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Stem Cell > Pluripotent Stem Cells > Flashcards

Pluripotent Stem Cells Flashcards

1
Q

What is special about stem cells?

A

They retain pluripotency

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2
Q

What gives a cell its identity?

A

Stable genetic profiles that are less likely to be modified by extracellular signals

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3
Q

What allows a cell to maintain its identity?

A

Gene regulatory network gives robustness, which can withstand pertubations and return to its stable state

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4
Q

Where are mESCs derived from?

A

Inner cell mass of blastocysts

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5
Q

How are mESCs initially cultured?

A

On MEF ‘feeders’ in serum-containing media

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6
Q

What are MEF “feeders”?

A

Cells used to support the growth

MEF cells secrete growth factors into the medium, which help maintain the pluripotency of ES cells.

MEF cells provide a cellular matrix for ES cells to grow on.

MEF cells are often mitotically inactivated so that they remain viable but cannot replicate and overgrow the ES cell culture.

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7
Q

What factor was found to be the key component provided by MEF ‘feeders’?

A

LIF

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8
Q

What is serum-containing media?

A

Fetal bovine serum (FBS), which is a common supplement added to cell culture media to provide essential growth factors and nutrients for cell proliferation and survival

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9
Q

How do we know ESCs are truly pluripotent?

A

Because when injected into tetraploid embryo an alive fetus was produced

Shows ESC have the potential to form ALL fetal cell lineages

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10
Q

What pathways does LIF activate?

A

STAT3 and ERK pathways

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11
Q

Explain the STAT3 pathway

A

LIF binds LIFR, which is coupled to co-receptor gp130

Activation of JAK = phosphorylates STAT3 on specific tyrosine residue

Activated STAT3 allows it to dimerize and translocate to nucleus

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12
Q

Explain the ERK pathway

A

LIF binds LIFR, which is coupled to co-receptor gp130

Ras is activated by converting GDP to GTP

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13
Q

What effect do the two pathways LIF activates have on differentiation?

A

STAT3 inhibitis differentiation

ERK promotes differentiation

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14
Q

What happens when ERK activation is supressed in mESCs?

A

mESCs require less LIF to self-renew

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15
Q

How does STAT3 inhibit differentiation in mESCs?

A

LIF binds LIFR, which is coupled to co-receptor gp130

Activation of JAK, which then phosphorylates STAT3 on speciifc Tyr residue

Activated STAT3 dimerizes and translocates to nucleus

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16
Q

How does ERK promote differentiation in mESCs?

A

LIF binds LIFR, which is coupled to co-receptor gp130

Ras activated by conversion of GDP to GTP

Phosphorylates Raf > MEK > ERK

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17
Q

What do mESC need to be maintained?

A

Shown that they can be maintained without serum in LIF + BMP4

18
Q

What is LIF needed for to maintain mESC?

A

Leukaemia inhibtory factor (LIF)

To induce STAT3 = inhibitor of differentiation

19
Q

What is BMP4 needed for to maintain mESC?

A

Bone morphogenetic protein (BMP)

To induce inhibitor-of-differentiation proteins

20
Q

How do STAT3 and ERK regulate gene expression?***

A
21
Q

What factors do pluripotent cell express?

A

Transcription factors = Sox2, Oct4, Klf4 and Nanog

22
Q

What is the effect of deleting pluripotency TFs?

A

Phenotype would change, causing stem cells to stop being pluripotent and start differentiating

23
Q

What is the effect of overexpressing pluripotency TFs?

A

Causes more resistnace to differentiation = more robustnuss

But this depends on WHICH factors

24
Q

Which pluripotency TFs are NOT targets of LIF pathway?

A

Sox2, Oct4 & Nanog

25
Q

Which pluripotency TFs are targets of LIF pathway?

A

Klf4 indirectly by LIF

26
Q

What is the role of Nanog in pluripotency?

A

It is important when MAKING pluripotent cells

But NOT needed to MAINTAIN pluripotency

27
Q

What is the “ground state” hypothesis?

A

The ability to grow mESC without serum because pluripotency is the default

So only need to inhibit differentiation signals to allows pluripotency to occur

Inhibit ERK and GSK3 signalling

28
Q

What conditions create ground state pluripotency?

A

2i = 2 inhibitors

Inhibit ERK and GSK3 signalling

29
Q

Define ground state pluripotency

A

Pluripotency does not need to be promoted

Cell require input to exit from it = default

30
Q

How are ground state cells different to those not grown under 2i?

A

More homogeneous = high level of Nanog

31
Q

How are hESC different to mESC?

A

Don’t respond to LIF

Die in 2i = no ground state

32
Q

What do hESC need to self-renew?

A

Activin and FGF

33
Q

How does FGF act on hESC copmared to mESC?

A

FGF causes hESC to self-renew

FGF activates ERK in mESC = promoting differentiation

34
Q

hESC react very differently to conditions than mESC, what does this tell us?

A

The way hESC and mESC maintain their identitiy is different!!!

35
Q

How does LIF work in mice vs humans?

A

In mouse embryos, LIFR = not required for early embryo development

hESC are LIF indepencent

36
Q

Why are LIFR not required for early mouse embryo development?***

A
37
Q

Is there are specific function of LIF signalling in mice that is not required in humans?***

A
38
Q

What is the epiblast?

A

A tissue of the post-implantation embryo that generates the embryo proper

At implantation, the embryo is composed of the epiblast, a pluripotent derivative of the inner cell mass

39
Q

What is the difference between mESCs and EpiSCs derivation?

A

mESC obtained from mouse embryo BEFORE impantation in uterus

EpiSCs derived from epiblast, which is tissue of post-implanted embryo

40
Q

What do naive and primed states mean for ESC?

A

These are pluripotency states

In mESC naive state goes before primed state then to differentiation

High Nanog = naive
Low Nanog = primed to differentiaate

41
Q

How is the naive mESC phenotype stabilized?

A

2i conditions

42
Q

What factors influence ESC phenotype?

A

Culture conditions = signalling

Developmental stage

Species differences

Heterogeneous, fluid state

Stem Cell (7 decks)

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