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In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called "cellular Rosetta stones" because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging, and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any disease.
The Rosetta stone analogy. The Rosetta stone is an archeological slab with the same text in three different languages; Hieroglyphics, Demotic, and Ancient Greek, and its discovery proved to be a turning point in understanding the Hieroglyphic language. By direct comparison of the three languages, it was possible to decipher previously unintelligible Hieroglyphics from the other two known languages. An analogy can be made with respect to stem cells: they can be thought of as “cellular Rosetta stones” because they are key to understanding the multi-faceted mysteries underlying human health and disease. Stem cells can be conceptualized as “cellular Rosetta stones” because they are enabling us to create and compare cells with diseases or cellular phenomena that are poorly understood (non-deciphered Hieroglyphics), with isogenic cells that are completely normal (well-understood, Ancient Greek or Demotic). This is the contribution of stem cells toward modeling and treating diseases and generating functional cell types. In addition, they are becoming quintessential for studying epigenetics, aging, cancer and regeneration. It won't be long before they emerge as the ultimate practical tool and make their mark on the conceptualization of biological science in terms of understanding molecular and cellular events and treating debilitating diseases. By using reprogramming as the means to make cellular Rosetta stones, it will be possible to form a universal understanding of currently unknown biological phenomena and develop an accurate philosophy for cellular processes, disease and therapy. (A) Illustrates the rationale behind the way the Rosetta stone was used to decode hieroglyphics. (B) Illustrates the analogously unexplained biological phenomena to which stem cells can be made with various characteristics on an isogenic background and used to understand them; for example (1) limb-regeneration (2) disease modeling at the cellular level (3) treating organismal aging (4) understanding epigenetic mechanisms underlying diseases (5) generating lineage specific cell types to treat degenerative and chronic diseases, or acute injuries.
… 
The epigenetics of induced pluripotency. The process of induced pluripotency using OSKM involves turning on pluripotency genes and turning off genes responsible for the maintenance of a differentiated somatic cell state. The timeline of formation of an iPSC can be divided into three stages; early, intermediate, and late, and involves the presence of different transcription factors during each of these phases. Transcription factors Oct4, Esrrb, and Sall4, are expressed during the early stage. Lin 28, suggested to be expressed in the early phase, is a controversial marker of early stages of reprogramming. Gdf3 is expressed during the intermediate stage, and Sox2 during the late stage. Lamins A/C and B are expressed in somatic cells but not in iPSCs, whereas telomerase expression is upregulated in iPSCs, but not in somatic cells. The color bars tapering toward either side of the timeline indicate a decline in expression or activity of the epigenetic regulator or epigenetic mark. The top panel shows the range of activity of some well-defined epigenetic regulators of chromatin in a somatic cell during the three phases. Methyl group-adding enzymes are shown on the very top, next is histone acetyltranferase that adds acetyl groups, followed by demethylases that remove methylation marks; the presence of histone variant: macroH2A, and finally chromatin remodeling complexes are depicted in the last two rows. The bottom panel includes the list of known epigenetic tags present on regulatory regions of pluripotency genes and their presence during the early, late and intermediate phases of induction in a somatic cell to pluripotency using reprogramming factors. DNA methylation on pluripotency genes decreases in the course of reprogramming because of a decline in the activity of Dnmt (DNA Methyltransferase), which is responsible for methylation. Histone acetylation increases during reprogramming due to the increased activity of histone acetyltransferase. Insufficient histone acetylation and hypermethylated DNA are the “epigenetic barriers,” which need to be overcome during reprogramming. Reprogramming leads to acquisition of active histone marks (e.g., H3K9ac) and loss of repressive histone marks (H3K4me2) on pluripotency genes, which facilitates the opening up of a compact chromatin structure and thereby allowing exposure of pluripotency gene promoters and binding of pluripotency factors like Oct4. (Buganim et al., 2012; Hansson et al., 2012; Loh and Lim, 2012; Apostolou and Hochedlinger, 2013; Liang and Zhang, 2013; Luna-Zurita and Bruneau, 2013; Papp and Plath, 2013).
… 
Importance of the “epigenetic landscape” in cellular reprogramming. An epigenetic landscape represents the process of cell fate decisions during development and is a graphical rendering of complex regulatory networks. The figure represents such an epigenetic landscape denoted by a “mountain” with its various “valleys.” The top of the mountain displays a totipotent cell that is completely undifferentiated and represents ultimate “stemness,” whereas at the bottom of the mountain are differentiated cells resulting from differentiation of their predecessors. Proceeding from the top of the landscape to the bottom, the cell changes from undifferentiated (totipotent and pluripotent), to partially differentiated (progenitor), to terminally differentiated (eg., fibroblast). (A–F) represent the different methods and their course on an epigenetic landscape, showing reversal of cellular identity to a more primitive state. There are four types of methods that are currently known for reprogramming to (A) totipotency, (B,C) near totipotency, and (D) pluripotency. (For simplicity, we have excluded reprogramming methods using only small molecules, cell-extract treatment, and cell fusion that can also result in ESCs-like cell formation). (A) SCNT gives rise to a totipotent cell capable of forming a complete organism. (B) In vivo reprogramming using OSKM generates iPSCs that are additionally capable of contributing to the trophoectoderm lineage and express embryonic and extra-embryonic markers. (C) In vitro reprogramming to ground state naïve pluripotency using NHSM media generates iPSCs more similar to mouse naïve ESCs. (D) Induced pluripotency generates iPSCs similar to but less pluripotent than (A–C). Other processes that also involve manipulation of the epigenetic regulatory networks include (E) transdifferentiation—one differentiated cell type (smooth muscle cell) is converted to another (pancreatic beta cell) and (F) regeneration—derivation of a progenitor-like cell from a differentiated cell during wound healing.
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Disease modeling. iPSCs are an excellent source for modeling genetic, epigenetic, and environmental diseases. Such cellular models representing diseased phenotypes can be used for understanding the interplay between the genetics, epigenetics and environment involved in the disease, and can expose unknown details about disease pathophysiology, and can be used for screening drugs. In the figure all green cells represent diseased cells, and all pink cells represent healthy cells. (A) Genetic diseases can be modeled by reprogramming diseased cells to iPSCs and then re-differentiating them to produce a diseased phenotype. Additionally, these iPSCs can be corrected for the genetic mutation involved in the disease using gene-editing technology. On re-differentiation, corrected iPSCs produce healthy cells that can be used as isogenic controls. (B) Epigenetic diseases can be modeled using healthy cells that are reprogrammed to iPSCs and then induced toward an epigenetic disease state by recapitulating an environment containing the epigenetic factor(s) contributing to the disease. If iPSCs retain an epigenetic mark when in culture, or after being redifferentiated to the desired cell type, it indicates that the epigenetic mark is permanent and is likely to be passed on to offspring or carried by germ-line cells. It can also mean that the particular cell type is predisposed to retaining that epigenetic mark. Patient-specific models can be used as special models, as they can involve known epigenetic factors contributing to the disease. (C) Acute environmental diseases can be modeled using healthy cells by exposing them to a disease-causing environment that results in genetic damage or instability in the cells. For disease modeling, such cells can be reprogrammed to iPSCs and redifferentiated to diseased phenotypes. All of the above models can help us gain better insight into the diverse factors affecting a complex disease in terms of susceptibility, prognosis as well outcomes.
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Treating disease. Stem cells (iPSCs, ESCs, and SSCs/Progenitors) can be used in the following ways to treat acute, chronic, and degenerative diseases. (A) In vitro reprogramming. iPSCs reprogrammed in vitro using different combinations of transcription factors can be further differentiated to specific lineages and these cells can be transplanted into the patient suffering from a degenerative disease or injury. For example, autologous iPSCs generated from fibroblasts of a patient suffering from Parkinson's disease can be differentiated into functional dopamine secreting neurons and can be transplanted into the patient's brain through surgery. In cases where solely transplanting functional cells is inadequate to completely cure a genetic disease, a permanent fix of the genetic mutation involved is required. Thus, an additional step of gene correction can be performed on the patient-specific iPSCs using gene-editing technologies like CRISPR/Cas. Allogeneic iPSCs might be useful to treat traumatic acute injuries like spinal cord injury, as they would be available immediately when required. (B) In vivo reprogramming. iPSCs can be generated in vivo at the site of non-functional or damaged tissue (by administration of reprogramming factors like OSKM) and can then be differentiated toward a specific lineage (by the administration of differentiation signals). For example, iPSCs generated in vivo from pancreatic cells of a patient suffering from diabetes can be differentiated into functional β cells secreting insulin and thus restore the lost function by repopulating the pancreatic tissue with functional β cells. (C) In vivo trans-differentiation. A somatic cell can be trans-differentiated in vivo into a different type of somatic cell using transcription factors, growth factors, etc. such that the new cell type helps to restore the lost function of the diseased or damaged tissue. This process is noteworthy as it avoids the intermediate step of reprogramming somatic cells to pluripotency; however this process is not yet well-established. (D) Epigenetic rejuvenation. The idea behind epigenetic rejuvenation is that epigenetic drugs can be used to convert senescent cells to young cells such that aging is reversed without affecting the differentiation or specialized function of the cell. This can be done in vivo directly or in vitro followed by transplantation of the rejuvenated cells into the patient. (E) In vivo regeneration/progenitor cell stimulation. Progenitors can be induced to differentiate into their successors by injecting differentiation signals like Wnt and Bmp proteins in order to achieve regeneration in vivo, which will restore lost function of damaged or diseased tissue. This process avoids procedures involving induction of pluripotency as well as transplantation into the patient. (F) Tissue Engineering. Patient-specific iPSCs (with optional gene correction step) or ESCs can be used to culture tissues and organs in vitro using specialized scaffolds and organ molds or using bioprinting. Such cultured organs can then be transplanted into a patient through advanced surgical procedures to restore the function of an entire organ or organs, in the case of chronic or systemic diseases, as well as to treat acute illnesses like myocardial infarction with cardiac tissue transplants.
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REVIEW ARTICLE
published: 12 November 2014
doi: 10.3389/fcell.2014.00067
Cellular reprogramming for understanding and treating
human disease
Riya R. Kanherkar1, Naina Bhatia-Dey1, Evgeny Makarev2and Antonei B. Csoka1*
1Epigenetics Laboratory, Department of Anatomy, Howard University, Washington, DC, USA
2InSilico Medicine, Emerging Technology Center, Johns Hopkins University Eastern, Baltimore, MD, USA
Edited by:
Dan S. Kaufman, University of
Minnesota, USA
Reviewed by:
In-Hyun Park, Yale University, USA
Shree Ram Singh, National Cancer
Institute, USA
George Scaria, University of
Minnesota, USA
*Correspondence:
Antonei B. Csoka, Epigenetics
Laboratory, Department of Anatomy,
Howard University, 520 W St. NW,
Mudd 431, Washington, DC 20059,
USA
e-mail: antonei.csoka@howard.edu
In the last two decades we have witnessed a paradigm shift in our understanding of cells
so radical that it has rewritten the rules of biology. The study of cellular reprogramming
has gone from little more than a hypothesis, to applied bioengineering, with the creation
of a variety of important cell types. By way of metaphor, we can compare the discovery
of reprogramming with the archeological discovery of the Rosetta stone. This stone slab
made possible the initial decipherment of Egyptian hieroglyphics because it allowed us
to see this language in a way that was previously impossible. We propose that cellular
reprogramming will have an equally profound impact on understanding and curing human
disease, because it allows us to perceive and study molecular biological processes
such as differentiation, epigenetics, and chromatin in ways that were likewise previously
impossible. Stem cells could be called “cellular Rosetta stones” because they allow
also us to perceive the connections between development, disease, cancer, aging, and
regeneration in novel ways. Here we present a comprehensive historical review of
stem cells and cellular reprogramming, and illustrate the developing synergy between
many previously unconnected fields. We show how stem cells can be used to create
in vitro models of human disease and provide examples of how reprogramming is being
used to study and treat such diverse diseases as cancer, aging, and accelerated aging
syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic
ovary syndrome. While the technology of reprogramming is being developed and
refined there have also been significant ongoing developments in other complementary
technologies such as gene editing, progenitor cell production, and tissue engineering.
These technologies are the foundations of what is becoming a fully-functional field of
regenerative medicine and are converging to a point that will allow us to treat almost any
disease.
Keywords: reprogramming, stem cells, aging, disease, epigenetics
INTRODUCTION
PARADIGM SHIFTS AND SEEING THINGS IN NEW WAYS
Stem cell biology is sometimes thought of as a field that offers
promises that can’t be kept; for example the potential to one day
treat heart attacks or repair spinal cord injuries. But focusing
on regenerative medicine’s so-far small impact on patient care,
misses a shift in our understanding of cells so radical that it has
rewritten the rules of biology in less than two decades.
How much have things changed? By way of analogy, the
Rosetta stone was a stone slab found in 1799 that bore parallel
inscriptions in Greek, Demotic characters, and Egyptian hiero-
glyphics that made possible an unprecedented decipherment of
the latter. We propose that the discovery of the mechanisms
for producing and modulating stem cells, in particular induced
pluripotent stem cells, will have an equally profound impact in
understanding human health and disease (Figure 1). Stem cells
can be conceptualized as “cellular Rosetta stones” because they
are enabling us to create and compare cells with diseases or
cellular phenomena that are poorly understood (non-deciphered
Hieroglyphics), with isogenic cells that are completely normal
(well-understood, Ancient Greek or Demotic). Because of this,
reprogrammed stem cells will be the quintessential tool for study-
ing epigenetics, aging, cancer, and regeneration. This new possi-
bility will open up opportunities to study cells that go awry in
diseaseandtoonedayusepatients’owncellstohealthem.
Stem cells are emerging as the ultimate tool for understand-
ing biological processes at the molecular and cellular level, as well
as treating debilitating diseases. By using reprogramming as the
means to make “cellular Rosetta stones,” it will be possible to form
an accurate understanding of currently unknown biological phe-
nomena, and perhaps eventually develop a universal synthesis of
cell biology (Figure 1).
In this review we present a comprehensive summary of the his-
tory of cellular reprogramming from its initial conception to the
present day. We also describe how these techniques are being used
to better understand basic biology and human disease, and lay
www.frontiersin.org November 2014 | Volume 2 | Article 67 |1
CELL AND DEVELOPMENTAL BIOLOGY
Kanherkar et al. Reprogramming for understanding and treating human disease
FIGURE 1 | The Rosetta stone analogy. The Rosetta stone is an
archeological slab with the same text in three different languages;
Hieroglyphics, Demotic, and Ancient Greek, and its discovery proved to be a
turning point in understanding the Hieroglyphic language. By direct
comparison of the three languages, it was possible to decipher previously
unintelligible Hieroglyphics from the other two known languages. An analogy
can be made with respect to stem cells: they can be thought of as “cellular
Rosetta stones” because they are key to understanding the multi-faceted
mysteries underlying human health and disease. Stem cells can be
conceptualized as “cellular Rosetta stones” because they are enabling us to
create and compare cells with diseases or cellular phenomena that are poorly
understood (non-deciphered Hieroglyphics), with isogenic cells that are
completely normal (well-understood, Ancient Greek or Demotic). This is the
contribution of stem cells toward modeling and treating diseases and
generating functional cell types. In addition, they are becoming quintessential
for studying epigenetics, aging, cancer and regeneration. It won’t be long
before they emerge as the ultimate practical tool and make their mark on the
conceptualization of biological science in terms of understanding molecular
and cellular events and treating debilitating diseases. By using
reprogramming as the means to make cellular Rosetta stones, it will be
possible to form a universal understanding of currently unknown biological
phenomena and develop an accurate philosophy for cellular processes,
disease and therapy. (A) Illustrates the rationale behind the way the Rosetta
stone was used to decode hieroglyphics. (B) Illustrates the analogously
unexplained biological phenomena to which stem cells can be made with
various characteristics on an isogenic background and used to understand
them; for example (1) limb-regeneration (2) disease modeling at the cellular
level (3) treating organismal aging (4) understanding epigenetic mechanisms
underlying diseases (5) generating lineage specific cell types to treat
degenerative and chronic diseases, or acute injuries.
the foundations for a fully functional discipline of regenerative
medicine.
Therefore, to begin, what exactly are stem cells?
STEM CELLS
Stem cells are undifferentiated cells that possess two unique
characteristics:
(1) The ability to self-renew through numerous cycles of cell
division while maintaining an undifferentiated state, and
(2) Pluripotent potential with the ability to generate progeni-
tors of multiple lineages (International Stem Cell et al., 2007;
Mitalipov and Wolf, 2009). These qualities confer unique
regenerative abilities upon stem cells, and make them a
desirable commodity in the endeavor to replenish, regen-
erate and repair human tissues (Gurtner et al., 2007; Wu
et al., 2007). There are essentially two types of stem cells in
mammals: somatic stem cells (SSCs, also sometimes called
progenitor cells), and embryonic stem cells (ESCs) (Niwa
et al., 2000).
SSCs are found in various adult tissues, such as bone marrow,
adipose tissue and blood (including umbilical cord blood) (Jiang
et al., 2002; Terai et al., 2006; Gimble et al., 2007; Luna-Zurita
and Bruneau, 2013). They are partially differentiated cells found
throughout the body that possess the capacity to divide in order to
replenish damaged tissue. They are able to differentiate into more
than one cell type, but unlike ESCs, they are restricted to a specific
Frontiers in Cell and Developmental Biology | Stem Cell Research November 2014 | Volume 2 | Article 67 |2
Kanherkar et al. Reprogramming for understanding and treating human disease
cellular lineage. The advantage of SSCs is that their production
doesn’t require the destruction of embryos, or reprogramming
(see below) (Gardner, 2002).
ESCs are produced from eggs derived from the inner cell mass
of fertilized embryos. Contrasted with SSCs, ESCs are pluripo-
tent, and can differentiate into all of the three primary germ
layers (ectoderm, endoderm, and mesoderm) and their deriva-
tives. ESCs are characterized by long-term self-renewal, and can
be grown in cell culture as an undifferentiated, pluripotent pop-
ulation. Regulation of pluripotency networks is important for
maintaining the undifferentiated state of such cells in culture, or
during differentiation to obtain desired cell types. The transcrip-
tion factor (TF), Oct 3/4 is the master regulator of pluripotency,
and its precise levels during development are responsible for
the differentiation of ESCs into specific lineages, whereas repres-
sion of Oct 3/4 results in loss of pluripotency and formation of
trophoectoderm (Niwa et al., 2000).
ESCs can be directed to differentiate into a particular cell
type through alteration of culture conditions and/or the sup-
plementation of differentiation signals. Understanding the dif-
ferentiation process has provided insights into de-differentiation
and trans-differentiation strategies as well. Dedifferentiation is
the formation of pluripotent or multipotent stem cells from ter-
minally differentiated somatic cells, i.e., reverting to a state of
increased developmental plasticity, and becoming ready to accept
a new identity (Halley-Stott et al., 2013). Transdifferentiation is
the process in which a particular somatic cell is switched from
one lineage-specific identity to a completely different identity
(Graf, 2011; Vierbuchen and Wernig, 2012); in other words, the
direct conversion of one type of somatic cell into another type,
bypassing the intermediate step of dedifferentiation.
The discovery of ESCs (Evans and Kaufman, 1981; Martin,
1981) eventually prompted the search for discovering artificial
dedifferentiation techniques to confer the properties of ESCs onto
somatic cells by altering epigenomic activity, such that the derived
cells are pluripotent and capable of giving rise to embryonic-
like stem cells. These techniques are collectively referred to as
cellular reprogramming. But before we describe these various
techniques, we will provide some background on the history of
how we arrived at today’s reprogramming technology.
HISTORY AND DEVELOPMENT OF CELLULAR
REPROGRAMMING
In 1909, Ethel Browne Harvey, who was known for her work
on sea urchins, was the first to show that cell transplants could
induce a secondary axis of polarity in the host. Harvey’s exper-
iments were the basis for the discovery of Spemann’s organizer
(Lenhoff, 1991). In 1928, Hans Spemann and Hilde Mangold, in
a quest to discover the factors responsible for embryonic deter-
mination and cell differentiation, performed classical embryol-
ogy experiments with salamanders and demonstrated cell-to-cell
induction, in which a group of cells or organizing centers sig-
nal differentiation in neighboring cells and hence regulate their
fate in the embryo (De Robertis, 2006). The cells responsi-
ble for this kind of phenomenon came to be known as the
Spemann organizer, which over subsequent decades led to many
experiments in molecular embryology aimed at finding inducing
factors responsible for early embryonic determination and cell
fate (Grunz, 2001). Further, Spemann had proposed an experi-
ment to determine whether differentiated cells could be restored
to an embryonic state, or if the cells continued to remain special-
ized (Subramanyam, 2013). Spemann reasoned that if a nucleus
from a differentiated cell implanted in a previously enucleated egg
developed into a normal embryo, this would prove that the trans-
planted nucleus retained a genome fully capable of directing all
types of differentiation. In other words, a differentiated nucleus
could still be totipotent.
SOMATIC CELL NUCLEAR TRANSFER
In 1938, Spemann published an account of his experiments with a
prototypical nuclear transfer technique (Spemann, 1938). Using a
piece of hair wrapped around a newly-fertilized salamander egg,
he separated the egg’s nucleus on one side, with the cytoplasm
on the other. After the nucleated side divided four times, creating
a 16-cell embryo, he removed the hair and allowed a nucleus to
slide back into the separated cytoplasm. Cell division now began
on this side as well, and by putting the hair loop back again tightly,
he broke the two embryos apart. The result was a twin set of
salamanders.
This important work showed that the nucleus remained
totipotent after undergoing four divisions, but Spemann won-
dered whether nuclei from much older embryos, or even adult
animals, had similar potential. He wrote that transplanting an
older nucleus into an egg would be a “fantastical experiment.
What he was proposing is what eventually became known as
somatic cell nuclear transfer (SCNT). However, for the next 14
years, scientists struggled with making it work.
But in 1952 Briggs and King performed the first successful
SCNT experiment showing the reversal of cellular identity. They
showed that the transfer of a frog nucleus from a blastula cell
to an enucleated egg gave rise to a cleaved blastula (Briggs and
King, 1952). This was followed by the pioneering experiments of
John Gurdon in 1962, involving transfer of nuclei from termi-
nally differentiated frog intestinal epithelium cells into enucleated
eggs to produce normal tadpoles, which proved that the nucleus
contains all the genetic information required to give rise to all
the differentiated cells in the organism (Gurdon, 1962). After
incremental improvements in SCNT techniques by various labo-
ratories over the intervening three decades, the cloning of “Dolly”
in 1996 was a major breakthrough, and the first ever success-
ful mammalian cloning experiment, which involved transplanting
quiescent nuclei from cultured adult sheep mammary gland cells
into enucleated sheep eggs (Campbell et al., 1996). The creation
of SCNT-derived human ESCs required further improvements in
technology and almost another two decades, but in 2013, after
a long and challenging pursuit, Mitalipov and colleagues finally
succeeded in creating SCNT-derived human embryonic stem cell
(hESC) lines (Tachibana et al., 2013).
SCNT has, over the decades, evolved as a technique, which can
be used for reproductive cloning as well as to produce lines of
hESCs. The attraction of using an egg for nuclear reprogramming
is that, in nature, it has near 100% efficiency in reprogramming
sperm nuclei (Gurdon and Melton, 2008; Teperek and Miyamoto,
2013). The egg uses molecular chaperones and enzymes to erase
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Kanherkar et al. Reprogramming for understanding and treating human disease
the epigenetic signatures during synkaryon formation and cell
division, resulting in extremely efficient reprogramming (Kikyo
and Wolffe, 2000). Variations in the technique used to produce
offspring through SCNT such as improvement of processes like
oocyte maturation, enucleation, donor nucleus transfer, activa-
tion and culture, along with prior epigenetic modification of
the donor nucleus, can improve the efficiency of the process
(Campbell et al., 2007).
INDUCED PLURIPOTENCY
Once SCNT in mammals was achieved, researchers wondered if
the same result could be achieved without using eggs. Thanks
to the pioneering experiments of Yamanaka, it is now possible
to create close equivalents of ESCs without using embryos by
engineering their creation in vitro. In 2006 and 2007, Yamanaka
and colleagues identified conditions that allowed somatic cells
to be genetically reprogrammed into induced pluripotent stem
cells (iPSCs) that are almost as potent as ESCs. This was a sig-
nificant breakthrough and was achieved by transfection of mouse
and human fibroblasts with the transcription factors (TFs) Oct4,
Sox2,Klf4,andc-Myc,(OSKM)usingretroviralvectorstoinduce
a “forced” expression of specific genes that results in iPSC for-
mation (Takahashi and Yamanaka, 2006; Okita et al., 2007).
Subsequent manipulation using different reprogramming factors
such as Nanog rendered the reprogramming process more effi-
cient (Wernig et al., 2007). There are now many different such
pathways of inducing pluripotency, because different combina-
tions of reprogramming factors can all achieve complete repro-
gramming (Takahashi, 2012). In addition to such reprogramming
factors, introduction of improvements such as p53 knockdown
and telomerase overexpression, along with small molecules that
exert epigenetic effects (see Section Small Molecules below) are
now being used routinely (Batista, 2014; Campos-Sanchez and
Cobaleda, 2014). Besides using different combinations of TFs,
iPSCs have also been induced using methods other than retroviral
vectors, such as DNA and RNA (integrating plasmids, episomal
plasmids and transposons), cell penetrating peptides, and small
molecules (see Small Molecules below) (Li et al., 2014).
At present, a primary concern is the ability to achieve
pluripotency in iPSCs while maintaining the functionality of
ESC’s—irrespective of the type of somatic cells used at the out-
set. For example, it is possible that less differentiated cells of the
hematopoietic lineage reprogram more efficiently than differen-
tiated cells, suggesting that the use of SSCs/progenitor cells as
starting material could maximize the efficiency of reprogram-
ming (Eminli et al., 2009). This may be because less differentiated
progenitors possess less condensed chromatin in specific regions,
which is more accessible to reprogramming factors. Also pro-
genitors in general may be more susceptible to the disruption of
their transcriptional networks, or possess a transcriptome that has
higher resemblance to the transcriptome of ESCs (Papp and Plath,
2011).
A very recent refinement to induced pluripotency has been
achieved which allows the in vitro reprogramming of cells back
to a ground state of pluripotency that is even closer to ESCs.
Novel human naïve stem cells have been derived by adding a
unique combination of cytokines and small molecule inhibitors
to the standard reprogramming cocktail (Gafni et al., 2013).
These ground state stem cells closely resemble mouse iPSCs and
ESCs (relatively more pluripotent than human ESCs or iPSCs),
exhibiting hallmarks of naïve pluripotency that include driv-
ing Oct 4 transcription by its distal enhancer, retaining a pre-
inactivation X chromosome state and a global reduction in DNA
methylation and an H3K27me3 repressive chromatin mark depo-
sition on developmental regulatory gene promoters (Gafni et al.,
2013). The epigenetic changes induced by naïve human stem
cell medium (NSHM) conditions indicated that naïve conditions
are likely to resolve previously described technical phenotypes
of epigenetic memory, lineage differentiation biases and aber-
rant reprogramming in human iPSCs and ESCs, thus providing
a stable source for treating and modeling diseases (Gafni et al.,
2013).
Also, recent studies in mice have shown that the in vivo transi-
tory induction of OSKM generated teratomas, indicating that in
situ reprogramming can give rise to iPSCs from a variety of cell
types, and these in vivo iPSCs more closely resembled ESCs than
in vitro generated iPSCs, with a more primitive and plastic state
(Abad et al., 2013)(Figure 3). These in vivo iPSCs can further
generate desired cell types in the presence of appropriate signals
for re-differentiation (shown in Figure 3).
The most attractive application of induced pluripotency using
the newest techniques is of course the production of patient-
specific iPSCs for replacement of damaged, aged, or non-
functional tissue, which will be discussed in later sections. While
both ESCs and iPSCs are desirable for reasons of potency, the
availability of ESCs is hindered by practical (egg availability) and
ethical concerns, while iPSC production still remains relatively
inefficient.
CELL FUSION
Another possible alternative to SCNT is cell fusion, in which two
cells fuse together in the presence of agents like Sendai virus,
polyethylene glycol (PEG), chimeric hemagglutinins, or an elec-
tric pulse to give rise to homokaryons from the same type of
cells or heterokaryons from different types of cell (Soza-Ried and
Fisher, 2012). In the case of heterokaryon formation, the domi-
nant cell is the larger and actively dividing partner that imposes
its own pattern of gene expression on the other. But cells fused in
this way do not usually proliferate well, limiting their therapeutic
value (Gurdon and Melton, 2008).
CELL-EXTRACT TREATMENT
In recent years, cell extracts (usually extracted from embryonic-
like stem cells) have been used to derive pluripotent cells that can
give rise to diverse cell lineages following differentiation (Patel
and Yang, 2010). The use of extracts derived from pluripotent cells
such as ESCs can also trigger the formation of ESC-like colonies
with the upregulation of pluripotency genes and the downregu-
lation of somatic genes such as Lamin A (LMNA)(Alberio et al.,
2006). Notably, remodeling of the nuclear lamina has been used as
a marker for reprogramming events in experiments involving the
incubation of Xenopus laevis somatic cells in egg-extract in which
reprogramming events could be tracked by the resulting nuclear
configuration which acted as an indicator of increased plasticity
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Kanherkar et al. Reprogramming for understanding and treating human disease
(Alberio et al., 2006). LMNA is also a marker of differentiation of
human ESC’s into somatic cells (Constantinescu et al., 2006).
SMALL MOLECULES
Another recent addition to the different types of reprogramming
techniques is the use of small molecules, with or without a com-
bination of TFs. It is thought that small molecules might lead
to a better clinical approach because they involve fewer genetic
manipulations than TF-based reprogramming (Pandian et al.,
2014). A recent breakthrough study showed that a combination
of just seven small-molecules was enough to chemically repro-
gram mouse embryonic fibroblasts (MEFs) such that Oct4 was
dispensable for reprogramming (Hou et al., 2013). Such chem-
ically induced pluripotent stem cells (CiPSCs) were similar to
ESCs in terms of gene expression profile and epigenetic state, and
did not require any exogenous expression of master pluripotency
genes at all (Hou et al., 2013).
TRANSDIFFERENTIATION/DIRECT REPROGRAMMING
Transdifferentiation techniques might make it possible to pro-
duce patient-specific cells in a faster and more efficient manner
by avoiding the intermediate stage of iPSC formation, and thus
preventing the risk of tumorigenesis and immunogenicity asso-
ciated with iPSCs (Ma et al., 2013)(Figure 3). For example,
transdifferentiation studies in vivo have already demonstrated the
conversion of adult murine pancreatic exocrine cells to β-cell-like
populations using the TFs Ngn3, Pdx1, and Mafa (Zhou et al.,
2008).
iPSC TF-based transdifferentiation is a new method of
transdifferentiation that uses the transient overexpression of
iPSC related TFs and cell-type-specific signals (growth factors,
cytokines, and small molecules) to reprogram somatic cells to
diverse lineage-specific cells or multipotent progenitors without
transitioning through the iPSC stage (Ma et al., 2013). An exam-
ple of such inter-lineage transdifferentiation is the generation of
expandable neural progenitors from fibroblasts by the transient
induction of OSKM along with appropriate signaling inputs (Kim
et al., 2011). Another example is the conversion of human dermal
fibroblasts to multipotent hematopoetic progenitors of myeloid,
erythroid and megakaryocytic lineages via induction of OCT4
and its specific binding to regulatory regions of hematopoetic-
specific genes along with cytokine supplementation (Szabo et al.,
2010).
REGENERATION
Finally, the process of regeneration involves local dedifferentia-
tion of somatic cells into a blastema composed of SSCs, which are
multipotent and capable of proliferating, and re-differentiating
into different lineages, and which can repopulate the damaged
or degenerated tissue with functional cells. In human adults
somatic cells cannot normally dedifferentiate and the process
of wound healing involves scar tissue formation caused by the
deposition of collagen, which is the most abundant part of the
extracellular matrix secreted during wound healing. Thus, as
compared to lower vertebrates like salamanders that are capable
of complete regeneration with very little or no scar tissue, human
adults have limited regenerative capacity. Regenerative capacity in
humans seems to have diminished during the course of evolution
compared to our lower vertebrate cousins as a mechanism to fight
cancer.
However, regeneration in humans is not completely non-
existent. The developing fetus is capable of scarless wound healing
and this capacity is lost in gestation. For example, in the case of
cardiac regeneration in neonates, the same extracellular matrix
that contributes to scar formation might be involved in signaling
an increase in proliferative capacity (Porrello and Olson, 2014).
Thus, the much-coveted mammalian fetal regeneration capacity
including wound healing without scar-tissue formation is a result
of the extracellular matrix modulators, growth factors as well as
cytokines (Colwell et al., 2005).
In spite of having wide-ranging information about the regen-
erative capacity of invertebrates, absolute knowledge of the lim-
its of mammalian regenerative capacity is obscure because our
understanding of mammalian stem cell biology usually comes
from isolated stem cells, rather than from actually studying them
in vivo. Given the fact that cells are dynamic entities and their
properties change according to the environment they are in, the
actual information on the number and functionality of stem cells
in vivo is not accurately known (Sanchez Alvarado and Yamanaka,
2014).
Apart from harvesting the intrinsic regenerative potential of
stem cells, mammalian regeneration can be induced through
the administration of TFs known to locally dedifferentiate cells
(transient) followed by re-differentiation. In a landmark study, it
was shown that the upregulation of a single transcription factor,
FOXN1, resulted in regeneration of the thymus in aged mice to an
extent that it restored thymopoesis, a ground breaking example of
in vivo mammalian regeneration (Bredenkamp et al., 2014). Such
recent efforts in regeneration research have helped to advance
our knowledge to the point where restoration of regenerative
potential in mammals is perhaps within reach.
All of the above cellular reprogramming methods remodel
chromatin and cause epigenetic changes in the target cells. But
before we describe the application of the above methods to under-
standing and treating disease, we will first provide an explanation
of what exactly is meant by “epigenetics,” with a brief summary of
this rapidly developing field.
EPIGENETICS
OVERVIEW OF EPIGENETICS
Epigenetics is the study of heritable changes in gene activity exclu-
sive of direct modification of the DNA sequence, and includes
DNA methylation and post-translational histone modification
(Berger et al., 2009). The epigenome is a collection of the DNA
methylation states and covalent modifications of histone proteins
along the genome, and it differs in each cell type. Epigenetic
mechanisms play an important role in the control of gene expres-
sion by organizing the nuclear architecture of chromosomes,
restricting or facilitating TF access to DNA, and preserving a
memory of past transcriptional activities (Rivera and Ren, 2013).
Epigenetic theory explains how the genome and environment
work in tandem, involving mechanisms that affect DNA by regu-
lating gene expression (Weinhold, 2006), and this interaction can
operate across the entire human lifespan (Kanherkar et al., 2014).
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Kanherkar et al. Reprogramming for understanding and treating human disease
Epigenetic modifications are of course crucially important for
driving a cell toward an appropriate function during differenti-
ation. The complexity of signaling during differentiation causes
a cell’s DNA to acquire specific epigenetic marks that restrict
the expression of specific genes, and this inactivation survives
cell division. DNA methylation is a signaling tool that occurs
naturally on cytosine bases at CpG island promoter sequences
and inactivates genes (Phillips, 2008). This type of epigenetic
modification is associated with regulation of gene transcription,
X-chromosome inactivation, and regulation of cellular develop-
ment and differentiation (Bird, 2007). Another method of gene
regulation is through the remodeling of chromatin. Remodeling
occurs by post-translational modification of the amino acids that
make up histone proteins via acetylation, methylation, phos-
phorylation and ubiquitination (Lunyak and Rosenfeld, 2008).
Such epigenetic signatures are maintained by histone modifying
enzymes such as histone acetyltransferases and histone methyl-
transferases (known as the “writers”) and histone demethylases
and histone deacetylases (known as the “erasers”) which act as
co-activators or co-repressors of OSKM respectively at different
stages of reprogramming, and thereby influence iPSC formation
(Apostolou and Hochedlinger, 2013)(Figure 2).
EPIGENETICS DURING CELLULAR REPROGRAMMING
Cellular reprogramming affects epigenetics at two different levels:
firstly, through histone modification at the level of chromatin, and
secondly through regulation of epigenetic marks on pluripotency
genes and lineage-specifying genes that promote differentiation
of various progenitors cells (Festuccia et al., 2013; Van Oevelen
et al., 2013; Watanabe et al., 2013)(Figure 2). A major mecha-
nism affecting epigenetic plasticity is genome-wide methylation
and its regulation (Watanabe et al., 2013); nevertheless, it has to
be accompanied by other epigenetic modulators that affect the
state of pluripotency, totipotency, and their erasure (Loh and Lim,
2012). These modulators include histone acetylases and deacety-
lases such as Brg1 and Baf155 (Figure 2). While initial efforts to
improve the reprogramming efficiency targeted the roles of tran-
scriptional regulators, current studies suggest the involvement of
signaling pathways in this process, and that the upregulation and
downregulation of major signaling pathways can help in improv-
ing pluripotency reprogramming, lineage reprogramming and/or
cell differentiation (Fritz et al., 2014).
The role of epigenetics during cellular reprogramming can be
shown figuratively as an “epigenetic landscape.” An epigenetic
landscape graphically represents the process of cell fate decision
during development. Such a landscape is in reality a product
of complex gene networks that are epigenetically regulated but
can be represented figuratively in the form of a “mountain” with
numerous “valleys.” Cells (represented by balls) reside in the val-
leys (epigenetically stable networks) and a cell can go down any
valley depending upon which stimuli it receives to attain a dif-
ferentiated state (Figure 3). Such epigenetic landscapes and the
related complex gene regulatory networks that regulate cell fate
can be used to predict the TFs associated with particular cell fates
(Lang et al., 2014).
Reprogramming cell fate works through the manipulation
of networks governing an epigenetic state. Different types of
reprogramming can result in producing undifferentiated cells
with varying degree of “stemness” by moving the cell back to
the top of the mountain. Four cellular reprogramming methods,
namely SCNT, and induced pluripotency (in vivo, in vitro, and
in vitro using a special media) are noteworthy and are described
below.
The totipotent cell resides at the very top of the landscape,
and currently SCNT is the only method capable of reprogram-
ming a somatic cell to a totipotent cell capable of forming a
complete organism (Figure 3A). Below this would be a near-
totipotent stem cell that has been generated through one of the
following two methods: in vivo reprogramming using OSKM that
generates iPSCs that are additionally capable of contributing to
the trophoectoderm lineage and express embryonic and extra-
embryonic markers (Abad et al., 2013)(Figure 3B), or in vitro
reprogramming to ground state naïve pluripotency using NHSM
media (see Section Induced Pluripotency above) that generates
iPSCs more similar to mouse naïve ESCs and demonstrates the
potential to overcome problems related to epigenetic memory and
lineage differentiation biases (Gafni et al., 2013)(Figure 3C). The
conventional in vitro method of TF-based reprogramming gener-
ates iPSCs that are similar to ESCs but often have problems related
to epigenetic memory and lack totipotent cell-like features (Papp
and Plath, 2011; De Los Angeles and Daley, 2013)(Figure 3D).
Other processes that also involve manipulation of the
epigenetic regulatory networks include transdifferentiation
(Figure 3E) and regeneration (Figure 3F). In transdifferentiation
one differentiated cell type is converted to another through
direct reprogramming (Takahashi, 2012), by avoiding going all
the way back up the mountain to an intermediate pluripotent
state. Regeneration is similar in that it results in derivation of
SSCs or progenitor-like cells from a differentiated cell during
wound healing, for example the formation of a blastema (an
undifferentiated cell mass) in amphibians (Gurtner et al., 2008)
that involves the cell going half-way back up the mountain.
A new framework has been developed in order to elucidate
the role of epigenetic landscapes in reprogramming that com-
bines the techniques of whole genome expression profiling and
spin glass physics (Lang et al., 2014). This model has verified that
partially reprogrammed cells co-express TFs associated with mul-
tiple cell fates, has reproduced known reprogramming protocols,
and has the potential to generate new reprogramming protocols
to create novel cell types (Lang et al., 2014). Defining epigenetic
landscapes can not only help to gain better insights into repro-
gramming processes, but also to design highly efficient protocols.
This can be done by targeting epigenetic modifiers involved in
the differentiation process at specific positions in the landscape,
as well as identifying easy access points in the hierarchy at which
reprogramming can be induced for high efficiency.
Now that reprogramming technology has been described in
detail we will move on to practical applications.
MODELING HUMAN DISEASE IN VITRO
We can group human diseases into three broad categories: genetic,
epigenetic and acute environmental (Cherry and Daley, 2012).
Modeling of all three types is possible in vitro using stem cells,
and an excellent way to study the intricate mechanisms and
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Kanherkar et al. Reprogramming for understanding and treating human disease
FIGURE 2 | The epigenetics of induced pluripotency. The process of
induced pluripotency using OSKM involves turning on pluripotency genes and
turning off genes responsible for the maintenance of a differentiated somatic
cell state. The timeline of formation of an iPSC can be divided into three
stages; early, intermediate, and late, and involves the presence of different
transcription factors during each of these phases. Transcription factors Oct4,
Esrrb, and Sall4, are expressed during the early stage. Lin 28, suggested to
be expressed in the early phase, is a controversial marker of early stages of
reprogramming. Gdf3 is expressed during the intermediate stage, and Sox2
during the late stage. Lamins A/C and B are expressed in somatic cells but
not in iPSCs, whereas telomerase expression is upregulated in iPSCs, but not
in somatic cells. The color bars tapering toward either side of the timeline
indicate a decline in expression or activity of the epigenetic regulator or
epigenetic mark. The top panel shows the range of activity of some
well-defined epigenetic regulators of chromatin in a somatic cell during the
three phases. Methyl group-adding enzymes are shown on the very top, next
is histone acetyltranferase that adds acetyl groups, followed by
demethylases that remove methylation marks; the presence of histone
variant: macroH2A, and finally chromatin remodeling complexes are depicted
in the last two rows. The bottom panel includes the list of known epigenetic
tags present on regulatory regions of pluripotency genes and their presence
during the early, late and intermediate phases of induction in a somatic cell to
pluripotency using reprogramming factors. DNA methylation on pluripotency
genes decreases in the course of reprogramming because of a decline in the
activity of Dnmt (DNA Methyltransferase), which is responsible for
methylation. Histone acetylation increases during reprogramming due to the
increased activity of histone acetyltransferase. Insufficient histone acetylation
and hypermethylated DNA are the “epigenetic barriers,” which need to be
overcome during reprogramming. Reprogramming leads to acquisition of
active histone marks (e.g., H3K9ac) and loss of repressive histone marks
(H3K4me2) on pluripotency genes, which facilitates the opening up of a
compact chromatin structure and thereby allowing exposure of pluripotency
gene promoters and binding of pluripotency factors like Oct4. (Buganim et al.,
2012; Hansson et al., 2012; Loh and Lim, 2012; Apostolou and Hochedlinger,
2013; Liang and Zhang, 2013; Luna-Zurita and Bruneau, 2013; Papp and Plath,
2013).
pathways underlying the etiology and pathophysiology of disease
(Figure 4). Stem cells in general are ideal for creating “disease-
in-a-dish” models because of their capacity for self-renewal and
differentiation, their potential for recapitulating disease patho-
genesis, and also their amenability for developing and testing
therapeutics (Sterneckert et al., 2014).
The first and simplest types of diseases are monogenic dis-
orders. For example Fanconi anemia (FA) has been studied
using patient-derived, integration-free iPSCs with and without
in situ gene correction (Liu et al., 2014). These FA-iPSCs are
also useful for drug screening and shortlisting compounds that
can trigger their hematopoietic differentiation potential (Liu
et al., 2014)(Figure 4A). Likewise, iPSCs have been derived from
cells obtained from patients suffering from a wide and rapidly-
growing number of diseases such as Hutchinson-Gilford Progeria
Syndrome (HGPS, see below), cystic fibrosis, Parkinson’s disease,
familial amyotrophic lateral sclerosis (ALS), dyskeratosis con-
genita, epilepsy, autism, spinal muscular atrophy, Huntington’s
disease, adenosine deaminase severe combined immunodefi-
ciency,sicklecellanemia,longQTsyndrometypeI,glycogen
storage disease type 1a, Alzheimer’s disease, diabetes, and Down’s
syndrome and can be differentiated toward disease-specific lin-
eages in order to study the genotype-phenotype interactions of
the disease, as well as to screen new drugs that could be capable of
reversing cellular pathology (Liu et al., 2011a; Unternaehrer and
Daley, 2011; Cherry and Daley, 2012; Ess, 2013; Miller et al., 2013;
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Kanherkar et al. Reprogramming for understanding and treating human disease
FIGURE 3 | Importance of the “epigenetic landscape” in cellular
reprogramming. An epigenetic landscape represents the process of cell fate
decisions during development and is a graphical rendering of complex
regulatory networks. The figure represents such an epigenetic landscape
denoted by a “mountain” with its various “valleys.” The top of the mountain
displays a totipotent cell that is completely undifferentiated and represents
ultimate “stemness,” whereas at the bottom of the mountain are
differentiated cells resulting from differentiation of their predecessors.
Proceeding from the top of the landscape to the bottom, the cell changes
from undifferentiated (totipotent and pluripotent), to partially differentiated
(progenitor), to terminally differentiated (eg., fibroblast). (A–F) represent the
different methods and their course on an epigenetic landscape, showing
reversal of cellular identity to a more primitive state. There are four types of
methods that are currently known for reprogramming to (A) totipotency,
(B,C) near totipotency, and (D) pluripotency. (For simplicity, we have excluded
reprogramming methods using only small molecules, cell-extract treatment,
and cell fusion that can also result in ESCs-like cell formation). (A) SCNT
gives rise to a totipotent cell capable of forming a complete organism.
(B) In vivo reprogramming using OSKM generates iPSCs that are additionally
capable of contributing to the trophoectoderm lineage and express
embryonic and extra-embryonic markers. (C) In vitro reprogramming to
ground state naïve pluripotency using NHSM media generates iPSCs more
similar to mouse naïve ESCs. (D) Induced pluripotency generates iPSCs
similar to but less pluripotent than (A–C). Other processes that also involve
manipulation of the epigenetic regulatory networks include (E)
transdifferentiation—one differentiated cell type (smooth muscle cell) is
converted to another (pancreatic beta cell) and (F) regeneration—derivation
of a progenitor-like cell from a differentiated cell during wound healing.
Abdelalim et al., 2014; Acimovic et al., 2014; Hibaoui et al., 2014;
Mohamet et al., 2014)(Figure 4A).
Monogenic diseases are easy to model in culture, but it is
becoming increasingly clear that most diseases are a manifesta-
tion of altered genetics and/or epigenetics, with factors like age
and environment complicating things further. It is necessary to
consider such interlinked effects on different cell types in the
body, especially in the case of late-onset disease, and for such
purposes iPSCs can serve as an excellent model (Csobonyeiova
et al., 2014). Thus, in addition to modeling monogenic diseases,
cellular diseased phenotypes derived from iPSCs can be use-
ful in studying complex diseases having genetic and epigenetic
components, or having unknown sporadic genetic or acute envi-
ronmental etiologies (Grskovic et al., 2011; Zhu et al., 2011).
Such cellular phenotypes are also useful for screening drugs
and understanding the pathophysiology of complex diseases
(Grskovic et al., 2011).
Epigenetic diseases can be subtly contrasted with environmen-
tal diseases based on the type and extent of damage caused by
the epigenetic or environmental factor. Specifically, when iPSCs
are exposed to epigenetic factors, the iPSCs and redifferentiated
cells may gain temporary or permanent epigenetic marks and
manifest the diseased phenotype. Modeling epigenetic diseases
in such a way can elucidate the trans-generational potential of
the epigenetic factors i.e., whether the epigenetic mark is strong
enough to pass on through the germ line to the next generation.
Compared to epigenetic diseases, acute environmental insults
do not cause heritable genetic or epigenetic effects, but rather
the effect is seen at the somatic DNA sequence or protein level
(Cherry and Daley, 2012). For example, an acute environmen-
tal disease like melanoma that is frequently caused by excessive
sunburn (ultraviolet ray damage) can lead to direct DNA dam-
age in the form of pyrimidine dimers (Anna et al., 2007). Such
sporadic environmental insults, including spontaneous deami-
nations, change the DNA sequence as well as alter the cellular
biochemistry through upregulation of DNA repair processes, alter
expression of cell cycle regulators, or possibly cause apoptosis
(Fulda et al., 2010).
Using patient-specific iPSCs we can recapitulate the suspected
effects of the environmental or epigenetic component known
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Kanherkar et al. Reprogramming for understanding and treating human disease
FIGURE 4 | Disease modeling. iPSCs are an excellent source for modeling
genetic, epigenetic, and environmental diseases. Such cellular models
representing diseased phenotypes can be used for understanding the
interplay between the genetics, epigenetics and environment involved in the
disease, and can expose unknown details about disease pathophysiology,
and can be used for screening drugs. In the figure all green cells represent
diseased cells, and all pink cells represent healthy cells. (A) Genetic
diseases can be modeled by reprogramming diseased cells to iPSCs and
then re-differentiating them to produce a diseased phenotype. Additionally,
these iPSCs can be corrected for the genetic mutation involved in the
disease using gene-editing technology. On re-differentiation, corrected
iPSCs produce healthy cells that can be used as isogenic controls. (B)
Epigenetic diseases can be modeled using healthy cells that are
reprogrammed to iPSCs and then induced toward an epigenetic disease
state by recapitulating an environment containing the epigenetic factor(s)
contributing to the disease. If iPSCs retain an epigenetic mark when in
culture, or after being redifferentiated to the desired cell type, it indicates
that the epigenetic mark is permanent and is likely to be passed on to
offspring or carried by germ-line cells. It can also mean that the particular
cell type is predisposed to retaining that epigenetic mark. Patient-specific
models can be used as special models, as they can involve known
epigenetic factors contributing to the disease. (C) Acute environmental
diseases can be modeled using healthy cells by exposing them to a
disease-causing environment that results in genetic damage or instability in
the cells. For disease modeling, such cells can be reprogrammed to iPSCs
and redifferentiated to diseased phenotypes. All of the above models can
help us gain better insight into the diverse factors affecting a complex
disease in terms of susceptibility, prognosis as well outcomes.
to have contributed to the patient’s disease in order to under-
stand its severity or mechanism of action before, during, or
after reprogramming (Figures 4B,C). Cells from any source can
be epigenetically manipulated to mimic epigenetic (Figure 4B)
or acute environmental (Figure 4C) alterations contributing to
disease. Such models can help us gain better insight into the
environmental or epigenetic factors affecting a complex dis-
ease in terms of susceptibility, prognosis as well as outcomes.
Patient-specific models can also be used as special models, as
they can involve known epigenetic changes contributing to the
disease.
iPSCs can also be used for modeling disease at the organ level
as well as understanding systemic diseases. For example human
iPSCs can be used for in vitro development of complex structures
like the retina by spatiotemporally recapitulating the in vivo steps
of retina formation and making it possible to test future therapies
in vitro (Zhong et al., 2014).
TREATING HUMAN DISEASE
Beyond modeling disease, reprogramming can also be used to
treat disease. While the technology of cellular reprogramming
has been developed and refined there have also been signifi-
cant ongoing developments in other complementary technolo-
gies such as gene editing, progenitor cell production, and tissue
engineering that we will describe below. These are converg-
ing to a point that will allow us to treat almost any disease.
Personalized medicine may soon become a reality, with cellu-
lar harvest and reprogramming, genetic engineering and tissue
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Kanherkar et al. Reprogramming for understanding and treating human disease
engineering becoming a routine procedure. These technologies
are the foundations of what is becoming a fully-functional field
of regenerative medicine.
IN VITRO REPROGRAMMING AND COMPLEMENTARY TECHNOLOGIES
Induced pluripotent stem cells
The greatest advantage of using in vitro reprogramming is that
we can generate patient-specific iPSCs (autologous) and eliminate
the threat of rejection, or have well-characterized iPSC cell-lines
(allogenic). Reprogramming in vitro is often more useful when
used with complementary technologies like gene editing to treat
genetic diseases by fixing the causative mutation and then repop-
ulating damaged or non-functional tissue with the desired healthy
cell type (Figure 5A).
Generation of iPSCs enables in vitro coordination of differ-
entiation into cells of all three germ layers (endoderm, meso-
derm, and ectoderm). Where the aim is simply to repopulate
the non-functional or damaged tissue with functional cells,
transplantation with iPSC-derived cells could cure the patient.
For example, mouse embryonic fibroblasts or mouse pancreas-
derived epithelial cells reprogrammed to iPSCs and then dif-
ferentiated to functional pancreatic beta cells were capable of
treating diabetes in mice (Jeon et al., 2012). These iPSC-derived
beta-like cells released insulin and on transplantation into non-
obese mice, normalized the blood-sugar level (Jeon et al., 2012).
If treating a disease involved fixing a genetic mutation perma-
nently, then gene-editing technologies (which are explained in
detail in section Gene editing using CRISPR/Cas9, ZFN, TALENs
etc) can be used as an additional step before differentiating
the iPSCs into the desired cell type. For example in mice, the
correction of a mutation in iPSCs by gene therapy using homol-
ogous recombination, followed by autologous transplantation
back into the mouse, has led to the replacement of diseased
cells and rejuvenation of functional tissue in single-gene defects
like sickle cell anemia. Such procedures have overcome prob-
lems associated with immune rejection and validated the role of
iPSCs in regenerative medicine (Hanna et al., 2007; Takahashi
and Yamanaka, 2013). However, autologous iPSCs will incur a
comparatively higher medical cost compared to allogenic ones,
and their creation could take a longer time compared to allo-
genic iPSCs. Also they might not be available immediately when
required for the treatment of acute injuries like spinal cord injury,
implying the need for HLA-matched donor allogenic iPSCs
(Takahashi and Yamanaka, 2013), unless they can be prepared
beforehand.
One of the most promising cell types derived from iPSCs
are neural cells (Svendsen, 2013). Neurons derived from iPSCs
demonstrate great promise in revitalizing and repopulating
the central nervous system, and have promise for use in the
development of neurologically based disease models such as
Huntington’s and Parkinson’s diseases (Kriks et al., 2011; Liu
et al., 2013b).
Potential risks associated with clinical iPSC applications
include the possibility that viral or other agents used for repro-
gramming could trigger harmful immune or inflammatory
responses (Zhao et al., 2011; Guha et al., 2013). The prevention of
vector or transgene integration into the host genome or residual
transgene expression once reprogramming has been achieved
is important in order to avoid such unwanted responses. This
problem has now been resolved with the use of non-integrating
episomal vectors to create integration-free iPSCs (Hu et al.,
2011; Su et al., 2013). The use of non-viral vectors such as the
transposon-based systems like Sleeping Beauty (SB) and piggy-
Bac (PB) offer a cost-effective and safe alternative (Talluri et al.,
2014). Several experiments to eliminate the use of more than one
or two reprogramming factors during induced pluripotency have
also been conducted (Radzisheuskaya and Silva, 2014). For exam-
ple easily accessible dermal papilla cells (specialized mesenchymal
cells endogenously expressing Sox2, Klf4, Myc) from the hair
follicles of skin have been reprogrammed with Oct4 alone and
without any other chemical compounds or small molecules (Tsa i
et al., 2011).
Gene editing using CRISPR/Cas9, ZFN, TALENs etc
As stated, the use of the combined techniques of reprogramming
and genetic engineering followed by cellular therapy can poten-
tially correct a genetic defect and repopulate a degenerated or
damaged tissue with the required cell type and cure a genetic dis-
ease. Such technology can also be used to manipulate and study
model organisms as well (Gaj et al., 2013).
Gene editing technologies are methods used to fix an endoge-
nous mutation, or to make a gene inactive for the purpose of
repairing or deleting a mutation. Recent progress in gene edit-
ing technology has made it possible to induce a site-specific DNA
cleavage followed by repair. This results in high precision genome
editing in cultured cells and whole organisms; useful in designing
therapeutic treatments and creating models in vitro and in vivo
(Kim and Kim, 2014).
Gene editing technology is based on the use of RNA sequences
or proteins that bind to specific DNA sequences and recruit nucle-
ases to introduce double strand breaks in the target sequence.
Once DNA damage occurs, homologous or non-homologous end
joining processes repair the DNA sequence. With homologous
recombination, the wrong nucleotides/s can be replaced with the
correct ones to repair a genetic mutation. Current precise gene
editing methods include the following three types depending on
the type of repair and nucleases involved: 1. Zinc finger nucleases
(ZFN), 2. Transcription activator-like effector nuclease (TALEN),
and 3. Clustered regularly interspaced short palindromic repeats
(CRISPR).
1. ZFN- This is the most commonly used type at present, and is
comprised of a eukaryotic zinc finger binding protein domain
coupled with a nuclease.
2. TALEN- This type similarly uses a transcription activator-like
effector (TALE) domain coupled with a nuclease.
ZNFs and TALENs include programmable, sequence specific
DNA binding modules linked to a non-specific DNA cleav-
age domain and are capable of introducing a variety of genetic
modifications (Gaj et al., 2013).
3. CRISPR- This is the newest type of gene editing technology
based on RNA interference and uses CRISPR and CRISPR
associated genes (Cas) from bacterial and archeal immune
systems. The CRISPR loci have short palindromic repeats
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Kanherkar et al. Reprogramming for understanding and treating human disease
FIGURE 5 | Treating disease. Stem cells (iPSCs, ESCs, and
SSCs/Progenitors) can be used in the following ways to treat acute, chronic,
and degenerative diseases. (A) In vitro reprogramming. iPSCs reprogrammed
in vitro using different combinations of transcription factors can be further
differentiated to specific lineages and these cells can be transplanted into the
patient suffering from a degenerative disease or injury. For example,
autologous iPSCs generated from fibroblasts of a patient suffering from
Parkinson’s disease can be differentiated into functional dopamine secreting
neurons and can be transplanted into the patient’s brain through surgery. In
cases where solely transplanting functional cells is inadequate to completely
cure a genetic disease, a permanent fix of the genetic mutation involved is
required. Thus, an additional step of gene correction can be performed on the
patient-specific iPSCs using gene-editing technologies like CRISPR/Cas.
Allogeneic iPSCs might be useful to treat traumatic acute injuries like spinal
cord injury, as they would be available immediately when required. (B) In vivo
reprogramming. iPSCs can be generated in vivo at the site of non-functional
or damaged tissue (by administration of reprogramming factors like OSKM)
and can then be differentiated toward a specific lineage (by the administration
of differentiation signals). For example, iPSCs generated in vivo from
pancreatic cells of a patient suffering from diabetes can be differentiated into
functional βcells secreting insulin and thus restore the lost function by
repopulating the pancreatic tissue with functional βcells. (C) In vivo
trans-differentiation. A somatic cell can be trans-differentiated in vivo into a
different type of somatic cell using transcription factors, growth factors, etc.
such that the new cell type helps to restore the lost function of the diseased
or damaged tissue. This process is noteworthy as it avoids the intermediate
step of reprogramming somatic cells to pluripotency; however this process is
not yet well-established. (D) Epigenetic rejuvenation. The idea behind
epigenetic rejuvenation is that epigenetic drugs can be used to convert
senescent cells to young cells such that aging is reversed without affecting
the differentiation or specialized function of the cell. This can be done in vivo
directly or in vitro followed by transplantation of the rejuvenated cells into the
patient. (E) In vivo regeneration/progenitor cell stimulation. Progenitors can
be induced to differentiate into their successors by injecting differentiation
signals like Wnt and Bmp proteins in order to achieve regeneration in vivo,
which will restore lost function of damaged or diseased tissue. This process
avoids procedures involving induction of pluripotency as well as
transplantation into the patient. (F) Tissue Engineering. Patient-specific iPSCs
(with optional gene correction step) or ESCs can be used to culture tissues
and organs in vitro using specialized scaffolds and organ molds or using
bioprinting. Such cultured organs can then be transplanted into a patient
through advanced surgical procedures to restore the function of an entire
organ or organs, in the case of chronic or systemic diseases, as well as to
treat acute illnesses like myocardial infarction with cardiac tissue transplants.
(24–37 base pairs) separated by unique spacer sequences of
equal length that can integrate foreign DNA, [which in the
source bacteria/archea can be used as a type of memory in
the case of a future attack by the same foreign DNA (Richter
et al., 2013)]. When a CRISPR sequence is transcribed, the
foreign DNA is transcribed along with it. The processed
RNA in the form of crRNA and tracrRNA is responsible for
sequence specific silencing by recognizing the target foreign
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Kanherkar et al. Reprogramming for understanding and treating human disease
DNA sequence and degrading it with Cas proteins that have
nuclease activity (Gaj et al., 2013).
This RNA-guided genome editing tool could be used to cor-
rect disease- associated genes in zygotes and human cell lines
(High et al., 2014). The CRISPR/Cas system can effectively
replace the traditional method of producing mutant mice by
sequential recombination since this technology allows a highly
efficient single-step approach for production of ES cells with
mutations in multiple genes, as well as generation of mutant
mice by direct embryo manipulation (Wang et al., 2013).
CRISPR- based genome editing is also useful in disease mod-
eling because it is very efficient at generating allelic isogenic
models to study diseases in vitro. For example the combina-
tion of CRISPR technology and an antibody-based method to
screen recombination events has been used to generate genet-
ically modified human cell lines for the study of Huntington’s
disease (An et al., 2014). The Type II CRISPR/Cas9 sys-
tem is gaining special recognition for its ability to accelerate
theproductionofcelllines,especiallyincombinationwith
reprogramming, and the production of animal models with
a desired genetic makeup for drug screening and pre-clinical
evaluation. Also it seems to be a desirable candidate for gene
therapy alone (Yingze Zhao, 2014).
IN VIVO REPROGRAMMING
Other potential ways of using iPSCs for treating diseases would
be by generating them in vivo. These in vivo iPSCs can fur-
ther generate desired cell types in the presence of appropriate
signals for re-differentiation (Figure 5B). As described in sec-
tion Induced Pluripotency and Epigenetics during cellular repro-
gramming, in vivo transitory induction of OSKM in mice led
to in situ reprogramming and gave rise to iPSCs from a vari-
ety of cell types (Abad et al., 2013). These iPSCs represented
totipotent-like features and they resembled ESCs more closely
compared to in vitro generated iPSCs (Abad et al., 2013). Another
in vivo study showed the generation of induced adult neurob-
lasts (iANBs) from resident astrocytes using a single TF Sox2 and
these iANBs formed mature neurons when targeted for differ-
entiation using noggin, BDNF or histone deacetylases inhibitor
(Niu et al., 2013).
IN VIVO TRANS-DIFFERENTIATION
Transdifferentiation is the process in which a particular somatic
cell is switched from one lineage-specific identity to a com-
pletely different identity (Graf, 2011; Vierbuchen and Wernig,
2012); in other words, the direct conversion of one type of
somatic cell into another type, bypassing the intermediate
step of dedifferentiation (Figure 5C). As described in Section
Transdifferentiation/direct reprogramming transdifferentiation
in vivo converted adult murine pancreatic exocrine cells to
β-cell like population using the transcription factors Ngn3, Pdx1,
and Mafa (Zhou et al., 2008). Another example is the in vivo
transdifferentiation of human peripheral blood CD34+cells
into non-hematopoietic lineages such as cardiomyocytes (Yeh
et al., 2003). However, it is important to note that such pro-
cesses occur only under the conditions of severe tissue damage
(Yeh et al., 2003).
EPIGENETIC REJUVENATION
Epigenetic rejuvenation is the concept of erasing an “aged”
epigenome to obtain an epigenome that corresponds to the one
seen in a “young” healthy cell along with maintaining its differen-
tiated state and rejuvenating its function (Manukyan and Singh,
2012). The idea behind epigenetic rejuvenation is to treat aged
cells from patients in vitro and transplant them back into the tis-
sue and replace aged or unhealthy, damaged cells (Manukyan and
Singh, 2012)(Figure 5D). (Please refer to Section Hutchinson-
Gilford Progeria Syndrome for additional details.)
IN VIVO REGENERATION/PROGENITOR CELL STIMULATION
In vivo regeneration involves the repair of tissue damage by stim-
ulation of endogenous stem cells (SSCs/progenitors) by growth
factors, cytokines, transcription factors, and second messen-
gers (Figure 5E). A promising strategy to treat degenerative and
chronic disorders resulting from loss of function of a particu-
lar type of cell, is through harnessing the intrinsic regenerative
potential of the progenitor cells which are responsible for cre-
ating the specific type of damaged cell and restoring their lost
function. Studies involving the use of regenerative factors like
developmental signals that play a role in the regeneration of a
damaged tissue, suggest that the latent/dormant intrinsic poten-
tial can be triggered to harness the in vivo regeneration capacity in
a disease or trauma-struck individual (Figure 5E). Such changes
during regeneration are accompanied by epigenetic modifica-
tions. A few examples of signaling pathways and differentiation
factors, which are being studied and could potentially activate
regeneration (thus should be considered as suitable targets) are
described below.
1. Wnt, a ligand working in conjunction with a receptor, involved
in the transcription of β- catenin promotes morphogenesis
and has the capacity to initiate the regeneration of broken bone
to treat bone deformities or injuries, as well as cause Muller
progenitors to differentiate into retinal progenitors, thereby
ameliorating retinal trauma, and possibly blindness (Minear
et al., 2010; Liu et al., 2013a).
2. Platelet-derived growth factor (PDGF) and insulin-like growth
factor (IGF) have been shown to stimulate the regenerative
capacity of mammalian skeletal and neural tissue (Rutherford
et al., 1992; Mason et al., 2000).
3. Bone morphogenic protein (BMP) is essential for ESC renewal
whereas basic Fibroblast growth factor (bFGF) increases
the differentiation potential of MSCs into chondrogenic,
osteogenic and adipogenic lineages and epidermal growth fac-
tor receptor (EGFR) increases the proliferative potential and
survival capacity of MSCs (Bhattacharyya et al., 2012).
4. Lin28 is expressed during embryogenesis as it plays an
important role in pluripotency and development, also its
overexpression promotes epidermal hair regrowth, and digit
repair in mice by altering the bioenergetic state during tissue
repair following enhanced translation of metabolic enzymes
that increase glycolysis and oxidative phosphorylation (Shyh-
Chang et al., 2013).
5. The transient inactivation of two tumor suppressors ARF
(alternative reading frame protein) and RB (retinoblastoma
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Kanherkar et al. Reprogramming for understanding and treating human disease
protein) in mammalian cells resulted in dedifferentiation
restoring regenerative capacity and produced differentiated
celltypeswhenculturedinthepresenceofpropersignaling
factors suggesting that terminally differentiated mammalian
cells can be induced for regeneration without neoplastic trans-
formation (Pajcini et al., 2010).
6. Hox genes play an important role in embryonic pattern for-
mation and can be programmed such that their previous
epigenetic memory is erased resulting in plasticity, as observed
in iPSCs, that can in turn be used to trigger regenerative
capability of injured tissue (Wang et al., 2009).
TISSUE ENGINEERING
Tissue engineering is the field of engineering tissues, or organs
grown from in vitro cultures of stem cell-derived differentiated
cells, followed by their transplantation (Figure 5F).
Tissue engineering is the de novo production of tissues or
organs using starting material like cells, extracellular matrix and
scaffolds for supporting the three dimensional process of tissue
formation (Fisher and Mauck, 2013). This technology has been
translated into actual therapies for skin replacement and cartilage
repair (Berthiaume et al., 2011).Thefirststepintissueengineer-
ing is to procure the desired cell types required for constructing
the tissue or organ. iPSCs as well ESCs serve as a good source for
producing the cells through directed differentiation. For exam-
ple, iPSC-derived multipotent neural crest stem cells (NCSCs)
have been used to engineer nerve conduits by seeding NCSCs into
nanofibrous tubular scaffolds and such nerve conduits were used
for regenerating sciatic nerve (Wang et al., 2011). Tissue engineer-
ing uses conventional methods for scaffold fabrication that are
probably inadequate (Seol et al., 2014), but the latest bioprinting
technology can construct 2-D and 3-D tissues and organs using
additive manufacturing technology that allows precise placement
of cells, biomaterial and biomolecules in an adequately fabricated
scaffold (Seol et al., 2014).
CLINICAL TRIALS FOR STEM CELL THERAPY IN HUMANS
The U.S. Food and Drug Administration defines somatic cell
therapy as the administration to humans of autologous, allo-
geneic or xenogeneic living non-germ line cells, other than
transfusion blood products, which have been manipulated, pro-
cessed, propagated or expanded ex vivo, or are drug-treated
(Health et al., 2001; Parson, 2006; Bieback et al., 2011; George,
2011).
With the FDA approving the first ESCs Phase I clinical tri-
als for acute spinal cord injury in 2011, the science behind stem
cell biology and regenerative medicine made its way to prac-
tical applications in the real world (Table 1 ). Extensive efforts
from the California Institute of Regenerative Medicine (CIRM),
National institute of Health (NIH) and other organizations
around the world has allowed stem cell therapies to progress
into clinical trials for a variety of diseases, many of which
have made significant progress. Clinical trials using mesenchy-
mal stem cells (MSCs) for treating bone and cartilage diseases,
neurodegenerative diseases, heart diseases, gastrointestinal dis-
eases, autoimmune/immune rejection diseases, and cancer have
shown a lot of progress with massive funding from CIRM,
their collaborating partners and others (Trounson et al., 2011)
(Table 1).
The most advanced clinical trials listed by EuroStemCell
include testing of therapies for bone, skin, corneal disease and
injury, the majority of which use mesenchymal stem cells. The
NIH lists two active clinical trials for the eyes diseases Age related
Macular Degeneration (AMD) and Stargardt’s muscular dystro-
phy using cells derived from human ESCs, being produced by
Advanced Cell Technology. Other sources like bone marrow stem
cells for treating cancer and blood disorders, human central ner-
vous system stem cells for AMD and human spinal cord stem cells
for ALS are also in clinical trials. Human MSCs are being used in
clinical trials by Osiris therapeutics for Type 1 diabetes, cardiomy-
opathies, Graft vs. host disease and Crohn’s disease. More details
about these ongoing clinical trials can be found through the links
for online resources provided in Tab l e 1 .
CANDIDATE DISEASES FOR TREATMENT USING
REPROGRAMMING
In this section, we provide specific examples of how reprogram-
ming and complementary technologies can be used to treat and
eventually cure human disease.
AGING AND AGE-RELATED DISEASES
A few decades ago, Richard Cutler proposed that aging was a
result of cells drifting away from their proper state of differ-
entiation due to changes in gene expression, through a process
Table 1 | Resources for tracking progress in the Field of Regenerative Medicine.
Sr. No. Topic Affiliation Access data
1. Current status of therapies undergoing
clinical trials
CIRM http://www.cirm.ca.gov/sites/default/files/files/about_stemcells/Portfolio%
20table_10-13.pdf
2. Publically and Privately supported Clinical
studies around the world
NIH http://clinicaltrials.gov/ct2/home
3. Clinical trials and stem cell treatment EuroStemCell http://www.eurostemcell.org/clinical-trials
4. Adult stem cell clinical trials Stem cell
research facts
http://www.stemcellresearchfacts.org
5. Disease information CIRM http://www.cirm.ca.gov/our-progress/disease-information
6. Current status of funding and research CIRM http://www.cirm.ca.gov/sites/default/files/files/funding_page/Portfolio%
20summary%2006-03.pdf
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Kanherkar et al. Reprogramming for understanding and treating human disease
he termed “dysdifferentiation” (Kator et al., 1985). This process
could be reinterpreted today as epigenetic drift. These changes
are so predictable that recently an “epigenetic clock” has been for-
mulated that can very accurately predict the age of an individual
based solely on the methylation of specific genes in genomic DNA
(Horvath, 2013). Indeed, epigenetic changes represent a major
aspect of aging, and epigenetic manipulation may allow reversal
of these damaging effects (Campisi and Vijg, 2009; Munoz-Najar
and Sedivy, 2011).
It has been proposed that if we can reverse the epige-
netic changes associated with aging, we could actually reverse
aging itself. This epigenetic approach to reversing aging has
been termed “epigenetic rejuvenation” (see Section Epigenetic
Rejuvenation and Figure 5D). Although the very existence of
animals cloned by SCNT proves that epigenetic rejuvenation is
possible in principle, only recently have in vitro experiments on
human cells shown that this is possible. Indeed, even centenarian
fibroblasts can be rejuvenated, and young fibroblasts cultured to
senescence can be reprogrammed to a pluripotent state (Lapasset
et al., 2011; Villeda et al., 2011; Loffredo et al., 2013). Recently,
very detailed experiments have shown that age-related and tissue-
specific DNA methylation patterns remain erased in mesenchy-
mal stem cells produced by induced pluripotency (Frobel et al.,
2014).
Epigenetic reversal also crucially includes reactivation of
telomerase expression, which resets the chromosome “aging
clock” back to a young state. Reprogramming “old” cells to
“young” cells will eventually enable the repopulation of an aged
and/or degenerated tissue with cells capable of extended prolif-
eration. Perhaps this strategy could eventually be applied to the
whole body.
An example of a type of epigenetic rejuvenation already prac-
tically applied is heterochronic parabiosis: experiments with two
animals of a different age joined together surgically, showed that
tissue-specific stem cells from an aged animal could be rejuve-
nated by exposing them to a “young” environment (Rando and
Chang, 2012; Conboy et al., 2013). This “young” environment
involves factors such as chemokine’s, cytokines and rejuvenat-
ingWntandTGFβsignaling pathways (Conboy et al., 2013). On
theotherhand“young”stemcellsadoptamoreagedstructure
and function (Conboy et al., 2005). Unlike reprogramming, het-
erochronic parabiosis maintains the differentiated states of the
cells and rejuvenates the regenerative potential of old cells in
the milieu of a young environment, thus uncoupling dediffer-
entiation and rejuvenation (Conboy et al., 2013). This type of
rejuvenation might have an epigenetic basis regulated by a switch
that could possibly convert a cell from “old” to “young” or vice
versa.
Aging also involves mitochondrial dysfunction as well as defec-
tive mitochondrial DNA. SCNT can be used to correct diseases
caused by defective mitochondrial DNA (mtDNA) because in
NT-ESCs the mtDNA exclusively comes from the oocyte, and only
the nucleus from the donor/patient, thus making it possible to
avoid defective mtDNA being carried over from the donor/patient
(Tachibana et al., 2013). Surprisingly, induced pluripotency reju-
venates mitochondira, but in a different way to SCNT (Suhr et al.,
2010).
Hutchinson-Gilford Progeria Syndrome
Hutchinson-Gilford Progeria Syndrome (HGPS) is a segmental
premature aging disease characterized by symptoms of premature
aging, with a life expectancy in the early teens or twenties. The sin-
gle point mutation responsible for causing this disease occurs in
position 1824 (cytosine is replaced with thymine) of the Lamin A
(LMNA) gene on chromosome 1. Lamin A is an intermediate fil-
ament protein that stabilizes the inner membrane of the nuclear
envelope. The mutation results in a defective Lamin A that pro-
duces an abnormal protein called Progerin. Ultimately, this results
in abnormally shaped, dysfunctional nuclei that in turn disrupt
cell division and other cellular functions (Eriksson et al., 2003;
McClintock et al., 2007; Korf, 2008).
Somatic cells from Progeria patients can be reprogrammed
to iPSCs and the LMNA mutation can be corrected using gene-
editing technology (see above). The fact that ESCs and iPSCs
do not express LMNA (Constantinescu et al., 2006), allows the
efficient correction of the mutation in iPSCs, and any disease-
related pathology can be avoided in iPSC-derived differentiated
cells. Notably, HGPS iPSCs exhibit downregulation of lamin A/C
and Progerin, causing a lack of progeroid features in the iPSC
population, rendering these iPSCs normal with respect to nuclear
morphology, pluripotency, and epigenetic profiles (Liu et al.,
2011a). HGPS iPSCs corrected for the LMNA mutation can then
be differentiated into normal somatic cells (Liu et al., 2011b).
These iPSC-derived differentiated cells could eventually be trans-
planted into patients using cellular therapy to repopulate the aged
tissue with healthy proliferative cells free of the original Progeria
mutation.
Age-related macular degeneration
Age-related macular degeneration (AMD) is a complex multifac-
torial diseases caused by the degeneration of the photoreceptors
and retinal pigment epithelium (RPE) of the eye (Yonekawa
and Kim, 2014). It is the most common cause of visual impair-
ment in the elderly (+60 years) and it is broadly classified into
two clinical categories, namely, the wet form (neovascular or
exudative) and dry form (Cook et al., 2008). AMD, which is pri-
marily known to be an age-related disease, is also affected by
other risk factors like genetics, patient history, smoking, trauma,
etc (Chakravarthy et al., 2010). Data from pre-clinical studies
suggests that transplantation with ESCs-derived RPE or patient-
specific iPSC-derived RPE (with correction of the gene mutation)
may prevent photoreceptor degeneration in animal models of
RPE degeneration (Schwartz et al., 2012; Heller and Martin,
2014). With the advent of techniques in in vivo reprogram-
ming, another promising approach to cure AMD is to target
the intrinsic potential of neural progenitors in a patient and
drive their differentiation into RPE through the administration
of appropriate differentiation signals. It has already been sug-
gested that retinal stem cells (RSCs) that are multipotent, rare
cells in the pigmented ciliary epithelium of peripheral retina
can be enriched an directed to differentiate into photorecep-
tors using factors influencing neural retinal development (Ballios
et al., 2012). Specialized knowledge of such differentiation and
proliferation signals with the development of advanced tech-
niques toward delivery and transplantation of stem cells or RPE
Frontiers in Cell and Developmental Biology | Stem Cell Research November 2014 | Volume 2 | Article 67 |14
Kanherkar et al. Reprogramming for understanding and treating human disease
lead us one step closer to the development of clinical therapies
for AMD.
EPIGENETIC DISEASES
Epigenetic diseases are caused by altered expression of genes
caused by abnormal changes in the epigenetic profile of a cell.
Reprogramming can be used to reset the epigenetic profile of any
cell so that the cell becomes pluripotent. It can then be differenti-
ated to obtain the cell type with a desired epigenetic profile. Such
procedures followed by cellular therapy might be a potential treat-
ment for epigenetic diseases caused by known epigenetic factors.
Let us consider the example of polycystic ovary syndrome (PCOS)
as an epigenetic disease.
PCOS is a heterogeneous endocrine disorder responsible for
a large proportion of female subfertility (Azziz et al., 2004).
The hallmarks of the disease are anovulation, excessive andro-
genic hormones, and insulin resistance. The PCOS phenotype
is characterized by hypersecretion of both luteinizing hormone
as a result of compromised hypothalamic sensitivity, and insulin
in response to increased abdominal adiposity (Escobar-Morreale
et al., 2005). Familial clustering of cases and the heritability of
endocrine and metabolic aspects of PCOS indicate a dominant
regulatory gene with incomplete penetrance and strongly indicate
a genetic etiology (Diamanti-Kandarakis et al., 2006).
As described in Section Epigenetics above, environmental fac-
tors can induce epigenetic alterations, which potentially involve
trans-generational inheritance of methylation patterns. The expo-
sure of female rats to a mixture of pesticides showed that the
first and third generation offspring were afflicted with PCOS
(Manikkam et al., 2012). This demonstrated that PCOS might
have an epigenetic basis caused by a differential methylation pat-
tern in the diseased offspring. In humans aberrant gene methyla-
tion has been reported in patients with PCOS, suggesting a role
for a bZIP TF, CEBPB, in insulin resistance and regulation of the
interleukin-1 response element in the IL-6 gene and other genes
important for the immune and inflammatory components of this
disease (Natsuka et al., 1992; Harries et al., 2012; Shen et al.,
2013). Inappropriate epigenetic reprogramming is a contributing
factor in PCOS. Epigenetic alterations of peroxisome proliferator-
activated receptor gamma 1 (PPARG1), nuclear co-repressor 1
(NCOR1), and histone deacetylase 3 (HDAC3) genes in granulosa
cells are induced by hyperandrogenism (Qu et al., 2012).
Recently, PCOS-derived hESCs have been successfully isolated
from the inner cell masses of blastocysts. These hESCs retain
pluripotency, which allows the dissection of the in vitro patho-
genesis of the disease. Eventually cell therapies will be developed
for this disease.
COMBINED GENETIC AND EPIGENETIC DISEASES
Some diseases long known to have genetic causes are now thought
to also have epigenetic bases. Just as with epigenetic diseases,
such diseases can be treated using reprogramming to re-establish
the normal epigenetic profile but with the addition of gene
correction. One of the most well-known such diseases is cancer.
Cancer
Cancer remains the leading cause of death worldwide with over
7.6 million deaths (13%) in 2008 (Jemal et al., 2011). Cancer
does of course have a partly genetic basis: most human can-
cers are caused by gene mutations in one or more components
of the p53 and/or the pRB pathway (Campisi, 2001). And most
tumor suppressor genes can be classified into two types; Caretaker
tumor suppressors which prevent DNA damage and Gatekeeper
tumor suppressors which eliminate potential neoplastic cells via
apoptosis (Campisi, 2005).
But besides the genetic mutations, cancer is also an epigenetic
disease. The dysdifferentiation hypothesis of aging and cancer
emphasizes the importance of instability of the differentiated
stateofcellsduetoimpropergeneregulationinthedevelop-
ment of cancer (Zs-Nagy et al., 1988). Cancer is comprised of the
following eight hallmarks, some of which may have a wholly epi-
genetic etiology: sustained proliferative signaling, evading growth
suppressors, enabling replicative immortality, inducing angio-
genesis, resisting cell death, activating invasion and metasta-
sis, reprogramming of energy metabolism and evading immune
destruction. (Hanahan and Weinberg, 2011). Reprogramming of
metabolic pathways contributes to the development of the disease,
which is evidently seen in cancer with upregulation of oxida-
tive phosphorylation and glycolysis-associated tarnscriptomes
(Lisanti et al., 2014). For example, cancer-associated fibroblasts
found in tumor microenvironments have a distinctive energy
metabolism reprogramming phenotype responsible for prolifer-
ation, migration, invasion and epithelial-mesenchymal transition
(Tang et al., 2014).
Cancer cells have altered DNA methylation profiles such that
the overall methylated DNA is less than that seen in normal
cells. A study testing epigenetics as a molecular marker sys-
tem for cancer noted 12 genes to be associated with promoter
hypermethylation causing silencing of their respective genes.
These were functional in cell cycle regulation, tumor suppres-
sion, DNA repair, metastatic potential and apoptosis and have
been linked to 15 major tumor types, making epigenetics a
significant tool in cancer detection and typing (Esteller et al.,
2001). Epigenetic changes in cancer are becoming well-noted and
epigenetic drugs like DNA methylation inhibitors and histone
deacetylase inhibitors have shown promising results in clinical tri-
als and are less toxic than the conventional chemotherapy drugs
(Chen et al., 2014). Epigenetic modulators may be used to inhibit
self-renewal and survival of quiescent cancer stem cells (CSCs) by
making them sensitive to targeted or conventional chemotherapy
(Li and Bhatia, 2011).
Other diseases
Genetic factors contributing to epigenetic variation like DNA
sequence variants, differential expressions of genes that regu-
late chromatin remodeling and DNA methylation, can contribute
toward an epigenetic etiology of a disease (Bjornsson et al., 2004).
Familial forms of neurodegenerative diseases like Alzheimer’s
disease, Huntington’s disease, Parkinson’s disease, and ALS are
known to have a genetic basis but increasing evidence points
toward epigenetic alterations like DNA methylation, histone tail
modifications, and RNA mediated mechanisms in sporadic forms
(Kleivi, 2006).
Some autoimmune diseases such as systemic lupus erythema-
tous, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes
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Kanherkar et al. Reprogramming for understanding and treating human disease
mellitus suggest the contribution of environment to a genetically
pre-disposed trait (Hewagama and Richardson, 2009). For exam-
ple, Immunodeficiency chromosomal-instability facial anomalies
syndrome (ICF) is one such disease, which is caused by mutation
in the gene for DNA methyl transferase (DNMT3B) that further
leads to abnormal methylation causing varied gene expression
(Ehrlich, 2003; Bjornsson et al., 2004; Rodenhiser and Mann,
2006).
OtherexamplesincludethedownregulationoftheReelin
gene (RELN) in schizophrenia which leads to the reduction of
RELN protein by 50% compared to a normal brain and this has
been linked to an epigenetic modulation of the promoter of the
RELN gene through increased expression of DNMT1 (Costa et al.,
2002; Sharma, 2005; Rodenhiser and Mann, 2006), and Rett syn-
drome, mostly caused by a mutation in the gene MeCP2 that
produces methyl-CpG- binding protein, is an X linked domi-
nant neurodevelopmental disorder (Bienvenu et al., 2000). The
loss of the MeCP2 protein results in loss of transcriptional repres-
sion and hence inappropriate transcription of downstream genes
during brain development (Van Den Veyver and Zoghbi, 2001;
Rodenhiser and Mann, 2006).
INFECTIOUS DISEASES
One of the most promising strategies to treat an infectious dis-
ease is to replace a receptor on a host target cell, which is used by
a pathogen to gain entry into the cell, with a mutated one. This
makes the receptors on the target cell non-functional and blocks
the entry of the pathogen into the cell, preventing its multipli-
cation and the progression of the disease. One important disease
that may eventually be universally treated and perhaps eradicated
this way is acquired immunodeficiency syndrome (AIDS). AIDS
is a disease of the human immune system caused by infection
with human immunodeficiency virus (HIV). It is a major pub-
lic health concern worldwide, with more than 30 million people
infected (Kallings, 2008; Unaids, 2013).Thereisnocureorvac-
cine at present, but antiretroviral treatment (ART) can slow the
course of the disease and result in near-normal life expectancy.
ART reduces the risk of death and complications from the dis-
ease, but is expensive and associated with side-effects. We need to
find better treatments.
Of note, one of the molecular mechanisms of HIV infection
and resulting AIDS is depletion of CD4+T cells. All cells suscep-
tible to HIV infection (CD4 T cells, macrophages, and dendritic
cells) are derived from hematopoietic stem cells. Following infec-
tion, most CD4+T cell loss occurs in the intestinal mucosa
which harbors the majority of the lymphocytes found in the body
(Mehandru et al., 2004).
C-C chemokine receptor type 5, also known as CCR5, is a
protein on the surface of white blood cells. It functions as a
receptor for chemokines allowing T cells to hone in on tar-
get tissues and organs. HIV often uses CCR5 to enter and
infect host cells. The majority of mucosal CD4+T cells express
the CCR5 protein, which HIV uses as a co-receptor to gain
access to the cells (Brenchley et al., 2004). HIV seeks out and
destroys CCR5-expressing CD4+T cells during acute infection.
However, certain individuals carry a mutation known as CCR5-
32, which renders the receptor non-functional and prevents
entry of HIV into the cells. In humans the CCR5 gene is located
on chromosome 3. A 32 mutation results in the genetic dele-
tion of a portion of the CCR5 gene. Homozygous carriers of
this mutation are resistant to certain strains of HIV-1 infection
(Samson et al., 1996). This mutation, although having neg-
ative effects on T-cell function, can protect these individuals
against HIV.
Future reprogramming technology may allow generation of
CCR5-deficient HSCs derived from hESCs/iPSCs, perhaps by cre-
ating iPSCs from a CCR5-deficient individual (Ledran et al.,
2008; Hutter et al., 2009), or even better from an existing
patient using gene editing technology. Diseases such as HIV
could potentially be cured by means of hematopoietic cell trans-
plantation whereby HSCs or even fibroblasts are first dediffer-
entiated into continuously growing iPSC lines using induced
reprogramming, followed by gene editing to introduce the 32
mutation and then directed differentiation toward the desired
hematopoietic lineage followed by bone marrow transplanta-
tion (Kambal et al., 2011). The use of engineered zinc finger
nucleases (ZFNs) to disrupt the CCR5 gene in human HSCs
in vivo suggested that ZFN mediated autologous HSC mod-
ification could provide a permanent supply of HIV-resistant
CD34+HSC progeny which as well would be capable of immune
reconstitution and long-term protection against viral replication
(Holt et al., 2010).
In principle this basic idea could be applied to other infectious
diseases that rely on a single or even multiple receptors to gain
entry into human cells.
CONCLUSION
The last decade has seen a boom in the field of stem cell biol-
ogy and regenerative medicine coupled with extensive studies
on the epigenetics involved in cellular reprogramming and dis-
ease. Nuclear reprogramming experiments beginning in the early
1950’s introduced the concept of generation of pluripotent cells
from somatic cells through nuclear transfer. The discovery of
hESCs in the late 1990’s acquainted us with a source for designing
therapies to treat a broad range of diseases. Following that came
the invention of iPSC technology, bringing stem cell production
and its use in medicine out of an ethical quandary. Today, stem
cell therapies have advanced into clinical trials and we predict
that it will not be long before regenerative medicine units become
established at health care centers.
Similarly, epigenetic studies have revolutionized the classical
genetic approach toward studying and treating hereditary dis-
eases. As we embark on this journey to understand every aspect of
the interlocking pathways and processes underlying aging, cancer,
regeneration, and repair, we still need to completely decipher the
epigenetic code in order to design and implement effective and
safe stem-cell based therapies to treat age-related diseases, acute
injuries and perhaps chronic disorders.
A full understanding of epigenetics, reprogramming, senes-
cence, cancer, and regeneration will drive remarkable progress
in achieving cellular and rejuvenation therapies as well as
designing drugs for diseases with an epigenetic basis. The
best approach is to embrace all of these fields in a unifying
synthesis.
Frontiers in Cell and Developmental Biology | Stem Cell Research November 2014 | Volume 2 | Article 67 |16
Kanherkar et al. Reprogramming for understanding and treating human disease
ACKNOWLEDGMENTS
The Authors would like to thank Dr. Donald Orlic for critical
review of the manuscript.
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Conflict of Interest Statement: The authors declare that the
research was conductedin the absence of any commercial or finan-
cial relationships that could be construed as a potential conflict of
interest.
Received: 18 June 2014; accepted: 27 October 2014; published online: 12 November
2014.
Citation: Kanherkar RR, Bhatia-Dey N, Makarev E and Csoka AB (2014) Cellular
reprogramming for understanding and treating human disease. Front. Cell Dev. Biol.
2:67. doi: 10.3389/fcell.2014.00067
This article was submitted to Stem Cell Research, a section of the journal Frontiers in
Cell and Developmental Biology.
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www.frontiersin.org November 2014 | Volume 2 | Article 67 |21
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Early life stress (ELS) induces long-term phenotypic adaptations that contribute to increased vulnerability to a host of neuropsychiatric disorders. Epigenetic mechanisms, including DNA methylation, histone modifications and non-coding RNA, are a proposed link between environmental stressors, alterations in gene expression, and phenotypes. Epigenetic modifications play a primary role in shaping functional differences between cell types and can be modified by environmental perturbations, especially in early development. Together with contributions from genetic variation, epigenetic mechanisms orchestrate patterns of gene expression within specific cell types that contribute to phenotypic variation between individuals. To date, many studies have provided insights into epigenetic changes resulting from ELS. However, most of these studies have examined heterogenous brain tissue, despite evidence of cell-type-specific epigenetic modifications in phenotypes associated with ELS. In this review, we focus on rodent and human studies that have examined epigenetic modifications induced by ELS in select cell types isolated from the brain or associated with genes that have cell-type-restricted expression in neurons, microglia, astrocytes, and oligodendrocytes. Although significant challenges remain, future studies using these approaches can enable important mechanistic insight into the role of epigenetic variation in the effects of ELS on brain function.
... Fifth, since the landmark recognition of induced pluripotent stem cells, there has been a series of discoveries of small numbers of transcription factors which control cell identity [20,21]. These have the potential for manufacturing cells for personalized and regenerative medicine [22,23], drug development [24] and disease modelling [25]. These special combinations of transcription factors give rise to a cascade event which ultimately controls the cell identity. ...