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BMS247 - Stem cells > In vitro stem cell models > Flashcards

In vitro stem cell models Flashcards

1
Q

What is a unipotent cell?

A

A cell that can only give rise to ONE specific lineage

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2
Q

What signals does the niche provide the stem cell with?

A

Signals that promote self-renewal

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3
Q

What happens when the stem exits from the niche?

A

There is exposure to differentiation signals (these are different to the self-renewal signals

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4
Q

Why are mice the best mammalian system to study developmental biology?

A
  • Takes 20 days to have a new born
  • Large litter
  • Can be manipulated
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5
Q

Which cells are pluripotent?

A

Cells found in the very early embryo, that can give rise to ALL the cells of the body, including the germ cells (but not the extra embryonic tissue)

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6
Q

Where are the pluripotent cells found?

A

Inner cell mass of the blastocyst stage embryo

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7
Q

What are 2 ways to test if a cell is a stem cell?

A

1) Descriptive test - cell expresses pluripotency factors

2) Functional test - cell causes teratoma formation

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8
Q

Describe the descriptional test of the proposed stem cells

A
  • Stem cells express a SPECIFIC set of pluripotency markers
  • Using in situ hybridisation - use to detect the RNA expression of these set of makers SPECIFICALLY CONFINED tot the the inner cell mass (where PS cells are found)
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9
Q

What are the main pluripotency markers)

A

Nanog
Oct4
Sox2

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10
Q

When do cells remain pluripotent until?

What do these cells express?

A

They are still pluripotent STRAIGHT after implantation of the mouse embryo into the uterus

Express pluripotency markers: Nanog, Oct4 and Sox2

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11
Q

Describe the functional test of the proposed stem cells

A
  • Graft PS cells (from a mouse embryo straight after implantation) into the kidney of a host mouse
  • Cells grafted should give rise to teratocarcinoma (a pluripotent tumour formed of ALL cell types)
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12
Q

Why is the proposed stem cell grafted into the kidney in the functional test?

A

Permissive environment - provides the right signals for the cell to divide into all the cells of the body

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13
Q

When do the pluripotent cells of the body begin to differentiate?

A

At GASTRULATION

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14
Q

When does gastrulation occur?

A

After implantation

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15
Q

What is gastrulation?

A

A highly morphogenetic process where 1 layer transforms to 3 layer, which are the first founders of all the cells that will make the embryo proper

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16
Q

What does the ectoderm give rise to?

A

Surface
Neural
Neural crest

(skin, peripheral and central nervous system)

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17
Q

What does the mesoderm give rise o?

A

Axial
Paraxial
Intermediate
Lateral

(blood, heart, kidney, muscle, bone)

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18
Q

What does the endoderm give rise to?

A

Gut
Internal organs

(liver, pancreas, intestine)

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19
Q

At what stage in development are the pluripotent cells extinct?

A

E7/E7.5 (end of gastrulation)

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20
Q

When do the neural stem cells form?

A

E9 at the future forebrain

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21
Q

What is the potency of neural stem cells?

What cells can they generate?

A

Bipotent - can generate 2 different stem cell types

Neurons and glia

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22
Q

What do glial cells do?

A

Surround the neuron

Provide support and nutrients

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23
Q

Why are ES cells hard to study in vivo?

What is the solution to this?

A

Not very accessible
Small in numbers
In utero development
Ethics

Solution - CAPTURE the stem cells in vitro

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24
Q

What can in vitro modelling of embryonic development be useful for?

A
  • Drug screening

- Cell replacement therapies

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25
Q

What signals must/must not be present to capture the self-renewing state of an ES cell?

A
  • Need to MIMIC the conditions in the NICHE

- Need to STOP the process of differentiation (block using INHIBITORS)

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26
Q

What are the critical signals in the mouse to maintain cells in a self-renewing, undifferentiated state?

A

LIF (leukaemia inhibitory factor)

BMP antagonists

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27
Q

What are the critical signals in the human to maintain cells in a self-renewing, undifferentiated state?

A

FGF2

TGF beta

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28
Q

What are the stages of capturing the ES cell in vitro?

A

1) Take blastocyst stage embryo (E4 in mouse) and DISSOCIATE the inner cell mass using a laser
2) Plate the inner cells on layer of FEEDER cells

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29
Q

What are ‘feeder cells’?

A

Irradiated stomal cells derived from the late embryos - support ES growth by providing the trophic factors

30
Q

What can the feeder cells be substituted with?

A

LIF and BMP

31
Q

As well as ES cells, what other stem cells can be captured in vitro?

A

Adult stem cells that have been REPROGRAMMED to a PLURIPOTENT cell fate

32
Q

How are adult stem cells REPROGRAMMED to a PLURIPOTENT cell fate?

A

1) Take biopsy from adult

2) OVER EXPRESS the genes that characterise pluripotent cells (Oct4, Nanog, c-myc, klf4)

33
Q

What are the advantages of iPS cells?

A
  • Can also differentiate into ALL the cells of the body
  • Bypasses the use of embryos - more ethical
  • Good approach for disease modelling
34
Q

How can we tell the ES cells we have captured are pluripotent?

A

2 HALLMARKS of pluripotency
Descriptive and functional tests

1) Express Oct4, Nanog and Sox2 under the conditions od LIF and BMP
- DON’T express genes indicative of differentiation

2) ES cells form TERATOCARCINOMAS when transplanted into a permissive environment (eg. kidney)

35
Q

What is another functional test for ES cells? (as well as the formation of teratocarcinomas)

A

When transplanted into the inner cell mass of a normal embryo - these cells contribute to NORMAL development

36
Q

When transplanting ES into the inner cell mass of a normal embryo, how can we visualise/follow the cell?

A

Using GFP labelled ES cells:
- Put into the inner cell mass of an UNLABELLED embryo

  • Take injected blastocyst into pseudopregnant mouse —> some mice labelled completely green
37
Q

What can the method of blocking differentiation and using signals to promote self-renewal be applied to?

What does this mean?

A

Applied to any other stem cell type (eg. multipotent, unipotent)

Means that ANY stem cell type can be captured in vitro

38
Q

Which signals mimic the niche environment of the NS cells of the body?

What happens when these signals are removed?

A

Cytokines: FGF2 and EGF

When removed - the cell undergoes DIFFERENTIATION

39
Q

What are the undifferentiated markers expressed by NS cells?

A

RC2

40
Q

What are the genes indicative of NS cell differentiation?

A

GFAP - glial

TUJ1 - neurons

41
Q

How can we make precise cell types from the differentiation of stem cells in vitro?

A

By mimicking GASTRULATION in vitro

By ensuring that the cells are expressing the right markers

42
Q

What are the 2 approaches of differentiation of the stem cells in vitro?

A

1) 3D approach

2) 2D approach

43
Q

What is the 3D approach of differentiation in vitro?

A

Removed signals that keep the cells in an undifferentiated state:

  • BMP, LIF (mouse)
  • FGF, TGFb (human)

Allowing the cells to differentiate on THEIR OWN

44
Q

What happens to the PS cells in vitro do if remove the signals that keep cells in an undifferentiated state?

A

SELF-ORGANISE and generate cell aggregates (EMBRYOID BODIES or ORGANOIDS)

To recapitulate the environment of the niche

Form FUNCTIONAL cells (eg. heart cells beat) and express the right markers in the right place

45
Q

What are the advantages of the 3D approach?

A
  • Recapitulation more accurately of the embryonic environment
  • Can see the cell interactions, which are similar to those happening in the embryo
46
Q

What are the disadvantages of the 3D approach?

A
  • Difficult to observe by microscopy

- Difficult to dissect the role of individual signals

47
Q

What is the 2D approach of differentiation in vitro?

A
  • Plate specific no. of cells on the right substrate that mimics embryonic environment
  • Remove signals that keep the cells in the differentiated state
  • Plate the cells in signals that push the cells into differentiation
48
Q

In the 2D approach, what signals push the cells into differentiation?

A

Signals that we know promote the differentiation into the 3 germ layers:
- APPROPRIATE levels of FGF, WNT

49
Q

What are the advantages of the 2D approach?

A

1) More controlled than the 3D approach (apply signals that push the cells into differentiate)
2) More traceable system (eg. for live images) - easier to test the role of specific signals, due to the 2D nature

50
Q

What are the disadvantages of the 2D approach?

A

1) Loss of cell interactions that may occur in vitro (not fully representative)
2) Differentiation in a CHAOTIC manner (hard to 100% control) - gives rise to IMPURITIES

51
Q

How can iPS cells be used in disease modelling?

A

Reprogram adult stem cells into iPS

  • Patient with genetic disease and reprogram their skin cells back into the PS state
  • Differentiate them into the lineages of affected cells
52
Q

What is microcephaly?

A
  • A neuro developmental disorder in infants

- Abnormally small brain

53
Q

What causes microcephaly?

A

Autosomal RECESSIVE mutation in the gene CDK5RAP2

54
Q

What are the symptoms of microcephaly?

A

Neurological defects

Seizures

55
Q

Why can mouse models not be used to study microcephaly?

A

Mouse mutants fail to recapitulate the condition

Cannot make models to show the phenotype

56
Q

How can microcephaly be studied?

A

1) Take skin cells from patient WITH the mutation
And take skin cells from a CONTROL sibling (without the mutation)

2) Generate iPS by expressing pluripotency factors
3) Use 3D approach to create a ‘mini brain’ in the petri dish
4) Look at the differences between the affected and not affected phenotype

57
Q

What was observed from the organoids in invitro modelling of microcephaly?

A

Affected:

  • Smaller no. of neurons and neural progenitors
  • Smaller cerebral organoids
58
Q

What is the potential application of organoids?

A

Small molecule screens using a variety of small molecules in order to find compounds that can REVERSE the phenotype

59
Q

What is familial dysautonomia?

A

Genetic disorder that affects the development and survival of the neurons controlling involuntary actions such as:

  • Digestion
  • Breathing
  • Tear production
  • BP regulation
  • Body temp

Also affects the sensory nervous system - controls activities relating to the senses:

  • Taste
  • Perception of pain, heat and cold
60
Q

What is the cause of familial dysautonomia?

A

Mutation in the IKBKAP gene

61
Q

What is the treatment of familial dysautonomia?

A

No treatment - 50% die before age 40

62
Q

How can familial dysautonomia be modelled using reprogramming?

A

1) Take skin cells from an affected patient and a healthy patient
2) Use these cells to make iPS cells
3) Differentiate the cells into neurons known to be affected by the disease
4) Measure the amount of cells that express Tuj1
5) Test molecules to try and reverse the phenotype

63
Q

What neurons are known to be affected by FD?

A

Neurons that innervate the visceral organs

64
Q

What is Tuj1?

A

A neuronal marker

65
Q

What happens when Tuj1 is quantitated in normal and affected cells in vitro?

A

No Tuj1 from the ND patients

No neurons from these patients

66
Q

What does Kinetin do when applied to iPS cells from FD affected patients?

What does this mean?

A

Significant increase in the number of in vitro derived neurons

67
Q

What is Parkinson’s?

What is it causes by?

A

A neurodegenerative disease

Caused by loss of dopaminergic neurons in the substansia nigra

68
Q

How can Parkinson’s be treated/not be treated with drugs?

A

Drugs treat the SYMPTOMS

But not treat the CAUSE of the disease

69
Q

How COULD Parkinson’s be treated?

A

1) Generating dopaminergic neurons in the petri dish using iPS
2) Inject back into the patient

70
Q

What are the markers found in the dopaminergic neurons in the SN?

A

TH

FOXA2

BMS247 - Stem cells (15 decks)

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