BBA - Molecular Cell Research 1864 (2017) 1359–1369

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BBA - Molecular Cell Research

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Review

Transdifferentiation and reprogramming: Overview of the processes, their MARK

similarities and differences
Artur Cieślar-Pobudaa,b,1, Viktoria Knoflachc,1, Mikael V. Ringhd, Joachim Starke, Wirginia Likusf,
Krzysztof Siemianowiczg, Saeid Ghavamih,i, Andrzej Hudeckij, Jason L. Greenk, Marek J. Łosl,⁎
a
Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland
b
Stem Cell Group, Nordic EMBL Partnership, Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo, Norway
c
Unit of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institute, Stockholm, Sweden
d
Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden
e
Department of Surgery, Länssjukhuset Ryhov, Jönköping, Sweden
f
Department of Anatomy, School of Health Science in Katowice Medical University of Silesia, Katowice, Poland
g
Department of Biochemistry, School of Medicine in Katowice, Medical University of Silesia, 18 Medyków Street, 40-752 Katowice, Poland
h
Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
i
Health Policy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
j
Institute of Nonferrous Metals, Gliwice, Poland
k
Duke University, School of Medicine, Durham, NC, USA
l
Małopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A str., 30-387 Krakow, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem
Reprogramming cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called “Yamanaka
Transdifferentiation factors” (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also
iPS overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This
Teratomagenesis
raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or
Yamanaka factors
-conversion), is a process in which one mature, specialized cell type changes into another without entering a
pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli.
Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent
teratomagenesis.

List of abbreviations while regions of chromosomal gain included oncogenes.

ANT altered nuclear transfer; an experimental reprogramming
technique developed to satisfy research-ethical concerns.
It employs nuclear transfer-based reprogramming, but
only after the somatic cell nucleus and/or the oocyte are
altered in order to inhibit totipotency and eliminate the
full embryonal developmental potential, while still
allowing the formed pseudo-embryo to generate
pluripotent stem cells.
CNV copy number variations; interpersonal variations in the
numbers of copies of certain genes. Recently, Laurent and
colleagues have described that deleted regions within the
iPS-genome often contained tumor-suppressor genes,
ES cells embryonic stem cells; pluripotent stem cells obtained
from the inner mass of a blastocyst, they are capable of
forming all tissues within an organism, but not extra-fetal
tissues like i.e. the placenta.
FACS fluorescence-activated cell sorting; versatile, flow
cytometry-based techniques commonly used for
quantification of expression of cellular proteins within
intact cells, and also for the measurement of various
biologic processes. It is for example used for the
detection and quantification of stemness markers or
hematologic markers in the clinic.
HIFs Hypoxia inducible factors; transcription factors that play
a role, among others, in the regulation of several genes
related to hypoxia, vascularization, and pluripotency in

⁎
Corresponding author at: Małopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A str., 30-387 Kraków, Poland.
E-mail address: bioappl@gmail.com (M.J. Łos).
1
These authors share first authorship.

http://dx.doi.org/10.1016/j.bbamcr.2017.04.017
Received 8 March 2017; Received in revised form 24 April 2017; Accepted 26 April 2017
Available online 28 April 2017
0167-4889/ © 2017 Elsevier B.V. All rights reserved.
A. Cieślar-Pobuda et al.
human embryonic stem cells cultured under hypoxic
conditions by upregulating the expression of Oct4, Sox2
and NANOG.
iPS induced pluripotent stem (cells); functionally-
comparable/equal to ES cells pluripotent cells, obtained
by expression of ‘Yamanaka factors’ (OSKM) in somatic
cells; please consult Boxes 1 and 2 for details. Unlike ES-
cells that are usually obtained by destruction of embryos,
generation of iPS is not ethically controversial.
lincRNAs long intergenic non-coding RNAs; long non-coding RNAs
that are transcribed from non-coding DNA sequences
between protein-coding genes (hence, intergenic); may
harbor transposable elements and may attach to
messenger RNA to block protein production.
miRNA microRNA; an ‘umbrella-term’ for several classes of
small, regulatory RNAs that may affect gene
transcription, messenger RNA (mRNA) maturation, the
stability of mRNAs, and their translation.
mtDNA mitochondrial DNA; mitochondrial genome, it codes for
some of the mitochondrial proteins.
MACS magnetic-activated cell sorting; a cell-separation
technique where cells are marked with antibodies
specific to selected proteins; the antibodies carry nano-
magnet particles attached and this allows for separation
of the cell population marked with such antibodies from
the rest.
OSKM Oct3/4, Sox2, Klf4, c-Myc; ‘classical’ set of transcription
factors required for reprogramming, discovered by S.
Yamanaka, hence they are also called “Yamanaka
factors” (please consult Boxes 1 & 2 for more details).
PEG polyethyleneglicol; a chemical reagent frequently used,
among others, to facilitate fusions between cells.
RISCs RNA-induced silencing complexes; large multi-protein
complex, containing mainly ribonucleoproteins, with the
protein ARGONAUT responsible for mRNA-cleavage;
they are a key part of the cellular mRNA-silencing
machinery regulated by miRNAs; RISC is also the key
component of an innate antiviral response that degrades
foreign (m)RNAs.
ROCK Rho-associated kinases; a family of a versatile signal-
transduction proteins involved, among others, in the
regulation of cell migration (cytoskeleton dynamics) cell
cycle, and apoptosis. ROCK inhibitors enhance the
efficiency of iPS generation (reprogramming), at least
partially by inhibiting single-cell dissociation-induced
apoptosis.
SCNT somatic cell nuclear transfer, an early reprogramming
(cloning) technique, used i.e. in 90s for experimental
(proof of principle) cloning of sheep (please consult Box
3 for details).
1. Reprogramming and transdifferentiation — key facts and major
differences
During ontogenesis, the cell fate is determined by a complex set of
transcription factors and epigenetic networks that governs the differ-
entiation program, which until recently was considered unidirectional
and irreversible. In the past decade, Yamanaka, and others, showed that
a cocktail of just four transcription factors (Oct4, Sox2, Klf4 and c-myc,
OSKM) is sufficient to reprogram murine- [1] and human [2,3]
fibroblasts into induced pluripotent stem (iPS) cells. The group of
Yamanaka was using the same transcription factors for the reprogram-
ming of mouse fibroblasts, whereas Yu et al. exchanged c-myc and Klf4
for Nanog and Lin28. Since then several protocols were established,
some only using two or three transcription factors, others utilizing small
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BBA - Molecular Cell Research 1864 (2017) 1359–1369
Induced-pluripotent stem cell
(iPS)
TRANSDIFFERENTIATION
Differentiated cell Another (partly) differentiated cell
Fig. 1. Basic difference between reprogramming and transdifferentiation.
Reprogramming (shown in a green arrow) requires a reversal change of a differentiated
cell into a pluripotent stem cell (i.e. iPS), which next may undergo a differentiation
process (shown in a blue arrow) into another differentiated cell. Transdifferentiation
(shown in a red arrow) does not require a full reversal into iPS cells in order to transform
into another cell type. In some cases transdifferentiation is a one-step process, while in
others the cells pass through an intermediate state(s). Note: the drawing is schematic and
the differences in size and shape of pictured cells do not exactly reflect the actual
differences.
molecular compounds in addition to transcription factors as well as
different delivery systems used. All of those methods have different
reprogramming efficiencies ranging from 0.00001 to > 1% [4–6].
Another route in generating cells of a different cell type is by
utilizing transdifferentiation (Fig. 1). This concept of lineage conver-
sion is not new; already in 1987 Lassar and colleagues showed that the
introduction of just one cDNA converts fibroblasts into myoblasts [7].
Instead of using transcription factors important in maintaining plur-
ipotency, lineage-specific transcription factors are introduced into the
cell. Recently, a predictive system was published, called Mogrify which
aims to predict the composition of transcription factors needed for
optimal transdifferentiation into a given cell type [8]. It combines gene
expression data with regulatory network information to predict the
transdifferentiation factors necessary to induce cell conversion.
Whereas iPS cell reprogramming is a rather time-consuming pro-
cess, transdifferentiation is often fast and highly efficient. In 2008 Zhou
OSKM
A SOMATIC CELL
vehicle
C
OSKM
OSKM
B
OSKM
iPS
iPS
iPS
Fig. 2. Cell reprogramming. A — Four genes (OSKM) are delivered into a nucleus of a
differentiated cell. Various methods discussed in the text use various types of carriers
(“vehicle”) to introduce them into the cells' nucleus. B — After the introduction of OSKM
the cell proliferates. C — Among new cells there are iPS cells (marked in blue), but also
cells that failed to (fully) reprogram, which will eventually die. Note: the drawing is
schematic and the differences in size and shape of pictured cells do not exactly reflect the
actual differences.
A. Cieślar-Pobuda et al. BBA - Molecular Cell Research 1864 (2017) 1359–1369

Table 1 injection into blastocysts [1]. Subsequently, different genes have been
Examples of in vitro cellular transdifferentiation. substituted for a subset of the OSKM genes, and other gene transfer
methods have been applied, all with their respective advantages and
Source cells Transdifferentiation inducing Target cells Ref.
factors disadvantages.
A major issue with the first iPS cells was that mice derived from
Fibroblasts MyoD Myocytes [7] them were prone to tumor development. All of the original OSKM
B-cells C/EBPα, C/EBPβ Macrophages [12]
factors to some extent possess oncogenic potential, and the tumorigen-
T-cells Bcl11b deletion NK cells [111]
Pancreatic Duct Pdx1 Beta cells [112] esis in iPS cell-derived mice has been especially attributed to c-Myc.
cells Combinations of reprogramming factors excluding c-Myc evaded tu-
Pancreatic Ngn3, Mafa, Pdx1 [9] morigenesis during the study period while maintaining efficiencies
exocrine cells similar to OSKM reprogramming [14,15]. However, a non-zero long-
Hepatocytes Exendin-4, Pdx1 [113]
term risk cannot be excluded for any combination of reprogramming
Fibroblasts Hnf4α, Foxa1 or Foxa2 or Hepatocytes [114]
Foxa3 factors.
Fibroblasts Ascl1 (also know as Mash1), Neurons [115] Retroviruses, the type of vectors initially used for reprogramming,
Brn2, Myt1l integrate into the host genome and thereby drive the expression of the
miR-124, Brn2, Myt1l [116]
reprogramming factors. There is a substantial risk of insertion-muta-
Astrocytes Pax6 neurogenin 2, Ascl1 [117]
Fibroblasts Ascl1, Nurr1, Lmx1a Dopaminergic [118] genesis as a consequence of retroviral integration, severely constraining
Ascl1, Brn2, Myt1l, Lmx1a, neurons [119] use in clinical applications. Retroviral transgenes are effectively
FoxA2 inactivated in iPS cells, while the expression of the endogenous
Fibroblasts Oct4, Sox2, Klf4 and c-Myc Cardiomyocytes [120] pluripotency genes maintains pluripotency [16]. Still, the possibility
Tbx5, Mef2c, Gata-4, Mesp1 [121]
of transgene reactivation and residual expression poses a risk, especially
with regard to the tumorigenesis related to c-Myc expression.
et al. succeeded in differentiating exocrine cells into β-cells by Effective retroviral delivery can only be achieved in dividing cells
introducing three transcription factors important in the embryonic [17]. Retrovirally reprogrammed murine autologous iPS cells were
development of the pancreas. First transdifferentiated cells appeared more immunogenic compared to iPS cells reprogrammed by a non-
already on day 3 and as much as 20% of the cells were successfully integrating episomal method and autologous embryonic stem cells [18].
transdifferentiated [9]. In 2013 the first successful transdifferentiation It is unclear whether this immunogenicity extends to all iPS cells, only
of human fibroblasts towards a cardiac fate was published [10]. We to those iPS that have been obtained by employing integrating viruses
have recently transdifferentiated human fibroblasts into corneal limbal as vectors, or retrovirally reprogrammed iPS cells only. The immuno-
cells [11]. In some cases transdifferentiation is a one-step process, while genicity study underscores the fact that iPS cells may cause immuno-
in others the cells pass through an intermediate state, as shown in a rejection in syngenic transplantation and that there may exist differ-
study to reprogram B cells into macrophages where the cells express ences in immunogenicity between iPS cells produced by different
low levels of both B cell-specific genes and macrophage-specific genes reprogramming methods. Lentiviral delivery of reprogramming factors
before they completely transdifferentiate into macrophages [12]. The is another successful approach, applied e.g. in one of the two first
most important examples of in vitro transdifferentiation and factors instances of human iPS generation [3] (Box 2).
needed for cellular conversions to specific cell types are presented in
Table 1. 2.1.2. Non-integrating viral approaches
The dedifferentiated state sometimes present during transdifferen- A major advantage of non-integrating methods is the evasion of
tiation is unnatural and therefore rather unstable, whereas iPS cells potentially oncogenic insertion-mutagenesis, transgene reactivation
could be cultured indefinitely without losing their pluripotent potential. and residual expression, but the trade-off of adenoviral reprogramming
One hallmark, and also a negative aspect of pluripotency is the is considerably lower efficiency, the requirement for high viral titers
formation of teratomas when transplanted into immune deficient mice. and sometimes the need for repeated transductions [19,20]. One
Therefore it must be ensured that the differentiation of iPS cells is possible contributing variable is the dilution of reprogramming factors
uniform and the maturating cells are not contaminated with a ‘leftover’ as cells multiply.
iPS progeny. Another issue is the reactivation of the transgenes which One such non-integrating approach is adenoviral reprogramming
could lead to tumorigenic tendencies as shown by Okita el al [13]. [20,21]. Although adenoviral vectors are generally considered non-
Although some transdifferentiation protocols include a dedifferentia- integrating and adenovirally reprogrammed iPS cells have been pro-
tion step, those cells do not go back to pluripotency, therefore lowering duced with the apparent absence of viral integration, including
or eliminating the risk of tumor formation. Despite that transdiffer- negative Southern blot analysis [19,20], adenoviral vectors have been
entiation was historically described much earlier, nowadays much more reported to integrate to a small extent [22]. Sendai-virus based delivery
is known about reprogramming. Below, we discuss various aspects of of reprogramming factors (currently the most popular reprogramming
both methods. Although at first glimpse they may seem very similar, method) has been summarized in Box 2.
both have some important differences, which impact their plausible use
in regenerative therapy. The molecular principles of reprogramming 2.1.3. Non-viral delivery of reprogramming factors
have been briefly outlined in Box 1. Non-viral vectors are safer, because they are free from dangers like:
transgene activation, residual expression, insertion-mutagenesis of
integrating viruses, as well as concerns regarding the presence of
2. Reprogramming techniques viruses per se. In addition, non-viral methods allow for reprogramming
without the need for virus production and reprogramming of cell lines
2.1. Introduction of reprogramming transcription factors not limited by viral tropism.
Conventional plasmids, self-replicating episomal plasmids and
2.1.1. Integrating viral approaches minicircle plasmids are some of the non-viral methods of introducing
Upon introduction of OSKM genes into murine fibroblast via retro- reprogramming factors to generate iPS cells. Conventional plasmids do
viral infection, the cells undergo reprogramming into a pluripotent not replicate in mammalian cells, thus requiring multiple rounds of
state. They could then be differentiated into all three germ layers, and transfection for successful reprogramming, and the reprogramming
as a proof of concept, led to the production of chimeric mice after efficiency is considerably lower than in viral methods [19,23,24].

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Box 1
Reprogramming — basic mechanism, and ‘Yamanaka factors’.

All cells of the body have the same genes. Pluripotent stem cells can differentiate into all cell types within the organism. This process is
accompanied by a limitation of genes that could be expressed in a differentiated cell. DNA is packed in the cell's nucleus and various
proteins associated with DNA regulate the degree of its “compression” and which genes could be expressed. In 2006, Yamanaka and co-
workers managed to generate iPS cells from fibroblasts by using a set of defined reprogramming factors [1] (Box 2, Fig. 2). The chromatin
structure in pluripotent ES cells has higher plasticity and is more open compared to differentiated cells [122] and heterochromatin
rearrangement is the first observable change in somatic cells undergoing reprogramming into iPS cells [123]. Telomeres in iPS cells have
the same epigenetic marks as ES cells, and are further elongated after reprogramming compared to their progenitors [124]. iPS generated
from the fibroblast of old donors, similarly elongate telomeres as iPS cells derived from young donor fibroblasts. Several long intergenic
non-coding RNAs (lincRNAs) have also been identified, which show increased expression in iPS cells compared to ES cells and may serve as
targets of pluripotency transcription factors [125].
Yamanaka and co-workers evaluated 24 candidate factors by co-expression in murine fibroblasts and after successive exclusion of
individual transcription factors four genes were identified as the minimal requirement for iPS cell generation. These defined factors
included Oct-3/4 (Pou5f1), Sox2, KLF4, and c-Myc (OSKM). They were then introduced into mouse fibroblasts by using retroviral vectors,
and in turn generated iPS cells [1]. These cells were morphologically similar to ES cells, including the ability to generate cells from all 3
germinal layers, and also showed comparable proliferation characteristics and expressed the same sets of surface markers. Several methods
exist to introduce Yamanaka factors to somatic cells, and more common examples are provided in Box 2.

Epstein-Barr virus-based episomal vectors, capable of self-replica-
tion, enable single transfection reprogramming but suffer from low
efficiency, and in several cases require more reprogramming factors in
addition to the OSKM factors [25–27]. It has been suggested that the
limiting variables are properties intrinsic to the vector itself, including
fast methylation silencing the vector sequence, infrequently established
stability despite replicative potential and common loss of “established”
plasmids with cell division upon removal of selective pressure
[25,28,29]. Interestingly, one study reported less immunogenicity of
episomally reprogrammed iPS cells compared to retrovirally repro-
grammed iPS cells [18].
Minicircle vectors are circular, supercoiled DNA elements, produced
by e.g. in vivo PhiC3-integrase based intramolecular site-specific re-
combination of conventional plasmids containing sequences of interest
[6,30]. Non-viral and devoid of a bacterial backbone sequence, mini-
circle vectors offer an interesting safety profile and higher transfection
efficiency along with longer and stronger expression in both dividing
and non-dividing cells, attributed to decreased silencing, when com-
pared to conventional plasmids [6,30,31]. Yet, compared to viral
methods, minicircle delivery of reprogramming factors have yielded
considerably lower reprogramming efficiencies even when multiple
consecutive transfections were employed [32,33]. Another non-viral
approach to introduce reprogramming factors is the use of transposon
systems (Box 2).
2.1.4. Non-transgene factor delivery
In order to circumvent the risks of insertion-mutagenesis and
oncogenic transformation, several non-transgene reprogramming tech-
niques such as i.e. delivering reprogramming factors in the form of
mRNA or protein have been developed. Sendai viral reprogramming is
also non-transgene, but the perks of delivering reprogramming factors
in the form of mRNA or protein is the avoidance of possible viral

Box 2
Examples of delivery methods of reprogramming factors.

Lentiviruses are integrating viruses of the Retroviridae family, and lentiviral vectors share many advantages and disadvantages with the
other retroviruses used in iPS cell production. Amongs others, the disadvantages of a lentiviral delivery method are insertional mutagenesis,
transgene activation, residual expression and immunogenicity. Lentiviruses differ from retroviruses by offering alternative tropism and are,
unlike retroviruses, capable of transducing non-dividing cells. Slower silencing of reprogramming factors makes lentiviral reprogramming
more efficient than retroviral reprogramming [16,25,36,126,127]. Differences in storage stability, the smaller maximum insert size of
lentiviruses and the safety concerns regarding lentiviruses being derived from immunodeficiency viruses are some disadvantages of
lentiviruses compared to retroviruses [128,129].
Sendai virus-mediated delivery becomes increasingly popular, as it evades the risks of insertion-mutagenesis, transgene activation and
residual expression, while still maintaining even higher reprogramming efficiencies than lentiviral methods [23,130]. As Sendai virus
constitutively replicates, concerns regarding remaining viral particles have been raised [131]. Sendai viruses are not pathogenic in humans,
but may infect epithelial cells, thus caution is advised, especially if oncogenes such as c-Myc are introduced [25,132]. Use of replication-
deficient-, and temperature sensitive Sendai viruses have shown some promise in mitigating this aspect [130,133].
Transposons are genetic mobile elements, which can be moved within a genome by a transposase, an enzyme catalyzing excision and
insertion of the transposon. The piggyBac transposon system has been used for reprogramming of both murine and human cells, and in
murine cell lines it was possible to tracelessly excise the exogenous transgene reprogramming factors when they were no longer needed for
reprogramming [134]. Another excision approach using the Cre-Lox system, where the reprogramming factors are flanked by lox sites and
excised by subsequent Cre recombinase transfection when no longer needed, may reprogram without permanent genetic alterations [90].
The excision of transgenes can prevent the risks of transgene activation and residual expression associated with many reprogramming
methods, though the risk of inefficient and imperfect excision exists. There is also the practical matter of an extra excision step. PiggyBac
reprogramming is further complicated by the fact that tranposases not only excise, but also insert transposons and screening is needed to
assess whether all transgenes have been effectively removed. Another concern is that the human genome contains piggyBac system-like
components, which potentially could interact with the piggyBac system [25,135].

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infections in patients and the lack of a need to handle viral particles.
Modified synthetic mRNA encoding reprogramming factors repro-
grams faster and with high efficiency as compared to other methods
[34,35]. Unlike the DNA-based delivery of reprogramming factors, RNA
is not epigenetically silenced [36]. However, foreign mRNA elicits
strong innate antiviral response and is targeted for degradation by RNA-
induced silencing complexes (RISCs), and the in vivo half-life of mRNA
is very short — it is degraded over the course of a few days. Therefore,
repeated transfections, and large quantities of modified, high quality,
long sequence mRNA, poses technical challenges (also high costs).
Another non-transgene-based method is the delivery of the repro-
gramming transcription factors into the cell in the form of proteins.
Successful protein reprogramming of both murine and human cells has
been reported, although the process was relatively slow and very
inefficient, especially in human cells [37,38]. The reprogramming of
murine cells was made with several rounds of transfection of recombi-
nant proteins produced in E. coli, and the human cells were repro-
grammed using cell extracts of HEK293 cells expressing recombinant
proteins. Bacterially produced proteins may be inferior in reprogram-
ming applications because of the lack of eukaryotic posttranslational
modifications and possible misfolding during purification/renaturation,
and cell extracts containing recombinant proteins produced in mam-
malian cells might have to be enriched for higher efficiency [6,25].
Recombinant protein delivery offers reprogramming without involve-
ment of any exogenous nucleic acids and no alterations of the genome
(aside from secondary epigenetic changes due to the reprogramming
process), and so it has an appealing safety profile. However, large
efforts for optimizing and improving efficiency are much needed.
2.2. Somatic cell nuclear transfer (SCNT)
As SCNT (Box 3) replaces the oocyte nucleus with a somatic nucleus,
the mitochondrial DNA (mtDNA) of the oocyte remains in the cell. This
requires interaction between the oocyte mtDNA and a foreign nucleus,
raising concerns of incompatibility. Successful SCNT proves that this
matter can be overcome to the extent of producing viable cells, embryos
and even full-grown animals. The lack of somatic cell donor mtDNA
transfer facilitates the therapeutic use of SCNT in diseases caused by
mtDNA mutations [39,40]. Although totipotency features have been
reported in in vivo induced pluripotent stem cells [41], SCNT is
currently the only known technique able to confer true totipotency
on somatic nuclei.
An approach intended to attenuate the ethical issues regarding
therapeutic SCNT is altered nuclear transfer (ANT). This technique
employs nuclear transfer, but only after the somatic cell nucleus and/or
the oocyte are altered in order to inhibit totipotency and eliminate the
full embryonal developmental potential, while still allowing the formed
pseudo-embryo to generate pluripotent stem cells [42].
There are other methods enabling the production of human
pluripotent stem cells without the use of neither oocytes nor embryonic
stem cells as starting materials, and which do not include formation or
destruction of pre-implantation state embryos. However, nuclear
transfer is still the only known method of reprogramming cells into
totipotency — a feature for which specific therapeutic applications
could theoretically be found in the future. It is also possible that the
resultant reprogrammed cells from nuclear transfer could differ from
cells reprogrammed using other methods. Furthermore, as non-human
cloning is already used to produce transgenic animals for research
purposes, along with foods and some medical products approved for
human use, nuclear transfer methods have found important applica-
tions.
2.3. Cell fusion
During cell-fusion-reprogramming (Box 3), somatic cells are fused
with embryonic stem cells, thereby exposing them to the reprogram-
ming milieu of the stem cell. The method has been employed using both
murine [43,44] and human [45,46] cells. Alternative methods of
inducing cell fusion include electrofusion and Sendai virus-induced
fusion. Both these methods have shown promise, though a comparative
study found PEG to be the superior fusion inducer [47].
Since it can be achieved using human embryonic stem cells, without
the specific need for oocytes, it makes cell fusion reprogramming a
practically and ethically interesting alternative for regenerative medi-
cine applications. However, following fusion, the resultant hybrid cells
contain nucleic acid material from both the somatic cell and the
embryonic stem cell used. Because nuclear factors are likely involved
in the process of reprogramming by cell fusion [48], removal of stem

Box 3
Cellular-factors based reprogramming methods.

Independently of the reprogramming method, involving factors identified by Yamanaka, which upon ectopic expression could drive
reprogramming, others have successfully reprogrammed cells using factors readily available in stem cells or oocytes. Below we show a few
examples:
Somatic cell nuclear transfer (SCNT), often referred to as “cloning”, is achieved by transfer of a nucleus from a somatic cell into an
enucleated unfertilized oocyte, after which the oocyte cytoplasmic environment confers reprogramming on the transferred nucleus and
subsequently activating embryo development [136,137].
Cell fusion: Most modern protocols use a 1:1 ratio of somatic cells:stem cells, and fusion is induced by addition of PEG, yielding
approximately 1% fusion efficiency [45]. The resulting hybrid cells expressed reactivated pluripotent markers Oct4, Nanog and Sox2, and
both in vitro and in vivo differentiation yielded cell types from all three germ layers [45,48]. Results from one study suggests that it is
nuclear factors rather than cytoplasmic factors that are responsible for cell fusion reprogramming as fusion with karyoplasts, but not with
cytoplasts, induced Oct4 expression in the somatic genome [48].
Cell extracts: Exposure to extracts of human or murine pluripotent cells has been shown to generate pluripotent stem cell-like phenotypic
and genotypic characteristics in human somatic cells [138,139]. Upregulated expression of pluripotency-related genes Oct4, Sox2, c-Myc
and Klf, as well as downregulated expression of LMNA, a marker of differentiation [140], were indicative of pluripotency in these studies.
The human cell extract reprogrammed cells were able to differentiate into mesoderm and ectoderm lineages, but they were not
demonstrated to differentiate into all three germ layers in vivo. This could suggest that the cells may have been only partially/incompletely
reprogrammed. Cell extract method offers possible reprogramming without genetic alteration of target cells, and avoids some of the
practical and ethical pitfalls of nuclear transfer and cell fusion. However, long-term expression profiles supporting pluripotency and in vivo
differentiation into all germ layers have not yet been demonstrated. However, with the establishment of reprogramming methods capable
of producing human pluripotent cells using only a few well-defined transcription factors, miRNAs or small molecule compounds, the
importance of such methods are profoundly diminished.

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Table 2
Notable advantages and disadvantages of different reprogramming methods.

Virus Method Advantages Disadvantages

Yes Retrovirus High efficiency Risk of insertional mutagenesis, transgene reactivation and residual
expression
Risk of presence of viral particles
Can only be used in dividing cells
Yes Lentivirus High efficiency Risk of insertional mutagenesis, transgene reactivation and residual
Can be used in both dividing and non-dividing cells expression
Risk of presence of viral particles
Yes Adenovirus No integration/small risk of integration Moderate efficiency
Need for repeated transductions
Risk of presence of viral particles
Yes Sendai virus High efficiency Risk of presence of viral particles
Non-transgene, no integration
No Conventional and episomal plasmids No integration Very low efficiency
No viral particles Need for repeated transfections
No Minicircle Efficiency higher than conventional plasmids Low efficiency (lower than viral methods)
No integration (Need for repeated transfections)
No viral particles
No bacterial backbone
No piggyBac transposon No viral particles Low efficiency
Theoretically possible to excise transgenes Extra excision step
Imperfect excision and transposition
Risk of insertional mutagenesis, transgene reactivation and residual
expression
Possible interactions between piggyBac system and endogenous
transposon systems
No mRNA Very high efficiency Need for repeated transfections
Non-transgene, no integration High cost
No viral particles
No Protein Non-transgene, no integration, no exogenous nucleic Very low efficiency
acids
No viral particles
No Small molecule compounds Non-transgene, no integration, no exogenous nucleic Not yet shown in human cells
acids Moderate efficiency
No viral particles
Moderate efficiency
No Direct transfection of mature miRNA Non-transgene, no integration Moderate efficiency
No viral particles
Moderate efficiency
Yes Lentiviral delivery of miRNA coding Very high efficiency Risk of insertional mutagenesis, transgene reactivation and residual
DNA expression

cell nucleic acids prior to fusion may not be a viable alternative. Other
approaches have aimed at selectively removing stem cell nuclei after
allowing time for reprogramming or selectively removing stem cell
chromosomes from hybrids. Such approaches have shown promise in
murine cells, but their feasibility in human cells is yet to be confirmed
[49,50]. Furthermore, hybrid cells also contain non-nuclear stem cell-
derived mtDNA, which could potentially pose a problem of incompat-
ibility. Thus, the presence of stem cell nucleic acids in the hybrid cells
and the possible immunological rejection in recipients are issues that
must be resolved before cell fusion reprogramming could be used in
clinical applications. Another issue is the low efficiency of cell fusion
reprogramming. Several optional methods using no embryonic cells,
but only addition of a few well-defined transcription factors, miRNA or
small molecule compounds exist. Cell extracts constitute another
method (Box 3).
3. Reprogramming without OSKM-factors delivery
Numerous studies have identified several molecules that can
enhance reprogramming and even replace individual OSKM factors by
epigenetic regulation. Various combinations of a subset of reprogram-
ming factors together with i.e. Rcor2, 5-mc-hydroxylase Tet1, valproic
acid, kenpaullone or various miRNAs may induce pluripotency
[51–53]. Finding a small molecule substitute for Oct4 long seemed
near impossible, but recently one study reported reprogramming of
murine somatic cells using only seven small molecule compounds [54].
Even though the reprogramming efficiency was modest, should this
result be confirmed by others and extended to human cells, the small
molecule-only technique offers an interesting alternative as many of the
safety concerns regarding other techniques involving viruses, trans-
genes and/or non-transgene exogenous nucleic acids are evaded. Still,
since the small molecule compounds affects epigenetic cell-cycle
regulation and related functions, caution must be taken not to unleash
uncontrolled proliferation or genome instability [55–57].
MicroRNAs (miRNAs) are short, non-coding RNAs that regulate
gene expression by enhancing degradation of mRNA and repressing
mRNA translation [58]. While down-regulation is the most widely
recognized function of miRNA, up-regulation of target mRNA transla-
tion by miRNA has also been reported [59]. Knockdown of key players
in the miRNA biogenesis results in loss of miRNA in cells and decreased
reprogramming efficiency [60,61]. Several miRNAs are able to enhance
factor-induced reprogramming, other miRNAs decrease reprogramming
efficiency, and OKSM reprogramming factors and reprogramming
relevant miRNAs are reciprocally regulated [61–64]. As is presumably
the case for most cellular processes, miRNAs clearly play an important
role in reprogramming and maintaining pluripotency. Recently, se-
lected miRNA clusters were used to carry reprogramming both murine
and human cells with high efficiency without the introduction of any
exogenous transcription factors or transcription factor coding nucleic
acids [52,65]. miRNA-based reprogramming offers an appealing safety
profile, especially direct transfection of mature miRNA which evades
the concerns of insertional mutagenesis, transgene activation, residual
expression and presence of viral particles associated with most other
reprogramming methods. Remarkably high reprogramming efficiency

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was reported when viral vectors were used to introduce miRNA coding
DNA [65]. The reprogramming efficiency of direct transfection of
mature miRNAs was substantially lower, though still comparable to
the reprogramming efficiency of viral delivery of reprogramming
factors [52]. One suggested rationale for the high efficiency is that
one single miRNA can regulate hundreds of targets, and when clusters
of miRNA are introduced, a huge number of targets can be regulated by
several miRNAs working in great synergy [60,66,67]. On the downside,
the stability of transfected miRNA is not very high and multiple
transfections were needed for complete reprogramming. Also, the
detailed mechanisms of miRNA reprogramming are far from entirely
outlined, and, as is the case with all reprogramming methods, more
research is needed before miRNA reprogramming can be considered
safe for clinical applications. The fact that one single miRNA can
regulate hundreds of targets adds to this concern, as many non-
reprogramming cellular processes could be simultaneously affected.
Table 2 summarizes the most important advantages and disadvantages
of the described methods.
4. Methods that enhance reprogramming
A plethora of methods of enhancement of reprogramming efficiency
has been proposed. p53 pathways are upregulated in OSKM factors
reprogramming, and knockdown of p53 enhances reprogramming
efficiency [56,68]. But, as p53 is utterly important in maintaining the
genomic integrity, it is unclear if p53 knockdown could safely be
applied in the production of iPS cells for clinical use, especially when
reprogramming itself could potentially compromise the genomic integ-
rity. Targeting some selected mediators in the p53 pathway could
provide a safer option [69].
Rho-associated kinases (ROCKs) are Rho-GTPase activated kinases
that serve many diverse roles, including regulation of cytoskeletal
dynamics, cell cycle control, and modulation of apoptosis [70].
Application of ROCK inhibitors could enhance the efficiency of iPS cell
production, at least partially by inhibiting single-cell dissociation-
induced apoptosis [71,72].
A novel way of enhancing reprogramming efficiency is mechan-
omodulation of cells' epigenetic profile by introducing microgrooves in
the cell-adhesive substrates [73]. The detailed mechanism is thought to
rely on decreased histone deacetylase activity and increased H3
methyltransferase activity, resulting in increased histone H3 acetylation
and methylation. Though ROCK is involved in cytoskeletal processes,
which are susceptible to mechanical stimuli, no role of ROCK was
proposed in this model. Application of tensile strain reduces expression
of pluripotency-related genes via ROCK activation, and upon ROCK
inhibition the gene expressions are recovered [74].
Enhancement of iPS cell generation by culturing under hypoxic
conditions has been described in both murine and human cells [75,76].
Hypoxia inducible factors (HIFs) play a role in regulation of pluripo-
tency in human embryonic stem cells cultured under hypoxic condi-
tions by upregulating the expression of Oct4, Sox2 and NANOG which
are some of the most commonly used reprogramming factors [77].
Several additional compounds have been used to promote and
enhance reprogramming, in some cases increasing reprogramming
efficiency > 100-fold when low-efficient reprogramming methods are
used. Various histone deacetylase inhibitors and histone methyltrans-
ferase inhibitors, SV40 T-large antigen and telomerase-reverse tran-
scriptase are some worth mentioning [78,79]. Knockdown of Mbd3, an
integral part of the Mbd3/NuRD repressor complex, allowed for very
fast, extraordinarily efficient and synchronized OSKM factor repro-
gramming fitting a deterministic model [80]. Standard OSKM factor
reprogramming is widely considered a stochastic process [81,82].
When multiple reprogramming transgenes are used, polycistronic
vectors allow reprogramming with less integrations than using one
vector for each factor [13,83]. At the very least one integration is still
necessary for reprogramming, but minimizing the number of integra-
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BBA - Molecular Cell Research 1864 (2017) 1359–1369
tions reduces the chance for insertional mutagenesis, transgene reacti-
vation and residual expression. From a practical point of view, the
production and handling of only one polycistronic construct could be
convenient. Also, the use of polycistronic vectors ensures that all
successfully transduced cells express all factors. A trade-off is negatively
influenced packaging efficiency of viral particles as the genome size
increases [25,36,83].
5. Reprogramming, transdifferentiation, and carcinogenesis
The major challenge of translating reprogramming and transdiffer-
entiation research into clinical applications is the risk of the develop-
ment of cancer. OKSM reprogramming factors themselves are proto-
oncogenes, and their expression commonly increases in metastatic
cancers. We have already discussed the dangers of insertional mutagen-
esis posed mainly by retro- and lentiviral vectors used in some
reprogramming protocols. iPS cells proliferate in an uncontrolled
manner similar to cancer cells. The transplantation of iPS-derived cells
containing any residual iPS may result in the formation of somatic
tumors.
Transdifferentiation is viewed as a safer alternative to reprogram-
ming. By directly converting differentiated cells into other types of
cells, one avoids the state of pluripotency. Hence, obtained by
transdifferentiation autologous cells do not inherently acquire the
ability to self-renew and proliferate uncontrollably as in reprogram-
ming. While this reduces the risk of cancer development, carcinogenesis
is still possible due to the genetic and epigenetic changes that could
occur during transdifferentiation.
5.1. Risk of carcinogenesis
The association between reprogramming and carcinogenesis is
mainly attributable to insertional mutagenesis, and genetic and epige-
netic alterations that could occur during reprogramming in addition to
the inherent tumorigenic capacity of pluripotent cells. The ability of iPS
cells to generate teratomas in immunocompromised mice is actually
used to verify their pluripotency. In vivo-teratoma formation occurs
quicker and more efficiently for iPS cells in comparison to embryonic
stem (ES) cells, indicating their increased propensity to form tumors
[84]. This is in part due to the factors used to induce reprogramming.
The primary method used for reprogramming is ectopic expression
of four transcription factors: Oct4, Sox2, c-Myc, and Klf4 [1]. Each of
these factors is associated with multiple forms of cancer. The method of
exogenous gene delivery into cells can also induce tumorigenesis. iPS
cells were initially generated by transduction of reprogramming factors
using integrating viral vectors. Integration of these genes near or within
gene encoding regions can inadvertently deactivate tumor suppressor
genes or activate oncogenes. Additionally, in vivo-reactivation of
integrated transgenes in iPS-derived cells can result in somatic tumor
development.
Given the potential tumorigenic properties of the reprogramming
factors (Box 4), ways to reduce their usage in reprogramming have been
sought. iPS cells have been generated without c-Myc and Klf4 through
replacement with alternative pluripotency factors such as Nanog and
Lin28 [3] but these proteins are also linked to different forms of cancer.
Reprogramming has also been completed using only Oct4 and Klf4 by
addition of glycogen synthase kinase-3 (GSK3) inhibitor CHIR 99021
[85]. However, this was done using murine embryonic fibroblasts only,
and so far not replicated in differentiated human cells. Even if
reprogramming of human cells occurs using Oct4 and Klf4 alone, two
genes associated with cancer development are still being overexpressed
in cells. In addition, consistent reprogramming with reduced transgenic
overexpression may only be feasible in cells with high endogenous
levels of omitted reprogramming factors.
A. Cieślar-Pobuda et al. BBA - Molecular Cell Research 1864 (2017) 1359–1369

Box 4
Carcinogenesis from reprogramming factors.

Oct4 is a transcription factor that is fundamental to reverting differentiated cells back to a pluripotent state during reprogramming.
However, its promotion of pluripotency is also involved in cancer development. For example, in breast cancer, Oct4 is expressed
significantly higher in cancerous tissue and has been proposed as a biomarker for the cancers initiation, and growth [141]. It is also
correlated with reduced differentiation and increased tissue invasion in gastric cancer, resulting in a more negative prognosis [142]. These
examples demonstrate that Oct4’s function in reprogramming overlaps with its role in cancer.
c-Myc is a well-established oncogene that is expressed at increased levels in a variety of cancers. Research has shown that it has at least a
partial role in higher frequency of tumorigenesis in iPS-derived tissues. A study analyzing tumor formation from iPS-derived cells found
that 20% of tumors were attributable to reactivation of the c-Myc transgene [13]. Reprogramming has been performed without ectopic
expression of c-Myc in human adult dermal fibroblasts, demonstrating that it is not necessary for reprogramming [15]. However, the
omission of c-Myc significantly reduces the efficiency of reprogramming.
Sox2 is a transcription factor that heterodimerizes with Oct4 to activate genes involved in the maintenance of pluripotency [143]. This
heterodimer is also overexpressed in multiple forms of cancer including neuroblastoma [144] and liver cancer [145]. Sox2 is critical factor
in the self-renewal and tumorigenicity of melanoma cells [146]. Reprogramming has occurred in adult cells with only expression of Oct4
and Klf4 but this was in dermal papilla cells which have high endogenous levels of c-Myc and Sox2 [147].
Klf4 is important for maintaining self-renewal in stem cells. It influences reprogramming through direct interaction with Oct4 and Sox2
[148]. In regards to carcinogenesis, it has a dual function as both a tumor suppressor and an oncogene depending on the type of cancer. As
an oncogene, it is highly expressed in over 70% of breast cancers and is required for the maintenance of breast cancer stem cells [149]. It is
also overexpressed in colon cancer stem cell populations and its reduced expression decreases the ability of these cells to generate tumors
[150]. The function of Klf4 in cancer stem cells likely overlaps with its role in iPS cells.

5.2. Incomplete differentiation
Clinical use of iPS-derived cells requires complete differentiation.
Any residual expression of genes related to pluripotency may increase
their tumorigenicity. Contamination of transplanted cells with iPS or
unsuccessfully reprogrammed cells cause cancer occurrence when
transplanted into patients. Achieving pure populations prior to intro-
duction into patients will require complete differentiation or complete
elimination of undifferentiated cells from the pool meant for transplan-
tation. Attempts to generate homogenous differentiated cell popula-
tions include elimination of pluripotent cells using cytotoxic antibodies
[86] and removal of undifferentiated cells based on cell surface
molecules using magnetic-activated cell sorting (MACS) and fluores-
cence-activated cell sorting (FACS) [87].
5.3. Transgenic reactivation
Reactivation of the reprogramming factors may induce carcinogen-
esis. As an example, ectopic expression of Oct3/4 [88] and Klf4 [89]
could trigger dysplasia in vivo. Transgene reactivation is mainly a risk
when genes are integrated into the cell genome. To reduce genomic
integration, reprogramming factors have been introduced using non-
integrating Sendai viral vectors [23], episomal vectors [19], and
piggyBac transposons [90]. Reprogramming has also been induced
without transgenes, by alternatively transducing purified recombinant
reprogramming proteins [38] or modified synthetic mRNAs [34].
However, these methods considerably reduce reprogramming effi-
ciency.
5.4. Mutations acquired in vitro
Therapeutic production of iPS cells requires a number of cell
divisions as cells are cultured in vitro for weeks at a time. During this
period genetic mutations may accumulate. In addition, reprogramming
likely elicits stress response pathways within cells, increasing their
susceptibility to genetic mutations. The majority of mutations should be
selected against but if mutations result in proliferative or antiapoptotic
abilities they can be selected for during reprogramming, resulting in iPS
cells with genetic abnormalities. Whole genome sequencing has re-
vealed that iPS cells have a higher number of copy number variations
(CNV) than do ES cells [91]. A separate study demonstrated that of
these CNV, deleted regions often contained tumor-suppressor genes,
while regions of chromosomal gain included oncogenes [92]. The rate
of mutation in iPS cells has also been estimated to be ten times more
frequent than in fibroblasts [93].
5.5. Epigenetic modifications
Reprogramming is an epigenetic process that does not directly alter
DNA sequence. During reprogramming, removal of epigenetic identities
of the source cells is combined with induction of epigenetic modifica-
tions similar to those of ES cells. Genetic alteration occurs only if
mutations occur during reprogramming or if transgenes integrate into
genomes. Epigenetic changes induced by reprogramming may contri-
bute to genomic instability. This could subsequently increase the rate of
genomic alterations, possibly in cancer-related genes. Accumulation of
epigenetic alterations, especially aberrant DNA methylation, increases
the risk of carcinogenesis, particularly those associated with chronic
inflammation [94,95]. iPS cells experience aberrant hyper-methylation
in early passages which is continuously detected throughout repro-
gramming [96].
6. Autophagy, reprogramming and stemness maintenance
Autophagy is a degradation process crucial for remodeling of
cellular homeostasis during cell and tissue development. Its deregula-
tion has been observed in some diseases, and several types of treatments
either trigger autophagy or use autophagy as a therapeutic target
[97–99]. A growing body of evidence shows that autophagy plays also
an indispensable role during reprogramming, differentiation and main-
tenance of cellular stemness [100,101]. In the light of recent studies it
appears that autophagy is particularly important for early phases of cell
reprogramming during the generation of iPS cells [100,102–104]. It has
been shown that autophagy induced during reprogramming is regulated
by the mechanistic target of rapamycin complex 1 (mTORC1) [102] and
that well-characterized mTOR inhibitors and autophagy activators
(PP242, rapamycin or resveratrol) notably improve the speed and
efficiency of iPS cells generation [103].
It is also well documented that autophagy is responsible for
maintenance of stemness by preventing cellular senescence
[105,106]. Studies performed by Garcia-Prat et al. shows that satellite
cells with impaired autophagy machinery enter a senescence state,

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which is accompanied with mitochondrial dysfunctions and oxidative
stress. Re-establishment of autophagy reverses senescence and restores
regenerative properties of satellite cells [106]. It is also suggested that
autophagy may play a relevant role in protecting cellular stemness and
self-renewal ability by reducing oxidative stress [107,108]. Induction of
autophagy by starvation or rapamycin, reduces ROS-induced DNA
damage and helps in maintaining the stemness [107]. Studies on
human mesenchymal stem cells (hMSCs) showed that elevated ROS
levels trigger FOXO3 induced autophagy and the knockdown of
autophagy-related 7 (ATG7) results in FOXO3 downregulation and
reduced capacity of hMSCs to handle elevated ROS levels. As a
consequence of defective autophagy hMSCs reduce their ability to
differentiate into osteoblasts [109]. The importance of autophagy
during differentiation and tissue regeneration was also confirmed in
the work of Saera-Vila et al. who showed that autophagy is activated in
response to zebrafish muscle injury, and blocking the autophagy using
chloroquine or Atg5 knockdown reduces the rate of regeneration [110].
Although, no one so far described the role of autophagy in
transdifferentiation, it can be presumed that its function is somehow
similar to that observed during generation of iPS cells through
reprogramming. Therefore, selective blocking or inducing autophagy
can be considered as a potential factor responsible of enhancing
processes of cellular conversion from one type of cell to another.
7. Transdifferentiation and carcinogenesis
Transdifferentiation does not carry as high a risk of carcinogenesis
as reprogramming. This is because direct cell-to-cell conversion does
not require an intermediate pluripotent state, which is the source of
much of tumorigenic propensity in iPS cells. Transdifferentiated cells
are generally cultured for a shorter time than iPS cells and therefore are
not as susceptible to the accumulation of genetic mutations during in
vitro culturing. Similar to reprogramming, transdifferentiation is pri-
marily induced by transgene introduction. These genes are usually
transcription factors determining the fate of the target cell type. As in
reprogramming, the characteristics of the vector used to overexpress
these transgenes could result in cancer by insertional mutagenesis or in
vivo-transgene reactivation. Similar gene delivery alternatives such as
non-integrating viruses, excisable vectors, and episomal vectors could
be used to reduce this risk. Transdifferentiation also involves epigenetic
processes and therefore, it may be subjected to the same epigenetic
abnormalities that may predispose iPS cells to form tumors. It would be
interesting to compare the cancer incidence between various implanted
tissues obtained by reprogramming with subsequent differentiation,
and by direct transdifferentiation.
8. Closing remarks
Nowadays regenerative medicine enters a new era. Reconstructive
plastic surgery, organ transplantation, various prosthesis and implants
are well known procedures. Generation of iPS cells offers to use patient-
specific somatic cells for tissue and organ regeneration. This new very
promising perspective gives hope to all patients who at present have
incurable diseases caused by the loss of a certain type of cell, i.e. type-1
diabetes and Parkinson's disease. It also offers hope for all people
suffering from paralysis due to spinal cord injury. The use of tissues
derived from autologous iPS cells is free from all disadvantages
associated with organ transplantation like immunosuppression, and
graft versus host disease.
Transdifferentiation seems to be a promising alternative for repro-
gramming as it could minimize the greatest risk associated with
reprogramming, a risk of malignant transformation. More basic re-
search is still needed to better understand all mechanisms involved in
these processes, factors limiting their efficiency and how to minimize
all risks associated with them. Several safety-related questions urgently
await answers through systematic studies.
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BBA - Molecular Cell Research 1864 (2017) 1359–1369
Transparency document
The http://dx.doi.org/10.1016/j.bbamcr.2017.04.017 associated
with this article can be found, in online version.
Acknowledgments
ACP thankfully acknowledges the support from GeCONiI
(POIG.02.03.01-24- 099/13). MJŁ and AH kindly acknowledge the
support from NCN grant #: 2016/21/B/NZ1/02812.
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