Chapter 8: Stem cells and differentiation (Lecture, main)

Question 1

What are the different subjects that will be discussed in the lecture? (you obv don’t have to learn this)

Answer 1

• 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

Question 2

What are the two characteristics of a stem cell?

Answer 2

• 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)

Question 3

How is the process called whereby stem cells become specialized to perform a particular function

Answer 3

Differentiation

Question 4

In what two ways can a stem cell divide?

Answer 4

Symmetrical (copy) and asymmetrical (differentiation)

Question 5

What are the different types of stem cells and their characteristics?

Answer 5

• 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).
• 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.

Question 6

Which cells are in heterochromatin, and which are in euchromatin form: stem cells or differentiated cells?

Answer 6

• Heterochromatin: differentiated cells (‘closed’ structure)
• Euchromatin: stem cells (‘open’ structure)

Question 7

Stem cells can also be divided into embryonic stem cells and adult stem cells. Can you explain what they mean?

Answer 7

1. 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
2. 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

Question 8

There are many different strategies for stem cell therapy but what are the three types that will be discussed?

Answer 8

• Embryonic stem (ES) cells
• Induced pluripotent stem (iPS) cells
• Nuclear transfer embryonic stem (NT-ES) cells

Question 9

How are embryonic stem (ES) cells ‘made’?

Answer 9

Embryonic stem cells are derived from inner cell mass (from embryo’s)

Question 10

How are induced pluripotent stem (iPS) cells ‘made’?

Answer 10

They are induced, by adding repogragramming factors (Yamanaka factors) to mature differentiated cells, so they become pluripotent stem cells

Question 11

How are nuclear transfer embryonic stem (NT-ES) cells ‘made’?

Answer 11

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.

Question 12

In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is in balance?

Answer 12

There is tissue homeostasis, a.k.a. normal tissue

Question 13

In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is shifted towards differentiation?

Answer 13

There is tissue aging and/or degeneration

Question 14

In the cell, there is a balance between self-renewal and differentiation. What happens when the cell is shifted towards self-renewal?

Answer 14

Cancer.

For an overview of the different ‘balances’, see this figure

Question 15

So, tumor cells are associated with self-renewal. In what ways can they acquire self-renewal?

Answer 15

1. Aberrant expression of (mutated) genes induce stem cell features
2. The initial/founder hit (driver mutation) is in a stem cell. Tumor has inherited the stem cell state/features of the cell of origin
3. Microenvironment imposes stem cell features onto the cancer cells

Question 16

What is the evidence that suggests that stem cells are the origin of cancer? (+ can you also name examples?)

Answer 16

• 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”

Question 17

There is additional evidence for stem cell origin of tumors… what are they?

(besides:

1. Self-renewal-long life of normal stem cells offers opportunities for mutations and thereby for cancer formation.
2. More differentiated normal cells obtain self-renewal capacity (mutation) “De novo stem cells”)

Answer 17

• 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

Question 18

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’?

Answer 18

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)

Question 19

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?

Answer 19

Examples are AML, CML and brain tumors, but as you can see in this figure there are many more

Question 20

In cancer, there is heterogeneity. What does this mean?

Answer 20

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

Question 21

How does heterogeneity occur? (3 answers)

Answer 21

1. Intrinsic differences among cancer cells caused by stochastic genetic and epigenetic changes, clonal evolution.
2. Extrinsic mechanisms lead to phenotypic and functional differences between tumor cells; microenvironment.
3. Stem cell model; in which stem cell-like cells “differentiate into non-stem cells.

Question 22

What is the definition of stemness and what three things belong to stemness?

Answer 22

Stemness refers to common molecular processes underlying the core stem cell properties of self-renewal and the generation of differentiated progeny.

1. Heterogenous mixture of genetic subclones (genetics)
2. Non-genetic determinants; epigenetic modifications (epigenetics)
3. Microenvironment

Question 23

True/false: The severity of cancer is directly related to the stemness of the cell

Answer 23

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

Question 24

Which of these two figures shows a normal bone marrow, and which shows (immature) blast cells?

Answer 24

The left figure is a blast cell, the right figure is a normal bone marrow

Question 25

In Acute Myeloid Leukemia (AML), there is a differentiation block. Where in this figure can such a differentiation block occur?

Answer 25

It can occur in many different stages, as indicated by the red lines

Question 26

What is important in the development of therapies in AML?

Answer 26

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)

Question 27

What is minimal residual disease (MRD)?

Answer 27

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.

Question 28

# 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

Answer 28

* 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

Question 29

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?

Answer 29

**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

Question 30

What are three important self-renewal signaling pathways in both normal and cancer cells?

Answer 30

1) WNT signaling 2) Hedgehog signaling 3) Notch signaling

Question 31

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?

Answer 31

1. WNT signaling: **Colon cancer, skin cancer** and **hepatocellular carcinoma** 2. Hedgehog signaling: **Medulloblastoma** 3. Notch signaling: **Breast cancer** and **Acute Lymphocytic Leukemia**

Question 32

Explain the process of the (canonical) WNT signaling pathway?

Answer 32

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*

Question 33

The WNT signaling pathway is enhanced by another pathway, how is this pathway called?

Answer 33

The Lgr-Rspondin pathway

Question 34

How does the Lgr-Rspondin pathway work?

Answer 34

R-spondin binds to the Lgr-receptor, which activates RNF43/ZNRF3. This complex blocks the ubiquitination of the Frizzled-receptor (and thus stabilizes it)

Question 35

Why is the WNT pathway so important in colon cancer?

Answer 35

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).

Question 36

What does Adenomatous polyposis coli (APC) do?

Answer 36

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.

Question 37

How does an APC mutation lead to colorectal cancer?

Answer 37

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

Question 38

In some tumors WNT signaling is aberrant, but not mutated. What genes can mutate to cause aberrant WNT signaling and in what tumor?

Answer 38

1. Colon tumors: mainly **APC mutations** 2. Skin tumors: activating **ß-catenin mutations** 3. Hepatocellular carcinoma: **Axin mutations** ## Footnote *Note: All give rise to enhanced self-renewal or a stem cell phenotype*

Question 39

What are the three Hedgehog homologues?

Answer 39

Desert (DHH), Indian (IHH), and Sonic (SHH)

Question 40

How does the Hedgehog signaling pathway work?

Answer 40

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

Question 41

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)*

Answer 41

1. **Gorlin’s syndrome**: germ-line mutation in Patched with predisposition for skin, cerebellar, muscle tumors (BCC medulloblastoma, rhabdomyosarcoma) 2. Inactivating mutation in Patched/activating mutation in **Smoothened sporadic BCC** (all have activated Hh and expression of Gli) 3. **Gliomas and gastrointestinal tumors**: Amplification of Gli and Hh overexpression. 4. **Feedback** via the microenvironment: Hh stimulates Gli in stroma paracrine signaling

Question 42

What is a natural Hedgehog inhibitor that is isolated from lilies (the flower)?

Answer 42

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

Question 43

What are polycomb proteins?

Answer 43

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).

Question 44

Of what two complexes do polycomb proteins exist and what do they do?

Answer 44

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

Question 45

Polycomb proteins are the '**guardians of stemness'**. How do they do this?

Answer 45

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 ## Footnote *Note: this is also the reason why it is an oncogenic potential!*

Question 46

There are also lineage-specific transcription factors that play a role in differentiation and in cancer. What do they do?

Answer 46

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

Question 47

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?

Answer 47

A differentiation block, because of the binding of corepressors (so there is an abnormal accumulation of immature granulocytes called promyelocytes)

Question 48

How can PML be treated?

Answer 48

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

Question 49

What happened to the survival rate after retinoid acid therapy was discovered?

Answer 49

* Before the retinoic acid therapy: 90 % death * After introduction of retinoic acid: 90% survival