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JSCRT Volume-1 | Issue-1 November, 2016
Journal of Stem Cells and Regenerative Therapy (JSCRT)
Modern Alchemy: Cellular Reprogramming and Transdifferentiation
*Summer A. Helmi 1, 2, Derrick E. Rancourt 1
1 Department of Biochemistry and Molecular Biology, University of Calgary, Canada
2 Department of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt.
Review Article
Received 01/11/2016 Accepted 22/11/2016 Published 23/11/2016 *For Correspondence Summer A. Helmi Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Canada Contact no: 001-403-220-2888 Email: summer.helmi@ucalgary.ca Keywords: Stem cell, Cellular reprogramming, Nuclear transfer, Cell fusion, transdifferentiation, Regenerative medicine.
ABSTRACT
The early conversion of a terminally differentiated somatic cell nucleus to totipotency by nuclear transfer and the consequent development of a whole organism pioneered the field of cellular reprogramming. Since then, several studies have demonstrated that the forced expression of lineage-specific transcription factors in adult cell types can convert cell fate from one lineage to another. The consequent breakthrough of generating induced pluripotent stem cells by reprogramming adult fibroblasts to pluripotency using the transduction of the Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc), opened new horizons in the fields of regenerative and personalized medicine. But the problems of reprogramming efficiency and tumor formation associated with iPSCs called for the development of other reprogramming techniques such as direct reprogramming both in vitro and in vivo. Direct cellular reprogramming in vivo, also known as in situ Transdifferentiation, is a new method to alter cell fate within the tissue that requires replacement or repair. This can be time and cost effective and may overcome some of the disadvantages associated with other methods of cellular reprogramming. In this article, we review the history of cellular reprogramming, highlighting the recent development of in situ transdifferentiation.
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INTRODUCTION
Alchemy was a protoscience that focused on the transmutation of common, base metals such as lead
into desirable, precious metals such as gold. Although the ability to generate gold from lead never
occurred, its tradition led to the disciplines of metallurgy, chemistry and nuclear physics. In biology, it
can be argued that a modern form of alchemy has arisen through the emerging field of cellular
reprogramming, wherein common, base cell types such as fibroblasts are transmutated into desirable,
precious cells such as pancreatic islets or cardiomyocytes. The early experiments of nuclear transfer
transformed older concepts that supported the irreversible silencing or deletion of genes during the
course of differentiation [1]. Following nuclear transfer, cellular reprogramming achieved wide steps; it
was crowned by the generation of induced pluripotent stem cells (iPSCs) [2]. This breakthrough came
with the hurdles of reduced reprogramming efficiency and tumor formation. Realizing cellular
reprogramming as a clinical approach led to the development of direct reprogramming, also known as
Transdifferentiation [3]. This review covers the history of cellular reprogramming beginning with the early
experiments in somatic cell nuclear transfer and ending with the introduction of in situ
Transdifferentiation.
CELLULAR REPROGRAMMING
Cellular Reprogramming via Nuclear Transfer
The ability of an oocyte to reprogram a somatic cell nucleus to totipotency and the subsequent
development of a new organism by nuclear transfer changed the long believed theory that cell
differentiation is the result of eliminating the genes that were silenced and keeping the genes that were
expressed in the tissue [1]. The early nuclear transfer experiments carried out by Briggs and King
successfully resulted in normal tadpoles in the frog Rana pipiens. This was achieved by transplanting a
blastula cell nucleus into an enucleated egg [4]. However, in a follow-on experiment, tadpoles failed to
develop normally after nuclear transfer of an endodermal cell nucleus derived from a neurula stage
embryo. They concluded that as development proceeds some genes needed for normal development
had either been lost or were irreversibly repressed [5].
Gurdon did similar experiments with the embryos of Xenopus laevi [1]. Unlike Rana pipiens, Xenopus
laevi has a shorter life cycle and can produce eggs all over the entire year by hormonal stimulation.
However, its most important characteristic is the anucleolate mutation, which in the heterozygous state
provided a live cell nuclear marker with which cells of a donor origin could be distinguished from those
that resulted from failed recipient egg enucleation [6]. Gurdon transferred the nuclei from blastula and
gastrula stages to eggs that proved to be of high quality, and that led to the development of normal
swimming tadpoles. However, the number of healthy tadpoles decreased as the stage of donor embryo
development progressed. Using similar results produced by Briggs and King, Gurdon concluded that
there is no exact relation between nuclear differentiation and tissue differentiation. He suggested that
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nuclear differentiation may be more related to the processes which take place when individual cells
become differentiated into their final functional state [7].
Nuclear transfer experiments later succeeded in mammals. Morula stage embryos were developed after
transferring labeled rabbit morula cell nuclei into enucleated rabbit eggs [8]. Early experiments in mouse
initially failed because somatic cell nuclei were transferred into enucleated zygotes instead of
unfertilized eggs. One exception was when live mice were produced by the transfer of a zygote donor
nucleus into an enucleated zygote [9]. The success of this latter experiment was thought to be due to the
same developmental state of donor and recipient cells. Afterward, donor cell nuclei from later
developmental stages failed to develop [10]. Although all experiments performed on amphibians used
unfertilized eggs, it was believed, perhaps incorrectly, that zygote cytoplasm could support development
better than unfertilized egg cytoplasm. Other nuclear transfer experiments using embryonic donor cell
nuclei from other mammalian species included pigs [11], cows [12] and monkeys [13]. In species where
nuclear transfer was problematic, nuclei were transferred into egg cytoplasm and, the next day, to
zygote cytoplasm [14].
Nuclear transfer experiments led to the idea of cloning livestock for purposes of genetic engineering. In
1996, Wilmut’s group demonstrated in sheep that an embryonic cell line nuclei could be used to
generate cloned sheep via transfer when fused to an enucleated egg [15]. A step forward used adult
mammary gland cells resulted in Dolly the sheep [16].
Serial cloning of mice to four and six generations in two independent lines was also claimed to be
successful. Although gross behavioral parameters showed no signs of premature aging, telomeres
appeared to increase slightly in length. In fact, only one cloned mouse was born in the sixth generation
from more than 1,000 nuclear transfers. This mouse was eaten by its foster mother, suggesting that it
was unhealthy [17].
Adult cloned mice were obtained from mature lymphocytes which carried differentiation-associated
immune-receptor rearrangements [18]. Genetically labeled post-mitotic olfactory neurons were also used
as nuclear donors to clone adult mice [19, 20]. Unfortunately, a very low number of the resulting animals
produced by transferring the nuclei of adult or differentiated cells resulted in normal offspring.
Developmental and physiological abnormalities were found in a large number of embryos obtained,
especially in the placentas. Because many of these abnormalities were not heritable, it is hypothesized
that they were not caused by deficiencies in chromosome replication, but by a failure to reprogram
epigenetic characteristics of somatic cells, especially imprinted genes [21, 22]. In support of this argument,
serial cloning of mice via pluripotent embryonic stem cells (ESCs; where ESC lines were derived from
each serially cloned embryo and used as donor cells) proved to be more efficient than cloning using
adult cells. Some of the produced clones further developed to fertile adults [23, 24].
The ability to derive ESC lines from cloned embryos made the idea of cloning humans using the
justification that “therapeutic cloning” could be used to generate patient-specific ESCs in order to
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generate cells and tissues that would, in theory, escape immune rejection. Advanced Cell Technology, an
American company conducted an unsuccessful experiment to produce cloned human embryos. Adult
cumulus cells in addition to skin fibroblasts were used as donor cells. With cumulus cells as donors,
embryos did not survive past the six- cell stage, while eggs reconstituted with adult fibroblasts
developed pronuclei but no cleavage was evident [25]. Another discredited study claimed that 11 human
ESC lines were created by somatic cell nuclear transfer using skin fibroblasts but were later shown to be
fraudulent [26]. With the advent of iPSCs that occurred shortly after this drama [27], the furor to generate
patient-specific ESCs via therapeutic cloning soon subsided.
Cellular Reprogramming via Cell Fusion
Fusing cells to create hybrids was also proposed as another promising method for cellular
reprogramming at the time. Embryonic cells were fused with adult cells to test the plasticity of
terminally differentiated cells. Findings showed that the resulting hybrids always shifted towards the
phenotype of the least differentiated cell forming the hybrid. Pluripotent PCC4aza1 embryonal
teratocarcinoma cells were fused with adult thymocytes. The resulting hybrids formed tumors
characteristic of embryonal carcinoma cells. These results suggested that the embryonal carcinoma cells
reprogrammed the adult thymocytes to form tumors [28]. In an embryonic germ cell ‐lymphocyte hybrid,
suppression of lymphocyte‐specific gene expression was apparent [29]. These and other studies argued
the repression of the adult somatic cell gene expression in hybrid cells containing pluripotent nuclei. For
example, it is possible that repression of the somatic nucleus occurs before epigenetic modifications
take place in order to suppress inappropriate gene expression while these modifications proceed [30].
Cellular fusion experiments were later directed towards the possibility of applying this approach to
human cells. Human embryonic stem cells were fused with human fibroblasts. The resulting hybrid cells
ma intained a stable tetraploid DNA content. The hybrids also had the morphology, antigen expression
and growth rate of human embryonic stem cells. Differentiation of these hybrids both in vitro and in vivo
yielded cells from the three germ layers. Results also showed that the somatic genome was
reprogrammed to an embryonic state. These results established that human embryonic stem cells are
capable of reprogramming the somatic nuclei to pluripotency [31].
Cellular Reprogramming using Transcription Factors
The idea of the egg cytoplasm being able to reprogram somatic cell nuclei regardless of low efficiency
opened the door for further advances in the field of somatic cell nuclear reprogramming. Mouse
fibroblast cell cultures treated with 5-azacytidine or 5-aza-2’-deoxycytidine contained functional
contractile striated muscle cells, adipocytes and chondrocytes capable of the producing cartilage-
specific proteins [32]. In a follow-on study, a transfected DNA locus, which was believed to respond to 5-
aza-2’-deoxycytidine (5-aza-c, converted fibroblasts into myoblasts. Screening done to identify the locus
narrowed the search to MyoA, MyoH and MyoD transcription factors. Only transfection with MyoD
cDNA converted fibroblasts to stable myoblasts [33]. The discovery of MyoD indicated that
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overexpression of a key transcription factor was enough to change and override the endogenous gene
expression pattern of a cell. MyoD was later used to convert chondroblasts, retinal-pigmented epithelial
cells and smooth muscle cells into mononucleated myoblasts and multinucleated myotubes. These data
showed that the four terminally differentiated phenotypes responded to MyoD in the same way.
Moreover, this response followed the same sequence observed during normal myogenesis [34]. These
observations generated the hypothesis that transcription factors, which determine cellular identity
during development, can alter cell fate when they are ectopically expressed in certain heterologous cells.
After the discovery of MyoD, several trials were carried out to change the phenotype of one blood cell
type to another. For example, splenic B cells were reprogramed to functional macrophages through the
forced expression of C/EBPα. This achievement was believed to be due to the inactivation of Pax5,
known to be responsible for the commitment of progenitors to become mature B lymphocytes [35].
Overexpression of C/EBPα in pre-T cells induced the formation of functional macrophages, while Gata-3
addition inhibited this C/EBPα-induced lineage conversion [36]. Gata-1 overexpressed in progenitors
committed for neutrophilic monocytes reprogrammed them into erythroid as well as basophilic and
eosinophilic cells [37]. Conditional Pax5 deletion in mice caused mature B cells from peripheral lymphoid
organs to dedifferentiate in vivo progenitors in bone marrow, which is believed to be the cause of the
increased T cell numbers in the thymus of T-cell-deficient mice [38].
Induced Pluripotent Stem Cells
Takahashi and Yamanaka first hypothesized that the transcription factors, which maintain embryonic
stem cell (ESC) pluripotency have the potential to turn somatic cells back to the pluripotent state. 24
candidate transcription factors known to be involved in pluripotency of experiments were reduced to
four factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM), able to reprogram mouse fibroblasts into an ESC-like
state [27]. Yamanaka and colleagues also reported that human-induced pluripotent stem cells (iPSCs)
could be reprogrammed using this same combination of factors [39]. Human iPSCs have pluripotent
characteristics that make them capable of giving rise to more than 200 cell types [27]. The phenomenal
success of Yamanaka and his team brought an end to nuclear transfer and cell fusion experiments,
especially when directed towards generating patient-specific pluripotent cells.
The Yamanaka factors were used to derive iPSCs in multiple species including rats and rhesus monkeys [40, 41]. Furthermore, several cell sources have been used for the generation of iPSCs such as neural cells [42], keratinocytes [43], liver cells [44] and terminally differentiated blood cells [45]. Reprogramming of
human fibroblasts to iPSCs has also been achieved using a different combination of reprogramming
factors. For example, Oct4, Sox2, Nanog and Lin28 [46].
Since the development of iPSCs, understanding chromatin changes and chromatin dynamics during the
process of reprogramming became essential. Initial studies have focused on the mechanism by which
the four key reprogramming transcription factors work. The state of ESC chromatin is known as “open”.
In open chromatin state, heterochromatin is disperse and dynamic, indicating a hyperactive
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transcriptional state [47]. A particular network of transcription factors with epigenetic proteins that
interact with DNA is necessary to maintain ESCs pluripotency. iPSCs and ESC share the same
characteristics; iPSCs have to sustain the same molecular structure as ESC. Moreover, they have to
overcome the epigenetic barrier of closed chromatin during the reprogramming process. Hence, the
reprogramming process involves a cascade of transcription factors that interacts with chromatin
modifier enzymes and histone related enzymes.
It has been suggested that the reprogramming process is initiated by the exogenous transcription
factors causing epigenetic changes. These transcription factors either interact with the DNA, via histone
modifiers or interact together with chromatin remodeling factors [39]. Thus, there are known time points
where the OSKM factors start the reprogramming process. Certain transcription factors are capable of
interacting with DNA or with a chromatin remodeling enzyme. The interaction depends on which gene is
activated. Briefly, the locus, the type of transcription factor and the context determine this molecular
mechanism [39]. Oct4 was found to be indispensable for reprogramming. In some studies, Oct4 alone is
sufficient for reprogramming to take place [48]. Furthermore, Oct4 also has a significant effect on the
reprogramming process when combined with Sox2.
Oct4 and Sox2 are known to form a heterodimer that interacts with some promoters. Besides, this
heterodimer was found to interact with Nanog. Nanog is a transcription factor that is not part of the
reprogramming cocktail presented by Yamanaka and his colleagues [39]. However, its importance lies in
participating in the ESC regulatory circuitry together with Oct4 and Sox2 to maintain pluripotency. When
Oct4 and Sox2 interact with promoters in DNA, Nanog is also involved in the interaction [49]. Oct4 and
Sox2 can activate transcription in a chromatin independent manner through interaction with other co-
activators [50]. In this context, studies showed that Oct4 and Nanog together interact with repression
complexes in mouse ESCs. These complexes are often histone deacetylases such as Mta1 [51].
c-Myc is an important participant as it recruits multiple chromatin modifiers, such as histone
acetyltransferases GCN5, p300 and histone deacetylases HDACs. c-Myc allows the augmentation of
methylation in H3K4me3 site and the global acetylation. During the reprogramming process, c-Myc,
activates its target before other core pluripotency transcription factors are activated, which facilitates
opening the chromatin for other factors [52]. An example of c-Myc’s potential in opening chromatin is its
association with Tip60-p400 a complex, which acetylates and remodels nucleosomes, respectively. p400
is a part of the Swi2/Snt2 family that is famous among the ATPase chromatin remodeling enzymes,
exchanging histones H2AZ-H2B within nucleosomes [53]. It also functions to release RNA polymerase
from a paused state from only about one-third of the genes that are being actively transcribed. This
activity might be enhancing the reprogramming of the cells [54].
Klf4 activates the transcription of Sox2, which participates in the pluripotency cascade through JAK-
STAT3, phosphatidylinositol 3-kinase (PI(3)K), also known as Akt and extracellular signal-regulated
kinases (Erk) pathway. LIF first reaches the receptor gp130 and sends a signal to JAK, which later
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phosphorylates Stat3 to activate Klf4. Klf4 will then activate Sox2, which in sequence activates Oct3/4,
thus promoting pluripotency [55].
Direct cellular reprogramming
In his famous model of the epigenetic landscape (Fig. 1), Waddington suggested that cells committed to
a specific lineage couldn’t be recommitted to another lineage or change channels due to energetics [56].
Yamanaka used the same analogy of channels from Waddington, proposing that cells could be pushed
back up the channel towards pluripotency. This stochastic reprogramming model also predicted that
through the process of partial reprogramming, cells could be redirected to down other lineages or
channels [57].
Figure 1. Stochastic Model of Cellular Reprograming Proposed by Yamanaka.
Besides undergoing the natural process of differentiation, a cell can be pushed up to the pluripotent state, or partway to a progenitor state where it is then free to differentiate towards another lineage.
Cells can also pass to senescence or apoptosis.
Transdifferentiation (Fig. 2) refers to the conversion of one terminally differentiated cell state to another
without regressing to pluripotency. It is also referred to as lineage reprogramming or direct conversion
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[58]. Transdifferentiation allows avoiding the iPSCs state, which can result in faster reprogramming with
less chance for tumor formation [59].
Figure 2: Transdifferentiation
Fibroblasts are turned into different cell types in vitro including hepatocytes, neurons, muscle cells,
blood cells and melanocytes.
The first Transdifferentiation attempt was achieved before the discovery of iPSCs when MyoD was used
to change the fate of fibroblasts to muscle cells [32]. MyoD’s discovery indicated that over-expression of a
key transcription factor was necessary and sufficient to change and override the endogenous gene
expression pattern of a cell. However, these experiments demonstrated that changes could only be
achieved within cells from the same germ layer, in this case, mesoderm [60].
Since then, many transcription factor cocktails have been discovered for transdifferentiating one cell
type to another, beyond germ lineages. For example, 13 transcription factors responsible for cardiac
differentiation were narrowed down to combining Gata4, Mef2c and Tbx5 turning mouse dermal
fibroblast directly into differentiated cardiomyocytes [61]. Similarly, 19 transcription factors deemed to
be essential for neural differentiation were narrowed down to Ascl1, Brn2 and Myt11 in order to convert
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mouse fibroblasts into functional neurons [62]. Other transcription factor cocktails and resulting cell types
are shown in Table 1.
Table 1: Direct cellular reprogramming conducted using transcription factors
Cell Source Transcription Factors Resulting type Reference
Mouse embryonic
fibroblasts.
Hnf4α plus Foxa1,
Foxa2 or Foxa3
Hepatic like cells [89]
Human fibroblasts Sox10, MITF, and
Pax3
Melanocytes [90]
Mouse fibroblasts. Erg, Gata2, Lmo2,
Rux1c, and Scl
Blood cells [91]
Human fibroblasts Klf4, c-Myc + chon-
drogenic factor SOX9
Chondroblasts
[92]
Human gingival
fibroblasts
Oct4,l-Myc, Runx2
and Osterix
Osteoblasts [93]
Human endothelial
progenitor cells
MYOCD Smooth muscle
cells
[94]
Another Transdifferentiation approach, proposed as an alternative to transcription factor screening,
uses the OKSM factors to partially revert cells so that they can be pushed forward either using chemicals
or growth factors. The OKSM factors are expressed regenerating tissues such as the zebrafish tail fin
blastema. This finding suggests that partial reprogramming may be necessary for regeneration [63]. Thus,
the presence of these pluripotency factors in a differentiated cell may have the same effect of loosening
chromatin arrangement and placing the cell in a plastic position where it can be directed to become any
type of cell [64]. However, rather than completely reverting cells this alternative Transdifferentiation
strategy interferes with the acquisition of pluripotency using inhibitors.
Leukemia Inhibitory Factor (LIF) is part of the interleukin (IL)-6 family of cytokines and is considered to
be a very important driver of pluripotency [65]. LIF controls pluripotency by targeting the JAK STAT3, JAK
PI3K and JAK Erk signaling pathways. These pathways cause activation of the core circuitry of
pluripotency transcription factors Oct3/4, Sox2, and Nanog [66]. Part of the OKSM Transdifferentiation
method involves blocking the reprogramming process before reaching pluripotency. This process can be
achieved through removal of LIF from the cell culture media and inactivating the JAK/STAT pathway
using JAK inhibitor (JI1) during the reprogramming process.
The second part of this method uses chemicals and/or growth factors to redirect differentiation towards
the desired cell lineage (Fig. 3). Successful Transdifferentiation of mouse embryonic fibroblasts into
beating cardiomyocytes was first achieved using this method. This study suggested that
Transdifferentiation using OSKM factors was more efficient in producing cardiomyocytes compared to
using iPSCs [67]. Neural progenitor cells have also been successfully produced using this same platform [48].
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Figure 3. Direct Reprogramming using OSKM factors.
JI and the absence of LIF puts the cells in a plastic state preventing them from returning to the iPSC state.
The use of transcription factors for cellular reprogramming have some safety concerns. These concerns
include mutagenesis and tumor formation. Understanding the molecular mechanisms by which
transcription factors work led to the identification of their downstream molecules. Moreover, figuring
out their role in lineage reprogramming, lead to one of the most promising potential solutions, which is
cellular conversion using small molecules. Small molecules have multiple important advantages over
using transcription factors for regulating cell fate: they can be more cost-effective and cell permeable.
Their synthesis, preservation, and standardization are easier. More importantly, the effects of small
molecules can be optimized by changing concentrations and combinations, providing a higher degree of
control over protein function [68].
The molecular mechanism of small molecules during the process of Transdifferentiation is not yet fully
understood. Some of the most common small molecules include histone deacetylase (HDAC) inhibitors.
These inhibitors cause chromatin decondensation and induce a short dedifferentiation state, which
changes the chromatin state [69]. 5-azacytidine (5-aza-C), a DNA methyltransferase inhibitor, is believed
to play a similar role in loosening the chromatin [68]. Glycogen synthase kinase-3 inhibitor (GSK3) which is
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a WNT signaling activator and TGF-β inhibitors may undergo regulating transition between
mesenchymal and epithelial state to promote cell reprogramming and help convert cell fate [71]. It is
interesting that treatment with the same small molecule can produce different cell types. For example,
cells treated with 5-aza-c with various concentrations for certain times can convert human fibroblasts
into functional neuronal precursors, pancreatic cells and muscle cells [70,72].
An interesting study showed that direct reprogramming of mouse embryonic fibroblasts to
cardiomyocytes was possible through small molecules. A two-stage protocol was used: exposure of
MEFs to a small-molecule cocktail: CRFVPTZ (C, CHIR99021; R, RepSox; F, Forskolin; V, VPA; P, Parnate; T,
TTNPB; and Z, DZnep) followed by cardiac reprogramming medium containing 15% fetal bovine serum
(FBS), 5% knockout serum replacement (KSR), N2 and B27 was used for the induction of the beating
colonies. Matrigel- coated dishes proved to better then non-coated and gelatin coated ones [73].
Micro-RNAs have also been used to transdifferentiate fibroblasts to cardiomyocytes. A combination of
miRNAs 1, 133, 208, and 499 induced direct reprogramming of fibroblasts to cardiomyocyte-like cells in
vitro. Here, JI1 treatment enhanced the reprogramming efficiency by ten folds than using miRNAs alone [74].
In Situ Transdifferentiation
The success of Transdifferentiation in vitro has encouraged the idea of in situ Transdifferentiation (Fig 4).
This process includes the direct introduction of lineage-determining transcription factors into a target
organ in vivo. If successful, this approach can present advantages over in vitro cellular reprogramming.
Less manipulation of the reprogrammed cells can be achieved making this process more clinically
efficient [75].
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Figure 4. In Situ Transdifferentiation
Vectors of choice transfer transcription factors into the desired tissue inside the animal to elicit in situ
Transdifferentiation. Examples include pancreas, heart and brain.
Adenovirus-mediated transduction of mouse liver with Pdx-1 activated the expression of insulin 1 and 2
and prohormone convertase 1/3 (PC 1/3). Although hyperglycemia in diabetic mice was ameliorated, no
phenotypic or morphological conversion into functional β-cells was detected [76]. A similar study using
the combination of Pdx-1 and Ngn3 in adeno-associated virus (AAV) serotype 8 however failed to
alleviate diabetes in streptozotocin-treated mice [77]. The same result occurred using a non-viral vector
approach [78]. However, Sox9-expressing hepatic duct cells, when reprogrammed in vivo using Ngn3, Pdx-
1 and Mafa secreted insulin, which restored normal glucose levels in streptozotocin-treated SCID mice.
Functional β-cells capable of producing insulin were also generated when exocrine pancreatic cells were
transduced in vivo with the transcription factors Ngn3, Pdx-1 and Mafa using adenovirus [79]. Although
the transdifferentiated cells could not arrange themselves into pancreatic islets, these studies
demonstrated that in vivo Transdifferentiation is possible which will stimulate further studies.
Resident non-myocytes in mice hearts have been reprogrammed into cardiomyocyte-like cells in vivo by
local delivery of Pdx-5 after coronary ligation to simulate myocardial infarction. Resulting cells were
contractile upon electrical stimulation resembling normal ventricular cells. Reprogramming was even
enhanced when Pdx-5 was accompanied by thymosin β4 in the target cells [80]. These results were
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confirmed when dividing non-myocytes were converted to functional cardiomyocytes in vivo after
overexpression of the transcription factors Gata4, Hand2, Mef2c and Tbx5. This study also reported that
fibrosis following myocardial infarction was reduced when Tbx5 was introduced to the non-dividing
myocytes [81].
Direct neural conversion in vivo was also achieved in the adult rodent brain. When transcription factors
Ascl1, Brn2a, and Myt11 were over-expressed in astrocytes of the striatum, functional neurons were
generated [82]. In vivo reprogramming was also achieved in the brains of mice when embryonic and early
postnatal colossal projection neurons of layer II/III were reprogrammed into Layer-V/VI corticofugal
projection neurons through Fezf2 overexpression. Here, reprogrammed neurons acquired the molecular
properties of corticofugal projection neurons [83]. Sox2 alone was able to directly reprogram resident
astrocytes into proliferative neuroblasts in the brains of adult mice [84]. A subsequent study showed that
the mechanism by which the reprogramming process takes place, as astrocytes were shown to passes
through proliferative intermediate progenitors to become differentiated neurons [85].
In vivo iPSCs were obtained through generating genetically engineered reprogrammable mice. In these
animals, overexpression of the OSKM factors could be induced upon doxycycline injection.
Unfortunately, teratomas formed in almost every organ in the bodies of these mice [86]. The in vivo
generated iPSCs were assumed to be totipotent. A follow-on study proved this assumption wrong.
Reprogrammable mice with an Oct4-GFP reporter gene allowing in vivo cell tracking demonstrated that
teratomas derived from in vivo iPSCs were morphologically indistinguishable from ESCs. They expressed
pluripotency markers, and could differentiate into tissues of all three germ layers. However, these in
vivo reprogrammed iPSCs did not contribute to placental tissues when introduced into eight-cell
embryos, suggesting that in vivo reprogrammed iPSCs were pluripotent and not totipotent [87].
FUTURE RESEARCH
Stem cell therapy has shown a remarkable outcome in treating certain diseases, such as leukemia,
lymphoma, and immunodeficiency. Nonetheless, utilizing stem cells for tissue transplantation, especially
iPSCs and trans-differentiated cells, has not yet been successful. Further investigation is needed to
understand the reasons behind an array of factors that hamper the clinical application of somatic cell
reprogramming in regenerative medicine. Some of these factors include as follows.
Safety: Safer cellular reprogramming processes can be achieved with a better understanding of the
ability of target cells to maintain new cellular properties and function over time. Studying the behavior
of transfected un-reprogrammed cells is also of great importance. Also, developing strategies to assess
damage to non-targeted cells and tissues inside the body is essential. Finally, it is important to find
potential alternatives to viral vectors that can be safer yet efficient in reprogramming. Library screening
and rational design approaches using chemicals, RNA, and CRISPR libraries will facilitate this endeavor.
Similarly, new delivery methods will help to improve efficiency.
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Microenvironment: Lineage tracing and live imaging studies can markedly enhance our understanding
of how tissues sustain appropriate cell types and numbers during homeostasis, and how they respond to
different injury stimuli [91]. Moreover, investigating mechanisms within cellular niches, wherein plasticity
is controlled in vivo, can cast light on new, efficient and safe reprogramming methods. This includes
studying cell interaction, and the role of the extracellular matrix, and cytoskeleton in influencing cell fate.
Similarly, a better understanding of developmental biology including that of model organisms will help
in understanding cellular plasticity.
Cell Communication: Although the signaling pathways involved in dedifferentiation, Transdifferentiation,
and reprogramming are not utterly understood, recent studies propose that these pathways may have a
critical role in the application of stem cell therapy. The TGF-β, PI3K/Akt, Wnt/β-catenin, Activin/Nodal,
and Jak-Stat signaling pathways all have a fundamental role in regulating pluripotency [92].
Understanding which signaling pathways play a crucial role during dedifferentiation, Transdifferentiation
and reprogramming can facilitate the development of new strategies to control these processes both in
vitro and in vivo.
Epigenetics: Epigenetic modifications have significant control over the cellular reprogramming process.
Controlling histone modifications, for example, through studying the control of the levels of H3K9
methyltransferases and H3K9 demethylases, in addition to, DNA demethylation and chromatin
remodeling can lead to a better outcome of the reprogramming process.
Immune Modulation: Evidence suggests that the immune system suppresses regeneration [88]. No
matter how great our advances become in directed reprogramming, a better understanding of how the
immune system influences cellular differentiation needs to occur before regenerative medicine can be
advanced. Understanding which elements of the immune system need to be suppressed to promote cell
specific differentiation will not only promote the application of in situ transdifferentiation but may also
facilitate natural endogenous repair.
As a potential disruptive technology, cellular reprogramming has undergone several important
advancements in the recent years. Although cellular reprogramming is not close to clinical application
yet, it is certainly a promising approach in cell-based regenerative medicine.
AWKNOWLEDGEMENT
I do thank the Ministry of the Higher Education, Egypt for providing the funding for the first two years of my PhD research.
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REFERENCES
1. Gurdon JB (1960) The developmental capacity of nuclei taken from differentiating endoderm
cells of Xenopus laevis. J Embryol Exp Morphol 8:505–526.
2. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors. Cell 126:663–76. doi: 10.1016/j.cell.2006.07.024
3. Chin MT (2014) Reprogramming cell fate: a changing story. Front cell Dev Biol 2:46. doi:
10.3389/fcell.2014.00046
4. Briggs R, King TJ (1952) Transplantation of Living Nuclei From Blastula Cells into Enucleated
Frogs’ Eggs. Proc Natl Acad Sci U S A 38:455–63.
5. King TJ, Briggs R (1955)Changes in the nuclei of differentiating gastrula cells, as demonstrated
by nuclear transplantation. Proc Natl Acad Sci U S A 41:321–5.
6. Elsdale TR, Gurdon JB, Fischberg M (1960) A description of the technique for nuclear
transplantation in Xenopus laevis. J Embryol Exp Morphol 8:437–44.
7. GURDON JB (1960) Factors responsible for the abnormal development of embryos obtained by
nuclear transplantation in Xenopus laevis. J Embryol Exp Morphol 8:327–40.
8. Bromhall JD (1975) Nuclear transplantation in the rabbit egg. Nature 258:719–22.
9. McGrath J, Solter D (1983) Nuclear transplantation in the mouse embryo by microsurgery and
cell fusion. Science 220:1300–2.
10. McGrath J, Solter D (1984) Inability of mouse blastomere nuclei transferred to enucleated
zygotes to support development in vitro. Science 226:1317–9.
11. Prather RS, Sims MM, First NL (1989) Nuclear transplantation in early pig embryos. Biol
Reprod 41:414–8.
12. Sims M, First NL (1994) Production of calves by transfer of nuclei from cultured inner cell mass
cells. Proc Natl Acad Sci U S A 91:6143–7.
13. Meng L, Ely JJ, Stouffer RL, Wolf DP (1997) Rhesus monkeys produced by nuclear transfer.
Biol Reprod 57:454–9.
14. Prather RS, Sims MM, First NL (1989) Nuclear transplantation in early pig embryos. Biol
Reprod 41:414–8.
15. Campbell KH, McWhir J, Ritchie WA, Wilmut I (1996) Sheep cloned by nuclear transfer from a
cultured cell line. Nature 380:64–6. doi: 10.1038/380064a0
16. Wilmut I, Schnieke AE, McWhir J, et al (1997) Viable offspring derived from fetal and adult
mammalian cells. Nature 385:810–3. doi: 10.1038/385810a0
17. Wakayama T, Shinkai Y, Tamashiro KL, et al (2000) Cloning of mice to six generations. Nature
407:318–9. doi: 10.1038/35030301
18. Hochedlinger K, Jaenisch R (2002) Monoclonal mice generated by nuclear transfer from mature
B and T donor cells. Nature 415:1035–8. doi: 10.1038/nature718
19. Eggan K, Baldwin K, Tackett M, et al (2004) Mice cloned from olfactory sensory neurons.
Nature 428:44–9. doi: 10.1038/nature02375
20. Li J, Ishii T, Feinstein P, Mombaerts P (2004) Odorant receptor gene choice is reset by nuclear
transfer from mouse olfactory sensory neurons. Nature 428:393–9. doi: 10.1038/nature02433
21. Tsunoda Y, Kato Y (2002) Recent progress and problems in animal cloning. Differentiation
69:158–61. doi: 10.1046/j.1432-0436.2002.690405.x
22. Hill JR, Burghardt RC, Jones K, et al (2000) Evidence for placental abnormality as the major
cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol Reprod 63:1787–94.
Zygoscient Research Insights
16
JSCRT Volume-1 | Issue-1 November, 2016
23. Eggan K, Akutsu H, Loring J, et al (2001) Hybrid vigor, fetal overgrowth, and viability of mice
derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A
98:6209–14. doi: 10.1073/pnas.101118898
24. Rideout WM, Wakayama T, Wutz A, et al (2000) Generation of mice from wild-type and
targeted ES cells by nuclear cloning. Nat Genet 24:109–10. doi: 10.1038/72753
25. Cibelli JB, Kiessling A a., Cunniff K, et al (2001) Somatic Cell Nuclear Transfer in Humans:
Pronuclear and Early Embryonic Development. e-biomed J Regen Med 2:25–31. doi:
10.1089/152489001753262168
26. Hwang WS, Roh S Il, Lee BC, et al (2005) Patient-specific embryonic stem cells derived from
human SCNT blastocysts. Science 308:1777–83. doi: 10.1126/science.1112286
27. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors. Cell 126:663–76. doi: 10.1016/j.cell.2006.07.024
28. Miller RA, Ruddle FH (1976) Pluripotent teratocarcinoma-thymus somatic cell hybrids. Cell
9:45–55. doi: 10.1016/0092-8674(76)90051-9
29. Ingraham HA, Lawless GM, Evans GA (1986) The mouse Thy-1.2 glycoprotein gene: complete
sequence and identification of an unusual promoter. J Immunol 136:1482–9.
30. Tada M, Tada T, Lefebvre L, et al (1997) Embryonic germ cells induce epigenetic
reprogramming of somatic nucleus in hybrid cells. EMBO J 16:6510–20. doi:
10.1093/emboj/16.21.6510
31. Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic cells
after fusion with human embryonic stem cells. Science 309:1369–73. doi:
10.1126/science.1116447
32. Taylor SM, Jones PA (1979) Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated
with 5-azacytidine. Cell 17:771–9.
33. Lassar AB, Paterson BM, Weintraub H (1986) Transfection of a DNA locus that mediates the
conversion of 10T12 fibroblasts to myoblasts. Cell 47:649–656. doi: 10.1016/0092-
8674(86)90507-6
34. Choi J, Costa ML, Mermelstein CS, et al (1990) MyoD converts primary dermal fibroblasts,
chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated
myoblasts and multinucleated myotubes. Proc Natl Acad Sci U S A 87:7988–92. doi:
10.1073/pnas.87.20.7988
35. Xie H, Ye M, Feng R, Graf T (2004) Stepwise reprogramming of B cells into macrophages. Cell
117:663–76.
36. Laiosa C V, Stadtfeld M, Xie H, et al (2006) Reprogramming of committed T cell progenitors to
macrophages and dendritic cells by C/EBP alpha and PU.1 transcription factors. Immunity
25:731–44. doi: 10.1016/j.immuni.2006.09.011
37. Heyworth C, Pearson S, May G, Enver T (2002) Transcription factor-mediated lineage switching
reveals plasticity in primary committed progenitor cells. EMBO J 21:3770–81. doi:
10.1093/emboj/cdf368
38. Cobaleda C, Jochum W, Busslinger M (2007) Conversion of mature B cells into T cells by
dedifferentiation to uncommitted progenitors. Nature 449:473–7. doi: 10.1038/nature06159
39. Takahashi K, Tanabe K, Ohnuki M, et al (2007) Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131:861–72. doi: 10.1016/j.cell.2007.11.019
40. Liu H, Zhu F, Yong J, et al (2008) Generation of induced pluripotent stem cells from adult
rhesus monkey fibroblasts. Cell Stem Cell 3:587–90. doi: 10.1016/j.stem.2008.10.014
Zygoscient Research Insights
17
JSCRT Volume-1 | Issue-1 November, 2016
41. Li W, Wei W, Zhu S, et al (2009) Generation of rat and human induced pluripotent stem cells by
combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4:16–9. doi:
10.1016/j.stem.2008.11.014
42. Kim JB, Zaehres H, Wu G, et al (2008) Pluripotent stem cells induced from adult neural stem
cells by reprogramming with two factors. Nature 454:646–50. doi: 10.1038/nature07061
43. Aasen T, Raya A, Barrero MJ, et al (2008) Efficient and rapid generation of induced pluripotent
stem cells from human keratinocytes. Nat Biotechnol 26:1276–84. doi: 10.1038/nbt.1503
44. Aoi T, Yae K, Nakagawa M, et al (2008) Generation of pluripotent stem cells from adult mouse
liver and stomach cells. Science 321:699–702. doi: 10.1126/science.1154884
45. Seki T, Yuasa S, Oda M, et al (2010) Generation of induced pluripotent stem cells from human
terminally differentiated circulating T cells. Cell Stem Cell 7:11–4. doi:
10.1016/j.stem.2010.06.003
46. Yu J, Vodyanik MA, Smuga-Otto K, et al (2007) Induced pluripotent stem cell lines derived
from human somatic cells. Science 318:1917–20. doi: 10.1126/science.1151526
47. Efroni S, Duttagupta R, Cheng J, et al (2008) Global transcription in pluripotent embryonic stem
cells. Cell Stem Cell 2:437–47. doi: 10.1016/j.stem.2008.03.021
48. Kim JB, Greber B, Araúzo-Bravo MJ, et al (2009) Direct reprogramming of human neural stem
cells by OCT4. Nature 461:649–3. doi: 10.1038/nature08436
49. Boyer LA, Lee TI, Cole MF, et al (2005) Core transcriptional regulatory circuitry in human
embryonic stem cells. Cell 122:947–56. doi: 10.1016/j.cell.2005.08.020
50. Fong YW, Inouye C, Yamaguchi T, et al (2011) A DNA repair complex functions as an
Oct4/Sox2 coactivator in embryonic stem cells. Cell 147:120–31. doi: 10.1016/j.cell.2011.08.038
51. Liang J, Wan M, Zhang Y, et al (2008) Nanog and Oct4 associate with unique transcriptional
repression complexes in embryonic stem cells. Nat Cell Biol 10:731–9. doi: 10.1038/ncb1736
52. Stadtfeld M, Hochedlinger K (2010) Induced pluripotency: history, mechanisms, and
applications. Genes Dev 24:2239–63. doi: 10.1101/gad.1963910
53. Kim J, Woo AJ, Chu J, et al (2010) A Myc network accounts for similarities between embryonic
stem and cancer cell transcription programs. Cell 143:313–24. doi: 10.1016/j.cell.2010.09.010
54. Bauwens C, Yin T, Dang S, et al (2005) Development of a perfusion fed bioreactor for
embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of
cardiomyocyte output. Biotechnol Bioeng 90:452–61. doi: 10.1002/bit.20445
55. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460:118–22. doi: 10.1038/nature08113
56. Waddington C (1957) The strategy of the genes a discussion of some aspects of theoretical
biology. Allen & Unwin, London
57. Yamanaka S (2009) Elite and stochastic models for induced pluripotent stem cell generation.
Nature 460:49–52. doi: 10.1038/nature08180
58. Guo J, Wang H, Hu X (2013) Reprogramming and Transdifferentiation shift the landscape of
regenerative medicine. DNA Cell Biol 32:565–72. doi: 10.1089/dna.2013.2104
59. Ma T, Xie M, Laurent T, Ding S (2013) Progress in the reprogramming of somatic cells. Circ
Res 112:562–74. doi: 10.1161/CIRCRESAHA.111.249235
60. Weintraub H, Tapscott SJ, Davis RL, et al (1989) Activation of muscle-specific genes in pigment,
nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S
A 86:5434–8.
61. Ieda M, Fu J-D, Delgado-Olguin P, et al (2010) Direct reprogramming of fibroblasts into
functional cardiomyocytes by defined factors. Cell 142:375–86. doi: 10.1016/j.cell.2010.07.002
Zygoscient Research Insights
18
JSCRT Volume-1 | Issue-1 November, 2016
62. Vierbuchen T, Ostermeier A, Pang ZP, et al (2010) Direct conversion of fibroblasts to functional
neurons by defined factors. Nature 463:1035–41. doi: 10.1038/nature08797
63. Christen B, Robles V, Raya M, et al (2010) Regeneration and reprogramming compared. BMC
Biol 8:5. doi: 10.1186/1741-7007-8-5
64. Meshorer E, Yellajoshula D, George E, et al (2006) Hyperdynamic plasticity of chromatin
proteins in pluripotent embryonic stem cells. Dev Cell 10:105–16. doi:
10.1016/j.devcel.2005.10.017
65. White UA, Stephens JM (2011) The gp130 receptor cytokine family: regulators of adipocyte
development and function. Curr Pharm Des 17:340–6.
66. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460:118–22. doi: 10.1038/nature08113
67. Efe JA, Hilcove S, Kim J, et al (2011) Conversion of mouse fibroblasts into cardiomyocytes
using a direct reprogramming strategy. Nat Cell Biol 13:215–22. doi: 10.1038/ncb2164
68. Li W, Li K, Wei W, Ding S (2013) Chemical approaches to stem cell biology and therapeutics.
Cell Stem Cell 13:270–83. doi: 10.1016/j.stem.2013.08.002
69. Maki N, Martinson J, Nishimura O, et al (2010) Expression profiles during dedifferentiation in
newt lens regeneration revealed by expressed sequence tags. Mol Vis 16:72–8.
70. Mirakhori F, Zeynali B, Kiani S, Baharvand H (2015) Brief azacytidine step allows the
conversion of suspension human fibroblasts into neural progenitor-like cells. Cell J 17:153–8.
71. Ichida JK, Blanchard J, Lam K, et al (2009) A small-molecule inhibitor of tgf-Beta signaling
replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5:491–503. doi:
10.1016/j.stem.2009.09.012
72. Pennarossa G, Maffei S, Campagnol M, et al (2014) Reprogramming of Pig Dermal Fibroblast
into Insulin Secreting Cells by a Brief Exposure to 5-aza-cytidine. Stem Cell Rev Reports 10:31–
43. doi: 10.1007/s12015-013-9477-9
73. Fu Y, Huang C, Xu X, et al (2015) Direct reprogramming of mouse fibroblasts into
cardiomyocytes with chemical cocktails. Cell Res 25:1013–24. doi: 10.1038/cr.2015.99
74. Jayawardena TM, Egemnazarov B, Finch EA, et al (2012) MicroRNA-mediated in vitro and in
vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes. Circ Res 110:1465–73. doi:
10.1161/CIRCRESAHA.112.269035
75. Sancho-Martinez I, Baek SH, Izpisua Belmonte JC (2012) Lineage conversion methodologies
meet the reprogramming toolbox. Nat Cell Biol 14:892–899. doi: 10.1038/ncb2567
76. Ferber S, Halkin A, Cohen H, et al (2000) Pancreatic and duodenal homeobox gene 1 induces
expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat
Med 6:568–72. doi: 10.1038/75050
77. Wang AY, Ehrhardt A, Xu H, Kay MA (2007) Adenovirus transduction is required for the
correction of diabetes using Pdx-1 or Neurogenin-3 in the liver. Mol Ther 15:255–63. doi:
10.1038/sj.mt.6300032
78. Wang AY, Ehrhardt A, Xu H, Kay MA (2007) Adenovirus transduction is required for the
correction of diabetes using Pdx-1 or Neurogenin-3 in the liver. Mol Ther 15:255–63. doi:
10.1038/sj.mt.6300032
79. Banga A, Akinci E, Greder L V, et al (2012) In vivo reprogramming of Sox9+ cells in the liver
to insulin-secreting ducts. Proc Natl Acad Sci U S A 109:15336–41. doi:
10.1073/pnas.1201701109
80. Qian L, Huang Y, Spencer CI, et al (2012) In vivo reprogramming of murine cardiac fibroblasts
into induced cardiomyocytes. Nature 485:593–8. doi: 10.1038/nature11044
Zygoscient Research Insights
19
JSCRT Volume-1 | Issue-1 November, 2016
81. Song K, Nam Y-J, Luo X, et al (2012) Heart repair by reprogramming non-myocytes with
cardiac transcription factors. Nature 485:599–604. doi: 10.1038/nature11139
82. Guo Z, Zhang L, Wu Z, et al (2014) In vivo direct reprogramming of reactive glial cells into
functional neurons after brain injury and in an Alzheimer’s disease model. Cell Stem Cell
14:188–202. doi: 10.1016/j.stem.2013.12.001
83. Rouaux C, Arlotta P (2013) Direct lineage reprogramming of post-mitotic callosal neurons into
corticofugal neurons in vivo. Nat Cell Biol 15:214–21. doi: 10.1038/ncb2660
84. Niu W, Zang T, Zou Y, et al (2013) In vivo reprogramming of astrocytes to neuroblasts in the
adult brain. Nat Cell Biol 15:1164–75. doi: 10.1038/ncb2843
85. Niu W, Zang T, Smith DK, et al (2015) SOX2 reprograms resident astrocytes into neural
progenitors in the adult brain. Stem cell reports 4:780–94. doi: 10.1016/j.stemcr.2015.03.006
86. Abad M, Mosteiro L, Pantoja C, et al (2013) Reprogramming in vivo produces teratomas and iPS
cells with totipotency features. Nature 502:340–5. doi: 10.1038/nature12586
87. Choi HW, Kim JS, Hong YJ, et al (2015) In vivo reprogrammed pluripotent stem cells from
teratomas share analogous properties with their in vitro counterparts. Sci Rep 5:13559. doi:
10.1038/srep13559
88. Aurora AB, Olson EN (2014) Immune modulation of stem cells and regeneration. Cell Stem
Cell 15:14–25. doi: 10.1016/j.stem.2014.06.009
89. Sekiya S, Suzuki A (2011) Direct conversion of mouse fibroblasts to hepatocyte-like cells by
defined factors. Nature 475:390–3. doi: 10.1038/nature10263
90. Yang R, Zheng Y, Li L, et al (2014) Direct conversion of mouse and human fibroblasts to
functional melanocytes by defined factors. Nat Commun 5:5807. doi: 10.1038/ncomms6807
91. Batta K, Florkowska M, Kouskoff V, Lacaud G (2014) Direct reprogramming of murine
fibroblasts to hematopoietic progenitor cells. Cell Rep 9:1871–84. doi:
10.1016/j.celrep.2014.11.002
92. Outani H, Okada M, Yamashita A, et al (2013) Direct induction of chondrogenic cells from
human dermal fibroblast culture by defined factors. PLoS One 8:e77365. doi:
10.1371/journal.pone.0077365
93. Yamamoto K, Kishida T, Sato Y, et al (2015) Direct conversion of human fibroblasts into
functional osteoblasts by defined factors. Proc Natl Acad Sci U S A 112:6152–7. doi:
10.1073/pnas.1420713112
94. Ji H, Atchison L, Chen Z, et al (2016) Transdifferentiation of human endothelial progenitors into
smooth muscle cells. Biomaterials 85:180–194. doi: 10.1016/j.biomaterials.2016.01.066
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