1
Studying gene networks in stem cells: the
potential of differentiation, transdifferentiation,
and dedifferentiation for therapeutic purposes
Ian Parrag

Abstract— Cellular structure and function is defined by gene
organization and expression.
Using systems biology to
understand gene networks, it is possible to completely
characterize the genetic states of cells.
By studying
developmental pathways, we can expand our knowledge of gene
networks and the dynamic events that occur during stem cell
differentiation. Insight gained in these studies may reveal
techniques to manipulate stem cells. Control of the genetic states
may allow us to stimulate specification of stem cells, provoke
adult stem cells to cross lineage barriers, and induce the
dedifferentiation of differentiated cells to renew stem cell
populations. Each of these methods holds tremendous potential in
regenerative medicine and the advancement of therapies to
combat diseases and injuries.
Index Terms—gene networks, stem cells, systems biology
I. INTRODUCTION
S
biology is an emerging field that attempts to
understand genetic or metabolic pathways from a systems
level approach [1]. Through this, a more comprehensive
identification of the biological system is revealed compared to
traditional molecular biology and genetic analyses.
Knowledge not only of the components of the pathway, but
descriptions of their interactions in a spatial-temporal manner
provides more complete and physiologically relevant data on
the workings of different pathways and organisms on a whole.
Although in a premature state, systems biology offers a
large potential in advances in several areas, including cell and
molecular biology, pharmaceutics, biomedical engineering,
and regenerative medicine to name a few. Using systems
theory to study cellular processes, new tools may be
constructed for synthesizing, testing, and refuting hypotheses,
explaining counterintuitive or contradictory data, and aiding in
experimental design [1]. To reach its potential, however,
requires an intimate knowledge of the systems under
investigation. This in turn necessitates the convergence of
experimental and theoretical approaches, both of which may
be limiting its universal use. As a result, systems biology
currently provides a conceptual framework for which to think
YSTEMS
and drive biological research.
In the post-genomic era and with the development of highthroughput devices, such as DNA micro-arrays, gene networks
are receiving much attention using systems biology techniques
[2]. As the mechanisms of gene expression and regulation
continue to become lucid, the mathematical models describing
these interactions are further advanced. Models of the
dynamic and complex interactions involved in gene expression
and regulation will not only help to gain a complete
understanding of genomics, but it may also reveal methods to
manipulate gene networks for therapeutic purposes.
The study of gene networks in stem cells provides insight
into understanding natural developmental and regenerative
strategies used by different organisms. Stem cell research
holds tremendous therapeutic potential for combating injuries
and diseases and is the basis of regenerative medicine.
Various changes in genetic networks accompanies cell
differentiation processes, ultimately leading to terminally
differentiated cell types. A systems level understanding of the
differentiation cascades, including changes in gene
organization and expression, may yield strategies for
controlling cell specification in addition to enhancing tissue
regeneration. Moreover, knowledge of gene networks in stem
cells may allow the manipulation of these cells to cross lineage
barriers, thus giving rise to cells they were conventionally
unable to produce, or even the reversal of lineage restrictions
in terminally differentiated cells, leading to the renewal of
stem cell populations and regenerative capacity.
This paper discusses gene networks in stem cells and the
promise this knowledge may have for manipulating cells. The
underlying principles of gene organization and expression are
first introduced. Next, an understanding of gene networks in
differentiation, revealed through studying developmental
pathways, is presented followed by a discussion of the
potential this comprehension could elucidate on mechanisms
for controlling differentiation, transdifferentiation, and
dedifferentiation.
II. BASICS OF GENE ORGANIZATION AND EXPRESSION
DNA stores information for the synthesis of all proteins and
thus provides the blueprints of life. Every cell in the body
contains the same genome, i.e. the ability to produce any
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protein, yet different cells vary dramatically in the type and
amount of proteins they synthesize. The expression of
different genes is what gives a cell a certain morphology and
function that distinguishes it from other cell types. This, in
turn, is determined by many factors including, for this
discussion, the way genes are organized and the factors
influencing the gene transcription.
A. Gene organization
The DNA encoding the entire genome is divided up into
chromosomes. Chromosomes consist of a single, enormously
long DNA molecule that is folded and packed into a more
compact arrangement [3]. Histone proteins are involved in
packing the DNA and taken together, the complex of DNA and
histone proteins is called chromatin. Two types of chromatin
exist within the cell. Euchromatin is a less condensed form of
chromatin and contains genes that are generally capable of
being expressed [3]. Heterochromatin, conversely, is a more
condensed form of chromatin and the genes contained here are
resistant to being expressed.
The type of chromatin in which genes reside is a function of
the organizational level that the DNA is compacted into.
Histone proteins successively coil and fold DNA into higher
and higher levels of organization. Nucleosomes and the 30 nm
chromatin fiber are the first levels of DNA organization. Both
of these structural motifs are seen in euchromatin.
Heterochromatin, on the other hand, is not well understood
structurally, involving additional levels of folding and proteins
other than the histones [3].
The complex organization of DNA within heterochromatin
prevents access to DNA sequences within it, thus inhibiting
gene expression. Similarly, the level of organization within
euchromatin may prevent gene expression, however, this
chromatin structure is dynamic and may change to allow
transcription to take place [3]. Histone tails can undergo
covalent modifications, including acetylation, methylation, and
phosphorylation, which can greatly affect their function in
regulating chromosomal organization. Chromatin remodeling
complexes are responsible for the modifications to the histone
proteins. These changes allow the DNA to be less tightly
bound to the histones, permitting access to the DNA for
transcription and thus gene expression.
B. Gene expression
While gene organization is one mechanism regulating gene
expression, there are many others, including processes
controlling transcription, RNA processing, RNA transport and
localization, translation, mRNA degradation, and protein
activity [3]. Transcription is especially important in gene
expression because it is the main point in which the cell
regulates what proteins are produced and at what rate.
Transcription is a complex process where a particular
portion of the DNA sequence is copied into an RNA sequence.
Several components, both within the DNA sequence and
additional proteins, are involved in transcription. RNA
polymerases are proteins that catalyze the formation of bonds
that link two nucleotides together. RNA polymerase binds to
the promoter region of the DNA, moves along the DNA in a
stepwise manner, and unwinds the DNA helix to expose the
template as it moves along. A complex of transcription
factors, transcriptional activators, and chromatin modifying
enzymes are required for the attachment of RNA polymerase
to the promoter region.
The various components that are necessary for the
transcription of each gene are being identified and the
interactions of all these molecules are being elucidated. Once
this happens, gene networks can be used to describe the
dynamic events associated with transcription and gene
expression.
III. REGULATION OF GENETIC NETWORKS IN DEVELOPMENT
During development, stem cells naturally progress through
differentiation cascades to produce organized and functional
tissues. By studying the development of organisms, it is
possible to take what nature has supplied and use it for
regenerative strategies. Understanding what genes are turned
on and off, the interactions causing these effects, and the
temporal aspects of these events are all necessary for
describing the gene networks involved. Elucidating these
mechanisms of development will yield cues and sequences for
inducing the same events outside of development, which can
then be used in cell-based therapies.
The environment surrounding stem cells helps determine the
fate of the cells [4]. Spatial cues, such as cell-cell contacts,
morphogenetic gradients, and other signaling molecules,
influence genetic networks driving the stem cells to a
particular cell type. Determining the signaling molecules
involved in development and their effects has been the focus of
decades of research, essentially creating the parts list for
system structure identification, referred to by Kitano [1].
More recently, the genetic networks behind these
developmental decisions are beginning to be defined. The
mechanisms allowing changes in gene organization during
differentiation are the basis for understanding modifications in
gene expression and are a key to identifying developmental
processes.
A. Epigenetic modifications
Alterations in gene expression without changes in DNA
sequence are referred to as epigenetic modifications [5]. The
mechanisms behind this involve heritable and potentially
reversible modifications to DNA organization. Proceeding
from a state of gene expression to a state of gene silencing
involves changes in chromatin structure.
Changes in
chromatin structure arise from DNA methylation and histone
acetylation patterns and determines whether genes will be
expressed or not [6]. The formation of stable chromatin
structures ensures efficient silencing of genes that are no
longer necessary for the determination of cell fate [5]. As a
result of this, only genes that define the properties of the
differentiated cell type are expressed.
Epigenetic control of gene expression is a fundamental
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feature of mammalian development. Epigenetic modifications
in chromatin structure are essential for stably maintaining
different functional states [6]. An example where epigenetic
control establishes the appropriate mechanisms for normal
development is seen in genomic imprinting, discussed below.
Genomic imprinting refers to the state of chromatin
organization of genes inherited from parental origin [5].
Paternal and maternal genomes exhibit epigenetic asymmetry,
resulting in the expression or repression of certain genes
derived from the mother and the expression or repression of
other genes derived from the father. Modifications in the
methylation of specific DNA sequences regulates the
expression of the imprinted genes. The parental imprints, i.e.
the ability of certain genes to be expressed or not based on
parental origin, remain stable throughout the lifetime of the
individual. It is therefore the genes that are not imprinted that
change in epigenetic states during development and
differentiation of stem cells.
Imprinted genes are being identified and may regulate
crucial aspects of mammalian physiology [5]. Consequently,
understanding both the epigenetic states of the stable imprinted
genes and more dynamic non-imprinted genes is necessary for
gene network analyses. Identifying specific epigenetic states
and determining how they can be changed or reprogrammed
may
yield
insight
into
inducing
differentiation,
transdifferentiation, and dedifferentiation.
B. Gene regulatory network in sea urchin development
From a systems perspective, development proceeds as a
progression of states of spatially defined regulatory gene
expression [7]. Through this process, stem cells become
specified, where cells express a given set of genes, and this is a
function of inter- and intracellular spatial cues. Cis-regulatory
elements respond to these signals by regulating the expression
of transcription factors necessary for the transcription of genes
involved in specification. These elements play a key role in
bringing the cells from one state in the differentiation cascade
to the next.
Developmental processes are difficult to model because they
go through successive stages of pattern formation from one
intricate state to another to generate complex morphologies. A
systems level approach to the developmental pathways would
provide a complete understanding of gene networks in these
processes. Davidson et al. [7] utilized a systems biology
technique for advancing the understanding of one aspect of the
developmental process. Based on the wealth of knowledge of
sea urchin development and perturbation analysis, the
regulatory steps involved in endomesoderm specification in
the sea urchin embryo have been modeled [7].
The systems approach to sea urchin development reveals
several important points about the gene network. First, while
there are a few genes implicated in the differentiation of stem
cells and in the production of signaling ligands, the majority of
the genes involved encodes a DNA sequence-specific
transcription factor used to regulate gene expression [7]. The
transcription factors, regulated by cis regulatory elements,
control the specification processes that can occur. Second, the
model provides an explanation for why target genes are
expressed where and when they are, thus highlighting the
capacity to define spatial-temporal aspects of developmental
events. Third, it could be inferred from the model when
signaling molecules are no longer necessary, therefore
indicating the completion of a specification process. Finally,
the network explains some phenotypes observed when given
processes are perturbed, providing an explanation for observed
data.
This systems biology approach yields direct evidence of the
potential of this method for uncovering genetic behavior. The
promise of this type of knowledge is presented in the next
section.
IV. POTENTIAL OF UNDERSTANDING GENE NETWORKS
Several regenerative strategies are being developed and
employed to enhance the regenerative capacity of mammals: 1)
transplanting stem cells, progenitor cells, or differentiated cells
into the injured tissue; 2) transplanting cell-seeded scaffolds
into the damaged site; 3) inducing endogenous regeneration by
activating the differentiation of resident stem cells; 4)
transplanting transgenic stem cells with the corrected genome
into injured region; and 5) targeting stem cells using gene
therapy to correct genetic defects. At the heart of these
strategies is the use of stem cells, defined as such by their
ability to undergo self-renewal and differentiate into one or
more specialized cell types. By fully characterizing the
genetic states of stem cells, we may synthesize methods for
controlling the genetic networks, a feat that would have a
tremendous impact in the clinical setting for combating
injuries and diseases.
A. Influencing specification
The developmental pathways give insight into the natural
specification of stem cells to a differentiated phenotype. The
mechanisms behind this are beginning to be elucidated and are
being used in the laboratory. For example, protocols have
been established for the in vitro differentiation of embryonic
stem cells into cardiomyocytes, skeletal muscle, neurons,
epithelium, and vascular smooth muscle cells [8]. In addition,
models are also being derived to predict stem cell behavior
and population dynamics in response to culture stimuli [9].
The result of these studies yields techniques to predict and
manipulate stem cells but optimizing these processes would
allow the production of all cell types and the appropriate
numbers for cell-based therapies.
Using a systems biology to study specification processes
reveals insight into the complexity of development and
differentiation. The sea urchin development model makes
explicit the small number of differentiation genes and the large
number of regulatory genes involved in this process. In
addition, it provides knowledge of the spatial-temporal events
required for the differentiation cascade to proceed. This
model highlights key nodes required for inducing
specification, including the identification of regulatory
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mechanisms and their interactions. This knowledge may be
used to define the different developmental states and
appropriate signals in a spatially-temporally relevant manner
to manipulate the genetic networks of stem cells and induce de
novo differentiation of stem cell populations.
B. Stem cell plasticity
Although most cells in the adult body are differentiated
cells, there remains a population of stem cells to replace
senescent cells or give rise to new cells in the case of tissue
injury. Conventionally, adult stem cells were believed to be
restricted, giving rise to cell types specific to the tissue in
which they reside. Evidence, however, suggests that adult
stem cells may possess the ability to cross lineage restrictions
to replace cell types different from the tissues where they
inhabit, reviewed by Watt [10] for the hematopoietic system.
This stem cell plasticity, or transdifferentiation, debated about
in the stem cell community, but the implications of these
findings could have huge impact on regenerative strategies.
Changes in the gene networks of adult stem cells, as with
any changes in morphology of cells, is the underlying principle
inducing transdifferentiation. The claim of several studies is
that the adult stem cells respond to external cues supplied by
their environment allowing changes in gene expression and the
generation of unrelated cell lineages [10]. Looking at this
from a systems perspective, the genetic state of the adult stem
cell could be identified and the changes to the genetic state,
brought about by external signals, can be mapped out. The
alterations in the gene networks of the stem cells could be
elucidated, resulting in knowledge of chromatin organization
of genes and expression patterns. Mechanisms for controlling
these changes may then be developed for manipulating stem
cell states and redirecting adult stem cells to regenerate
damaged tissues with no inherit regenerative capabilities.
C. Dedifferentiation of cells to renew stem cell populations
While mammals possess some capacity to regenerate
damaged tissues, there is a huge discrepancy between the
regenerative abilities compared to other organisms. The
salamander, for example, is capable of regenerating their
limbs, tails, and jaws through a process involving cellular
dedifferentiation [11]. In dedifferentiation, differentiated cells
reverse the normal developmental processes thus renewing
stem cell populations that can proliferate and redifferentiate to
produce viable, fully functional tissue. Identifying the genetic
networks regulating cellular dedifferentiation could lead to
methods for inducing similar processes in mammals.
The concept of dedifferentiation of mammalian cells is not
such a far-fetched, science-fiction perspective. Proof that cells
can reverse the epigenetic modifications accumulated during
the development comes from the restoration of totipotency by
transplanting the nucleus of a somatic cell into an enucleated
oocyte [5]. Studies in determining how this occurs, i.e. the
different elements involved in altering gene organization, as
well as understanding the complications due to genomic
imprinting are necessary to fully characterize the steps
involved in this process.
V. CONCLUSIONS
The knowledge gained from a systems level understanding
of gene networks in stem cells may provide the necessary
information to control gene organization and expression. Cells
could
be
manipulated
to
induce
differentiation,
transdifferentiation, or dedifferentiation. This would yield
optimal therapeutic strategies for regenerating diseased or
damaged organs and tissues.
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