Adult stem cells Flashcards
Describe the regenerative capacity of humans/mammals?
Limited (unable to regenerate limbs)
BUT can regenerate some tissues
What are the fast regenerating tissues of mamals?
- Skin
- Gut epithelium
- Blood
What is the lifespan and proliferative ability of the blood cells?
What does this mean?
VERY short lifespan (each day lose ~10^11 red blood cells)
BUT they are constantly being replenished
–> MUST be a pool of progenitor cells in the blood that are able to regenerate the different blood cell types
What is the stem cell that gives rise to the blood cells
The hematopoietic stem cell
What happens if irradiate a mouse?
What happens if inject an irradiated mouse with bone marrow from a healthy mouse?
Irradiated mouse will die as there is no longer the production of blood cells
HOWEVER, if inject bone marrow from healthy mouse –> irradiated mouse will SURVIVE and is able to produce ALL of the blood cell types
What did Till and McCulloch do (1961)?
What did they identify?
Investigated the spleens of the RESCUED irradiated mice and noticed COLONIES form on the spleens:
- The more cells injected from the bone marrow –> the more colonies they get
1) Due to the discreteness of the colonies, they concluded they are most likely to develop from a SINGLE CELL
2) Due to the LINEAR relationship between injection of the cells and the amount of colonies –> suggests the cells from the BONE MARROW are forming these colonies
What confirmed the findings of Tim and McCulloch?
Genetic markers
What did Tim and McCulloch call the cells forming the colonies?
COLONY-FORMING UNITS
Why are the COLONY-FORMING UNITS seen as stem cells?
They form colonies on the spleen containing:
1) Differentiated blood cells - showing differentiation
2) New ‘colony forming units’ - showing self-renewal
This is the definition of a stem cell (must be able to self-renew AND differentiate)
How was it seen that the colonies formed by the colony-forming units contain undifferentiated cell types (new colony forming units)?
Injection of the colony-forming units from the spleen into the irradiated mouse
–> Mouse survived
What is the CLASSICAL model of the HSC lineage commitment?
Hierarchy:
- Pluripotent stem cells that give rise to the hematopoetic tissue (as well as the 3 germ layers)
- Multipotent progenitors (HSC)
- Oligopotent progenitors
- Lineage restricted progenitor
- Effector cells
What is the functional capacity of the HSC?
Multipotent (only give rise to the cells of the blood)
Give rise to the MESODERM only
What are oligopotent progenitors?
Examples?
Able to differentiate into a FEW cell types (less than multipotent)
Eg. Lymphoid and myeloid stem cells
Which lineage-restricted progenitors do the common myeloid progenitor cells give rise to?
MEP (megakaryocyte erythrocyte progenitors)
GMP (granulocyte macrophage progenitors)
What are megakaryocytes?
What do the MEPs give rise to?
Large bone marrow cell
Give rise to:
- Erythrocytes
- Platelets
What are granulocytes?
What do the GMPs give rise to?
White blood cells of the immune system
Give rise to:
- Granulocytes
- Macrophages
What effector cells do the common lymphoid progenitor cells give rise to?
T cells
B cells
What are the ‘effector cells’ of the blood?
The functional DIFFERENTIATED cells of the blood system
How do we prove that a cell is a stem cell?
Do functional assays to prove it has stem cell properties (can differentiate and self-renew)
- In VITRO
- In VIVO
What is the in vivo functional test for a HSC?
Inject into irraditated mice and assess the ability to recapitulate the blood system
SERIALLY
How can stem cells be IDENTIFIED?
Looking at surface markers that define a stem cell population:
- Looking for the PRESENCE and ABSECE of markers
- Looking at the COMBINATION of markers
How can stem cells be ISOLATED from a heterogenous population of cells?
Using FACS (based on the cell surface antigens of the cells):
- Label the cells with antibodies (against specific markers)
- Cells with presence/absence of certain markers will be separated
How do cells adopt different cell fates?
- Cells change their gene expression profile as they differentiate
- Different terminally differentiated effector cell types have different gene expression profiles
- Gene expression profile is determined by differential expression of TF
Why do different terminally differentiated effector cell types have different gene expression profiles?
Express genes that are SPECIFIC for THEIR function
Repress genes that are SPECIFIC to OTHER cell types/other cell functions
What do TF do?
1) Determine gene expression patterns by binding to DNA
2) Recruit ACTIVATORS/ COREPRESSORS to the ares of transcription
How are TF often found?
As components of multiprotein complexes q
What are the TF that influence stem cell fate?
What can these 4 factors do?
- Sox2
- Oct4
- Myc
- Klf4
They can REPROGRAM somatic cells –> ES cell-like fate (iPSC)
What is a master regulator?
A TF that appers to control most of the regulatory activities of other TF that cause a cell to adopt a particular fate
At the top of the hierarchy of transcriptomic regulation
What are the requirements of a TF to be a master regulator of stem cell fate? (3)
1) REQUIRED for the development into a specific lineage
2) Can CHANGE the fate of the cells they are introduced to
3) Can ANTAGONISE the opposite lineage programmes
What is considered to be the master regulator of the Erythroid cell fate?
Why?
GATA-1
Fulfils the 3 requirements:
1) If delete Gata-1 from the MEP progenitors –> no development of the erythroid lineages
–> REQUIRED
2) If introduce Gata-1 into the GMP progenitors –> changes the fate of the cells into MEP and the development of the E lineage
–> can CHANGE the fate
3) Gata-3 up regulates erythroid markers at the same time as down regulating the myeloid markers
–> ANAGONISES the opposite lineage programme
What is considered to be the master regulator of the myleoid cell fate?
PU.1
How does GATA-1 mediate the antagonism of PU.1?
Normally:
- PU.1 binds to DNA
- Binds to c-Jun (co-factor) through its Ets domain
- -> Regulate target genes
In the presence of GATA-1:
- Outcompetes c-Jun and binds to the Ets domain of PU.1 (through its carboxyterminal zinc finger domain)
–> PU.1 no longer bound to the c-Jun cofactor and cn no longer drive the expression of PU.1 target genes
How does PU.1 mediate the antagonism of GATA-1?
Normally:
- GATA1 binds to GATA sequence in the DNA (through its carboxyterminal zinc finger domain)
–> Drives expression of GATA1 target genes
In the presence of PU.1:
- Bind to GATA1 (through its TAD domain to the czf GATA1 domain) and displaces GATA1 from the DNA
–> GATA1 no longer drives expression of GATA1 target genes
How can TF A and TF B cross-antagonise? (3)
*Model for GATA1 and PU.1
1) Negatively influence each other
2) Auto regulate each other
3) Positively and negatively regulate target genes
(down regulate the expression of opponents through inhibiting the expression of target genes)
Describe the levels of TF A and TF B in a undifferentiated/uncommitted cell
What does this mean?
(*Model for GATA1 and PU.1)
EQUAL amounts
Meaning:
- NO expression of the target genes (both are inhibiting each other)
What happens in undifferentiated cell when there is a stochastic event/signalling cue?
- Get higher expression of either TF A or TF B
- Through a series of loops –> the TF auto regulates itself (increase expression) and REPRESSES the transcription of the other TF
- Cell turns into a specific cell type due to having more TF of A or B (can make 2 alternative cells)
Why was the classical stem cell models questioned?
As it suggests that every time we take a HSC in a transplantation experiment –> should ALWAYS get the SAME functional outcome
HOWEVER, this is not the case - HSCs appear to be heterogeneous (not all equal)
In what aspects are HSCs heterogeneous?
With regards to:
1) Self-renewal upon transplantation
2) Cell cycle properties
3) Differentiation
How are HSCs heterogeneous with regards to self-renewal upon transplantation?
If take HSCs and put into irradiated mice - SHOULD be able to SERIALLY transplant them over many generations of mice
BUT, the ability of the HSCs to serially transplant is variable (not all have equal self-renewal capacity)
How are HSCs heterogeneous with regards to the cell cycle properties?
Some HSCs are dormant/quiescent/not cycling
Whilst others are progressing through the cell cycle
How are HSCs heterogeneous with regards to differentiation?
IF the classical model is true…
When take HSC - should be balanced in differentiation and should get EQUAL amounts of all the erythroid and myeloid lineages
BUT, there is a LINEAGE BIAST for HSCs to preferentially make more myeloid or erythroid cells
How can heterogeneity be masked? (may have been the case in previous experiments that proposed the classical model)
Population-based cell studies
Why?
Get the SAME measure of gene expression profile in a population of cells that do not have equal transcript levels (average out) as a population of cells that may have the same number of transcripts per cell (uniform)
Why is it advantageous to do SINGLE cell studies of stem cells?
To understand properly the properties of the cell
As population-based studies can mask certain properties (Eg. heterogeneity)
When looking at the gene expression of single multi potent cell types, what was seen?
What is this idea called?
Although found at the LOW gene expression level,
undifferentiated, multipotent cell ALREADY express come genes that are SPECIFIC for their differentiated cell types
LINEAGE PRIMING
When are lineage programmes initiated?
How?
Initiated in the HSC compartment
- HSCs express specific lineage markers –> more prone upon the differentiation cue to give rise to that specific lineage
What can still occur in the HSC compartment, even though the cells may be lineage primed?
They can still inter-covert between different lineages (change the genes they are expressing)
What are the 3 different HSC lineage commitment models?
Haas S et al (2018) Cell Stem Cell 22:627-638
1) Classical
2) Early split
3) Continuous Waddingotn-like
What is the ‘early split’ model of HSC lineage commitment?
?
What is the ‘continuous Waddingotn-like’ model of HSC lineage commitment?
- HSCs are still at the ‘top’ of the hierarchy but are not a particular entity
HSCs are more like a state
- Cells exist in this state but as go down the epigenetic landscape, they become more restricted
- Cells progress through a continuum, rather than being discrete entities
What are the causes of HSC heterogeneity?
1) Niche localisation
2) Genetic heterogeneity
3) Epigenetic heterogeneity
4) Biological processes
5) Segregation of cell fate determinants
6) Stochasticity (randomness)
How does niche localisation cause HSC heterogeneity?
In VIVO, HSCs exist in different locations in the body –> receive different signalling cues –> different impacts on the cell
How does genetic heterogeneity cause HSC heterogeneity?
Intrinsic differences of the SC arise due to inherent errors into the genome when the SC divide
(Eg. DNA mutation, chromosome translocation)
How does epigenetic heterogeneity cause HSC heterogeneity?
Different cells differ in DNA methylation profiles
How do biological processes cause HSC heterogeneity?
Not all the stem cells are actively cycling –> are in different stages of the cell cycle
- Cells in early G1 will differentiate into a different cell type to the cells in late G1 if you force them to differentiate
How does the segregation of cell fate determinants cause HSC heterogeneity?
A dividing cell needs to divide all of its cytoplasmic components:
- Cytoplasmic components may be not equally distributed when the cells divide
- -> cells become different
How does stochasticity cause HSC heterogeneity?
Random encounter of molecules into the cell may occur
Transcription is leaky
What are the consequences of HSC heterogeneity?
1) Contribution to hematopoiesis - which cells contribute to the cells of the blood system??
2) Emergency hematopoiesis - do lineage primed cells allow this process to occur rapidly when this is needed??(eg. blood loss)
3) Ageing
4) Hematologic malignancies
