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Molecular Mechanisms > HSCs > Flashcards

HSCs Flashcards

1
Q

what is haematopoiesis? how long does it take?

A

The production of blood
- 11 days in mice
- 5 weeks in humans

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2
Q

what organs are involved in haematopoiesis?

A
  • aorta-gonad-mesonephros
  • fetal liver
  • yolk sac
  • placenta
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3
Q

what are the first stages of haematopoesis?

A

First stage of haematopoiesis occurs in yolk sac at day 7
- Formation of blood islands composed of haematopoietic cells and mature erythrocytes
- More poises in AGM - form myeloid and lymphoid lineages

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4
Q

what mouse model can be used to study the onset of haematopoiesis?

A

Use of KI/KO mice
- KI of RUNX1-LACZ mouse – HSCs produced in AGM and yolk sac.
- Generate mouse where runx1 promoter drives expression of LacZ (manipulate mouse genome)
- whenever runx1 is expressed, lacz is expressed
- stain for LacZ expression to see RUNX1
- Not all the cells positive for LacZ will be HSCs, but all the HSCs will be LacZ positive

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5
Q

how are HSCs established during haematopoiesis?

A

Process:
- Start with hemangoblast – precursor of vasculature or haematopoetic cells
- pre-HSC is formed from hemangoblast in yolk sac
- In AGM – maturation of HSCs = acquires HSC properties: self-renewal, engraftment, survival
- In foetal liver: expansion of HSCs
- Reach steady state: quiescence and self-renewing capacity of HSCs in the bone marrow (BM)

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6
Q

describe the haematopoietic hierarchy:

A

at the apex: most self-renewing
- LT, ST HSCs and MMPs (multi-potential progenitors)

middle: progenitors
- common myeloid, lymphoid, MEP (meg/e lineage), GMP (granylocyte/monocyte lineage), CMP
- these are highly proliferative and slightly more committed - less self-renewal capacity

bottom: mature, committed cells

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7
Q

is this haemotopoetic model accurate?

A

The process is more plastic than what the hierarchy shows
- scRNA-seq shows that it is not a classical model, but is non-linear
- Intermediates can shift to either fates

other studies have shown that hierarchical model doesn’t actually occur in foetal liver
Progenitors in foetal liver are not derived from HSCs

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8
Q

how can the self-renewal properties of HSCs be studied?

A

Lethally irradiate mice and inject bone marrow HSCs by IV
- Animal does not die, and recovers full blood – stem cell population given by IV reconstitutes full bone marrow

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9
Q

what are HSCs supported by?

A

the bone marrow niche
- contains stromal/mesenchymal cells
- These can differentiate to osteoblasts, chondrocytes and adipocytes

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10
Q

what cell types make up the bone marrow niche?

A
  • Some are hematopoietic e.g. megakaryocytes, macrophages, T cells
  • Osteoblasts
  • endothelial cells
  • Adipocytes
  • Non-myelinating schwann cells - sympathetic nerve fibres
  • perivascular cells
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11
Q

what is the role of the bone marrow niche?

A

These cell types provide cytokines and contact interactions which dictate fate of HSCs
- e.g. Non-myelinating schwann cells produce TGFb to induce quiescence
- E.g. adipocytes produce adiponectin to prevent HSC growth

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12
Q

what are the two types of bone marrow niche?

A
  1. endosteal niche
  2. perivascular (central niche)

both niches are important and coexist

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13
Q

what are the properties of the endosteal niche?

A

endosteal = close to bone
- 15% of HSCs are here – these are the most quiescent HSCs
- mostly made up of osteoblasts
- hypoxic
- high calcium - maintains quiescence
- provide long-term storage of HSCs for whole life to repopulate blood

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14
Q

what are the properties of the perivascular niche?

A

perivascular = outside of bone
- majority of HSCs are located here which are more proliferative
- mostly made up of endothelial cells
- normoxic
- less calcium
- short-term HSC storage to repopulate quickly upon injury/infection

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15
Q

how do the HSCs and osteoblasts interact in the endosteal niche?

A

via cellular interactions:
- Osteoblasts produce angiopoetin-1
- HSC has receptor for angiopoetin-1 called Tie2 expressed on their surface
- Tie2 expression defines LT-HSCs
- C-kit on HSC binds with SCF on osteoblast
- N-cad binds with N-cad

Interactions affect fate of HSC

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16
Q

what are the different fates of HSCs?

A
  1. quiescence
  2. apoptosis
  3. division
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17
Q

what are the 3 ways in which HSCs can divide?

A

Expansion – parental HSC makes 2 identical clones which are HSCs and can self-renew

Depletion – HSC doesn’t produce clones of HSC, both daughters are committed to a fate and are proliferative

Homeostatic self-renewal – one daughter is a cloned HSC, one daughter is more committed and proliferative

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18
Q

what support does the niche provide for HSCs?

A

self-renewal
quiescence
proliferation
engraftment
homing

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19
Q

what does dysregulation in haematopoiesis lead to?

A

Blood disorders

e.g. AML:
- Myeloid lineage affected e.g. CMP, GMP, MEPs
- leads to excessive proliferation
- self-renewal capacity where progenitors act as a leukaemic stem cell

e.g. ALL
- lymphoid progenitors can also become self-renewing

20
Q

how do malignant HSCs persist?

A

Treatments kill bulk of cells, but leukaemic stem cells can remain
- Malignant HSCs/progenitors change the niche to their own advantage

21
Q

how do malignant HSCs change their niche for survival?

A

Immune evasion – express PDL1 to switch off T cells

Deplete osteoblast and gain adipocytes from mesenchymal stem cells
- Adipocytes produce fatty acids to be uptaken for energy – allows cancer survival

Deplete sympathetic nerve fibres

Produce VEGF to induce angiogenesis

22
Q

how can HSCs be isolated by flow cytometry?

A

using cell surface markers:
1. KSL – 3 components of all HSCs (kit positive, sca-1 positive, lineage negative)
- Markers for LT, ST and MPP
- Lineage marker is a cocktail of markers of more mature cells

  1. SLAM – CD150 positive, CD48 negative
  2. Side population – Hoechst 33342 staining
23
Q

how can HSCs be isolated using KSL sorting?

A
  • Combine BM with cocktail of antibodies for lineage markers, sca-1 and kit
  • Lineage cocktail: Ter119 (erythrocyte), Mac1 (macrophage), B220 (B cell), CD5 and CD8a (T cell), Gr1 – mature cell markers, HSCs should be negative for these
  • Green fluorescence vs FSC (size of cell)
  • Gating must incorporate lineage negative population – HSCs are not committed so won’t express these lineage markers
  • Red rectangle gating made from lineage negative HSC population – positive for KIT and SCA1 - contains LT, ST and MPP HSCs - see lec slide 27
24
Q

how can LT, ST HSCs and MPPs be further defined?

A

CD34 and flt-3 define these HSC populations further:
- LT is negative for both
- ST is positive for CD34 and negative for flt-3
MPP positive for both

25
Q

how can HSCs be isolated using SLAM?

A

SLAM: Antibodies for CD48 and CD150
- CD150 positive and CD48 negative are the most long-term HSCs

26
Q

how can HSCs be isolated using side population (SP)?

A

Hoechst 33342 dye:
- HSCs express ABC transporters which efflux the dye

27
Q

can the flow cytometry markers be combined for HSC isolation?

A

yes: Can refine by using KSL with SP
- Cells at tip of SP are the HSCs which are more quiescent

28
Q

how can different colonies be identified following differentiation?

A

Colony assay
- take bone marrow and put cells in semi-solid medium
- Makes columns of cells – white spots
= Microscope can see how morphology is different between colonies
- CFU-E (erythrocytes) have haemoglobin – red so easily detected
- Sparse growth = monocytes
- Compact growth = granulocytes

Can look at how mutations affect different lineage growth/proliferation in the colony
- If colony isn’t forming, the mutated gene must be important

29
Q

how can the repopulation capacity of HSCs be studied?

A

bone marrow transplants using congenic mice which differ in their CD45 subtype allele
- host mouse is CD45.1 positive
- donor mouse is CD45.2 positive
- use antibodies to detect specific CD45 subtype alleles to see which cells were orginally in the animal, and which have been donated

30
Q

how is a repopulation capacity assay conducted?

A
  • Lethal irradiation of CD45.1 mouse
  • Inject donor CD45.2 BM cells via IV in the tail
  • Detect cells from donor and cells from host using antibodies for .1 and .2
  • Competitor/reference – cells from mouse which express both .1 and .2
31
Q

what is a competitor/reference mouse?

A

Competitor/reference mouse is a positive control which has HSCs expressing both CD45.1 and CD45.2
- Lethal irradiation of mice – deplete whole HSC population
- Control checks that the blood was injected into correct vein – don’t want to miss the vein
- Also keeps the animals alive

32
Q

what are the potential outcomes of the repopulation capacity study?

A

If CD45.2 cells only engraft, the mice will survive

If they die, we don’t know why – cells may be non-functional, or too much irradiation

33
Q

how can homing of HSCs be studied?

A
  • intrafemural transplants
  • immunofluorescence
  • IVIS imaging
  • intravital microscopy
34
Q

how are intrafemural transplants used to study homing of HSCs?

A

Intrafemural transplants:
- lethally irradiate mouse
- inject donor CD45.2 HSCs into the right knee
- isolate bone marrow cells (CD45.1/CD45.2) and KSL cells (CD45.2)
use flow cytometry to analyse

35
Q

what are the possible outcomes of HSC intrafemural homing studies?

A

Injection into right knee:

  • If cells aren’t seen in the bone marrow of the right knee…
    There is a problem with BM retention

If in right knee but not left knee…
- Problem with migration/homing

36
Q

how can we tell that there are issues in HSC homing in intrafemural studies?

A

If the CD45.2 cells of interest are not there, but the reference cells are there, then the gene of interest that is K/O must be important for homing to the bone marrow from the blood

Or cells are not reaching bone marrow from IV injection

Bone marrow produces cytokines for chemotaxis of cells – gene may impact cell reception of chemokines

37
Q

how can immunofluorescence be used to study HSC homing?

A

Inject with IV, sacrifice mouse after 18 hours, take BM section, immunofluorescence study

Stain HSCs with CD150 = red
Mesenchymal cell: Nesting-GFP = green
Lineage cocktail and CD48 = blue – excludes differentiated, mature cells
Determine if red HSCs can migrate to endosteal niche

38
Q

how can IVIS imaging be used to study homing?

A

Anaesthetise mice
- Inject cells with luciferin - glow
- can see luciferase glow in thymus and spleen

39
Q

how can intravital microscopy be used to study homing?

A

Record movement of HSCs in vivo migrating into osteoblasts
- Determine timing of movement
- Compare to leukaemic cells for example

40
Q

how can a conditional knockout be generated?

A

Conditional KO – induce mutation whenever wanted in specific tissue of interest, for example, c-myc:
- Cre enzyme recognises palindromic sequence called floxP which has 2 loxP sites either side of gene of interest – Cre excises DNA in between loxP sites to create a KO
- Cre only expressed in presence of interferon – induces KO under interferon conditions
- Crossed MxCre mice with loxP-cMyc-loxP mouse in intron 1 after exon 3
- Cre deletes cMyc – K/O

41
Q

how is c-myc implicated in haematopoesis?

A

When c-myc is K/O, bones of animal were paler = little haematopoises occuring
- Mutant organs lack HSCs population – low HSC count over weeks post c-myc deletion

KI67 is marker for quiescence:
- Only proliferative cells will be positive for KI67, whereas HSCs will be negative
- More quiescent cells when c-myc was K/O

Lineage cocktail with KIT and SCA1:
- Increased lineage negative cells with c-myc KO
- KSL cells increased under c-myc KO

42
Q

how does c-myc expression change during division of HSCs?

A

Interactions with N-cad, ICAM, integrins which maintain cells in quiescence, where they express low levels of cmyc
- Cell divides via mitogenic signals:
- Expansion – 2 daughter HSCs which have low c-myc levels - Homeostatic – 1 daughter cell has low c-myc, other is more committed with more c-myc expression
- Depletion – both daughter cells are committed with high c-myc

43
Q

how is c-myc implicated in blood cancer?

A

LT HSCs have low c-myc:quiescent and self-renewing
- Mitogenic signal leads to high c-myc expression in LT HSC, so it becomes ST HSC
ST HSCs undergo expansion of c- myc for proliferation - maturing of cells means c-myc is switched off

in c-myc K/O mice:
- LT-HSCs continue to expand and cannot mature to MPPs as c-myc levels are too low
- haematopoesis cannot progress, leading to cancer at level of too many LT-HSCs

in high c-myc expression:
- LT-HSCs continue to differentiate and are depleted
- super expansion of progenitors which cannot switch off c-myc as it is overexpressed
- cant produce mature cells - cancer at level of too many progenitors

44
Q

how are sympathetic nerve fibres important for HSC niche?

A

Schwann cells surround HSC niche to help against myeloproliferative neoplasms (high number of mature cells which don’t function)
- Mutation in JAK2 in 80% cases
- humanised mouse with JAK2 mutant shows reduced mesenchymal stem cells and absence of sympathetic fibres and schwann cells

JAK2 mutant depletes niche

45
Q

how can myeloproliferative neoplasms be treated to restore sympathetic nerve fibres in the HSC niche?

A

beta3-adrenergic agonist can recover schwann cells
- mature cell levels come down to baseline and are now functional
- fibrosis is reduced when treated

46
Q

how can sympathetic nerve fibres be destroyed in the HSC niche? how can this be treated?

A

Mutant HSCs produce IL-1b which destroy the sympathetic fibre glial cells
Inflammation destroys mesenchymal stem cells – cytokines cause fibrosis and gliois

B3-adrenergic agonist protects glial cells
- More CXCL12 to recover normal haematopoiesis

47
Q

how has studies of HSCs advanced?

A

use of bone marrow organoids in 3D derived from iPSCs
- See interactions of components in the niche between leukaemic stem cells and normal HSCs

Molecular Mechanisms (8 decks)

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