Lecture 28

Question 1

What is meant by a stem cell niche

Answer 1

A stem cell niche refers to the environment in which a stem cell is present. This usually includes the stem cells and its surrounding support cells. The stem cell is situated and held in its location by a tight microenvironment

Question 2

What is the role of support cells in a stem cell niche

Answer 2

Support cells release signals to maintain stem cells in a quiescent state as well as later triggering its differentiation

Question 3

Endothelial cells of the vasculature are frequently support cells, T or F

Answer 3

T

Question 4

Which key signalling pathways have been found to be released by the support cells

Answer 4

BMP, Wnt and Notch signalling

Question 5

What is the main problem with using stem cells as a treatment of disease

Answer 5

Many tissue are less amenable and more complex than we thought and there are many problems with using stem cells due to the difficulty in controlling their proliferation and differentiation

Question 6

Which is the best studied stem cell niche in the body and has proved the gold standard in stem cell application

Answer 6

Gut stem cells of the intestinal crypt

Question 7

Where are the stem cells of the gut located

Answer 7

Gut stem cells are located in the gut crypt at the base of the villi

Question 8

How do the cells of the intestinal crypt differ from proximal to distal

Answer 8

The stem-like crypt cells are located at the distal end and the differentiating cells are found at the proximal end

Question 9

The villi and crypt cells are squamous epithelial cells, T or F

Answer 9

F – they are columnar epithelial cells

Question 10

What cells are found alongside the stem cells at the base of the intestinal crypt

Answer 10

Paneth cells

Question 11

What are the transit amplifying cells of the gut crypt

Answer 11

The transit amplifying (TA) cells are a stem cell progeny that have moved upwards from the base of the crypt and have begun to differentiate

Question 12

What is the specific name given to the stem cells at the base of the gut crypt

Answer 12

Crypt base columnar cells

Question 13

What type(s) of gene expression is seen by the stem cells at the base of the crypt

Answer 13

Wnt target genes

Question 14

What genes are expressed by the TA cells and those cells that have differentiated

Answer 14

BMPs and Notch

Question 15

How do the stem cells prevent their own differentiation despite BMPs coming from the proximal cells that supress stem fates

Answer 15

The stem cells express BMP antagonists such as noggin and gremlin to refine the BMPs coming from the differentiated cells

Question 16

Which cells of the gut crypt express wnt ligands

Answer 16

Paneth cells

Question 17

How do the CBCs response to wnt signalling

Answer 17

They begin to express the wnt target gene LGR5 as well as making EGF receptor and notch ligands. Wnt signalling together with EGFs and Notch act to maintain a stem cell fate

Question 18

BMPs inhibit the stem cell fate, T or F

Answer 18

T

Question 19

What happens as the cells move up the gut crypt, how does this account for differentiation of CBCs to mature gut cells

Answer 19

As cells move up the crypt the BMP antagonist levels reduce and BMP signalling begins to repress the stem cell fate. This accounts for the differentiation seen proximally in the gut crypt

Question 20

The LGR5 receptor is only expressed by Paneth cells of the gut crypt, T or F

Answer 20

F – LGR5 is only expressed by the stem cells

Question 21

All signals from the Paneth cells bind to the promoter of the LGR5 gene to upregulate its expression, T or F

Answer 21

T

Question 22

Describe a genetic technique that could be used to determine the location of the LGR5 positive CBCs at the base of the gut crypt

Answer 22

As the LGR5 gene is only expressed by the stem cells, these will be the only cells to show reporter construct expression for this gene which is upregulated by signals from the Paneth cells. Firstly, isolate the mouse LGR5 promoter and replace the coding region for LGR5 with the sequence for GFP. This creates a reporter construct in which the GFP coding region under control of LGR5 promoter. This transgene is then inserted into a mouse to create a transgenic organism. The expression of LGR5 can then be visualised under a microscope by viewing the fluorescent reporter in UV light

Question 23

What are gut organoids and how can we use gut stem cells to make them

Answer 23

Gut organoids are mini guts derived from stem cells of the gut crypt. Firstly the cells of the gut crypt are dissociated and then the cells with positive reporter construct staining for LGR5 are isolated. These cells are placed into a fluorescent activated cell sorter which separates the fluorescent population. This population can differentiate into all the cells of the intestinal crypt and villi and so are placed in a medium containing the correct substances for growth. Finally, the single cells are encouraged to proliferate and then differentiate to give rise to all the cells of the gut

Question 24

How can we use organoids developed from gut crypt cells therapeutically

Answer 24

Gut organoids can be used in the treatment of wound beds in the gut that have resulted from injury, surgery or abuse. Implantation of organoids into the wound bed leads to a regeneration of gut structures in mouse models

Question 25

Explain the three categories of organoid usage which could prove useful

Answer 25

Experimental tool to isolate and interrogate intestinal stem cells. Therapeutic tool to treat post-endoscopic mucosal resection and ulcerative colitis. Diagnostic tool to diagnose CF, metabolic disorder and diabetes by isolating stem cells and grow organoids to diagnose inherent mechanistic abnormality