Adult Stem Cells in the Gut

Question 1

What pathways are normally altered in cancer?

Answer 1

Developmental pathways

Question 2

How does the lining change along the GIT?

Answer 2

Stratified epithelium = mouth/pharynx/esophagus

Columnar epithelium = stomach + intestines

Stratified epithelium = rectum & anus

Question 3

Where is the stem cell compartment in the gut?

Answer 3

Intestinal crypts = where stem cells reside

Very proliferative
High turnover

Question 4

What other cells are found in the intestinal crypts?

Answer 4

At the very base stem cells and Paneth cells alternate

When the crypt is no longer curved = transit-amplyifying cells

Goblet and enteroendocrine cells are found as well
Absorptive cells are most numberous

Question 5

What are the 2 epithelium stem cell pools found in the small intestine?

Answer 5

Columnar Lgr5-expressing cells = present at crypt base

Bmi1-expressing cells = largely reside ABOVE crypt base

Question 6

Features of Lgr5 and Bmi1 stem cell population cycles?

Answer 6

Bmi1 slow cycling

Lgr5 rapidly cycling

Question 7

How did they ablate Lgr5 cells in mice and what happened?

Answer 7

Used a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus.

We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population

Question 8

What does the fact that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium tell us?

Answer 8

Other cell types can compensate for elimination of this population.

After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells.

Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium.

Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool.

The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.

Question 9

What does suprabasal describe?

Answer 9

Where the curvature of the crypt stops

Question 10

What are the cell types that intestinal stem cells directly differentate into?

Answer 10

Enterocyte

Secretory

Question 11

What cells do secretory cells differentate into?

Answer 11

Paneth/goblet

Enteroendocrine

Question 12

What does Notch regulate in intestinal stem cells?

Answer 12

The stem versus secretory fate decision as well as further fate choice and differentiation events in the crypt

Question 13

What is Ascl2?

Answer 13

Proneural bHLH transcription factor Ascl2 is associated with stemness and is absolutely required for intestinal stem cell maintenance

Question 14

How does Notch affect Ascl2?

Answer 14

Active Notch is required for Ascl2 expression and its loss results in precocious crypt cell differentiation

Question 15

What is Atoh1?

Answer 15

Acts as a master regulator of fate specification of the secretory lineage

Question 16

How does Notch affect Atoh1?

Answer 16

Ascl2 expression is maintained by active Notch signalling that also acts to suppress Atoh1. Expression of Atoh1 is cell-autonomously inhibited by Hes proteins and in the absence of Notch signalling, crypt stem cells precociously differentiate into secretory goblet cells

Question 17

Explain the role of Paneth cells in secretory and non-secretory fate

Answer 17

Paneth cells express Delta

So stem cells near Paneth cells express Notch = maintain undifferentiated state because express Ascl2 and supress Atoh1

As cell migrate they lose contact with Paneth cells so don’t express Notch as much = cells become poised between secretory and non-secreotry fate

Question 18

How do stem cells become enterocytes ? ***

Answer 18

As cells move away from Paneth cells, they lose direct Delta input from Paneths, but Notch signaling is maintained in some cells through lateral inhibition, where neighboring progenitor cells express Delta ligands and reinforce Notch-Hes1-Atoh1 repression, pushing cells toward absorptive enterocyte fate.

As they move away from stem cell niche = decrease in Wnt and increase in BMP causing the cells to differentiate and not self-renew

Hight Notch = High HEs1 = No Atoh1 = Absorbtive Enterocyte Fate

Question 19

What role does Wnt play in intestinal stem cell differentiation?***

Answer 19

Wnt signaling helps to maintain a balance between stem cell self-renewal and differentiation

Active Wnt signaling keeps ISCs in an undifferentiated state by promoting the expression of genes involved in stem cell identity, such as Lgr5.

As cells move away from crypt = Wnt signals decrease so cells can differentiate

Question 20

What role does BMP play in intestinal stem cell differentiation?***

Answer 20

Works antagonistically to Wnt

BMP signaling is primarily involved in inducing differentiation of intestinal epithelial cells, particularly in the upper regions of the crypt. As cells migrate up from the base of the crypt to the villus, BMP signaling becomes more active, promoting the transition from proliferating stem cells to differentiated cell types

Question 21

What does the stem cell niche provide?

Answer 21

Signalling = grwoth factors, immune cells and cytokines

Metabolic nutrients and blood supply

Structural cues

Mechanical regulation = forces in different directions

Question 22

Describe the diversity of the intestinal stem cell population

Answer 22

Phenotypically diverse

Stem cell markers overlap and interconvert

Slightly different states of stemness in the crypt

Question 23

What experimental technique did they use to study the intestinal stem cell niche?***

Question 24

What determines the probability a stem cell exits the niche?

Answer 24

When stem cell exits niche = stops being a stem cell

Position in the niche determines this = further away from Goldilocks spot means more likely to leave niche

Question 25

What is the Goldilocks spot?

Answer 25

Optimal position for being a stem cells = base of intestinal crypt

Question 26

What is the difference between small-intestinal epithelium and colon epithelium?

Answer 26

The shorter large intestine, and colon, lack villi and largely specializes in water reuptake

Question 27

How were label retaining cells (LRC) discovered?

Answer 27

Induction causes all cells to express the marker: histone 2B-YFP (yellow) within 24h except in non-cycling Paneth cells After turning off induction = label should be diluted with each division After 3 weeks very few cells still have the marker Stem cells are often quiescent or slow-cycling, meaning they divide much less frequently than transient amplifying cells. If you label all dividing cells, the fast-dividing progenitors will quickly dilute that label, but slow-dividing stem cells will retain it over time.

Question 28

Are label retaining cells Paneth cells and how do we know?

Answer 28

No they are not Paneth cells Paneth cells can be marked by UEA but these could not be

Question 29

How are label retaining cells related to Paneth cells?

Answer 29

LRCs are secretory cells = precursors to Paneth and Goblet cells Looking that their transcriptomic profiles we can see they have overlapping but distinct profiles to Paneth cells They eventually do become Paneth cells

Question 30

Can label retaining cells return to being stem cells?

Answer 30

YES When stem cells are depeleted (potentially due to injury) these secreotry precurosrs can be reactivated to become stem cells again

Question 31

To build a tumour, what do cancer cells have to compete with?

Answer 31

Non-cancer cells

Question 32

What process do cancer-associated mutations affect?

Answer 32

The ability of cancer cells to compete with non-cancer cells to build a tumour

Question 33

Why is clone size important in cancer?

Answer 33

Clone size is used as an indicator of how competitive a particular mutation is Cancer happens when cells start to grow uncontrollably. The reason this happens is that certain mutations allow cancerous cells to outcompete normal cells in their environment. If a mutated cell is able to form a large clone, it could eventually take over the tissue and cause problems, like in cancer. This is why scientists look at clone size to understand how cancer might start and how easily mutated cells can take over a tissue.

Question 34

What is the role of the Kras gene?

Answer 34

The Kras gene is part of a family of genes that make proteins involved in sending signals inside cells. These signals help regulate important cell functions like growth, division, and survival When the Kras gene mutates, it can cause the Kras protein to become permanently "on." This means that the signals for the cell to keep growing and dividing don't turn off, even when they should

Question 35

What happens in the Kras mutation compared to WT cells?

Answer 35

Clones become bigger faster with Kras mutation = meaning the cells are better at taking over the entire tissue Kras has a larger average clone size than WT cells Kras mutants are more competitive and can take over crypts

Question 36

What is the role of Apc gene?

Answer 36

APC protein helps regulate the Wnt signaling pathway, a pathway that controls cell growth and the process of cells deciding whether to grow or stop growing. The APC protein helps degrade certain molecules that promote cell growth. When APC is functioning properly, it stops cells from growing too fast or forming tumor

Question 37

What happens when APC is mutated, compared to WT cells?

Answer 37

Same as in Kras mutations Apc mutant cells are also more competitive and can take over crypts APC normally inhibits Wnt by degrading beta-catenin No APC, means beta-catenin is expressed and permenantly expresses Wnt

Question 38

How do Kras and APC mutations differ?

Answer 38

While both APC mutations and Kras mutations lead to uncontrolled cell growth, they work in slightly different ways. APC mutation = Loss of a "brake" that stops cell growth, leading to early cancer changes. Kras mutation = Constant "gas pedal" that tells cells to grow even when they shouldn’t, making cells divide uncontrollably. Both mutations contribute to cancer, but APC is more about losing control over growth, while Kras is about constantly driving growth forward.