Stem cell niche I Flashcards
In order to be able to sustain haematopoiesis throughout lives what must HSC do?
Carefully BALANCE/tightly regulate self-renewal with differentiation
What happens if there is excessive self-renewal of HSC (at the expense of differentiation)?
Why?
Cancer
What happens if there is excessive differentiation of HSC (at the expense of self-renewal)?
Why?
Many blood deficiencies (eg. anemia, immune deficiency, inability of the blood to clot)
As differentiated cells have a LIMITED regenerative capacity –> stem cells will eventually run out
What is the self-renewal vs differentiation decision regulated by?
Different INTRINSIC and EXTRINSIC factors
What are the INTRINSIC factors that influence SC behaviour?
Transcription factors
What are examples of the EXTRINSIC factors that influence SC behaviour?
Blood loss/immune reaction –> causes the HSCs to produce the relative cel types
In stem cell biology, what 2 things must a niche have?
1) A DEFINED anatomical location
2) Functional dimension
What is the definition of a stem cell niche?
A LOCAL tissue ENVIRONMENT that:
1) HOSTS and
2) INFLUENCES the
BEHAVIOURS/characteristics of stem cells
How did the concept of a stem cell niche emerge?
From Till and McCulloch experiments in 1961:
- Injected bone marrow into irradiated mice (with no blood cells) –> mouse survived
- Injected bone marrow cells hung onto the spleen of the mouse and created colonies
- Some of the colonies contained UNDIFFERENTIATED stem cells (proven by transplantation experiments into irradiated mice)
Although the undifferentiated cells taken from the colonies of the spleen can rescue an irradiated mouse phenotype, what is the issue with this?
What does this show?
Not able to rescue the mice upon many SUBSEQUENT transplantations
Shows:
- These cells in the colonies of the spleen are more restricted in their self-renewal capacity
- These cells need to reside in the bone marrow to have infinite self-renewal
What did Schofield propose (1978)?
From what observation?
The stem cell niche hypothesis
From his observation that HSCs need to reside in the bone marrow to retain their ‘infinite’ potential
What is the stem cell niche hypothesis proposed in 1978 by Schofield)?
The stem cell niche:
1) Has a DEFINED anatomical location
2) Regulates SELF-RENEWAL (in the niche - cells are UNDIFFERENTIATED)
3) Removal of the niche results in stem cell DIFFERENTIATION
What was the first evidence for the existence of a stem cel niche? (before any experimental work)
The fact that irradiating patients helped the bone marrow transplant to graft
Idea that there is competition between the endogenous bone marrow cells and the grafted bone marrow cells
–> compete to occupy the niche??
What is the motivation to study stem cell niches?
1) In order to produce better clinical strategies (better than irradiation)
2) Currently unable to maintain adult SC for prolonged periods of time in vitro
–> In vitro niche may help to maintain the SC in vitro in order to be able to study the SC (eg. research and devise new clinical studies)
What is the difference between the behaviour of ES cells and adult SC in vitro?
ES cells - proliferate INDEFINITELY
Adult SC - Unable to proliferate indefinitely (cease self-renewal, differentiate or undergo apoptosis)
In what organism did experimental evidence show the existence of a stem cell niche?
When?
The drosophila germ line system:
- Testies
- Ovaries
In the 90s
What is the structure of the testis?
Tube-like structure that is closed at one end (at the apex)
Where is the testis stem cell niche?
What is the structure?
At the apex
Structure:
- Hub cells that are in close contact with 2 stem cells: germ line stem cells and the somatic (cyst) stem cell
- Gonialblast cells
What are the hub cells in the testis stem cell niche?
10 NON-mitotic cells
The niche producing cells (maintain the stem cell niche)
Where are the somatic (cyst) stem cells in the testis stem cell niche?
Surround the germ line stem cells and are in contact with the hub cells (niche producing cells)
Where are the germ line stem cells in the testis stem cell niche?
In direct contact with the hub cells (niche producing cells)
What happens when the germ line stem cells in the testis stem cell niche divide?
One daughter:
- Stays in contact with the stem cell niche
The other daughter:
- Moves posteriorly away from the hub cells (loses contact)
- Differentiates to become a gonialblast
What are the gonialblast cells in the testis stem cell niche?
What do they do?
- Differentiated germ line stem cells that have migrated away from the hub cells
- Undergo 4 mitotic divisions to produce spermatogonia that will eventually undergo meiosis to form the sperm
What is the local signalling that occurs in the drosophila testes niche?
- Hub cell (in contact with the GSC and CySC produces Upd (UNPAIRED) ligand
- Upd activates JAK-STAT signalling in both SC types
- JAK-STAT signalling is required for the adhesion of the SC to the hub cell
- The hub AND the CySC cell also secrete Gbb/Dpp, which activates BMP in the GSC
- In the GSC: BMP activates pMad which suppresses the expression of Bam (a differentiation factor)
–> Causing the GSC to remain undifferentiated
What occurs at the signalling level when the GSC divides in the drosophila testes niche?
One daughter is displaced OUT of the niche:
- No longer can reach the signals from the hub cell
–> JAK-STAT signalling and BMP signalling no longer activated
–> Contact adhesions to the hub cell are lost and Bam is no longer inactivated
–> Differentiation can occur (no longer are stem cells)
What is the difference between the need of JAK-STAT signalling in the GSC and in the CySC?
JAK-STAT signalling is required and sufficient for the self-renewal of the CySC
But not sufficient for the self-renewal of GSC –> requires BMP signalling activation by Gbb/Dpp
What is the structure of the drosophila ovaries?
- Consists of 10-16 ovarioles
- Each with a germanium at the anterior and a series of differentiating egg chambers
What is the structure of the drosophila ovarian niche?
- Stack of somatic filament cells are the very anterior of the niche (hub cells)
- Cap cells in contact with the germ line stem cells
- Only 1 type of SC
What cells in the drosophila ovarian niche are the niche producing cells?
The cap cells - keeps the GSC undifferentiated
How are GSC regulated in the ovarian niche?
NO requirement for JAK-STAT signalling
BMP activation - through ligands Gbb and Dpp
–> Activates Mad –> Represses Bam (differentiation signal)
How do stem cells orient their division in the stem cell niche?
When the cell is about to divide:
- Centrosome of the stem cell is duplicated
- Daughter centrosome moves to the opposite side of the cell (opposite the mother centrosome - next to the hub cell)
- Then, when division takes place (between the 2 centrosomes) - it occurs PERPENDICULAR to the hub cell
- Therefore, when the cell divides - one cell remains close to the hub cell and the other is displaced
What are the other components of the stem cell niche that help to keep the stem cell retained at the niche?
PHYSICAL cell-cell adhesions to the niche producing cell
In males:
- Both GSCs and CySCs require Ecadherin-mediated adhesion to the hub
Females:
- Adheres junctions to the cap cells
What occurs when overexpress integrin in the CySC in the drosophila testis niche?
OUTCOMPETES the GSC from the niche
What is the difference between the main site of haematopoiesis (HSC niche) between humans and the mouse?
Why is there a difference?
Humans:
- Axial skeleton
Mice:
- Long bones
In humans, the bone marrow in the long bones is replaced
What is the structure of the bone marrow?
Cortical bone on the outside (compacted bone)
Spongey on the inside (trabecular bone)
Lining of the bone
What is the lining of the bone (between the bone tissue and the bone marrow) called?
Endosteum
What cells were previously thought to be the main drivers of the haematopoietic stem cell niche?
Why?
The bone cells
Due to their proximity to the bone marrow
Describe the claim that the bone cells are the main drivers of the haematopoietic stem cell niche
Experiment has show that if increase the number of bone cells –> influence HSC number
BUT, there were many limitations with this experiment (overexpression of the gene to a level that is not physiologically relevant –> misinterpretation of results)
What is now being considered as the creator of the HSC niche?
Why?
Looking at the role of the vasculature
As the bone marrow is highly vascularised
AND
Due to technological advances that give the ability to look at cells at the SINGLE cell level
(Can do in vivo imaging and lineage tracking)
Why is there constant bone turnover?
Due to the action of:
- Osteoblasts (bone forming cells)
and
- Osteoclasts (bone resolving cells)
What are the blood vessels present in the bone marrow?
Structure
How was this seen?
- Arterioles
- Sinusoids (large in number in the centre)
- Transition zone vessels (on the outside
Seen using staining experiments
What is the function of sinusoids?
Take the venous blood away from the bone marrow
Where do the HSCs associate/cluster in the bone marrow?
What does this suggest?
Cluster in the centre of the bone marrow with ~80% associating with sinusoids
Suggests a notion that sinusoids have a functional role in the maintenance of HSCs
Describe the behaviour of the HSC in the bone marrow
- Exist in a niche
- SC in the niche are mainly quiescent and non-proliferative
- Until triggered to divide and produce TAC (progenitors)
- -> progenitors do most of the proliferation
Why are the HSC in the niche mainly quiescent?
To protect themselves from genetic insult
As every time a cell divides - there is a chance of a mutation
How were the signals that maintain the HSC niche identified?
What did they show?
Using ABLATION experiments
Showed different cell types that influence HSCs:
1) Non-hematopoietic cells
2) HSC progeny in a feedback loop
What are the non-hematopoietic cells that influence the HSCs?
1) Endothelial cells
2) Perivascular stromal cells
3) Non-myelinating Schwann cells
4) Adipocytes
How do endothelial cells influence the HSCs?
- Line the blood vessel
- Produce factors that drive HSC maintenance
What factors are produced by the endothelial cells that drive HSC maintenance
- CXCL12
- Notch
- Pleiotrophin
- SCF (stem cell factor)
How do perivascular stromal cells influence the HSCs?
- They are an important source of CXCL12
How do non-myelinating schawnn cells influence the HSCs?
NOT ESSENTIAL for HSC maintenance
BUT
- Drive some of the BEHAVIOUR (eg. circadian release of HSC into the peripheral blood)
- Some parasympathetic/sympathetic neurons go through the bone marrow
How do adipocytes influence the HSCs?
Negatively regulate the HSCs
What factors do the stem cell progeny produce?
CXCL4
TGFbeta
What are the stem cell progeny that influence the HSC in a feedback loop?
What do they associate with?
Megakaryocytes
Macrophages
Associate with the sinusoids
What is SCF (stem cell factor) required for?
HSC maintenance
What type of molecule is SCF?
Soluble OR membrane bound
What does SCF do?
Soluble SCF binds to KIT (expressed by HSCs)
Why is is thought that HSC associate with siusoids?
Perivascular stromal cells that are associated with sinusoids express SCF which is critical for HSC maintenance
What does triple labelling of: Arterioles Sinusoids SCF stomal cells Show?
SCF stomal cells associate with sinusoids MORE than arterioles
What does CXCL12 do?
Maintain and retain HSC in the bone marrow
Where is CXCL12 expressed?
Perivascular stromal cells and endothelial cells
What does CXCL12 do?
- Binds and activates CXCR4 (receptor) that is expressed by HSCs
What are the 3 key cell interactions in the stem cell niche?
Cell:cell interactions
Cell:ECM interactions
Cell:soluble interactions
What are the cell:cell interactions in the niche?
1) SC and other cells
2) SC and the niche cells (support)
3) SC and their own progeny
What are the potential therapeutic implications of using the knowledge of the HSC niche?
Describe this and how the knowledege of the niche environment helped us to devise this strategy
Nervi et al (2016) J Cell Biochem
Autologous cell transplants - Want to mobilise the HSC into the peripheral blood
Able to use AMD3100 (A CXCR4 inhibitor) to do this:
- CXCR4 on the HSC no longer able to bind CXCL12 in the ECM of the niche environment
- HSCs no longer retained at the niche –> mobilised
Knowledge of what retained the cell at the niche helped us to devise this strategy
REARCH PAPER
What did the paper ‘A Microenvironment-Induced Myeloproliferative Syndrome Caused by Retinoic Acid Receptor γ Deficiency
Walkley et al’ show?
For the FIRST TIME that the destruction of the stem cell niche can negatively influence stem cell behaviour to make them behave as malignant cells
What are myeloproliferactive syndromes (MPS)?
Heterogenous subclass of non-lymphoid hematopoietic neoplasms
Considered to be INTRINSIC to hematopoietic cells
What was unclear about the development of MPS before the work of Walkley et al?
Whether the hematopoietic microonvironment plays an active role in promoting/supporting the development of MPS
Where is the RARg receptor expressed?
In HSCs
What did Walkey et al do in their experiments?
What did they show?
KO RARg receptor
Showed:
- Loss of RARg in mice–> perturbed hematopoieses (with significantly elevated granulocytes)
- Overtime, these mice developed a profound myeloproliferative-like disease
What could the development of the profound myeloproliferative-like disease in RARg KO mice be due to?
How did Walkely et al test this?
Results?
Lack of the gene in HSC?
OR
HSCs are NORMAL but the lack of the gene in the environment makes them behave abnormally
To test:
1) Took bone marrow from RARg-/- mice –> health mice
- No disease
2) Took bone marrow from healthy mice –> RARg-/- mice - Cells proliferate abnormally
What were the conclusions from Walkley et al’s research and why?
- RARg null microenvironement creates the abnormal behaviour of the SCs
- NOT due to a cell intrinsic change (mutation) as previously thought
How can the SC niche be targeted for therapy?
1) Increasing the efficiency of BM engraftment
2) Drive the production of certain cell types (alter the outcome of SC differentiation)
How can the niche be used to increase the efficiency of BM engraftment?
Idea that can maybe expand the SC niche by increasing the no of niche support cells/increasing the supportive function of the niche
To improve the efficiency of engraftment of exogenous bone marrow (no longer needs to compete with the exogenous bone marrow for the niche)
How can the niche be used to drive the production of certain cell types (alter the outcome of SC differentiation)
1) Healthy SC behave abnormally in the wrong environment - can tweak the niche environment to drive a particular behaviour of the cells
2) Niches influence stem cell fate decisions - modification of the signals -> changes cell fate
