What is a
Stem Cell?
In the popular media and even in some medical
circles, stem cells are presented as miracle cells that can do anything. When
administered to a patient with some serious disease they will rebuild the
damaged tissues and make the patient young again. Alas, in reality there are no
such cells. However, there are cells that exhibit stem cell behavior and the
future of regenerative medicine will undoubtedly be built on a good scientific
understanding of their properties. In this chapter these properties are briefly
outlined, and in the remainder of the book each of them will be underpinned by
an explanation of the relevant areas of science and technology.
A list of characteristics of stem cell behavior that is generally agreed
upon is the following:
●
Stem cells reproduce themselves.
●
Stem cells generate progeny destined to
differentiate into functional cell types.
●
Stem cells persist for a long time.
●
Stem cell behavior is regulated by the immediate
environment (the niche).
This is shown diagrammatically in Figure 1.1. The first two items on the
list indicate the key abilities of self‐renewal and of generation of
differentiated progeny. As will be explained below, these abilities may be
shown at a cell population level rather than by every single stem cell at every
one of its divisions. Also, the second item indicates “destined to differentiate”
meaning that cell division may continue for a while before differentiation, but
not indefinitely. Cells derived from stem cells that proliferate for a limited number of
cycles are called progenitor cells or transit amplifying cells. The third item
on the list means that if the stem cell population is one of those that exists
in tissue culture then it should be capable of indefinite growth, while if it is
part of an organism it should be very long lasting, normally persisting for the
whole life of the organism. The fourth characteristic indicates that all stem
cells exist in a specific micro-environment that controls their program of
division and differentiation. This may seem at first sight only to apply to
stem cells within the body and not to those grown in vitro, but in order to get
them to grow, the cells in vitro are always provided with specialized medium
ingredients that, in effect, mimic the components normally provided in the
niche.
This fourfold definition involves not just intrinsic properties of stem
cells, but also properties that depend on aspects of their environment such as
the lifespan of the animal, the nature of the niche, or the composition of the
culture medium. This emphasizes the fact that the goal of stem cell biology is
understanding the behavior and not just the intrinsic nature of stem
cells. To achieve this, the characteristics of the stem cell environment are
just as important as the properties of the stem cells themselves. Moreover,
understanding stem cell behavior means understanding various aspects of cell
and developmental biology which are not always familiar to workers in stem cell
laboratories.
The above definition is of value in indicating the special
characteristics of stem cell behavior, but is also helpful in indicating what is not stem cell
behavior. For example, most of the cells in the body that are dividing are not
stem cells. In particular cells in the embryo that differentiate after a
certain period of time, such as the earliest cells formed by division of the
fertilized egg, are not stem cells. Nor are differentiated cells that divide
during postnatal life to generate more of themselves, such as hepatocytes or tissue‐resident
macrophages. A common term found in the literature is “stem/progenitor cell”.
This is a singularly unhelpful designation as it conflates two entirely
different cell behaviors. Progenitor cells are precisely those that
differentiate into functional cell types after a finite period of
multiplication. They include the transit amplifying cells that arise from stem
cells (Figure 1.1) and also cells of the embryo and of the growing individual
that are destined to differentiate after a certain time.
Real stem cells comprise two fundamentally different types: pluripotent
stem cells that exist only in vitro, and tissue‐specific stem cells that exist
in vivo in the postnatal organism. Pluripotent stem cells comprise embryonic
stem cells (ESC) and induced pluripotent stem cells (iPSC). There are various subdivisions that will
be considered later, but the essential features of these cells are first that
they can be propagated without limit in vitro, and second that, under appropriate
culture conditions, they are able to give rise to a wide variety of cell types,
perhaps all the cell types in the normal organism except for the
trophectoderm of the placenta. By contrast, tissue‐specific stem cells exist
within the body and generate progeny to repopulate the tissue in question.
Well‐studied tissue‐specific stem cells include those of the hematopoietic
(blood‐forming) system, the epidermis, the intestinal epithelium and the
spermatogonia of the testis. Under normal circumstances, tissue‐specific stem
cells do not produce cells characteristic of other tissue types. There are also
some well‐characterized stem cells that do not undergo continuous division, but
seem to be kept in reserve to deal with tissue regeneration when required. A
good example is the muscle satellite cells, which are normally quiescent but
are able to be mobilized to divide and fuse to form new myofibers following
injury. This type of stem havior is sometimes called facultative.
Many criteria for identifying stem cells have been proposed and used.
These are briefly listed here and the concepts and technologies wil in later
chapters of the book.
Stem Cell
Markers
Very often a cell is said to be a stem cell because it expresses one or
more gene products associated with stem cells. However, there is no molecular
marker that identifies all stem cells and excludes all non‐stem cells. Those
components required for general cell metabolism and cell division are certainly
found in all stem cells, but they are also found in many other cell populations
as well.
Pluripotent stem cells (ESC and iPSC) express an important network of
transcription factors which are necessary for maintenance of the pluripotent
state (see Chapter 6). Transcription factors are the class of proteins that
control the expression of specific genes. A key member of the pluripotency
group is the POU‐domain transcription factor OCT4 (also known as OCT3 and
POU5F1). The presence of OCT4 is certainly necessary for the properties of
pluripotent stem cells. However it is not expressed in any type of
tissue‐specific stem cells except at a low level in spermatogonia.
A component that might be expected to be found in all stem cells is the
telomerase complex. At the end of each chromosome is a structure called the
telomere, made up in vertebrate animals of many repeats of the simple sequence
TTAGGG. Because of the nature of DNA replication, the double helix cannot be
copied right up to the end, so a part of the telomere is lost in each cell
cycle. After enough cycles, the erosion of chromosome ends activates the
system which senses DNA double‐stranded breaks and causes death of the cell.
This process is an important reason for the limited survival time of most
primary tissue culture cell lines, which undergo senescence after a certain
number of population doublings in vitro. Obviously there must be a mechanism for repairing
telomeres in vivo, and this is provided by the telomerase complex, of which the
most important components are an RNA‐dependent DNA polymerase called TERT, and
an RNA called TERC which contains the template CCCTAA for the telomere
sequence. High levels of telomerase are found in germ cells, ensuring the
survival of full length chromosomes for the next generation. Telomerase is also
upregulated in permanent (“trans- formed”) tissue culture cell lines and in
most cancers. However most types of somatic cell have little or no telomerase.
Tissue‐specific stem cells do contain some telomerase; generally enough to
maintain cell division for a normal lifetime, but not enough to fully reverse
the erosion of the telomeres. In situations such as repeated transplantation of
hematopoietic stem cells from one mouse to another, there is an upper limit to
the number of possible transplants and this is determined at least partly by telomere
erosion. The presence of telomerase can be considered to be a stem cell marker,
although it is also found in permanent tissue culture lines, early embryos and
most cancers.
In human or animal tissues, various markers have been advanced as
characteristic of all stem cells. For example the cell surface glycoprotein
CD34 is found on human hematopoietic stem cells (HSCs) and can be used to
enrich them from bone marrow by fluorescence‐activated cell sorting (FACS).
However it is also found on other cell types, such as capillary endothelial
cells, and it is unclear whether it is actually necessary for the stem cell
behavior of the HSC. In fact, since it is not found on mouse HSC, which are
generally similar in behavior to human HSC, it is probable that it is not
necessary. CD34 is not found on human embryonic stem cells or on most
epithelial stem cell types, indicating that it is not a generic stem cell
marker. A molecular marker which is known to be required for stem cell function
is LGR5. This is an accessory receptor for the Wnt family of signaling
molecules (see Chapter 7) and is found on stem cells in the intestine, hair follicle, mammary
gland and stomach. These types of stem cell all depend on Wnt signaling from
their environment for continued cell division, so the presence of the LGR5 is
really necessary. However it is not found on other types of stem cell, so is
also not a universal marker.
An interesting type of marker is that offered by dye exclusion, in
particular exclusion of the Hoechst 33342 dye. This is a bisbenzimide dye,
excited by UV light to emit a blue fluorescence. It is widely used as a
DNA‐binding reagent, but it is also actively pumped out of some cell types. If
a subgroup of cells has lost more dye than the rest of the population, then it
appears in flow cytometry as a cluster of cells showing less blue fluorescence
than average. This is called a side population. The side population is enriched
for stem cells in some situations, especially in murine bone marrow where it
provides a similar degree of enrichment of hematopoietic stem cells to FACS
using a panel of cell surface markers (Figure 1.2). The dye exclusion property
is due to the activity of cell membrane transporter molecules including the
P‐glycoprotein (MDR1) and transporters of the ABC class. Dye exclusion is
indicative of an increased capacity for export of all hydrophobic small molecules, many of which are
toxic to cells. Although useful to the investigator, it is unlikely that this
capacity is really important for stem cell behavior. For example, mouse
embryonic stem cells show dye exclusion while human embryonic stem cells do
not.
In summary, there is no single gene product which is found in all stem
cells and not in any non‐stem cell. Many so‐called stem cell markers are probably
not necessary for stem cell behavior. Of those gene products which are
necessary for stem cell behavior, some, such as the cell division machinery and
telomerase, are found in stem cells and in some non‐stem cells. Others, such as
OCT4 or LGR5, are but not all, types of stem cell.
Label‐Retention
When a cell population is exposed to a DNA precursor, such as the
nucleoside bromode- oxyuridine (BrdU), which is metabolized by cells in the
same way as thymidine, all cells undergoing DNA synthesis will incorporate it
into their DNA and so become labeled. The BrdU in the cell nuclei can be
detected by immunostaining. After the BrdU supply is withdrawn, so long as cell
division is continuing, then the level of BrdU in the DNA will halve with every
subsequent S phase and become undetectable to immunostaining after about six
divisions. If a cell divides slower than average, it will retain detectable
BrdU for longer. This label‐retaining behavior is often considered to be a
characteristic of stem cells. In Figure 1.C.1 is shown an image of a
hematopoietic stem cell (HSC) visualized with an antibody to the cell surface
marker CD150. It retains a DNA precursor (EdU) label from a pulse given 30 days
previously. Likewise, muscle satellite cells, that enable muscle regeneration
following damage, are usually in a quiescent state. This relatively quiescent
behavior is considered necessary to maintain regenerative function of some
types of stem cell over a lifetime. If the mechanisms of quiescence are disturbed in mice by knocking
out key components, then hematopoietic stem cells or muscle satellite cell
populations have been shown to become exhausted during the lifetime of the
animal, because they are dividing too much. Relative quiescence also serves to
protect the stem cells against the oxidative damage which results from continuous
growth with its associated oxidative metabolism.
Slow division is the cause of label retention in stem cells, but it must
be remembered that not all label retention is due to stem cell behavior. In
particular differentiation to a completely non‐dividing (post‐mitotic) cell
type leads to permanent label retention. This property has been used especially
to establish the differentiation time of neurons in embryonic development, and
the final mitosis is often referred to as the cell birthday. Moreover, label
retention is by no means universal among stem cells. For example, it is not
shown by intestinal or epidermal stem cells. It is also, of course, not shown
by the pluripotent stem cells (ES or iPS c hich undergo rapid division in
culture.
The Niche
The concept of a stem cell niche arose in the 1970s to explain the fact
that the spleen colony‐forming cells from the bone marrow had a lesser
differentiation potency than hematopoietic stem cells in vivo (see Chapter 10).
The idea is that stem cells require continuous exposure to signals from
surrounding cells in order to maintain their stem cell behavior. This was first
proved experimentally using the fruit fly Drosophila. In the Drosophila
ovary there are female germ cells called cystoblasts (Figure 1.3). These
lie in contact with somatic cells called cap cells, which secrete a TGFĪ²‐like
molecule called Decapentaplegic (Dpp). Dpp maintains the cystoblasts in
mitosis. But as they divide, some of the cysto- blast progeny become displaced
from contact with the cap cells, and are then exposed to less of the Dpp. This
fall in Dpp lifts a repression
on the oocyte maturation process and enables the cystoblast to differentiate.
It then undergoes a fixed differentiation program, dividing four times to
generate a post‐mitotic complex of one oocyte and 15 supporting nurse cells.
This illustrates the behavior of a niche very nicely. The cystoblasts
continue to divide so long as they are in contact with the niche, and they
differentiate when they are no longer in contact. If a cystoblast is removed
experimentally, its position may be taken by a progeny cell which would
normally have differentiated, but because of its renewed occupancy of the niche
it remains a dividing cystoblast.
Probably all the stem cells types in the mammalian body exist within
specific niches like this which control their behavior. For example the
intestinal stem cells lie adjacent to Paneth cells which supply WNT, and
spermatogonial stem cells lie adjacent to Sertoli cells that supply them with
glial derived neurotrophic factor (GDNF). In both cases the signaling molecules
are needed to maintain the stem cells in mitosis, and removal from the niche
brings an end to cell division unless the factors are provided experimentally.
In the bone marrow,
there has been controversy about the exact nature of the niche, but
hematopoietic stem cells are often found adjac blood vessels, as shown in
Figure 1.C.1.
Asymmetric Division
and Differentiated Progeny
It is often thought that all stem cells must undergo asymmetric
divisions, with one daughter being a stem cell and the other des- tined to
differentiate. This does sometimes occur, but it is also possible for stem
cells to have a less rigid program of cell division with some divisions
producing two stem cells, some two progenitor cells, and some producing one of
each. Statistically a steady state requires that the stem cell number remains
constant, although there may be occasions where it needs expanding, such as
during normal growth of the organism or following injury. In the intestine for
example it has been shown by cell labeling and by direct visualization that
symmetric divisions predominate (see Chapter 10).
By definition, stem cells must produce differentiated progeny, but how
many differentiated cell types do they actually produce? The answer is very variable
and depends on the tissue concerned. In the intestine, stem cells produce
absorptive, goblet, tuft and Paneth cells, together with several types of
enteroendocrine cells. In the bone marrow, the hematopoietic stem cells produce
all the cell types of the blood and immune system. At the other end of the
scale, the spermatogonia of the testis produce only sperm. Epidermal stem cells
are often said to produce only keratinocytes, but they can also form a type of
neuroendocrine cell called the Merkel cell, responsible for touch sensitivity.
The examples of both the intestine and the epidermis indicate that
neuroendocrine cells can arise from epithelial stem cells quite distinct from
the central or peripheral nervous systems, but they are not indicative of a wider
potency enabling other tissue types to be formed.
Around the year 2000 there was a rash of papers indicating that
hematopoietic stem cells
(HSCs) were able to repopulate many, or perhaps all, other tissue types in the
body following transplantation. This phenomenon was known as
“transdifferentiation” (a term more usefully reserved for changes of differentiation
type between fully differentiated cells). However further investigation showed
that the phenomenon could mostly be explained by donor cells lodging within
other tissues but not actually differentiating into them, or by cell fusion
with cells of other tissues. Unfortunately the idea of very wide plasticity of
tissue‐specific stem cells became established in many people’s minds at this time
and has helped promote the present worldwide industry of “stem cell therapy”
much of which has no scientific rationale or real clinical effectiveness.
Clonogenicity and
Transplantation
It is often supposed that stem cells are those which grow rapidly and can
form large clones in vitro. This perception came from early studies on
epidermal stem cells, where the proportion of such cells (holoclones) does
correlate well with the estimated proportion of stem cells in the basal layer
(Figure 1.4). Sometimes, “spheres”, such as neurospheres or mammospheres, which
contain both stem cells and their differentiated progeny, can be grown from
tissue samples in suspension. However, stem cell behavior depends both on the
cells and their environment, and it is well‐known that cell behavior can be
greatly changed by the environment of in vitro culture. For example,
neurospheres can be grown from parts of the central nervous system in which
there are no stem cells in vivo. As another well‐known example, cells of the
mammalian embryo epiblast, which rapidly develop into other cell types in vivo,
can give rise to pluripotent embryonic stem cells in vitro, which continue to
divide indefinitely in an appropriate medium.
Transplantation behavior also looms large in thinking about stem cells.
The ability to rescue
irradiated animals with bone marrow transplants was the original discovery
that, decades later, led to the identification of hematopoietic stem cells. It
is generally felt that a hematopoietic stem cell is defined by the ability to
repopulate the entire blood and immune system of an irradiated host. This is
certainly an important property, although a single‐minded focus upon it has
tended to obscure the important distinction between cell behavior in an extreme
regeneration situation and that in normal homeostasis. Some authors even
suggest that a cell is not a bona fide stem cell unless it, as a single cell,
can repopulate an entire tissue following transplantation. While this has
been done a few times and may be a theoretical possibility for all stem cell systems,
there are always practical limits to transplantation. All adult vertebrate
animals have highly sophisticated immune systems that, as a by‐product to their
role in defending against infection, cause the rejection of cell and tissue
grafts from other individuals. This is a very complex subject, but in general
grafting between adults is
only possible between genetically identical individuals (e.g. identical twins
or inbred mouse strains), or following immuno- suppression with drugs, or by
using highly immunodeficient strains of animal as hosts.
A type of stem cell defined almost entirely by transplantation is the
so‐called cancer stem cell. These are subsets of cells from tumors, isolated
using various stem cell markers, which will generate tumors in immunodeficient
hosts following grafting, under conditions where the majority of cells from the
same tumor do not. Cancer stem cells are discussed in Chapter 11.
In Vivo Lineage
Labeling
This is the most reliable method for establishing the existence of stem
cell behavior in vivo because it can provide direct visualization. So far it
has only been widely used in mice, but the wide availability of CRISPR‐ Cas9
technology will soon make it available for other organisms as well. The
principle is to use a DNA recombinase enzyme (Cre) to impart a permanent
genetic label to a cell in vivo that expresses a particular gene, or, more
precisely, has a particular promoter highly active. The label is subsequently
heritable on cell division and is unaffected by any differentiation events
occurring in the progeny cells. A modification of the Cre recombinase to make
it activatable by estrogen‐like hormones (CreER) has been widely used in mice
and enables the labeling to be initiated at a specific time. This method is
described in Chapter 3. Once it has been labeled a stem cell will produce a
sector of labeled tissue in which all its dividing and differentiated progeny
carry the label. The labeled sector will grow initially as cells divide and
mature and will eventually reach a steady state in which addition of new
labeled cells is balanced by the removal of dead ones. This pattern should then
remain unchanged in the long term. An example is shown in Figure 1.5, showing
intestinal stem cells labeled using the Lgr5 promoter. These cells
reside in the intestinal
crypts and generate a file of cells up the crypt and onto the neighboring
villus. Near the villus tip the cells die and are then lost into the intestinal
lumen. Some other examples of stem cell labeling are shown in Figures
10.C.1–10.C.6.
Because it can provide visualization of individual stem cell domains,
cell lineage labeling has provided the data for an influential model of stem
cell behavior which may be called stochastic. Here the idea is that the stem
cells have a certain chance of dividing to form two stem cells, two transit
amplifying cells or one of each (Figure 1.6). If the out-come is 50% of new
stem cells and 50% of transit amplifying cells, then this gives quantitatively
the same outcome as a situation of obligatory asymmetric division in which
every division yields one stem cell daughter and one transit amplifying
daughter. However it gives different predictions about the behavior of labeled
stem cell clones. In the situation of the obligatory asymmetric division,
labeled stem cell clones will each comprise one stem cell plus all their
descendants. When the steady state has been reached, the labeled clones should
persist for life and stay the same size. However in the stochastic model, clones may be
lost if their stem cell divides to form two transit amplifying cells. They may
also increase in size if the stem cell divides to form two stem cells. This
situation has been modeled mathematically and it predicts that the number of
labeled clones should steadily decline with time while the size of clones
should become progressively more disparate, with the average size increasing.
This means that the proportion of the tissue occupied by the labeled clones
remains constant, but the number of labeled clones becomes progressively fewer
and their size more varied. In fact this behavior is precisely what is observed
when lineage labeling data are analyzed quantitatively, at least for the
epidermis, spermatogonia and intestinal epithelium. In particular there is a
property called “scaling behavior” which means that the frequency distribution
of labeled clone sizes, divided by the average clone size, stays the same over
time. Under such circumstances, which may turn out to be the norm for mammalian
stem cell systems, the key stem cell properties of self‐renewal, persistence
and differentiation are still maintained, but they exist at a cell population
leve her than as the properties of a single cell.
