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Induce Pluripotent Stem Cell Methods, Development and Advances

Niketan Deshmukh1 and Reshma Talkal2
ABSTRACT- The self renewal and Pluripotency of Embryonic stem cell can be used in different medical purposes. But due to ethical
issues we cannot use them up to the mark. However there is a good alternative of embryonic stem cell namely induced Pluripotent
stem cells (iPSCs). The generation of iPSCs from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a
Pluripotent state by the enforced expression of a few embryonic transcription factors. In addition, iPSC technology has provided
researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders In
this review, we summarize the progress that has been made in the iPSC field.
Key words: Self-renewal, Pluripotency, Embryonic stem cell, iPSCs.

INTRODUCTION
Embryonic stem cells offer hope for new therapies,
but their use in research has been hotly debated.
Different countries have chosen to regulate
embryonic stem cell research in very different ways.
Mention embryonic stem cells in the pub and the
topic still divides opinion. Embryonic stem cell
research poses a moral dilemma. It forces us to
choose between two moral principles:

The duty to prevent or alleviate suffering

The duty to respect the value of human life

In the case of embryonic stem cell research, it is

impossible to respect both moral principles. To
obtain embryonic stem cells, the early embryo has to
be destroyed. This means destroying a potential
human life. However this limitation can be
successfully overcome with the help Induced
Pluripotent Stem Cell. Induced pluripotent stem cells
are similar to natural pluripotent stem cells, such
as embryonic stem (ES) cells, in many aspects, such
as the expression of certain stem cell genes and
proteins,
Author: Niketan Deshmukh, Mtech (Biotech),
Department of Biotechnology,Lovely Professional
University
Phagwara
144401,
India.
Email:
niketan@hotmail.co.in
Co-Author:
Reshma
Talkal,
Department
Biotechnology, Sindhu Mahavidyalaya Center
Biotech, Nagpur 440005 India.

of
of

DNA methylation patterns, doubling time, embryoid

body formation, and potency and differentiability, but
the full extent of their relation to natural pluripotent
stem cells is still being assessed. So iPSCs represent
a powerful tool for biomedical research and may
provide source for replacement therapies.
Are iPSCs truly Equivalent to ESCs?
ESCs and iPSCs are created using different
strategies and conditions, leading researchers to ask
whether the cell types are truly equivalent. To assess
this issue, investigators have begun extensive
comparisons to determine pluripotency, gene
expression, and function of differentiated cell
derivatives. Ultimately, the two cell types exhibit
some differences, yet they are remarkably similar in
many key aspects that could impact their application
to regenerative medicine. Future experiments will
determine the clinical significance (if any) of the
observed differences between the cell types.
Other than their derivation from adult
tissues, iPSCs meet the defining criteria for ESCs.
Mouse and human iPSCs demonstrate important
characteristics of pluripotent stem cells, including
expressing stem cell markers, forming tumors
containing cell types from all three primitive
embryonic layers, and displaying the capacity to
contribute to many different tissues when injected
into mouse embryos at a very early stage of
development. Initially, it was unclear that iPSCs were
truly pluripotent, as early iPSC lines contributed to

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mouse embryonic development but failed to produce

live-born progeny as do ESCs. In late 2009, however,
several research groups reported mouse iPSC lines
that are capable of producing live births,1,2 noting that
the cells maintain a pluripotent potential that is "very
close to" that of ESCs.2 Therefore, iPSCs appear to
be truly pluripotent, although they are less efficient
than ESCs with respect to differentiating into all cell
types.2 In addition, the two cell types appear to have
similar defense mechanisms to thwart the production
of DNA-damaging reactive oxygen species, thereby
conferring the cells with comparable capabilities to
maintain genomic integrity.3
Undifferentiated iPSCs appear molecularly
indistinguishable from ESCs. However, comparative
genomic analyses reveal differences between the two
cell types. For example, hundreds of genes are
differentially expressed in ESCs and iPSCs,4 and
there appear to be subtle but detectable differences
in epigenetic methylation between the two cell
types.5,6 Genomic differences are to be expected; it
has been reported that gene-expression profiles of
iPSCs and ESCs from the same species differ no
more than observed variability among individual
ESC lines.7 It should be noted that the functional
implications of these findings are presently unknown,
and observed differences may ultimately prove
functionally inconsequential.8
Recently, some of the researchers who first
generated human iPSCs compared the ability of
iPSCs and human ESCs to differentiate into neural
cells (e.g., neurons and glia).9 Their results
demonstrated that both cell types follow the same
steps and time course during differentiation.
However, although human ESCs differentiate into
neural cells with a similar efficiency regardless of the
cell line used, iPSC-derived neural cells demonstrate
lower efficiency and greater variability when
differentiating into neural cells. These observations
occurred regardless of which of several iPSCgeneration protocols were used to reprogram the
original cell to the pluripotent state. Experimental
evidence suggests that individual iPSC lines may be
"epigenetically unique" and predisposed to generate
cells of a particular lineage. However, the authors
believe that improvements to the culturing techniques

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may be able to overcome the variability and

inefficiency described in this report.
These findings underpin the importance of
understanding the inherent variability among discrete
cell populations, whether they are iPSCs or ESCs.
Characterizing the variability among iPSC lines will
be crucial to apply the cells clinically. Indeed, the
factors that make each iPSC line unique may also
delay the cells' widespread use, as differences among
the cell lines will affect comparisons and potentially
influence their clinical behavior. For example,
successfully modeling disease requires being able to
identify the cellular differences between patients and
controls that lead to dysfunction. These differences
must be framed in the context of the biologic
variability inherent in a given patient population. If
iPSC lines are to be used to model disease or screen
candidate drugs, then variability among lines must be
minimized and characterized fully so that researchers
can understand how their observed results match to
the biology of the disease being studied. As such,
standardized assays and methods will become
increasingly important for the clinical application of
iPSCs, and controls must be developed that account
for variability among the iPSCs and their derivatives.
Additionally, researchers must understand
the factors that initiate reprogramming towards
pluripotency in different cell types. A recent report
has identified one factor that initiates reprogramming
in human fibroblasts,10 setting the groundwork for
developing predictive models to identify those cells
that will become iPSCs. An iPSC may carry a genetic
"memory" of the cell type that it once was, and this
"memory" will likely influence its ability to be
reprogrammed. Understanding how this memory
varies among different cell types and tissues will be
necessary to reprogram successfully.
Principle of Reprogramming Somatic Cell
It is found that the difference between in a somatic
cell and embryonic stem cell is present at genomic
level. Expression of some specific genes is
responsible for maintained the property of self
renewal and pluripotency in embryonic stem cell. But
when the cell move from uncommitted stage to
committed stage i.e. differentiation, the expression of
these genes is stop. The cloning of Dolly

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demonstrated that nuclei from mammalian

differentiated cells can be reprogrammed to an
undifferentiated state by trans-acting factors present
in the oocyte11, and this discovery led to a search for
factors that could mediate similar reprogramming
without somatic cell.

dysregulated growth in the absence of a defensive

immune system. Optimization of bioengineered stem
cells will likely produce specialized properties that
improve stress tolerance, streamline differentiation
capacity, and increase engraftment/survival to
improve regenerative potential.

Recently, four transcription factors (Oct4, Sox2, cmyc, and Klf4) were shown to be sufficient to
reprogram mouse fibroblasts to undifferentiated,
Pluripotent stem cells (termed induced pluripotent
stem (iPS) cells) 1215. The process of reprogramming
requires controlled, stoichiometric expression of
transgenes for a transient period of time.16-20 .
Multiple sources of tissue such as ordinary
fibroblasts,21
keratinocytes,22
hematopoietic
lineages,23 or adipose tissue24 have been successfully
reprogrammed. Ectopic stemness factors are
sufficient to induce telomere elongation,25 histone
modifications,26 secondary gene expression profiles,27
and cellular metamorphosis that collectively reestablish
a
self
stabilizing
phenotype.28
Reprogramming occurs typically within weeks,
following exposure to trans-acting factors that can be
delivered to the nucleus either by plasmids, viruses,
or bioengineered proteins. Thus, transgene expression
initiates a sequence of reprogramming events that
eventually transforms a small fraction of cells (<
0.5%) to acquire an imposed pluripotent state
characterized by a stable epigenetic environment
indistinguishable from the blastocystderived natural
stem cell milieu. The converted pluripotent ground
state results in the maintenance of the unique
developmental potential with the ability to
differentiate into all germ layers.

METHODS OF IPSC PRODUCTION

Induced pluripotent stem cell platforms bypass the

need for embryo extraction to generate genuine
pluripotent stem cells from self-derived, autologous
sources. In the mouse, bioengineering strategies have
yielded iPS cells sufficient for complete de novo
embryogenesis as the highest evidence of pluripotent
stringency.33,34 In humans29,30,31,32, iPS cells have
ensured
comprehensive
multi-lineage
tissue
differentiation by demonstrating the ability to give
rise to all three germ layers in teratoma formation.
Self-derived iPS cells are recognized within the
transplanted hosts as native tissue due to their
autologous status and thus require protection from

Retroviral metod of iPSC Production

In the first works on murine and human
iPSC production, either retro or lentiviral vectors
were used for the delivery of Oct4 , Sox2 , Klf4 ,
and cMyc genes into somatic cells. The efficiency of
transduction with retroviruses is high enough,
although it is not the same for different cell types.
Retroviral integration into the host genome requires a
comparatively high division rate, which is
characteristic of the relatively narrow spectrum of
cultured cells. Moreover, the transcription of
retroviral construct under the control of a promoter
localized in 5LTR (long terminal repeat) is
terminated when the somatic cell transform switches
to the pluripotent state 36. This feature makes
retroviruses attractive in iPSC production.
Nevertheless, retroviruses possess some properties
that make iPSCs that are produced using them
improper for cell therapy of human diseases. First,
retroviral DNA is integrated into the host cell
genome. The integration occurs randomly; i.e., there
are no specific sequences or apparent logic for
retroviral integration. The copy number of the
exogenous retroviral DNA that is integrated into a
genome may vary to a great extent 35. Retroviruses
being integrated into the cell genome can introduce
promoter elements and polyadenylation signals; they
can also interpose coding sequences, thus affecting
transcription. Second, since the transcription level of
exogenous Oct4 , Sox2 ,Klf4 ,
and cMyc in
the
retroviral construct decreases with cell transition into
the pluripotent state, this can result in a decrease in
the efficiency of the stable iPSC line production,
because the switch from the exogenous expression of
pluripotency genes to their endogenous expression
may not occur. Third, some studies show that the
transcription of transgenes can resume in the cells
derived from iPSCs 36. The high probability that the
ectopic Oct4 , Sox2 , Klf4 ,
and cMyc gene

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expression will resume makes it impossible to apply

iPSCs produced with the use of retroviruses in
clinical trials; moreover, these iPSCs are hardly
applicable even for fundamental studies on
reprogramming
and
pluripotency
principles.
Lentiviruses used for iPSC production can also be
integrated into the genome and maintain their
transcriptional activity in pluripotent cells. One way
to avoid this situation is to use promoters controlled
by exogenous substances added to the culture
medium, such as tetracycline and doxycycline, which
allows the transgene transcription to be regulated.
iPSCs are already being produced using such systems
37
.
Another serious problem is the gene set
itself that is used for the induction of pluripotency 38.
The ectopic transcription of Oct4 , Sox2 , Klf4 ,
and cMyc can lead to neoplastic development from
cells derived from iPSCs, because the expression
of Oct4 , Sox2 , Klf4, and cMyc genes is associated
with the development of multiple tumors known in
oncogenetics . In particular, the overexpression
ofOct4 causes murine epithelial cell dysplasia 39, the
aberrant expression of Sox2 causes the development
of serrated polyps and mucinous colon carcinomas 40,
breast tumors are characterized by elevated
expression of Klf4 [2 , and the improper expression
of cMyc is observed in 70% of human cancers 41.
Tumor development is oberved in ~50% of murine
chimeras obtained through the injection of retroviral
iPSCs into blastocysts, which is very likely
associated with the reactivation of exogenous c
Myc 42,43.
Non-retroviral Methods of iPSC Production
A huge barrier to the eventual use of iPSCderived treatments is the use of retroviruses to force
the expression of the four key genes, discussed
above, and activating their transcription factors.
Retroviruses can carry target DNA that is inserted
into a host cells genome upon injection, making
them ideal for incorporating the four genes into target
cells. However, this DNA and the rest of the viruses
genomes remain in the host genome, which can lead
to transcription of unwanted genes and greatly
increases the risk of tumors. The expression of the
four
transgenes
must
be
silenced
after

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reprogramming to avoid harmful gene expression. cMyc, a tumor-promoting gene, especially must be
silenced after cellular reprogramming or the risk of
tumor development becomes too great for clinical
use. These retroviral methods in which the transgenes
are still present in the pluripotent cells pose a danger
to safety, and also are less closely related to ESCs in
gene
expression
than
their
non-retroviral
alternatives. Methods of reprogramming iPSCs
without transgene expression in the reprogrammed
cell are therefore essential not only for potential
therapies and clinical applications, but also for
reliable
and
accurate in vitro models
of
diseases. Yet, the low efficiency of alternatives
remains a worry. Whether these methods will be
viable for human clinical use remains to be seen.
Excisions strategy of iPSC generation
The excision strategy (transient transfection)
of iPSC generation allows the transgenes to briefly
integrate into the genome but then removes them
once reprogramming is achieved. An example of this
site/enzyme combination is the loxP site and the Cre
enzyme. In a study of Parkinsons disease (PD),
specific iPSCs, this loxP/Cre combination was used
to generate the iPSCs. Neural differentiation was then
induced on the iPSCs to test whether they could
differentiate into dopaminergic neurons, the cells
harmed in PD. The differentiation was successful,
indicating the transgenes had been excised. However,
a loxP site remains in iPSC genome as does some
residual viral DNA, so there is still a small potential
for
insertional
mutagenesis.
The piggyBac site/enzyme system on the other hand
is capable of excising itself completely, not leaving
any remnants of external DNA in the iPSC
genome. The piggyBac system also was much more
efficient than other non-retroviral methods, with
comparable efficiency to retroviral methods, but with
the added benefits of safety and ease of application.
Adenoviral Methods
Adenoviral methods do not pose the same
threats as retroviral methods of generating iPSCs.
Adenoviruses work like all viruses by hijacking their
hosts cellular machinery to replicate their own
genome and reproduce, but unlike retroviruses they
do not incorporate their genome into the host DNA.

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Because the transgenes are never even incorporated

into the hosts genome they do not have to be
excised. Instead, the genes are expressed directly
from the virus genome. iPSCs created by adenoviral
methods demonstrated pluripotency, but have
extremely
low
reprogramming
efficiency. Viral genomic material could not be
detected in any of the iPSCs, and no tumor formation
was reported. This suggests that the use of nonintegrating adenoviral methods substantially lowers
the threat of tumorgenesis. The successful creation of
iPSCs from adenoviral methods proves definitively
that safer, non-retroviral methods can also
successfully reengineer cells.
Non-DNA methods of iPSC generation
Recent studies have implied that
perhaps genetic material is not required for iPS
cellular reprogramming. The substitution of
transgenes with small molecules that promote iPSC
generation would be a safe, clinically appropriate
way of creating iPSCs, though it remains to be seen if
small molecules will be able to completely replace
genetic methods of iPSC generation or are just useful
as supplementary aids to the process. Protein
transduction is a different method shown to entirely
replace gene delivery. In this method fusion proteins
are created, which fuse each of the transgenes to a
cell-penetrating peptide sequence that allows it to
cross the cellular membrane
Reprogramming
without DNA intermediates should eliminate the risk
of tumorgenesis and distorted gene expression due to
the reactivation of the transgenes.
Methods
for
iPSC
production
modification of the cell genome.

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CreloxP Mediated Recombination.

To prepare iPSCs from patients with Parkinsons
disease, lentiviruses were used, the proviruses of
which can be removed from the genome by Cre
recombinase. To do this, the loxP site was
introduced into the lentiviral 3LTRregions
containing separate reprogramming genes under the
control of the doxycyclineinducible promoter.
During viral replication, loxP was duplicated in the
5LTR of the vector. As a result, the provirus
integrated into the genome was flanked with
two loxP sites. The inserts were eliminated using the
temporary transfection of iPSCs with a vector
expressing Cre recombinase 44.
In another study, murine iPSCs were
produced
using
a
plasmid
carrying
the Oct4 , Sox2 , Klf4I, and cMyc genes in the same
reading frame in which individual cDNAs were
separated by sequences encoding 2 peptides, and
practically the whole construct was flanked
with loxP sites 45. The use of this vector allowed a
notable decrease in the number of exogenous DNA
inserts in the host cells genome and, hence, the
simplification of their following excision 46. It has
been shown using lentiviruses carrying similar
polycistronic constructs that one copy of transgene
providing a high expression level of the exogenous
factors Oct4, Sox2, Klf4, and cMyc is sufficient for
the reprogramming of differentiated cells into the
pluripotent state47,48.The drawback of the CreloxP
system is the incomplete excision of integrated
sequences; at least theloxP site remains in the
genome, so the risk of insertion mutations remains.

without
Plasmid Vectors.

As was mentioned above, for murine and

human iPSC production, both retro and lentiviruses
were initially used as delivery vectors for the genes
required for cell reprogramming. The main drawback
of this method is the uncontrolled integration of viral
DNA into the host cells genome. Several research
groups have introduced methods for delivering
pluripotency genes into the recipient cell which
either do not integrate allogenic DNA into the host
genome or eliminate exogenous genetic constructs
from the genome.

The application of lentiviruses and plasmids

carrying the loxP sites required for the elimination
of transgene constructs modifies, although
insignificantly, the host cells genome. One way to
avoid this is to use vector systems that generally do
not provide for the integration of the whole vector or
parts of it into the cells genome. One such system
providing a temporary transfection with polycistronic
plasmid vectors was used for iPSC production from
mEFs.
A
polycistronic
plasmid
carrying
the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as a
plasmid expressing cMyc , was transfected into

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mEFs one, three, five, and seven days after their

primary seeding. Fibroblasts were passaged on the
ninth day, and the iPSC colonies were selected on the
25th day. Seven out of ten experiments succeeded in
producing GFPpositive colonies (the Gfp gene was
under the control of the Nanog gene promoter). The
iPSCs that were obtained were similar in their
features to murine ESCs and did not contain inserts
of the used DNA constructs in their genomes.
Therefore, it was shown that wholesome murine
iPSCs that do not carry transgenes can be
reproducibly produced, and that the temporary
overexpression ofOct4 , Sox2 , Klf4 , and cMyc is
sufficient for reprogramming. The main drawback of
this method is its low yield. In ten experiments the
yield varied from 1 to 29 iPSC colonies per ten
million fibroblasts, whereas up to 1,000 colonies per
ten millions were obtained in the same study using
retroviral constructs.
Episomal VectorsHuman iPSCs were successfully produced
from skin fibroblasts using single transfection with
polycistronic episomal constructs carrying various
combinations
of Oct4 , Sox2 ,Nanog , Klf4 , c
Myc , Lin28 , and SV40LT genes. These constructs
were designed on the basis of the oriP/EBNA1
(EpsteinBarr nuclear antigen1) vector 48. The
oriP/EBNA1 vector contains the IRES2 linker
sequence allowing the expression of several
individual cDNAs (encoding the genes required for
successful reprogramming in this case) into one
polycistronic mRNA from which several proteins are
translated. The oriP/EBNA1 vector is also
characterized by lowcopy representation in the cells
of primates and can be replicated once per cell cycle
(hence, it is not rapidly eliminated, the way common
plasmids are). Under nonselective conditions, the
plasmid is eliminated at a rate of about 5% per cell
cycle 49. In this work, the broad spectrum of the
reprogramming factor combinations was tested,
resulting in the best reprogramming efficiency with
cotransfection with three episomes containing the
following
gene
sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + S
V40LT + Klf4 , and cMyc + Lin28 .SV40LT ( SV40
large T gene ) neutralizes the possible toxic effect
of overexpression 50. The authors have shown

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that wholesome iPSCs possessing all features of

pluripotent cells can be produced following the
temporary expression of a certain gene combination
in human somatic cells without the integration of
episomal DNA into the genome. However, as in the
case when plasmid vectors are being used, this way
of reprogramming is characterized by low efficiency.
In separate experiments the authors obtained from 3
to 6 stable iPSC colonies per 106 transfected
fibroblasts. Despite the fact that skin fibroblasts are
wellcultured and accessible, the search for other cell
types which are relatively better cultured and more
effectively subject themselves to reprogramming
through this method is very likely required. Another
drawback of the given system is that this type of
episome is unequally maintained in different cell
types.
PiggyBacTranspositionOne promising system used for iPSC
production without any modification of the host
genome is based on DNA transposons. So
called PiggyBac transposons
containing
2
linkered reprogramming genes localized between the
5 and 3terminal repeats were used for iPSC
production from fibroblasts. The integration of the
given constructs into the genome occurs due to
mutual transfection with a plasmid encoding
transposase. Following reprogramming due to the
temporary expression of transposase, the elimination
of inserts from the genome took place 51,52. One
advantage of the PiggyBac system on CreloxP is
that the exogenous DNA is completely removed
53
.However, despite the relatively high efficiency of
exogenous DNA excision from the genome
byPiggyBac transposition; the removal of a large
number of transposon copies is hardly achievable.
Nonintegrating Viral VectorsMurine iPSCs were successfully produced
from hepatocytes and fibroblasts using four
adenoviral vectors nonintegrating into the genome
and carrying the Oct4 , Sox2 ,Klf4 , and cMyc genes.
An analysis of the obtained iPSCs has shown that
they are similar to murine ESCs in their properties
(teratoma formation, gene promoter DNA
methylation, and the expression of pluripotent
markers), but they do not carry insertions of viral

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DNA in their genomes 54. Later, human fibroblast

derived iPSCs were produced using this method 55.
The authors of this paper cited the postulate
that the use of adenoviral vectors allows the
production of iPSCs, which are suitable for use
without the risk of viral or oncogenic activity. Its
very low yield (0.00010.001%), the deceleration of
reprogramming, and the probability of tetraploid cell
formation are the drawbacks of the method. Not all
cell types are equally sensitive to transduction with
adenoviruses.
Another method of gene delivery based on
viral vectors was recently employed for the
production of human iPSCs. The sendaivirus (SeV)
based vector was used in this case 56. SeV is a single
stranded RNA virus which does not modify the
genome of recipient cells; it seems to be a good
vector for the expression of reprogramming factors.
Vectors containing either all pluripotency factors
or three of them (without ) were used for
reprogramming the human fibroblast. The construct
based on SeV is eliminated later in the course of cell
proliferation. It is possible to remove cells with the
integrated provirus via negative selection against the
surface HN antigen exposed on the infected cells.
The authors postulate that reprogramming technology
based on SeV will enable the production of clinically
applicable human iPSCs 56.
Cell Transduction with Recombinant ProteinsAlthough the methods for iPSC production
without gene modification of the cells genome
(adenoviral vectors, plasmid gene transfer, etc.) are
elaborated, the theoretical possibility for exogenous
DNA integration into the host cells genome still
exists. The mutagenic potential of the substances
used presently for enhancing iPSC production
efficiency has not been studied in detail. Fully
checking iPSC genomes for exogenous DNA inserts
and other mutations is a difficult task, which becomes
impossible to solve in bulk culturing of multiple
lines. The use of protein factors delivered into a
differentiated cell instead of exogenous DNA may
solve this problem. Two reports have been published
to date in which murine and human iPSCs were
produced using the recombinant Oct4, Sox2, Klf4,
and cMyc proteins 57,58 . T he method used to deliver

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the protein into the cell is based on the ability of

peptides enriched with basic residues (such as
arginine and lysine) to penetrate the cells membrane.
Murine iPSCs were produced using the recombinant
Oct4, Sox2, Klf4, and cMyc proteins containing
eleven Cterminal arginine residues and expressed
in E. coli . The authors succeeded in producing
murine iPSCs during four rounds of protein
transduction into embryonic fibroblasts. However,
iPSCs were only produced when the cells were
additionally treated with 2propylvalerate (the
deacetylase inhibitor). The same principle was used
for the production of human iPSCs, but protein
expression was carried out in human HEK293 cells,
and the proteins were expressed with a fragment of
nine arginins at the protein Cend. Researchers have
succeeded in producing human iPSCs after six
transduction rounds without any additional treatment.
The efficiency of producing human iPSC in this way
was 0.001%, which is one order lower than the
reprogramming efficiency with retroviruses. Despite
some drawbacks, this method is very promising for
the production of patientspecific iPSCs.
Potential Medical Applications of iPSCs
iPSCs have the potential to become
multipurpose research and clinical tools to
understand and model diseases, develop and screen
candidate drugs, and deliver cell-replacement therapy
to support regenerative medicine. This section will
explore the possibilities and the challenges that
accompany these medical applications, with the
caveat that some uses are more immediate than
others. For example, researchers currently use stem
cells to test/screen drugs or as study material to
identify molecules or genes implicated in
regeneration. Conducting experiments or testing
candidate drugs on human cells grown in culture
enables researchers to understand fundamental
principles and relationships that will ultimately
inform the use of stem cells as a source of tissue for
transplantation. Therefore, using iPSCs in cellreplacement therapies is a future application of these
cells, albeit one that has tremendous clinical
potential. The following discussion will highlight
recent efforts toward this goal while recognizing the
challenges that must be overcome for these cells to
reach the clinic.

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Reprogramming technology offers the

potential to treat many diseases, including
Alzheimer's
disease,
Parkinson's
disease,
cardiovascular disease, diabetes, and amyotrophic
lateral sclerosis (ALS; also known as Lou Gehrig's
disease). In theory, easily-accessible cell types (such
as skin fibroblasts) could be biopsied from a patient
and reprogrammed, effectively recapitulating the
patient's disease in a culture dish. Such cells could
then serve as the basis for autologous cell
replacement therapy. Because the source cells
originate within the patient, immune rejection of the
differentiated derivatives would be minimized. As a
result, the need for immunosuppressive drugs to
accompany the cell transplant would be lessened and
perhaps eliminated altogether. In addition, the
reprogrammed cells could be directed to produce the
cell types that are compromised or destroyed by the
disease in question. A recent experiment has
demonstrated the proof of principle in this
regard,59 as iPSCs derived from a patient with ALS
were directed to differentiate into motor neurons,
which are the cells that are destroyed in the disease.
Although much additional basic research will be
required before iPSCs can be applied in the clinic,
these cells represent multi-purpose tools for medical
research. Using the techniques described in this
article, researchers are now generating myriad
disease-specific iPSCs. For example, dermal
fibroblasts and bone marrow-derived mesencyhmal
cells have been used to establish iPSCs from patients
with a variety of diseases, including ALS, adenosine
deaminase deficiency-related severe combined
immunodeficiency, Shwachman- Bodian-Diamond
syndrome, Gaucher disease type III, Duchenne and
Becker muscular dystrophies, Parkinson's disease,
Huntington's disease, type 1 diabetes mellitus, Down
syndrome/trisomy,
and
spinal
muscular
atrophy.60,61 iPSCs created from patients diagnosed
with a specific genetically-inherited disease can then
be used to model disease pathology. For example,
iPSCs created from skin fibroblasts taken from a
child with spinal muscular atrophy were used to
generate motor neurons that showed selective deficits
compared to those derived from the child's unaffected
mother.62 As iPSCs illuminate the development of
normal and disease-specific pathologic tissues, it is
expected that discoveries made using these cells will

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inform future drug development or other therapeutic

interventions.
One particularly appealing aspect of iPSCs
is that, in theory, they can be directed to differentiate
into a specified lineage that will support treatment or
tissue regeneration. Thus, somatic cells from a patient
with cardiovascular disease could be used to generate
iPSCs that could then be directed to give rise to
functional
adult
cardiac
muscle
cells
(cardiomyocytes) that replace diseased heart tissue,
and so forth. Yet while iPSCs have great potential as
sources of adult mature cells, much remains to be
learned about the processes by which these cells
differentiate. For example, iPSCs created from
human63 and murine fibroblasts64,65 can give rise to
functional cardiomyocytes that display hallmark
cardiac action potentials. However, the maturation
process into cardiomyocytes is impaired when iPSCs
are usedcardiac development of iPSCs is delayed
compared to that seen with cardiomyocytes derived
from ESCs or fetal tissue. Furthermore, variation
exists in the expression of genetic markers in the
iPSC-derived cardiac cells as compared to that seen
in ESC-derived cardiomyocytes. Therefore, iPSCderived
cardiomyocytes
demonstrate
normal
commitment but impaired maturation, and it is
unclear whether observed defects are due to technical
(e.g., incomplete reprogramming of iPSCs) or
biological barriers (e.g., functional impairment due to
genetic factors). Thus, before these cells can be used
for therapy, it will be critical to distinguish between
iPSC-specific and disease-specific phenotypes.
However, it must be noted that this
emerging field is continually evolving; additional
basic iPSC research will be required in parallel with
the development of disease models. Although the
reprogramming technology that creates iPSCs is
currently imperfect, these cells will likely impact
future therapy, and "imperfect" cells can illuminate
many areas related to regenerative medicine.
However, iPSC-derived cells that will be used for
therapy will require extensive characterization
relative to what is sufficient to support disease
modeling studies. To this end, researchers have
begun to use imaging techniques to observe cells that
are undergoing reprogramming to distinguish true
iPSCs from partially-reprogrammed cells. The

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potential for tumor formation must also be addressed

fully before any iPSC derivatives can be considered
for applied cell therapy. Furthermore, in proposed
autologous therapy applications, somatic DNA
mutations (e.g., non-inherited mutations that have
accumulated during the person's lifetime) retained in
the iPSCs and their derivatives could potentially
impact downstream cellular function or promote
tumor formation (an issue that may possibly be
circumvented by creating iPSCs from a "youthful"
cell source such as umbilical cord blood). Whether
these issues will prove consequential when weighed
against the cells' therapeutic potential remains to be
determined. While the promise of iPSCs is great, the
current levels of understanding of the cells' biology,
variability, and utility must also increase greatly
before iPSCs become standard tools for regenerative
medicine.
CONCLUSIONS
Stem cell biology has been an area of great
interest and intense debate since its inception, and
iPSC technology has furthered this research and
created hope for potential therapeutic applications.
While there are still many barriers to the clinical use
of stem cells, iPSCs may help elucidate the nature of
both pluripotent stem cells and of many disease
pathologies to reach an eventual concrete connection
between the two. Reprogramming adult tissues to
embryonic-like states has countless prospective
applications to regenerative medicine, drug
development, and basic research on stem cells and
developmental processes. To this point, a PubMed
search conducted in April 2010 using the term
"induced pluripotent stem cells" (which was coined
in 2006) returned more than 1400 publications,
indicating a highly active and rapidlydeveloping
research field.
However, many technical and basic science
issues remain before the promise offered by iPSC
technology can be realized fully. For putative
regenerative medicine applications, patient safety is
the foremost consideration. Standardized methods
must be developed to characterize iPSCs and their
derivatives. Furthermore, reprogramming has
demonstrated a proof of-principle, yet the process is
currently too inefficient for routine clinical

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application. Thus, unraveling the molecular

mechanisms that govern reprogramming is a critical
first step toward standardizing protocols. A grasp on
the molecular underpinnings of the process will shed
light on the differences between iPSCs and ESCs
(and determine whether these differences are
clinically significant). Moreover, as researchers delve
more deeply into this field, the effects of donor cell
populations can be compared to support a given
application; i.e., do muscle-derived iPSCs produce
more muscle than skin-derived cells? Based on the
exciting developments in this area to date, induced
pluripotent stem cells will likely support future
therapeutic interventions, either directly or as
research tools to establish novel models for
degenerative disease that will inform drug
development. While much remains to be learned in
the field of iPSC research, the development of
reprogramming techniques represents a breakthrough
that will ultimately open many new avenues of
research and therapy.
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