REVIEW

Direct Reprogramming into Desired Cell Types
by Defined Factors
Masaki Ieda1–3

1Department of Clinical and Molecular Cardiovascular Research, School of Medicine, Keio University,

Tokyo, Japan

2Department of Cardiology, School of Medicine, Keio University, Tokyo, Japan
3The Japan Science and Technology Agency, CREST, Tokyo, Japan(Received for publication on December 9, 2012)(Revised for publication on January 8, 2013)(Accepted for publication on January 18, 2013)(Published online in advance on June 21, 2013)

In the field of developmental biology, the concept that cells, once terminally differentiated, are fixedin their cell fate was long believed to be true. However, Dr. Gurdon and colleagues challenged thisfundamental doctrine and demonstrated that cellular reprogramming and cell fate conversion are possible by somatic nuclear transfer and cell fusion. The Weintraub laboratory discovered in the 1980s
that a single transcription factor, MyoD, can convert fibroblasts into skeletal muscle cells, and subsequent studies also demonstrated that several transcription factors are lineage converting factors
within the blood cell lineage. In 2006, Takahashi and Yamanaka discovered that transduction of thefour stem cell-specific transcription factors Oct4, Sox2, Klf4, and c-Myc can reprogram mouse fibroblast cells into a pluripotent state. In 2007, they demonstrated that the same four factors similarly
reprogram human somatic cells into pluripotent stem cells. These discoveries by Dr. Yamanaka andcolleagues fundamentally changed research in the fields of disease modeling and regenerative medicine and also inspired the next stage of cellular reprogramming, i.e., the generation of desired cell
types without reverting to stem cells by overexpression of lineage-specific transcription factors.
Recent studies demonstrated that a diverse range of cell types, such as pancreatic β cells, neurons,
neural progenitors, cardiomyocytes, and hepatocytes, can be directly induced from somatic cells by
combinations of specific factors. In this article, I review the pioneering works of cellular reprogramming and discuss the recent progress and future perspectives of direct reprogramming technology.
(doi: 10.2302/kjm.2012-0017-RE; Keio J Med 62 (3) : 74–82, September 2013)

Keywords: cardiomyocytes, induced pluripotent stem cell, direct reprogramming, transcription
factors

Introduction

clear transfer. The birth of Dolly the sheep was reported
in 1997, and many other species of cloned mammals were
subsequently reported.3–11 Moreover, identification of
MyoD, a master gene for skeletal myocytes, demonstrated
that cell fates can be changed simply through the overexpression of specific transcription factors.12–18 Thesestudies suggested that cell fates are more plastic than previously expected and that dormant gene expression pro-In the 1960s, John Gurdon and colleagues demonstrated
that the nucleus of a differentiated frog cell could be reprogrammed back to that of a totipotent cell when transferred into an enucleated egg. Such cells can then giverise to a whole new frog,1,2 and this research lead to thesuccess of clone generation in mammals by somatic nu-

Reprint requests to: Masaki Ieda, MD, PhD, Department of Clinical and Molecular Cardiovascular Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan, E-mail: mieda@z8.keio.jp
Copyright © 2013 by The Keio Journal of Medicine
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Fig. 1 Generation of desired cell types via induced pluripotent stem cells (iPSCs) and by direct lineage reprogramming.
Transduction of Oct4, Sox2, Klf4, and c-Myc converts fibroblasts into iPSCs. Terminally differentiated cells, including neurons, cardiomyocytes, and hepatocytes, can be generated from iPSCs. Alternatively, neurons, cardiomyocytes, and hepatocytes can be directly
induced from fibroblasts by overexpression of lineage-specific transcription factors.

grams can be activated in terminally differentiated cells
by defined factors.2–4,19 This pioneering research led to
the landmark discovery by Takahashi and Yamanaka in2006 that reprogramming mouse fibroblasts into pluripotent stem cells is possible by transduction of four stem
cell-specific transcription factors. Sir John Gurdon and
Dr. Shinya Yamanaka received the 2012 Nobel Prize inPhysiology or Medicine as a result of these achievements.
The generation of induced pluripotent stem cells (iPSCs) by Yamanaka factors has revolutionized the fieldof regenerative medicine and has triggered studies oncell therapy, disease modeling, and personalized medicine.20–30 Moreover, the discovery of iPSCs has delivered
a new approach for the generation of desired cell types
called lineage reprogramming, which is the direct conversion of one adult cell type into another using combinations of lineage-specific transcription factors or microRNAs without the cell passing through the pluripotent stem
cell state (Fig. 1). Recent studies have demonstrated that
direct lineage reprogramming can yield a diverse range of
medically relevant cell types, such as pancreatic β cells,
neurons, cardiomyocytes, and hepatocytes.31–35Here, I
will give an overview of the history and recent progressof cellular reprogramming and its potential future applications in regenerative medicine.

MyoD Converts Fibroblasts into Skeletal Myocytes
MyoD is a classic example of a master gene for cell differentiation in the sense that transduction of this gene issufficient to activate the whole genetic program of muscle
differentiation in non-muscle cells.36,37 In 1979, Taylor
and Jones found that treatment of the mouse embryonic
fibroblast cell line with 5-azacytidine (5-azaC) induced
differentiation of these cells into skeletal muscle cells.38,39
This observation led to the hypothesis that DNA demethylation and the resulting activation of unidentified genes
were responsible for the conversion to muscle cells.40 A
subtractive hybridization experiment comparing untreated and 5-azaC-treated fibroblasts led to the identificationof MyoD.12 When overexpressed in primary fibroblasts orin a wide variety of other cell types, including pigment,
nerve, fat, and liver cells, MyoD can convert these cells toskeletal muscle cells.15,16 These findings provided the firstevidence that a single gene, acting as a master switch, caninitiate a complex program of differentiation.
The ability of MyoD to convert somatic cells into skeletal muscle cells suggested that it might have a central
role in myogenesis, and subsequent studies sought to determine its biological roles and molecular mechanisms
in muscle development.41 The MyoD protein contains
a basic helix-loop-helix (bHLH) motif that is common

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to a large family of transcription factors.41–43 In addition to MyoD, the highly related proteins Myf5, Mrf4,
and myogenin are also expressed in skeletal muscle, andeach sequentially and synergistically plays a crucial rolein muscle cell specification and differentiation during
development in vivo.44–46 These observations led to theidea that lineage-associated transcription factors that determine cellular identity during development can change
cell fate when ectopically expressed in certain heterologous cells. Following the discovery of MyoD, conversionof one cell type into another was demonstrated within hematopoietic cell lineages by overexpression of transcription factors, including Gata1 and C/EBPα, or deletion of
Pax5.13,14,47–53
Oct4, Sox2, Klf4, and c-Myc Reprogram Fibroblasts
into Pluripotent Stem Cells
In 2006, Takahashi and Yamanaka achieved a breakthrough by demonstrating that the overexpression of four
embryonic stem cell (ESC)-specific transcription factors,
Oct4, Sox2, Klf4, and c-Myc, can convert fibroblasts
into pluripotent stem cells, or iPSCs.54 Using retroviral
vectors, they expressed 24 candidate genes and selected
for reprogrammed cells by incorporating neomycin resistance and β-galactosidase reporter genes into Fbx15,
a gene specifically expressed in pluripotent stem cells.55
The combination of 24 factors activated Fbx15 and induced the formation of drug-resistant colonies with characteristic ESC morphology. Successive rounds of elimination of individual dispensable factors led to identificationof the minimally required core set of four genes: Oct4,
Sox2, Klf4, and c-Myc, now known as Yamanaka factors.
Following this finding, many laboratories reproduced
their results, improved iPSC generation techniques, anddemonstrated that murine iPSCs share all their featureswith naive mouse ESCs, including expression of pluripotency markers, reactivation of both X chromosomes, andthe ability to generate chimeric mice.56–63
iPSCs can be derived from a number of different species, including humans, rats, and rhesus monkeys, byexpression of the four Yamanaka factors.64–68 Similarly,
iPSCs have been derived from other somatic cell populations, such as keratinocytes, neural cells, stomach andliver cells, and melanocytes, as well as from terminallydifferentiated blood cells, demonstrating the universality of induced pluripotency by these factors.69–73 Importantly, reprogramming can be induced not only by Oct4,
Sox2, Klf4, and c-Myc but also by alternative combinations that employ Nanog, Lin28, ESRRB, NR5A2, andother genes that establish the core transcriptional circuitry of stem cells.65,74–76

Ieda M: Direct Reprogramming by Defined Factors

Reprogramming of Pancreatic Exocrine Cells into
Endocrine β cells in Vivo
The generation of iPSCs sparked a new idea: the conversion of mature cell types directly into another cell type
without passing through a stem cell state by combinationof several lineage-specific factors.31,33,35,77,78 Zhou et al.
reported that adenoviral gene transfer of a combinationof three transcription factors, Ngn3, Pdx1, and Mafa, canefficiently reprogram pancreatic exocrine cells into functional β cells in mice.35 The three pancreatic reprogramming factors, Ngn3, Pdx1, and Mafa, are known to beimportant in the embryonic development of pancreas andβ cells.79 The induced β cells were indistinguishable fromendogenous islet β cells in structure and they expressed
genes essential for β cell function. Importantly, induced
β cells can ameliorate hyperglycemia in type 1 diabetic
mice. Although the new β cells were not reorganized into
islet structures (and this may limit their effectiveness),
this study provides the first evidence of cellular reprogramming in vivo by defined factors.
Reprogramming of Mouse and Human Fibroblasts
into Neural Cells
It is conceivable that cell type conversion within thesame lineage might be easy, but conversion to other lineages may be challenging. Vierbuchen et al. reported
that neuronal lineage-specific transcription factors Ascl1,
Brn2, and Myt1l efficiently convert mouse dermal fibroblasts into functional neurons in vitro.33 These induced
neuronal (iN) cells expressed multiple neuron-specificproteins, generated action potentials, and formed functional synapses. The same group also reported that whencombined with the bHLH transcription factor NeuroD1,
these three factors could also convert human fibroblasts
into iN cells.80 Human iN cells also showed typical neuronal morphologies and expressed multiple neuronalmarkers, even after downregulation of the exogenoustranscription factors. Subsequently, it was reported that a
combination of other transcription factors, Ascl1, Nurr1,and Lmx1a, was able to generate functional dopaminergic neurons from mouse and human fibroblasts without
reverting to a progenitor cell stage.81 This study also
showed that dopaminergic neuronal cells can be generated from cells from patients with Parkinson’s disease by
the same factors. These studies first revealed that lineage
conversions are not restricted to within the same lineage
or germ layer, since fibroblasts are mesodermal in origin
and neurons are derived from ectoderm. Recently, Wernig’s group also reported that terminally differentiatedhepatocytes, derived from endoderm, can be converted into iN cells by overexpression of Ascl1, Brn2, andMyt1l.82

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Fig. 2 Screening for cardiac reprogramming factors by fluorescence-activated cell sorting (FACS).
FACS analyses for α-MHC-GFP+ cell (cardiac reporter) induction from fibroblasts. Transduction of 14 factors into fibroblasts induced
1.8% of GFP+ cells, and serial removal revealed that the combination of Gata4, Mef2c, and Tbx5 induced 17% of GFP+ cells 1 week
after transduction.34

Fig. 3 Induced cardiomyocytes expressed cardiac genes in vitro.
Immunofluorescent staining for α-MHC-GFP, α-actinin, and DAPI (nuclei staining) in the Gata4, Mef2c, and Tbx5-transduced fibroblast cells. Induced cardiomyocytes expressed cardiac genes and exhibited sarcomeric organization.34 Bar 100μm.

Direct Reprogramming into Cardiomyocytes by
Gata4, Mef2c, and Tbx5
Although embryonic mesoderm can be induced to differentiate into cardiomyocytes, no master regulator of
cardiac differentiation has been identified, despite muchresearch inspired by the discovery of MyoD.12,83,84 We
hypothesized that no single gene can directly convert
fibroblasts into cardiomyocytes, but that a combinationof key developmental cardiac genes might achieve direct conversion. We selected 14 genes as candidates for
cardiac reprogramming; these candidate genes are specifically expressed in embryonic cardiomyocytes andare critical for cardiac cell fate specification, as demonstrated by knockout studies in mice.85 Cardiac fibroblasts
were isolated from transgenic mice expressing enhanced
green fluorescent protein (EGFP) under a cardiac-specific alpha myosin heavy chain (αMHC) promoter, andfibroblast cells that did not express EGFP were used forscreening.86,87 Transduction of all 14 factors into fibroblasts induced 1.8% of GFP+ cells, and serial reductionof individual factors demonstrated that a combination of
three factors, Gata4, Mef2c, and Tbx5, were sufficientfor GFP+ cell induction (around 15%, Fig. 2). We designated these GFP+ cardiomyocyte-like cells induced cardiomyocytes (iCMs).34 The three cardiac reprogramming
factors, Gata4, Mef2c, and Tbx5, are core cardiac transcription factors in early heart development88–90 and are
known to interact with one another, to coactivate cardiac
gene expression, and to promote cardiomyocyte differentiation.91–93
We determined the molecular features of iCMs by genetic and epigenetic analyses and by cell fate mapping
studies. The iCMs expressed several cardiac-specificgenes and had sarcomeric structures (Fig. 3). Microarray
analyses established that the global gene expression profile of iCMs is similar to that of neonatal cardiomyocytes,
but different from that of the original fibroblasts. For
some cardiac gene promoters, the histone modifications
and DNA methylation patterns of iCMs were also similar
to cardiomyocytes.94 A subset of iCMs exhibited intracellular Ca2+ transients and contracted spontaneously after

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Ieda M: Direct Reprogramming by Defined Factors

Fig. 4 Direct cardiac reprogramming in infarcted mouse hearts by Gata4, Mef2c, and Tbx5.
Gata4, Mef2c, Tbx5, and GFP were directly injected into the mouse hearts after myocardial infarction. Gata4, Mef2c, Tbx5, and GFPtransduced fibroblasts expressed cardiac gene α-actinin and exhibited sarcomeric structures.102 Bars 50μm.

4 weeks of culture. Lineage-mapping experiments with
Mesp1-Cre/R26R-YFP and Isl1-Cre/R26R-YFP reporter
mice suggested that the fibroblasts were directly reprogrammed to the differentiated cardiomyocyte fate without
reverting to a cardiac mesoderm/progenitor stage.84,95–97
This study was the first to represent the global gene expression profiles and epigenetic status of directly induced
cells and the first to demonstrate cell fate conversion
without the cells reverting to stem/progenitor states using reporter mice. Following our report, several othergroups also demonstrated cardiac reprogramming fromfibroblasts using the same factors and microRNAs.98–100
In 2012, we and two other groups reported that genetransfer of Gata4, Mef2c, and Tbx5 with or without Hand2
can convert resident cardiac fibroblasts into cardiomyocyte-like cells in infarcted mouse hearts (Fig. 4).99,101,102
We found that expression of Gata4, Mef2c, and Tbx5 usinga polycistronic vector enhanced cardiac maturation in vivo
compared with injecting the three vectors independently.102 Srivastava and colleagues and Olson and colleagues
demonstrated not only in vivo cardiac reprogramming but
also improvement of heart function after myocardial infarction by gene transfer of cardiac reprogramming factors.99,101 These results are striking and may provide a
potential new strategy for regenerative medicine. It will
be important to understand the molecular mechanisms ofcardiac reprogramming utilizing in vitro culture models toenhance cardiac induction efficiency in vivo.

Induction of Hepatocyte-like Cells by Defined
Factors
Overexpression of lineage-specific transcription factors
can directly convert terminally differentiated cells into
the cells of some other lineages; however, until recently it remained unclear whether transplantation of these
induced cells could improve the function of damaged
organs. In 2011, two groups demonstrated that induced
hepatocyte-like (iHep) cells that were directly generated
from fibroblasts could restore damaged hepatic tissues
after cell transplantation.31,32 Huang et al. demonstrated
the direct induction of proliferative functional iHep cells
from mouse tail-tip fibroblasts by transduction of Gata4,
Hnf1a, and Foxa3 and inactivation of p19Arf.31 The iHep
cells showed typical epithelial morphology, expressed hepatic genes, and acquired hepatocyte functions. Notably,transplanted iHep cells repopulated the damaged livers inmice and spared almost half of the recipients from deathby restoring liver functions. Sekiya et al. established that
Hnf4a plus Foxa1, Foxa2, or Foxa3 can convert mouseembryonic and adult fibroblasts into iHep cells.32 TheiriHep cells were also proliferative, functional, and rescued
damaged hepatic tissues after cell transplantation. Thesestudies provide novel strategies to generate functional
hepatocyte-like cells for the purpose of liver engineeringand regenerative medicine. However, iHep cell transplantation experiments showed that the rescue was partial andKeio J Med 2013; 62 (3): 74–82

that the cells were not identical to bona-fide hepatocytes.
Further modifications to the process of iHep cell generation might be needed to enable this approach to be appliedin clinical settings.
Conclusions
Progress in the field of cellular reprogramming has
been extensive and has the potential to change the face ofregenerative medicine in the future.26,34,103,104 Pluripotent
reprogramming with directed differentiation is theoretically able to produce all desired cell types, as has beenshown in ESC studies.105,106 A potential advantage of using iPSCs is that pluripotent stem cells have nearly unlimited capacity to proliferate in culture and may provide
a large number of desired terminally differentiated cells.
In contrast, directly induced cells appear to quickly exit
the cell cycle by lineage reprogramming, and the utilityof reprogrammed cells in vitro might be limited in someinstances. Direct induction of progenitor cells, as shownin neural stem/progenitor cell reprogramming, may be analternative approach to solving this issue.107,108 It is conceivable that lineage reprogramming techniques might beutilized in in vivo reprogramming, i.e., converting endogenous cells directly into desired cell types in situ by genetransfer of defined factors, as has been demonstrated inpancreatic β cell and cardiomyocyte induction.35,99,101,102
If this is possible, it has several advantages: first, theprocess is simple and quick; second, the avoidance of reprogramming to pluripotent cells before lineage differentiation might greatly lower the risk of contaminationof immature cells; and third, direct injection of defined
factors can avoid cell transplantation in which long-term
cell survival might be challenging in some organs, suchas the heart.109–112 Detailed analyses of the properties of
directly induced cells and understanding of the molecular mechanisms of lineage reprogramming will likely benecessary to advance this technology for future clinicalapplications.
Acknowledgments
The author is supported in part by research grants fromJST CREST, JSPS, The Mitsubishi Foundation, AstraZeneca, Takeda Science Foundation, Banyu Life Science,
The Uehara Memorial Foundation, Kimura Memorial
Heart Foundation, Japan Research Foundation for Clinical Pharmacology, Nateglinide Memorial Toyoshima
Research and Education Fund, and SENSHIN Medical
Research Foundation.

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References
1. Gurdon JB: From nuclear transfer to nuclear reprogramming: Thereversal of cell differentiation. Annu Rev Cell Dev Biol 2006; 22:
1–22. [Medline] [CrossRef]
2. Gurdon JB: The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 1962; 10: 622–640. [Medline]
3. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH: Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 810–813. [Medline] [CrossRef]
4. Hochedlinger K, Jaenisch R: Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 2002; 415:
1035–1038. [Medline] [CrossRef]
5. Eggan K, Baldwin K, Tackett M, Osborne J, Gogos J, Chess A,
Axel R, Jaenisch R: Mice cloned from olfactory sensory neurons.
Nature 2004; 428: 44–49. [Medline] [CrossRef]
6. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M,
Rideout WM, Yanagimachi R, Jaenisch R: Hybrid vigor, fetal
overgrowth, and viability of mice derived by nuclear cloning andtetraploid embryo complementation. Proc Natl Acad Sci USA
2001; 98: 6209–6214. [Medline] [CrossRef]
7. Inoue K, Wakao H, Ogonuki N, Miki H, Seino K, Nambu-Wakao
R, Noda S, Miyoshi H, Koseki H, Taniguchi M, Ogura A: Generation of cloned mice by direct nuclear transfer from natural killer T
cells. Curr Biol 2005; 15: 1114–1118. [Medline] [CrossRef]
8. Li J, Ishii T, Feinstein P, Mombaerts P: Odorant receptor genechoice is reset by nuclear transfer from mouse olfactory sensory
neurons. Nature 2004; 428: 393–399. [Medline] [CrossRef]
9. Gurdon JB, Byrne JA, Simonsson S: Nuclear reprogramming andstem cell creation. Proc Natl Acad Sci USA 2003; 100(Suppl 1):
11819–11822. [Medline] [CrossRef]
10. Gurdon JB, Byrne JA: The first half-century of nuclear transplantation. Proc Natl Acad Sci USA 2003; 100: 8048–8052. [Medline][CrossRef]
11. Tamashiro KL, Wakayama T, Akutsu H, Yamazaki Y, Lachey JL,
Wortman MD, Seeley RJ, D’Alessio DA, Woods SC, Yanagimachi R, Sakai RR: Cloned mice have an obese phenotype not transmitted to their offspring. Nat Med 2002; 8: 262–267. [Medline][CrossRef]
12. Davis RL, Weintraub H, Lassar AB: Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987; 51:
987–1000. [Medline] [CrossRef]
13. Xie H, Ye M, Feng R, Graf T: Stepwise reprogramming of B cells
into macrophages. Cell 2004; 117: 663–676. [Medline] [CrossRef]
14. Laiosa CV, Stadtfeld M, Xie H, de Andres-Aguayo L, Graf T:
Reprogramming of committed T cell progenitors to macrophages
and dendritic cells by C/EBP alpha and PU.1 transcription factors.
Immunity 2006; 25: 731–744. [Medline] [CrossRef]
15. Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer
H: MyoD converts primary dermal fibroblasts, chondroblasts,smooth muscle, and retinal pigmented epithelial cells into striatedmononucleated myoblasts and multinucleated myotubes. Proc
Natl Acad Sci USA 1990; 87: 7988–7992. [Medline] [CrossRef]
16. Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA,
Lassar AB, Miller AD: Activation of muscle-specific genes inpigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA 1989; 86: 5434–5438.
[Medline] [CrossRef]
17. Nerlov C, Graf T: PU.1 induces myeloid lineage commitment
in multipotent hematopoietic progenitors. Genes Dev 1998; 12:2403–2412. [Medline] [CrossRef]
18. Nerlov C, McNagny KM, Doderlein G, Kowenz-Leutz E, Graf
T: Distinct C/EBP functions are required for eosinophil lineage

80
commitment and maturation. Genes Dev 1998; 12: 2413–2423.
[Medline] [CrossRef]
19. Blau HM: How fixed is the differentiated state? Lessons from heterokaryons. Trends Genet 1989; 5: 268–272. [Medline] [CrossRef]
20. Wu SM, Hochedlinger K: Harnessing the potential of induced
pluripotent stem cells for regenerative medicine. Nat Cell Biol
2011; 13: 497–505. [Medline] [CrossRef]
21. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura
A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ: Diseasespecific induced pluripotent stem cells. Cell 2008; 134: 877–886.
[Medline] [CrossRef]
22. Amabile G, Meissner A: Induced pluripotent stem cells: Current
progress and potential for regenerative medicine. Trends Mol Med
2009; 15: 59–68. [Medline] [CrossRef]
23. Kamp TJ, Lyons GE: On the road to iPS cell cardiovascular applications. Circ Res 2009; 105: 617–619. [Medline] [CrossRef]
24. Carvajal-Vergara X, Sevilla A, D’Souza SL, Ang YS, Schaniel C,
Lee DF, Yang L, Kaplan AD, Adler ED, Rozov R, Ge Y, Cohen
N, Edelmann LJ, Chang B, Waghray A, Su J, Pardo S, Lichtenbelt
KD, Tartaglia M, Gelb BD, Lemischka IR: Patient-specific induced pluripotent stem-cell-derived models of leopard syndrome.
Nature 2010; 465: 808–812. [Medline] [CrossRef]
25. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L,
Dorn T, Goedel A, Hohnke C, Hofmann F, Seyfarth M, Sinnecker
D, Schomig A, Laugwitz KL: Patient-specific induced pluripotent
stem-cell models for long-QT syndrome. N Engl J Med 2010; 363:
1397–1409. [Medline] [CrossRef]
26. Yoshida Y, Yamanaka S: Recent stem cell advances: Induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation 2010; 122: 80–87. [Medline] [CrossRef]
27. Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman O, Gepstein A, Arbel G, Hammerman H, Boulos M, Gepstein L: Modelling the long QT syndrome with induced
pluripotent stem cells. Nature 2011; 471: 225–229. [Medline][CrossRef]
28. Nelson TJ, Terzic A: Induced pluripotent stem cells: An emerging
theranostics platform. Clin Pharmacol Ther 2011; 89: 648–650.
[Medline] [CrossRef]
29. Tiscornia G, Monserrat N, Belmonte JC: Modelling long QT syndrome with iPS cells: Be still, my beating heart. Circ Res 2011;108: 648–649. [Medline] [CrossRef]
30. Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer
J, Dolmetsch RE: Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 2011; 471:
230–234. [Medline] [CrossRef]
31. Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui
L: Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature. 2011; 475: 386–389.
32. Sekiya S, Suzuki A: Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 2011; 475: 390–393.
[Medline] [CrossRef]
33. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC,
Wernig M: Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2010; 463: 1035–1041. [Medline][CrossRef]
34. Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y,
Bruneau BG, Srivastava D: Direct reprogramming of fibroblasts
into functional cardiomyocytes by defined factors. Cell 2010; 142:
375–386. [Medline] [CrossRef]
35. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA: In vivo
reprogramming of adult pancreatic exocrine cells to beta-cells.
Nature 2008; 455: 627–632. [Medline] [CrossRef]
36. Berkes CA, Tapscott SJ: MyoD and the transcriptional control of
myogenesis. Semin Cell Dev Biol 2005; 16: 585–595. [Medline][CrossRef]

Ieda M: Direct Reprogramming by Defined Factors
37. Tapscott SJ: The circuitry of a master switch: MyoD and the regulation of skeletal muscle gene transcription. Development 2005;
132: 2685–2695. [Medline] [CrossRef]
38. Constantinides PG, Jones PA, Gevers W: Functional striated muscle cells from non-myoblast precursors following 5-azacytidine
treatment. Nature 1977; 267: 364–366. [Medline] [CrossRef]
39. Taylor SM, Jones PA: Multiple new phenotypes induced in 10T1/2
and 3T3 cells treated with 5-azacytidine. Cell 1979; 17: 771–779.
[Medline] [CrossRef]
40. Lassar AB, Paterson BM, Weintraub H: Transfection of a DNA
locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts. Cell 1986; 47: 649–656. [Medline] [CrossRef]
41. Pownall ME, Gustafsson MK, Emerson CP: Myogenic regulatory
factors and the specification of muscle progenitors in vertebrate
embryos. Annu Rev Cell Dev Biol 2002; 18: 747–783. [Medline][CrossRef]
42. Ledent V, Vervoort M: The basic helix-loop-helix protein family:
Comparative genomics and phylogenetic analysis. Genome Res
2001; 11: 754–770. [Medline] [CrossRef]
43. Ledent V, Paquet O, Vervoort M. Phylogenetic analysis of thehuman basic helix-loop-helix proteins. Genome Biol. 2002;3:RESEARCH0030.
44. Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J, Meilhac S, Montarras D, Rocancourt D, Relaix F: The formation ofskeletal muscle: From somite to limb. J Anat 2003; 202: 59–68.
[Medline] [CrossRef]
45. Molkentin JD, Olson EN: Defining the regulatory networks formuscle development. Curr Opin Genet Dev 1996; 6: 445–453.
[Medline] [CrossRef]
46. Perry RL, Parker MH, Rudnicki MA: Activated MEK1 binds thenuclear MyoD transcriptional complex to repress transactivation.
Mol Cell 2001; 8: 291–301. [Medline] [CrossRef]
47. Kulessa H, Frampton J, Graf T: GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev 1995; 9: 1250–1262. [Medline] [CrossRef]
48. Visvader JE, Elefanty AG, Strasser A, Adams JM: GATA-1 but
not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J 1992; 11: 4557–4564. [Medline]
49. Heyworth C, Pearson S, May G, Enver T: Transcription factormediated lineage switching reveals plasticity in primary committed progenitor cells. EMBO J 2002; 21: 3770–3781. [Medline][CrossRef]
50. Zhang P, Iwasaki-Arai J, Iwasaki H, Fenyus ML, Dayaram T,
Owens BM, Shigematsu H, Levantini E, Huettner CS, LekstromHimes JA, Akashi K, Tenen DG: Enhancement of hematopoietic
stem cell repopulating capacity and self-renewal in the absence ofthe transcription factor C/EBP alpha. Immunity 2004; 21: 853–
863. [Medline] [CrossRef]
51. Nutt SL, Heavey B, Rolink AG, Busslinger M: Commitment to
the B-lymphoid lineage depends on the transcription factor Pax5.
Nature 1999; 401: 556–562. [Medline] [CrossRef]
52. Rolink AG, Nutt SL, Melchers F, Busslinger M: Long-term in vivo
reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 1999; 401: 603–606. [Medline] [CrossRef]
53. Cobaleda C, Jochum W, Busslinger M: Conversion of mature B
cells into T cells by dedifferentiation to uncommitted progenitors.
Nature 2007; 449: 473–477. [Medline] [CrossRef]
54. Takahashi K, Yamanaka S: Induction of pluripotent stem cells
from mouse embryonic and adult fibroblast cultures by defined
factors. Cell 2006; 126: 663–676. [Medline] [CrossRef]
55. Tokuzawa Y, Kaiho E, Maruyama M, Takahashi K, Mitsui K,
Maeda M, Niwa H, Yamanaka S: Fbx15 is a novel target of Oct3/4
but is dispensable for embryonic stem cell self-renewal and mouse
development. Mol Cell Biol 2003; 23: 2699–2708. [Medline][CrossRef]
56. Boland MJ, Hazen JL, Nazor KL, Rodriguez AR, Gifford W,
Martin G, Kupriyanov S, Baldwin KK: Adult mice generated

Keio J Med 2013; 62 (3): 74–82
from induced pluripotent stem cells. Nature 2009; 461: 91–94.
[Medline] [CrossRef]
57. Kang L, Wang J, Zhang Y, Kou Z, Gao S: iPS cells can support
full-term development of tetraploid blastocyst-complemented embryos. Cell Stem Cell 2009; 5: 135–138. [Medline] [CrossRef]
58. Zhao XY, Li W, Lv Z, Liu L, Tong M, Hai T, Hao J, Guo CL,
Ma QW, Wang L, Zeng F, Zhou Q: iPS cells produce viable micethrough tetraploid complementation. Nature 2009; 461: 86–90.
[Medline] [CrossRef]
59. Okita K, Ichisaka T, Yamanaka S: Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448: 313–317.
[Medline] [CrossRef]
60. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K,
Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K: Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem
Cell 2007; 1: 55–70. [Medline] [CrossRef]
61. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R: In vitro reprogrammingof fibroblasts into a pluripotent ES-cell-like state. Nature 2007;
448: 318–324. [Medline] [CrossRef]
62. Stadtfeld M, Maherali N, Breault DT, Hochedlinger K: Defining
molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2008; 2: 230–240. [Medline][CrossRef]
63. Maherali N, Hochedlinger K: Guidelines and techniques for thegeneration of induced pluripotent stem cells. Cell Stem Cell 2008;
3: 595–605. [Medline] [CrossRef]
64. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda
K, Yamanaka S: Induction of pluripotent stem cells from adulthuman fibroblasts by defined factors. Cell 2007; 131: 861–872.
[Medline] [CrossRef]
65. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget
J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R,
Slukvin II, Thomson JA: Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318: 1917–1920.
[Medline] [CrossRef]
66. Park IH, Lerou PH, Zhao R, Huo H, Daley GQ: Generation ofhuman-induced pluripotent stem cells. Nat Protoc 2008; 3: 1180–
1186. [Medline] [CrossRef]
67. Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, Hao E, Hayek A, Deng
H, Ding S: Generation of rat and human induced pluripotent stem
cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 2009; 4: 16–19. [Medline] [CrossRef]
68. Liu H, Zhu F, Yong J, Zhang P, Hou P, Li H, Jiang W, Cai J, Liu M,
Cui K, Qu X, Xiang T, Lu D, Chi X, Gao G, Ji W, Ding M, Deng
H: Generation of induced pluripotent stem cells from adult rhesus
monkey fibroblasts. Cell Stem Cell 2008; 3: 587–590. [Medline][CrossRef]
69. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K,
Chiba T, Yamanaka S: Generation of pluripotent stem cells fromadult mouse liver and stomach cells. Science 2008; 321: 699–702.
[Medline] [CrossRef]
70. Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez
F, Vassena R, Bilic J, Pekarik V, Tiscornia G, Edel M, Boue S,
Izpisua Belmonte JC: Efficient and rapid generation of induced
pluripotent stem cells from human keratinocytes. Nat Biotechnol
2008; 26: 1276–1284. [Medline] [CrossRef]
71. Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, Nakata
H, Tohyama S, Hashimoto H, Kodaira M, Okada Y, Seimiya H,
Fusaki N, Hasegawa M, Fukuda K: Generation of induced pluripotent stem cells from human terminally differentiated circulating
T cells. Cell Stem Cell 2010; 7: 11–14. [Medline] [CrossRef]
72. Kim JB, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V,
Arauzo-Bravo MJ, Ruau D, Han DW, Zenke M, Scholer HR:
Pluripotent stem cells induced from adult neural stem cells by

81
reprogramming with two factors. Nature 2008; 454: 646–650.
[Medline] [CrossRef]
73. Hanna J, Markoulaki S, Schorderet P, Carey BW, Beard C, Wernig
M, Creyghton MP, Steine EJ, Cassady JP, Foreman R, Lengner
CJ, Dausman JA, Jaenisch R: Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell
2008; 133: 250–264. [Medline] [CrossRef]
74. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S: The homeoprotein Nanog is required for maintenance of pluripotency in mouse
epiblast and ES cells. Cell 2003; 113: 631–642. [Medline] [CrossRef]
75. Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh
KM, Carter AC, Di Giorgio FP, Koszka K, Huangfu D, Akutsu
H, Liu DR, Rubin LL, Eggan K: A small-molecule inhibitor of
tgf-beta signaling replaces sox2 in reprogramming by inducing
nanog. Cell Stem Cell 2009; 5: 491–503. [Medline] [CrossRef]
76. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP,
Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK,Melton DA, Jaenisch R, Young RA: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122:
947–956. [Medline] [CrossRef]
77. Szabo E, Rampalli S, Risueno RM, Schnerch A, Mitchell R,
Fiebig-Comyn A, Levadoux-Martin M, Bhatia M: Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 2010; 468: 521–526. [Medline] [CrossRef]
78. Kanazawa H, Ieda M, Kimura K, Arai T, Kawaguchi-Manabe H,
Matsuhashi T, Endo J, Sano M, Kawakami T, Kimura T, Monkawa T, Hayashi M, Iwanami A, Okano H, Okada Y, Ishibashi-Ueda
H, Ogawa S, Fukuda K: Heart failure causes cholinergic transdifferentiation of cardiac sympathetic nerves via gp130-signaling
cytokines in rodents. J Clin Invest 2010; 120: 408–421. [Medline][CrossRef]
79. Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton
DA: A multipotent progenitor domain guides pancreatic organogenesis. Dev Cell 2007; 13: 103–114. [Medline] [CrossRef]
80. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang
TQ, Citri A, Sebastiano V, Marro S, Sudhof TC, Wernig M: Induction of human neuronal cells by defined transcription factors.
Nature 2011; 476: 220–223. [Medline]
81. Caiazzo M, Dell’Anno MT, Dvoretskova E, Lazarevic D, Taverna
S, Leo D, Sotnikova TD, Menegon A, Roncaglia P, Colciago G,
Russo G, Carninci P, Pezzoli G, Gainetdinov RR, Gustincich S,
Dityatev A, Broccoli V: Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 2011;
476: 224–227. [Medline] [CrossRef]
82. Marro S, Pang ZP, Yang N, Tsai MC, Qu K, Chang HY, Sudhof
TC, Wernig M: Direct lineage conversion of terminally differentiated hepatocytes to functional neurons. Cell Stem Cell 2011; 9:
374–382. [Medline] [CrossRef]
83. Takeuchi JK, Bruneau BG: Directed transdifferentiation ofmouse mesoderm to heart tissue by defined factors. Nature 2009;459: 708–711. [Medline] [CrossRef]
84. Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M,
Kyba M, Blanpain C: Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell 2008;
3: 69–84. [Medline] [CrossRef]
85. Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM,
Srivastava D: Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell 2009; 16: 233–
244. [Medline] [CrossRef]
86. Ieda M, Kanazawa H, Kimura K, Hattori F, Ieda Y, Taniguchi M,
Lee JK, Matsumura K, Tomita Y, Miyoshi S, Shimoda K, Makino
S, Sano M, Kodama I, Ogawa S, Fukuda K: Sema3a maintains
normal heart rhythm through sympathetic innervation patterning.
Nat Med 2007; 13: 604–612. [Medline] [CrossRef]

82
87. Gulick J, Subramaniam A, Neumann J, Robbins J: Isolation andcharacterization of the mouse cardiac myosin heavy chain genes.
J Biol Chem 1991; 266: 9180–9185. [Medline]
88. Zhao R, Watt AJ, Battle MA, Li J, Bondow BJ, Duncan SA: Lossof both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice. Dev Biol 2008; 317: 614–619.
[Medline] [CrossRef]
89. Srivastava D: Making or breaking the heart: From lineage determination to morphogenesis. Cell 2006; 126: 1037–1048. [Medline] [CrossRef]
90. Olson EN: Gene regulatory networks in the evolution and development of the heart. Science 2006; 313: 1922–1927. [Medline][CrossRef]
91. Ghosh TK, Song FF, Packham EA, Buxton S, Robinson TE,
Ronksley J, Self T, Bonser AJ, Brook JD: Physical interaction between TBX5 and MEF2C is required for early heart development.
Mol Cell Biol 2009; 29: 2205–2218. [Medline] [CrossRef]
92. Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, Butler CA, Rothrock CR, Eapen RS, Hirayama-Yamada K, Joo K,
Matsuoka R, Cohen JC, Srivastava D: GATA4 mutations cause
human congenital heart defects and reveal an interaction with
TBX5. Nature 2003; 424: 443–447. [Medline] [CrossRef]
93. Morin S, Charron F, Robitaille L, Nemer M: GATA-dependent
recruitment of MEF2 proteins to target promoters. EMBO J 2000;
19: 2046–2055. [Medline] [CrossRef]
94. Li B, Carey M, Workman JL: The role of chromatin during transcription. Cell 2007; 128: 707–719. [Medline] [CrossRef]
95. Wu SM, Fujiwara Y, Cibulsky SM, Clapham DE, Lien CL, Schultheiss TM, Orkin SH: Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.
Cell 2006; 127: 1137–1150. [Medline] [CrossRef]
96. Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S,
Lin LZ, Cai CL, Lu MM, Reth M, Platoshyn O, Yuan JX, Evans S,
Chien KR: Postnatal isl1+ cardioblasts enter fully differentiatedcardiomyocyte lineages. Nature 2005; 433: 647–653. [Medline][CrossRef]
97. Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki J,
Inoue T: MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 1999;
126: 3437–3447. [Medline]
98. Jayawardena TM, Egemnazarov B, Finch EA, Zhang L, Payne JA,
Pandya K, Zhang Z, Rosenberg P, Mirotsou M, Dzau VJ: MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac
fibroblasts to cardiomyocytes. Circ Res 2012; 110: 1465–1473.
[Medline] [CrossRef]
99. Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A,
Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R,
Olson EN: Heart repair by reprogramming non-myocytes withcardiac transcription factors. Nature 2012; 485: 599–604. [Medline] [CrossRef]
100. Protze S, Khattak S, Poulet C, Lindemann D, Tanaka EM, Ravens
U: A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells. J Mol Cell
Cardiol 2012; 53: 323–332. [Medline] [CrossRef]

Ieda M: Direct Reprogramming by Defined Factors
101. Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D: In vivo reprogramming of murine
cardiac fibroblasts into induced cardiomyocytes. Nature 2012;
485: 593–598. [Medline] [CrossRef]
102. Inagawa K, Miyamoto K, Yamakawa H, Muraoka N, Sadahiro T,
Umei T, Wada R, Katsumata Y, Kaneda R, Nakade K, Kurihara C,
Obata Y, Miyake K, Fukuda K, Ieda M: Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c,
and Tbx5. Circ Res 2012; 111: 1147–1156. [Medline] [CrossRef]
103. Efe JA, Hilcove S, Kim J, Zhou H, Ouyang K, Wang G, Chen
J, Ding S: Conversion of mouse fibroblasts into cardiomyocytes
using a direct reprogramming strategy. Nat Cell Biol 2011; 13:
215–222. [Medline] [CrossRef]
104. Hochedlinger K: From MYOD1 to iPS cells. Nat Rev Mol Cell
Biol 2010; 11: 817. [Medline] [CrossRef]
105. Yamashita JK: ES and iPS cell research for cardiovascular regeneration. Exp Cell Res 2010; 316: 2555–2559. [Medline] [CrossRef]
106. Bu L, Jiang X, Martin-Puig S, Caron L, Zhu S, Shao Y, Roberts
DJ, Huang PL, Domian IJ, Chien KR: Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 2009; 460: 113–117. [Medline] [CrossRef]
107. Matsui T, Takano M, Yoshida K, Ono S, Fujisaki C, Matsuzaki
Y, Toyama Y, Nakamura M, Okano H, Akamatsu W: Neural stem
cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency. Stem Cells 2012; 30:
1109–1119. [Medline] [CrossRef]
108. Han DW, Tapia N, Hermann A, Hemmer K, Hoing S, ArauzoBravo MJ, Zaehres H, Wu G, Frank S, Moritz S, Greber B, Yang
JH, Lee HT, Schwamborn JC, Storch A, Scholer HR: Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 2012; 10: 465–472. [Medline] [CrossRef]
109. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate
JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S,
O’Sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S,
Yuan C, Gold J, Murry CE: Cardiomyocytes derived from humanembryonic stem cells in pro-survival factors enhance function of
infarcted rat hearts. Nat Biotechnol 2007; 25: 1015–1024. [Medline] [CrossRef]
110. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE: Cardiomyocyte grafting for cardiac repair: Graft cell death and antideath strategies. J Mol Cell Cardiol 2001; 33: 907–921. [Medline][CrossRef]
111. Caspi O, Huber I, Kehat I, Habib M, Arbel G, Gepstein A, Yankelson L, Aronson D, Beyar R, Gepstein L: Transplantation ofhuman embryonic stem cell-derived cardiomyocytes improves
myocardial performance in infarcted rat hearts. J Am Coll Cardiol
2007; 50: 1884–1893. [Medline] [CrossRef]
112. Blum B, Benvenisty N: The tumorigenicity of human embryonic stem cells. Adv Cancer Res 2008; 100: 133–158. [Medline][CrossRef]