Chapter 8: Stem cells and differentiation (Lecture, main) Flashcards
What are the different subjects that will be discussed in the lecture? (you obv don’t have to learn this)
- Stem cells and differentiation
- Epigenetic mechanisms regulating self-renewal and differentiation
- Different kind of stem cells and how to make them
- Cancer cells with self-renewal capacity: cancer stem cells
- Leukemia and leukemic stem cells
- Self-renewal signaling pathways: Notch, Wnt and Hedgehog
- The polycomb pathway
- Lineage specific transcription factors
What are the two characteristics of a stem cell?
- Ability to divide (self replicate) for indefinite periods throughout the life of an organism (self renewal)
- Ability to differentiate to the many different cell types upon different signals (pluripotency)
How is the process called whereby stem cells become specialized to perform a particular function
Differentiation
In what two ways can a stem cell divide?
Symmetrical (copy) and asymmetrical (differentiation)
What are the different types of stem cells and their characteristics?
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Totipotent Stem Cell: the potential to create any type of cell necessary for embryonic development
- (extraembryonic membranes and tissues, the embryo itself, and all postembryonic tissues and organs).
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Pluripotent Stem Cell (PSC): the capability of developing cells of all germ layers
- (endoderm, ectoderm, and mesoderm).
- Multipotent Stem Cell: give rise to cells that have a function dependent of the embryonic germ layer they derived from.
Which cells are in heterochromatin, and which are in euchromatin form: stem cells or differentiated cells?
- Heterochromatin: differentiated cells (‘closed’ structure)
- Euchromatin: stem cells (‘open’ structure)
Stem cells can also be divided into embryonic stem cells and adult stem cells. Can you explain what they mean?
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Embryonic stem cells – they are pluripotent – indefinite self-renewal
- Embryonic stem cells of the inner cell mass are able to differentiate to generate primitive ectoderm which differentiate during gastrulation into endoderm, ectoderm and mesoderm
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Adult stem cells – they are multipotent – limited self-renewal
- They are present in every tissue and involved in regeneration of these tissues.
- Examples: hematopoietic stem cells (in the bone marrow), hair follicle stem cells, breast stem cells and intestinal stem cells
There are many different strategies for stem cell therapy but what are the three types that will be discussed?
- Embryonic stem (ES) cells
- Induced pluripotent stem (iPS) cells
- Nuclear transfer embryonic stem (NT-ES) cells
How are embryonic stem (ES) cells ‘made’?
Embryonic stem cells are derived from inner cell mass (from embryo’s)
How are induced pluripotent stem (iPS) cells ‘made’?
They are induced, by adding repogragramming factors (Yamanaka factors) to mature differentiated cells, so they become pluripotent stem cells
How are nuclear transfer embryonic stem (NT-ES) cells ‘made’?
This was how Dolly was made, a cytoplasmic donor with enucleated cell/oocyte and a somatic nuclear donor cell is needed. After the cell and the DNA are ‘merged’, it is injected in a third animal that will carry the blastocyst.
In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is in balance?
There is tissue homeostasis, a.k.a. normal tissue
In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is shifted towards differentiation?
There is tissue aging and/or degeneration
In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is shifted towards self-renewal?
Cancer.
For an overview of the different ‘balances’, see this figure
So, tumor cells are associated with self-renewal. In what ways can they acquire self-renewal?
- Aberrant expression of (mutated) genes induce stem cell features
- The initial/founder hit (driver mutation) is in a stem cell. Tumor has inherited the stem cell state/features of the cell of origin
- Microenvironment imposes stem cell features onto the cancer cells
What is the evidence that suggests that stem cells are the origin of cancer? (+ can you also name examples?)
- Self-renewal-long life of normal stem cells offers opportunities for mutations and thereby for cancer formation (Example: skin).
- More differentiated normal cells obtain self-renewal capacity (mutation) (example: acute and chronic myeloid leukemia and colon cancer): “De novo stem cells”
There is additional evidence for stem cell origin of tumors… what are they?
(besides:
- Self-renewal-long life of normal stem cells offers opportunities for mutations and thereby for cancer formation.
- More differentiated normal cells obtain self-renewal capacity (mutation) “De novo stem cells”)
- Tumor specific mutations are present in the normal stem cell compartment (Example for leukemia)
- Presence of similar cell surface markers in normal- and cancer stem cells
Earlier, we noticed that tumor specific mutations that are present in the normal stem cell compartment are additional evidence that stem cells are the origin of tumors. How is this phenomenon called and how does it ‘work’?
Clonal hematopoiesis (CHIP): aging related phenomenon in which hematopoietic stem cells (HSCs) contribute to the formation of a genetically distinct subpopulation of blood cells (example: acute and chronic myeloid leukemia)
Earlier, we noticed that the presence of similar cell surface markers in normal and cancer stem cells is evidence that stem cells are the origin of cancer. What are some examples of diseases where these markers can be used?
Examples are AML, CML and brain tumors, but as you can see in this figure there are many more
In cancer, there is heterogeneity. What does this mean?
Tumor heterogeneity refers to observations that although cancer formation is believed to be a clonal process beginning with a single transformed cell, not all malignant cells within a tumor are the same
How does heterogeneity occur? (3 answers)
- Intrinsic differences among cancer cells caused by stochastic genetic and epigenetic changes, clonal evolution.
- Extrinsic mechanisms lead to phenotypic and functional differences between tumor cells; microenvironment.
- Stem cell model; in which stem cell-like cells “differentiate into non-stem cells.
What is the definition of stemness and what three things belong to stemness?
Stemness refers to common molecular processes underlying the core stem cell properties of self-renewal and the generation of differentiated progeny.
- Heterogenous mixture of genetic subclones (genetics)
- Non-genetic determinants; epigenetic modifications (epigenetics)
- Microenvironment
True/false: The severity of cancer is directly related to the stemness of the cell
True! This also means that the patient survival can be predicted upon the factors that impinge on stemness, and also the treatment is less effective if the cell has more of these features
Which of these two figures shows a normal bone marrow, and which shows (immature) blast cells?
The left figure is a blast cell, the right figure is a normal bone marrow
In Acute Myeloid Leukemia (AML), there is a differentiation block. Where in this figure can such a differentiation block occur?
It can occur in many different stages, as indicated by the red lines
What is important in the development of therapies in AML?
The leukemic stem cells (LSCs) have to be eliminated and the hematopoietic stem cells (HSCs) have to be spared (because treatment after relapse is hard to treat, but there has to be enough HSCs after the treatment for the patient to recover)
What is minimal residual disease (MRD)?
Minimal residual disease (MRD) is the name given to small numbers of leukaemic cells (cancer cells from the bone marrow) that remain in the person during treatment, or after treatment when the patient is in remission (no symptoms or signs of disease). It is the major cause of relapse in cancer and leukemia.
Fill in:
- Cancer with high number of cancer stem cells: drug … and thus … outcome
- Cancer with low number of cancer stem cells: drug … and thus … outcome
- Cancer with high number of cancer stem cells: drug insensitivity and thus poor outcome
- Cancer with low number of cancer stem cells: drug sensitivity and thus good outcome
We now know that it is important to eliminate LSCs while sparing HSCs. To identify these specific cells gene expression profiling has been done. What was the outcome of this?
IGFBP7 was identified that induces differentiation and loss of survival of human AML-SCs without affecting normal normal hematopoiesis (so apoptosis is induced in the stem cells!). A visual representation of this mechanism is shown in this figure
What are three important self-renewal signaling pathways in both normal and cancer cells?
1) WNT signaling
2) Hedgehog signaling
3) Notch signaling
In cancer, there are often mutations in the self-renewal signaling pathways. What are the corresponding cancers that are related to the Wnt signalling, Hedgehog signalling and Notch signalling?
- WNT signaling: Colon cancer, skin cancer and hepatocellular carcinoma
- Hedgehog signaling: Medulloblastoma
- Notch signaling: Breast cancer and Acute Lymphocytic Leukemia
Explain the process of the (canonical) WNT signaling pathway?
A WNT ligand binds to a Frizzeld (FRZ)-receptor. Upon activation of the receptor, a signal is sent to the phosphoprotein Dishevelled (Dsh). This results in the stabilization of ß-catenin that acts as a transcription factor for self-renewal (e.g. cell proliferation, tumorigenesis and stem cell maintenance).
Note that there are about 20 different WNT-ligands and 10 different FRZ-receptors. Also, the stabilization of ß-catenin is one of many pathways, but it’s the most common
The WNT signaling pathway is enhanced by another pathway, how is this pathway called?
The Lgr-Rspondin pathway
How does the Lgr-Rspondin pathway work?
R-spondin binds to the Lgr-receptor, which activates RNF43/ZNRF3. This complex blocks the ubiquitination of the Frizzled-receptor (and thus stabilizes it)
Why is the WNT pathway so important in colon cancer?
As can be seen in this figure, the crypt base columnar (CBC) cells are mainly driven by the Lgr (and thus the WNT) pathway, which is involved in the maintenance of the ‘stemness’. The cells that are in the villi don’t express these LGR-receptors (as you can see in the figure left-bottom).
What does Adenomatous polyposis coli (APC) do?
Adenomatous polyposis coli (APC), a tumor suppressor gene, is a negative regulator that controls beta-catenin concentrations by targeting it for degradation and interacts with E-cadherin, which are involved in cell adhesion.
How does an APC mutation lead to colorectal cancer?
A deletion/mutation of APC in the stem cells leads to a tumor. Interestingly, a deletion/mutation of APC in transit-amplifying cells (orange in figure) does not lead to a tumor formation
In some tumors WNT signaling is aberrant, but not mutated. What genes can mutate to cause aberrant WNT signaling and in what tumor?
- Colon tumors: mainly APC mutations
- Skin tumors: activating ß-catenin mutations
- Hepatocellular carcinoma: Axin mutations
Note: All give rise to enhanced self-renewal or a stem cell phenotype
What are the three Hedgehog homologues?
Desert (DHH), Indian (IHH), and Sonic (SHH)
How does the Hedgehog signaling pathway work?
In the absence of Hh (Figure a), Patched (PTCH) inhibits the Smoothened (SMO) receptor. When extracellular Hh is present (Figure b), it binds to and inhibits Patched, allowing Smoothened to accumulate and activate the GLI transcription factors, so that Hh target genes (e.g. stem cell genes) are activated
What are the four different syndromes/cancers that are associated with the Hedgehog signaling?
(I assume you don’t have to know this by heart, so that’s why there is only one card about it… not sure though)
- Gorlin’s syndrome: germ-line mutation in Patched with predisposition for skin, cerebellar, muscle tumors (BCC medulloblastoma, rhabdomyosarcoma)
- Inactivating mutation in Patched/activating mutation in Smoothened sporadic BCC (all have activated Hh and expression of Gli)
- Gliomas and gastrointestinal tumors: Amplification of Gli and Hh overexpression.
- Feedback via the microenvironment: Hh stimulates Gli in stroma paracrine signaling
What is a natural Hedgehog inhibitor that is isolated from lilies (the flower)?
Cyclopamine. There are also many analogs identified, which are shown in the orange box (don’t study that please). GDC-0449 is used in advanced basal cell carcinoma (most common skin cancer) as clinical treatment
What are polycomb proteins?
Polycomb-group proteins can remodel chromatin such that epigenetic silencing of genes takes place.
(Polycomb-group proteins are well known for silencing Hox genes through modulation of chromatin structure during embryonic development in fruit flies).
Of what two complexes do polycomb proteins exist and what do they do?
It exists of a PRC2 and PRC1 complex. The PRC2-complex methylates K27 on histone 3. Because the K27 is methylated, PRC1 can bind to it and will ubiquitinate K119 on histone 2a. This modulates transcription of genes
Polycomb proteins are the ‘guardians of stemness’. How do they do this?
By silencing gene expression, especially tumor suppressor genes are blocked. The mechanism for this is the formation of PcG repressive complexes: inhibition of transcription, methylation and chromatin compaction
Note: this is also the reason why it is an oncogenic potential!
There are also lineage-specific transcription factors that play a role in differentiation and in cancer. What do they do?
They dictate what type of cell a stem cell becomes. In this figure we see (in the hematopoietic system) an important lineage specific transcription factor: Pu.1 that very early in the development/differentiation determines what type of hematopoietic cells are made. Also C/EBPa is very important in this process
In acute promyelocytenleukemie (PML, a subtype of AML) there is a chromosomal translocation involving the retinoic acid receptor alpha (RARα or RARA) gene. What does this translocation lead to?
A differentiation block, because of the binding of corepressors (so there is an abnormal accumulation of immature granulocytes called promyelocytes)
How can PML be treated?
With retinoid acid, which removes the co-repressors, so that co-activators can bind to the gene. In this way, differentiation is activated and the disease is cured
What happened to the survival rate after retinoid acid therapy was discovered?
- Before the retinoic acid therapy: 90 % death
- After introduction of retinoic acid: 90% survival
