Gut organoids Flashcards
What are progenitors cells?
The first daughters from a stem cell that are heading in the differentiation route
How do we know which cells are in the stem cell niche?
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
Why is the gut stem cell highly described?
- Has a MARKER of the stem cell, which is NOT expressed in ANY of its immediate progenitor cells
- Marker is highly conserved
What gave us our understanding about the cells and signals operating in stem cell niches to GOVERN stem cell behaviour?
Studies in DROSOPHILA:
- Make MUTANTS with particular signals/signalling components MISSING
Eg.
- BMP
- JAK/STAT
- WNT
- Notch/Delta
What is armadillo (Arm)
A HIGHLY CONSERVED, key component of the Wnt signalling pathway
What happens when cells receive certain level of Wnt signalling?
Why?
These cells become STEM CELLS
These cells turn on specific genes at this level of Wnt signalling that are involved in stemness
Why do we want to harness stem cells?
To be able to multiply them:
For use in future therapies, drug screening, understanding of a disease process, understanding aging
How can we identify stem cells in the vertebrate adult, in order to harness them?
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)
What is one of the best-defined adult stem cell niches?
Where is this present?
The mammalian gut crypt
Present at the bottom of each finger like vili that project into the lumen of the gut
What lies at the bottom of the mammalian gut crypt?
What surrounds this? What do these do?
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
What are the support cells in the mammalian gut crypt called?
PANETH cells
What are the gut stem cells and Paneth cells a part of?
The gut epithelium
What does the gut epithelium lie adjacent to?
Mesenchymal cells and blood vessels
What is collectively known as the ‘gut stem cell niche’?
The gut epithelium (containing the stem cell and the Paneth cells) and the adjacent mesenchymal cells and blood vessels
How many gut stem cell niches are there?
MANY
- 4 sit at the bottom of the crypt of each vili
What is the speed of turn over in the gut?
Very high
What happens when the gut stem cell divides asymmetrically?
What happens to these cells?
Gives rise to:
- 2nd stem cell –> retained at the niche
- Daughter that begins to differentiate –> Leaves the microenvironment and ASCENDS up the crypt
What are the first progenitors from the gut stem cell called?
What do these cells do?
Transit amplifying cells
Undergo many SYMMETRICAL divisions to give rise to MANY progenitor cells that move up the crypt and form the differentiated cells
Where is wnt signalling high in the crypt?
At the BASE
What is a target gene of Wnt signalling?
Where are the cells expressing this gene found?
Lgr5
4 Lgr5+ cells sit in the NICHE - each surrounded by Paneth cells
What is the original name for the Lgr5+ cells?
CBC cells (crypt base columnar cells)
What was it determined that the CBC (Lgr5+ cells) are?
Why?
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)
What experiments can be done to prove that the Lgr5 expressing cell in the crypt is the stem cell?
ECTOPIC TRANSPLANTATION:
- This cell should be able to SELF-RENEW
- As well as DIFFERENTIATE to give rise to vili
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?
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
How is a transgenic reporter line made?
- 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)
How can many different cells be visualised using the transgenic reporter line at the same time?
Using many different colours from jelly fish
All under the regulatory influences from different genes
When using the transgenic reporter line and transplanting the Lrg5 expressing cell into a WT organism, what is seen?
What does this prove?
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
What is an organoid?
A 3D multicellular in vitro tissue construct that mimics its corresponding in vivo organ
Mini organ
What can a gut organoid be useful for?
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
How is a gut organoid made?
- 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
