Stem cell niche II Flashcards
What are the disadvantages of a 2D culture of cells?
- Simplistic
- Often fails to recapitulate the in vivo phenotype (cells lose their phenotype and behaviour due to the unnatural environment)
- Drug responsiveness/disease modelling may not be predictive of the in in vivo context
What are the properties of a 2D cell culture?
- ABSENT soluble gradients (cells see an isotropic gradient)
- FORCED apical-basal polarity
- Unconstrained spreading and migration in x-y
- Adhesions restricted to X-y (not 3D)
- High stiffness of the plastic (hardness of bone/tissue in the in vivo environment is much lower)
- Layer of matrix coats the dish –> presented as a continuous layer of matrix
Why is the 3D approach to cell culture a more representative model to culture cells?
More of a realistic representation of how cells exist in the tissue:
- SOLUBLE gradients present
- No prescribed polarity
- Discrete matrix fibrils (not a continuous layer of matrix)
- Spreading and migration is sterically hindered
- Adhesions distributed in ALL 3 dimensions (3D)
- Low stiffness of the culture
What method allows us to take cells from the in vivo environment and grown them in vitro to enable them to do the same functions/exist how they would in the body?
Form ORGANOIDS
How are organoids formed?
- Take pluripotent (or adult stem cells)
- Aggregate the cells and give them:
Soluble cues
Biomaterials - The cells then SELF-ORGANISE and create 3D structures that recapitulate some of the major features of the organs they come from
Order the biological model systems from low physical relevance to high
2D cell culture
3D cell culture
Organoids
Model organisms
Humans
Order the biological model systems from high experimental tractability to low
2D cell culture
3D cell culture
Organoids
Model organisms
Humans
What comes with an increase in physiological relavence?
A decrease in experimental tractability
What is the definition of an organoid?
What does this imply?
A structure that resembles an organ, implying:
1) Multiple specific cell types (as are found in the organ in vivo
2) Capable of recapitulating some specific function of the organ (eg. excretion, filtration, neural activity, contraction)
3) The multiple cell types of the organoid must be grouped together and spatially organised similar to an organ
What does organoid formation recapitulate?
Both the major features of SELF-ORGANISATION during development:
1) Cell sorting
2) Spatially-restricted lineage commitment
What is ‘self-organisation’?
When a group of HOMOGENOUS cells can SPONTANEOUSLY create fine-tuned structures without major EXTERNAL influences
What is the classical example of a ‘self-organising’ structure?
Experiment to show this?
Sea-sponges
- Dissociate the cells into single cells
- Cells then spontaneously rebuild the basic body structure in the correct SPATIALLY organised manor when they are reaggregated
What experiment did Townes and Holtfreter 10 (1955) do that showed self-organisation?
- Took presumptive epidermal cells (from one embyro)
- Took neural plate cells (from another embryo)
Dissociated the cells and allowed them to spontaneously reaggregate
–> Created a correct body plan (with epidermal cells on the outside and neural cells on the inside)
What hypotheses are there that may explain how the cells know where to go to when they self-aggregate?
1) Differential adhesion hypothesis
2) Spatially restricted lineage commitment
What is the differential adhesion hypothesis based on?
Describe this
Based on the differentiation cell adhesion properties:
- Different cells have different CAMs on their surface
- When put cells together –> cells will organise in a manner where the similar cells form more bonds with each other rather than the cells of a different cell type
What is the spatially restricted lineage commitment hypothesis?
Progenitor cells give rise to more differentiated progeny due to:
1) SPATIAL CONSTRAINTS of the tissue
2) Cells orienting their division
This causes cells to be forced into a more superficial position and further away from the self-renewing signals –> causing the cells to change fate
What cell types have organoids been derived from?
- Pluripotent stem cells
- Adult stem cells
What adult-stem cell derived organoid is critical to gain knowledge of the intestinal stem cell and niche?
The gut organoid
What is the turnover of the intestinal epithelium?
4-5 days
What are the features of the intestinal epithelium?
- Villi (small protrusions) that project into the lumen
- Many differentiated cell types
- Also contain stem cells and transit amplifying cells (lining the crypt)
What is the function of the vili in the gut?
Increase SA for absorption
What are the different differentiated cell types of the intestinal epithelium and where are they?
1) Enterocytes (cover the surface of the vili and extend into the crypt
2) Paneth cells (at the bottom of the crypt)
3) Goblet cells
4) Entereoendocrine cells
What is the function of Enterocytes?
Absorption of nutrients/water from the lumen
What is the function of paneth cells?
1) Secretion of anti-microbial compounds - important in innate immunity
2) Maintenance of the intestinal stem cells
What is the function of goblet cells?
Secret mucin and other products
Help to move material across the gut
Where is the stem cell located in the gut epithelium?
At the bottom of the crypt
Between the Paneth (support) cells
What do the stem cells at the bottom of the crypt do?
1) They divide to make copies of THEMSELVES
2) They divide to make TRANSIT AMPLIFYING CELLS (progenitors)
What do the transit amplifying cells do?
What happens as they do so?
Proliferate RAPIDLY and move up the walls of the crypt (with each division - eventually giving rise to differentiated cells)
As they the cells migrate upwards:
1) They begin to differentiate into the cells of the gut (goblet cells and enterocytes)
2) Carry out the essential roles of the intestine
What happens when the differentiated cells reach the top of the vilus?
They undergo APOPTOSIS and SHED into the lumen of the small intestine
How long does the process of outmigration and death of the cells of the intestinal crypt take?
3-4 days
What are the intestinal stem cells called?
What factor do they express?
Crypt base columnar cells
Express LGR5 (A leucine rich repeat G protein-coupled receptor 5)
How often do the crypt base columnar cells divide?
Once every 24 hrs
What factors do the Paneth cells secrete/express?
What are these factors important for?
Secrete:
Wnt, EGF
Express:
Notch ligands
Important for the maintenance of the LGR5+ crypt base columnar cells
What does EGF do?
- Binds to the EGF receptor
- Impacts ont he Ras/MEK pathway
What do the LGR5+ crypt base columnar cells also rely on?
BMPs
R-spondin
Where is R-spondin secreted from in the intestinal stem cell niche?
Unclear
What does R-spondin do?
Binds to Lgr4/5 and potentiates Wnt signalling
How did KO mouse studies benefit us in understanding the intestinal stem cell niche?
- Helped us to work out the hierarchy
Helped us to gain knowledge of the different transcription factors that influence both the:
1) Secretory (paneth, goblet, enteroendocrine)
2) Absorptive (enterocyte)
Lineages
And the signalling pathways important in the maintenance of the stem cell niche
What is the prolonged proliferation of adult stem cells in vitro associated with?
The cells undergoing SENESCENCE or TRANSFORMATION
RESEARCH PAPER!!
What was the AIM of the research paper ‘Single Lrg5 stem cells build crypt-villus structures in vitro without a mesenchymal niche’ aim to do?
What was the RATIONALE of the study?
Sato et all (2009)
Rebuild the crypt-villus structures using the organoid models
AIM - to design a long-term culture of the intesitinal system
RATIONALE:
- Long term cultures could be designed by
incorporating previously elucidated components of the intestinal epithelium niche
(given the knowledge of the system –> should be able to rebuild in vitro)
- Recapitulating the niche in vitro should incorporate the
signalling molecules as well as the appropriate matrix
support
(niches rely on MANY different interactions in the niche - not just cell:cell)
What should long term cultures of the intestinal stem cell involve?
Why?
1) Active WNT signalling
2) EGF signalling
3) Noggin expression
4) Overcome anoikis
Knew these signals were important in VIVO
What is anoikis?
When does this occur in the intestinal stem cell niche?
Programmed cell death that occurs in anchorage-dependent cells when they DETACH from the surrounding ECM
Occurs when the epithelial cells come to the top of the vili and are released
Why is Wnt signalling needed for long term cultures of the intestinal stem cells?
For crypt proliferation
Why is EGF signalling needed for long term cultures of the intestinal stem cells?
For enterocyte proliferation
Why is Noggin expression needed for long term cultures of the intestinal stem cells?
For the expansion of crypt numbers
What experimental approach did Sato et al (2009) use in attempt to recreate the niche in vitro?
Was this successful?
1) Isolation of the mouse intestinal crypts
2) Crypt preparations were suspended in Matrigel
3) Optimisation of culture medium by the titration of the key signalling molecules in the medium to find the OPTIMUM CONCENTRATION (EGF, R-spondin, Noggin)
Was succesful - could see the creation of crypts
How did Sato et al (2009) isolate the mouse intestinal crypts?
Using Lgr5-GFP+ maker
–> allowed to FACS (cells that are fluorescing are Lgf5+
What is Martigel?
What does it do?
A protein mixture derived from mouse sarcoma cells
Enriched in ECM proteins - supports the cells by mimicking the ECM
How did Sato et al (2009) identify the formation of the crypts was succesful?
They could identify:
- Paneth cells
- Lgr5+ cells (glow green from GFP)
What is the evidence that organoids be created from single cells?
Why must further experiments be done?
Sato et al (2009)
1) Take a single crypt and dissociate them into single cells
2) FACS analysis - cells of interest can be sorted, harvested and placed into culture
Cells built crypt-like structure (resemble a crypt morphologically)
Further experiments must be done as to be considered an organoid - they must resemble the organ they cam from (must represent in vivo crypts)
What are the validation experiments that must be done on the crypt-like structures formed from single cells?
Sato et al (2009)
1) Immunohistochemistry - use markers that they know should be expressed in the relevant cell types
2) Electron microscopy
3) Histology?
What were the conclusions of the Sato et al (2009) paper?
The gut organoids generated and the crypts within the organoids are:
1) Virtually indistinguishable from the in vivo cells
2) Contain differentiated cell types
3) Paneth cells and SCs are located at the bottom of the crypt
What is the summary of the Sato et al (2009) paper?
1) Organoids can be generated from single Lgr5+ stem cells
2) Organoids show polarisation and differentiation
3) Orgnaoids can be propagated for at least 1.5 years (due to the formation of the niche)
- Before this is wasn’t possible
What was the Sato et al (2009) paper so important?
It was the FIRST study to start the organoid field
Reinspired research into this area
What is the structure of the gut organoid?
A circular shape with a central lumen lined by villus-like epithelium and several surrounding crypt-like domains
What are the potential applications of organoids in relation to medicine?(what tools)
Enables us to study organisms on a miniscale in vitro:
1) Experimental tool
2) Diagnostic tool
3) Therapeutic tool
How can organoids be used as an experimental tool?
Can KO/over express genes and analyse the effect on:
1) The function of the stem cells in the organoid
2) The differentiation of other cell types
How can organoids be used as a diagnostic tool?
- Used to interpret the mutations seen in patients
- Can analyse which gene in the genome is causing a phenotype
How can organoids be used as a therapeutic tool?
Can see the effect of a drug in trying to correct a disease phenotype
How has the gut organoid been used as a therapeutic tool?
- Depletion of the intestinal epithelium in mice (to resemble some of the human conditions in the disease)
- Then, deliver gut organoids (using colonic infusion) to the gut –> able to show the organoids can implant and recreate the intestinal epithelium
What are some examples of congenital disorders that gut organoids have been used to model?
- Cystic fibrosis
- Autism
- Microcephaly
What are some examples of acquired disorders that gut organoids have been used to model?
- Cancer
- Zika virus infection
Describe modelling of the zika virus infection using organoids
What are the next steps?
- Babies are normally born with microcephaly (small brains)
- Make brain organoids (using human pluripotent cells) and infect the organoids in vitro
–> Infected brains were smaller (shows recapitulation of the Zika virus infection
–> Shows the virus to affect the development of certain types of neurons
- Can THEN look are the molecular mechanisms that cause this to happen (cannot do this in human patients)
Describe the testing of CFTR function using organoids
What can be done to the organoids?
From HEALTHY controls:
- Functioning CFTR channel allows the influx of salt and fluid
- -> Organoid is spherical (looks like an inflated ball of cells)
In CF organoids:
- Dysfunctional channel
- No influx of fluid
- -> Organoid is small and compressed
Can then:
- Apply DRUGS to the organoids in high throughput screens
- To see which of the drugs CORRECT the phenotype (to give the appearance of the healthy control)
What is the ‘personalised medicine approach’?
Why is it needed?
Samples taken from patients –> can then tailor the drugs specifically to the patient
Needed because:
- Patient have different mutations and other things in the genome that can MODIFY how the particular mutation manifests in the patient (modifiers)
What is thought to be the reason why some patients respond differently to the same medicine?
Modifiers in the genome
For personalised medicine to be feasible, what must be done?
Organoids need to be able to be REPRODUCED and ROBUSTLY formed (currently issues with this)
Why are snake organoids of interest?
100,000 of people die from snake bites each year –> want to be able to study to get the anti-venom
Difficult to get venom from snakes to make antivenom so instead make orgnoids of the snake venom
How were venom gland organoids made?
- Dissect out the venom glad from snakes
- Get SINGLE CELL suspensions which they can then aggregate to from organoids
- Condition the media (using R-spondin)
- Reduced temperature from 37–>32 degress
- Formation of venom gland organoid
How were the venom gland organoids characterised?
???
Compare the organoids to the actual venom gland and analyse what factors are needed for proliferation
Withdraw the growth factors from the medium and keep the factors needed for proliferation:
–> Organs differentiated (less proliferation)
–> Organoids now contained the secretory vesicle (as would see in the venom gland)
How do we determine if the secretory vesicle from the organoid actually produces venom?
What was seen?
1) Using RNA extraction and transcription analysis:
- At the RNA level - see a spectrum of toxins as observed in the venom glands (in the venom glans- see heterogeneity in toxin expression)
2) Single-cell transcriptiome analysis
- See different cell types contribute the the different toxin production (rather than one cell type producing many different toxins)
Describe the process and conclusions of the functional study of the venom gland organoids
- Allow the organoids to differentiate
- Get the in vitro secretion of toxin in the organoids –> harvest the venom
- Apply the venom to a model for muscle in vitro
- -> shows a similar phenotype to muscle paralysis that is seen with the toxin –> functional
What is the model for muscle in vitro?
Functional mytotubes - see Ca waves through
What cell types have organoids been developed from?
Pluripotent stem cells and tissue-specific stem cells
