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A Stem cell biology > Embryonic stem cells and self-renewal II > Flashcards

Embryonic stem cells and self-renewal II Flashcards

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1
Q

What 2 properties do pluripotent cells have?

What are these properties determined by?

A

1) The ability to self-renew
2) Ability to generate the lineages from the 3 germ layers

Determined by:
- A SET of molecules expressed together in a CAREFULLY controlled balance (must be expressed at the right level)

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2
Q

Describe the formation of the mouse epiblast

A

1) Morula stage (early stages of division) - ball of cells
2) Morula produces the early blastocyst with 2 main cell types:

  • Inner cell mass
  • Trophectoderm/trophoblast

3) Gives rise to the late stage blastocyst

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3
Q

What does the inner cell mass give rise to?

A

Gives rise to:

  • The EPIBLAST - that ultimately gives rise to the embryo (and the endoderm inside the embryo, during gastrulation)
  • The HYPOBLAST (primitive endoderm) - forms the membranes around the embyro (before gastrulation)
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4
Q

What does the trophectoderm/trophoblast give rise to?

A

The placenta

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5
Q

What does the hypoblast give rise to?

A
  • The visceral endoderm
  • The parietal endoderm

(Endoderm OUTSIDE of the embryo)

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6
Q

Describe the battlefield model of pluripotency

A

Continual conflict between pluripotency transcription factors that seek to direct ESC differentiation to OPPOSING LINEAGES

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7
Q

What TF are important to create the cell lineages seen during gastrulation?

What do these TF do?

A

Sox2 - ectoderm

Oct4 - mesoderm (and a little ectoderm)

Nanog - endoderm

These TF try and push the cells into a particular direction

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8
Q

What happens if Sox2, Oct4 and Nanog are expressed all toghether?

A

They cancel out each other (trying to push they the cells into a particular direction)

–> No direction into particular lineage

–> Get the state of pluripotency

–> Keeps the cells undifferentiated with the potential to create different lineages

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9
Q

When do pluripotent cells get directed into particular lineage?

A

When the pluripotency factors (SOX2, OCT4 and Nanog) become UNBALANCED

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10
Q

What type of molecules are Sox2, Oct4 and Nanog

A

DNA binding proteins (have DNA binding domain)

–>TF

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11
Q

What family is SOX2 a part of?

A

HMG DNA binding domain family

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12
Q

What family is Oct4 a part of?

A

Pou DNA binding domain family

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13
Q

What family is Nanog a part of?

A

Homeodomain DNA binding domain family

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14
Q

How do Sox2 and Oct4 sometimes interact?

A

Through the TAD domain (outside of the DNA binding domain)

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15
Q

What is Oct4 essential for in vivo?

A

To maintain the pluripotency potential of an embryonic stem cell

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16
Q

What happens when KO Oct4 (in vivo)

A
  • The embyro FAILS to acquire the potential to produce the different lineages of the germ layers (fails to produce the inner cell mass and following epiblastic cells)
  • ONLY produces the extra embryonic tissue (trophoectoderm)
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17
Q

What happens when KO Oct4 in vitro?

A

Differentiation into trophoectoderm

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18
Q

What happens if upregulate Oct4 levels at an early time point?

At a later time point?

What does this show?

A

At an early time point - Formation of the hypoblast (extra-embryonic endoderm)

At a later time point- get mesoderm

Shows:
- If the timing of the upregulation/downregulation changes –> cells are directed down a different fate as a consequence

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19
Q

How does Oct4 prevent the formation of the trophoblast?

A

Controls the expression of Cdx2 (TF) that normally causes lineage/specification into TROPHOECTODERM

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20
Q

What type of factor is Cdx2?

A

A transcription factor

NOT a pluripotency factor

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21
Q

How do Oct4 and Cdx2 interact?

A

In a RECIPROCAL REPRESSION loop:

- Mutually bind in a complex to inhibit each others transcriptional activity

22
Q

What does the down regulation of Sox2 in the embryo cause?
How is this seen?

What is this similar to? What does this show?

A

1) Faliure to produce the epiblast
2) Production of the trophoectoderm

Seen using beta geo reporter

Also seen in vitro

Similar to the effects of downregulating Oct4 (both Oct4 and Sox2 are required in the cell lineage leading to epiblast and in their absence, trophoectoderm is formed)

23
Q

What is nanog essential for?

A

Self-renewal

24
Q

How was Nanog identified?

A

In 2 independent screens by:

  • Mitsui et al
  • Chambers et al
25
Q

How did Mitsui et al identify Nanog?

A

Using an in silico screen:
1) Looking at genes highly expressed in the ES cell population using differentially digital forensics

  • Predicted if a gene is important, it is expressed in high levels in the ES cell
    2) Selected the highly expressed genes and looked for their expression in OTHER tissues
  • Saw Nanog to be highly and neatly expressed in ES cells
26
Q

How did Mitsui et al prove that Nanog was a pluripotency gene?

What was the control?

A

Hypothesis that cells need LIF to remain proliferative/pluripotent:

  • If a factor is TRULY conferring pluripotency, they should be able to take over the effect of LIF (in the presence of the factor and the absence of LIF, the cell should REMAIN PLURIPOTENT)
  • So, they took cells and transfected them with Nanog and cultured them in the PRESENCE and ABSENCE of LIF
  • When removed LIF - cells remain undifferentiated with proper ES cell colonies

Control:

  • Cells with LIF show typical ES cell morphology
  • Remove LIF –> cells differentiate
27
Q

How did Chambers et al discover Nanog?

A

1) Took genes from a library (all the genes expressed in the ES cells) and put them into cells
2) Using an alkaline phosphatase marker as a marker of pluripotent ES cells
- Looking at the combination of genes that confer ES cell properties to the cells –> cells up regulate the reporter
3) Identified the colonies that were up regulating alkaline phosphatase and sequenced their cDNA
4) Repeated the experiment with another selection of genes until the final sequence was identified
5) Put the cDNA sequence into cells without the LIF receptors

  • -> Cells remain pluripotent in the absence of LIF
  • -> showing this particular sequence of Nanog to be a pluripotency gene
28
Q

What happens if KO Nanog in ICM and ES cells?

A

1) Lose pluripotency
2) No formation of the ectoderm
3) Differentiation into extra-embryonic endoderm (visceral/parietal)

29
Q

What happens if overexpress Nanog?

A

Overcomes the requirement for LIF (and BMP/serum)

30
Q

What does Nanog drive the formation of?

What is antagonistic to this?

A

The epiblast

Gata6 antagonises this

31
Q

What does Gata6 drive the formation of?

A

The primitive endoderm (visceral and parietal)

32
Q

What does Tbx3 drive the formation of?

A

The hypoblast

33
Q

What is a key concept of the stem cell populations?

A

Stem cell populations have a low level of heterogeneity (disorder)

34
Q

Describe the heterogeneity in the stem cell compartment with regards to Oct4 and Nanog

A

Oct4:

  • Homogenous expression
  • Most of the cells in the colony are +ve for Oct4

Nanog:
- Expressed in some cells at HIGH levels

  • Expressed in other cells at LOW levels

–> Patched appearance

35
Q

What is FACS?

Describe the process

???

A

Fluorescent Activated Cell Sorting :

  • Have a population of cells that at labelled with different fluorescent markers
  • Put into a system that produces a controlled flow of the single cells
  • Pass these cells in front of a laser with a particular colour intensity –> laser differentially activates/charges the cell with a different colour
  • Cells go the the plate that is +ve or -ve –> pool these cells
  • Data displayed on a HISTOGRAM showing a certain number/percentage and fluorescence intensity
36
Q

What is the evidence for differential expression of Nanog?

A

1) GFP targeted to Nanog in ES cells

2) GFP+ and GFP- cells could be separated by FACS
- -> evidence there is differential expression of Nanog in the ES cells

37
Q

What was done after 6 days culturing the GFP+ and GFP- cells separately?

What did this show?

What was the conclusion of this experiment?

A

FACS sorting repeated

Results:
- GPF+ cells generated a GFP- population of cells

  • GPF- cells generated a GFP+ population of cells

Conclusions:

  • The different populations of cells are not SET into 2 different pathways
  • The cells containing Nanog are still pluripotent (can generate the other cell types) but the cells are slightly different
38
Q

What does the evidence suggest Nanog provides?

A

Provides variable resistance to differentiation

39
Q

What behaviours do the cells expressing Nanog have?

A

Cells transient between highly expressing Nanog (Nanog+) and low expressing Nanog (Nanog-)

Cells can cycle between the 2 different states of Nanog expression

40
Q

What predisposes cells to differentiate?

A

Very low concentrations of Nanog in the cell (very -ve)

41
Q

Why is heterogeneity in the stem cell compartment important?

A

Cells from different states may be ‘primed’ for particular lineages

42
Q

Describe the analogy that cells in different stem cell states may be ‘primed’ for different lineages

A
  • Undifferentiated cells sit in a CENTRE of a crater at the top of a hill
  • Cells can move around in the crater and convert between the different positions in the crater
  • Energy is required to push the cells over the top of the crater and one of 2 alternative vallies
  • But the cell will very easily go down the hill and sit in a valley at the bottom of the hill (cells will move from one stable state to another stable state)
  • BUT, cells may be predisposed into going into a specific valley (cells closest to valley A –> valley A, cells closest to valley B –> valley B)
43
Q

Why is it useful to understand heterogeneity?

A

May facilitate the production of different cellular subsets if we can understand which is the heterogeneous population

44
Q

As well as levels of Nanog, what are the other ways that heterogeneity can be acheived?

A

Cell surface markers:
- Reflect the change in cell state

  • As cells move up the hill from the centre of the crater, the cells express different markers
45
Q

What marker is marker of the very early ES cell types (niave)?

A

SSEA3

46
Q

What is TRA-160 a marker of?

A

Movement of the cell further up the hill, closer to commitment but the cell still remains undifferentiated

47
Q

What are SSEA3- TRA-60- cells?

A

Cells that are very high to the top of the crater and just about to go over the edge (become committed)

48
Q

What 3 things can a stem cell do?

A

1) Self-renew
2) Differentiate
3) Die

49
Q

How is genetic stability affected by culturing stem cells in vitro?

What are the advantages and disadvantages of this?

A

Preventing the stem cells from differentiating or dying has a direct impact on the cells to self-renew/proliferate

Mutations that favour self-renewal are selected for by the culture (differentiation and death are prevented)

Disadvantages:
- Detrimental for therapeutic application (unable to use)

Advantages:
- Could provide insight into genes that control proliferation and self-renewal

–> give the cells competitive advantage over the other cells

50
Q

What are the common genetic abnormalities in hES cells?

What do these mutations allow?

A

Gains on:

Chromosome 12

Chromosome 17

Chromosome 20

Chromosome X

  • Hotspots
  • Confer competitive advantage
  • Shows there may be genes on these chromosomes that are critical for pluripotency and expansion of cell culture
51
Q

What is seen in chromosome 4 in hES cells?

What does this show?

A

Nothing gained

Shows:
- No genes on these chromosomes that are critical for pluripotency and expansion of cell culture

  • OR the genes on this chromosome are so important that a mutation in this chromosome would KILL the cell all together –> conferring no competitive advantage

A Stem cell biology (16 decks)

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