Vol.2, No.3, 61-73 (2013) Open Journal of Regenerativ e Medicine
hiPSCs: Reprogramming towards
cell-based therapies
Phillip E. Woolwine
Independent Author; phillipwoolwine@gmail.com
Received 6 May 2013; revised 13 June 2013; accepted 15 July 2013
Copyright © 2013 Phillip E. Woolwine. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Stem cell therapies show great potential for use
in regenerative medicine, though advancements
in safe stem cell technology need to be realized.
Human induced pluripotent stem cells (hiPSCs)
hold an advantage over other stem cell types for
use in cell-based therapies due to their potential
as an unlimited source of rejuvenated and im-
munocompatible SCs which do not elicit the
ethical and moral debates associated with the
destruction of human embryos. Towards reali-
zation of this potential this review focuses on
the recent progress in DNA- and xeno-free re-
programming methods, particularly small mole-
cule methods, as well as addresses some of the
latest insights on donor cell gene expression,
telomere dynamics, and epigenetic aberrations
that are a potential barrier to successful wide-
spread clinical applications.
Keywords: iPSC; Reprogramming; Small
Molecules; Oct4; Epigenetics; Regenerative
Medicine; Cli nica l Reg ulatio ns
The promise of stem cell-based rejuvenation therapy
has long been heralded and has recently been previewed
with interim reports of success by companies such as
StemCell, Inc. In a Phase I/II trial using their proprietary
human neural stem cells (HuCNS-SCs), StemCell, Inc.
was able to show allogeneic SCs could improve the sen-
sory function of chest-level complete spinal cord injuries
[1]. Thus far the results have shown the treatment to be
safe owing to the low immunogenicity of fetally-derived
SCs, though the patients remain temporarily immuno-
suppressed. In another Phase I study these same alloge-
neic HuCNS-SCs were shown safe for injection in hu-
man brains suffering from Palizaeus-Merzbacher disease
(PMD) [2]. There is currently no cure for PMD and chil-
dren normally die of this disease around ages 10 - 15 due
to severe neurological dysfunction owing to defective
oligodendrocytes which fail to myelinate axons (review-
ed in [3]). With cautious optimism, early results show
that within nine months after the procedure transplanted
regions had increased myelination and neurological func-
These reports support the great expectations of animal-
based SC research translated into human therapies [4];
however, fetal SCs have limited availability and the use
of these and related human embryonic SCs (hESCs) still
carry many ethical considerations as harvesting these
viable embryos destroys potential life [5]. Moreover,
though ESCs have low immunogenicity due to their im-
muno-suppressive capacity and do not readily provoke a
T-cell response, as they differentiate and express more
MHC molecules on their surface allogeneic ESCs can
provoke an immune response [6] that could necessitate
lifelong immuno-suppression o r even make the cond ition
worse. While an immunocompatible and potentially
unlimited source of autologous hESCs for cell therapy
can be derived through somatic cell nuclear transfer
(SCNT), hESC generation by SCNT is technically chal-
lenging [7] and not only elicits the same ethical debate
concerning the destruction of a viable embryo but also
that of human cloning—globally banned by the United
Nations [8,9]. Though therapeutic cloning is permitted in
some countries and US states such as California and
Massachusetts [10], public perception and political cli-
mates restrict funding for development of this technology.
Adult mesenchymal stem cells (MSCs) do not have the
ethical or moral complications associated with ESCs, are
more readily available, and have already shown efficacy
in cell therapy trials to treat myocardial infarction, dia-
betes, bone lesions, cartilage damage, and skin burns,
among others [11]. However, while MSCs have been
shown to have low immunogenicity due to secretion of
immune modulators such as IL-10, HLA-G5, and TGFβ1,
allogeneic MSCs can become immunogenic upon termi-
Copyright © 2013 SciRes. OPEN A CCESS
P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73
nal differentiation [12,13]. Moreover, while autologous
MSCs are immunocompatible there are not only known
differences in quality between early and late passage
MSCs but also between MSCs derived from “youthful”
and “aged” donors which can limit safe and efficacious
use: aged and late-passage MSCs have less proliferation
ability and are more prone to genetic [14], proteomic
[15], and phenotypic abnormalities [1 6,17] and are th ere-
fore more limited in their capacity to be expanded into
clinically relevant, efficacious, and safe numbers. There-
fore, hiPSC technology which carries no ethical or moral
debate and is capable of generating potentially unlimited
numbers of immunocompatible pluripotent SCs (PSCs)
with both rejuvenated bioenergetics and replicative life-
spans from patients own cells has been under aggressive
Indeed, hiPSCs have been successfully created in
many labs using a variety of reprogramming techniques;
however, iPSC technology still has significant advances
to be made in safety and efficiency for viable use in
regulatory-compliant, clinical-scale human therapies—a
focus in this review. Takahashi & Yamanaka [18] first
defined the core transcription factors (TFs) for inducing
pluripotency in somatic cells of mice as Oct3/4, Sox2,
Klf4, and c-Myc (OSKM) using gammaretroviruses with
high transduction efficiencies and then in human dermal
fibroblasts [19] using lentiviruses; however, these meth-
ods resulted in more than 20 retroviral integrations per
clone and oncogenic po tential too high for clinical thera-
peutics. This is a significant hurdle to regulatory ap-
proval as the FDA code of federal regulations (21 CFR
Part 1271) and compliance program (7341.002) requires
that the iPSCs be xeno- and foreign DNA-free, free of
growth abnormalities and mutagenesis, noncontaminated,
and consistently manufactured according to cGMP be-
fore regulatory approval will be awarded [20]. Thus,
advances in iPSC derivation have been made using non-
integrating ad enoviru ses [21], len tiviral vector s [22], epi-
somal vectors [23], minicircle vectors [24], piggyBac
(PB) transposons [25,26], Sendai Virus [27], mRNA [28,
29], miRNAs [30,31], proteins [32], and small molecules
[33-43]. Moreover, though hiPSC reprogramming does
manifest some epigenetic aberrations [44,45] currently
limiting safe clinical ap plication, these cells appear to be
rej uven ated, having both “youthful” telomere lengths [4 6]
and mitochondria [47,48]. While there has also been
great success in animal-based iPSC research, the focus of
this review will be on safe and efficient hiPSC methods
and genomics.
2.1. Adenoviral, Cre-Lox, PiggyBac
Since Takahashi & Yamanaka’s pivotal research on
reprogramming terminally differentiated cells to pluri-
potency, a number of vector and reprogramming factor
variations and improvements have been made. In 2009,
Zhou & Freed [21] showed that much more genomically
stable hiPSCs could be generated using OSKM in nonin-
tegrating, transient adenoviral transfection, for example.
These reprogrammed fibroblasts were then shown to
readily differentiate into neural dopaminergic cells. Re-
grettably, however, the method had low efficiency
(~0.0002%). An improvement in efficiency of induction
of pluripotency (0.005% - 0.01%) was described by
Soldner et al. [22] using a self-excising Cre-recombinase
method with doxycycline (DOX)-inducible lentiviral
vectors containing LoxP sites in the 3’ LTRs. The self-
excision of oncogenic genes such as c-Myc decreased the
oncogenic potential; however, though the transgenes are
excised from the genome, residual LoxP sites still remain.
While a great system for mouse engineering, the residual
foreign DNA is a human safety issue not likely to gain
regulatory acceptance in the near future. The use of
DOX-inducible PB transposition of OSKM was another
novel use of a vector requiring only terminal inverted
repeats and transient expression of the transposase en-
zyme in order to catalyze insertion or excision of the
transgenes [25]. Though verification of complete exci-
sion of the tr ansgen es and vecto r sequen ces can b e labor-
intensive and cost-prohibitive on an industrial scale,
Woltjen et al. note that the their use of established plas-
mid preparation techniques and commercial transfection
technology under xeno-free conditions is an improve-
ment over limited-lifetime, xeno-biotic viral methods.
2.2. Episomal, Minicircle, RNA Sendai
Further improvement in transgene and vector free
hiPSC reprogramming methods were made using epi-
somal vectors with a cis-acting oriP element and trans-
acting EBNA1 gene derived from the Epstein-Barr virus
[23]. This method required only a single transfection to
reprogram human fibroblasts using OSKM as well as
NANOG, LIN28, and an IRES2 for coexpression and did
not require viral packaging. In terestingly, the addition of
the SV40 large T gene (SV40LT) gene was required and
was thought to have coun tered the toxic effects of c-Myc
expression. Plasmid free clones can easily be drug-se-
lected over time as these vectors only replicate once per
cell cycle and ~5% of plasmids are lost each cycle. Effi-
ciency, however, wa s very low (0.000006%) , SV40 is an
oncogene, the EBNA1 protein may elicit an immune
response if the transgene is not completely removed, and
there is still the possibility of integration when using
DNA-based methods even though this DNA is extra-
chromosomal. Another plasmid-transfection based but
nonviral minicircle vector of supercoiled DNA using
Oct4, Sox2, Lin28, and NANOG in a single, primarily
eukaryotic expression cassette further improved on trans-
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P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73 63
gene and vector free hiPSC reprogramming [24]. Trans-
fection efficiencies (~10.8% ± 1.7%) were approx. an
order of magnitude higher than with the aforementioned
episomal vectors and bene fitted from reduced exogen ous
silencing leading to longer ectopic expression; repro-
gramming efficiencies were (~0.005%) were also much
higher than other transgene and vector-free methods.
While this decreased labor associated with reprogram-
ming, FDA-required screening for possible DNA inte-
gration can still be labor intensive. Improving safety fur-
ther, a move towards DNA-free methods without the risk
of integration was described by Fusaki et al. [27] using
an RNA Sendai virus (SeV) which does not replicate
using a DNA phase. Reprogramming efficiencies (1%)
using the SeV vector carrying OSKM were much im-
proved over retroviral methods. Additionally, the SeV
genome is naturally depleted from the cytoplasm by the
cell over time. However, SeV can activate innate anti-
viral mechanisms; anti-HN protein antibody negative se-
lection can easily purify for viral free clones but does
add extra labor.
2.3. RNAs
Truly xeno-, transgene-, vector sequence-, and DNA-
free methods to reprogram human somatic cells without
the risk of residual integration using mRNA were first
published by Yukabov et al. [28]. Yukabov et al. showed
that transfecting in-vitro produced mRNA coding Oct4,
Sox2, Lin28, and NANOG could successfully induce
human fibroblasts to pluripotency, albeit with low effi-
ciency (0.0005%). While RNA reprogramming methods
have been known for some time, innate immune re-
sponses such as those effected by the RNA helicase reti-
noic acid inducible gene I (RIG-I) which detects viral
RNA [49] have generally limited success. Research by
Angel & Yanik [50] first showed that knockdown of in-
nate immune system-related genes such as Ifnb1 and
Eif2ak2 using a standard siRNA cocktail could suppress
this response and improve RNA delivery methods. Fur-
ther advancements were made using synthetic mRNA
developed by Warren et al. [29] and delivered using
RNAiMAX (Invitrogen) cationic lipid delivery vehicles.
Knowing that ssRNA can activate cellular anti-viral
mechanisms the authors modified the mRNA by remov-
ing the 5’ phosphates with phosphatases; this attenuated
interferon signaling. Additionally, by mimicking normal
mRNA editing through substitution of either 5-methyl-
cytidine (5mC) for cytidine or pseudouridine (psi) for
uridine, increased transcript and cell viability was ob-
served. The addition of a type I interferon receptor decoy
receptor improved viability further. This combination
greatly decreased the innate antiviral responses and ini-
tial toxicity normally encountered when using unedited
RNA. Under these conditions, reprogramming human
somatic cells using OSKM as well as Lin28 mRNAs
resulted in much improved efficiencies of 1.4% versus
0.04% using retroviral methods; timeframes for deriva-
tion (16 days) were approximately half that using retro-
viral methods. However, Warren et al. noted that the
mRNA reprogramming required daily (16) transfections
in order to maintain high levels of ectopic expression,
though some reported optimizations in reprogramming
factor cocktails and the use of an Oct4-MyoD fusion
protein mRNA are capable of reducing required daily
transfections to approximately 1 week [51].
Indeed, moderation of innate immune activities may
benefit the use of directly transfected mature double-
stranded miRNAs such as mir-200 c, mir-302 s, and mir-
369 which induce reprogramming by global demethyla-
tion, indirectly promoting expression of pluripotency TFs
Oct4 and Sox2 [30,31], for example. miRNAs have
shown feasibility in reprogramming human cells but ef-
ficiency has suffered, presumably due to detection by
RIG-I. While these methods present a much more viable
method capable of meeting the strict requirements for
safety and regulatory approval while being commercially
feasible, the industrial production of modified RNAs
could increase costs considerably.
2.4. Directed Delivery of Proteins
Employing bioprocesses already in place for the pro-
duction of recombinant proteins may improve the com-
mercialization of hiPSC technology. Such methods
would also bypass the inherent risks of DNA-based ge-
nome manipulation and the added complexity of RNA-
based methods. It has already been shown feasible that
the direct reprogramming of human somatic cells with
OSKM proteins is possible, for example, as performed
by Kim et al. [32], but with low efficiency (0.001%). A
major hurdle to improving protein-based methods is the
ability to deliver them across the cell membranes; Kim et
al. have made progress in overcoming this hurdle by
taking advantage of the viral HIV transactivator of tran-
scription (TAT) protein containing a high proportion of
basic amino acids known as cell penetrating peptides
(CPPs). These CPPs are capable of efficiently entering
the cell and nucleus—a quality Kim et al. exploited by
anchoring them to OSKM proteins. Further advances in
protein-based methods have aimed at stabilizing the pro-
tein in culture and improving endosomal release upon
uptake. In an optimization of direct protein delivery
methods, Their et al. [52] employed media containing
KnockOut D-MEM medium supplemented with 2% fetal
calf serum, 7.5% serum replacement, and 2.5% lipid rich
Albumax to confer enhanced protein stability and trans-
duction efficiencies. This media supported recombinant
Oct4-TAT reprogramming efficiencies only slightly re-
duced and on the same order of magnitude as viral meth-
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P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73
ods using transduction of the Oct4 gene. Using similar
media, Their et al. [53] have shown recombinant Sox2-
TAT is reprogramming competent, though getting the
proteins delivered to the right parts of the cell for effi-
cient expression needs improvement.
2.5. Small Molecules
The use of small molecules represents an advanta-
geous approach to consistent and safe derivation of
hiPSCs: using small molecules not only circumvents the
need for laborious assays proving the absence of adventi-
tious agents and/or foreign genetic elements required for
FDA approval, particularly if the molecules are already
part of an FDA-approved drug library, but also takes
advantage of existing industrial drug development infra-
structure. Moreover, small molecule platforms are ame-
nable to clinical- and industrial-scale high-throughput
(HT) platforms that not only include automated cell cul-
ture systems such as the CompacT SelecT (TAP Biosys-
tems) used by StemCell, Inc. to manufacture their line of
HuCNS-SCs for human cell therapy [54] but also HT,
label-free microfluidic platforms capable of separating
hiPSCs based on their unique adhesion signature [55]—
greatly reducing labor while increasing reliability and
regulatory standardization. Many compounds which in-
crease hiPSC reprogramming efficiency such as through
reduced extrinsic and intrinsic apoptosis and modulation
of master TFs involved in pluripotency have already
been identified, for example.
Inh ibition of Caspase3-mediated Rho kinases (ROCKs)
which mediate caspase cascades, cell detachment, mem-
brane blebbing, and nuclear fragmentation have shown
beneficial to cell survival [56]. Thiazovivin, a ROCK
inhibitor, is just one example of a small molecule which
increases hiPSC survival and colony derivation after cell-
cell and cell-substratum detachment d uring sp litting [3 9].
Vitamin C (Vc) is a cheap, readily available, and FDA
approved molecule which has also been shown to in-
crease survival of viral OSKM reprogrammed somatic
(fibroblast) cells [33], for example. Here, Estaben et al.
showed Vc could decrease senescence of hiPSC through
suppression of p53, research that is supported by [57]
who have shown that suppression of the p53-p21 path-
way increases virally-based generation of hiPSCs. The
exact mechanisms of Vc in cell reprogramming were
further elucidated by Wang et al. [41] who showed that
Vc promotes the histone demethylase (HDM) activities
of Jhdm1a/1b in Oct4 transduced mouse fibroblasts;
Jhdm1a/1b (kdm2a/b) not only demethylated H3K36-
me2/3 marks in the Ink4/Arf locus—repressing it and
senescence while promoting cell cycle progression—but
also activated miRs 302/367 in conjunction with Oct4.
Estaben et al’s hypothesis that Vc can modulate dioxy-
genase hypoxia inducible factor (HIF) target genes as a
cofactor for HIF reactions is supported in research by
Yoshida et al. [58] whom had first shown that hypoxic
(5% O2) culture conditions could improve hiPSC repro-
gramming efficiency; HIF-2α is known to directly bind
hypoxia response elements (HREs) located in Oct4 pro-
moter but also the conserved regions 3 & 4 of the Oct4
distal enhancers (DE) known to drive expression in the
ICM and in ES cells [59]. Building on this discovery, a
small molecule activator of 3’-phosphoinositide-depen-
dent kinase-1 (PDK1) discovered by Zhu et al. [60],
PS48, has been shown to activate Akt and stimulate a
transition from aerobic to glycolytic metabolism. This
transition to anaerobic metabolism is also hypothesized
to improve reprogramming through a reduction in mito-
chondrial oxidation-associated ROS. Supporting research
on this change in bioenergetics, mt morphology and en-
ergetics analysis [47,48] have shown that the cristae and
energetic capacity of hiPSCs mts undergo a transition
and are rejuvenated to a youthful ESC-like state during
reprogramming. The addition of the glycolytic interme-
diate L-lactate can improve this mt metabolic shift and
reprogramming efficiency [36].
Small molecule modulation or replacement of master
TFs involved in pluripotency has also been shown to
benefit iPSC reprogramming efficiencies. While not
shown in human iPSCs, Ichida et al. [34] first showed
that Sox2 and C-Myc can be replaced by a small mole-
cule inhibitor of TGF-β signaling (E-616452, RepSox)
through induction of Nanog in retrovirally reprogram-
med mouse embryonic fibroblasts (MEFs). Other re-
search using commercially available small molecule in-
hibitors of the pan-Src family kinase (SFK) have also
shown that activation of Nanog can replace Sox2 to
virally reprogram MEFs [61]; SFk inhibitors have not
been adequately assayed in hiPSC reprogramming but
have been shown to promote epithelial differentiation in
hESCs, however [62]. Moreover, it has been shown that
removal of C-Myc can increase OKS lentiviral hiPSC
reprogramming efficiency in combination with other
small molecule inhibitors of TGF-β receptor kinase (SB
431542) as well as inhibitors of MEK signaling (PD
0325901), GSK3β signaling (CHIR 99021), and ROCK
inhibition with Thiazov ivin [40]. Indeed, Valamehr et al.
showed that using Thiazovivin to decrease apoptosis in
combination with small molecules that inhibit TGF-β,
MEK, and GSK3β signaling increases hiPSC reprogram-
ming efficiency over methods without Thiazovivin-me-
diated ROCK inhibition or just ROCK inhibition alone.
Interestingly, lithium (Li) was found to be able to replace
some core factors in O alone, OK, and OS transduced
hiPSCs through partial inhibition of GSK3β signaling,
increased transcriptional activity of Nanog, and through
inhibition of the H3K4 HDM Kdm1a (LSD1) [42].
However, complete replacement of all reprogramming
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P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73 65
factors with nongenetic, small molecule methods is de-
sirable. Oct4 is known to be the most important factor
[18] and can be used alone to reprogram cells, though
with greatly reduced efficiency as compared to OSKM.
Thus, selecting cells which endogenously express some
of the master TFs OSKM may be a consideration. Hu-
man fetally-derived neural SC (NSC) which endoge-
nously express Sox2 and c-Myc were reprogrammed by
Kim et al. [63] with just retroviral transduction of Oct4,
for example; however, fetally-derived NSCs are not a
reliable supply of cells. In the previously mentioned re-
search by Zhu et al. [60], human keratinocytes—a clini-
cally feasible source of cells for patient-specific hiPSCs
which endogenously express Klf4 and c-Myc—were
successfully reprogrammed using just retroviral trans
duction of Oct4 and the small molecule PS48. Small
molecules which can increase the endogenous expression
of Oct4 through interactions with epigenetic modifier s of
pluripotency which reduce the suppressed state and in-
crease activation have also been shown to increase
hiPSC reprogramming efficiency. Valproic acid (VPA) is
a histone deacteylase (HDAC) inhibitor (such as H3K9ac
in mESCs [64]) which increases access of the transcrip-
tional machinery to the Oct4 promoter [65]; sodium bu-
tyrate (NaBu) is another HDAC inhibitor that has been
shown to increase OS reprogramming efficiencies in
human fibroblasts [37]. Wang et al. [66] have further
shown VPA cooperates with Klf4 to increase the activity
of the histone acetyltransferase (HAT) EP300 and the
H3K27me2/3 HDM Kdm6b (JMJD3) in the proximal
promoter (PP) of Oct4 while the H3K4me2/3 HDM
Kdm5a (Jarid1a) and H3K27 HDM Kdm6a (Utx) activ-
ity are increased at the Oct4 PP and proximal enhancers
(PEs). Likewise, BIX-01294-mediated inhibition of the
H3K9 histone methyltransferase (HMT) KMT1C (G9a)
has been shown to increase OK reprogramming effi-
ciency in MEFs [67] by inhibiting both G9a-mediated
heterochromatinization and H3K9 trimethylation at the
Oct4 promoter [68]. Benefiting safe derivation with
chemical methods, UNC0638 is a small molecule inhibi-
tor of G9a/GLP in human cells with higher potency and
lower cytotoxicity than BIX-0129 4 [69]; Chen et al. [70]
have shown UNC0638-mediated inhibition of G9a/GLP
in human hematopoietic stem and progenitor cells
(HSPCs) repressed lineage-specific genes and supported
“stemness” during expansion. Shi et al. also showed that
inhibition of the pluripotency gene silencing DNA me-
thyl transferases (DNMTs) 3a/3b (responsible for DNA
methylation during differentiation of ES cells) by 5-aza-
cytidine and RG108 can act synergistically to enhance
OK-transduced MEF reprogramming. Relevant to clini-
cal hiPSC production, RG108 has higher potency and is
less cytotoxic than 5-azacytidine in human cells [71].
Moreover, RG108 does not result in covalent trapping of
DNMTs and may have another advantage over other
inhibitors in that RG108 seems to support stability of
satellite DNA and centromere methylation states that are
commonly found perturbed in hiPSCs. Silencing line-
age-specific gene expression is also critical to successful
hiPSC reprogramming; inhibition of the H3K79 HMT
Dot1L and somatic gene expression can be improved
with the small molecule EPZ004777 [38]. While there
are currently no validated small molecules which can
substitute for the human Oct4 reprogramming factor,
recent HT screens of heterocyclic chemical libraries have
described Oct4-activating cpds (OACs) capable of pro-
moting Oct4 expression through direct interactions with
the Oct4 promoter [35]. Indeed, it is this reviews opinion
that future hiPSC reprogramming research focus on
identifying small molecules which activate endogenous
expression of OSK pl uripotency factor s.
In the following the author of this review presents a
theoretical nongenetic, small molecule reprogramming
recipe for hiPSCs which focuses on activating endoge-
nous Oct4 expression. Fibroblasts are a reprogrammable
and clinically viable source of donor cells which can be
obtained from a patient with relatively little nuisance;
thus, these will be the cell types considered in the fol-
lowing recipe. It is likely that the assay will require sig-
nificant development as normal Oct4 expression pro-
moting pluripotency must be maintained: repression of
expression by half will result in trophoectoderm while
twofold overexpression will result in endoderm and
mesoderm differentiation [72]. This methodology posits
that early reprogramming (Figure 1, (a)-(j)) steps should
focus on decreasing epigenetic repression with DNMT
inhibitors (e.g. RG108), HMT G9a inhibitors (e.g. UNC
0638), and HDAC inhibitors (e.g. VPA, NaBu). OACs
should be added next. It is likely that an initial direct
delivery of the Oct4 protein (e.g. Oct4-TAT) may sub-
stantially jumpstart Oct4 expression: it has been shown
that Oct4 alone is capable of binding its own DE and
maintaining an active and transcriptionally competent
nucleosome-depleted region (NDR) once cytosines have
been demethylated [73]. Supporting Oct4 enhancer bind-
ing stability after cytosine demethylation is research by
Bhutani et al. [74] showing that activation-induced dea-
minase (AID) is only required within the first 72 hrs of
lentiviral transduced OSKM MEFs. AID overexpression
only leads to a 2-fold increase in efficiency, however,
and small molecule activators of AID may not be as ad-
vantageous as other targets. Additionally, Sox2 upstream
enhancers are known to be a downstream target of Oct4
while Sox2 reciprocally regulates transcription of Oct4
via Oct4-Sox2 elements in the DE [75]; therefore, epi-
genetic derepression of these TFs along with initial Oct4
proteins and TF substitutes is hypothesized to be suffi-
cient for robust activation of Oct4 and the pluripotency
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P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73
Copyright © 2013 SciRes.
positive, express surface antigens SSEA-3 & 4, TRA-1-
60 & TRA-1-81, show in vitro differentiation capacity,
and teratoma assays showing the ability of the cells to
differentiate into ectodermal, endodermal, and mesoder-
mal lineages [76]. hiPSCs are characterized similarly and
resemble hESCs under these definitions; however, while
hiPSCs display globally similar gene expression profiles
they often show persistent donor cell gene expression
signatures not completely silenced [77] as well as epige-
netic differ ences [44,45].
network. Simultaneously, one should begin silencing
lineage-specific somatic gene expression (e.g. EPZ
004777). While Onder et al. [38] found that early and
middle stage Dot1L inhibition could not only substitute
for Klf4 and c-Myc in OS transduced human fibroblasts
but could also increase expression of Nanog and Lin28,
interestingly, TGF-β inhibitors did not increase effi-
ciency with this combination and are not a part of this
cocktail. Addition of the GSK3β and HDM LSD1 in-
hibitor—Li—should be considered. ROCK inhibitors
(e.g. Thiazovivin) as well as Vc should be included in
this initial cocktail an d throughou t reprogramming. Mid-
stage reprogramming (Figure 1, (a)-(m)) should con-
tinue using the same cock tail but should include a transi-
tion from early stage aerobic culture conditions which
promote cell cycle progression to hypoxic culture condi-
tions (5% O2) as well as the PDK1 activator PS48 and
lactate to further promote a shift to glycolytic metabo-
lism. Late-stage reprogramming should continue using
the same cocktail, though it would be interesting to fur-
ther optimize the assay for a reduction and/or complete
discontinuation of HMT, HDM, DNMT, and HDAC
modifiers so as to limit the potential for global epigen etic
perturbance, to be discussed next.
3.1. Potential Aberrant Gene Expression
In transcriptome analysis Ghosh et al. [77] discovered
significant residual fibroblastic gene expression signa-
tures such as those involved in remodeling the extracel-
lular matrix (ECM) (PLAT and PLAU) as well as those
in cell migration (CXCL1) in fibroblast-derived hiPSCs,
adipose-specific gene expression such as PALLD and
COL1A1 in adipose-derived hiPSCs, and keratinocyte-
specific protein expression such as keratins and prote-
olytic enzymes in keratinocyte-derived hiPSCs. Fur-
thermore, many genes such as LEFTY1 and others in-
volved in maintaining hESC pluripotency and an undif-
ferentiated state were found to be downregulated in
hiPSCs. These donor cell expression signatures can not
only be found in hiPSCs reprogrammed by integrating
retroviral transduction but also in cells reprogrammed
with nonintegrating episomal vectors and by the directed
delivery of defined reprogramming proteins. Addition-
ally, culture conditions can play a role in genomic het-
erogeneity: it has been found that the use of feeder-layer
Even with safer xeno- and DNA-free methods and ef-
ficient reprogramming of somatic cells improving, fur-
ther characterization of altered epigenetic signatures and
differentiation potential of hiPSCs is required. hESC
pluripotency is often characterized as Oct4- and Nanog-
Figure 1. Stimulating Oct4 and the pluripotency network. (a) DNMT inhibitors (e.g. RG108), (b) H3K9 HMT G9a inhibi-
tors (e.g. UNC0638), (c) H3K9ac HDAC inhibitors (e.g. VPA, NaBu), (d) promoters of H3K27me3 and H3K4me3 HDMs
JMJD3, Jarid1a, Utx (e.g. VPA), and (e) Oct4 protein (e.g. Oct4-TAT) should be included first in order to render the Oct4
gene transcriptionally competent and kickstart the pluripotency network; (f) OACs should be added throughout early, mid-
dle & late reprogramming; (g) H3K79 HMT Dot1L inhibitor (e.g. EPZ004777) helps silence lineage-specific gene expres-
sion during early and middle stage reprogramming; (h) H3K4me3 HDM LSD1, GSKβ inhibitor (e.g. Li) and the cell sur-
vival promoting (i) ROCK inhibitors (e.g. thiazovivin) and (j) Vc (also a H3K36m2/3 HDM promoter) should be added
during early, middle, and late reprogramming; Stimulators of glycolytic metabolism (k) 5% O2, (l) Lactate, and (m) PS48
should be included through mid and late reprogramming.
P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73 67
culture of hiPSC contributes to DNA replication and
cell-cycle variances in hiPSCs [78]; however, in support
of xeno-free generation of hiPSCs it was found that de-
fined, feeder-free culturing of hiPSCs on Matrigel (BD
Biosciences) and mTeSR1 (StemCell Technologies) more
closely resembled hESCs than those cultured with feed-
ers. TeSR2 (StemCell Technologies) is closely related to
mTeSR1 but contains no animal proteins and has been
shown capable of maintaining hESCs in clinical-grade
conditions [79].
It is long known that prolonged cell passaging can lead
to abnormalities. In hESCs this manifests as karyotypic
aberrations, most commonly in chromosomes 12, 17, and
X [80]. These abnormal cells often show an increased
ability to proliferate and mirror malignant transforma-
tions in vivo. Indeed, a rather large meta-analysis of 66
hiPSC lines from 38 independent studies conducted by
Mayshar et al. [81] revealed that 20% of these lines con-
tained chromosomal aberrations after prolonged culture,
particularly trisomy of 12p—a region which includes the
pluripotency genes NANOG and GDF3—as also found
in hESCs, and general functional enrich ment in cell cycle
genes. In contrast to long-term passaging, mutations aris-
ing during early passaging and isolation were mostly
limited to subchromosomal duplications or deletions.
Mutations associated with the derivation method includ-
ed trisomies of chromosome 1 and 9 in lines reprogram-
med with both retroviral transduction and the directed
delivery of factor proteins. Such high mutation rates re-
gardless of integrating or nonintegrating (episomal and
mRNA) methods were also found in the exomes of 22
hiPSC lines analyzed by Gore et al. [82]. Harboring
about 6 mutations per iPSC line, Gore et al. found that
most (83/124) were miss-sense with 53 predicted to alter
protein function while 50 were known cancer-related,
such as ATM, NTRK1, and NTRK3. Copy number va-
riations (CNV) were also found to occur in common
fragile sites and sub-telomeric regions at twice the rate in
early-passage hiPSC lines derived by retroviral transduc-
tion and by PB transposon s than in hESCs or the original
fibroblasts; however, both the number and size decreased
with passaging as these mutations are neg atively selected
for [83]. In ensuring the safe application of hiPSC-based
therapies, HT qPCR assays of pluripotency gene expres-
sion will need to not only include Oct4 and other master
TFs but also assays of donor cell gene expression, triso-
mies, and other common cancer-related mutations, in-
cluding those that may have been acquired over the life-
time of the donor.
3.2. Potential Aberrant Epigenetics
Perturbations associated with epigenetic memory are
potentially tumourigenic [84,85] and are therefore safety
issues limiting regulatory approval. While genome-scale
single base resolution has shown hiPSC epigenetic sig-
natures are similar to ESCs, differentially methylated
regions (DMRs) do occur [44,45]. Assaying cell lines of
varying somatic type from multiple labs reprogrammed
with both integrating and nonintegrating methods and
using HT methylC-Seq and ChIP-Seq methods, Lister et
al. discovered 1175 CG dinucleotide DMRs (CG-DMRs)
totaling 1.68 Mb that were either a failure to reprogram
somatic memory (44% - 49%) or (51% - 56%) were par-
ticular to the hiPSC reprogramming process (iDMRs),
80% were in CG islands (CGI-DMRs) and near or within
genes (62%), and 92% were hypomethylated in iPSCs
and indicated insufficient methylation during repro-
gramming. Moreover, these CG-DMRs and iDMRs were
transmitted with high frequency during differentiation
and were not removed by passaging as can occur with
CNVs. Common motifs included DMRs for Klf4, a re-
programming factor (that may be substituted) which may
be contributing to aberrant methylation, and FOXL1 and
could be useful biomarkers for complete reprogramming,
for example. Lister et al. also discovered 29 large
nonCG-DMRs comprising 32.4 Mb, 22 of which were
associated with hypomethylation and H3K9me3 enrich-
ment proximal to centromeres and telomeres. These
nonCG-DMRs were associated with transcriptional dis-
ruption with 33 of 50 downregulated genes found per-
turbed by more than a 2 fold lower tran script abundance,
64% of which also harbored CG dinucleotides hyper-
methylated at the TSS. This transcriptional downregula-
tion was associated with aberrant loss of H3K27me3.
While not only po tentially tumourigen ic, these transcrip-
tional aberrations in comparison to ESCs can lead to
functional heterogeneity [45] and reduced efficacy in
cell-based therapies.
The polycomb group proteins 1 and 2 (PRC1 and 2)
are evolutionary conserved epigenetic regulators whose
perturbations are known to reduce or prevent repro-
gramming (reviewed by Watanabe, Yamada, & Yama-
naka [86]). PRC1 is largely responsible for maintaining
the repressed transcriptional state that PRC2 initiates
through H3K27 trimethylation, for example. It is known
that both Oct4 and Nanog regulate and increase expres-
sion of DNMT1 through direct binding to DNMT1’s
promoter [87] while Sox2 has been shown to regulate
miR-29b-catalyzed repression of DNMT3A/3B during
reprogramming [88]. However, increased understanding
of DNA methylation interactions suggests histone modi-
fications regulate DNMT activity (reviewed in [89]):
DNMT3A/3B-catalyzed DNA methylation of pericentric
satellite repeats is dependent on HMT Suv39h-mediated
H3K9 methylation [90], the PRC2 protein HMT EZH2
(Kmt6a) which catalyzes H3K27 trimethylation can
physically direct DNMTs and CpG methylation [91], and
Copyright © 2013 SciRes. OPEN A CCES S
P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73
histone tails lacking H3K4 methylation h ave been shown
to allosterically activate de novo DNA methylation by
DNMT3A [92], for example. Thus, opportunity for un-
derstanding aberrant epigenetics should focus on the his-
tone code hierarchy. Indeed, another required epigenetic
regulator of reprogramming, Utx, has recently been iden-
tified [93]. In mediating demethylation of repressive
H3K27me2/3 chromatin marks Utx globally regulates
approximately 500 genes, is required to sufficiently acti-
vate many ES-associated genes, and physically interacts
with OSK in mediating reprogramming to a state of
pluripotency. In coordinating gene repression and activa-
tion Utx also forms a protein complex with the Trithorax
group (TrxG) HMT MLL2/3 (MLL2/3 normally adds the
activating tri-methylation mark of H3K4me3 while the
HDM Jarid1a removes it), potentially functioning in bi-
valent domain regulation. Such suggestions are rein-
forced in research showing Utx complexes with MLL
2/3/4 during development in murine erythroleukemia
(MEL) cells [94]. In mouse ESCs, Chaturvedi et al. have
also shown crosstalk between G9a as well as Jarid1a; a
significant amount of gene silencing is maintained by the
repressive dimethylation of H3K9 and H3K27 mediated
by G9a and the demethylation of activating H3K4me3 by
Jarid1a. The coordination between these HMTs and
HDMs could play a role in timely repression of lineage-
specific genes and maintaining optimal stoichiometric
ratios of TFs such as Oct4 during hiPSC reprogramming.
It is already known that reprogramming factor stoichio-
metry can affect iPSC reprogramming and epigenetic
states [95], for example. Moreover, perturbation of Utx
has been shown to contribute to aberrant epigenetic re-
programming both in vitro and in vivo. Thus, considering
Mansour et al’s [93] and Chaturvedi et al’s [94] findings,
it is this reviews opinion that suboptimal correlation be-
tween levels of OSKM expression and levels of epige-
netic regulators in global crosstalk and feedback mecha-
nisms important for pluripotency and development is
likely contributing to aberrant epigenetics such as the
hypomethylation of H3K27 and H3K9 that Lister et al.
observed. Further related insight is found in recent re-
search by Parsons [96] showing that the globally acety-
lated state of hESCs is at least partially mediated b y lev-
els of Oct4 which influence HDAC activity; Parsons
found that decreased levels of Oct4 lead to hyperacetyla-
tion and induction of differentiation. Perturbations in this
crosstalk can not only lead to inefficient activation of
H3K27me2—repressed pluripotency-associated genes
but can also lead to H3K9me3 enrichment which pre-
vents OSKM TF target binding [97]—another possible
culprit in the low efficien cy currently observed in hiPSC
reprogramming. Finally, though not yet proven, pertur-
bations in this crosstalk are also a potential culprit in
H3K4me3 enrichment and the induction of and/or resid-
ual donor cell gene expression. Further elucidation of
epigenetic crosstalk should benefit the precise applica-
tion of reprogramming technologies which do not aber-
rantly perturb the delicate balance of epigenetic regula-
tors. In light of the still en igmatic crosstalk and potential
perturbations inherent to the reprogramming process,
validation of hiPSCs to be used in cell therapies should
include routine HT methylome analysis to ensure safe,
efficacious, and nontumourigenic application of hiPSCs.
3.3. Telomere Rejuvenation
Despite aberrant methylation of subtelomeric regions,
advocates of hiPSC research for use in regenerative
medicine can remain optimistic with research showing
that telomere lengths are rejuvenated in a number of cell
types [46], thus ameliorating some concerns about cellu-
lar senescence when using cells from aged donors. Also,
this is another advantage over using MSCs which may be
prone to senescence from aged donors and/or prolonged
passaging due to inactive telomerase, though telomere-
induced senescence can be avoided with ectopic expres-
sion of hTERT [98]. hTERT is stably expressed during
hiPSC reprogramming [99], though there is some het-
erogeneity in length found among hiPSC lines that could
be related to suboptimal ratios of pluripotency TFs and
the regulatory loops governing telomere length. It is
known that Oct4 and Nanog bind the promoters of the
telomerase RNA component (TERC) locus and upregu-
late transcription and lengthening of telomeres in dyske-
ratosis congenita (DC) cells [100]. Recently, Hoffemeyer
et al. [101] have shown that the Tert promoter is a target
of β-catenin and Klf4 in human carcinoma and mouse ES
cells. Interestingly, Klf4 is only required for β-catenin to
bind the Tert promoter: β-catenin actually drives Tert
expression, possibly by recruiting HMTs. Wnt/β-catenin
is also a target of Tert expression, however, and this may
also form a regulatory loop governing telomere length in
hiPSCs that is perturbed by suboptimal correlations of
the pluripotency factors. hTERT and alternative length-
ening of telomeres (ALT) is required for full telomere
rejuvenation and true pluripotency [102], however. ALT
lengthens telomeres in association with epigenetic modi-
fiers such as DNMT3A/3B and HMTs Suv39h1/h2; the
aberrant hyper- & hypo-methylation of subtelomeric
regions that Lister et al. [44] identified may be associ-
ated with aberrant crosstalk between these epigenomic
modifiers, possibly by the use of associated inhibitors
[99]. It can be hypothesized these aberrations may even
be an artifact of the previously shortened state and/or
ALT. Still, Yehezkel et al. [99] have shown successful
hiPSC reprogramming elongates telomeres on average
by approximately 10 kb ; hTERT and telomere elong ation
is then stably repressed upon differentiation, allowing
Copyright © 2013 SciRes. OPEN A CCES S
P. E. Woolwine / Open Journal of Rege nerative Medicine 2 (2013) 61-73 69
normal telomere shortening. However, a safe and effica-
cious replicative lifespan fo r hiPSCs used in cell therapy
must be shown: this review suggests routine telomere
assays not only include an assay of hTERT expression
but also assays of absolute telomere length such as with
modified Cawthon HT qPCR [103].
SCs for use in cell-based therapies have recently
shown interim clinical viability and hold great potential
for use in regenerative medicine. There are many regula-
tory and biological concerns to be resolved before com-
mercialization of SCs for clinical therapy can be achie-
ved, however: the use of hESCs in such therapies not
only carries great ethical debate but also poses an immu-
nogenicity risk; SCNT technology is technically chal-
lenging and also controversial; allogeneic MSCs may
still pose an immunogenicity risk while autologous MSCs
may be susceptible to senescence. In contrast, autologous
hiPSCs have all the potential of hESCs and are a source
of potentially unlimited, immunocompatible SCs with
rejuvenated bioenergetics and replicative lifespans for
patient-specific cell-based therapies that pose no ethical
dilemma; however, iPSC reprogramming methods still
suffer from safety, efficiency, and efficacy concerns,
though these obstacles are being surmounted. Since Ta-
kahashi & Yamanaka first defined the core OSKM TFs
required to reprogram somatic cells to iPSCs using inte-
grating viral transduction methods, much progress has
been made in developing safer nonintegrating-, RNA-,
protein-, and small molecule-based methods. Towards
the goal of clinically viable hiPSC technology, strategies
exploiting modifiers of cell survival, endogenous pluri-
potent TF expression, and epigenetic regulation have
been developed to increase hiPSC reprogramming safety,
efficiency, and efficacy. However, epigenetic memory of
hiPSCs still poses a safety and efficacy concern. This
review has discussed the latest discoveries benefiting an
increa sed understand ing of plurip otent TF and ep igenetic
regulator crosstalk perturbed during reprogramming.
This knowledge is paramount to developing reprogram-
ming technology which completely silences lineage-
specific gene expression, maintains telomere integrity,
and circumvents aberrant epigenetic methylation. Per-
turbations in hiPSC methylomes may be a result of
suboptimal correlations between pluripotency factors and
epigenetic regulators during hiPSC reprogramming and
more research is needed in this regard. The use of non-
genetic small-molecule methods to very precisely restart
the endogenous expression of required TFs such as OSK
may ameliorate the epigen etic scars of forced OSKM TF
expression characteristic of other methods; however,
there is currently insufficient genomics data concerning
the epigenetic landscape of hiPSCs reprogrammed using
still nascent small molecule—only methods to know at
this time. Nevertheless, it is this review’s final opinion
that DNA- and xeno-free small molecule methods hold
the most potential as a clinically viable and relatively
lower-cost HT technology capable of generating hiPSCs
in a safe, regulatory-compliant, and efficacious platform.
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