6. Tissue homeostasis Flashcards
Explain tissue homeostasis
Tissue homeostasis - long term turnover of adult tissues by stem cells
What is a stem cell?
Stem cell:
- cell that renews a continously turning over tissue
- cell that can renew a tissue in regeneration after injury
Why are stem cells needed?
To renew tissues - tissues which constantly experience insults from environment - need expendable cells - replace them with new ones (ex: gut villi)
What are the examples of high turnover tissues?
- blood (blood cells short lived)
- gut (lining of the gut due to environment)
- skin (dead cells on the surface needed)
Why stem cells are used to renew tissues instead of differentiated cells?
Some differentiated cells:
- so highly specialised - lack nucleus -> can’t restore themselves with new cells
- lose DNA parts in differentiation
Stem cells - reservoir of original/unchanged cells
Why stem cells are used instead of highly proliferative cells to renew tissues?
Due to chance of mutation - if random mutation occurs - will be inherited by many cells (very fast dividing daughter cells) - cancer
Final differentiated cell may be completely non-proliferative
=> stem cells are slow dividing and potentially immortal = protected from mutations
What kind of choice does a stem cell have in division?
Stem cell can divide to give:
- a copy of itself
- differentiated cell
Also: symmetric / asymmetric division
What kind of divisions can a stem cell undergo?
- symmetric division
- asymmetric division
How can asymmetry in division be generated?
Assymetry generated by:
- the environment (neighbouring cells, ECM, GFs)
- inside the cell (localised cell somponents)
How were stem cells discovered?
From radiation effects - Till and McCulloch effects of whole body irradiation on haematopoietic system - linear relationship
How was it determined where haematopoietic cells are produced?
Haematopoietic cells produced in the spleen:
irradiated mouse was transplanted with bone marrow cells - many spleen colonies formed - # of spleen colonies directly proportional to # of transplanted bone marrow cells
How can it be determined if one transplanted bone marrow cell forms one spleen colony or not?
Need to use** several markers** for transplanted bone marrow cells:
- if spleen colonies only of **one marker **/ no marker -> each transplanted cell = a spleen colony
- if different markers observed -> a single transplanted cell can’t form one spleen colony
Marker: radiation - induces chromosomal breaks -> abnormal chromosomes - can be detected
Result => **all spleen colonies were pure **- one bone marrow cell = one spleen colony (single clonal haematopoietic cells) - differentiate into many cell types
Explain stem cell therapy for leukemia
Stem cell therapy:
- irradiation introduced to kill patient’s all bone marrow cells
- healthy bone marrow stem cells transplanted
Why are stem cells difficult to purify?
- slow division
- hidden in tissues rather than form tissues themselves
What proves that stem cells are a good solution to tissue growth?
Same stem cell mechanism in animals and plants (shoot and root meristems) - developed independently -> stem cells a good solution for tissue growth
What are the characteristics of adult stem cells?
- slow dividing
- potentially immortal
- have restricted differentiation potential (ex: heamatopoietic cells can only differentiate into one of the heamatopoietic cell types)
Two examples of assymetric stem cell division in organisms
- Drosophila reproductive system stem cells
- Mammalian gut stem cells
Where do reproductive stem cells divide in female Drosophila?
Germline stem cells divide in germarium
What is the structure of female Drosophila germarium?
In a single germarium, structure:
- Terminal filament
- Cap cells
- Germline stem cells
- Cytoblast
Take Cat Get Crap
How can germarium be manipulated?
- possible to remove cellls / organelles
- possible to introduce DNA
- possible to transplant different parts into other Drosophila
What is a stem cell niche?
Niche - the environment of a stem cell that provides needed factors (ex: signalling molecules) for stem cell maintenance
Niche can be other cell rather than whole environemnt
Explain the composition of female Drosophila germline stem cell division
GSC niche: cap cells - maintenance and number regulation:
2-3 GSCs per germarium undergo asymmetric division (intracellular components influence) - produce cytoblast - divides 4 times to produce 15 progenitor nurse cells and 1 oocyte
Explain Drosophila male vs female GSC location
*No need to know
What is the example of a gene regulating Drosophila GCS renewal (regulates numbers of stem cells)?
- Bam (Bag-of-marbles) - regulates numbers of germ cells - secreted by niche - cap cells
- mutants of Bam have excessive numbers of germ cells
- When Bam is off - GSC can’t differentiate
How are asymmetric divisions achieved in GSCs in female Drosophila?
Asymmetry in GSCs generated:
Inside the cell:
Spectrosome - organelle of spectrin, contractile protein which mediates cell adhesion - spectrosome responsible for anchoring mitotic spindle - anchores GSC to the niche - necessary for signalling for GSC to remain GSC
Cell divisions are oriented with respect to the niche - oriented division - asymmetric division - one cell inherits spectrosome - remains a GSC - remains in contact with niche (cap cells) - the other develops
From environment:
- secreted signalling molecules (Bam) from cap cells
Explain the structure of mammalian gut villi in small intestine
Small intestine contains villus:
- villi - absorb nutrients
- crypts - contain gut stem cells (CBC) for gut renewal
What are the crypt based columnar cells (CBCs)?
Crypt based columnar cells (CBC) - small intestine stem cells (renew shedded villi cells) - in the crypt part of gut villi - express Lgr5 gene
Explain the experiment how can it be determined that Lgr5 expressing cells are stem cells
Permanently mark Lgr5 expressing cells - mark their descendants:
- engineer Lgr5 promoter for recombinant enzyme - also expression of LacZ in all descendants
- to choose the time when you want to see the descedants - engineer for the recombinant enzyme only to act when drug tamoxifen is present (can control when you want to add the drug)
What is the niche for crypt based columnar cells (CBC) in small intestine?
Niche for CBC (gut stem cells) (express Lgr5) - Paneth cells
What signalling do Paneth cells secrete for CBC gut stem cells?
**Paneth cells **- niche for CBC gut stem cells - secrete Wnt3 gene - allows CBC cell survival
How was it determined that Paneth cells are sufficient to sustain CBC gut stem cell survival?
CBC gut stem cells can form gut organoids in artificial matrix
- when CBC and Paneth cells grown together - higher survival rate - higher potential to form gut organoids
How was it determined that Wnt3 is sufficient to sustain CBC gut stem cell survival?
Wnt3 is sufficient to sustain CBC survival - not whole Paneth cell needed - when only Wnt3 added - same survival potential as when whole Paneth cell was present
Result: CBC (Lgr5) and Paneth cell doublet = same survival = CBC (Lgr5) doublet + Wnt3
What kind of division do CBC gut stem cells undergo?
**CBC **gut stem cells divide symmetrically - identical daughter cells
What was the experiment used to determine if CBC gut stem cells divide symmetrically or asymmetrically?
Cell labelling: asymmetrically dividing cells should retain one daughter cell in crypt - the other differentiate into villus:
- CBC cells randomly labelled 4 colours - tamoxifen added => each crypt started as multicoloured - **turned single coloured **after 8 weeks => symmetric division
Compare how Drosophila germarium maintain GSC stem cells and how mammalian gut villus maintain CBC gut stem cells?
Drosophila GSC divide asymmetrically - one cell remains a stem cells attached to niche
Gut CBC stem cells divide symmetrically (stem cell held by repulsive interactions between Ephb/EphrinB - flanking cells)
=> more than one way to maintain stem cells
Give examples of organs with high cell turnover rate
Organs where cell death is high:
- gut
- skin
- hematopoietic system
Give examples of organs with medium cell turnover rate
Turnover only when there is loss of cells due to external reasons:
- muscle (loss of cells due to mechanical stress)
- liver
Give examples of organs with low cell turnover rate
Organs regenerate poorly:
- lens
- brain
- heart
How does medium-dose radiation affect the hematopietic system and gut cells?
Hematopoietic system:
- kills bone marrow cells - stem cells
Gut cells:
- kills stem cells (CBC cells expressing Lgr5) - delayed failure in gut function - after 2-3 weeks
What happens to Lgr5+ CBC cells after irradiation?
Lgr5+ stem cells killed in radiation - restored by previously non Lgr5+ cells - regenerate from transit amplifying cells (stem cell recovery cells)
How can it be figured out what happens to Lgr5+ CBC cells after irradiation?
Lgr5+ CBC cell lineage tracing:
- label Lgr5+ cells with lacZ - labelled descendants
- irradiation - no lacZ label - all Lgr5+ cells killed
=> BUT mouse survives - gut regenerates Lgr5+ cells from previously Lgr5- cells - transit amplifying cells (stem cell recovery)
Explain gut regeneration from organoids grown in vitro
Healthy gut stem cells - grown into organoid in vitro - transplanted into damaged gut - gut regenerated - engrafted cells function normally
Explain how can assisted regeneration be used for skin regeneration
Basal layer cells cultured - formed 3 colonies: holoclone, meroclone, paraclone - only holoclone able to divide long-term - the stem cells - grown in vitro into tissues - transplanted
=> in vitro grown skin stem cells cnan be used for full recovery of 3rd degree burns (basal layer destroyed) - when restored only missing hair folicles and sweat glands
Explain the structure of epidermis
- Cornified layer - dead cells which are constantly shed
- Suprabasal layer - differentiating cells
- Basal layer - undifferentiated cells that are dividing (stem cells)
Explain how skin grows and shed cells are regenerated
Basal layer cells - dividing stem cells - push cells into suprabasal layer - cells differentiate (lay protein matrix, enucleate and die providing barrier against pathogens / env) into epidermis cells - travel into cornified layer
What are transit amplifying cells?
Transit amplifying cells - undifferentiated cells in transition between stem cells and differentiated cells
How is muscle tissue regenerated?
Muscle tissue - satellite cells (muscle stem cells) - at edge of muscle fibers - in between basal membrane and ECM - Pax3+ expressing
How are muscle stem cells called?
Satellite cells
How can satellite cells be used for muscle regeneration?
Satellite cells (Pax3+) grafted into mouse damaged muscle (ex lacking dystrophin) - incorporate into tissue - healthy cells recovered
What the are examples of organisms which have great regenerative powers?
- Hydra
- Planarian worms
- Axolotl (salamander)
All use different regeneration mechanisms
Explain the mechanism how hydra regenerates
When halved - generates two organisms
Mechanism: cells sense their position in organism - adjust gene expression
Dynamic gradients of diffusable molecules (ex Wnt) that specify head-foot regions => after halving gradient is restored before growth depending on the position of cells in organism
Hydra cells re-specify their cells
Explain the mechanism how planarians regenerate
Many different outcomes fop cutting:
- If halved - generates 2 organisms
- cut into three equal pieces - generates 3 organisms
- a cut piece is too thin - two heads - no morphogen gradient - abnormal regeneration
- head cut first, then tail - the thin middle part forms 1 organism - time lag introduced morphogen gradient
Planarians use multipotent stem cells for regeneration - all cell types in regenerating body can be made
Explain the mechanism how axolotl regenerates
Set organs which can be fully regenerated: limbs, tail, jaw, lens, heart
Limb regeneration: each cell types de-differentiates (all gene expression patterns lost), proliferates and re-differentiates (new gene expression patterns created)
Compare the regeneration mechanisms used by hydra, planarians and axolotl
Hydra: re-specifies cells
Planarians: use multipotent stem cells
Axoltl: de-differentiate and re-differentiate cells
What are the 3 situations where cells undergo controlled differentiation?
- stem cell
- regenerating cell
- embryo cell
Compare cell differentiation in stem cells, regenerating cells and embryo cells
Not all genes involved in embryo cells in development are used by regenerating cells in regeneration (development vs regeneration in limbds, gut)
=> adult stem cells + regenerating cells - not preserved embryonic cells BUT embryos have stem cells (ESC)
Explain the function of Paneth cells in gut development vs maintenance
Development: Paneth cells **not needed **for survival (not confined in villus crypts) - embryonic gut proliferates without
Maintenance: CBC stem cells need Paneth cells (confined in villus crypts) for survival in adult gut crypts
=> CBC-Paneth cell relationship and villus structure differs in embryonic development vs adult life
How can embryo development differentiation be defined (type fo differentiation)?
Progressive differentiation
What gene is involved in maintaining cell pluripotency?
Nanog - expressed in inner cell mass (ICM) cells in pre-implantation blastocyst - gives rise to embryonic tissues - pluripotency - ICM source of ESCs
How are ESCs derived and differentiated in vitro?
IVF - development - ESCs taken from ICM - cultured in vitro - needed factors introduced for specific development
How does ESC culture halt embryo development / maintained in vitro?
ESC maintained on layer of ‘feeder’ cells - inactivated - don’t divide - provide signalling (LIF) for ESC maintenance
What is LIF signal function in the embryo?
LIF scereted from extraembryonic part (trophoblast) - ICM have receptors for LIF - LIF maintains totipotency during diapause
What is diapause? What is its function?
Diapause - a period of halted embryo development
Used to maximise breeding performance
Explain mouse diapause
Mice recently given birth can ovulate - new litter of fertilised embryos held in diapause inside mother uterus without implanting - until mother looks after previous litter - embryos implant => maximise breeding efficiency
While in diapause ICM cells maintained by LIF - LIF receptor lacking cells not maintained in diapause
Diapause in hybernating animals is controlled by daylight
Do embryos contain true stem cells?
Yes, ESC, true stem cells, are present in embryos but only transiently (12-24h in mice) - unless organism enter diapause (few weeks longer in mice as development is halted)
Explain the experiment used to prove that ES cells are stem cells
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ES cells need to show stem cells properties: unlimited self-renewal, ability to differentiate (if pluripotency - all cell types):
add trypsin to form single cell solutions (dissociate tissues) - transfer to culture dish - isolate single cells from colonies using trypsin again => observe descendants - see if properties remain: - unlimited self-renewal: cells should constantly divide in vitro if proper env.
- differentiation: reintroduce cells to embryos -> make up adult cells (developed organisms - chimeras)
What stages in embryo development contain cells which can be arrested in a proliferative, non-differentiating state?
Where are neural stem cells found in the embryo? What do they differentiate into?
Around the neural tube - can differenitiate to make neurons / glial cells
What are the applications of human ESCs?
- cell therapies: ESC replaces damaged cells (ex: Parkinson’s disease - loss of cells in Substantia nigra, diabetes - loss of beta cells in pancreas)
- ESC for testing new pharmaceuticals
What is the biggest challenge for stem cell differentiation in vitro?
To make stem cells differentiate into the target tissue - giving factors not enough - various cell types develop - difficult to create homogenous tissues
