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Stem cells Flashcards

1
Q

What is a totipotent cell and when is this?

A

A cell that can produce an entire organism. Only a zygote and blastomere from 2-8 cell stage embryo belong to this category

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2
Q

What is a pluripotent cell?

A

A cell that is able to give rise to derivatives of all three germ layers –> Embryonic stem cells (=epiblast). NOT extra-embryonic tissue.

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3
Q

What is a multipotent cell?

A

A cell that can give rise to multiple cell types, but that is restricted to a specific sublineage or argan.
Most adult/somatic stem cells belong to this category.

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4
Q

What is a unipotent cell?

A

A cell that can give rise to only one cell type

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5
Q

What is the zona pellucida and its function?

A

The zona pellucida is a glycoprotein layer surrounding the plasma membrane of mammalian oocytes –> remains until implantation into the womb
Function: protection of oocyte.

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6
Q

What are cumulus cells and what is its function?

A

Cumulus cells are somatic cells surrounding the oocyte. They interact with the oocyte which is crucial for the maturation of the oocyte.

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7
Q

How can genetic testing of the embryo be done?

A

Before implantation one cell can be removed for genetic testing. Cleavage stage embryos are flexible and this does not harm the embryo.

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8
Q

What is the morula?

A

Stage of embryo where the first differentiation happens. Difference between the inner and outer cells –> Inner cells only has different cells as neighbour while the outer cells have an inner and outer cells as neighbour.

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9
Q

What is activated in the outer cells but not in the inner cells and how does this happen?

A

Outer cells: YAP is a transcription factor for cdx2

Inner cells: YAP is phosphorylated and destroyed.

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10
Q

What are markers for the trophectoderm and ICM?

A

Trophectoderm: Cdx2

Inner cell mass: Oct4

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11
Q

What are markers for the epiblast (EPI) and primitive endoderm/hypoblast (PE)?

A

Epiblast: Nanog

Primitive endoderm: Gata6

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12
Q

What does MAPkinase?

A

MAPK induces Gata6 expression and represses Nanog

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13
Q

What will the primitive endoderm form and what is its function?

A

PE will form the yolksac which is important for providing nutrients and oxygen in the embryo.

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14
Q

How can it be tested that a cell is totipotent?

A

1 cell can give rise to whole organism

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15
Q

How can it be tested that a cell is pluripotent?

A

1) Use chimaera
Mix potential pluripotent stem cells with ICM of embryo and implant in womb - see if potential stem cells form all germ layers.
For humans: to this in an immunocompromised mouse and a tumor will be formed. If cells are pluripotent is will be teratome (= tumor consisting of three germ layers)

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16
Q

What happens in the different weeks of development?

A

Week 1: Embryo reaches uterus and implants
Week 2: Embryo grows into many cells
Week 2-4: Placentation - exchange gas and nutrients between fetus and mother

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17
Q

What are developmental difference between humans and other animals?

A
  • Placenta looks different
  • Implantation in humans is very invasive - is not the case in all animals
  • In the mouse the location of amnion, embryo and yolk sac is different.
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18
Q

What is nuclear reprogramming (also called nuclear transfer) ?

A

1) Take oocyte and remove nucleus
2) Take nucleus from somatic cell
3) Combine this oocyte and nucleus
4) Activate oocyte with small electrical pulse or chemically –> cause calcium flow that activates active cell division
5) Embryo will form
6) Place embryo in pseudopregnant mother
7) Clone of animal from which nucleus of somatic cell is taken will form.

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19
Q

What does nuclear reprogramming show?

A

That an adult cells:

1) Contains all the information of a complete animal
2) The information can be retrieved by putting the nucleus in egg cytoplasm

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20
Q

What is needed for reprogramming of somatic cells to a pluripotent cells and how are these cells called?

A

Factors: Oct4, Sox2, Klf4 and C-myc

Cells are called induced pluripotent stem cells (iPS cells)

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21
Q

What are pros and cons of doing somatic-cell nuclear transfer and making iPS cells (=direct reprogramming)?

A 
Somatic-cell nuclear transfer:
- Many oocytes needed
- Ethically debatable
- Very complex
iPS cells: 
- Can be done with all differentiated cells
- Inefficient
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22
Q

What is the difference between ESC and EpiSC?

A

ESC lines are from pre-implantation

EpiSC are from post-implantation

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23
Q

What are the properties of embryonic stem cells:

A

1) Self-renewal (longevity)

2) Able to differentiate into all germ layers (multipotency)

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24
Q

What can be used to culture ESC in vitro?

A

1) Use fibroblast feeder cells –> support the undifferentiated propagation of stem cells
2) Use Leukaemia inhibitory factor (LIF) in medium with serum –> serum contains BMP

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25
Q

What does LIF do?

A

Enables ESC self-renewal by inhibiting differentiation. Does this through phosphorylation of the transcription factor STAT3 which translocates to the nucleus and activates genes.

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26
Q

How do ESC look like in vitro?

A

Dense colony, big nuclei, little cytoplasm, prominent nucleoli

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27
Q

What does the bone morphogenetic protein (BMP) do?

A

BMP can block neural differentiation by inducing transcription of Id genes.

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28
Q

What do Id proteins do?

A

Id proteins sequester (=isolate) ubiquitous pro-neural bHLH proteins into non-functional dimers.

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29
Q

What do LIF & BMP together do?

A

Block germ layer differentiation.
BMP –> induce Id genes –> block neuroectoderm differentiation
LIF –> induce Stat3 genes –> block mesoderm and endoderm differentiation

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30
Q

What are the core pluripotency transcription factors in ESCs and what is their structure?

A

Nanog, Oct4 and Sox2
Consist of:
- DNA binding domain (HD, HMG, POU)
- Transactivating domain (TAD)

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31
Q

What happens when Oct4 OR Sox2 is deleted?

A

Trophectodermal differentiation –> cannot specify ICM

THUS both essential for specification of pluripotency in vivo

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32
Q

What do OCT & Sox recognize and how is their relative orientation?

A

Oct and Sox recognize palindromic DNA sequences
Their orientation is next to each other —> —>
Probably because side-chains of S domain of Oct bind to side chains of HMG domains of Sox
–> TF Oct and Sox act combinatorially to direct gene expression

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33
Q

What does nanog do?

A
  • Suppress differentiation of TE and PE
  • Induce efficiency of self-renewal
  • -> Nanog is not essential - ESCs without Nanog can self-renew but with reduced efficiency
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34
Q

How are Oct4 and Sox2 expressed and how is nanog expressed?

A

Oct4 and Sox2 are homogenous expressed

Nanog is heterogenous expressed

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35
Q

What happens to the cell fate at different concentration levels of Oct4?

A

Low concentration Oct4 –> trophectoderm
Intermediate concentration –> pluripotent stem cells
High concentration –> endoderm or mesoderm.

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36
Q

What can FGF do?

A

FGF can drive loss of naive pluripotency

–> Fgf4 is an Oct/Sox target gene that is expressed in ESCs.

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37
Q

What is the mechanism of action of FGF?

A
  • FGF binds to FGFR
  • Activated FGFR activates Ras
  • Ras stimulates a phosphorylation cascade operating via MEK/ERK kinases
  • -> erk2-null ESCs have impaired differentiation capacity.
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38
Q

What does the inhibition of MEK and GSK3B do?

A

It circumvents the ESC requirement for BMP

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39
Q

What is ‘2i /LIF’ and what happens?

A

In 2i/LIF a combination of MEK inhibition, GSK3B inhibition and LIF is used for maintaining pluripotency of ESCs.
ESC are more rounded, clearly differentiated cells are absents, expression of Nanog is homogenous.
–> ESCs are trapped in a so-called ‘Ground State; in which pre-implantation specific TFs are robustly expressed.

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40
Q

How can pluripotency be resuscitated (make active again)?

A

By Oct4 expression.

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41
Q

What are the difference between monogenic and sporadic diseases?

A 
Monogenic:
- Rare
- Clear genetics
- Often display robust phenotype
Sporadic, late onset diseases:
- Common and medically most relevant
- No clear genetic basis
- Likely a more subtle phenotype
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42
Q

What can ESC be used for?

A

To study development and disease

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43
Q

What can iPS cell be used for and what is an issue with this?

A

For disease modeling, drug screening and cell therapy
For example:
Take control iPS cell and iPS cells from patient with ALS and perform chemical screening
Issue: control cells for iPS
There are unpredictable difference between individual iPS cells due to genetic background. Is a subtle phenotype disease related or rather due to the system-immanent iPSC-iPSC variation?

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44
Q

What is the solution for the issue of control cells for IPSC?

A

Isogenic control with identical genetic background.
Get iPSC from 1 person
Two options:
1) take healthy iPSCs and induce disease
2) take disease iPSCs and repair mutation with Crispr/Cas

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45
Q

How to use isogenic control in monogenic diseases and how in sporadic diseases?

A

Monogenetic: Correct the mutation in sick iPSCs or induce mutation in healthy iPSCs.
Sporadic: –> is combination of age, genetic susceptibility, environmental risk and positive family history –> first identify risk loci in GWAS
When risk loci is identified then correct or induce mutation .

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46
Q

What is the problem for gene targeting/editing in humans?

A

Homologous recombination is very inefficient in human ES / iPS cells –> Crispr/Cas already improved this a lot

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47
Q

What are the issues of gene editing in the embryo/germ line?

A

1) Scientific issues

2) Ethical issues

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48
Q

What are possible applications of gene editing in the human embryo/germ line and its issues ?

A

1) Correction of disease causing mutation
Issues: Any manipulation will alter genes in 50% (dominant genes) or 75% (recessive genes) of normal embryos –> cannot distinguish mutant and normal embryos by genotyping.
2) Disease resistance by inactivation of a susceptibility gene or expression of protective gene
Alternative: specific tissue cells can be manipulated in postnatal individuals by somatic gene editing –> thus, germ line editing may not be needed
3) Enhancement –> Example insertion of growth hormone gene
Ethical issues

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49
Q

What is Waddington’s epigenetic landscape?

A

Illustrates a very potent cell that is on top of the hill and goes down while making choices. This reduces the amount of possibilities –> when choice is made it cannot go back. When the ball is at the bottom of the hill it represents a differentiated cell.

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50
Q

Which ways of epigenetic regulation are there?

A
  • Accessibility of genes is in a heritable fashion.
    Modification of chromatin in 4 ways:
    1) DNA methylation
  • Double configuration - on both strands
    2) Histone modifications or use of special histone variants
    3) Association of ATP-dependent complexes (polycomb/tritorax proteins)
  • Change distance between nucleosomes
    4) Association with non-coding RNAs
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51
Q

What are important DNA methyltransferases and what do they do?

A

DNMT3a and DNMT3b
Both involve in de novo methylation - not known how methylation pattern is determined

DNMT1
Involved in maintenance of methylation during cell division

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52
Q

How does DNA demethylation occur?

A

Not sure yet but two hypothesis:

1) Passively in the absence of DNMT1
2) Actively by among others ten-eleven translocation (TET) enzymes –> transfer to hemi methylated (5hmC) and then demethylate

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53
Q

What is a big difference between histone and DNA methylation?

A

DNA methylation involves many enzymes while histone methylation only involves a few enzymes.

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54
Q

What is euchromatin and what is heterochromatin?

A 
Euchromatin = very open --> gene expression 
Heterochromatin = closed --> gene silincing
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55
Q

How is the X chromosome silenced?

A

1) Xist (and Tsix) bind to DNA –> non coding RNA.
2) Attract polycomb complexes
3) This puts the mark H327me3 on gene
4) Histone variant macroH2A comes in
5) DNA methylation happens
- -> All epigenetic mechanisms are involved in silencing of X

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56
Q

How does X chromosome inactivation in mice work?

A

1) Early zygote: both Xs are inactive
2) Preimplantation development: imprinted X inactivation occurs of paternal X
3) In ICM, paternal X becomes active - mouse embryonic stem cells have 2 active X chromosomes
4) Epithelialized embryo: random X inactivation
- -> Pluripotency in mice corresponds to having two activated X chromosomes

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57
Q

How does X chromosome inactivation in human work?

A

In humans there might not be imprinted X inactivation –> inactive till the implantation
Random inactivation occurs, when this happens is unsure.
In human ESC three classes can be distinghuised.
Class I: XaXa –> XaXi –> when brining primed cells to naive cells
Class II: XaXi –> XaXi –> primed cells
Class III: XaXe –> XaXe –> eroded X when cells start being transcribed

58
Q

What is the differentiation bias of human embryonic stem cells?

A

1) Differences in differentiation propensity / different cell lines
2) Genetic and epigenetic instability during long-term culturing
3) Heterogeneity within the self-renewing cell population

59
Q

What are the two pluripotent states ?

A

1) Naive

2) Primed

60
Q

What are the advantages of using naive pluripotent stem cells over primed pluripotent stem cell?

A 
Naive pluripotent stem cells have:
- No differentiation bias
- Are amenable to gene targeting
- Blastocyst chimeric competency 
- Higher developmental potential 
The major safety issue with primed cells is that they are genetically unstable!
61
Q

What is the morphology of naive and primed cells and on which signal do they depend?

A

Primed PSCs:
Flattened morphology and depend on Activin and FGF

Naive PSCs:
Dome-shaped morphology and depend on LIF and BMP

62
Q

How to establish a culture condition for human naive PSCs?

A

1) Screen for small molecules that could activate the Oct4 distal enhancer –> with reporter system
2) Further identify candidates that relieve human PSCs from TGFbeta dependency –> add candidate small molecules; select for domed colonies; inhibit TGFbeta; select for colonies that remained domed.

Results:LCDM cocktail

63
Q

What is LCDM?

A

LCDM is a medium that enable naive-like stem cell derivation in humans and mice.
LCDM-derived mESC could integrate both embryonic and extraembryonic tissue –> Extend Pluripotent stem cells (EPS)

64
Q

What are Extend Pluripotent Stem cells?

A

Stem cells that can differentiate into the embryo AND the placenta and yolk sac

65
Q

What is the highly stringent assay and how does it work?

A

An assay for chimeric competency of a single donor cell.

1) Label a mouse EPS cell with a fluorescent marker
- In culture: a single EPS cell can differentiate into TE and ICM in chimeric blastocyst –> check functionality with FACS sorting and qPCR for trophoblast markers
- in Vivo: a single mouse EPS can contribute to both embryonic and extraembryonic lineages

66
Q

What should you look at to determine whether EPS-cell-derive trophoblasts are functional or not?

A

1) Expression of trophoblast markers
- -> Is upregulated in EPS cells
2) Functional characteristics

67
Q

Why would you want to create single mEPS cell-derived postnatal chimeric mice?

A

With a single cell you have higher failure rate but most definitive way of showing that it is a pluripotent cell.

68
Q

What is the difference between 2i-ESC and EPS?

A

EPS have a superior efficiency for chimeric mice and germline transmission compared to 2i ESC cells.

69
Q

What are possible causes for limited interspecies chimerism?

A

hEPS cells show interspecies chimeric competency and integrate into both embryonic and extraembryonic lineages.
However limitations due to interspecies chimerism:
- Mismatched developmental stages
- Evolutionarily non-conservative

70
Q

How can genetics in EPS cell prove that they can form both embryonic and extraembryonic lineages?

A

1) There is an upregulation of a collection of genes that is upregulates in embryonic cells early during preimplantation.
2) EPS cells possess unique epigenetic features that are different from known pluripotent stem cells
3) EPS cells have long-term genome stability while culturing
- -> Different compare to PSCs

71
Q

What is an important regulator in the maintenance of EPS cell potency?

A

Inhibition of Parp1

Inhibition of histamine and muscarinic receptors that normally induce MAPK pathways –> so inhibit MAPK pathways

72
Q

What are the advantages of EPSCs?

A
  • Higher developmental potential
  • Faster proliferation
  • Higher single cell survival
  • Increased stability
73
Q

What are the functions of the polar, proximal and distal mular?

A

Polar: Initiation formation of the placenta –> stem cells of the TE (next to ICM cells)
Proximal mural: Function of the fluid filled area is separating the polar from the distal mural
Distal mural: Attaches to uterus

Cells between polar and distal mural that block embryonic induction

74
Q

What is embryonic induction?

A

The concept that one group of cell directs the development of a neighboring other

75
Q

What induces the proliferation of polar cells (stem cell for trophoblast cells) and trophoblast cells ?

A

Embryonic induction of the ICM

–> embryonic cells fuel trophoblast cells

76
Q

What is the function of the trophoblast?

A

1) Form the placenta –> polar

2) Implant in the uterus –> distal mural

77
Q

What happens when Oct4 is knocked out and how can this be resolved?

A

With Oct4 KO less trophoblast cells are formed –> blastocyst is small and never implants
Can be resolved with adding FGF4 –> number of trophoblast increases again

Oct4 regulates expression of Fgf4

78
Q

What are two factors for expansion?

A

1) LIF –> expands embryonic cells

2) Fgf4 –> expands trophoblasts

79
Q

What are blastoids and what are trophospheres?

A

Blastoid: Combined trophoblast stem cells and ESC to form kind of blastocyst
Trophosphers: Only trophoblast without ICM

80
Q

What is a deciduae?

A

Blastoids can implant in the utero and form a cocoon that is called the deciduae.

81
Q

What can the blostoid do and what not?

A
  • Uterus can form cocoon around the blastoid –> implants in utero
  • Blastoid will proliferate, elongate and differentiate
  • Blastoid does not form an embryo.
82
Q

How can you compare blastoid and blastocysts?

A

By looking at the transcriptome

83
Q

What are the key transcription factors driving the trophoblast?

A

Elf5, Eomes and Gata3

84
Q

How are blastoids formed?

A

1) ESCs aggregate and become embryonic bodies
2) Trophoblasts are formed with TSCs and TFs
3) Trophosheres start expressing following factors to initiate differentiation into the placenta:
- Basp1
- Tead 3
- Gata2

85
Q

How can be studied whether embryonic induction regulates proliferation?

A

1) Quantify amount of trophoblast. This amount is larger in blastoid compared to trophospheres –> SO more proliferation
2) Harvest single cells and put in a disc and see whether they form a colony. Blastoids form better colonies than trophospheres –> SO more self-renewal
Conclusion: The presences of ESCs induces proliferation and self-renewal

86
Q

Nodal and BMP4 are expressed in embryonic cells. What happens if you put BMP4 and Nodal in trophosphers?

A

Diameter of trophospheres increases –> sweling cavity - trophoblast cells pump the water in the cavity - ligating the trophoblast together
Conclusion: Embryonic induction cause epithelial morphogenesis

87
Q

What does embryonic induction cause?

A

1) Maintain trophectoderm core TF/preven differentiation
2) Regulation trophectoderm proliferation, self-renewal and epithelial morphogenesis
3) All together, embryonic inductions maintain the potential to implant

88
Q

Which two options are there in mammalians when there is a wound?

A

1) Wound healing –> Results in scar tissue formed by the epidermis that closes over the wound
2) Full restoration of lost body parts –> regeneration

89
Q

Which signalling pathway is important for regeneration?

A

ERK is important for regeneration

Inhibition of MAPK/ERK signalling does NOT prevent wound healing

90
Q

What are the two types of wounds?

A

1) Tissue loss –> amputation

2) No tissue loss –> incision

91
Q

What is dependent on ERK?

A

Wound healing is ERK independent

Regeneration of tissue loss is ERK dependent

92
Q

By what signals is regeneration activated?

A

Regeneration is activated by generic wound healing signals, rather than specific amputations signals. –> Because regeneration does not take place without a wound

93
Q

What is needed for regeneration?

A

1) Initiation of generic wound healing signals

2) Positional information

94
Q

How can be determined what genes play a role in asymmetrical regeneration?

A

1) Cut the piece of interest in small pieces
2) Extract RNA and do Tomo-seq
3) Compare expression of genes in different pieces
In acomys (spiny mouse) there is high expression of Dkk3 at proximal site (close to the head) = high regeneration and low expression of Dkk3 at distal side (far from the head) = low regeneration

95
Q

What do signalling pathways control during embryonic development?

A

1) Stem cell behaviour –> self-renew or differentiate.
2) Symmetry breaking –> axis of the embryo
3) Pattern formation & maintenance –> where which tissue will develop

96
Q

How do signalling pathways control embryonic development?

A

1) By different signalling gradients
2) By signalling dynamics
3) By different duration of the signal controls

97
Q

What is the clock-and-wavefront model?

A

There is oscillation of signals going on and there is regression of the differentiation front which causes different differentiation at different places

98
Q

Which reporters can be used to visualize real-time signalling dynamics?

A

1) Visualize the ligand which binds to the receptor
2) Visualize the signal transduction cascade
3) Visualize induced gene expression

99
Q

What causes the oscillation in signalling?

A

The pathway is activated and there is a delay in the feedback which gives an inhibitor which causes oscillation.

100
Q

How can be studied whether each cell is an independent oscillator or not?

A

1) Cut the tissue up and dissociate the cells
2) Mesh the cells back together
4) Individual oscillating cells will resynchronize over time and start to form segments
- -> Cells are independent oscillators and couple to each other allowing traveling waves of activity through the tissue.

101
Q

How can be studied whether signalling dynamics is functional?

A

Have tissue in a chip and flush liquids through that influence oscillation. Do not inhibit a pathway but bring a pathway out of phase with a drug. Then observe the pathways with a reporter.
–> Example: It was shown that Wnt and Notch signalling is coupled - when they are brought out of phase everywhere then the whole tissue stopped oscillating which influences segmentation

102
Q

Is in or out phase oscillation needed in regions with stem cells?

A

Out of phase oscillation is needed in regions with stem cells while is needs to change into in phase oscillation in the anterior for proper segmentation.

103
Q

What are the advantages of studying molecular mechanisms in an embryo-like model?

A

1) Generate a large number (overcome a low number of cell input)
2) Easy genetic manipulation (do not need to knockout the mice)
3) Fewer animals (overcome ethical issues)
4) Allow studying the mechanism of signaling dynamics

104
Q

What is a gastruloid?

A

An embryo model that represent the gastrulated embryo.

105
Q

How to study signaling dynamics in multicellular systems?

A

1) Visualization of dynamics
2) Functional analysis
3) Analysis of the molecular mechanism
4) Modeling

106
Q

What is genetic lineage tracing?

A

A technique used to identify and track cell populations in vivo. Generally performed by combining an inducible Cre expressed in a cell type of interest with a Cre-activated reporter usually expressing a fluorescent protein.

107
Q

What is neural drift?

A

In the crypt of intestinal stem cells there is a neural drift towards crypt clonality. The space in the stem cell niche is restricted and the cells divide symmetrically. Every time some cells are pushed out which causes accumulation of clones.

108
Q

What are the niche cells and stem cells in the intestine?

A

Niche: Paneth cells
Stem: LGR5+ cells

109
Q

What are characteristics of organoids?

A

1) Unlimited expansion
2) Non-transformed
3) Genetically stable
4) Cryo-conservation
5) Single cell cloning
6) Phenotypically stable

110
Q

What are the applications or organoids?

A

1) Drug screening
2) Personalized drug treatment
3) Cell engineering/replacement therapy
4) Disease modeling

111
Q

How are neural brain organoids made and why?

A

From iPSCs because there are no adult stem cells found in the brain.

112
Q

Why can brain organoids survive so long in culture?

A

Since they follow the development as a blastocyst in vivo. The brain is very lately vascularized so that is probably the reason that a brain organoid can survive so long without vascularization.

113
Q

What are 3 sorts of 3D models of the brain and what are the pros and cons?

A

Cerebral organoids
–> not directed, gives nice structures but is unpredictable

Forebrain organoids
–> more direct, creates pure forebrain tissue

Cortical spheroid
–> Most directed, gives only cortical tissue

114
Q

What are the pros and cons of complicated and simple organoids?

A

Complicated organoids:
Pro: Recapitulate brain better and is really heterogenous
Con: Cannot be directed, unpredictable which regions will develop

Simple organoids:
Pro: Can be directed, is very homogenous
Con, Cannot be directed and does not recapitulate brain as good

–> choose best model for you research

115
Q

How can mature, progenitor and stem hematopoietic cells be identified and how long does it take?

A

Mature hematopoietic cells –> Have clear marker:

1) Morphology
2) Flow cytometry analysis (after staining with antibody marker)
- -> 1 afternoon

Progenitor –> Can identify with multiple markers
1) Clonogenic assay (CFU-C)
Formation of colonies into semi-solid medium with cytokines and serum –> identify cell types in colonies and see from which progenitors they come from.
–>Takes 12 days
2) Complex multi-parameter flow cytometric isolation
Using different lineage markers

Stem cells –> no specific marker
1) In vivo transplantation –> takes 4 months

116
Q

How to test multipotency and self-renewing capacity in mice?

A

Multipotency:
Inject cells into a mouse with the cell you want to test marked with GFP. Perform PCR in hematopoietic organs, if all organs types have marked cells then it indicates different hematopoietic lineages.

Self-renewal:
Remove cells from the recipient mouse (will have marked en non-marked cell) and inject them in different mice. If they can self-renew than the transplanted cells will also survive in a 2nd recipient.

117
Q

What is a limitation in HSC production in vtiro?

A

They have a low yield and have limited multi-potent and self-renewal capacity.

118
Q

How do HSC develop?

A

First HSC appear at the developing aorta in clusters that both express hematopoietic and endothelial cells. Then they also go to the yolk sac AGM (aorta-gonad-mesonephros) and placenta. Then they will colonize and expand in the liver after which they will go to the BM. In the BM the system is formed and is quiescent.
–> different GF in different locations

119
Q

Where to all HSCs derive from?

A

From hemogenic endothelial cells via an endothelial to hematopoietic transition (EHT)

120
Q

What is the composition of the clusters that form HSCs?

A
  • About 700 cells
  • Clusters contain very functions HSC, about 3 per aorta
  • There are about 12 HSC progenitors per aorta
  • Clusters have pre-HSC (type I and II) ; pre-HSCs are NOT functional
  • All clusters move to the fetal liver where pre-HSCs mature and become functional
121
Q

What is tomo-seq?

A

A technique that combines cryosectioning and low input RNA sequencing to generate a high-resolution genome-wide 3D atlas of gene expression patterns.
–> can get gene expression profiles of different pieces

122
Q

What does Adm-Ramp2 do?

A

Might provide a signaling route that senses hemodynamic shear stress in the environment and transfers this signal to the hemogenic endothelium and/or to HSPCs.

123
Q

What is the function of the Thymus?

A

Essential for late T-lymphocyte development

124
Q

Why are HSCs attracted to the bone marrow?

A

Due to excretion of Cxcl12 by the niche.

–> limited space in the nice; for treatment first chemo or irradiation needed

125
Q

How can transplanted HSCs be traced?

A

Using the HLA alleles.

126
Q

What are niche constituents?

A 
Endothelium:
- Sinusoids
- Arterioles
Mesenchymal
- Skeletal stem/progenitor cells
- Osteoblasts
- Adipocytes
Sympathetic nerve fibres
Haematopoietic 
- Megakaryocytes
- Macrophages
127
Q

What is the role of the endosteum in HSCs?

A
  • Transplanted HSCs prefer migration to endosteum
  • Ca2+ reservoir, regulator of HSC metabolism
  • Niche for lymphoid progenitors
128
Q

What are the two cell systems of the HSCs niche and their function?

A

1) Skeletal stem cells
- Multilineage differentiation: osteoblasts, adipocytes and chondrocytes.
- Regulates formation of niches due to differentiation
- Secrete niche factors (Scf, Cxcl12)
- Highly heterogenous population
- Peri-vascular localization

Peri-vascular niche:
conformed by sinusoids and SSCs.

129
Q

Which marker can be used to identify HSCs in situ?

A

SLAM markers: CD150, CD48

130
Q

How does Cre-LoxP tracing system work?

A

Insert two LoxP sequences flanking both sides of the essential exon of the gene of interest.
LoxP can be recognized by the recombinase Cre.
Cre makes a loop on the DNA which inactivates the gene.

Furthermore, it can also be used by putting a stop codon between the LoxP flanks, when Cre is added the stop codon will not be expressed and what is behind it will be expressed.

Can be used for finding which cells are important for a niche.

131
Q

How can HSC dynamics in the bone marrow be studies in vivo?

A

By using intra-vital imaging of the bone marrow for in vivo imaging of HSCs. Kind of open window in the head of the mouse –> use microscopes with multiphoton lasers with high penetrance for image through bone.

132
Q

What is the role of the HSC niche in leukemia progression?

A

Leukemia can remodel the HSC niche and make it more beneficial to promote leukemia development. –> reduction of amount of osteoblasts and healthy HSCs

133
Q

Which cells are in the liver?

A

1) Parenchymal cells
- Hepatocytes
- Cholangiocytes
2) Non-parenchymal cells
- Kupffer cells, sinusoidal endothelial cells and stellate cells

134
Q

What is the function of hepatoblasts at early developmental stage

A
  • -> are liver progenitor cells
  • Clonally expand and express both hepatocytes and cholangiocytes
  • Form duct-like structures in vivo
  • Did not contribute in cholangiocytes in vivo
135
Q

Why are hepatoblasts not liver stem cells?

A
  • Since they change markers over developmental time
  • -> Stem cells should retain their identity over time and thus should express the same cell surface markers –> stems cells are persistently self-renewable
136
Q

Which cells expand during normal homeostasis of the liver and which cells expand during injury of the liver?

A

Homeostasis: Axin2+ cells

Injury: Sox9+ cells

137
Q

What does the mesenchymal and hematopoietic stem cell lineages give rise to?

A

Mesenchymal –> give rise to osteoblasts

Hematopoietic –> give rise to osteoclasts

138
Q

How are MSCs defined?

A

1) Adherence to plastic
2) Specific surface antigen expression
3) Multipotent differentiation potential

139
Q

What can MSCs differentiate to?

A

Mesenchymal lineages and some other tissues

140
Q

Where do MSCs reside?

A

In the pericyte niche

141
Q

What influences MSCs heterogeneity and potency ?

A

Heterogeneity: Gender, age and tissue source
–> Different CD markers

Potency: tissue source

Stem cells (1 decks)

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