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Stem Cell and Cancer > L4 - In Vitro Stem Cell Models > Flashcards

L4 - In Vitro Stem Cell Models Flashcards

1
Q

Why is the mouse a goof model to test the effect of ESCs?

A

Can be genetically modified

Short gestation period

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2
Q

Where are early embryonic cells found?

A

In inner cell mass

They are pluripotent

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3
Q

What two things show that a cell is pluripotent?

A

Expression of pluripotency factors (only expressed in inner cell mass)
- Nanog
- Oct4
- Sox2
Teratocarcinoma formation
- Pluripotent cells grafted onto kidney of host mouse - give rise to teratocarcinomas
- They are tumours containing all cell types

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4
Q

What evidence is there that stem cell potency diminishes as the embryo develops?

A

At E4 we find pluripotent cells

At E9 we find neural stem cells (bipotent)

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5
Q

What does the ectoderm form?

A

Skin

CNS

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6
Q

What does the mesoderm form?

A

Blood

Muscle

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7
Q

What does the endoderm form?

A

Gut

Lung

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8
Q

Why are stem cells in vivo difficult to study

A

Small cell numbers
In utero development
Ethics

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9
Q

What is the importance of capturing cell in vitro in petri dishes?

A

In vitro modelling of embryonic development can lead to production of clinically relevant cell populations

  • Drug screening
  • Cell replacement therapies
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10
Q

How are embryonic stem cells captured in vitro?

A
  1. Signals to maintain cells in self-renewing, undifferentiated state and can replace feeders
    a. Mouse - Leukaemia Inhibitory Factor, BMP
    b. Human - FGF2, TGFβ
  2. Plate single E4 cells on layer of feeder cells - irradiated stromal cells derived from later embryos which support ES cell growth
  3. Once embryonic cells have divided a few times, disaggregate and re-plate
  4. ES cells express a transgene encoding GFP
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11
Q

How do you reprogram adult somatic cells to a pluripotent cell fate?

A
  1. Get skin or biopsy from an adult with a disease or normal
  2. Ectopically overexpress reprogramming factors
  3. Reverses cells back into embryonic stem cell state
    More ethical than using embryos
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12
Q

What do embryonic stem cells express?

A

Express the pluripotency factors Oct4, Nanog, Sox2

Resemble the cells present in the inner cell mass of early embryo

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13
Q

When transplanted into permissive environments what do ESCs form?

A

Teratocarcinomas

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14
Q

What happens if mouse ES cells are reintroduced to normal mouse embryos?

A

They contribute to normal development

  • Mice derived from donor embryonic cells
  • Shown using GFP
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15
Q

How do you capture neural stem cells in vitro?

A
  1. Dissociate cells
  2. Plate on laminin in the presence of the cytokines FGF2 and EGF
  3. NS cells express undifferentiated markers - RC2
  4. No expression of genes indicative of differentiation
    a. Glia - GFAP
    b. Neuron - TUJ1
  5. A single cell can generate identical daughter cells - stem cell
  6. Can differentiate into glia and neurons after removing FGF2, EGF
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16
Q

What are the two main approaches to in vitro differentiation?

A

3D

2D/adherent

17
Q

What is the 3D approach to in vitro differentiation?

A

Remove signals that keep cells in an undifferentiated state
- Mouse – BMP and LIF
- Human - FGF2, TGFβ
Grow in aggregates (embryoid bodies/organoids) in presence or absence of signals

18
Q

What are the advantages of the 3D approach?

A

Recapitulates more accurately embryonic environment

19
Q

What are the disadvantages of the 3D approach?

A

Difficult to observe role of individual signals

20
Q

What is the 2D approach to in vitro differentiation?

A

Plate a defined number of cells on the right substrate or extracellular matrix
Remove signals that keep cells in an undifferentiated state
- Mouse – BMP and LIF
- Human - FGF2, TGFβ
Grow in defined medium with appropriate amounts of signals - FGF, WNT

21
Q

What are the advantages of the 2D approach?

A

More tractable system – live imaging
- E.g. GFP expression reflects T(Bra) (early mesodermal marker) expression during ES cell Easier to test the role of specific signals

22
Q

What are the disadvantages of the 2D approach?

A

Loss of cell interactions that may occur in vivo

23
Q

What is microcephaly

A

Neurodevelopmental disorder
Infants are born with an abnormally small brain
Neurological defects and seizures

24
Q

What is the cause of microcephaly?

A

Due to various autosomal recessive mutations

Mouse mutant fail to recapitulate the condition

25
Q

What is the possible treatment for microcephaly?

A

Taken skin biopsy’s from infected patient and a normal sibling
Generated induced pluripotent stem cells by expressing pluripotency factors
Generated mini brains in petri dishes
- Smaller number of neurons and get smaller number of cerebral organoids

26
Q

What is the potential application for microcephaly on the development of mini brains?

A

Small molecule screens using mutant differentiated cells can help identify suitable drugs

27
Q

What is familial dysautonomia?

A

Genetic disorder that affects
Development and survival of neurons that control involuntary actions - digestion, breathing, tears, regulation of blood pressure and body temperature
Sensory nervous system - taste and the perception of pain, heat, and cold

28
Q

Familial dysautonomia facts

A

Problems related to this disorder first appear during infancy
No treatment - 50% of the affected individuals by age 40
Usually caused by mutation in IKBKAP gene

29
Q

What is the possible treatment for familial dysautonomia?

A

Took skin biopsy from FD patient and healthy individual
Produced induced pluripotent stem cells
Differentiated these into neurones we know are affected by FD

30
Q

What is the role of Kinetin in familial dysautonomia?

A

Candidate drug testing to discover molecules that reverse the phenotype
Kinetin treatment induces an increase in number of in vitro derived neurons

31
Q

What are the symptoms of Parkinson’s?

A 
Tremor 
Slowness of movement
Rigidity
Dementia
Progressive loss of dopaminergic neurons
32
Q

What is the potential treatment for Parkinson’s?

A

Generate dopaminergic neurones in petri dish using hES cells
Tyrosine hydroxylase - enzyme involved in dopamine synthesis
Neurones transplanted into mice which showed evidence of improvement in motor function

33
Q

How can you treat macular degeneration?

A

Development of retinal pigment epithelial cells from hPSCs

Stem Cell and Cancer (15 decks)

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