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

Embryonic stem cells and self-renewal I Flashcards

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1
Q

What study highlighted the carcinogenesis of stem cells?

A

The study of teratomas in strain 129 mice (mice that produce this tumour)

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2
Q

What is a teratoma?

A

A large tumour that gives rise to many different tissues (eg. hair, bone, nerves)

Very differentiated tissues from different origins

Sometimes in an organised way

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3
Q

What are cancerous teratomas called?

A

Teratocarcinomas

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4
Q

What did Kleinsmith et al show in 1964?

How?

A

The complexity of the tissues in the teratoma can be originated to a single cell (pluripotent cells - produces the different tissues of the different germ layers)

Using clonal experiments:
- Take single cell from animal with the tumour and put into another animal –> recreates the tumour

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5
Q

What are embryonic carcinoma (EC) cells?

What do they resemble?

How was this shown?

A

The stem cells of the teratocarcinomas

Resemble true pluripotent embryonic cells

Shown by transplanting EC cells (from a pigmented animal) into the blastocyst of an albino mouse
–> cells contribute to the whole developing animal

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6
Q

What did the independent work of Evans and Martin in 1981 show?

How?

What did they show?

A
  • The ability to isolate and characterise truly pluripotent cells from the developing mouse by using the knowledge acquired from working with EC cells (how to culture and manipulate them
  • Showed that mouse ES cells grow better on a layer of feeder cells
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7
Q

What are feeder cells?

A
  • Support cells (eg. fibroblasts) that condition the media to support the growth of the stem cells
  • They are INACTIVATED with a drug or radiation that stop them from dividing
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8
Q

Where are ES cells extracted from?

A

From the inner cell mass of the blastocyst

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9
Q

What tests are used to see if a cell is truly pluripotent?

A

1) Creation of a teratoma when the cells injected into the mouse
2) If inject into the blastocyst of a mouse embryo –> contributes to the different tissues in that animal

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10
Q

What tissues should a pluripotent stem cell be able to give rise to?

A

All of the different cells and germ layers, including the germ cells (sperm and egg)

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11
Q

What are the 9 properties of an ES cell?

A

1) Derived from the ICM of a blastocyst
2) Non-transformed (normal)
3) Indefinite proliferative potential (immortal)
4) Stable diploid karyotype
5) Clonogenic
6) Pluripotent
7) Incorporation into chimaeras (contribution to the cells of many different tissues)
8) Germline transmission into chimeras
9) Permissive to genetic manipulation (eg. KO, replace mutated genes, over-express genes)

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12
Q

What can happen to ES cells as they are extensively cultured in vitro?

Why is this a problem?

A

They can acquire abnormalities in the genome

A problem because ES cells must be non-transformed and have a stable diploid karyotype

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13
Q

What does clonogenic mean?

A

Can originate a culture from a single cell

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14
Q

What are the issues when determining hES using the properties of ES cells?

A
  • Difficult to achieve a stable diploid karyotype
  • Difficult to clone hES from single cells
  • Cannot test the contribution to all the different cell types in vivo (eg. contribution to the gametes)
  • Not practical to test the incorporation of the stem cells into chimeras due to ethical considerations
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15
Q

To what extent has chimerism with hES been demonstrated?

A

hES into the mouse blastocyst

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16
Q

What happens when inject mouse ES cells into a blastocyst?

How is this seen?

A

The cells will incorporate into all embryonic cell types:

  • Seen when looking at a mouse foetus produced form ES cells where one of the copies of a particular gene (of the ES cells) is replaced with an enzyme (eg. beta-galactoside)
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17
Q

How is beta-galactoside used?

A

As a reporter gene:

  • Turns blue when the right substrate is added
  • Can see where the gene is expressed

(Used to visualise ES cells that have it incorporated into the genome of the cell)

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18
Q

How can you create a transgenic mouse?

A

By genetically manipulating ES cells that are incorporated into the animal

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19
Q

What are the extrinsic factors that allow us to maintain cells in their pluripotent, proliferative state?

A

LIF (Leukaemia Inhibitory Factor)

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20
Q

Where is LIF secreted from?

A

Initially from the feeder cells

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21
Q

What happens when LIF is withdrawn?

A

Proliferation continues but DIFFERENTIATION is induced

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22
Q

Describe the LIF pathway

A
  • LIF interacts with the LIF receptor (LIFR) that combines with Gp130 (glycoprotein co-receptor)
  • LIFR and corresponding Gp130 will activate the JAK pathway
  • JAK pathway upregulates STAT3 (that is critical to maintain the proliferative state of the cell)
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23
Q

As well as the LIF pathway, what other pathway does LIF act on?

Describe this pathway

A

The SHP2 pathway:

  • LIF binds to the LIFR (with co-receptor Gp130) –> causing the activation of SHP2
  • SHP2 activates ERK1/2 (a kinase)
  • ERK1/2 then blocks STAT3 (therefore blocking self-renewal/pluripotency) and ACTIVATES proliferation/differentiation
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24
Q

Describe the balance between the LIF and the SHP2 pathways

A

Balance between the 2 (self-renewal and differentiation) is critical

  • HOWEVER, the LIF and SHP2 pathways are not evenly balanced
  • In the absence of LIF (not activating either pathway) –> differentiate
  • LIF above a certain THRESHOLD –> force the cells to self-renew
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25
Q

When is the ONLY time LIF drives self-renewal in mES cells?

A

When grown in SERUM

in serum-free conditions, LIF alone is INSUFFICIENT to maintain pluripotency or block neural differentiation

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26
Q

What is needed in serum-free conditions to sustain self-renewal and pluripotency?

A

LIF and BMP4 (or BMP2)

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27
Q

What media must cells be put into in order to drive neuralisation?

A

N2B27 media

28
Q

What marker do neurons express?

A

A neuronal marker - Tuj1

29
Q

What happens if express LIF in the background that is driving neuralisation?

How is this shown?

A

Can prevent neutralisation and maintain pluripotency

Shown by the expression of a pluripotency marker Oct4 (using a Oct4-GFP reporter line)

30
Q

What happens if put cells with LIF and serum? (in terms of gene expression)

A

Have NO Tuj1 expression

Have Oct4 expression

31
Q

What can serum be substituted for?

A

Presence of GDF6 or BMP4 (with LIF keeps the cells pluripotent

32
Q

What does BMP do?

A

1) Induce a gene that BLOCKS the differentiation into neural lineages via Smads

AT the SAME time:

2) Induce differentiation into MESODERM/ENDODERM

33
Q

What does LIF/Stat do? (in regards to BMP?

A

INHIBITS BMP from producing mesoderm/endoderm

34
Q

How does LIF and BMP maintain self renewal?

A

Competing actions in combination to sustain self-renewal:

  • BMPs block neural differentiation and promotes mesodermal/endodermal differentiation through SMADS
  • LIF blocks BMP from inducing meso/endodermal differentiation via STAT3
  • -> Both lineages are blocked
  • -> Cell remains undifferentiated and pluripotent
35
Q

What is important in order to achieve the right cell from a stem cell?

A

Application of the right factors in the right combinations at the right time/order at the right concentrations

In order to block different pathways at different points

36
Q

Who was the first to isolate hES cells?

What were the limitations of this?

A

Thomson et al (1998)

Limitations:

  • Ethical considerations
  • Biological - cannot isolate in the same way as mouse ES cells
37
Q

What are some of the differences between mES and hES cells?

A

1) Shape of colonies
- mES cells - form SMALL colonies that project UPWARDS (forming domes)
- hES cells - flat colonies with very well-defined edges
2) Different markers (surface antigens) expressed

  • mES - SSEA-1 (absent in hES)
  • hES - SSEA-4 (absent in mES)

3) LIFR-gp130
- hES DO NOT respond to LIF (are not dependent)
4) Different substances required to isolate the hES cells

  • Activin/nodal signalling
  • FGF signalling

5) Cell cycle rates and cell death
6) Rex1 (variable expression in human ES cell lines)

38
Q

What pathways are CONSERVED between mES and hES cells?

A
  • Stat3 signalling
  • Nanog
  • Oct-sox
  • FGF signalling
  • TGFb signalling
  • BMPR1a
  • MicroRNAs
  • Methylation - X inactivation
  • Cell cycle
  • Igf2-H19
39
Q

What is the hES phenotype?

A

SSEA1-

SSEA3+
SSEA4+
TRA-1-60+
GCTM2+
Thy1+
MHC+

ALP+
OCT4+
NANOG+

40
Q

What is the mES phenotype?

A

SSEA1+

SSEA3-
SSEA4-
TRA-1-60-
GCTM2-
Thy1-
MHC-

ALP+
OCT4+
NANOG+

41
Q

What does further work show about the maintenance of pluripotency in mES cells?

A

LIF can maintain pluripotency in mES when combine them with 2 inhibitors:

  • Inhibition of MAPK /ERK1/2 pathway
  • Inhibition of GSK3b pathway
42
Q

What is the most common way of culturing mES cells?

A

LIF with 2 inhibitors

  • ERK1/2 inhibitor
  • GSK3b inhibitor
43
Q

What is needed to allow self-renewal of hES cells?

A

FGF2 and Activin

44
Q

What is needed to understand the differences between the mES and hES cells?

A

Need to understand the differences between the very early stages of embryogenesis

45
Q

How does the human epiblast develop?

** unsure this is right???**

A
  • Formation of a PRE-IMPLANTATION blastocyst (not yet grafted into the wall of the uterus)
  • Inner cell mass of this differentiates into the epiblast (flat dish of cells)
46
Q

How does the mouse epiblast develop?

** unsure this is right???**

A
  • Epiblast forms as a flat sheet and then forms a cup (cylindrical shape) that is seen when the embryo is IMPLANTED
47
Q

What does the epiblast contain?

A

Contains truly pluripotent cells (ES cells)

48
Q

SO, what is different about the timings of the extraction of ES cells from the human and the mouse blastocysts?

What question did this propose?

A

hES: post-implantation-like cells (epiblast stage)

mES: pre-implantation-like cells (inner cell mass stage)

Proposed the question:
Are hES cells really more equivalent to mouse epiblastic stem cells (EpiSCs) than mouse ES cells?

49
Q

What did Brons et al and Tesar et al do?

A

Independently isolated mouse EpiSCs and shows they are very similar to hES cells

50
Q

What are niave cells?

What do they require to remain pluripotent?

Example?

A

The PRE-IMPLANTATION, inner-cell mass stage cells

Require:

  • LIF and STAT2 activation
  • ERK and GSK inhibition

Example:
- Mouse ES cells

51
Q

What are prime cells?

What do they require to remain pluripotent?

Example?

A

The POST-IMPLANTATION, epiblastic stage cells

Require:
- FGF and Activin

Example:

  • hES cells
  • Mouse epiblastic cells
52
Q

What happens to differentiative potency during development?

What is the first stage of this loss?

A

It is gradually lost

First stage is the divergence of the extraembryonic/trophoblastic cells from the inner cell mass and ES cells

53
Q

What are the 2 stages of pluripotency?

A

1) Niave/ground state (ICM-like)

2) Primed state (epiblastic-like)

54
Q

Can we capture the ground state/true first stage of human pluripotency?

A

It is difficult but throughts that we can capture it by:

1) Deriving new cells

OR

2) Pushing an established line back to become niave

55
Q

How was it identified how to create a niave hES cell?

What did this identify?

A

Using an Oct4-GFP readout (pluripotent cells will turn green) and applying a different combination of factors to define the factors that cause the change of a primed –> niave cell

Identified:

  • A pool of 8 factors (NHSM) that when applied to primed ES cells caused the colonies to change to a more domed-like, raised colony (typical of mouse ES cells)
  • -> To create NHSM-derived pluripotent stem cells
  • When these factors are applied to cells isolated from a human blastocyst –> look more like mES cells
56
Q

How is Oct4 expression controlled in mouse ES cells (niave) compared to hES cells (prime)?

A

Different enhancers used

57
Q

How can mES cells be converted to mEpi cells?

mEPi –> mES?

A

Addition of Activin and FGF

mEpi –> mES using 2i and LIF

58
Q

How are mEpi cells cells isolated?

A

Activin and FGF (same as human ES cells)

59
Q

What is shown about the Oct4 promoter in niave and primed ES cells using flow cytometry?

How?

A

The niave and primed cells control the expression of Oct4 using alternative enhancers:

  • Niave cells use the distal enhancer
  • Primed cells use the proximal enhancer

As fluorescence is LOST when mutate these enhancers in these cells, whereas if mutate the other enhancer, the fluorescence of Oct4-GFP is maintained

60
Q

What does the injection of niave hES cells into the blastocyst of the mouse show?

A

The formation of chimeras (cells are incorporated into different mature tissues of the mouse)

61
Q

What are the characteristics of the niave/ICM-like pluripotent cell state?

A

?

62
Q

What are the characteristics of the primed/epiblast-like pluripotent cell state?

A

?

63
Q

Why are niave hES important?

A
  • Fundamental to the understanding of pluripotency
  • Easier to modify genetically (more efficient homologous recombination) to use cells as models
  • Integration into the blastocyst can be done with niave cells but NOT with primed cells
64
Q

Why do we want to integrate niave hES cells into a blastocyst ?

A
  • Provide confirmation of human-mouse chimerism (to produce ‘humanised’ organs for transplant) –> potential application??
  • Idea of putting hES into the blastocyst of another organism (eg. pig) –> get humanised heart without the worry of rejection
65
Q

Describe the different mouse ES cell lines

How are they different to human ES cells?

A

Mouse ES cell lines look quite similar

Different human ES cell lines have much genetic variability:
- Most cells differentiate into most tissues (because they are pluripotent but they do this with a different efficiency and efficacy

66
Q

Why are are mouse ES cell lines very similar to each other?

A

Mice are homogeneous/inbred strain

67
Q

Why are human ES cell lines very different from each other?

A

Each ES cell line represents a particular individual/embryo

A Stem cell biology (16 decks)

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