L5 - Adult Stem Cells In The Gut

Question 1

What do stem cell niches provide?

Answer 1

Provide support cells that keep the stem cell in their undifferentiated state
Learnt a lot from comparison of niches in simple animal models

Question 2

How can you detect the a stem cell from a normal cell?

Answer 2

The stem cell expresses particular genes not shown in other cells
Can detect these by
- In situ hybridisation – detects if the mRNA is translated
- Antibodies
- Only bind to particular cells expressing particular genes
Very few markers only expressed in stem cells and not in the progenitor cell daughters

Question 3

Which four signalling pathways regulate stem cells in their niche were discovered through Drosophila?

Answer 3

Notch/delta
Jack/stat
Wnt
- Armadillo is a key component
- If cells receive a certain amount of Wnt signalling they become stem cells
- They turn on genes only found in stem cells
Bmp

Question 4

What is the potential future for stem cells?

Answer 4

Harness stem cells for therapies
- Can be grown in a petri dish to create a large source of cells
Gut stem cells show the best practice for developing stem cells for future application

Question 5

Where are gut stem cells found?

Answer 5

One adult stem cell niche is in the mammalian gut crypt - at the base of the villi

• You have millions of these niches in your gut
• 1 niche for each villi

Question 6

Where are the different cells found within the crypt of the gut?

Answer 6

The crypt is a tube of cells arrayed on a basement membrane

• Stem-like and Paneth cells in a niche at the distal end
  
  • They are part of the gut epithelium
• Differentiating cells at the proximal end

Question 7

What are transit amplifying cells?

Answer 7

Stem cell progeny (transit amplifying cells) move upwards and differentiate

Question 8

What helps regulate stem cell activity in the gut?

Answer 8

Signals from underlying mesenchymal cells

Question 9

How were crypt base columnar cells identified?

Answer 9

Through expression of Wnt target genes
Studies identified factors that regulate proliferation and differentiation in the crypt
High levels of Wnt and Notch expression at distal/ventral end of the crypt
Identified cells that respond to Wnt signals and express Wnt target genes
- Respond to Wnt signalling by turning on Lgr5 gene

Question 10

How was the role of Lgr5 tested?

Answer 10

Lineage tracing analysis

Question 11

How does lineage tracing analysis work?

Answer 11

Transgenic reporter lines - make a Lgr5-GFP transgenic mouse
- All the Lgr5 positive cells express this GFP - report gene in real time
Expression of Lgr5 controlled by promoters and enhancers upstream of the coding sequence
Use restriction enzymes to cut a section of the DNA that contains the promoters of Lgr5
Clone GFP sequences downstream of this section of DNA
- GFP under the control of mouse Lgr5 cell promoters
Can then dissect the cell, containing Lgr5, out and transplant it ectopically

Question 12

How are mini-guts made from gut stem cells?

Answer 12

Lgr5+ CBC cells genetically labelled by GFP are sorted and imbedded in Matrigel
- Culture medium consists of EGF, Noggin and R-spondin
FACs - fluorescence-activated cell sorting machine
- Separates out the cells that are fluorescing
- This cell continues to grow forming an organoid - a symmetric cyst structure
- The symmetry is broken by bud formation
- The budding structure resembles a crypt

Question 13

What are mini organoids used to better understand?

Answer 13

Used to better understand the factors used to organise the crypt

Question 14

What is the importance of engraftment of intestinal organoids?

Answer 14

Organoids adhere to de-epithelialised wound bed

They form a flat epithelial layer followed by crypt reconstruction

Question 15

What are the 3 main uses for mini-organoids?

Answer 15

Experimental tool
Diagnostic tool
Therapeutic tool

Question 16

How are mini organoids used as an experimental tool?

Answer 16

Research for 
- Intestinal stem cell
- Intestinal differentiation 
- Epithelial functions 
Genetic manipulation 
Gene expression analysis 
Live imaging

Question 17

How are mini-organoids used as a diagnostic tool?

Answer 17

Cystic fibrosis
Mutational analysis in CRC
Drug absorption/metabolism
Patient derived epithelial mini-guts recapitulate in vivo intestinal epithelial functions and genetic signatures
Expansion of pure epithelial cells provides a source for deep sequencing or functional assays

Question 18

How are mini-organoids used as a therapeutic tool?

Answer 18

Potential regenerative therapy for

• Microvillous inclusion disease
• Ulcerative colitis
• Post-endoscopic mucosal resection