Stem Cells

Question 1

What are tissue stem cells?

Answer 1

AKA Adult stem cells, these are multipotent stem cells (form a number of vaguely similar cell types, often being able to produce anything within a single germ layer) that reside in tissues to replace cells.

These are considered to be rare, and are difficult to isolate.

Like any stem cells, they are defined by their ability to regenerate a tissue.

Question 2

What factors regulate stem cells?

Answer 2

External Signalling
o Systemic factors (hormones, growth factors, immune response etc.), which all act on the stem cells
o Local regulators (local GF, cell contacts, polarisation, oxygen, metabolism)
o Niche

Intrinsic programs
o Transcription factors:
o Networks and master regulators
o Epigenetic landscape

Question 3

What is a stem cell niche?

Answer 3

An anatomical structure, including cellular and acellular components, that integrates local and systemic factors to regulate stem cell proliferation, differentiation, survival and localization.

The necessity for stem cells to be interacting with the right kind of surrounding cells may be part of the reason adult stem cells are so difficult to isolate - mimicking the niche to allow for expansion is an area of intense research.

Question 4

What evidence of stem cell heterogeneity is provided by HSCs?

Answer 4

In a repopulation assay and a limit dilution experiment, in which you dilute the bone marrow, some of the recipients don’t receive any stem cells.

o You can have myeloid biased stem cells that produce more myeloid cells but only few lymphoid cells
o You can have lymphoid biased stem cells that produce more lymphoid cells but few lymphoid cells
o Some cells renew more than others and have a greater proliferative capacity than others
o Biased stem cells are still multipotent

Question 5

What does transcriptomics reveal about stem cell heterogeneity?

Answer 5

When looking at single cell analysis of RNA within a population of stem cells, you can see that cells express different amounts of different genes, even though they are supposed to be the same cell type.

Although this can fluctuate through time, it appears that biases in stem cell function are stable, so can be inherited by daughter cells; all of the stem cells
produced from that stem cell have a lineage bias – it is not a fluctuating unstable heterogeneity.

Question 6

Why do some think that stem cell heterogeneity is inevitable?

Answer 6

Inevitable fluctuation - the idea that it would be too difficult and energetically expensive to regulate potency so tightly that no heterogeneity exists, and since they are all still multipotent and can regenerate the tissue there is little issue.

Question 7

What advantages might stem cell heterogeneity have?

Answer 7

It may increase the robustness of a system and allow for gradation of response; if a signal is present that induces the stem cell pool to differentiate into a single cell type, some being more resistant to this than others protects the stem cell pool from exhaustion.

Question 8

What are the models of differentiation induction regulation?

Answer 8

The instructive, the stochastic/selective and the combined.

Question 9

What is the instructive model of differentiation regulation?

Answer 9

Signals act on the multipotent cells and direct them in particular lineages.

In a stem cell capable of differentiating into cell type A or B, when it receives signal A, the cell only produces A; when it receives signal B, the cell only produces B.

Question 10

What is the stochastic/selective model of differentiation regulation?

Answer 10

The stem cell population are heterogenous and randomly form progenitors of different lineages. Signalling factors act on these to stabilise the desired cell’s progenitor or destroy progenitors of other cell lines.

This is thought to be how a lot of cytokines and growth factors work in that they maintain particular recommitted cell types and then the other cells die off.

Question 11

What is the combined model of differentiation regulation?

Answer 11

There is an instructive situation where you get a signal that tells the cell which route to take.

However, because this could lead to stem cell exhaustion e.g. if you’ve got too much of signal A and all cells become A, there is also a situation where the cell recommits in a random way to each of the lineages and then gets selected via signals downstream.

Question 12

What is the epigenetic landscape?

Answer 12

The “Gravity” of landscape represents the directionality of differentiation and likely lineage paths.

There is a gravity in the epigenetic landscape, which means that the cells will always go in one direction. Self-renewing stem cells at the top commit to differentiate down one particular lineage as they go down the branching routes. This directionality results from differences in entropy/energy.

Question 13

What is an attractor basin?

Answer 13

In the epigenetic landcape model, “Attractor basins” at points along the furrows represent positions of stability during differentiation.

These represent a point in a lineage when a cell can transit through a semi-stable state (they are not completely stable so they won’t stay in that situation for long), which allows them to explore different options – as these often connect to other furrows and pathways acting as points of semi-confluency.

Question 14

How are decisions at attractor basins thought to be regulated?

Answer 14

The reason why progenitor cells can explore branch points here is because the lineage decisions are made by transcription factors cross antagonisms; two transcription factors (eg A and B) which reinforce their own expression and inhibit the other, producing a natural balance - the semi-stable state.

Stochastic or signalling factor induced changes in the levels of A or B can drive changes in the ratio of A cell production to B cell production.

Question 15

Provide an example of cross antagonism at an attractor basin.

Answer 15

In the haematopoietic stem cell lineage, the PU1/GATA1 switch acts at an early attractor basin within the early myeloid lineage.

PU1 stimulates myeloid gene expression (producing macrophages, granulocytes etc), while GATA1 induces erythroid-megakaryocyte lineage gene expression.

Question 16

How can differentiation down the epigenetic landscape be misregulated in cancer?

Answer 16

• Differentiation block – e.g. APML (PML-RARA – TF)
o The cell gets stuck on one of the semi-stable progenitor states and cannot go down any of the routes

• Failure of apoptosis – e.g. follicular lymphoma (Bcl2)
o The cell fails to die at the end, increasing expansion of that cell type

• Over proliferation (with some limited differentiation– e.g. CML (BCR-ABL – TK)

• Non-physiological route/attractor basin
o The cell takes a different route to how it would normally go, due perhaps to misexpression of two transcription factors not normally coexpressed in the same cell, leading to lineage confusion inability to differentiate and proliferate

Question 17

How does AML provide an example for collaborative mutations affecting stem cell fate?

Answer 17

Acute myeloid leukemia is thought to arise from a class I mutation, which occurs in signalling factors e.g. tyrosine kinase receptors, causing enhanced self renewal, and a class II mutation, which is usually in a transcription factor that corrects differentiation, causing a block in differentiation.

Both are needed (two hits), as differentiation prevents the cells from taking over the bone marrow, blocking both is needed to flourish and inhibit the activity of normal cells.

Question 18

What was the previous view on intratumour heterogeneity?

Answer 18

This been portrayed as reasonably homogeneous cell populations until relatively late in the course of tumor progression, when hyperproliferation combined with increased genetic instability spawn distinct clonal subpopulations.

Question 19

What are cancer subclonal populations individually responsible for?

Answer 19

Cancers often contain regions demarcated by various degrees of differentiation, proliferation, vascularity, inflammation, and/or invasiveness.

There is now new evidence of a hitherto-unappreciated subclass of neoplastic cells within tumors, termed cancer stem cells (CSCs).

Question 20

How abundant are cancer stem cells thought to be?

Answer 20

Although the evidence is still fragmentary, CSCs may prove to be a common constituent of many if not most tumors, albeit being present with widely varying abundance.

CSCs were initially implicated in the pathogenesis of hematopoietic malignancies (Bonnet and Dick, 1997) and then years later were identified in solid tumors, in particular breast carcinomas and neuroectodermal tumors.

Question 21

How are CSCs defined?

Answer 21

CSCs are defined operationally through their ability to efficiently seed new tumors upon xenografting into immunodeficient host mice.

This functional definition is often complemented by including the expression in CSCs of markers/transcriptional profiles that are also expressed by the normal stem cells in the tissue-of origin.

Question 22

How can CSCs be identified?

Answer 22

Fractionation of cancer cells on the basis of displayed cell-surface markers has yielded subpopulations
of neoplastic cells with a greatly enhanced ability, relative to the corresponding majority populations, to seed new tumors upon implantation in immunodeficient mice.

Question 23

Where do CSCs come from?

Answer 23

The origins of CSCs within a solid tumor have not been clarified.

Tissue stem cells may serve as the cells-of-origin that undergo oncogenic transformation to yield CSCs, or partially differentiated transit-amplifying cells may suffer
the initial oncogenic transformation thereafter assuming more stem-like character.

Question 24

What is the role of CSCs?

Answer 24

CSCs, like their normal counterparts, may self-renew as well as spawn more differentiated derivatives; in the case of neoplastic CSCs, these descendant cells form the great bulk of many tumors.

Question 25

What is the root of the CSC hypothesis?

Answer 25

That despite genetic homogeneity cancer cells are organised as a differentiation hierarchy at the top of which is a pool of self renewing CSCs with the properties of other adult stem cells but with the ability to produce various cancer subclonal regions.

Question 26

How do the different models of cancer heterogeneity explain the frequent inability of cancer sup-populations to re-initiate tumours in xenografting?

Answer 26

In the stochastic model, where the subclonal populations arise without CSCs, the fact that only some of the cells are able to cause tumour growth is due to intrinsic fluctuations in the expression of stem-like genes.

In the CSC model, only a few cells in the tumour, the CSCs, are capable of regenerating the tumour while all the rest of the more differentiated subclonal populations cannot.

Question 27

What controls the extent of differentiation seen within a tumour?

Answer 27

This depends on the stringency of the differentiation arrest produced by oncogenic mutation and hence can vary between different cancers.

Often as a cancer becomes more aggressive this correlates with an increase in the number of these CSCs within that tumour. In some tumours such as melanoma it has been suggested that essentially all the cells can act as CSCs.

It is also thought that that the phenotypic plasticity within tumors may produce bidirectional interconversion between CSCs and non-CSCs, resulting in dynamic variation in the relative abundance of CSCs.

Question 28

How might conversion from non-CSCs to CSCs be mediated?

Answer 28

The metastatic EMT program enables cancer cells
to physically disseminate from primary tumors and
confers on such cells the self-renewal capability required for clonal expansion at sites of dissemination.

The heterotypic signals that trigger an EMT, such as those released by an activated, inflammatory stroma, may also be important in creating and maintaining CSCs.

Question 29

How might conversion from CSCs to non-CSCs be mediated?

Answer 29

An EMT can also convert epithelial carcinoma cells into mesenchymal, fibroblast-like cancer cells that may well assume the duties of cancer-associated fibroblasts (CAFs).

Glioblastoma cells (or possibly their associated CSC subpopulations) can transdifferentiate into endothelial-like cells that can substitute for bona fide host-derived endothelial cells in forming a tumor-associated neovasculature.

Thus tumors may acquire stromal support by inducing some of their own cancer cells to undergo various types of metamorphosis to produce stromal cell types rather than relying on recruited host cells to provide their functions.

Question 30

What implications on cancer therapy does the CSC model have?

Answer 30

Increasing evidence in a variety of tumor types suggests that cells with properties of CSCs are more resistant to various commonly used chemotherapeutic treatments.

Their persistence may help to explain the almost-inevitable disease recurrence following apparently successful debulking of human solid tumors by radiation and various forms of chemotherapy, and may mediate cancer dormancy.

Thus CSCs would be the primary target of any chemotherapy, as killing them is both necessary and sufficient for tumour eradication.

Question 31

What are the sources of cancer cell phenotypic variability?

Answer 31

The discovery of CSCs and biological plasticity in tumors indicates that a single, genetically homogeneous population of cells within a tumor may nevertheless be phenotypically heterogeneous due to the presence of cells in distinct states of differentiation.

However, an equally important source of phenotypic variability may derive from the genetic heterogeneity within a tumor that accumulates as cancer progression proceeds.

Question 32

Why might CSC-mediated phenotypic variability be more harmful than clonally selected subpopulations?

Answer 32

Thus, elevated genetic instability operating in later stages of tumor progression may drive rampant genetic diversification that outpaces the process of Darwinian selection, generating genetically distinct subpopulations far more rapidly than they can be eliminated.

This genetic diversification may enable functional specialization, producing subpopulations of cancer cells that contribute distinct, complementary capabilities, which then accrue to the common benefit of overall tumor growth as described above.

Question 33

What controversy is there concerning the CSC model?

Answer 33

Better mouse xenografting assays suggest they are not so rare or phenotypically restricted - more cells than previously thought may be able to reproduce the tumour.

The majority of the work has been done in blood cancers, there is reason to believe other tissues are less hierarchical.

The differences in cancer propagating capacity may actually be due to sub-clonal genetic variegation increasing or decreasing potency.