lecture 15: gastrointestinal stem cells Flashcards
1
Q
Which tissue regenerates more rapidly than any other?
A
- intestinal epithelium
- small intenstine characterised by villous projections
- at the base of the villi are crypts
- this is where the stem cells are located
- between nondividing differentiated paneth cells
- previously thought they were located between nondividing and rapidly dividing cells
- knowledge in the field is moving really fast
2
Q
What are the four types of differentiated cells located in the gut epithelium?
A
- enterocytes - absorptive
- goblet cells - secrete mucus
- enteroendocrine cells - secrete hormones
- paneth cells - immune cells and recently found to be niche cells (only in small intestine)
3
Q
Where are the stem cells found?
A
- stem cells are found in the crypts - crypt based columnar (CBC) cells and “+4” cells
- also transet amplifying cells
4
Q
How is the intestinal crypt a stem cell niche?
A
- intestinal stem cells drive normal cell turnover and maintenance
- all cell types in the epithelium are derived from stem cells
- stem cells generate a population of proliferating transit amplifying (TA) cells
- the behaviour of stem cells is controlled by the surrounding environment or “niche”
5
Q
What are the two models of intestinal stem cells?
A
- +4 cell model
- if you radioactively label the intestinal epithelium you see long term label retaining cells
- this is suggestive of cells that proliferate slowly
- the cells were identified in this +4 position
- suggested by analogy (cf epidermal stem cells) that maybe these slowly cycling cells are the stem cells
- stem cell zone model
- suggested in 2007
- identified a population of rapidly proliferating cells found squeezed in between the paneth cells
- histologists hadn’t really taken much notice of these cells
- difficult to see unless you are really looking for them
- crypt base columnar cells
- able to show through a lineage tracing experiment that these cells can produce all the cells found in the intestinal epithelium
- a number of molecules have now been isolated that mark these cell populations but which of these models is correct?
- there is experimental evidence for both models
- controversy in the literature
- +4 american, CBC european
- some evidence suggests that +4 are simply a subpopulation of CBCs, not necessarily as a seperate cell type in their own right
6
Q
What are models and markers of intestinal stem cells?
A
- stem cell zone model:
- CBC stem cell markers:
- LGR5 (wnt target)
- OLFM4
- ASCL2 (wnt target)
- CBC stem cell markers:
- +4 cell model
- Bmi1
- suggested that this is also expressed in the CBCs
- wnt signalling maintains the stem cell pool
7
Q
What happens with increased Wnt signalling in the intestinal epithelium?
A
- results in adenomatous polyps that develop into colorectal carcinoma
8
Q
What is mutated in familial adenomatous polyposis?
A
- a protein in the wnt signalling pathway - APC
- without Wnt Signal
- LRP and frizzled not active
- inactive dishevelled
- beta catenin rapidly destroyed
- recruited by APC molecule into a complex with the axin molecule and a couple of kinases (CK1 and GSK3)
- phosphorylated
- ubiquitylated and degraded in proteasomes
- if destroyed, not present in nucleus, groucho represses wnt target genes
- with wnt signal
- activated frizzled → recruits LRP → activated
- activated dishevelled
- recruits axin, GSK3, Ck1
- breakup of the destruction complex
- beta catenin stabilised
- unphosphorylated beta-catenin accumulates, migrates to nucleus, displaces groucho and associates with coactivator to initiate transcription of Wnt target genes
- if APC is mutated, no protein will bring beta catenin into the destruction complex → never destroyed
- continually activating wnt targets leading to over proliferation
9
Q
What experimental strategies are there to identify intestinal stem cells?
A
- genetic strategy
- candidate stem cells are genetically marked
- introduced marker allows stem cells and differentiated progeny to be observed
- the ability to genetically manipulate mice has permitted these studies to be employed to study intestinal stem cells
- isolation and culture strategy
- candidate cells are isolated and cultured
- cultured cells should have the capacity to form all differentiated cell types of the tissue in vitro
- upon transplantation cells should be able to replace the tissue
10
Q
What can Lgr5 positive cells do?
A
- they can generate all cell types in the intestinal epithelium
- need a Cre protein that is driven by a particular promoter in a cell type of interest and then you need the marker protein
- rosa promoter driving a stop sequence that has little FRT sites on either side
- lacZ downstream
- two seperate transgenes in the mouse
- if the cre protein is activated it will cut out the stop sequence allowing the rosa promoter to drive expression of Lac z
- in this model:
- Lgr5 promoter (Lgr5 was identified as a molecule that was only present in those stem cells, therefore should be promoting Cre only in those stem cells)
- particular kind of Cre variant that is only activated after addition of tomoxifin (CreERT2)
- this construct was also made to include GFP as a way of marking the cells
- If you add tomoxifin you get the rosa promoter activating lacZ in the Lgr5 cells
- image shows panel over a few weeks
- after a few days see marking of stem cell populations in the base of the crypts
- if you let these animals grow you see ribbons of lacZ cells growing up the length of the crypts and into the villi until they eventually reach the tops
- one the stem cells become blue they are permanently expressing lac Z
- as they produce TAs and differentiated cells (paneth cells, goblet cells, enteroendocrine cells) all become blue
- in other words the blue stem cells are producing all the cell types of the mature intestinal epithelium
- this is the experiment that really showed CBCs are stem cells
- didn’t address whether + 4 cells are also stem cells
11
Q
From which cells do colorectal cancers generally originate?
A
- stem cells
- Loss of APC in TA cells (Oral Ah-Cre) results in small adenomas that do not progress
- loss of APC in CBC cells (Lgr5-Cre) results in rapidly proliferating adenomas
12
Q
What is the role of Wnt and Notch signalling?
A
- key regulators of intestinal stem cells
- for maintenance of the stem cell population within the crypts you need both notch and wnt signalling to be present
- wnt signalling plays an important role in keeping the cells proliferative
- through a process call lateral inhibition
- a way that cells can sort out amongst themselves whether they will be responsive to notch signalling or not
- end up within the TA zone two populations of cells: one is responsive to notch and one is not
- notch activated in some cells and inactive in others
- as they leave the crypt and the proliferative capacity given to them by wnt signalling, these two populations of cells behave very differently
- those in which notch is active go on to become absorptive cells
- inactive → secretory cells
- still haven’t quite figured out the direct interaction between these two pathways and how they function together
13
Q
What signals combine to regulate the intestinal stem cell niche?
A
- many
- Wnt, Hedgehog, BMP
- classic organ in which all the major signalling pathways that we know about are present
- hedgehog and BMP signalling play an important role in the process of segregating villi and crypt
- crypt cells secrete the ligands for hedgehog and wnt signalls → cause BMP4 expression in the villus core
- BMP4 has a role in preventing the formation of villous crypts / ectopic crypts
14
Q
What happens with disruption of BMP signalling in the gut?
A
- results in ectopic crypts forming along the sides of the villi
15
Q
How do most Lgr5 expressing stem cells divide?
A
- most divide symmetricaly and stochastically adopt stem cell or TA cell fates
- this causes populations of stem cells in the crypt to drift towards clonality over a 1-6 month period
- experiment with four fluorescent proteins found that within each crypt eventually all stem cells were of the same colour even if initially all four were expressed by chance
- one of the stem cells takes over → dominant stem cell present in each crypt, maybe proliferating at a slightly greater rate, slightly more genetically fit?, drift to towards clonality
- characteristic of the system
16
Q
Is the niche an absolute requirement?
A
- Lgr5 positive stem cells can be isolated and cultured into organoids in vitro
- single Lgr5 stem cells build crypt-billus structures in vitro without a mesenchymal niche
- brings into question the purpose of the niche
- most likely still has an important role
17
Q
What is the role of paneth cells in the niche?
A
- paneth cells are niche components for Lgr5 stem cells in intestinal crypts
- paneth cells and stem cells develop next to each other
- paneth cells secrete Wnt3a (ligand for Wnt pathway) and DII4 (ligand for Notch pathway)
- stem cells express Fzd7 receptor and Notch 1
- factors secreted by paneth cells play an important role in maintaining the stem cells
- if you grow a stem cell on its own one of the first things it will do is produce a daughter paneth cell
- some sort of self-organizing capacity
- far from the whole story
18
Q
What can transplanation of cultured organoids do?
A
- cultured organoids were transplanted into the bowel of mice where the epithelium was damaged in a way that mimics colitis
- the organoids were incorporated into the sites of damage and repaired the lesions
