Induced pluripotent stem cells:
Induction of pluripotency and the molecular mechanism of partial
reprogramming
Master thesis
Tom van den Hoeven, 3019144
Master: Molecular and Cellular Life Sciences
Supervisor: Dr. L.H.K. Defize
August, 2014
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Contents
Abstract ................................................................................................................................................... 3
Introduction ............................................................................................................................................ 4
iPS: methods for reprogramming ........................................................................................................... 6
Technical approaches .......................................................................................................................... 6
iPS: phases of reprogramming differentiated cells ............................................................................... 9
Epigenetic modifications ................................................................................................................... 10
Epigenetic profiles in ESCs and iPSCs ................................................................................................. 11
Epigenetic memory in iPSCs............................................................................................................... 11
Partial reprogramming: identification of the intermediate reprogramming stage ......................... 12
The role of transcription factors in pre-iPSCs .................................................................................... 12
Pre-iPSCs: potentials, advantages and disadvantages ...................................................................... 13
Discussion.............................................................................................................................................. 14
Acknowledgements .............................................................................................................................. 17
References............................................................................................................................................. 18
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Abstract
Induction of pluripotency is a process that reverses the phenotype of terminally differentiated cells.
Somatic cells, induced by specific transcription factors, are reprogrammed to a pluripotent state. The
resulting cells, called induced pluripotent stem cells, have activated pluripotency-related genes and
silenced genes of the somatic programme. Many studies have been done in order to elucidate the
molecular mechanisms of reprogramming. Experimental data will increase our understanding of the
reprogramming process and help to improve reprogramming strategies. Reprogramming somatic
cells occurs in phases. Pre-induced pluripotent stem cells (pre-iPSCs) have been identified as an
intermediate stage and will allow the study of the molecular pathways of reprogramming. Pre-iPSCs
and iPSCs hold potential for regenerative medicine as well as the modelling of diseases.
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Introduction
Multicellular organisms are composed of many different types of cells. Each cell is specialised to
perform a specific function within a certain tissue. These cells acquire their function through
differentiation, a process that sets undifferentiated cells on a path to attain a certain fate. The
pathway involves many molecular changes, including altered gene expression patterns. Gene
expression affects the cell fate as it determines which proteins are synthesized that are necessary to
perform the function. All cells express a standard set of genes that are essential for survival or
maintenance. Aside from these householding genes, somatic cells only express genes that are
required for the function of the cell. In other words, from the large pool of genes that exist in the
genome, only a subset is activated. All other genes are effectively down-regulated or inactivated. This
concept of genetic restriction accompanies the formation of specific cell types. The expression of
genes is under epigenetic control. Epigenetics is the modification and modelling of DNA in the cell
and transcription factors, which affects gene transcription. Key players in this process are histones,
transcription factors and miRNAs which are affected by external queues. Their importance will be
discussed in following chapters.
A stem cell is a unique cell type and is defined by its ability to self-renew. This means that a stem cell
forms at least one new stem cell. The self-renewal ensures the maintenance of a pool of
undifferentiated cells, that may differentiate into other cell types if required. Differentiation is
usually initiated when cells or tissues become damaged and need replacing or repair. Also tissues
with a high turnover rate of cells have very active stem cells, like bone marrow for producing blood
cells or the epithelial lining of the intestines. Stem cells can be roughly divided into two types:
embryonic stem cells (ESCs) and adult stem cells (Bayart and Cohen-Haguenauer, 2013). ESCs are
able to generate cells that form all three germ layers and are therefore referred to as pluripotent.
They are present only in the earliest stages of the development of multicellular organism, at a time
when proliferation and differentiation are tightly regulated to ensure proper development. Adult
stem cells arise later in development and are less potent. They can differentiate only into cells of a
certain lineage (Bayart and Cohen-Haguenauer, 2013). Even though ESCs have great potential, they
are not used for therapeutic applications, because of the small number of cells and the difficulty to
isolate them. On top of that it is ethically sensitive. In contrast, adult stem cells are easier to obtain
and because they are harvested from adult tissues with consent, there are (almost) no ethical
dilemmas. Adult stem cells however are less potent in terms of differentiation. Although these points
limit the use of stem cells in regenerative medicine, great progress has been made in the last decade.
In fact, researchers have been able to revert terminally differentiated cells back into a pluripotent
state. This process is entails reverting the genetic restriction by removing epigenetic modifications on
the genome.
In the 1960’s, experiments with reprogramming frog cells by John Gurdon led to the hypothesis that
the silencing of genes could be undone and that the differentiated state of cells is not fixed. This
hypothesis was controversial at first because the experiments could not be replicated for other
species. The first real success in the field of cloning was obtained by Ian Wilmut and colleagues in
1997. By fusing an oocyte, from which the nucleus had been removed, with a mammary cell, they
had succeeded in cloning Dolly the sheep and provided the evidence that the fate of specialised cells
can be reversed (Wilmut et al, 1997). In 2006, Yamanaka and co-workers were able to revert mouse
fibroblasts to a pluripotent state with ES-like characteristics. This was achieved by using a cocktail of
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the transcription factors Oct3/4, Sox2, Klf4 and c-Myc, inducing the fibroblasts to return to a ground
state of pluripotency (Takahashi and Yamanaka, 2006). In essence, the cells are reset by erasing the
active epigenetic network (Schambach et al., 2010) and therefore change gene expression. The
epigenetic network consists of all the modifications established on the genome. Erasing these
modifications therefore allows the activation of pluripotency-related genes (Djuric et al., 2010). Such
genes are important for the functioning of the embryonic stem cell, by maintaining differentiation
potential and the ability to self-renew. Genes most referred to in literature are the ones that encode
for the ‘Yamanaka’-factors, nanog and LIN28. This new approach of reprogramming was coined
induced pluripotent stem cells (iPS). After their publication, the amount of new papers expanded
rapidly and describe successes in the field of developmental biology. The proof of principle of iPS has
driven many research groups to replicate the experiments and adapt them to human pluripotent
stem cells. Only a year later, human somatic cells were successfully reprogrammed using the same
set of transcription factors (Takahashi et al., 2007).
There are numerous papers that describe successful reprogramming of both mouse and human cells,
however much remains to be researched. Key focus points are the molecular mechanisms behind the
reprogramming (Plath and Lowry, 2011) and the differences and similarities between iPSCs and ESCs.
Many studies indicate that they are actually not exactly the same and that complete reprogramming
requires passing multiple barriers. The most important aspect is change of gene transcription and
thus changes in the epigenomic landscape. Epigenetic modifications play an important part in
reprogramming somatic cells, which became apparent in the analysis of the transition to iPS cells.
Failure to properly silence and activate essential genes could result in cells not reaching the full
pluripotent state. These non-pluripotent cells are generally referred to as partial reprogrammed cells
or pre-iPSCs. These cells represent neither the initial somatic cell nor a completely reprogrammed iPS
or ES cell, rather reflecting an intermediate stage (Silva et al., 2008). The pre-iPSCs offer a chance to
study the molecular mechanisms and the function of transcription factors in the reprogramming
pathway.
Stem cells are studied for scientific purposes and also for practical applications. With the onset of iPS
technology, the field of regenerative medicine has advanced greatly, for example cell therapy seems
to be a step closer. IPS cells are also useful for modelling diseases and the discovery of new drugs.
Clearly, iPS offers great potential, however one must consider many aspects and pitfalls with regard
to application in humans, including safety, efficiency and cost. The next step that has to be taken is
unravelling the pathways and molecular mechanisms of iPS cell generation. What are the key
epigenetic changes involved in this partial reprogramming and why do these form a barrier to a
complete iPS transition? What are the major differences between iPS and pre-iPS and what
usefulness do they have for medicinal applications? In order to answer these question, this thesis will
give an overview of the iPS reprogramming process and the current available methods, the changes
in gene expression and epigenomic properties of iPSCs. Further, several aspects of the pre-iPSCs
phenomenon will be explored with discussions on their stability, safety and usefulness in therapeutic
applications.
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iPS: methods for reprogramming
Reprogramming somatic cells ultimately leads to the generation of a new type of cell, an iPSC. The
process of converting somatic cells into pluripotent ones depends on several factors like initial cell
type (Polo et al., 2010) and reprogramming methods. Techniques to reprogram terminally
differentiated cells were already available before the discovery of iPS. As stated briefly before, the
reprogramming process essentially consists of ‘resetting the cell’ by making changes in gene
expression and therefore inherently changes in the epigenome. Techniques described in literature to
induce such changes include nuclear transfer, cell fusion and transduction with transcription factors
(Yamanaka and Blau, 2010). The first two methods are described briefly below.
Nuclear transfer is the transfer of a nucleus from a differentiated somatic cell to an ‘empty’ oocyte,
also referred to as somatic cell nuclear transfer (SCNT). Introduction of the donor nucleus into the
oocyte initiates nuclear reprogramming. The resulting cell can fully grow into an adult which is
essentially a clone from the original donor (Yamanaka and Blau, 2010). Embryonic stem cells can be
harvested from a growing embryo, though the efficiency of generating a successful clone is generally
very low and abnormalities in gene expression are common (Yamanaka and Blau, 2010). These
abnormalities presumably arise from a problem with erasing epigenetic modifications on the DNA,
also referred to as ‘epigenetic memory’. This term will be discussed in detail in a following chapter.
The cell fusion technique has been around for decades. With cell fusion, two or more cell types are
fused together to form either a heterokaryon or a hybrid cell (Yamanaka and Blau, 2010). Fusing an
embryonic stem cell and a somatic cell in the right condition can lead to a situation in which the
pluripotent state is dominant (Sumer et al., 2014). Such experiments imply that previously silent
genes can become activated by factors that were already present (Yamanaka and Blau, 2010), as has
been shown for mouse (Tada et al., 2001) as well as human cells (Cowan et al., 2005).
Technical approaches
The methods described above led to the hypothesis that certain factors in undifferentiated cells
confer pluripotency properties (Takahashi and Yamanaka, 2006). The initial breakthrough of
reprogramming by transcription factors led to the development of an array of different approaches.
In concept, the somatic cell is reprogrammed by ectopically expressing pluripotency-related
transcription factors (Takahashi and Yamanaka, 2006). These transgenes later become silenced and
endogenous pluripotency genes become up-regulated, a characteristic that marks iPSCs as fully
reprogrammed (Ramos-Mejía et al., 2012). The usual cocktail of transcription factors consists of
OCT4, SOX2, KLF4 and c-MYC. Some research groups use combinations of transcription factors that
are slightly different from the four previously mentioned or leave some factors out of the cocktail
(Huangfu et al., 2008; Shi et al., 2008). Other reports mention a combination of transcription factors
and other small molecules that efficiently reprogram somatic cells (Shi et al., 2008). The genes that
code for the transcription factors need to be delivered to the target somatic cells and each delivery
method has its own advantages and disadvantages. A division can be made into viral- and non-viral
transduction and both approaches either integrate or do not integrate DNA into the target cell’s
genome (Figure 1).
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Lentiviruses and retroviruses are vectors that are very efficient in transduction and integrate the
transgenes they carry into the genome of the target cell. Retroviruses can transduce only actively
dividing cells, like epithelial cells. Over time, transgenes integrated by retroviruses become silenced,
caused by de novo methylation of viral DNA (Stewart et al, 1982), and endogenous pluripotency
genes up-regulated to maintain pluripotency. Insertional mutagenesis, caused by random integration
of genes is known to occur and can result in malignancies (Bayart and Cohen-Haguenauer, 2013).
Lentiviruses can transduce most cells as long as they are metabolically active and have not been
reported to cause mutations. The transgene silencing however is much less and could result in
continuous expression, leading to unwanted effects. Other viral delivery systems are adeno- and
sendaïviruses. These viruses do not integrate DNA into the genome and can transduce a broad range
of cell types. The DNA of adenoviruses remains in the nucleus as episomes, therefore mediating good
transgene expression. Episomes will be reviews in the non-viral strategies. Still, viral expression might
not be long enough in most cells, contributing to the low efficiency of generating (Stadtfeld et al.,
2008, a). Although in theory adenoviruses do not integrate into the genome, it has been reported
that this does happen (Stephen et al., 2008). The sendaïvirus is more promising and has already been
shown to reprogram human somatic cells. The virus can be completely eliminated once the iPS is
generated, via several rounds of divisions (Bayart and Cohen-Haguenauer, 2013).
Figure 1: Schematic overview of the different approaches of iPS generation, divided into viral and non-viral strategies. The most efficient
systems are highlighted in the square box. See text for details on the methods. (Bayart and Cohen-Haguenauer, 2013)
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The second branch of Figure 1 is comprised of the non-viral strategies. Transfection of plasmid DNA is
a good alternative to viruses and also incorporates transgenes into the genome. Polycistronic
plasmids have been designed that can express all induction factors with a single promoter and have
been shown to be successful in the generation of iPSCs from mouse cells (Kaji et al., 2009). Non-viral
vectors are mostly used for transient expression and the use of an origin of replication permits
maintenance as stable episomes. Episomes are circular DNA molecules, replicated in the nucleus. A
further advancement comes in the form of minicircles, which are essentially episomes lacking
prokaryotic sequences. This increases the transfection efficiency and enables longer expression due
to less activation of silencing mechanisms (Chen et al., 2003). A report has shown the reprogramming
of human adipose stem cells (hASCs) by using a minicircle vector carrying four (Oct4, Sox2, Lin28,
Nanog) reprogramming factors (Jia et al., 2010).
The final two non-viral delivery systems make use of RNA and proteins. Both systems avoid the
problem of bringing genetic material into the cell that can integrate into the genome. When the RNA
transduction technique was developed, mRNAs that coded for five reprogramming factors were
introduced into human fibroblasts, however did not achieve complete reprogramming (Plews et al.,
2010). The technique was improved by producing synthetic mRNAs with a strong translation
initiation signal and a poly-A tail (Warren et al., 2010). These mRNAs where also better protected
against immune responses by substituting cytidine and uridine with modified bases. This method
works for a broad range of somatic cell types with a higher efficiency of reprogramming than other
systems. The downside however is that RNA is still less stable than DNA, which is why several rounds
of transfection are required that also bring higher costs.
In order to reprogram cells with direct protein delivery, recombinant transcription factors are
produced by extracted from E. coli. Somatic cells are then exposed to protein extracts. Mouse iPS cell
lines have been readily produced in single rounds of transfection (Cho et al., 2010). Producing human
iPS cell lines is somewhat problematic, though one group has succeeded in generating colonies after
many protein exposures (Kim et al., 2009). The lysates used to reprogram the target somatic cells
may contain other factors that could contribute to unwanted effects (Plews et al., 2010).
A problem with integration of transgenes into the target genome is the possible reactivation once
cells have been differentiated. Especially the expression of oncogene-related factors pose a risk,
which may lead to tumour formation (Wernig et al., 2007). The most simple solution would be to
leave out such factors (Nakagawa et al., 2008). Re-expression is preventable by using the correct
inducible promoter. A second problem with integration is the introduction of mutations, caused by
random insertions of the transgenes (Varas et al., 2009). Vectors that are polycistronic reduce the
amount of integrations per cell significantly, due to a lower vector copy number (Carey et al., 2009).
Although improvements have been made with non-integrative strategies, a very low chance of DNA
integration may remain. Safety must always be a primary goal in generating iPSCs and the next step
was taken with the introduction of excisable vectors. The transgenes can be delivered via
lentiviruses, plasmids or transposon elements (Bayart and Cohen-Haguenauer, 2013). Integrated
genes are flanked with loxP sites that allow recombination upon expression of Cre recombinase
protein. Human iPSCs have been generated successfully this way and maintain their pluripotent state
(Soldner et al., 2009). Although the resulting iPSCs are transgene free, they genomes still contain the
insertion sites. In addition, if Cre recombinase is not tightly controlled there might be non-specific
recombination events that could lead to genomic instability. Transposons have been used in the
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generation of iPSCs from human fibroblasts and, depending on the type of transposon, do not leave
any genetic elements behind when excised (Kaji et al., 2009). Further, the transposons can be used in
a broad range of somatic cell types. However the transposition event is not always precise and as
long as transposase is expressed the transposon can move from site to site. The latter could cause
unwanted rearrangements in the genome. Taken together, it is difficult to make the choice of which
delivery system to use. Genetic rearrangements and insertional mutagenesis are unwanted features
that pose a great risk for use in cell-based therapeutics. There is no perfect method yet, however
some approaches look very promising and will need to be refined and carefully tested to generate
stable and correct iPSCs.
iPS: phases of reprogramming differentiated cells
The cellular processes underlying the generation of iPSCs are very complex. We have only just begun
to understand each of the steps of reprogramming. Reprogramming occurs in phases and during
each phase only a small percentage complete the transition, making it hard to study the function of
reprogramming factors. Also during the phases of reprogramming a multitude of things may go
wrong, including incorrect epigenetic modifications, changes in the copy number of genes and the
introduction of mutations. Genetic and epigenetic abnormalities may lead to apoptosis, senescence
or cell-cycle arrest (Theunissen and Jaenisch, 2014). The transition of somatic cell to stem cell
requires many changes. Figure 2 presents the multistep process of generating iPSCs. Concerning the
workings of reprogramming factors, the generation of iPSCs can be roughly divided into two stages.
In the first stage, transcription factors need to be expressed in order to initiate reprogramming with
continued expression until reaching a pluripotent state. In the second stage, the expression of
exogenous factors is repressed and the cells maintain a pluripotent state in which they proliferate
and do not differentiate, indicating a stable conversion (Stadtfeld et al., 2008, b). The transition of
somatic cell to iPSC occurs in three phases: early, middle and late (Plath and Lowry, 2011). Most
obvious are the phenotypical changes. Somatic cells obtain epithelial-like characteristics by becoming
small and round fairly early and acquiring an ESC-like cell cycle. This is also the reason why it is easier
to reprogram somatic epithelial cells than mesenchymal cells for example. Mesenchymal cells first
have to make the Mesenchymal to Epithelial Transition (MET) during reprogramming (Plath and
Lowry, 2011). The key transcriptional changes in the early phase are down-regulation of somatic
gene expression and the up-regulation of proliferation and DNA replication genes. In the middle
phases there is up-regulation of genes that confer epithelial properties followed by up-regulation of
transcriptional and developmental regulators of the pluripotent state in the late phase. Gene
regulation inherently requires modifications of the epigenomic landscape. Key epigenetic
modifications in the generation of iPSCs are DNA methylation, histone modifications and the activity
of microRNAs. To understand the relation between iPS and epigenomics, first the types of
modifications will be briefly reviewed.
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Figure 2: Schematic representation of the phases of reprogramming. In the early phase somatic genes are silenced and
genes related to proliferation and metabolism are up-regulated. Pluripotency-related genes become activated in the late
phase. Fully reprogrammed cells are not dependant on exogenous expression of transcription factors to maintain
pluripotency. Pre-iPSCs represent an intermediate state where pluripotency genes have not been activated. Further
reprogramming to iPSC can be induced. Refer to text for detailed information. (Adapted from Plath and Lowry, 2011)
Epigenetic modifications
Methylation is the process of attaching methyl groups to DNA or to histones, catalysed by DNA
methyltransferases, and thereby controlling gene expression. DNA methylation occurs on adenine or
cytosine nucleotides, typically in CpG sites. A CpG site reflects a cytosine residue directly followed by
a guanine. Demethylation is primarily a passive process in which maintenance of methylation system
is disabled or down-regulated resulting in loss of methyl groups. Active removal entails the excision
of entire methylated bases and subsequent repair (Lee et al., 2014), however it is not clear yet which
components are involved in this process.
Histones can be methylated as well as acetylated. Methyl groups are attached to or removed from
lysine or arginine residues so that parts of the chromatin can become active or inactive. The
responsible enzymes histone methyltransferase and histone demethylase attach or remove the
moieties and need to be tightly regulated (Koch et al., 2007). The modifications facilitate or block
binding of transcription factors or other proteins that access the DNA. Arginine can be methylated
twice and lysine up to three times. Methylated lysines are best understood as specific lysines can be
correlated with gene expression states. For example, methylation of lysines 4 and 36 on histone 3
(H3K4 and H3K36) represent transcriptional activation. In contrast, lysines H3K9 and H3K27 promote
transcriptional repression when di- ore tri-methylated. Histones can also be acetylated or
deacetylated by histone acyltransferases and deacetylases respectively (Koch et al., 2007). Chromatin
is less packed at the sites of acetylated residues. Therefore, addition of an acetyl group to lysines on
histones is associated with transcriptional activation and removal with transcriptional repression.
Acetylation of H3K9 and H3K14 are examples of acetylated lysines at the sites of actively transcribed
promoters.
Knowing how the epigenetic modifications affect gene expression, we can take a look at the changes
on the chromatin level. Pluripotency genes in somatic cells are repressed because of enriched
methylation at H3K9 and/or H3K27. In the transition to iPSC, these methylation marks are lost in the
late phase leading to activation of pluripotency-related genes. It was found that during
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reprogramming, the promoter and enhancer regions of many pluripotency genes already gain
histone marks at H3K4 in the early phase. This is strange, since the up-regulation of these genes
occurs much later in the reprogramming process (Koche et al., 2011). Even though these marks are
applied, the repressive marks that were already present in the surrounding regions are not lost. It is
possible that these epigenetic changes, caused by the reprogramming factors, prepare the cell for an
organised, temporal order of events (Koche et al., 2011).
Epigenetic profiles in ESCs and iPSCs
Quite a few studies have tried to map the differences and similarities between ESCs and iPSCs.
Extensive analysis of DNA methylation patterns has been performed by Kim et al (2010a) in ESCs and
two derived iPS cell lines from mice, resulting in the identification of several differentially methylated
regions (DMRs). DMRs are regions with a different methylation status among samples. Even though
very similar in their epigenetic profiles, iPSCs acquire specific features during the reprogramming
process. The two mouse iPS cell lines showed 3349 and 516 DMRs compared to ESCs. Additionally,
Kim and colleagues found that some methylated regions in iPSCs were associated with genes that
were active in the initial somatic cell type. In other words, the genes are still active where they are
expected to be silenced during reprogramming. This suggests that those genes were not ‘reset’
during the reprogramming, contributing to the concept of epigenetic memory. Culture conditions
and duration of culturing affect the number of epigenetic markers. It is postulated that these
markers could be passively lost during DNA replication (Vaskova et al. 2013). A study for human cell
lines was also done, in which it was shown that the methylation pattern of 90% of CpG sites was
similar in ESCs, iPSCs and donor somatic cell lines (Nishino et al., 2011). This suggests that only a
small number of CpG sites is reprogrammed. The epigenetic differences in human iPSCs were caused
by aberrant methylation in early passages and diminished with continuous passaging. The consensus
seems to be that a higher number of passages lowers the number of epigenetic differences in iPSCs,
indicating there might be a passive loss of somatic markers associated with DNA replication (Vaskova,
2013). The number of passages needed also seems to depend on the initial somatic cell type (Polo et
al. 2010; Kim et al., 2012).
Epigenetic memory in iPSCs
A term that has become increasingly important in the reprogramming process is epigenetic memory.
It refers to epigenetic features that are inherited from the parent cell and are retained in the
transition of somatic cell to stem cell. In essence, some of the genes that were active in the initial
differentiated cell have not become inactivated, also apparent from the examples of mouse and
human iPSCs discussed before. In addition, the up-regulation of pluripotency genes does not take
place, effectively stalling progression to the pluripotent state and completion of the induced
reprogramming. Nuclear transfer seems to be more efficient in erasing the epigenetic markings
probably because DNA demethylation starts immediately upon nucleus transfer (Santos et al., 2002).
Demethylation is slower and inefficient in the process of iPS generation (Kim et al., 2010a),
contributing to un-reprogrammed DNA or epigenetic memory in other words. IPSCs reprogrammed
from different somatic cell lines show variability in their transcriptome and therefore logically will
have a different epigenetic memory. The epigenetic memory makes it more difficult to reset to
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pluripotency as it forms a barrier to complete reprogramming. This barrier may affect the
differentiation of iPSCs. In fact, iPSCs with some epigenetic memory can differentiate more easily
into the lineage they originate from then other lineages (Kim et al., 2010a, Kim et al., 2012).
Problems with epigenetic memory include limitations of differentiation and its usefulness in in vivo
studies. One study attempted to develop animals from established iPS cell lines only, however failed
to do so (Stadtfeld et al., 2010). It was found that some genes, required for the growth and
differentiation of some tissues, were repressed and that this was caused by a lack of specific
microRNAs in the iPSCs. In addition, acetylation and methylation levels were lower in the locus of
those genes. This is a good example of how epigenetic memory can have an impact on the
characteristics of iPSCs.
Partial reprogramming: identification of the intermediate
reprogramming stage
Literature from the past few years has begun to describe the phenomenon of partially
reprogrammed cells, also known as pre-iPSCs. At first, pre-iPSCs may seem like an unwanted byproduct from failed reprogramming. Certainly many researchers consider partially reprogrammed
cells imperfect as they have undesirable features, especially their limited differentiation potential.
Normally, the transition to completely reprogrammed iPSCs will result in a very small pool of cells
which makes the process exceedingly difficult to study. Isolating intermediate stages like pre-iPSCs,
which can proliferate, has allowed the study of intermediate and late steps of the reprogramming
process (Sridharan et al., 2009). Figure 2 illustrates the pre-iPSCs as an intermediate stage.
Experiments performed by Silva and colleagues in 2008 with mouse embryonic fibroblasts have
yielded good results concerning the identification of pre-iPSCs. Their findings suggest that using
transcription factors to induce pluripotency primarily generates cells that are undifferentiated and
non-pluripotent. These type of cells are further characterised by the fact that there is also
maintained expression of transgenes and epigenetic silencing of X-chromosomes. The hypothesis
that somatic cells first pass through an intermediate phase can account for the low efficiency of
obtaining iPSCs. It is conceivable that during induced reprogramming only a small portion of cells
dedifferentiates completely and that most cells merely get stuck in the pre-iPS state because of their
inability to activate pluripotency genes. This in turn could be caused by modifications on the DNA
that prevent these events. In the same paper, Silva and colleagues were able to induce the transition
of pre-iPSCs to iPSCs. The idea was based on the blocking of signals that cause loss of pluripotency
and stimulating a pathway that promotes the maintenance of pluripotency. Pre-iPSCs were cultured
in the presence of two small molecules that inhibit the Mek/Erk and GSK3 pathways (2i) in
combination with Leukaemia Inhibitory Factor (LIF), resulting in cells that obtained full pluripotency
and are indistinguishable from ESCs.
The role of transcription factors in pre-iPSCs
So to recap, inducing pluripotency in somatic cells with transcription factors Oct3/4, Sox2, Klf4 and cMyc results in the generation of iPSCs, with pre-iPS as stable (Mikkelsen et al., 2008) intermediate
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stage. The targets of c-Myc are predominantly genes that function in proliferation, metabolism and
protein synthesis (Kim et al., 2010b) in an early stage. Accordingly, many c-Myc targets are already
bound in pre-iPSCs (Sridharan et al., 2009). In addition, c-Myc is not involved in the up-regulation of
pluripotency genes during the final step of reprogramming. Some studies report that c-Myc is not
even required at all, but it does enhance the reprogramming process (Nakagawa et al. 2008, Wernig
et al., 2007). The other transcription factors, Oct4, Sox2 and Klf4, target the transcriptional and
developmental regulators that establish the pluripotency network in the late phase of
reprogramming. In contrast to c-Myc, these factors are not bound to their target genes in pre-iPSCs
(Sridharan et al., 2009). These findings suggest that those three factors do not have access to their
target genes early on. Two models have been proposed to account for this (Plath and Lowry, 2011).
The first is that the intermediate stage could lack additional transcription factors that are required to
bind Oct4, Sox2 and Klf4. The support for this statement comes from experiments with transcription
factor Nanog. Nanog is able to bind many proteins, including Oct4 and Sox2 (Wang et al. 2006), and
could enhance the reprogramming process by lowering barriers. Indeed, overexpression of Nanog in
pre-iPSCs derived from mouse embryonic fibroblasts induces pluripotency (Theunissen et al., 2011),
suggesting that Nanog mediates the binding of the transcription factors to their targets. The other
model proposes that the state of the chromatin, in the form of repressive moieties at promoters of
pluripotency genes, does not accommodate the binding of transcription factors.
Pre-iPS clones derived from different somatic cell types have similar transcription profiles, suggesting
that they stop reprogramming at a similar barrier. Indeed, a study has shown that the methylation of
H3K9 presents a barrier for complete reprogramming (Chen et al., 2013). Addition of vitamin C
induced the demethylation of H3K9, leading to further reprogramming. Improved iPSC generation by
vitamin C complementation was shown before and the data indicated that p53 levels were reduced
in addition to alleviated cell senescence (Esteban et al, 2010).
Pre-iPSCs: potentials, advantages and disadvantages
Even though the differentiation potential of pre-iPSCs may be limited, they still hold great potential
for regenerative medicine, specifically personalised therapy. Looking at pre-iPSCs in a different
perspective, differentiation towards the original somatic cell type is more efficient than
differentiation into another lineage. In addition, pre-iPSCs are less prone to develop cancerous
characteristics because they acquire less mutations or epigenetic aberrations during reprogramming
(Plath and Lowry, 2011). These characteristics could prove useful for the field of tissue engineering.
Tissue engineering is the growing of new tissue in vitro up till the level of proper functioning. The
following example provides a better understanding. In today’s world there is an increasing demand in
donor organs, due to organ failures or tissue damage for example. With the recent development in
iPS technology, one can obtain a sample from the target organ and follow reprogramming protocols.
Induction of pluripotency by specific transcription factors induces cells to reach a pre-iPS state, then
the cells are grown in vitro and subsequently differentiated into the same cell type as the donor cell.
Since the cells are derived from the patient, there is no risk of rejection when transplanted. Heart,
liver, skin or any other easily accessible tissue are good candidates for this type of therapy. While this
is a solid strategy, it will be difficult, if not impossible, for tissue that is not easily accessible, like the
brain and epithelium of some organs (Vaskova, 2013). Although this effectively limits this strategy, in
theory another closely related cell type from the same lineage could be used. Blood cells for
13
example that are reprogrammed differentiate more easily into any other blood lineage (Plath and
Lowry, 2011). Another option is to resort to completely reprogrammed iPSCs, however they require
more time to generate and possibly also to differentiate.
Discussion
Stem cells have been under study for a long time. Known for their great differentiation potential,
researchers found ways to reprogram terminally differentiated cells and discovered that the fate of
somatic cells is not fixed. Reprogramming somatic cells has allowed the study of the molecular
mechanisms behind the differentiation process as well as making progress towards practical
applications. With the discovery of induced pluripotent stem cells, stem cell technology has taken a
big leap forward and continued development will benefit the modelling of diseases, discovery of new
drugs and regenerative medicine.
IPS cells can be generated in many ways, though a perfect method still has to be established. The
ultimate goal will be the generation of iPSCs that are easy to reprogram with high efficiency, free of
any transgenes and safe to use for humans. There is still much that needs to be understood before
such a goal can be realised. Gene expression and epigenetic control are the major players that need
to be mapped clearly and controlled tightly. Yet to date, only a small portion of pluripotency-related
genes and epigenetic modifications have been studied. Regardless, such studied have already
provided great insight into the different phases of reprogramming. IPS generation is a clearly
described phenomenon since its discovery, however it seems that partial programming has not been
receiving much attention yet. Studies have provided data that indicate that the pre-iPSC is an
intermediate stage and can either be fully reprogrammed to a ground state of pluripotency or redifferentiated into a somatic cell type of the lineage it originated from. The current literature does
not provide insights into the extent of partial reprogramming. It is therefore unclear how pre-iPSCs
can be defined. If we imagine the transition of different somatic cell types to iPSCs on a certain
timeline, there is a range in which cells lose their somatic gene expression and become more
proliferative up to a point where they converge and have similar characteristics (Figure 3). Before this
convergence point each pre-iPSC is unique; a B-type pre-iPSC is different from a C-type pre-iPSC. Is
this convergent point the definition of a pre-iPSC or is there a range in which they are called pre-iPSC
(Figure 3, indicated between arrows)? It seems that the convergence point represents a ‘true’ preiPSC, because independent of the original lineage cells stall at the same epigenetic barrier (Mikkelsen
et al., 2008). Before this point, they are simply too different to put them under the same banner.
However, it must be noted that even these differences can be lost over time by passaging of the cells,
ultimately generating a uniform pool of cells. Other questions that arise concern the selectivity of
pre-iPSCs. Longer reprogramming means a greater proliferation rate, however more somatic cell
characteristics will be lost. Will it possible to select for a cell at a certain time point, essentially
optimizing proliferation while keeping great differentiation potential? The growth conditions will play
a major role in this aspect, like the amount of passages, but also the method used to induce
reprogramming. Addressing this question will require extensive analysis of pre-iPSCs, but will give a
better understanding of the reprogramming process in addition to the benefits for therapeutic
applications.
What is the relation between pre-iPSCs and epigenetic memory? Pre-iPSCs are defined by their lack
of pluripotency gene activation, which are normally up-regulated in embryonic stem cells to maintain
14
differentiation potential and self-renewal capabilities. This is not necessarily caused by epigenetic
memory, however this does not exclude the possibility that there is some form of memory. Earlier
stages of pre-iPSCs most likely have some epigenetic memory as they retain a potential to redifferentiate.
Figure 3: Model of transcription factor induced reprogramming of somatic cells. Somatic cells make the transition to iPS
and converge to an intermediate state. The cells gain increasingly proliferative capacity and lose their somatic markers
over time. Pre-iPSCs can be induced to reprogram further and become full pluripotent iPSCs. (Adapted from Yamanaka
and Blau, 2010)
In the paper of Silva and colleagues from 2008, pre-iPSCs were successfully reprogrammed to iPSCs.
This was accomplished by ectopic expression of nanog, though the authors report there was already
a low endogenous expression of nanog. This can lead to two possible scenarios. Either the
endogenous Nanog levels are too low to induce proper reprogramming or pre-iPSCs will reprogram
themselves completely given enough time. Further research could provide data to resolve this
question. The authors also attempted to generate chimeras from pre-iPSCs, however did not
succeed. Further reprogrammed cells could however contribute to healthy chimeras when implanted
in a blastocyst (Silva et al., 2008).
Safety should always be a primary goal in the development of new approaches for iPS generation for
therapeutic use. Progress has been made with the use of vectors that leave no trace of transgenes or
genetic artefacts. Also, reprogramming with different transcription vectors reduces the risk for
bringing oncogenes into cells. In this regard, several groups have been able to generate stable iPS
cells without c-Myc, though with much lower efficiency. Also, the possible accumulation of erroneous
15
epigenetic modifications or mutations in the reprogramming process is a factor that should be
accounted for. Each generated cell line will therefore always have to be submitted to careful genetic
analysis to exclude such problems.
A step forward has been made with the start of a clinical trial this year in Japan. The trial will be a
pilot study to assess the safety of iPS intervention. Patients with age-related macular degeneration
will have their skin cells reprogrammed and differentiated into retinal pigment epithelial cells. The
study is a very early-stage form of clinical research and it is not expected that the patients will
experience dramatic improvements to their vision (RIKEN Center for Developmental Biology, Press
release 2013).
The iPS technology develops at a high speed and numerous papers are published every year. In the
future it might be well possible to apply the pre-iPS technology in situ. Development from an embryo
is in essence a case of in situ differentiation. It would be extremely useful if damaged tissues could be
repaired directly in a patient without growing cells in vitro first. In addition, the cells are patient
specific without the risk of tissue rejection. Of course this brings some limitations as well as the risk
of complications. It will require a very high efficiency rate and precise control over the
reprogramming process.
To conclude this review, it remains challenging to cover all the aspects of reprogramming somatic
cells into pluripotent stem cells. Luckily, this will also allow us to learn more about this fascinating
phenomenon as well as improving current medicinal technology.
16
Acknowledgements
I would sincerely like to thank my supervisor, Dr. Defize, for guiding me during this thesis and for the
open discussions.
17
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