Modifying Cell Identity through Reprogramming Katelyn Foley
Submitted to the Department of Molecular and Cellular Biology
In partial Fulfillment of the Requirements
For a Bachelor of Arts Degree with Honors
In the Subject of Molecular and Cellular Biology
Harvard University
Cambridge, Massachusetts
March 2010
Copyright © 2010 Katelyn Foley
I. Development and Differentiation
An embryo’s development into an adult organism is a unidirectional process associated
with increasingly limited cell potential. The fusion of egg and sperm leads to the formation of a
single-cell embryo called a zygote, which has the potential to form an entire organism, as well as
extraembryonic tissues. The zygote and its progeny cells divide several times to form a
blastocyst, or a pre-implantation embryo structured like a hollow ball of cells with an inner cell
mass (ICM). Cells in the ICM are pluripotent, meaning that they can give rise to all cell types in
an organism (Fig. 1) (Surani et al. 2007).
The process by which less specialized cells become increasingly specialized is called cell
differentiation. During development, pluripotent inner cell mass cells give rise to multipotent
stem cells that are more committed to specific lineages. These multipotent stem cells then give
rise to progenitor cells, which have limited potential and self-renewal. Finally, differentiated
cells are generated from committed progenitors (Fig. 2). In some cases, tissue-specific stem cells
are present in an adult organism in order to maintain and repair certain types of tissues. The
process of cell differentiation can be mimicked in culture when pluripotent embryonic stem (ES)
cells are isolated from the inner cell mass of a blastocyst.
II. Epigenetic Changes Drive Differentiation
As cells become more specialized, they undergo epigenetic changes that reinforce a new
cell identity. Epigenetic changes are non-genetic changes that affect the reading of DNA without
altering the DNA sequence (Kouzarides, 2007). Covalent chemical modifications of DNA and
histones influence gene expression by affecting a gene’s physical accessibility to transcription
factors. Catalyzed by the DNA methyltransferase family of enzymes, the addition of methyl
groups to certain cytosine bases hinders physical contact between a promoter and the
transcription factors required for transcription initiation (Fig. 3) (Attwood, 2002).
Posttranslational histone modifications also play a role in gene expression by causing histones to
loosen or tighten DNA and by recruiting proteins that initiate chromatin remodeling. At least
eight different classes of modifications have been observed. For example, acetylation of histone
amino termini can lead to activation of gene expression by decreasing the affinity of histones for
DNA (Fig. 4) (Kouzarides 2007). Histone methylation is a versatile regulator that has been
associated with gene activation and repression, depending on the locations of methylated lysine
residues (Kouzarides 2007; Mikkelsen et al. 2007; Vakoc et al. 2005; Vakoc et al. 2006).
Epigenetic marks represent transcriptional activity and gene expression for a genomic
region (Aoto et al. 2006). Chromatin is found in two forms in interphase nuclei: transcriptionally
active euchromatin and highly condensed, inactive heterochromatin. Both chromatin variants
have a specific epigenetic signature based on the histone code, or the global chemical profile of
histone amino termini (Jenuwein & Allis 2001). While euchromatin is characterized by
hyperacetylation of histones H3 and H4 and by methylation of H3K4, H3K36, and H3K79,
heterochromatin displays low acetylation and methylation of H3K9, H3K27, and H4K20 (Aoto
et al. 2006; Kouzarides 2007).
The differentiation of stem cells is driven by epigenetic changes that reinforce a new
transcriptional program. Previous research indicates that stem cells are characterized by global
acetylation of histone H3 (Kimura et al. 2004; Lee et al. 2004) and by global hypomethylation
(Jackson et al. 2004; Zvetkova et al. 2005), suggesting that chromatin might be in a more
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permissive state in undifferentiated cells. In addition, a deficiency in DNA methyltransferase
greatly hinders stem cell differentiation, resulting in abnormal development and embryonic
lethality (Li et al. 1992; Okano et al. 1999). Aligned with these findings are data showing that ES
cells express 40 to 60% of the genes in a genome, whereas only 10 to 20% are expressed in
differentiated cells (Abeyta et al. 2004). From this perspective, differentiation may involve the
conversion of active euchromatin into stably silenced heterochromatin (Gasser 2002).
On a gene-specific level, ES-cell-specific genes are associated with activating epigenetic
marks and transcription machinery in ES cells. Oct-3/4 and Nanog, which have been implicated
in the maintenance of stem cell characteristics, are unmethylated and associated with histone
acetylation in ES cells (Delcuve et al. 2009). Although RNA Polymerase-II binding to the Oct3/4 promoter has been observed in ES cells, it is lost in neural precursor cells and post-mitotic
neurons (Aoto et al. 2006). These findings suggest that differentiation status is based on an
epigenetic signature determined by DNA methylation patterns and histone modifications.
III. Nuclear Reprogramming
The 220 cell types in the mammalian body are established and maintained by specific
patterns of gene expression. Although cells do not naturally express genes that are specific to
unrelated cell types, experimental procedures have been identified that allow dramatic switches
in gene expression and cell identity (Fig. 5). The cloning of amphibians and mammals from
differentiated cells (Briggs & King 1957; Gurdon 1962; Wilmut et al. 1997) has shown that cell
differentiation is caused by reversible epigenetic changes (Hochedlinger & Jaenisch 2006). With
the exception of antibody-producing cells, all cells in the body are genetically identical, even
though their distinct morphology and behavior is determined by a specific pattern of gene
expression (Fig. 6). Nuclear reprogramming describes a change in the nuclear gene expression of
one cell type to that of an unrelated cell type or embryo (Fig. 7) (Gurdon & Melton 2008). The
genetic equivalence across cell types makes nuclear reprogramming feasible.
At the molecular level, nuclear reprogramming is driven by epigenetic changes that alter
gene expression and cell identity. For example, the conversion of a differentiated cell into a stem
cell is associated with histone modifications that reverse cell differentiation. As somatic cells
return to a pluripotent state, lysine residues of histones H3 and H4 become hyperacetylated, and
lysine 4 of H3 becomes hypermethylated, corresponding to the permissive state of chromatin in
ES cells (Kimura et al. 2004). Other work shows that this transition also involves changes in
histone and DNA methylation patterns (Maherali et al. 2007; Okita et al. 2007; Wernig et al.
2007). Complete epigenetic reprogramming of cancer cells underscores the important role that
epigenetic changes play in generating cell heterogeneity (Hochedlinger et al. 2004). Taken
together, these data support the epigenetic basis of cell identity and reveal a high level of
epigenetic plasticity.
There are currently three methods for achieving reprogramming: somatic cell nuclear
transfer, cell fusion, and direct reprogramming.
IV. Significance
Nuclear reprogramming not only presents a framework for investigating the mechanisms
that govern epigenetic memory and the maintenance of cell identity but it also provides a model
for the generation of patient-specific cells for transplantation (Gurdon & Melton 2008). Somatic
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cells from a patient can be reprogrammed into immune-compatible cells that behave like
pluripotent stem cells. These cells can be expanded and induced to differentiate into a needed
cell type in vitro, providing patient-specific cells for replacement of diseased or damaged tissues
(Fig. 8). A needed cell type may also be directly generated from patient cells without the need
for intermediate pluripotent cells (Fig. 9). In addition, nuclear reprogramming enables the
generation of diseased cell lines, allowing observation of disease progression and drug screening.
V. Somatic Cell Nuclear Transfer
Early experiments in amphibians showed that cells across the differentiation spectrum
can be reprogrammed when their nuclei are injected into enucleated eggs (Briggs & King 1957;
Gurdon 1962). In somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is injected
into an egg that has had its own chromatin removed. Although some of the somatic proteins are
transferred with the nucleus, the large volume of egg cytoplasm dilutes the somatic
transcriptional program, allowing the embryonic transcriptional program to dominate (Fig. 10)
(Gurdon & Melton 2008). The reprogramming of somatic chromatin is associated with
epigenetic changes that allow reactivation of early embryonic genes, such as Oct-3/4 (Simonsson
& Gurdon 2004).
In mammals, metaphase II oocytes (Wilmut et al. 1997) and mitotic zygotes (Egli et al.
2007) have been shown to reprogram somatic nuclei. Somatic cell nuclear transfer can lead to the
development of a normal blastocyst, from which embryonic stem cells can be derived for
transplantation therapies. This process is termed therapeutic cloning. In some cases, SCNT can
support the full-term development of an adult organism. Cloned from an adult mammary gland
cell, Dolly the sheep is a famous example of reproductive cloning (Wilmut et al. 2002). At this
point in time, a number of ethical concerns and technical limitations surround human SCNT (see
Jaenisch 2004 for review).
Nuclear transfer experiments have yielded valuable information about the nature of the
epigenetic changes that drive differentiation. Thirty percent of nuclear transfer experiments give
rise to a normal adult when the donor nucleus is embryonic, but only 1 to 2% are successful
when the donor nucleus is from a differentiated cell (Tecirlioglu et al. 2006). However, even
terminally differentiated cells, such as certain post-mitotic neurons (Eggan et al. 2004; Li et al.
2004) and mature B and T cells (Hochedlinger & Jaenisch 2002), have been shown to support
the full-term development of mammalian clones. These data indicate that even the most
differentiated states are reversible but that the epigenetic modifications that characterize highly
specialized cells are more difficult to undo.
VI. Cell Fusion
Fusion of unrelated cell types has been used to investigate cell plasticity for decades
(Blau & Blakely 1999). When two cells are fused, a heterokaryon with two distinct nuclei is
formed. In some cases, nuclei from fused partner cells merge, leading to a stable hybrid cell that
is directed by the transcriptional program of one of the partner cells (Fig. 11). Previous work has
shown that a partner cell’s ability to impose its pattern of gene expression on a heterokaryon or
hybrid cell is dependent on its cell division activity and size (Gurdon & Melton 2008).
Unspecialized cells, which generally have active cell cycles, have been implicated in
transcriptional dominance after fusion with specialized cells (Hochedlinger & Jaenisch 2006).
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ES cells can reprogram somatic nuclei when fused with somatic cells, in both the mouse
(Tada et al. 2001) and human (Cowan et al. 2005) models. ES-somatic fusion can lead to the
demethylation and reactivation of ES-cell-specific genes from somatic chromosomes.
Reprogrammed cells that display embryonic stem cell morphology, growth, and gene expression
have been isolated by using a genetic marker that indicates reactivation of somatic Oct-3/4 (Fig.
12). In addition, ES-somatic hybrids can form teratomas that express genes from all three germ
layers, a hallmark of pluripotency (Cowan et al. 2005; Tada et al. 2003). However, it is important
to note that global gene expression analysis has revealed differences between the ES cell and ESfibroblast hybrid transcriptomes, although major regulators of pluripotency are similarly
expressed (Ambrosi et al. 2007).
The fusion of differentiated cell types has also suggested that the transcriptional program
of one partner cell can dominate a heterokaryon or hybrid. When hepatocytes are fused with
myotubes, non-muscle nuclei in the resulting heterokaryons express muscle genes, indicating
that the muscle phenotype is dominant (Fig. 13) (Pomerantz et al. 2009). Similarly, the fusion of
B cells and myotubes causes B cell nuclei to become physically structured like muscle nuclei and
to express muscle-specific genes in a sequence that resembles muscle development. The
establishment of muscle nuclear gene expression is associated with the silencing of lymphocyte
genes, suggesting loss of B cell epigenetic memory (Terranova et al. 2006). In both embryonicsomatic and somatic-somatic fusions, it will be necessary to separate chromatin from each
partner cell in order to understand whether transient transcriptional domination or true
reprogramming has occurred.
VII. Direct Reprogramming
Direct reprogramming is achieved by ectopic expression of specific genes that lead to a
switch in cell identity. Although the exact mechanisms of reprogramming are still being
elucidated, this switch is associated with epigenetic changes, stepwise silencing and activation of
endogenous genes, and sustained reprogramming after ectopically expressed genes are silenced
(Jaenisch & Young 2008). Direct reprogramming encompasses pluripotent reprogramming, or
the generation of induced pluripotent stem (iPS) cells from more specialized cells, and lineage
reprogramming, or the conversion of cells from one lineage to another.
The first direct reprogramming experiments showed that forced expression of the muscle
transcription factor MyoD in a number of cell types, including differentiated hepatocytes and
adipocytes, could lead to the activation of muscle-specific genes (Fig. 14) (Weintraub et al.
1989). The most famous breakthrough in the field came when Takahashi and Yamanaka showed
that retroviral transfection of Oct-3/4, Sox2, Klf4, and c-Myc can reprogram murine fibroblasts
into iPS cells (2006). The same factors (Takahashi et al. 2007), as well as a different quartet of
Oct-3/4, Sox2, Nanog, and Lin28 (Yu et al. 2007), can reprogram human fibroblasts (Fig. 15). In
both cases, endogenous ES-cell-specific genes are reactivated and remain active even after
viruses have been silenced (Fig. 16). Follow-up work has yielded iPS cells that are more ES-like
(Maherali et al. 2007; Okita et al. 2007; Wernig et al. 2007) and more clinically applicable due to
the use of nonintegrating adenoviruses (Okita et al. 2008; Stadtfeld et al. 2008b). iPS cells can be
generated from a wide range of cell types, including terminally differentiated B cells (Hanna et
al. 2007) and pancreatic β cells (Stadtfeld et al. 2008a), and then induced to differentiate into
cells from all three germ layers. The broad applications of pluripotent reprogramming support its
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therapeutic potential. However, it is important to note that genome-wide expression profiles
suggest that iPS cells are not identical to wild-type ES cells (Mikkelsen et al. 2008).
Recent lineage reprogramming experiments have shown that differentiated cells and
specialized progenitors can take on the nuclear gene expression patterns of other lineages,
suggesting that intermediate pluripotent cells might not be required for some switches in cell
identity. Adenoviral infection with Ngn3, Pdx1, and Mafa can lead to in vivo reprogramming of
pancreatic exocrine cells into insulin-producing β-cells (Zhou et al. 2008). In addition, in vitro
studies have shown that forced expression of C/EBPα or C/EBPβ can cause B cells to convert
into phagocytic macrophages with 100% efficiency (Bussmann et al. 2009). The use of similar
strategies to successfully convert more distantly related cell types will require a detailed
understanding of cell lineages and the transcription factors that establish and maintain specific
cell identities.
VIII. Mechanisms behind Nuclear Reprogramming
The specific mechanisms behind somatic cell nuclear transfer, cell fusion, and direct
reprogramming seem to be different in each scenario. Both SCNT and cell fusion involve a
competition between two transcriptional programs, but the inherent reprogramming capabilities
and large size of eggs increase the efficiency of SCNT. Unlike SCNT and cell fusion, which
introduce nuclei to a complex cocktail of transcription factors from another cell, direct
reprogramming is driven by the forced expression of select transcription factors. An
investigation of the timing of expression of iPS cell factors Nanog, Klf4, Myc, Lin28, and Oct4,
showed that only Lin28 and Oct4 are expressed in the early embryo. Nanog, Klf4, and Myc,
which have been implicated in iPS generation, are expressed at later stages, suggesting that they
do not mediate reprogramming in SCNT (Esikov et al. 2004; Esikov et al. 2006). Furthermore,
the addition of Nanog to the Oct-3/4/Sox2/Klf4/c-Myc quartet of iPS cell factors has not been
shown to increase the efficiency of pluripotent reprogramming (Silva et al. 2008). In contrast, the
fusion of Nanog-overexpressing ES cells with fibroblasts or thymocytes can significantly
increase the number of ES-like hybrid cells (Silva et al. 2006).
Despite these differences, there are commonalities between the reprogramming processes
observed for each method. Egg cytoplasm, whole cells, and select transcription factors can bring
about epigenetic changes when introduced to nuclei from unrelated cell types. In SCNT, the
treatment of oocytes with a histone deacetylase inhibitor leads to more faithful reprogramming
and a 100% increase in the number of normal blastocysts (Rybouchkin et al. 2006). Compared to
non-treated oocytes, treated oocytes display more ES-like levels of chromatin structure- and
DNA methylation-related genes (Li et al. 2008). Cell fusion has shown that histone deacetylases
have a cell-type-specific effect on reprogramming. While HDACs are required for erasure of
lymphocyte identity in B cell-myotube heterokaryons (Terranova et al. 2006), they promote
muscle gene expression in hepatocyte-myotube heterokaryons (Pomerantz et al. 2009). In direct
reprogramming, histone deacetylase inhibitors (Huangfu et al. 2008a; Huangfu et al. 2008b), and
demethylation-promoting agents (Mikkelsen et al. 2008) can increase the efficiency of iPS cell
generation. Although epigenetic changes are a common feature of reprogramming, they vary
according to the differentiation states of the nucleus and incoming transcriptional program.
In addition to epigenetic changes, SCNT, cell fusion, and direct reprogramming involve a
competition between transcriptional programs. The introduction of a cellular cocktail or select
transcription factors challenges a cell’s ability to self-reprogram with its own set of factors
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(Gurdon & Melton 2008). During the process of reprogramming to a pluripotent state, egg
cytoplasm, ES cells, and defined ES-cell-associated transcription factors override a somatic
transcriptional program by activating the Oct-3/4/Nanog/Sox2 autoregulatory loop. Oct-3/4,
Nanog, and Sox2 co-occupy a significant number of their target genes, including their own genes
(Boyer et al. 2005). Although special reprogramming powers have been attributed to eggs, recent
research suggests that DNA methylation patterns in sperm and ES cells are surprisingly similar,
except for methylation of Oct-3/4 and Nanog in sperm (Farthing et al. 2008; Imamura et al.
2006). This indicates that post-fertilization reprogramming events might not be as extensive as
previously thought and are instead concerned with demethylation of key pluripotency regulators
(Albert & Peters 2009). Similarly, Oct-3/4 is required for reactivation of Oct-3/4, Nanog, and
Sox2 in lymphocyte nuclei when ES cells are fused with B cells. Heterokaryon-based
reprogramming occurs after nuclear translocation of ES-derived Oct-3/4, suggesting that Oct-3/4
directly activates ES core transcriptional circuitry in lymphocyte nuclei (Pereira et al. 2008).
Although factors other than Oct-3/4, Nanog, and Sox2 have been implicated in iPS cell
generation, treatment of transfected cells with a histone deacetylase inhibitor results in
pluripotent reprogramming with only Oct-3/4 and Sox2 (Huangfu et al. 2008b). It has been
suggested that Nanog is dispensable for pluripotent reprogramming because it is sufficiently
activated by Oct-3/4 (Jaenisch & Young 2008). Taken together, these data indicate that onset of
ES cell identity in a somatic nucleus is associated with activation of the Oct-3/4/Nanog/Sox2
autoregulatory loop.
It is important to note that the temporal relationship between epigenetic changes and
activation of a new transcriptional program may be different for each method. Epigenetic
changes can be directed by the regulatory activity of transcription factors (Lu et al. 2009).
However, in reprogramming, chromatin reconfiguration may be necessary for synthesis of
transcription factors that mediate additional epigenetic changes and activate cell identity-defining
genes. Reprogramming by SCNT, including demethylation of the Oct-3/4 promoter, can occur in
the absence of DNA replication and RNA/protein synthesis, suggesting that existing maternal
factors induce epigenetic changes (Simonsson & Gurdon 2004). Cell fusion-mediated
reprogramming, which can take place during the heterokaryon stage, has been associated with
diffusible trans-acting regulators that initiate expression of regulatory genes in the reprogrammed
nucleus (Blau & Blakely 1999). Although lineage reprogramming can be mediated by one
lineage-specific transcription factor, pluripotent reprogramming requires ES-cell-associated
transcription factors and genes or chemicals implicated in chromatin modification. This suggests
that conversions between more distantly related cell types involve a dynamic interaction between
epigenetic changes and gene expression in the case of direct reprogramming. These differences
might account for some of the variance in duration and efficiency that has been observed for
different reprogramming methods.
IX. Nuclear Reprogramming Exploits Inherent Cell Plasticity
Although cell differentiation is normally a unidirectional, irreversible process, organisms
rely on some level of cell plasticity for environmental responses. While the controversial
transdifferentiation events reported by a number of studies are most likely the result of cell
fusion or stress-induced cellular look-alikes that express only limited markers of another cell
type (Jaenisch & Young 2008), certain evidence indicates that some cell types might not follow
traditional lineage paths (Slack 2008). A number of mysteries remain in the hematopoietic
7
system, in which tissue-specific stem cells that are not present in the early embryo are found later
in development (Orkin & Zon 2008). In addition, recent work has shown that a small percentage
of ES-derived endothelial cells loosen their tight junctions, gather together, and express erythroid
and monocytic hematopoietic antigens in culture (Eilken et al. 2009). In this context, the efficient
switch from B cell to macrophage identity in vitro could imply a mechanism for generating
macrophages in vivo. Specific pathogens could induce this reprogramming event by triggering
expression of C/EBPα or C/EBPβ when they infect B cells (Xie et al. 2004). In order to identify
true transdifferentiation events in vivo, it will be necessary to mark homogenous cell populations
with a label that rigorously defines cells lineages.
Epigenetic status and transcription factor activity also reveal natural cell plasticity.
Consistent with their pluripotency, embryonic stem cells have epigenetic marks that allow them
to efficiently commit to cell fates. In ES cells, developmentally significant transcription factors
are associated with bivalent domains that contain repressive and activating histone modifications
(Bernstein et al. 2006). These domains, which are mostly monovalent in differentiated cells, may
demonstrate readiness to take on a specific cell fate. On a transcriptional level, the activity of
transcription-associated proteins is paused during cell division. Displacement of transcription
factors, chromatin remodeling complexes, and RNA polymerases from mitotic chromosomes has
been observed for a range of cell types (Delcuve et al. 2008). Asymmetric segregation of these
displaced factors can allow daughter cells to inherit different transcriptional programs from a
parent cell. Thus, mitosis represents a weak link in a cell’s ability to preserve epigenetic
memory, temporarily causing vulnerability to transcriptional changes. In interphase, ES cells are
also amenable to large-scale transcriptional changes because of lower protein binding constants
than in differentiated cells. Recent work has shown that ES cell proteins have a looser, more
transient association with DNA, while proteins in differentiated cell types can be associated with
DNA for several minutes (Meshorer et al. 2006; Phair et al. 2004).
Taken together, these data suggest that reprogramming may be dependent on a
combination of epigenetic and transcriptional plasticity. An understanding of the characteristics
that support cell plasticity may elucidate new strategies for efficient reprogramming of highly
differentiated cells.
X. Clinical Progress in the Field of Reprogramming
Nuclear reprogramming has yielded immune-compatible cells for transplantation
therapies in the rodent model, as well as human iPS cell lines from individuals with a variety of
diseases. The isolation of ntES cells, or embryonic stem cells that are derived from nuclear
transfer blastocysts, has enabled further investigation of therapeutic cloning (Wakayama et al.
2001). One study showed that ntES cells with the genotype of immune-deficient mice could be
repaired using homologous recombination and then induced to differentiate into hematopoietic
stem cells for transplantation. This treatment led to partial rescue of immune function in
immune-deficient mice (Rideout et al. 2002). In another study, ntES-cell-derived dopaminergic
neurons were transplanted into mice with Parkinson’s disease, resulting in alleviation of
behaviors associated with the disease (Barberi et al. 2003).
Pluripotent reprogramming has also been used to generate immune-compatible cells for
transplantation. Recent work in the rat and mouse models has shown that iPS-based cell
transplantation therapies can improve the symptoms of Parkinson’s disease (Wernig et al. 2008)
and sickle cell anemia (Hanna et al. 2007). However, it is important to note that some of the
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Parkinson’s diseased animals developed tumors after treatment, possibly due to the presence of
undifferentiated cells in the graft. In addition, patient-specific iPS cell lines have been generated
from humans with a variety of diseases, including diabetes, Parkinson’s disease, and
Amyotrophic Lateral Sclerosis (ALS) (Dimos et al. 2008; Park et al. 2008). In the future, these
cell lines could facilitate disease modeling and drug screening.
Currently, lineage reprogramming is the only method of reprogramming that has potential
for in vivo cell conversion, since pluripotent stem cells do not have direct therapeutic
applications and could not be reliably differentiated in vivo without risk of tumorigenesis.
Lineage reprogramming can be used to convert an existing specialized cell into a needed cell
type in the body. The recent study in which pancreatic exocrine cells were reprogrammed into
insulin-producing β cells in vivo showed that these cells could improve hyperglycemia by
secreting insulin (Zhou et al. 2008).
To date, immune-incompatibility and high DNA ploidy of hybrid cells limit the clinical
applications of cell fusion. Tetraploidy is thought to promote genetic instability that can lead to
aneuploidy and tumorigenesis in vivo (Ganem et al. 2007). Although ES cell chromosomes have
been successfully targeted and eliminated from ES-somatic hybrids using a chromosome
elimination cassette (Matsumura et al. 2007), this technique could lead to global chromosomal
instability in hybrids (Jaenisch & Young 2008). In order to generate clinically applicable patientspecific cells using cell fusion, it will be necessary to efficiently and completely remove ES cell
chromatin before or after fusion.
XI. Conclusion
Nuclear reprogramming takes advantage of inherent epigenetic and transcriptional
plasticity in a range of cell types. An understanding of the molecular mechanisms that support
this plasticity can support the development of reprogramming strategies that exploit natural cell
behaviors. One crucial question that remains is whether the introduction of a transcriptional
program to permissive chromatin can trigger appropriate epigenetic changes. A future direction
could be to elicit epigenetic changes by exposure to small molecules before introducing a new
transcriptional program to a nucleus. In the case of cell fusion, it would be ideal to transfer
mitotic chromatin from a patient cell to a mitotic cytoplast derived from a needed cell type,
although this remains technically challenging. This strategy would minimize competition
between transcriptional programs by exploiting mitotic detachment from transcriptional identity.
However, the dramatic nature of certain cell conversions might make it necessary for a cell to
return to the branch point between two lineages before being induced to differentiate into a
needed cell type. To date, experimental methods can recapitulate cell differentiation for a range
of pathways, including hematopoietic, neural, and mesenchymal.
Even though nuclear reprogramming has demonstrated clinical value in the mouse model,
it is important to recognize that reprogrammed cells are not exactly the same as their wild-type
counterparts. Reprogrammed cells seem to stably take on the nuclear gene expression pattern of
a specific cell type. However, it remains to be seen whether we can generate reprogrammed cells
with the epigenetic signature, global gene expression, and cell architecture of corresponding cells
in the body. Ultimately, it may be that reprogramming will be most successful when different
reprogramming methods are applied to specific cell type conversions. By understanding the
molecular mechanisms behind reprogramming, we can develop more efficient, direct strategies
for generating patient-specific cells for transplantation (Fig. 22).
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Figure 1. Cells in the inner
cell mass are pluripotent,
meaning that they can give
rise to all three germ layers:
neurons (ectoderm), red
blood cells (mesoderm), and
pancreatic cells (endoderm).
Figure 2. Cell
differentiation is associated
with loss of cell potential
and decreased cell division
activity.
Figure 3. DNA methylation,
or the addition of methyl
groups to specific cytosine
bases, can repress or silence
gene activity by preventing
transcription factors from
binding to a gene’s
promoter.
Figure 4. Histone
acetylation can decrease the
affinity of histones for DNA,
allowing transcription
factors to access and bind to
a gene’s promoter.
Figure 5. Although the
naturally occurring process
of cell differentiation is
unidirectional, experimental
procedures have been
identified that allow
dramatic switches in gene
expression and cell identity.
Somatic cell nuclear transfer
(top), cell fusion (middle),
and direct reprogramming
(bottom) are the three main
methods.
d
Figure 6. With the exception
of antibody-producing cells,
all cells in the body are
genetically identical, even
though their distinct
morphology and behavior is
determined by a specific
pattern of gene expression.
Figure 7. Nuclear
reprogramming describes a
change in the nuclear gene
expression of one cell type
to that of an unrelated cell
type or embryo.
Figure 8. Nuclear
reprogramming provides a
model for the generation of
patient-specific cells for
transplantation. Somatic
cells from a patient can be
reprogrammed into immunecompatible cells that behave
like pluripotent stem cells.
These cells can be expanded
and induced to differentiate
into a needed cell type in
vitro, providing patientspecific cells for
replacement of diseased or
damaged tissues.
Figure 9. A needed cell type
may be directly generated
from patient cells without
the need for intermediate
pluripotent cells.
Figure 10. In somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is injected into an egg that has had its own
chromatin removed. Although some of the somatic proteins are transferred with the nucleus, the large volume of egg cytoplasm
dilutes the somatic transcriptional program, allowing the embryonic transcriptional program to dominate.
Figure 11. When two cells
are fused, a heterokaryon
with two distinct nuclei is
formed. In some cases,
nuclei from fused partner
cells merge, leading to a
stable hybrid cell.
Figure 12. In a small
percentage of hybrid cells,
stem cell-specific genes that
were previously silenced in
the somatic cell are
reactivated from somatic
chromosomes.
Reprogrammed cells that
display embryonic stem cell
morphology, growth, and
gene expression have been
isolated by using a genetic
marker that indicates
reactivation of stem-cellspecific genes.
Figure 13. When
hepatocytes are fused with
myotubes, non-muscle
nuclei in the resulting
heterokaryons express
muscle genes, indicating that
the muscle phenotype is
dominant.
Figure 14. Liver cells, as
well as other cell types, have
been redirected to a musclelike transcriptional program
through forced expression of
the muscle transcription
factor MyoD.
Figure 15. Retroviral
transfection of Oct-3/4,
Sox2, Klf4, and c-Myc can
reprogram murine and
human fibroblasts into
induced pluripotent stem
(iPS) cells. This process is
termed pluripotent
reprogramming.
Figure 16. In iPS cells,
endogenous ES-cell-specific
genes are reactivated and
remain active even after
viruses have been silenced.
Figure 17. Adenoviral
infection with Ngn3, Pdx1,
and Mafa can lead to in vivo
reprogramming of pancreatic
exocrine cells into insulinproducing !-cells.
Figure 18. A. Interphase ES cell showing dispersed chromatin, intact nuclear envelope. B. M phase ES
cell (treated with Nocodazole) showing condensed chromatin, porous nuclear envelope.
Figure 18. By understanding
the molecular mechanisms
behind reprogramming, we
can develop more efficient,
direct strategies for
generating patient-specific
cells.