Review
This Review is in a thematic series on Stem Cells: From Basic Biology to Clinical Applications, which includes the
following articles:
Direct Cardiac Reprogramming: Progress and Challenges in Basic Biology and Clinical Applications
Vascular Wall Progenitor Cells in Health and Disease
Use of Mesenchymal Stem Cells for Therapy of Cardiac Disease
Pluripotent Stem Cells for Cardiovascular Applications
Myocardial Stem Cells
Stem Cells for Myocardial Regeneration: Experimental Studies & Clinical Applications
Direct Cardiac Reprogramming
Progress and Challenges in Basic Biology and Clinical Applications
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Taketaro Sadahiro, Shinya Yamanaka, Masaki Ieda
Abstract: The discovery of induced pluripotent stem cells changed the field of regenerative medicine and inspired the
technological development of direct reprogramming or the process by which one cell type is directly converted into
another without reverting a stem cell state by overexpressing lineage-specific factors. Indeed, direct reprogramming
has proven sufficient in yielding a diverse range of cell types from fibroblasts, including neurons, cardiomyocytes,
endothelial cells, hematopoietic stem/progenitor cells, and hepatocytes. These studies revealed that somatic
cells are more plastic than anticipated, and that transcription factors, microRNAs, epigenetic factors, secreted
molecules, as well as the cellular microenvironment are all important for cell fate specification. With respect to the
field of cardiology, the cardiac reprogramming presents as a novel method to regenerate damaged myocardium
by directly converting endogenous cardiac fibroblasts into induced cardiomyocyte-like cells in situ. The first
in vivo cardiac reprogramming reports were promising to repair infarcted hearts; however, the low induction
efficiency of fully reprogrammed, functional induced cardiomyocyte-like cells has become a major challenge and
hampered our understanding of the reprogramming process. Nevertheless, recent studies have identified several
critical factors that may affect the efficiency and quality of cardiac induction and have provided new insights
into the mechanisms of cardiac reprogramming. Here, we review the progress in direct reprogramming research
and discuss the perspectives and challenges of this nascent technology in basic biology and clinical applications. (Circ Res. 2015;116:1378-1391. DOI: 10.1161/CIRCRESAHA.116.305374.)
Key Words: fibroblasts
■
induced pluripotent stem cells
D
espite great advances of modern medical therapy, cardiac
disease remains a leading cause of death worldwide. As
adult cardiomyocytes have little regenerative capacity, extensive myocardial injury such as myocardial infarction leads to
scar formation, cardiac remodeling, and heart failure, which
are associated with high mortality.1 Heart transplantation is
an established therapy for severe heart failure, but remains
plagued with several issues, including a limited number of
donor organs, chronic immunosuppressant therapy, and the
■
myocytes, cardiac
subsequent development of graft vasculopathy.2 Thus, advances in cardiac regenerative therapy would provide an avenue to
overcome these hindrances.
Currently, there are several potential strategies for heart
regeneration. One such approach is to generate new cardiomyocytes from endogenous cell sources in situ. This includes
differentiation of resident cardiac progenitor cells into cardiomyocytes and renewal of pre-existing adult cardiomyocytes in
damaged hearts.3–7 Alternatively, new cardiomyocytes could
Original received January 9, 2015; revision received February 24, 2015; accepted February 26, 2015. In February 2015, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13.9 days.
From the Department of Cardiology, Keio University School of Medicine, Japan Science and Technology CREST, Tokyo, Japan (T.S., M.I.); Japan
Science and Technology CREST, Tokyo, Japan (M.I.); Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (S.Y.); and
Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.).
Correspondence to Masaki Ieda, MD, PhD, Department of Cardiology, Keio University School of Medicine, Japan Science and Technology CREST, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail [email protected]
© 2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.116.305374
1378
Sadahiro et al Progress and Challenges of Direct Reprogramming 1379
Nonstandard Abbreviations and Acronyms
α-MHC-GFP
CF
cTnT
ESCs
GHMT
GMT
iCMs
iHep cells
iN cells
iPSCs
MiRNAs
TGFβ
TTFs
α-myosin heavy chain promoter–driven enhanced green
fluorescent protein
cardiac fibroblasts
cardiac troponin T
embryonic stem cells
Gata4, Hand2, Mef2c, and Tbx5
Gata4, Mef2c, and Tbx5
induced cardiomyocyte-like cells
induced hepatocyte-like cells
induced neuronal cells
induced pluripotent stem cells
microRNAs
transforming growth factor β
tail-tip fibroblasts
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also be supplied from exogenous sources by cell transplantation, in which embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs) might be most promising
candidates to remuscularize failing hearts.8–10 However, several issues remain to be resolved before using iPSC-derived
cardiomyocytes in the clinic, such as the heterogeneous and
relatively immature phenotype of cardiomyocytes generated
from stem cells, the possibility of teratoma formation by iPSC
contamination, and the poor survival of transplanted cells in
the injured heart.11,12
The discovery of iPSCs in 2006 by Takahashi and
Yamanaka13–15 inspired a new approach that generates specific
cell types without needing to transition through a stem cell
state by introducing combinations of lineage-specific factors,
called direct reprogramming. Recent studies demonstrate
that direct reprogramming has the potential to yield a diverse
range of cell types from fibroblasts, including neurons, cardiomyocytes, endothelial cells, hematopoietic stem/progenitor
cells, and hepatocytes (Figure 1).16–22 These findings indicate
that cell fate plasticity is much wider than previously anticipated, and that direct reprogramming may offer a new system
to study the mechanisms underlying cell fate decisions during
development, which is currently a major focus of investigation
in basic biology.
We found that a cocktail of 3 cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), was the minimum needed to reprogram mouse cardiac fibroblasts (CFs)
into induced cardiomyocyte-like cells (iCMs) directly in
vitro.21 Subsequently, Srivastava and Olson laboratories independently reported that endogenous CFs could be directly
reprogrammed into iCMs to improve the cardiac function
of infarcted mouse hearts by gene delivery of GMT with or
without Hand2.23,24 Later reports also demonstrated that human fibroblasts could be also reprogrammed into iCMs with
various combinations of cardiac transcription factors in the
presence or absence of microRNAs (miRNAs), albeit at a
lower efficiency compared with that observed in mice.25–27
These results suggest that direct reprogramming technology
may hold immense potential for heart regeneration through
the generation of new myocytes from fibroblasts and improve
cardiac function after myocardial injury. However, the reprogramming efficiency of fully reprogrammed iCMs was low,
and reproducibility of the cardiac induction with this nascent
technology has been controversial.28,29 Moreover, the mechanisms of cardiac reprogramming and the properties of newly
generated iCMs were obscure. Since our discovery of cardiac
reprogramming, multiple groups have improved the iCM reprogramming efficiency, which could help answer to some of
these important questions. Furthermore, lessons from direct
reprogramming studies in other fields may facilitate our understanding of the cardiac reprogramming process. Here, we
first briefly review the progress in other direct reprogramming
fields, and then discuss the recent advances and challenges
of cardiac reprogramming for basic biology and its future in
clinical applications.
Reprogramming Into Pluripotency by Oct4,
Sox2, Klf4, and c-Myc
Although the prototype of direct reprogramming or transdifferentiation was described many years ago by the demonstration of MyoD, a master gene of skeletal myocyte,30 the field of
direct reprogramming started after the epoch making discovery of iPSCs. Two pioneering studies mark the advent of this
developing field. Gurdon31,32 first demonstrated pluripotent
reprogramming by somatic cell nuclear transfer in the 1960s,
and recently Takahashi and Yamanaka15 achieved a breakthrough in this field by overexpressing 4 ESC-enriched transcription factors, Oct4, Sox2, Klf4, and c-Myc (also known as
the Yamanaka factors), in mouse fibroblasts to induce a pluripotent state. Many laboratories including Yamanaka group
have since improved iPSC generation techniques to show that
mouse iPSCs share all defining features with bona fide mouse
ESCs, including the expression of pluripotency markers, DNA
methylation patterns, and the ability to generate chimeric mice
and all-iPSC mice, the latter of which is the most stringent
criteria for pluripotent stem cells.33–40 Subsequently, iPSCs
have been successfully derived from several different species, including humans, rats, and rhesus monkeys, either by
expression of the 4 Yamanaka factors or combinations of other
factors.14,41–46 Similarly, iPSCs have been derived from other
differentiated cell populations, such as keratinocytes, neural
cells, stomach cells, liver cells, and terminally differentiated
lymphocytes, demonstrating the universality of induced pluripotency.47–51 Although the initial induction efficiency of mouse
iPSCs and human iPSCs under the original conditions was
0.1% and 0.01%, respectively, this has been greatly improved
with the modification of transcription factors, miRNAs, small
molecules, and epigenetic modifiers.52–56 One study even
claimed 100% efficiency of reprogramming in mouse and human, which they designated deterministic reprogramming.56
Recently, extensive efforts have been directed toward safe cell
therapies using iPSCs, which include the appropriate selection
of safe and high-quality iPSC lines, generation of integrationfree iPSCs with episomal vectors, Sendai viruses, and chemical compounds, and efficient differentiation and purification
methods of differentiated cells from iPSCs.11,49,57 First-in-man
clinical trial using iPSC-derived cells was undertaken by
Takahashi and colleagues57a in 2014, in which the autologous
iPSC-derived retinal pigment epithelium cell sheets were
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Figure 1. Summary of direct reprogramming by
lineage-specific transcription factors. A diverse
range of cell types, including pluripotent stem cells,
various subtypes of neurons, cardiomyocytes,
endothelial cells, hematopoietic stem/progenitor
cells, hepatocytes, skeletal muscle cells, and
pancreatic β cells, can be directly induced
from heterologous cells by overexpression of
transcription factors. BAM indicates Brn2, Ascl1,
and Myt1l.
transplanted in the patient with exudative age-related macular degeneration. Thus, the progress of iPSC field has grown
enormously in past years, although this will not be discussed
in detail because of the space limitation.
Direct Reprogramming Into Pancreatic β-Cells,
Neural Cells, and Hepatocytes
Taking the concept of pluripotent reprogramming with the
Yamanaka factors, we, along with others, demonstrated that
combinations of several lineage-specific factors could convert mature cell types directly into a different cell type without needing to first become a stem cell. Zhou et al22 showed
that adenoviral gene transfer of the transcription factors,
Neurogenin 3, Pdx1, and Mafa, could efficiently reprogram
pancreatic exocrine cells into functional β-cells in mouse
pancreas and ameliorated hyperglycemia in diabetic mice by
secreting insulin from newly generated β-cells. This study
provided the first evidence of in vivo reprogramming by defined factors. More recently, Baeyens et al58 reported that just
transient administration of 2 growth factors, epidermal growth
factor and ciliary neurotrophic factor, to adult mice efficiently
converted terminally differentiated acinar cells to functional
β-cells in vivo and reinstated normal glycemic control in
chronic hyperglycemia mouse without genetic manipulation.
Thus, the generation of pancreatic β-cells by in vivo direct
reprogramming may represent a new therapeutic strategy for
diabetes mellitus.
Neural reprogramming is one of the most advanced research
fields in direct reprogramming. Vierbuchen et al20 has shown
that mouse dermal fibroblasts (mesodermal cells) can be converted into functional excitatory neurons (ectodermal cells)
in vitro using combination of the neuronal lineage-specific
transcription factors, Brn2, Ascl1, and Myt1l. These induced
neuronal (iN) cells express multiple neuron-specific proteins,
generate action potentials, and can form functional synapses, suggesting that direct reprogramming is possible even
between different lineages or germ layers. Mechanistically,
Ascl1 acts as a pioneer factor by immediately occupying most
cognate genomic sites in fibroblasts to open the chromatin
structure and allow the recruitment of Brn2 and Myt1l to target sites genome wide.59,60 Subsequently, several groups reported on neural reprogramming in human cells using mouse
reprogramming factors in combination with other transcription factors and miRNAs.61,62 The induction efficiency of human iN cells with Brn2, Ascl1, and Myt1l plus NeuroD1 in
the original conditions (2%–4%) was ≈10-fold lower than that
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in mouse (20%), while a recent study demonstrated that small
molecule–based inhibition of glycogen synthase kinase-3 β
and Smad signaling in combination with the transcription factors could convert postnatal human fibroblasts into functional
iN cells with yields up to >200% and neuronal purities up to
>80%.62,63 Induction of clinically relevant subtypes of iN cells
has been succeeded with the addition of subtype-specific transcription factors to the neural reprogramming factors. Four
factors (Lhx3, Hb9, Isl1, and Ngn2) in combination with the
Brn2, Ascl1, and Myt1l factors generated motor neuronal cells
from mouse embryonic fibroblasts,64 and Ascl1, Nurr1, and
Lmx1a could generate functional dopaminergic neurons from
mouse and human fibroblasts, as well as from patients with
Parkinson disease.65–67 Ring et al68 reported on the generation
of induced neural stem cells from mouse and human fibroblasts through the overexpression of a single factor, Sox2.69
Thus, steady progress has been made in the neural reprogramming field during the past years.
Two groups independently demonstrated that mouse fibroblasts can be directly reprogrammed into functional induced
hepatocyte-like (iHep) cells by defined factors.18,19,70–72 Sekiya
and Suzuki19 demonstrated the direct induction of functional
iHep cells from mouse fibroblasts by transduction of Hnf4α
and Foxa1. These iHep cells showed typical epithelial morphology, expressed hepatic genes, and acquired hepatocyte
functions. However, using CellNet analyses, Morris et al73
recently demonstrated that the induced cells generated with
the method reported by Sekiya and Suzuki were endoderm
progenitors rather than differentiated hepatocytes. They found
that the induced cells have the capacity to differentiate into
either hepatocyte-like cells or intestine-like cells in vitro,
depending on the expression levels of Hnf4α and Foxa1 and
the combination with other transcription factors. Intriguingly,
transplantation of the induced cells into liver and colon led
to differentiation of the cells into mature hepatocytes and
colonic epithelium, respectively, suggesting that the microenvironment is critical for cell fate determination. The human iHep cells were directly generated with a combination
of hepatic fate conversion factors, Hnf1A, Hnf4A, and Hnf6,
together with the hepatic maturation factors, Atf5, Prox1, and
Cebpa.74,75 The resultant human iHep cells were functionally
mature hepatocytes with drug metabolic function, which is
difficult to obtain from human PSCs. Thus, ectopic expression
of both cell fate determination and functional maturation factors could generate various types of mature cells.
Direct Reprogramming Into Hematopoietic
Stem/Progenitor and Endothelial Cell Lineages
The hematopoietic stem-like cells capable of multilineage
long-term engraftment were successfully generated from
committed lymphoid and myeloid progenitors and myeloid
effector cells by transient ectopic expression of 6 transcription
factors, Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37, which
are selectively expressed in hematopoietic stem/progenitor
cells.17 The successful generation of engraftable hematopoietic stem cells required in vivo bone marrow environment,
a supportive niche for hematopoietic stem/progenitor cells,
­emphasizing the importance of microenvironment for cell fate
conversion. Another approach to generate hematopoietic stem/
progenitor cells is the induction of hematopoietic progenitors
mediated through an endothelial cell intermediate, mimicking the developmental process in embryos in which definitive
hematopoiesis emerges via an endothelial-to-hematopoietic
transition. Batta et al76 demonstrated that mouse fibroblasts
can be efficiently reprogrammed to multilineage hematopoietic progenitors by the ectopic expression of the transcription
factors Erg, Gata2, Lmo2, Runx1c, and Scl. Generation of
hematopoietic cells was preceded by the appearance of hemogenic endothelium, recapitulating the normal embryonic
program of blood development. These results suggest that a
simple combination of transcription factors is sufficient to
induce a complex, dynamic, and multistep developmental
program in vitro and that direct reprogramming may provide
a platform to dissect the molecular mechanisms of cell fate
determination during development.
Han et al16 recently reported that adult mouse fibroblasts
could be directly reprogrammed into functional differentiated
endothelial cells, using a cocktail of 5 transcription factors,
Foxo1, Er71, Klf2, Tal1, and Lmo2. The newly generated
induced endothelial cells functionally behaved like mature
endothelial cells in that they released nitric oxide on stimulation with acetylcholine or vascular endothelial growth factor.
Transplantation of induced endothelial cells enhanced angiogenesis and limb perfusion in a murine model of hindlimb
ischemia, demonstrating the therapeutic potential for regenerative purposes.
Direct Reprogramming of Mouse Fibroblasts
Into Cardiomyocytes by GMT
Although embryonic mesoderm can be induced to generate
cardiomyocytes, no master regulator of cardiac differentiation
has been identified.30,77,78 We hypothesized that a combination
of key developmental cardiac transcription factors is required
to directly convert fibroblasts into cardiomyocytes. In an attempt to identify cardiac reprogramming factors, we first developed a screening system in which the induction of cardiac
genes could be analyzed quantitatively by reporter-based fluorescence-activated cell sorting. We generated α-myosin heavy
chain promoter–driven enhanced green fluorescent protein
(α-MHC-GFP) transgenic mice, in which cells with activated
cardiac programs express GFP. After retroviral expression of
14 candidate genes in neonatal CFs, we detected cells with
cardiac morphologies and expression of cardiac markers, suggesting that such a conversion may indeed be possible. Serial
reduction of individual factors revealed that the 3 cardiac transcription factors, GMT, were minimally required to induce
iCMs.21 Transduction with GMT in CFs activates a cardiac reporter, α-MHC-GFP, in ≈15% to 20% of the cells, and cardiac
protein expression in 5% of the fibroblasts after 1 week; however, few cells became spontaneously beating iCMs after 4
weeks (only 0.01%–0.1% of the starting fibroblasts), suggesting the vast majority of the α-MHC-GFP+ cells were partially
reprogrammed iCMs (Figure 2). The efficiency of generation
of beating iCMs by GMT was similar to that in iPSC generation, with the original conditions. Despite the heterogeneity of the iCM populations, produced as above, global gene
expression analyses revealed the expression of a broad range
of cardiac genes, with silencing of the fibroblast program in
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Figure 2. Modifications of reprogramming factors alter the efficiency and quality of cardiac reprogramming. Time course and
efficiency of cardiac induction from mouse fibroblasts by Gata4, Mef2c, and Tbx5, with the original conditions, are shown (upper). Lower,
Several interventions could change the yield of partially reprogrammed induced cardiomyocyte-like cells (iCMs) and functional iCMs from
mouse fibroblasts. *The studies were conducted in adult cardiac fibroblasts or tail-tip fibroblasts. The functional improvement by addition
of Nkx2.5 and transforming growth factor (TGF)-β inhibitor was measured by spontaneous calcium activity in the iCMs. α-MHC indicates
α-myosin heavy chain; and cTnT, cardiac troponin T.
fluorescence-activated cell sorting–purified ­
α-MHC-GFP+
iCMs. The histone and DNA epigenetic marks in several
cardiac gene promoters in the iCMs were similar to those in
neonatal cardiomyocytes but were different from those in the
original CFs. GMT transduction also induced cardiac gene
expression and spontaneous intracellular calcium transients
in tail-tip fibroblasts (TTFs), but the TTF-derived iCMs did
not beat spontaneously. The lineage-tracing experiments using
Isl1-Cre-yellow fluorescent protein and Mesp1-Cre-yellow
fluorescent protein mice demonstrated that neither Isl1 nor
Mesp1 gene was activated during cardiac reprogramming,
suggesting that fibroblasts were converted directly into cardiomyocytes without passing through a progenitor cell state.
Our study was the first to represent the global gene expression profiles and epigenetic status of directly induced cells and
demonstrates the route of reprogramming unambiguously using several reporter mice.
Although the achievement of direct cardiac reprogramming
is exciting in the cardiac research, the induction efficiency of
iCMs remained variable depending on the protocol. Chen et
al28 reported that the lentiviral expression of GMT did not efficiently induce cardiac reprogramming in adult mouse CFs and
TTFs. The GMT-transduced fibroblasts expressed several cardiac genes and exhibited voltage-dependent calcium currents,
but spontaneous action potentials and cellular contractions
were not observed, suggesting the cells were in incomplete
reprogramming. The culture conditions for fibroblasts and
iCMs, the starting cell types used for reprogramming, and titers of viruses expressing the reprogramming factors may be
critical for successful cardiac reprogramming, as they are for
the induction of iPSCs.79–81 The expression of GMT by lentiviruses might be different from that by retroviruses, and the
starting fibroblasts used in our study were mainly neonatal
CFs, while they used adult CFs and TTFs, which were more
resistant to reprogramming.29
The low and variable reprogramming efficiency by GMT
may be because of the stoichiometry of reprogramming factors
in the cells transduced with separate vectors.80,82 To address this,
Wang et al83 recently generated all possible polycistronic vectors of GMT and demonstrated that the stoichiometry of reprogramming factors is indeed critical for cardiac reprogramming.
They found that the 2 polycistronic vectors, MGT and MTG,
which expressed a high level of Mef2c and low levels of Gata4
and Tbx5, enhanced cardiac reprogramming, whereas the other
vectors reduced reprogramming efficiency (Figure 2). This need
for precise stoichiometry of factors for successful cardiac reprogramming demonstrated that subtle differences in experimental
conditions may result in failure of cardiac reprogramming. It
is conceivable that the inefficient reprogramming reported by
Chen et al may have been caused by higher Gata4 and Tbx5
expression and lower Mef2c expression in their transduced
cells. Although the findings by Wang et al provided important
insights into the variable reprogramming efficiency reported by
different laboratories, they did not compare the cardiac gene
expression in the iCMs generated by the polycistronic vectors
with those in neonatal cardiomyocytes or adult cardiomyocytes.
Further investigation is needed to determine the similarity of the
MGT-induced iCMs to bona fide cardiomyocytes.
Sadahiro et al Progress and Challenges of Direct Reprogramming 1383
Direct Reprogramming Into Cardiomyocytes
by Different Factor Combinations
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Addition or modifications of transcription factors, miRNAs,
and small molecules may promote the cardiac reprogramming efficiency, as shown in the other reprogramming fields
(Figure 2). Song et al24 sought to investigate the optimal combination of the cardiac transcription factors to reprogram
adult CFs and TTFs into functional iCMs. They also used
­α-MHC-GFP reporter mouse and screened 6 cardiac transcription factors (Gata4, Hand2, Mef2c, Tbx5 [GHMT], Mesp1,
and Nkx2.5) as candidate factors. Addition of Hand2 to GMT
led adult CFs and TTFs to be reprogrammed into functional
iCMs more efficiently than did any other combinations, including GMT. GHMT induced ≈9% of starting populations
to express both α-MHC-GFP and cardiac troponin T (cTnT)
cardiac genes after 1 week, which was higher than those induced with GMT (3%), suggesting that Hand2 promoted early
stage of reprogramming process. Notably, after 5 weeks of
GHMT transduction into adult CFs and TTFs, the functional
properties, including calcium transients, action potentials,
and spontaneous contractions, were observed in a subset of
iCMs, suggesting that GHMT may be more potent than GMT
to overcome the barriers for cardiac reprogramming in TTFs.
More recently, Nam et al84 analyzed the iCM properties induced with GHMT in more detail. Although 10% to 30% of
the GHMT-transduced fibroblasts expressed cTnT, the iCMs
revealed remarkable degree of sarcomeric structural diversity and only a small portion of the cells (≈1% of fibroblasts)
formed well-organized sarcomere structure that correlated
with spontaneous beating activity (≈0.16%). Moreover, they
found that GHMT generated all 3 types of immature form of
myocytes, atrial, ventricular, and pacemaker cells, at roughly
equal proportions, determined by multiplex immunostaining
and electrophysiology. Thus, Hand2 promoted cardiac reprogramming at least in part by increasing the cardiac marker–
expressing cells at early stage, and the GHMT-iCMs consisted
of heterogeneous populations including all 3 cardiac subtypes.
Increasing the transactivation activity of transcription factors may also promote cardiac reprogramming. To address
this, Hirai et al85 fused a powerful transactivation domain of
MyoD to GHMT and transduced these genes in various combinations into mouse embryonic fibroblasts. Transduction of
the chimeric Mef2c with the wild-type forms of the other 3
genes generated more cardiac protein–expressing cells and
beating clusters of iCMs with an efficiency of 3.5%, which
was 15-fold greater than the wild-type gene combinations.
Intriguingly, transduction of other combinations of chimeric
genes reduced the number of beating iCMs, suggesting that
increasing the transcriptional activity in only Mef2c is critical for successful cardiac reprogramming, which is consistent
with the results by Wang et al83 using polycistronic vectors.
MiRNAs have numerous targets related to signaling pathways, transcription factors, and epigenetic regulation and
play important roles in cell fate specifications and embryonic
development. Jayawardena et al86 introduced a combination
of miRNAs (miR-1, miR-133, miR-208, and miR-499) into
neonatal CFs to succeed in creating functional iCMs. As miRNAs are not incorporated into host chromosome by transient
expression, miRNA-mediated cardiac reprogramming may be
safer applications in clinic. This article also revealed that culture conditions are vital to enhance the reprogramming efficiency and quality, as expression of α-MHC-cyan fluorescent
protein increased ≈10-fold, and spontaneous cell contractions
reached to 1% to 2% of the starting cells by addition of a JAK
inhibitor to the culture media.
A new approach to identify optimal combinations of reprogramming factors to generate functional iCMs was proposed
by Addis et al.87 They used a reporter system in which the calcium indicator GCaMP was driven by the cTnT promoter for
the screening of functional iCMs.88 They found that addition
of Nkx2.5 to GHMT resulted in the most efficient combinations for cardiac reprogramming and reported that the fibroblasts were stably reprogrammed into calcium transient+ iCMs
after 15-day exposure to GHMT with Nkx2.5 using doxycycline-inducible lentiviral system. More recently, the same
group used this reporter system to identify small molecules
sufficient to enhance cardiac reprogramming and demonstrated that the transforming growth factor β (TGFβ) inhibitor, SB431542, was capable of increasing the conversion of
both mouse embryonic fibroblasts and adult CFs into calcium
transient+ iCMs up to 5-fold.89 Inhibition of TGFβ signaling at
early in the reprogramming process led to the greatest increase
in the conversion of fibroblasts to iCMs.
Although multiple groups promoted cardiac reprogramming
by adding transcription factors or small molecules, the molecular mechanisms of direct reprogramming remained undefined. Recently, we demonstrated that addition of miR-133 to
GMT promoted cardiac reprogramming via Snai1 repression
and silencing the fibroblast gene signature. Cotransduction of
miR-133 with GMT increased the number of cells expressing
cardiac markers by 9-fold at the early stage of reprogramming,
leading to the generation of 7-fold more beating iCMs compared with GMT treatment alone; this treatment also shortened the duration required to induce beating iCMs from 30 to
10 days.90 Mechanistically, miR-133 directly targeted Snai1, a
master regulator of epithelial-to-mesenchymal transition, and
Snai1 knockdown suppressed fibroblast genes, upregulated
cardiac gene expression, and induced more contracting iCMs,
recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced
cells maintained fibroblast signatures and inhibited generation
of beating iCMs, suggesting that miR-133–mediated Snai1
repression is critical for cardiac reprogramming. Thus, many
laboratories improved the reprogramming efficiency by modifying or adding several factors to GMT,91–93 which in turn may
help our understanding of the reprogramming process, as we
will discuss later.
Direct Reprogramming Into Cardiomyocytes
In Vivo
The most exciting potential of direct cardiac reprogramming
lies in the possibility of in vivo use of this new technology, in
which a large pool of endogenous CFs could be directly reprogrammed into cardiomyocyte-like cells solely through gene
delivery. Moreover, it is conceivable that the in vivo cardiac
environment may help the reprogramming process to generate
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high-quality iCMs compared with those in vitro. To test the
possibility of direct cardiac reprogramming in vivo, we, along
with others, investigated whether the gene transfer of reprogramming factors into infarcted mouse hearts could convert
resident CFs into cardiomyocytes in situ.23,24,94,95
Qian et al23 directly injected GMT retroviruses into mouse
hearts after coronary ligation and demonstrated in vivo cardiac
reprogramming with functional improvement. They used fibroblast lineage-tracing mice (Fsp1-Cre and Periostin-Cre) to
demonstrate that iCMs originated from CFs or nonmyocytes
and used α-MHC-MerCreMer mice to determine whether the
newly generated iCMs arose from cell fusion with pre-existing
cardiomyocytes. Using these lineage-tracing approaches, they
showed that ≈35% of cardiomyocytes in the border/infarct
zone were newly generated iCMs derived from resident CFs
rather than cell fusion of fibroblasts with pre-existing cardiomyocytes. Of the in vivo iCMs, 50% had well-organized sarcomeric structures and exhibited functional characteristics of
adult ventricular cardiomyocytes, including cellular contraction, electrophysiological properties, and functional coupling
to other cardiomyocytes. The hearts also contained intermediate stages of partially reprogrammed iCMs, and time course
experiments revealed a progressive reprogramming process in
which a more complete cardiac phenotype arose over time.
These observations suggested that in vivo reprogramming
might yield mature iCMs more efficiently than in vitro conditions. Functional studies with cardiac MRI and echocardiography and histological analyses revealed that retroviral
GMT gene transfer significantly improved cardiac function
and reduced fibrosis until at least 3 months after myocardial
infarction.
Song et al24 also reported that GHMT retroviral injection
into mouse infarct hearts converted endogenous CFs into
functional iCMs in situ and improved cardiac function after
myocardial infarction. They used Fsp1-Cre and inducible
Tcf21-iCre mice as fibroblast lineage-tracing mice and found
that 2% to 6% of cardiomyocytes in the border/infarct area
were newly generated iCMs with clear striations and functional properties similar to endogenous ventricular cardiomyocytes, as determined by electrophysiology, Ca2+ transients,
and cell contractility. The discrepancy of cardiac induction
rate between 2 studies may be because of the difference of
reprogramming factors and lineage-tracing mice used in their
studies. Song et al also demonstrated that the ejection fraction was increased by 2-fold in GHMT-treated mice compared
with controls, and that the scar size was reduced by 50% at 12
weeks after myocardial infarction.
We demonstrated that GMT gene transfer could generate new iCMs in vivo; however, our approach to test the in
vivo cardiac reprogramming differed from those used by the
Srivastava and Olson groups.94 They used mainly fibroblast
lineage-tracing mice to demonstrate in vivo cardiac reprogramming, whereas we took an alternative approach of using
cotransduction of retroviral GMT and reporter gene to determine cardiac induction from nonmyocytes. We first confirmed
that retroviruses infected only proliferating cells, which are
mainly CFs in an infarcted myocardium. In addition, different from that reported in the previous 2 articles, we used
immunosuppressed mice to promote the survival of virally
transduced cells. We found that separate GMT gene transfer induced mostly immature α-actinin+ cells in vivo, with a
conversion rate of ≈1% in the transduced cells. To improve
the transduction of the 3 factors, we then generated a polycistronic retrovirus expressing GMT from the same promoter using 2A peptides.94,96 Gene transfer of this polycistronic GMT
retrovirus induced morphologically more mature iCMs in fibrotic tissues than those generated by injecting the separate
GMT vectors, suggesting that polycistronic systems could be
valuable tools for in vivo reprogramming.94,97
Mathison et al95 demonstrated that intramyocardial administration of vascular endothelial growth factors into the
infarcted myocardium 3 weeks before GMT gene delivery enhanced functional recovery and reduced scar size in rat hearts
after myocardial infarction compared with that observed with
the use of GMT or vascular endothelial growth factor alone.
These beneficial effects of vascular endothelial growth factor might be because of neovascularization and some other
mechanisms, including better survival of the newly generated
iCMs in the infarcted hearts.
Jayawardena et al98 demonstrated that direct administration of a combination of lentiviral miR-1, miR-133, miR-208,
and miR-499 into mouse infarcted hearts converted resident
CFs into iCMs and improved cardiac function after myocardial infarction. They used Fsp1-Cre mice for linage tracing of
nonmyocytes and found that 12% of cardiomyocytes in the
border/infarct area were newly generated iCMs. The iCMs
generated by in vivo reprogramming expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction
coupling, and action potentials characteristic of mature ventricular cardiac myocytes. Serial echocardiography revealed
that there was a time-delayed and progressive improvement
in ventricular function, beginning 1 to 2 months post surgery
and was enhanced at 3 months, which was similar to the period reported for reprogramming with transcription factors.
For clinical applications, the development of a nonviral delivery method, including chemically synthesized miRNA mimics, may be an attractive therapeutic option, as they do not
integrate into the host chromosomes. Together, these results
suggest that the abundant pool of endogenous CFs could be a
cell source for new cardiomyocytes by direct reprogramming
and that this new technology can improve cardiac function and
reduce scar size after myocardial infarction. Moreover, the in
vivo iCMs are more mature than those in vitro, suggesting
the in vivo environment may improve the quality of cardiac
reprogramming.
Direct Cardiac Reprogramming in Human
Fibroblasts
To advance the direct cardiac reprogramming technology toward clinical applications, it would be necessary to translate
the mouse system into human. Nam et al25 found that GHMT
reprogramming factors in mouse fibroblasts were ineffective
in activating cardiac gene expression in human fibroblasts,
and that the additional factor, Myocd, was required for human cardiac gene expression. Furthermore, adding 2 musclespecific miRNAs, miR-1, and miR-133, further improved the
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myocardial conversion of human fibroblasts and eliminated
the requirement for Mef2c in cardiac induction from human
neonatal foreskin fibroblasts, adult CFs, and adult dermal fibroblasts.99 The induction efficiency of cTnT-expressing cells
from fibroblasts was 10% to 20%, but only a small subset of
the cells derived from adult CFs exhibited spontaneous contractility after 11 weeks of culture, suggesting most human
iCMs were in a partially reprogrammed state.
We also found that GMT was not sufficient for cardiac
reprogramming in human fibroblasts. Thus, we screened 11
additional factors in combination with GMT and found that
addition of Mesp1 and Myocd upregulated a broader spectrum
of cardiac genes more efficiently in human CFs compared
with that observed with GMT alone.26 Myocd upregulated sarcomeric genes and Mesp1 increased intracellular Ca2+ oscillations, suggesting Myocd and Mesp1 differentially regulate
cardiac gene expression. The human CFs and dermal fibroblasts transduced with GMT, Mesp1, and Myocd changed
the cell morphology from spindle to rod-like or polygonal
shape, expressed multiple cardiac-specific proteins, increased
a broad range of cardiac genes and concomitantly suppressed
fibroblast genes, and exhibited spontaneous Ca2+ oscillations.
Although the human iCMs did not beat spontaneously, the
cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. The
EdU (5-ethynyl-2-deoxyuridine) assay revealed that the iCMs
generated did not pass through a mitotic cell state, suggesting
GMT, Mesp1, and Myocd directly reprogram human fibroblasts toward a cardiac fate. More recently, we also found that
addition of miR-133 to GMT, Mesp1, and Myocd promoted
cardiac reprogramming in human fibroblasts.90 The induction of cardiac markers, α
­ -actinin and cTnT, was increased
from 2% to 8% to 23% to 27% by addition of miR-133.
Mechanistically, miR-133–mediated Snai1 repression was
also critical for human cardiac reprogramming, which is in
accordance with the results in mouse data.
Fu et al27 reported that GMT plus Esrrg, Mesp1, Myocd,
and Zfpm2 induced global cardiac gene expression and phenotypic shifts to a cardiac state in human fibroblasts derived
from ESCs, fetal heart, and neonatal skin. Although most cells
were partially reprogrammed, a subset of human iCMs had
sarcomere formation, calcium transients, and action potentials. They demonstrated that the epigenetic status of human
ESC resembled that of hESC-derived cardiomyocytes in DNA
and histone methylation status, and that the iCMs were stably
reprogrammed to a cardiac state without the need for continuous expression of reprogramming factors. Furthermore,
they found that TGF β signaling was important for cardiac
reprogramming and improved the efficiency of human iCM
generation.
Islas et al100 reported that transient expression of Ets2 and
Mesp1, followed by activin A and bone morphogenetic protein
2 treatment, could reprogram human dermal fibroblasts into
cardiac progenitor–like cells. The induced cardiac progenitor–
like cells expressed several cardiac progenitor markers and
differentiated into cardiomyocyte-like cells that expressed
cardiac genes, sarcomeric structures, and Ca2+ activities in a
prolonged culture. Although these findings of human cardiac
reprogramming represent an important step toward potential
clinical applications, the process was slower and less efficient
than mouse reprogramming.14,62,101
Partial Reprogramming With Transient
Expression of Pluripotent Factors
Direct reprogramming of somatic cells to a pluripotent state
using Yamanaka factors entails extensive genetic and epigenetic resetting. Thus, it is conceivable that Yamanaka factors
function to erase cell identity by epigenetic mechanisms and
that subsequent exposure to external lineage-specific signals
may direct lineage specification and terminal differentiation.
Efe et al102 showed that transient overexpression of Oct4,
Sox2, Klf4, and c-Myc and subsequent exposure to the cardiogenic media, including bone morphogenetic protein 4 and
JAK inhibitor, converted mouse fibroblasts into spontaneously
contracting cardiomyocytes, via a cardiac progenitor cell state
with no pluripotent intermediate. More recently, the same
group reported that only a single transcription factor, Oct4, in
combination with the small molecule cocktails, consisting of
SB431542 (ALK4/5/7 inhibitor), CHIR99021 (GSK3 inhibitor), parnate (LSD1/KDM1 inhibitor), and forskolin (adenylyl cyclase activator), enabled cardiac conversion of mouse
fibroblasts without traversing the pluripotent state.103 A similar approach was also applied to generate neural progenitor
cells and blood progenitor cells from mouse fibroblasts.104,105
Kim et al105 demonstrated that the transient expression of the
Yamanaka factors and subsequent exposure to the cell culture
conditions favorable for neural precursor cells induced neural
progenitor cells from fibroblasts without reverting to iPSCs.
To apply the partial reprogramming approach toward heart
regeneration, it should be determined whether the cardiomyocyte foci do not form teratoma and functionally integrate into
the host myocardium after transplantation, and the same approach could be applied in human cells.
Direct Cardiac Reprogramming: Challenges
and Future Directions
Although the recent progress in cardiac reprogramming is
promising, the low reprogramming efficiency of fibroblasts
into iCMs in vitro is a major challenge. We first reported
that just 3 transcription factors rapidly and efficiently induced fibroblasts to a cardiac fate (activated cardiac genes
and suppressed fibroblast genes); however, the reprogramming to generate beating iCMs in vitro was inefficient and
slow. Since then, many researchers have demonstrated that
modifications of reprogramming factors and culture conditions could improve cardiac induction (Figure 2). Addition
of Hand2 to GMT promoted cardiac reprogramming at least
in part by increasing the number of cells expressing cardiac
markers and proteins at the early stage. Further investigations
using a doxycycline-inducible system for transient expression of Hand2 may clarify the role of Hand2 at the late stage
of reprogramming. MiR-133 promoted cardiac induction by
Snai1 suppression and silencing the fibroblast signature at the
early stage. Given that TGFβ is an activator of Snai1, it is
conceivable that TGFβ inhibitor-mediated cardiac reprogramming in mouse fibroblasts, reported by Ifkovits et al,89 might
be partly mediated through suppression of Snai1 and silencing fibroblast signatures. Intriguingly, this process is similar
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to the mesenchymal-to-epithelial transition process mediated
through Snai1 suppression in iPSC generation.106,107 In contrast, Fu et al reported that TGFβ signaling promoted human
cardiac reprogramming, suggesting that the TGFβ/Snai1
pathway may affect cardiac reprogramming paradoxically
depending on the species and cell lines, as seen in iPSC generation.108 High transcriptional activity of Mef2c and stoichiometry of transcription factors (high expression of Mef2c and
low expression of Gata4 and Tbx5) increased cardiac induction. Although the precise mechanisms remain unclear, this is
consistent with the view that precise dosage of transcription
factors is critical for proper development and cell fate specification. Addition of Nkx2.5 to GMT or GHMT increased the
generation of functional iCMs, determined by the spontaneous calcium activity. In contrast, we, along with others, previously reported that Nkx2.5 reduced the number of cardiac
marker–expressing cells,21,24 such as cTnT and α-MHC-GFP,
suggesting that Nkx2.5 may increase the proportion of functional iCMs without increasing the partially reprogrammed
iCMs. However, it should be determined whether addition of
Nkx2.5 indeed increased the number of beating iCMs, which
is the more stringent criterion for functional iCMs. Thus, we,
along with others, have identified several important molecules
that enhance cardiac induction at the early stage of reprogramming; however, the factors critical for the transition of partially
reprogrammed iCMs into spontaneously beating iCMs at the
late stage remain unclear. As the reprogramming efficiency at
early stage may not always correlate with functional reprogramming at late stage, the measurement of cardiac functional
outcomes, such as the efficiency of beating iCMs, will identify such molecules. Standardized conditions for efficient and
reproducible cardiac induction are needed for the screening,
and factors identified in the mouse system might be applied
to in vivo reprogramming and human cardiac reprogramming.
Recently, the Daley and Collins groups developed a new
biological platform, CellNet, which can compare gene regulatory networks in engineered cell populations with those in in
vivo counterparts and identify factors that may improve cellular reprogramming.73,109 Cahan et al109 analyzed the genomewide transcriptional profiles of α-MHC-GFP+ iCMs generated
with GMT,21 several cell types, and ESC-derived cardiomyocytes using CellNet. They found that the α-MHC-GFP+ population was exclusively classified as heart, but not other cell
types including skeletal muscle and fibroblasts, confirming a
global shift in the gene expression profile toward a cardiac
fate. They also demonstrated that the α-MHC-GFP+ cells were
less similar to neonatal cardiomyocytes than ESC-derived
cardiomyocytes, but this is not surprising, given that the majority of the cells were nonbeating, partially reprogrammed
iCMs (Figure 2). Some heart-related genes, including Gata6
and Tbx20, were incompletely activated in the α-MHC-GFP+
population compared with cardiomyocytes, and addition of
these factors might improve cardiac reprogramming. Cahan
et al also demonstrated that the in vivo iCMs were similar
to bona fide cardiomyocytes and established the heart gene
regulatory network more completely than ESC-derived cardiomyocytes. Development of CellNet for single-cell analyses
will likely lead to demonstration of the variability of reprogramming among individual iCMs generated with different
stoichiometries of reprogramming factors and also identify
new targets that enhance the quality of cardiac reprogramming.
As cardiomyocytes consist of several types of myocytes, including sinoatrial nodal cells, atrial myocytes, and ventricular
myocytes, it would be important to understand the mechanism
of cardiac subtype specification and navigate the cardiac reprogramming toward the desired mature cell types.110,111 The
beating phenotype in some in vitro iCMs seems to be nonrhythmic, unlike that in postnatal cardiomyocytes or ESC-derived
cardiomyocytes that is rhythmic and uniform across the entire
cell. Given that murine cardiomyocytes contract irregularly
at the early embryonic stages (embryonic days 9.5–11.5),112
this phenotype may be because of the generation of immature
iCMs or partially reprogrammed iCMs, in which the expression of some ion channel genes is not sufficiently high. The
detailed characterization of iCM functions, such as contractile
force generation and ion channel expression, will reveal the
properties and maturation level of iCMs. We demonstrated
that iCMs generated in vitro with GMT or GMT/miR-133
were mostly atrial-type myocytes, whereas GHMT induced all
3 types of myocytes in nearly equal proportions.84,90 It would
be intriguing to determine the phenotypes of myocytes induced with various reprogramming factors, which may reveal
key factors for cardiac subtype specification during development. Kapoor et al113 reported that overexpression of Tbx18,
a gene critical for sinoatrial node specification, could convert
murine ventricular cardiomyocytes to pacemaker cells in vitro
and in vivo.114,115 Similarly, forced expression of Tbx3 or activation of Notch signaling in working cardiomyocytes was sufficient to generate conduction cells in vitro and in vivo.116,117
Given that GHMT generate all 3 types of immature form of
cardiomyocytes from fibroblasts,84 it is conceivable that addition of these transcription factors or molecules to GHMT
may convert fibroblasts directly into the desired cardiac cell
types, which has been demonstrated in the iN and iHep cell
reprogramming.
In contrast to the reprogramming in vitro, the in vivo cardiac
reprogramming could generate more mature iCMs and repair
infarcted myocardium. Injection of the reprogramming factors
directly into the damaged heart may convert the endogenous
CF population, which represents >50% of all cardiac cells,
into new functional iCMs. This in vivo reprogramming approach may have several advantages over cell transplantation–
based therapy. First, the process is simple; second, avoiding
the induction of pluripotent cells before cardiac differentiation
would greatly lower the risk of tumor formation; and third,
direct injection of defined factors obviates the need for cell
transplantation, for which long-term cell survival remains
challenging (Figure 3).118–121
However, many questions and hurdles remain to be overcome to realize the success of this new technology.122 First,
it is not clear how many iCMs would remain in the infarct
hearts over a longer term, and to what extent the iCMs would
integrate with the native cardiomyocytes and contribute to the
observed improvement in cardiac function. Although it is challenging to count the total number of iCMs generated in vivo,
it will be important to expect the number of newly generated
cardiomyocytes and anticipate its effect on overall cardiac
function. Given that the cardiac reprogramming efficiency is
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Figure 3. Future cardiac regenerative therapy. The lost myocardial tissue is replaced by fibrous tissue that mainly consists of cardiac
fibroblasts and extracellular matrix. The cell transplantation–based approach using induced pluripotent stem cell (iPSC)–derived
cardiomyocytes is shown (upper). Direct cardiac reprogramming approach may convert endogenous cardiac fibroblasts directly into
cardiomyocytes by defined factors in situ (lower).
relatively low, the increased ejection fraction and reduced fibrosis after injection of the reprogramming factors into mouse
hearts seem to be greater than expected. Some other mechanisms, such as alteration of fibroblast profile, promotion of
angiogenesis, and prevention of apoptosis in cardiomyocytes
by paracrine factors, may contribute to increased cardiac function. Understanding the mechanism of functional improvement by in vivo reprogramming may identify new targets for
cardiac repair. Second, it is possible that the immature iCMs
generated in situ may have a propensity to trigger arrhythmias
in the damaged heart. There is also a risk of random genomic
integration of virally overexpressed transgenes with the current protocols, which may result in adverse effects. Therefore,
improving the quality of cardiac reprogramming, optimizing
gene delivery systems, and development of nonintegrating
vectors or small molecules to replace some or all transcription factors are required to advance this strategy toward future
regenerative therapies.49,123 Third, the low efficiency of human
cardiac reprogramming may raise concerns on whether this
approach will be feasible in the near future. Feedback from
mouse reprogramming and further modifications of factors
and culture conditions will enhance the human cardiac reprogramming. Given that in vivo reprogramming demonstrated
functional benefits and higher reprogramming efficiency compared with in vitro reprogramming in mice, it is conceivable
that the delivery of human cardiac reprogramming factors into
damaged heart may repair cardiac tissues and improve cardiac
function. Experiments in larger animals in vitro and in vivo
will assess the safety and efficacy of the direct reprogramming approach. Moreover, all in vivo studies thus far were
performed in the acute stage of myocardial infarction, and
it remains to be determined whether in vivo reprogramming
could be applied to chronic heart failure models, in which regenerative medicine is in high demand. Last, it would be intriguing to investigate the molecular mechanisms underlying
cardiac reprogramming and cell fate determination. Why are
cardiac reprogramming factors different in mice and humans,
in contrast to the Yamanaka factors in iPSCs? The genetic and
epigenetic networks and time periods required for development differ among species, which may affect the reprogramming efficiency and factors needed for reprogramming. It is
conceivable that the iPSC paradigm might be an exception
rather than the rule in cell fate conversion, considering that the
reprogramming factors in mouse and human are also different in other direct reprogramming fields. Identification of the
mechanisms that inhibit human reprogramming by transcriptomic as well as epigenetic analyses will enhance our understanding of the gene regulatory networks during development
and advance this new technology toward clinical applications.
Conclusions
Although still in a nascent stage, direct cardiac reprogramming has undergone great advances and attracted considerable
attention. From a basic biology standpoint, it may become a
new methodology in the developmental and molecular biology. It offers a new way to understand the transcription factor
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function and to study the complex interplay among transcriptional regulators, miRNAs, and epigenetic regulators.
Moreover, the generation of iCMs represents a novel way to
study the mechanisms of cell fate decisions during cardiac development and postmitotic cardiac maturation. From a clinical
point of view, direct cardiac reprogramming holds great potential for cardiovascular disease research and treatment, including patient-specific drug screening, cardiac disease modeling,
and regenerative purposes. When comparing the ability to
generate ex vivo cardiomyocytes through the differentiation of
iPSCs versus direct reprogramming, it is clear that the former
strategy is far more advanced at this stage. Expandability is
obviously a major advantage of iPSCs over postmitotic iCMs,
and efficiency of cardiac induction, particularly in humans,
is still low in direct reprogramming. Nevertheless, direct
reprogramming in vivo possesses several theoretical advantages, which may resolve many of the challenges and issues
associated with cell therapies. As the retroviral delivery of
reprogramming factors will be a concern in clinical applications, new methods to generate integration-free iCMs would
be required. Further studies, including optimization of cardiac
reprogramming in human fibroblasts and demonstration of the
therapeutic efficacy and safety of in vivo reprogramming approach in chronic heart failure and larger animal models, are
needed. As the demand is high for new regenerative therapies,
the opportunities and the potential benefits of this direct cardiac reprogramming approach are significant.
Acknowledgments
We are grateful to members of the Ieda laboratory for valuable discussion and contributions.
Sources of Funding
M. Ieda was supported by research grants from Japan Science and
Technology Agency (JST) CREST, Japan Society for the Promotion
of Science (JSPS), Banyu Life Science, The Uehara Memorial
Foundation, Takeda Science Foundation, and SENSHIN Medical
Research Foundation.
Disclosures
S. Yamanaka is a scientific advisor of iPS Academia Japan without
salary. The other authors report no conflicts.
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Direct Cardiac Reprogramming: Progress and Challenges in Basic Biology and Clinical
Applications
Taketaro Sadahiro, Shinya Yamanaka and Masaki Ieda
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Circ Res. 2015;116:1378-1391
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