MEDG520
Block 3
Stem Cells
Concepts:
 DEFINITION OF STEM CELL: THE HEMATOPOIETIC PARADIGM
 Properties of Stem Cells:
 HAEMATOPOIETIC STEM CELLS AND PROGENITORS
 ARE HSCs A GOOD PARADIGM FOR OTHER ADULT STEM CELLS?
 Prospective Isolation: its advantages and disadvantages
o Proposed cell-surface markers of undifferentiated HSCs
o Prospective Isolation
o Advantages
o Disadvantages
 KINETICS OF STEM CELL ACTIVITY in vivo – HOW MANY SCs ARE
ACTIVE AT ANY GIVEN TIME?
 ES CELLS: WHY ARE THEY UNIQUE AMONG STEM CELLS?
 ES CELLS IN THERAPY: THERAPEUTIC CLONING AND
REGENERATIVE THERAPIES (also major problems and potential
solutions)
 Working with a limited subset of ES cell lines: pros and cons
 How many mutations does it take for the development of cancer?
o Knudson’s Two-hit Hypothesis
o Multi-hit Model
 Cancer Stem Cells
o How are stem cells and tumor cells related?
o Why is self-renewal a key concept to understand?
o How can cancer arise from normal stem cells?
o What are cancer stem cells?
o What evidence is there for cancer stem cells?
o How does the concept of cancer stem cells affect current therapeutic
strategies?
 Studying human cells in animal models: the NOD-SCID mouse
 Problems with the NOD-scid model
DEFINITION OF STEM CELL: THE HEMATOPOIETIC PARADIGM
 A minority population of undifferentiated precursor cells which give rise to
the mature differentiated cells of the tissue
Properties of Stem Cells:
1. they are capable of self-renewing
2. they can give rise to specialized cell types (multipotency)
HAEMATOPOIETIC STEM CELLS AND PROGENITORS
HSCs are typically:
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Small quiescent cells.
Found primarily in bone marrow, but are also present in peripheral blood,
umbilical cord blood and in low numbers in the liver and spleen.
Generate multiple haematopoietic lineages through a successive series of
intermediate progenitors. These include common lymphoid progenitors (CLPs),
which can generate only B, T and Natural Killer cells, and common myeloid
progenitors (CMPs), which can generate only red cells, platelets, granulocytes and
monocytes. Thus all of the mature blood cells in the body are generated from a
relatively small number of HSCs and progenitors.
HSCs from bone marrow have capacity of long-term repopulation of
hematopoietic system of irradiated transplant recipients.
ARE HSCs A GOOD PARADIGM FOR OTHER ADULT STEM CELLS? NO!
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The hematopoietic stem cell, is one of the best-characterized stem cells in the
body and the only stem cell that is clinically applied in the treatment of diseases
such as breast cancer, leukemias, and congenital immunodeficiencies.
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Besides bone marrow, SC are present in several different tissues throughout the
body: skin , gut, brain, liver…All these SC models have different properties due
to different microenvironment, function and specific requirements of the tissue
where they reside. For example, brain, gut and bone marrow SC give rise to
multiple highly specialized cell types, but skin SC don`t, brain SC do not replicate
often (4/day), whereas bone marrow SC give rise to billion cells per day in order
to fulfill high cell turnover…
Prospective Isolation: its advantages and disadvantages
 Cells express distinct assortments of molecules on their cell surfaces, commonly
known as CD proteins (for cluster of differentiation). Many of these proteins
reflect either different stages of the lineage-specific differentiation of the cell or
different states of activation or inactivation. These cell surface markers can be
used to distinguish different types of cells.
Proposed cell-surface markers of undifferentiated HSCs – as cells begin to
differentiate into distinct cell lineages, the cell surface markers can no longer be
identified:
mouse
CD34 low/SCA-1 +
Thy +/low
CD38 +
C-kit +
Lin -
human
CD34 +
CD59 +
Thy +
CD38 low/C-kit -/low
Lin -
Prospective Isolation
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technique to separate certain types of cells based on the surface proteins they
display
uses monoclonal antibodies against particular markers to purify a certain type of
cell from a population of cells in a sample
it is common to purify by either positive or negative selection, usually with a
magnet:
o in positive selection, incubate cells with an antibody against the cell type you
want to purify, then add a magnetic particle that will bind to the antibody.
When you place the cells in the magnet, cells that will stick to the magnet are
those that are bound to the antibody and particle. All other cells can be
washed away.
o in negative selection, the cells are incubated with a cocktail that contains
antibodies against all of the cell types in the sample except the one you want
to purify. Cells are incubated with a magnetic particle, which binds to all
antibodies. When placed in a magnet, all cells except those that you want will
stick to the magnet. You can then pour out the cells you want to keep.
purified cells are then stained with an antibody conjugated to a fluorescent
stain such as PE or FITC, and then run through a fluorescence activated cell
sorter (FACS), which uses a laser to sort the cells
dense-medium cell separation is also a common method of prospective isolation
that also uses antibodies, but uses centrifugation over a dense medium rather than
a magnet
Advantages
 relatively quick and easy process to end up with a cell population that is highly
purified
 can select for more than one type of marker at one time by combining separation
with FACS
 FACS also allows you to determine the purity of your sample
 lots of kits available so little trouble-shooting is required
Disadvantages
 the groups of cells sorted by surface markers are heterogeneous and include some
cells that are true, long-term self-renewing stem cells, some shorter-term
progenitors and some non stem cells – non of the markers are tied to unique stem
cell functions or truly define the stem cell - functional assay is absolutely
necessary
 purification is only as good as the antibodies used in the process. Many
antibodies exist that do not detect their targets well – this could lead to low
purities after separation.
 some antibodies recognize more than one distinct cell surface structure on cells
that have structurally heterogeneous splice variants. Different variants may have
identical protein domains, and antibodies recognizing these shared domains could
cross-react with more than one cell variant
KINETICS OF STEM CELL ACTIVITY in vivo – HOW MANY SCs ARE
ACTIVE AT ANY GIVEN TIME?
A hematopoietic stem cell can replicate (self-renew to maintain the HSC pool),
differentiate or die. Once HSC commits to a differentiation pathway, it gives rise to a
clone that contributes to hematopoiesis until exhaustion. The fate of an individual HCS
dependes on the chance that it interacts with specific accessory cells in the marrow
microenvironment, the presence or quantity of cytokines it uniquely encounters, its cellsurface expression receptors and the integrity of its signal-transduction pathways.
However, commitment and self-renewal decisions are made by single HSCs and thus
require examination on the clonal level. Clonal approaches, such as injecting limited
numbers of purified or retrovirally marked HSCs, revealed extensive heterogeneity in the
HSC compartment. HSC heterogeneity can be visualized by the kinetics of repopulation
in transplantation assays: primitive HSCs need several proliferation and differentiation
steps until they give rise to mature progeny. This causes a delay in the appearance of
mature progeny in the periphery. However, the large clone size generated through the
multiple steps leads to long-term, sustained repopulation. In contrast, less primitive HSCs
require fewer steps, generating small clones. These less primitive HSCs contribute early
to peripheral hematopoiesis, but their contribution declines over time. Primitive and less
primitive HSCs can be separated to some extent, demonstrating that these different
functions are derived from distinct subsets of HSCs. When hosts receive mixtures of
several types of HSCs, the kinetics of repopulation will be a combination of the behaviors
of the individual HSCs.
ES CELLS: WHY ARE THEY UNIQUE AMONG STEM CELLS?
Mouse embryonic stem (ES) cells are undifferentiated pluripotent cells derived from
early mouse embryos. They exhibit two important properties:
o the ability to self-renew and give rise to new pluripotent ES cells, and
o the ability to differentiate into all specialized cell types found in the adult mouse.
ES cells are isolated from the inner cell mass of pre-implantation embryos or
blastocysts at day 3.5 of mouse development. These cells are considered pluripotent as
they can be maintained (by co-culture on irradiated mouse fibroblasts or in the presence
of leukemia inhibitory factor – LIF) indefinitely in the undifferentiated state in culture
and when injected back into a mouse blastocyst, have the ability to contribute to all of the
tissues, including the germ cells of the mouse.
Upon withdrawal of LIF and stromal contact, mouse ES cells will spontaneously
differentiate into complex, three dimensional cell aggregates called embryoid bodies
(EBs). Differentiation within EBs occurs in a well-defined temporal manner with the
initial formation of all three embryonic germ layers (ectoderm, mesoderm, endoderm)
followed by further differentiation to terminally differentiated cell types, similar to in
vivo embryogenesis.
ES CELLS IN THERAPY: THERAPEUTIC CLONING AND REGENERATIVE
THERAPIES (also major problems and potential solutions)
Human ES cells represent great source of cells that could potentially be used for
transplantation therapy. Since ES cell lines are immortal and pluripotent and can be
generated readily from human preimplantation embryos, they provide a renewable
source of any type of body cell. Thus, they can treat wide range of severe debilitating
disease whose underlying pathology involves cell degeneration, death or acute injury.
This concept was used to successfully treat mouse models of diabetes, Parkinson’s
disease, myocardial infarction, spinal injury and severe genetic immune disorder.
However, there are significant challenges to be overcome before these approaches can be
applied in the clinic: to generate sufficient numbers of the desired cell type in a pure
form, to understand what cell type to supply in order to correct a specific pathology, how
to deliver it and how to escape rejection by the host immune system. A number of
solutions to this have been considered: from creation of large SC banks representing a
wide array of histocompatibility backgrounds to therapeutic cloning – a procedure that
combines cloning by somatic cell nuclear transfer with ES technology, to create SCs that
are a custom match to a patients own cells. Practical difficulties with therapeutic cloning
include ethical and safety issues related to wide variety of developmental defects in
coned animals. However, there is evidence indicating that adult bone marrow stem cells
might have properties similar to those of their embryonic counterparts defined as adult
SC plasticity (the ability of a cell committed to a particular cell lineage to, under certain
micro-environmental conditions, acquire the ability to differentiate into cells of a
different tissue). This offers even greater potential (in contrast to ES cells, adult SCs
could be used as autologous grafts) for using SCs in treating degenerative or genetic
disorders of different organs, as well as in tissue repair and regeneration. It’s likely to
assume that in situ mobilization of SCs from the bone marrow and their migration to
various tissues is a normal physiological process of regeneration and repair, so that
therapeutic benefits can be generated with less invasive regimens than the removal an reinjection of SCs, through the stimulation of normal SC migration.
Working with a limited subset of ES cell lines: pros and cons
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a limited number of ES cell lines are available due to political and ethical
opposition to their use in research
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the one of the biggest obstacles in nuclear cloning using ES cells is the low
frequency of viable clones (most either die during gestation or soon after birth).
factors affecting cloning efficiency are related to the ES cells used in the
procedure; they include the following issues:
o genetic background of the ES cells – inbred donor ES cells have decreased
survival rates
o loss of imprinting during culture over long periods (as below)
o passage number – high-passage ES cells are related to a progressive loss of
epigenetic markers associated with imprinted genes
o accumulated genetic damage of the donor cells could occur over long periods
of culture
o ability of the oocyte to epigenetically reprogram the donor cell nucleus affects
cloning efficiency
o ability of ES cells to generate viable cloned offspring might be correlated with
their degree of polymorphism at different markers
o initial development of clones to the blastocyst stage may be dependent on the
compatibility between the cell cycles of the donor nucleus and the oocyte
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having access to only a few lines of ES cells influences most of these factors
(cons)
o research is restricted to limited genetic material (in the case the ES cell nuclei
are used in donor somatic cells), and therefore ES cells may not be
representative of the general population
o ES cell lines have likely been passaged many, many times, so there could be
loss of imprinting
o since ES cells have high plasticity, culture over long periods could cause other
cellular changes as the ES cells adapt to the culture environment
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on the other hand, working with a limited number of ES cell lines quiets the
debate over the use of ES cells somewhat
this debate centers on the following themes:
o some individuals view the human preimplantation embryo as a person or
subject with rights and interests – they believe that that the intentional
destruction of an embryo is equivalent to murder
o this view conflicts with the widely held philosophical and moral view that
holds that status as a person or as an entity with interests requires, at the very
least, a nervous system capable of sentience, if not also of cognition and
consciousness
o many people reject the view that the embryo is a person but believe that the
embryo is different from ordinary human tissue because of the unique
potential it has to develop into a new human being. This attitude towards
human embryos shows our respect for human life generally. For example,
destroying embryos that are left over from IVF procedures to develop cellreplacement therapies should be ethically acceptable because the goal of
treating disease and saving life justifies the symbolic loss that arises from
destroying embryos in process. By contrast, selling human embryos or using
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them in cosmetic-toxicology testing seems to be disrespectful of the symbolic
meaning that many people attach to embryos.
How many mutations does it take for the development of cancer?
Knudson’s Two-hit Hypothesis
Alfred Knudson suggested that two mutations or “hits” were sufficient for the
development of a retinoblastoma and that the inheritance of one of these mutations could
account for the earlier onset and frequent bilateral occurrence of the hereditary form of
this tumor.
Thus, in hereditary retinoblastoma, individuals begin life with a constitutional mutation
that inactivates one allele of the RB1 gene. The second “hit” occurs somatically and
usually involves loss of all or part of the chromosome containing the normal RB1 allele.
Multi-hit Model:
more Recent model.
Here, neoplastic development is attributed to the expansion of a clone of cells that have
accumulated 3-7 somatic mutations. This may be benign or malignant, depending on its
ability to metastasize. This model is based on progressive selection:
1. The 1st mutation in a somatic cell confers a slight growth advantage to its
progeny.
2. One of the progeny acquires a further proliferative advantage through a 2nd
mutation.
3. One of its cellular progeny acquires a 3rd mutation, possibly forming a benign
tumor.
4. Subsequent mutations in the descendants of these cells may permit them to escape
apoptosis or other growth control mechanism or to become genetically unstable,
increasing the likelihood for a tumor to progress. Examples of genes that normally
control cell cycle and cell death include ATM and p53. The gene products of
these genes are believed to play a major role in maintaining the integrity of the
genome such that alterations in these gene products may contribute to increased
incidence of genomic changes such as deletions, translocations and
amplifications, which are common during oncogenesis.
Cancer Stem Cells.
How are stem cells and tumor cells related?
1. similar mechanisms regulate self-renewal
2. tumor cells might arise from normal stem cells
3. tumors might contain “cancer stem cells” - rare cells with indefinite proliferative
potential that drive the formation and growth of tumors.
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Most research has focused on hematopoeitic system because both stem cells and
cancer cells are well characterized for this system.
Why is self-renewal a key concept to understand?
 crucial to stem cell function because many stem cells must survive for the entire
life of the organism.
 Cancer cell proliferation could be caused by unregulated self-renewal.
How can cancer arise from normal stem cells?
1) Signaling pathways that normally regulate stem cell self-renewal are disregulated,
leading to tumorigenesis.
 newly arising cancer cells appropriate the machinery for self-renewal that is
normally only expressed in stem cells.
 Many pathways classically associated with cancer may also be involved in
regulating normal stem cell development.
o eg. bcl-2, Notch, Sonic hedgehog, and Wnt are all implicated in both
oncogenesis and stem cell renewal. These genes are likely necessary for stem
cell self-renewal but if they become disregulated can lead to cancer.
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Stem cells themselves are target of transformation.
 because stem cells already have self-renewal machinery activated, it might be
simpler (ie. Require less mutations) to maintain self-renewal than activate it
ectopically in a more differentiated cell.
 Because stem cells are self-renewing they often persist for long time periods and
thus are more likely to accumulate mutations than a short-lived progenitor cell or
differentiated cell.
 A progenitor cell could inherit some mutations from a stem cell and then suffer
the last mutation to cause transformation.
 A variety of leukemia studies have indicated that the cancer may arise from
mutations that occur in HSCs or their progeny.
What are cancer stem cells?
 Cancer research has focused on the effects of particular mutations on proliferation
of model cells. But, we know little about the effects of these mutations on the
actual cells involved in the cancer.
 A tumor can be viewed as an aberrant organ initiated by a tumorigenic cell that
acquired the capacity for indefinite proliferation through accumulated mutations.
 From this viewpoint, normal stem cell biology can be applied to the tumor.
 Both normal stem cells and tumorigenic cells have extensive proliferative
potential, and give rise to phenotypically heterogeneous cells that exhibit various
degrees of differentiation.
 Tumorigenic cells can be thought of as cancer stem cells that undergo aberrant
and poorly regulated organogenesis in a similar process to normal stem cells.
What evidence is there for cancer stem cells?
 only a small subset of cancer cells are capable of extensive proliferation.
 It has been shown with AML (acute myeloid leukemia) cells that only a small,
predictable subset are consistently enriched for the ability to proliferate and
transfer disease.
How does the concept of cancer stem cells affect current therapeutic strategies?
 Understanding the molecular basis of the unregulated self-renewal of cancer cells
will allow the design of more effective therapies.
o Presently, all of the phenotypically diverse cancer cells are treated as though
they have unlimited proliferative potential and can acquire the ability to
metastatize. If cancer stem cells exist, then the targeted elimination of only
these cells might be required to destroy the cancer.
o We could isolate prospectively these cancer cells and identify diagnostic
markers and therapeutic targets expressed by the stem cells.
 The existence of cancer stem cells could explain the ineffectiveness of many
therapies which shrink metastatic tumors but do not completely kill them. If a
cancer stem cell is missed the tumor will simply grow again.
o Cancer stem cells could be naturally more resistant to chemotherapies.
Normal stem cells seem to be. This could be due to the higher expression of
anti-apoptotic genes.
o Thus, therapies that are more specifically directed at cancer stem cells could
be much more effective.
 Gene expression studies could focus on cancer stem cells to categorize cancer
rather than using samples containing a mixture of normal cells, highly prolierative
cells, and cells with low proliferation.
o This composite profile could obscure important differences between tumors
because the cells actually driving the tumorigenesis are in the minority.
o Purification of cancer stem cells will allow “sharper” gene expression profiles
and more efficient identification of new therapeutic and diagnostic targets.
Studying human cells in animal models: the NOD-SCID mouse
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the true functional measure of a long-term renewable stem cell is the capacity to
engraft myleoablated recipients, repopulate their hemopoietic systems, and sustain
long-term multi-lineage hemopoiesis in vivo
in 1988, the first reports of the engraftment of human cells into homozygous scid
mice appeared – these reports included engraftment of human peripheral blood
mononuclear cells following intraperitoneal injection into unirradiated recipients
and transplantation of fetal bone marrow and thymus fragments under the renal
capsule
the catalytic subunit of a DNA-dependent protein kinase encoded by a gene
termed “protein kinase DNA activated catalytic polypeptide” (Prkdc) has been
identified as the gene disrupted by the scid allele – the allele contains a nonsense
mutation which causes the insertion of a termination codon
mice homozygous for the scid mutation lack both humoral and cell-mediated
immunity due to the absence of mature T or B lymphocytes – this results in their
inability to express rearranged antigen receptors
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the term “scid-repopulating cell” (SRC) has been coined to describe the human
stem cell that engrafts in irradiated scid mice following intravenous injection
the SCID mouse model has been used to study human stem cell phenotypes and
differentiation, human gene therapy protocols, human T-cell function and
differentiation, and the homing of myelona cells to bone marrow
Limitations of the SCID mouse model:
o Engraftment levels of human cells are low, representing only 0.5%-5% of the
total scid recipient marrow population – it is thought that this could be due to
NK cell activity
o Human stem cells cannot utilize many of the murine hemopoietic growth
factors produced by their hosts due to the lack of species cross-reactivity of
these cytokines – this results in a cytokine-deficient environment that fails to
support human stem cell proliferation and differentiation
More recently, the NOD-scid mouse was investigated as a model
Levels of human peripheral blood mononuclear cell (PBMC) engraftment in
NOD-scid mice were always 5- to 10-fold higher than in any of the other genetic
stocks of scid mice examined
The NOD mouse is an animal model of spontaneous autoimmune T-cell-mediated
insulin-dependent diabetes mellitus
Inbred NOD strains have multiple defects in innate immunity: they have low NK
cell activity, display defects in myeloid development and function and lack the C5
component of complement so that they cannot generate either the classical or
alternative pathways of haemolytic complement activation
The NOD-scid mouse model has the characteristics of the individual strains;
however, NOD-scid mice lack an adaptive immune system, so due to the absence
of T cells, they do not develop autoimmune IDDM and remain insulitis and
diabetes-free throughout life
Less then 10% of NOD-scid mice develop detectable levels of circulating
immunoglobulin due to “leakiness”
Additional characteristics of NOD-scid mice that may relate to their enhanced
ability to be engrafted with human hemopoietic cells are:
o Their approximate two-fold reduction in bone marrow cell counts compared to
the NOD strain – this may increase the available “niches” in the marrow for
human stem cells
o They have a slight reduction in erythrocyte mean cell volumes, which results
in the expression of a mild macrocytic anaemia
Peak engraftment levels tend to be observed 4-8 weeks after transplantation, but
high levels of human cell engraftment in marrow remain detectable for ~4 months
Problems with the NOD-scid model:
o NOD-scid mice have been found to have a mean lifespan of only ~8 months
o they exhibit an unusually high incidence of thymic lymphomas – the
development of thymic lymphomas is imparted by the presence of the scid
mutation, since NOD mice rarely develop thymic lymphomas
o the scid mutation also imparts extreme radiosensitivity to the NOD-scid
mouse, such that as little as 400 rads of gamma-irradiation can cause death
o the microenvironment in which the human stem cell engrafts is currently
unknown, but is likely to be of predominantly murine origin – the lack of
appropriate cell-cell interactions due to non-species cross-reactivity, including
interaction with appropriate adhesion molecules or the lack of human
cytokines, may continue to impede engraftment and development
o although the NOD-scid mouse exhibits multiple defects in innate immunity, it
still displays low NK cell activity, disparate MHC loci and intact cytolytic
mechanisms.