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Cell and Developmental Biology > STEM CELLS > Flashcards

STEM CELLS Flashcards

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1
Q

Define Stem Cells

A

A single cell that can replicate itself or differentiate into many cell types

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2
Q

What are the 2 types of Stem Cells and what can they differentiate into?

A

Embryonic Stem Cells - can differentiate into almost any cell type in the body except from placenta (pluripotent)
Adult Stem Cells - can differentiate into a subset of related cell types (multipotent)

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3
Q

What type of cell can differentiate into any cell including the placenta?

A

Totipotent

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4
Q

What part of embryonic stem cells can be removed and grown in culture?

A

Inner cell mass

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5
Q

How can you use embryonic stem cells to genetically engineer mammals (use mice as example)?

A
  1. extract mouse blastocysts
  2. genetically modify the cells and harvest and culture them
  3. insert back into another blastocyst
  4. transplant into a mother
  5. mother will have babies
    - will be chimeric
    - some cells will be modified and some will not
    - can then selectively reproduce and create generations with the mutation wanted
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6
Q

Name one strategy for gene targeting in mice

A
  1. Start off with mouse blastocysts
  2. Harvest embryonic stem cells
  3. Create a targeting vector
    - Need 2 arms of homologous DNA
    - 1000 bases to match the dna wanted
    - There is a resistant antibiotic section and a Negative selection bit
  4. Introduce plasmid into embryonic stem cells
    - Hope some stem cells will take up plasmid
  5. To select for it - add puromycin
    - Kills off any non transfected cells
    - End up with population of pure embryonic stem cells
    - Targeted construct is not incorporated into one of the stem cells.
    - Homologous recombination occurs where we lose the negative selection marker
  6. Take modified embryonic stem cell where one of the copies of the gene has the resistance introduced - interrupting gene function
  7. Introduce this into a blastocyst of a mouse
  8. Use coat colour to see how well this is working
    - Modified cells being put back into a pseudo pregnant mother
    - She will have a mosaic / chimeric mice
    - The more pink she is, the more genes contributed and the more of the modified stem cells available
  9. Mate one of the mice with a pink male mouse
    - Creates knock out mice
    knock out mice = both copies are mutated and can now target with CRSPR/Cas9
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7
Q

Name the 4 transcription factors that maintains the ES cell state

A

Myc
Oct4
Sox2
Klf4

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8
Q

What does transcription factor Nanog do?

A

Up regulate embyronic stem cell genes

down regulate the differentiation of genes

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9
Q

What does transcription factors Myc do?

A

Up regulate cell proliferation

Loosens the chromatin structure

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10
Q

What does activation of telomerase do?

A

Prevents cellular senescence

We lose parts of chromosomes without telomerase

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11
Q

What are Induced pluripotent stem cells (iPS)

A

Cells that reprogrammed back into pluripotent stem cells.

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12
Q

Where doe iPS’ come from?

A

Made from the patients own cells - typically skin cells or monocytes taken from blood

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13
Q

How do you create iPS cells?

A
  1. Isolate cells from patient (skin/monocytes)
  2. Introduce the 4 transcription factors
  3. Wait a few weeks
  4. Change culture conditions to stimulate cells to specialise the cells
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14
Q

How do you select iPS cells?

A
  1. Take fibroblast
  2. Introduce reporter construct that will allow to select for IPS cells
  3. Reporter has a promotor which is only active in embryonic stem cells
    - Therefore is turned off in the fibroblast
    - Will turn on once is specialised into the pluripotent cell
  4. Add resistance marker for the antibiotic G418
    - Wont be turned on in the fibroblast
  5. Add the 4 transcription factors
  6. In vast majority of cells it wont work
    - Cell will remain a fibroblast
    - The reporter will not turn on
    - The resistance gene is not expressed
    - When the antibiotic is introduced, the cell will die
  7. If it does work (rare)
    - Promoter will become active
    - The resistance gene is expressed
    When the antibiotic is introduced, the cell will survive
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15
Q

Where do somatic stem cells reside in?

A

Blood Vessels
Teeth
Liver
Bone Marrow

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16
Q

What are the 2 well characterised adult stem cell types?

A

Stem Cells in the epithelia of small intestine

Haematopoietic stem cells

17
Q

How does the gut lining renew itself?

A

Dividing cells push themselves up the crypt
- the transit amplifying cells moving up and still specialises
- is slothed off the top of the Villi
creates 4 types of differentiated cells

18
Q

What 4 types of differentiated cells come from the stem cells in the crypt/ bottom of the Villi?

A 
Absorptive Cell
- takes up nutrients from gut lumen
Goblet Cell
- secrete protective coat of mucus
Paneth Cell
- Secrete proteins to kill bacteria
Enteroendocrine Cell
- Secrete serotonin and peptide hormones to regulate growth, proliferation and digestive activities of other cells
19
Q

How do daughter stem cells know whether to stay as an embryonic stem cell or to proliferate?

A

Independent Choice model

20
Q

What is the independent choice model?

A

Random
- When stem cell divided it could cause either:
○ 2 stem cells
○ 2 differentiated cells or 1 of each

21
Q

What is the Asymmetric division model?

A

Some sort of localised determinant that marked the cell
- So when division of the stem ells occurred, they would be different
- One would have the determinant and one would not
- The one that did not have the determinant would differentiate
- The one that did have the determinant would stay as the stem cell
This would predict these outcomes

22
Q

What experimental system was used to find out whether gut stem cells are multipotent?

A

Made a reporter construct which had a stem cell specific promoter
- Only active in the stem cells
- Will label stem cells green
- And produces the yellow CreERT2 enzyme
○ Enzyme is inactive until tamoxifen is added
- Activated form of CreERT2 recognises DNA sequences and causes recombination events to occur
○ Flip out a region of the dna sequence that is surrounded by recombinase sites (the blocks in pink)
- Another reporter construct is driven by ubiquitous promoter which is continuously turned on
- Expression of LacZ is blocked by blocking sequence
- Activated recombinase removes the blocking sequence allowing LacZ to be expressed

23
Q

What keeps the stem cells in its place and maintains its stem cell character?

A

Paneth cells

24
Q

How do Paneth cells create the stem cell niche?

A

There are Paneth cells surrounding the stem cells at the base of each crypt (in Villi in Gut cells).
These Paneth cells generate signals to activate pathways in stem cells.
Paneth cells release ligands that activate either the Wnt or Notch signalling pathway.

25
Q

What does the Wnt signalling pathway do?

A

Maintains proliferation

- in adenomas, the Wnt signalling cannot be turned off so keep dividing

26
Q

What does the Notch signalling pathway do?

A

Inhibits differentiation

27
Q

What are Haematopoietic stem cells?

A

Stem cells that only give rise to blood cells
- give rise to all the cells in the blood
(replenish the immune system)

28
Q

How did a transfusion of bone marrow cells rescue an irradiated mouse?

A
  1. Irradiation halts blood cell productions (mouse would die)
  2. inject bone marrow cells from healthy donor
  3. mouse survives - the injected stem cells colonise its hematopoietic tissues and generate a steady supply of new blood cells
    (similar procedure used to treat leukaemia)
29
Q

What cells do haemotopoietic stem cells depend on? And how?

A

Stromal cells occurs through binding through receptor ligand interactions.

30
Q

How can cancer stem cells be responsible for the growth of some tumours?

A

Cancer stem cells proliferate at a much slower rate. Therapies that target fast multiplying cells (such as radiotherapy or chemotherapy) do not destroy the stem cells and so they continue to replicate and a new tumour grows

Cell and Developmental Biology (16 decks)

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