Module 3

Cell Differentiation and Stem Cells

Learning objectives:
At the end of the module, you are expected to :
1. Understand the different processes involved with the control of gene
expression
2. Learn how different models of cell differentiation and stem cells
3. Comprehend the plasticity of the differentiated cell state

Embryonic cells start out morphologically similar to each other but eventually
become different, acquiring distinct identities and specialized functions. As such, many
developmental processes like the early specification of germ layers, involve transient
changes in cell form, patterns of gene activity, and protein synthesis. Subsequently, the
process of cell differentiation involves the gradual emergence of cell types that have a
clear-cut identity in the adult, such as nerve cells, red blood cells, and fat cells. Initially,
embryonic cells fated to become different cell types and can be distinguished from each
other only by small differences in patterns of gene expression particularly on proteins
produce. Differentiation is a progressive restriction in fates that occurs over successive
cell generations as cells gradually acquire special structural features associated with
their specialized functions.

3.1. The Control of Gene Expression

Every nucleus in the body of a multicellular organisms is derived from the single
zygotic nucleus in the fertilized egg and thus carries the same genome, which in
humans comprises around 20,000 distinct genes. But the subsets of these that are
active in differentiated cells vary enormously from one cell type to another. The egg
itself has a pattern of gene activity that is different from that of the embryonic cells to
which it give rise. So how do distinct and unique combinations of genes come to be
expressed in each different cell type? Although there are only a few hundred
morphologically recognizable basic mammalian cell types, there are in fact millions of
functionally distinct cells in the mammalian body, each with its particular pattern of gene
activity. As such, there are fewer genes in the genome than types of differentiated cell
that need to be specified, and therefore each distinct cell type must be defined by the
expression of a combination of genes rather than by a single gene (Figure 3-1). This
raises the question of how the particular pattern of gene activity in a differentiated cell is
specified and how it is inherited.
Figure 3-1. Different cell types are the result of the expression of different combinations
of genes. To make the most efficient use of the relatively modest numbers of genes in
the genome, different functional cell types are specified by the expression of different
combinations of genes. This enables many more cell types to be specified than if each
gene was only able to uniquely specify one cell types. This hypothetical example shows
how a set of three genes (X, Y, and Z) could specify eight different cell types by the
unique combinations of those genes that are active (red bars) and inactive (gray bars) in
each cell type.

We can define differential gene expression as the process by which cells

become different from one another based upon the unique combination of genes that
are active or “expressed.” By expressing different genes, cells can create different
proteins that lead to the differentiation of different cell types. There are three
postulates of differential gene expression:
1. Every somatic cell nucleus of an organism contains the complete genome
established in the fertilized egg. In molecular terms, the DNA of all differentiated
cells are identical.
2. The unused genes in differentiated cells are neither destroyed nor mutated; they
retain the potential for being expressed.
3. Only a small percentage of the genome is expressed in each cell, and a portion of
the RNA synthesized in each cell is specific for that cell type.

To understand the molecular basis of cell differentiation, we first need to know how a
gene can be expressed in a cell-specific manner (Figure 3-2).
Figure 3-2. The central dogma of biology.
A simplified schematic of the key steps in
the process of gene and protein
expression. (1) Transcription. In the
nucleus, a region of the genomic DNA is
seen accessible to a RNA polymerase,
which transcribes an exact
complementary copy of the gene in the
form of a single-stranded nuclear RNA
molecule. The gene is now said to be
“expressed.” (2) Processing. The nRNA
transcript undergoes processing to make
a finalized messenger RNA strand, which
is transported out of the nucleus (3). (4)
Translation. mRNA complexes with a
ribosome, and its information is translated
into an ordered polymer of amino acids.
(5) Protein folding and modification.
This polypeptide adopts secondary and
tertiary structures through proper folding
and potential modifications (such as the
addition of a carbohydrate group as seen
here). (6) Carry out function. The
protein is now said to be “expressed” and
can carry out its specific function (such as
functioning as a transmembrane receptor).

3.1.1 Control of Transcription involves both general and tissue-specific

transcriptional regulators
Most of the key genes in development are initially in an inactive state and require
activating transcription factors, or activators, to turn them on. These activators bind to
sites in the cis-regulatory regions in the DNA surrounding the gene (the “cis” simple
refers to the fact that the regulatory region is on the same DNA molecules as the gene it
controls). The site to which an individual transcription factor binds is a short-stretch of
nucleotides with a sequence specifically recognized by the protein. Sites to which
transcription factors bind to switch on a gene are often clustered within regulatory
regions called enhancers. The importance of of regulatory regions in tissue-specific
gene expression was clearly demonstrated by which the control region of one tissue
specific gene was replaced by the control region of another (Figure 3-3.)
In eukaryotic cells, most regulated protein-coding genes are transcribed by RNA
polymerase II. The polymerase binds to a control region in the gene called the
promoter, where it is positioned so that it can start transcription in the correct place.
The binding of RNA polymerase II to the promoter requires the cooperation of a set of
Figure 3-3. Tissue-specific gene expression is controlled by regulatory regions. Growth
hormone is normally made in the pituitary and the enzyme elastase I only in the
pancreas. To demonstrate that the regulatory regions of a gene determine its tissue-
specific expression, the control region of the mouse elastase I gene was joined to a
DNA sequence coding for human growth hormone. This DNA construct was injected
into the nucleus of a fertilized mouse egg, where it becomes integrated into the genome.
When the mouse develops, human growth hormones is made in the pancreas, A 213-
base-pair stretch of DNA that includes the elastase I promoter and other regulatory
regions is sufficient to drive growth hormone expression in the pancreas.

general transcription factors, so called because they are required by the genes
transcribed by RNA polymerase II. They form a transcription initiation complex with the
polymerase at the promoter. For highly regulated genes, such as those involved in
development, this complex cannot be formed and activated without additional help. This
is provided by the binding of regulatory regions of the gene, which help to attract the
general transcription factors and polymerase thus, position them correctly on the
promoter (Figure 3-4). Some regulatory sites, including some enhancers, are
alternatively bound by repressors, gene-regulatory proteins that prevent a gene being
expressed.
Figure 3-4. Gene expression is regulated by the coordinated action of gene-regulatory
proteins that bind to regulatory regions in DNA. Most protein-coding genes in eukaryotic
cells, including all highly regulated developmental genes, are transcribed by RNA
polymerase II. The transcriptional machinery itself, composed of the polymerase and its
associated general transcriptional factors (the core transcriptional machinery; for
example TFIID and the Mediator complex), recognizes the promoter region by binding
to specific sequences within it that are common to all genes transcribed by RNA
polymerase II. These core promoter sequences are platforms for the assembly of the
core transcriptional complex, and determine the nucleotide at which transcription will
start (the transcription start site, TSS). Other control sites are gene specific, and can be
occupied by specific activator or repressor proteins together with their associated co-
factors. These proteins interact with the polymerase and associated proteins to either
initiate or prevent trancsription. Thus, the particular combination of activators and
repressors occupying the control sites determines whether a gene is expressed or not in
a particular cell. These control sites can be located adjacent to the promoter, or further
upstream, and sometimes downstream, of the coding region of the gene. For some
genes, control sites can be many kilobases away from the start point of transcription.
The proteins bound to these distant sites are brought into contact with the transcription
machenery by looping of the the DNA, the RNA polymerase II complex, and this is
effected by a large multi-protein complex called Mediator. The fully operational initiation
complex activates the RNA polymerase and release it to start transcription.

The combinatory action of gene-regulatory proteins is a key principle in the

control of gene expression and is fundamental to the exquisite control and complexity of
the gene expression that drives development. Another important call of gene-regulatory
proteins comprises the co-activators and co-repressors, which do not bind DNA
themselves but link the transcriptional machinery to the DNA-binding activator and
repressor proteins (Figure 3-4). Moreover, transcriptional regulators fall into two main
group : those that are required for the transcription of a wide range of genes, and which
are found in many different cell types, and those that are required for a particular gene
or set of genes whose expression it tissue restricted, or tissue-specific, and which are
only found in one or a very few cell types. We shall encounter both types of transcription
factors in the following sections, when we look at the regulation of muscle-specific
genes in muscle precursor cells and the differentiation of red blood cell precursors. In
general, it can be assumed that activation of each gene involves a unique combination
of transcription factors.
As noted earlier, developmental genes generally have complex control regions,
containing binding sites for a range of activating or inhibitory transcription factors, so
whether or not a gene is activated depends on the precise combinations, and levels, of
these transcription factors, and their associated co-activators and co-repressor, in the
cell. The particular combination of gene-regulatory proteins in a given embryonic cell at
a given time is the result of its developmental history so far, and determines the next
step it its development.
3.2. Models of Cell Differentiation and Stem Cells
In this part of the module, we will first look at some particularly well-studied
examples of mammalian cell differentiation, which between them illustrate many of the
principles underlying differentiation. We start with the molecular basis of the
differentiation of a single cell type- mammalian striated skeletal muscle- form
undifferentiated embryonic mesodermal cells in the somites.

Muscle Differentiation is Determined by the MyoD Family of Transcription Factors

Skeletal muscle cells derive from the myotome of the somites. Somatic cells
becomes committed to form muscle by the activity of the homeodomain transcription
factors Pax3 and Pax7. These committed, but as yet undifferentiated, cells are called
myoblasts. This will synthesize muscle-specific proteins such as actin, myosin II, and
tropomyosin, which are part of the contractile apparatus, and muscle-specific enzymes
such as creatine phosphate kinase. As such, myoblasts also undergo structural
change during differentiation. They first become bipolar in shape as a result of
recognition of the microtube cytoskeleton, and then fuse to form multinucleate
myotubes (Figure 3-5).

Figure 3-5. Differentiation of striated muscle in culture, Myoblasts are cells that are
committed to becoming muscle but have yet to show signs of differentiation. In the
presence of growth factors, they continue to multiply but do not differentiate. When
growth factors are removed and myoblasts stop dividing ; they align and fuse into
multinucleated myotubes, which develop into muscle fibers that contract spontaneously.

MyoD is a member of a family of basic helix-loop transcription factors that are

expressed only in muscle precursors and muscle cells The proteins in this family can be
considered as key controlling transcription factors for muscle differentiation, as they
switch on muscle-specific genes and lead to differentiation. As well as the myoD gene,
the family includes three (3) other genes - mrf4, myf5, and myogenin. If transfected
into fibroblasts and some other non-muscle cells, which do not normally express either
these genes or muscle-specific structural proteins, they can still induce differentiation
into muscle cells. mrf4, myf5 and myoD are the first genes to be switched on in muscle
precursors in mammals and they act as muscle-determination genes (Figure 3-6). They
are also activated by the transcription factor Pax3, whose expression becomes
restricted to muscle-cell precursor. Once turned on, these factors maintain their own
expression by means of positive-feedback loops of the type illustrated in Figure 3-6. The
Mrf4, Myf5 and MyoD proteins can all activate the myogenin gene, which is involved in
muscle structural differentiation and functional maturation. They are expressed in
proliferating, undifferentiated myogenic cell, whereas myogenin is only expressed in no-
dividing, differentiating cells.

Figure 3-6. Key features of the differentiation of vertebrate skeletal muscle. Extracellular
signals lead to activation of the genes mrf4, myf5 and myoD and the initiation of muscle
differentiation. One of these genes tends to be expressed preferentially (depending on
the species) and their activity is self-sustaining. The transcription factor products of
these genes Mrf4, Myf5 and MyoD, activate muscle-differentiation genes such as
myogenin, which in turn activate expression of muscle-specific proteins, such as
proteins that make up the contractile apparatus.

All Blood Cells are Derived from Multipotent Stem Cells

Hematopoiesis, or blood formation, is particular well-studied example of
differentiation, partly because hematopoietic cells at all stages of differentiation are
relatively accessible in adult animals and can be grown in culture as well as its
importance in the field of medicine. As such, in hematopoiesis, we are looking at the
differentiation of a multipotent stem cell - a stem cell that can give rise to a large but
limited range of differential cell types.
Figure 3-7. Blood cells derived from multipotent hematopoietic stem cells. Multipotent
stem cells (the hematopoietic stem cells) in the bone marrow give rise to all cells that
circulate in the blood, as well as to some cells that reside in tissue. As well as renewing
themselves, the hematopoietic stem cells give rise to progenitor cells with more
restricted potential, which in turn give rise to the committed precursors of the three
blood-cell lineages. The erythroid lineages produces erythrocytes and megakaryocystes.
The myeloid lineage produces the monocytes and granulocytes that circulate in the
blood as well as the macrophages that differentiates from monocytes in tissues, tissue
mast cells, and dendritic cells of the immune system, and osteoclasts. The lymphoid
lineage produces the T lymphocytes and B lymphocytes and the natural killer cells (not
shown here).

The Epidermis of Adult Mammalian Skin is Continually Being Replaced by

Derivative of Stem Cells
Mammalian skin is composed of two cellular layers - the dermis, which mainly contains
fibroblasts, and the protective outer epidermis, which contains mainly keratin-filled cells
called keratinocytes. We are concerned here with the epidermis, which is renewed
from stem cells. Damaged to the dermis is repaired by proliferation of fibroblasts and
regrowth of damaged blood vessels. A basal lamina or basement membrane
composed of extracellular matrix separates the epithelium of keratinocytes that provides
a tough, watertight and resilient barrier, protecting internal tissues from outside
environment (Figure 3-8). Associated with this epithelium are evenly spaced hair
follicles, sebaceous and sweat glands. The epidermis between the hairs is known as the
interfollicular epidermis. Cells are being continuously lost from outer surface of the
epidermis and must be replaced-every four week we have a brand new epidermis. New
cell of interfollicular epidermis are produced throughout life by the differentiation of stem
cells that reside in basal layer of the epidermis in contact with the basement membrane.
Each hair has its own system of support in the form of stem cells at the base of each
follicle (Figure 3-8)

Figure 3-8. Section through human skin showing epidermal structures. The skin is
composed of an outer epidermis (pale orange) separated from the underlying dermis by
a basement membrane. Structures that are epidermal in origin are shown in color here;
dermal structures are in gray. The locations of stem cells are shown in red. In the case
of injury, the stem cells at the base of the hair follicles and in the sebaceous gland can
migrate to contribute to a new basal layer.

Moreover, basal-layer cells depend on stimuli received from the fibroblasts in

the dermis and the extracellular molecules of the basement membrane to remain
proliferative. The stem cell niche for epidermal stem cells therefore comprises the
basement membrane and dermis. Once a cell leaves this stem-cell niche, it becomes
committed to differentiate. After which, the epidermal cells differentiate further after
leaving the basal layer. One terminal differentiation pathway culminates in the
production of keratinocytes, which form the outermost, cornified epidermal layers that
provide the skin’s protective covering (Figure 3-9).

Figure 3-9. Differentiation of keratinocytes in human epidermis. Descendants of basal-

layer cells, which will become the keratinocytes of the epidermis, detach from the
basement membrane, divide several times, and leave the basal layer before beginning
to differentiate. The basal-layer cells express the intermediate filament proteins keratin
14 and keratin 5, and also the integrin α6 β4 required for hemidesmosome formation and
adhesion to the basement membrane. Differentiation into keratinocytes involves a loss
of integrin expression, a change in the keratin genes expressed and the production of
large amount of keratin 1 and keratin 10. In the intermediate layers, the cells are still
large and metabolically active, whereas in the outer layer of the epidermis, the cells lose
their nuclei, become filled with keratin filaments, and their membranes become insoluble
owing to deposition of the protein involucrin. The dead cells are eventually shed from
the skin surface.

Stem Cells Use Different Modes of Division to Maintain Tissues

The precise identification of stem cells in the basal layer of the epidermis, and
whether all the cells within the basal layer can, under certain circumstances such as
injury, act as stem cells, is still not resolved. Basal epidermal cells are heterogeneous,
with some cells proliferating rapidly and other dividing far less frequently. It has been
suggested that the slowly cycling cells are the stem cells and that the surrounding
proliferating basal cells, known as transmit-amplifying cells or sometimes as
progenitor cells, are committed to terminal differentiation. These observations fit the
classical model of stem-cell division, which is one of invariant asymmetric cell division.
Each stem cell division results in one stem cell and one cell committed to differentiate,
which then undergoes several rounds of amplifying divisions before differentiating
(Figure 3-10).

Figure 3-10. Different models of stem cells division contribute to tissue homeostasis. In
the “classical” model of stem cell division, the stem cell (red) always divides
asymmetrically to give rise to another stem cell and a “transit-amplifying cell” cell
(orange) that is committed to divide several times and then differentiate (blue). In the
population asymmetry model, a progenitor cell (green) can divide in the three models as
indicated. In the combined model, very slowly diving stem cells (undergoing only few
divisions per year) produce progenitor cells which then divide much more rapidly
following the rules of population asymmetry to renew the epidermis. To achieve tissue
homeostasis, the proportions of each type of division must be correctly balanced: in
particular, the symmetric divisions must occur in equal proportions. Thus, if a stem cells
(S) can divide in any of the three ways to give S + S, D + D, or S + D, where D is a cell
that will go on differentiate, then as long as the number or S + S divisions is equal to the
number of D + D divisions, the system will remain at a constant size. The percentage
shown on the figure represent the like hood that this particular division will occur within
the starting population of cells.

Embryonic Stem Cells can Proliferate and Differentiate into Many Cell Types in
Culture and Contribute to Normal Development in vivo

The multipotent stem cells that are responsible for tissue renewal in adults are
limited in the range of different cell types they give rise to, and require a specific stem
cell niche to maintain their stem cell, such as the bone-marrow stroma that supports
hematopoietic stem cells or basement membrane and the underlying dermis in the case
of epidermal stem cells. By contrast, pluripotent early embryonic cells can differentiate
into cell types of all three germ layers and do not require a special niche.
The pluripotent stem cells in mammalian embryos are the embryonic stem cells
(ES cells), which can be obtained from the inner cell mass of the blastocyst. Mouse
ES cells have been studied intensively in culture. They are clonal derived and can be
maintained in culture or long periods, apparently indefinitely, but if injected into a
blastocyst that is then returned into the uterus, they can contribute to all the types of
cells in that embryo, but not to extra embryonic structures. Such experiments have
shown that just two or three ES cells can make a whole embryo.

3.3. The Plasticity of the Differentiated State

We have seen several examples of naturally multipotent adult stem cells that
both self renew and give rise to a range of different sell types. If such stem cells could
be produced reliably and in sufficient numbers, it might be possible to use them to
replace cells that have been damaged or lost by disease or injury. This is one of the
main aims of the field of regenerative medicine. The therapeutic use of stem cells will
depend on understanding precisely how gene activity can be controlled in stem cells to
give the desired cell type.
As such, transplantation of nuclei from different animal cells into fertilized eggs,
and cell fusion studies, show that the pattern of gene expression in the nucleus of a
differentiated cell can often be reversed, implying that it is determined by factors
supplied by the cytoplasm and that no genetic material has been lost. Although the
differentiated state of an animal cell in vivo is usually extremely stable, some cases of
differentiation is reversible. Transdifferentiation of one differentiated cell type into
another has been shown to occur during regeneration in some cases and in culture cells.
Both amphibians and some mammals can be cloned by nuclear transplantation from
adult somatic cell into an enucleated egg. Stem cells are self-renewing and can
differentiate into wide range of cell types. They are being investigated with a view to
being used in cell-replacement therapy. It has been found that the introduction of four
transcription factors expressed in embryonic stem cells into adults differentiated cells
induces these cells to become pluripotent. These induced pluripotent cells and induced
transdifferentiation provide alternative approaches for producing cells for cell-
replacement therapy.

References

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2. Hickman, et.al. (2014). Integrated Principles of Zoology. New York: McGrawHill
3. Wolpert, L., Beddington, R., Jessell, T., Lawrence, P., Meyerowitz, E, and Smith,
J. (2002) Principles of Development, Second Edition. Oxford University Press:
Oxford
4. Wolpert, L., Tickle, C., & Arias, A. M. (2015). Principles of Development. Oxford
University Press, USA.