seen between osteoblasts and what?

A

vessels

as soon as the bone cavity forms, vessels enter and along with HSCs

78
Q

in KO experiments where osteoblasts are affected, what are also affected?

A

the vessels

79
Q

what cells are preferentially localise near osteoblasts? and what was this made in comparison to?

A

HSC

MMP and committed progenitors

80
Q

how is the distribution of more differentiated cells through the BM?

A

they are more distributed in the BM – the distance from osteoblasts does not matter to them

81
Q

what was the initial working model of where HSC?

A

quiescent HSC residue near osteoblasts and their progeny migrate towards the central BM

82
Q

how have the ways we able

to asses where HSC are in the BM changes? and what do they now show?

A

> they used to all be based on injecting SC and following where they go
now we can take whole bone and do a variety of stains
they now show that lots of stem cells are in close proximity to the bone
combining this with single cell omics can allow for determining where certain populations of cells are preferentially found

83
Q

immunofluorescence on bone section using lineage makers shows HSC close to what and what did people suggest from this?

A

vessels

>there is an endosteal (vascular membrane that lines the bone cavity) niche and a vascular niche

84
Q

what is the most current working model of where HSC are found in BM?

A

sinusoids vessels are found very close to the endosteal that seem to be associated with HSC
>they are also surrounded by mesenchymal cells

85
Q

how is the BM vascularised?

A

> arteries come into the bone – they break into smaller and smaller vessels as they go towards the endosteal
sinusoids vessels are highly permeable after which the blood become venous

86
Q

what are the second type of stem cells found in the BM called? and what do they give rise to in vivo?

A

mesenchymal stem cells
>cells of the mesenchymal lineage
>bone, cartilage and fat in BM

87
Q

what can MSC give rise to in vitro?

A

tendons, ligaments, CNS, skin and muscle

in addition to bone, cartilage and fat

88
Q

it has been hard to identify precise markers of MSC, what is the best way to isolate them?

A

put BM cells in culture and select for cells that adhere to plastic well

89
Q

MSC have been shown to make more in culture than they do in vivo, what has been suggested from this? and what benefit might this have?

A

the fact that they can differentiate into more things may be a result of how we culture them. we might be able to use these cells for regenerative medicine.

90
Q

not a lot is known about MSC and how they differentiate, what are people starting to do more of now?

A

lineage tracing and identify key promoters

91
Q

describe three therapeutic applications of MSC

A
  • they improve BM engraftment
  • improve results of several therapies like stroke therapies
  • MSC have potent immuno-modulatory effects/anti-inflammatory function
92
Q

when MSC are used therapeutically what is seen and why?

A

when they are intravenously administered MSC are trapped in the lungs and spleen as they are not very good at engrafting

93
Q

in terms of having an anti-inflammatory effect of tissue, how can MSC be used therapeutically?

A
  • reduce inflammation so that repair can take place
  • they have been successfully applied to reverse Grafts-versus-host disease in patients receiving BM transplants
  • shown improvement in MS and ALS
94
Q

where do MSC have a tendency to home to?

A

sites of damage

95
Q

tumour can be regarded as wounds that never heal and MSC have been seen to home to sites of damage. how can this be used therapeutically?

A

MSC that are either de novo mobilised or exogenously administered have been found to migrate to tumours
>they can be engineered with tumour killing agents such as INFα, IFNβ, IL-12 and TRAIL
>MSC also being developed to deliver nanoparticles to enhance their tumoricidal effects

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