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BMS247 - Stem cells > Gut organoids > Flashcards

Gut organoids Flashcards

1
Q

What are progenitors cells?

A

The first daughters from a stem cell that are heading in the differentiation route

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2
Q

How do we know which cells are in the stem cell niche?

A

1) Express SPECIFIC genes that are RESTRICTED to the stem cell and not expressed in other cells
2) In situ hybridisation - detect if mRNA transcribed
3) Immunostaining (using antibodies) - detects the protein made as a product of the transcript
4) Take SECTIONS through the embryo and apply ANTISENCE probes or ANTIBODIES - only bind to specific cells expressing the genes

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3
Q

Why is the gut stem cell highly described?

A
  • Has a MARKER of the stem cell, which is NOT expressed in ANY of its immediate progenitor cells
  • Marker is highly conserved
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4
Q

What gave us our understanding about the cells and signals operating in stem cell niches to GOVERN stem cell behaviour?

A

Studies in DROSOPHILA:
- Make MUTANTS with particular signals/signalling components MISSING

Eg.

  • BMP
  • JAK/STAT
  • WNT
  • Notch/Delta
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5
Q

What is armadillo (Arm)

A

A HIGHLY CONSERVED, key component of the Wnt signalling pathway

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6
Q

What happens when cells receive certain level of Wnt signalling?

Why?

A

These cells become STEM CELLS

These cells turn on specific genes at this level of Wnt signalling that are involved in stemness

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7
Q

Why do we want to harness stem cells?

A

To be able to multiply them:

For use in future therapies, drug screening, understanding of a disease process, understanding aging

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8
Q

How can we identify stem cells in the vertebrate adult, in order to harness them?

A

Cutting thin sections of the tissue and looking for cells that:

1) Occupy a particular microenvironment
2) Clearly RESPOND to specific signals (WNT, BMP, JAK/STAT, Notch)

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9
Q

What is one of the best-defined adult stem cell niches?

Where is this present?

A

The mammalian gut crypt

Present at the bottom of each finger like vili that project into the lumen of the gut

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10
Q

What lies at the bottom of the mammalian gut crypt?

What surrounds this? What do these do?

A

Cells that respond the most SPECIFICALLY to Wnt/BMP/Jak/stat signalling at the very bottom

Surrounded by a hub of support cells that provide the Wnt signalling and activate the Wnt signalling pathway in the the responding cells –> turn on genes associated with STEMNESS

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11
Q

What are the support cells in the mammalian gut crypt called?

A

PANETH cells

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12
Q

What are the gut stem cells and Paneth cells a part of?

A

The gut epithelium

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13
Q

What does the gut epithelium lie adjacent to?

A

Mesenchymal cells and blood vessels

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14
Q

What is collectively known as the ‘gut stem cell niche’?

A

The gut epithelium (containing the stem cell and the Paneth cells) and the adjacent mesenchymal cells and blood vessels

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15
Q

How many gut stem cell niches are there?

A

MANY

- 4 sit at the bottom of the crypt of each vili

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16
Q

What is the speed of turn over in the gut?

A

Very high

17
Q

What happens when the gut stem cell divides asymmetrically?

What happens to these cells?

A

Gives rise to:
- 2nd stem cell –> retained at the niche

  • Daughter that begins to differentiate –> Leaves the microenvironment and ASCENDS up the crypt
18
Q

What are the first progenitors from the gut stem cell called?

What do these cells do?

A

Transit amplifying cells

Undergo many SYMMETRICAL divisions to give rise to MANY progenitor cells that move up the crypt and form the differentiated cells

19
Q

Where is wnt signalling high in the crypt?

A

At the BASE

20
Q

What is a target gene of Wnt signalling?

Where are the cells expressing this gene found?

A

Lgr5

4 Lgr5+ cells sit in the NICHE - each surrounded by Paneth cells

21
Q

What is the original name for the Lgr5+ cells?

A

CBC cells (crypt base columnar cells)

22
Q

What was it determined that the CBC (Lgr5+ cells) are?

Why?

A

Stem cells

Stem cell behaviour:

  • High levels of Wnt signalling
  • Cell responding to the high level of Wnt signalling by turning ON the Lgr5 gene

(this is characteristic of other stem cells in model organisms)

23
Q

What experiments can be done to prove that the Lgr5 expressing cell in the crypt is the stem cell?

A

ECTOPIC TRANSPLANTATION:

  • This cell should be able to SELF-RENEW
  • As well as DIFFERENTIATE to give rise to vili
24
Q

During ectopic transplantation of the stem cell, how do you know which cell is the stem cell transplant? (no longer in a niche)

Why can in situ hybridisation not be used?

A

Lineage tracing analysis by making a transgenic reporter line:

  • Using GFP
  • Artificially make transgenic mouse where all the Lgr5+ cells fluoresce green is shine blue light onto them

NOT ISH - can only occur on DEAD, FIXED tissues

25
Q

How is a transgenic reporter line made?

A
  • Remove GFP from the DNA sequence using restriction enzyme
  • Insert GFP in its place
  • GFP now controlled by the enhancers/promoters/TF that normally drive the cell specific expression of Lrg5
  • Insert this DNA into an organism at the 2 cell stage –> inserts into host DNA (forms a transgenic organism)
26
Q

How can many different cells be visualised using the transgenic reporter line at the same time?

A

Using many different colours from jelly fish

All under the regulatory influences from different genes

27
Q

When using the transgenic reporter line and transplanting the Lrg5 expressing cell into a WT organism, what is seen?

What does this prove?

A

See ALL the cells of the villi are GREEN –> all derived from the single Lrg5 expressing cell

Proves that this Lrg5 expressing cell is the stem cell

28
Q

What is an organoid?

A

A 3D multicellular in vitro tissue construct that mimics its corresponding in vivo organ

Mini organ

29
Q

What can a gut organoid be useful for?

A

Study aspects of the organ in tissue culture:

  • Can COMPARE the organoids between diseased and normal individuals
  • Regenerative tool for ulcers, bed wounds etc
  • Better understand the signalling and CAMs involved in organising the crypt
  • Better understand the gut stem cell (how it differentiates and gets appropriate organisation)
  • Cell therapy
  • Test the effects of medicines/viruses
30
Q

How is a gut organoid made?

A
  • Take the crypt and DISSOCIATE the cells into single cells (stem, Paneth, mesenchymal)
  • Isolate the stem cell through fluorescent activated sorting
  • Culture single stem cell (expressing Lrg5) in VITRO in an appropriate medium –> form organoid

BMS247 - Stem cells (15 decks)

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