Abstract
Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms.
Introduction
Stem cell technology has systematically advanced from purely theoretical to applied biomedical science with significant progress towards practical applications anticipated in the upcoming decade. Naturally derived stem cells, including embryonic, umbilical cord blood and adult stem cells, contribute to organ development in utero and tissue renewal throughout adulthood.Citation1–Citation11 Beyond natural sources that are limited by stem cell availability, immune intolerance, and lineage specification, the latest platform of recently developed bioengineered stem cells is rapidly enriching the armamentarium of regenerative medicine. This overview highlights state of the art bioengineered stem cell technology, referred to as induced pluripotent stem cells (iPS), and underscores the emerging advances in iPS-based therapeutic applications.
Advances in bioengineered stem cell technology
By exploiting the ability to reprogram ordinary self-derived tissue sources, the innovation of bioengineered stem cells offers an unlimited supply of progenitor cells for virtually all cell types and tissues of the adult body (). Through control of the epigenetic environment within common cell types, nuclear reprogramming reverses cell fate, converting mature cells back to the embryonic ground state.Citation12 Advancement of nuclear reprogramming has materialized through the pioneering work of somatic cell nuclear transfer techniques that established the conserved ability of transacting environment, within the mammalian oocytes, to reprogram somatic cell nuclei to an undifferentiated state.Citation13,Citation14 Somatic cell nuclear transfer (SCNT), defined as therapeutic cloning, transplants the nuclear content of a somatic cell into an enucleated donor egg to engineer a blastocyst genetically identical to the parental source and derive pluripotent embryonic-like stem cells (). In this way, SCNT has resulted in cloned embryonic stem cells from mammalian somatic cell biopsies.Citation15–Citation18 However, SCNT still requires an embryonic host environment to direct the reprogramming of somatic cells. The search for factors sufficient to induce complete nuclear reprogramming has provided the more recent breakthroughs for successful embryo-independant iPS technologies.
Science of nuclear reprogramming
Nuclear reprogramming of ordinary somatic tissue through the ectopic introduction of stemness factors is a streamlined approach to coerce an embryonic stem cell-like phenotypeCitation19–Citation22 The transcription factors sets, Oct4, Sox2, c-Myc, and Klf4 or alternatively Oct4, Sox2, Nanog, and Lin28,Citation23 are sufficient to reprogram somatic cells through a sequential reversal into a pluripotent phenotype (). The process of reprogramming requires controlled, stoichiometric expression of transgenes for a transient period of time.Citation24–Citation30 Multiple sources of tissue such as ordinary fibroblasts,Citation31 keratinocytes,Citation32 hematopoietic lineages,Citation33 or adipose tissueCitation34 have been successfully reprogrammed. Ectopic stemness factors are sufficient to induce telomere elongation,Citation35 histone modifications,Citation36 secondary gene expression profiles,Citation37 and cellular metamorphosis that collectively re-establish a self-stabilizing phenotype.Citation38 Reprogramming occurs typically within weeks, following exposure to trans-acting factors that can be delivered to the nucleus either by plasmids, viruses, or bioengineered proteins. Thus, transgene expression initiates a sequence of reprogramming events that eventually transforms a small fraction of cells (<0.5%) to acquire an imposed pluripotent state characterized by a stable epigenetic environment indistinguishable from the blastocyst-derived natural stem cell milieu. The converted pluripotent ground state results in the maintenance of the unique developmental potential with the ability to differentiate into all germ layers (). Thereby, iPS cells should largely eliminate the concern of stem cell shortage, immune rejection of non-autologous sources, and inadequate capacity for lineage specification.Citation39–Citation41 Moreover, iPS-based technology will facilitate the production of patient-specific cell line panels that closely reflect the genetic diversity of a population enabling the discovery, development and validation of diagnostics together with therapeutics tailored for each individual.Citation42
Induced pluripotent stem cell platforms bypass the need for embryo extraction to generate genuine pluripotent stem cells from self-derived autologous sources. In the mouse, bioengineering strategies have yielded iPS cells sufficient for complete de novo embryogenesis as the highest evidence of pluripotent stringencyCitation43,Citation44 In humans, Citation23,Citation26,Citation28 iPS cells have ensured comprehensive multi-lineage tissue differentiation by demonstrating the ability to give rise to all three germ layers in teratoma formation (). Self-derived iPS cells are recognized within the transplanted hosts as native tissue due to their autologous status and thus require protection from dysregulated growth in the absence of a defensive immune system. Optimization of bioengineered stem cells will likely produce specialized properties that improve stress tolerance, streamline differentiation capacity, and increase engraftment/survival to improve regenerative potential.
Theoretical models of reprogramming
There are two proposed models that describe the mechanism of the reprogramming process: an “elite model” in which a small number of partially preprogrammed progenitor cells are capable of responding to transgenic stemness factors or alternatively, a “stochastic model” in which virtually any ordinary cell type can be reprogrammed with the proper combination of conditions depending on both the nature and environment of the target cell.Citation22 Both being plausible and supported by documented observations, the “stochastic model” has been further strengthened by evidence presented that parental sources not contaminated by progenitor cells, such as mature lymphoid cell types validated according to V(D)J recombination, are capable of dedifferentiating into stable pluripotent stem cells.Citation45,Citation46 The data supports the model that cell fate is indeed fully reversible even from mature tissue sources upon exposure to the proper intracellular and extracellular environments.
Original iPS technology
Gene delivery to somatic cells through retroviral or lentiviral vectors () provided the initial strategy for ectopic expression, and establishes the technological basis of nuclear reprogramming.Citation19,Citation23,Citation24,Citation32,Citation47–Citation52 The potential for oncogenesis due to insertional mutagenesis that is inherent to stable genomic integration has been identified as a limitation. However, it is important to recognize that distinct advantages of the retroviral-based vector systems enabled critical insight into the fundamental mechanisms of nuclear reprogramming. Retroviral and lentiviral systems have built-in sequences within the vector systems that silence the transcriptional machinery upon successful pluripotent induction. Therefore, persistent exposure to ectopic gene expression through these vectors is inhibited at the time of pluripotency re-induction, enabling an essential observation, in that successful self-maintenance of the pluripotent ground state is possible without long-term transgene expression. Thereby, next generation vectors and gene delivery systems for transient expression of stemness related genes have been designed to improve safety and ultimately efficacy of nuclear reprogramming (). The feasibility study of genomic modification free strategies was achieved by nonintegrating viral vector systems, such as adenovirus,Citation53 and confirmed by repeated exposure to extra-chromosomal plasmid-based transgenes.Citation54 Importantly, these reports established the evidence that expression of stemness related factors was required for only a limited timeframe – defined by the ability of progeny to develop autonomous self-renewal, establishing nuclear reprogramming as a bioengineered process that resets a sustainable pluripotent cell fate independent of permanent genomic modifications. The inefficiency of nonintegrated technologies has, however, hindered broader applicability and provoked the search for more efficient methodologies.
Optimization of iPS technology
The innovative advances that propel iPS-based products towards clinical applications is dependent on genome modification-free approaches equipped for high efficiency delivery of transgenes and subsequent nuclear reprogramming.Citation55,Citation56 One of these emerging approaches has utilized short sequences of mobile genetic elements that can integrate transgenes into host cell genomes and yet provide a genetic tag to “cut and paste” flanked genomic DNA sequences.Citation57 The prototypic piggyBac (PB) system couples enzymatic cleavage with sequence specific recognition using a transposon/transposase interaction to ensure high efficiency removal of flanked DNA without any footprint. Importantly, this technology achieves a traceless transgenic approach in which nonnative genomic sequences, that are transiently required for nuclear reprogramming, can be removed upon induction of pluripotency. Using the PB transposition system with randomly integrated stemness-related transgenes, recent studies have demonstrated that disposal of ectopic genes could be efficiently regulated upon induction of self-maintaining pluripotency according to expression of the transposase enzyme without infringement on genomic stability.Citation56 This state of the art system allows safe integration and removal of ectopic transgenes, and advances the technology by improving the efficiency of iPS production utilizing a minimally invasive strategy. Furthermore, the security of genetically unmodified interventions can be achieved with non-integrating episomal vectors.Citation58 Collectively these recent strategies () accelerate translation towards clinical applicability with progenitor cells that have acquired the capacity of pluripotency without compromise to the genomic stability of the parental cell source.
Additionally, advances in bioengineering technology have produced high stringency iPS cells with only proteins in the absence of any genetic or DNA material.Citation59,Citation60 The protein only approach has successfully induced reprogramming with either whole cell extract enriched in four stemness factors used in combination with pharmacological induction of cell permeability or with stemness factors modified by a cell permeating poly-arginine tag.Citation59 Although the reprogramming efficiency is reduced compared to original genetic based methodologies, there are emerging strategies that complement the influence of stemness factors exposure within somatic cells, namely, small molecules targeting histone modifications have improved the overall reprogramming efficienciesCitation61 along with the latest discovery that the tumor suppressor gene p53 is a roadblock that spontaneously inhibits the reprogramming process.Citation62–Citation66 Thereby, transient knockdown of p53 according to small interfering RNA (siRNA) strategies targeting the breakdown of mRNA or overexpression of MDM-2, to increase p53 protein degradation, have proven to successfully increase the overall efficiency one to two orders of magnitude with up to 20% of selected cells undergoing bona fide reprogramming.Citation62–Citation66 Together, these rapid advancements in nuclear reprogramming have brought bioengineered pluripotent stem cell platforms closer to the milestones required for possible clinical applications.
Therapeutic applications for bioengineered stem cells
Regenerative medicine aims to provide novel solutions for patients suffering from a spectrum of chronic degenerative diseases often triggered by a specific underlying genetic predisposition. Due to progressive cellular destruction and loss of functional tissues, degenerative diseases are largely responsible for chronic disabilities suffered throughout a lifespan. This creates an ever growing need for new therapies to apply a curative paradigm to repair underlying pathophysiology with corrupted cellular architecture. The emergence of regenerative medicine platforms expands the therapeutic options by establishing new approaches to address disease management needs unmet by traditional palliative strategies. In this way, stem cell-based regenerative medicine is expected to drive the evolution of medical sciences from palliation, which mitigates symptoms, to curative therapy aimed at treating the root cause of degenerative and genetic diseases.Citation67,Citation68 Uniquely, stem cell populations demonstrate an aptitude to differentiate into lineage specific progenitors, and form new tissue.Citation69 Cell-based strategies that promote, augment, and reestablish repair are at the core of translating the science of stem cell biology into the practice of regenerative medicine.Citation70–Citation76
The major impediments from the discovery to the application of stem cell technologies, have been based on two formidable challenges. First, immune intolerance between stem cells and the host environment inherent to allogeneic stem cell sources; and second the inability to secure definitive tissue specific differentiation from stem cells for in situ repair. With the advent of iPS technology, these limitations are addressed by the pluripotent potential of bioengineered stem cells that are derived from autologous sources (). Thereby, the ability to reproducibly generate unlimited self-derived progenitors that avoid immune intolerance is a unique feature. Furthermore, all lineages of the adult body have become viable targets for replacement, utilizing iPS-based technology. Finally, iPS cells enable the ability to genetically repair sequence defects through homologous recombination, which then produces healthy stem cells devoid of the original disease causing genetic impairment. These defining characteristics of iPS cells thus offer a new trajectory for advancing regenerative medicine; yet they also present new challenges that have only partially been addressed with previous natural stem cell sources. The unlimited differentiation potential of iPS is similar to embryonic stem cells, and thus the risk of dysregulated growth and teratoma formation requires stringent safeguards. Ensuring proper differentiation of pluripotent stem cells has been addressed in embryonic stem cells by either growth factor guidance of lineage-specific differentiation or physical selection of established lineage-specific progenitors.Citation77,Citation78 However, beyond the common challenges of natural pluripotent stem cells, iPS cells may also contain genetic modification as a consequence of the strategy used for reprogramming or spontaneously acquired cytogenetic abnormalities due to extensive in vitro manipulation. The long-term implications of nuclear reprogramming have yet to be determined as this technology is in the early stages of development.
The broad scope of therapeutic potential for iPS has been demonstrated in proof of principle studies for 4 diverse conditions to date (), namely sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease.Citation79–Citation82 Efficient in vitro differentiation of the tissue-specific lineage was the first required milestone for each of these conditions. The validated iPS clones were demonstrated to produce hematopoietic lineages, neural precursor cells giving rise to neuronal and glial cell types, and functional cardiac tissue prior to therapeutic application. Upon transplantation of iPS progeny into target organs ranging from fetal brain to adult post-ischemic heart tissue, progenitor cells migrated into microenvironments and differentiated in situ into target tissues. Collectively, these experimental models of diseases provide a proof-of-principle for therapeutic benefit of iPS-based strategies.
Sickle cell anemia
Sickle cell anemia is an inherited disease that affects millions of individuals worldwide, often producing life threatening symptoms. The disease is based on inadequate red blood cell production in the bone marrow, which is limited to replenishing circulating blood cells every 120 days. However, sickle cell anemia causes fragile red blood cells that are unable to survive more than 20 days. Thus, the bone marrow is unable to keep up with the high-demand of continuous cell production that ultimately results in low oxygen carrying capacity, accumulation of waste products, and risk of hypoxia throughout the body. This common disease has no known cure and patients are managed for symptomatic relief. A humanized mouse model for sickle cell anemia was used to determine the repair potential of progenitor cells derived from autologous iPS cells.Citation79 Diseased mice were the source of starting tissues that were reprogrammed into iPS clones. These acquired stem cells then underwent gene correction of the sickle hemoglobin gene through gene-specific targeting. Upon transplantation with hematopoietic progenitors obtained in vitro, the pathognomonic features of the disease were averted. Specifically, kidney defects due to red blood cell destruction in renal tubules with reduction in renal blood flow, and the systemic deterioration as demonstrated by decreased body weight and increased respiratory rate were rescued upon iPS therapy.Citation79 Thus, the first therapeutic application of iPS technology illustrated the advantages of both regeneration of a degenerative disease as well as gene-specific correction of an inheritable defect.
Parkinson’s disease
Parkinson’s disease is a debilitating neurodegenerative disease that affects 1–2 individuals per 1,000 due to the loss of dopaminergic neurons in a specialized region of the substantia nigra, that projects from the basal ganglia to the striatum, and is responsible for regulation of body movement. Muscle rigidity, resting tremor, and a generalized slowing of physical movements characterize the disease that is chronic and progressive in most patients. Standard of care is guided by the principle goal of increasing the dopamine concentration within the brain through daily titration of medications. Utilizing iPS technology in an animal model of Parkinson’s disease, the goal was to determine therapeutic efficacy with bioengineered stem cells that acquired the ability to differentiate into dopamine-producing progeny.Citation80 Electrophysiological recordings and morphological characterization demonstrated successful engraftment of transplanted iPS-derived neurons with functional neuronal activity. Notably, iPS progeny demonstrated characteristics of midbrain neurons with dopamine production.Citation80 The presence of these de novo cells enabled the improvement of symptoms in a model of Parkinson’s disease, with little risk of tumor formation from the engrafted cells. These results established the therapeutic reparative potential of nuclear reprogramming for neurodegenerative diseases.
Hemophilia A
Hemophilia A is a common inheritable disease that affects the levels of a single protein required for normal clotting of the blood. The disease that affects 1 in 5,000 males is caused by mutations within the Factor VIII (FVIII) gene and leads to decreased protein levels and subsequently life-threatening bleeding. Gene therapy attempts have failed for multiple reasons not limited to immune rejection of the recombinant protein. Utilizing validated iPS cells derived from a mouse model, endothelial progenitor cells were produced through spontaneous differentiation according to a standardized method. The resulting progeny expressed cell-specific markers, including FVIII protein, prior to transplantation into the liver of immunodeficient hemophilia A mice. Chimeric cohorts circumvented life-threatening bleeding, in dramatic contrast to vulnerable diseased cohorts.Citation81 As predicted, increased FVIII protein levels were associated with iPS treatment and beneficial outcome. Thereby, this study further established the evidence for successful iPS based therapy in the context of a genetic disorder, demonstrating the feasibility of achieving a targeted outcome.
Ischemic heart disease
Ischemic heart disease results when the arteries that carry oxygenated blood to the heart muscle are restricted or blocked. The heart muscle and vasculature are then unable to sufficiently rejuvenate themselves, which collectively culminates into massive tissue destruction with loss of billions of cells in the setting of acute infarction that eventually leads to decreased functional performance of the heart. The subsequent lack of blood flow then directly affects the rest of the body and precipitates overt heart failure symptoms. This disease is estimated to affect 1 in 100 people and limits functional activity in more than 14 million individuals in the United States with increasing prevalence throughout the world. Nuclear reprogramming provides an emerging strategy to produce de novo cardiac tissues from patient-specific somatic sources. In proof of principle studies, fibroblasts were transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC and converted into pluripotent stem cells that acquired the ability to contribute to normal embryonic heart development.Citation82 Upon intramyocardial delivery into adult infarcted hearts, cardiogenic iPS progeny properly engrafted without disrupting host tissues.Citation82 Notably, the parental fibroblasts that had not undergone the reprogramming process lacked the ability to engraft and when transplanted into post-ischemic hearts were associated with progressive heart dilation, with worsening heart function over the 4-week follow-up period.Citation82 Importantly, iPS-based transplantation restored post-ischemic cardiac performance with evidence of increased left ventricular thickness, and improved electrical stability following in situ regeneration of cardiac, smooth muscle, and endothelial tissue throughout the 4-week follow-up period. Furthermore, cardiogenic iPS clones are able to contribute to healthy adult chimeric animals with normal cardiac function.Citation83 Thereby, nonreparative fibroblasts reprogrammed by human stemness factors have demonstrated the potential for in situ regeneration of heart smooth muscle tissue following injury (such as acute myocardial infarction) to establish iPS-derived progeny in the treatment of heart disease.
Clinical perspective
Regenerative medicine, built on emerging discoveries of stem cell biology,Citation57,Citation67,Citation70 has begun to define a new perspective of future clinical practice. Regenerative medicine and stem cell biology integrate multiple disciplines of medicine and surgery to establish a universal paradigm of curative goals based on scientific discovery and clinical translation. Building on the foundation of transplant medicine with further advances in delivery systems,Citation84 regenerative medicine will continue to expand and implement new technologies to treat diseases at earlier stages. Individualized treatment applications for regenerative medicine will first require quantification of the inherent reparative potential of the patient to determine the scope of benefit from a targeted stem cell therapy.
Bioengineered nuclear reprogramming offers a revolutionary strategy for embryo-independent derivation of autologous pluripotent stem cells from an ordinary adult source which remain incompletely validated when compared to the gold standard embryonic stem cell counterparts.Citation85 Applying this technology, iPS progeny have to date attained similar differentiation capacity previously demonstrated only by natural embryonic stem cells, which now present new challenges to ensure reproducibility of safe and effective reprogramming throughout the bioengineering process. Furthermore, adult somatic cells from multiple tissue sources may provide variations in the overall efficiencies for iPS bioengineering as indicated recently by the degree of heterogeneity even within individual primary fibroblast cultures.Citation86 Therefore, continued mapping of the innate characteristics of the starting somatic source and the reprogrammed iPS-derived progeny should pave the way to optimize outcome. Thus, the uniqueness of iPS cells has established a new paradigm for personalized therapeutics across a diverse spectrum of chronic degenerative diseases, including incurable genetic disorders (). With “on demand” tissue repair now possible, regenerative medicine is entering a new era of research and discovery focused on applying optimization strategies to facilitate the acceleration of novel products and services to improve the value of patient care.
Acknowledgments
Supported by National Institutes of Health (R01HL083439, T32HL007111, R01HL085208, R56AI074363), American Heart Association, American Society for Clinical Pharmacology and Therapeutics, Caja Madrid Graduate Program, Marriott Individualized Medicine Program, Marriott Heart Disease Research Program, and Mayo Clinic.
Disclosures
The authors have no conflicts of interest that are directly relevant to the content of this review.
References
- NagyAGóczaEDiazEMEmbryonic stem cells alone are able to support fetal development in the mouseDevelopment19901108158212088722
- NelsonTJMartinez-FernandezATerzicAKCNJ11 knockout morula re-engineered by stem cell diploid aggregationPhilos Trans R Soc Lond B Biol Sci200936426927618977736
- YamadaSNelsonTJBehfarAStem cell transplant into preimplantation embryo yields myocardial infarction-resistant adult phenotypeStem Cells2009271697170519544428
- QuainiFUrbanekKBeltramiAPChimerism of the transplanted heartN Engl J Med200234651511777997
- TorellaDEllisonGMMéndez-FerrerSIbanezBNadal-GinardBResident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regenerationNat Clin Pract Cardiovasc Med20063Suppl 1S8S1316501638
- KuboHJaleelNKumarapeliAIncreased cardiac myocyte progenitors in failing human heartsCirculation200811864965718645055
- CopelanEAHematopoietic stem-cell transplantationN Engl J Med20063541813182616641398
- NelsonTJBehfarAYamadaSMartinez-FernandezATerzicAStem cell platforms for regenerative medicineClin Trans Sci20092222227
- KlimanskayaIRosenthalNLanzaRDerive and conquer: sourcing and differentiating stem cells for therapeutic applicationsNat Rev Drug Discov2008713114218079756
- MorrisonSJSpradlingACStem cells and niches: Mechanisms that promote stem cell maintenance throughout lifeCell200813259861118295578
- SuraniMAMcLarenAStem cells: a new route to rejuvenationNature200644328428516988700
- JaenischRYoungRStem cells, the molecular circuitry of pluripotency and nuclear reprogrammingCell200813256758218295576
- BeyhanZIagerAECibelliJBInterspecies nuclear transfer: Implications for embryonic stem cell biologyCell Stem Cell2007150251218371390
- HendersonJTLazarus’s gate: challenges and potential of epigenetic reprogramming of somatic cellsClin Pharmacol Ther20088388989318388874
- HallVJStojkovicMThe status of human nuclear transferStem Cell Rev2006230130817848717
- YangXSmithSLTianXCNuclear reprogramming of cloned embryos and its implications for therapeutic cloningNat Genet20073929530217325680
- ByrneJAPedersenDAClepperLLProducing primate embryonic stem cells by somatic cell nuclear transferNature200745049750218004281
- FrenchAJAdamsCAAndersonLSDevelopment of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblastsStem Cells20082648549318202077
- TakahashiKYamanakaSInduction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factorsCell200612666367616904174
- YamanakaSPluripotency and nuclear reprogrammingPhilos Trans R Soc Lond B Biol Sci20083632079208718375377
- YamanakaSA fresh look at iPS cellsCell2009137131719345179
- YamanakaSElite and stochastic models for induced pluripotent stem cell generationNature2009460495219571877
- YuJVodyanikMASmuga-OttoKInduced pluripotent stem cell lines derived from human somatic cellsScience20073181917192018029452
- MeissnerAWernigMJaenischRDirect reprogramming of genetically unmodified fibroblasts into pluripotent stem cellsNat Biotechnol2007251177118117724450
- MaheraliNSridharanRXieWDirectly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contributionCell Stem Cell20071557018371336
- TakahashiKTanabeKOhnukiMInduction of pluripotent stem cells from adult human fibroblasts by defined factorsCell200713186187218035408
- YamanakaSStrategies and new developments in the generation of patient-specific pluripotent stem cellsCell Stem Cell20071394918371333
- ParkIHLerouPHZhaoRHuoHDaleyGQGeneration of human-induced pluripotent stem cellsNat Protoc200831180118618600223
- ParkIHAroraNHuoHDisease-specific induced pluripotent stem cellsCell200813487788618691744
- NelsonTJMartinez-FernandezAJYamadaSInduced pluripotent reprogramming from promiscuous human stemness-related factorsClin Transl Sci2009211812620161095
- TakahashiKOkitaKNakagawaMYamanakaSInduction of pluripotent stem cells from fibroblast culturesNat Protoc200723081308918079707
- AasenTRayaABarreroMJEfficient and rapid generation of induced pluripotent stem cells from human keratinocytesNat Biotechnol2008261276128418931654
- LohYAgarwalSParkIGeneration of induced pluripotent stem cells from human bloodBlood20091135476547919299331
- SunNPanettNJGuptDMFeeder-free derivation of induced pluripotent stem cells from adult human adipose stem cellsProc Natl Acad Sci U S A2009106157201572519805220
- MarionRMStratiKLiHTelomeres acquire embryonic stem cell characteristics in induced pluripotent stem cellsCell Stem Cell2009414115419200803
- DengJShoemakerRXieBTargeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogrammingNat Biotechnol20092735336019330000
- MikkelsenTSHannaJZhangXDissecting direct reprogramming through integrative genomic analysisNature2008454495518509334
- SilvaJNicholsJTheunissenTWNanog is the gateway to the pluripotent ground stateCell200913872273719703398
- NishikawaSGoldsteinRANierrasCRThe promise of human induced pluripotent stem cells for research and therapyNat Rev Mol Cell Biol2008972572918698329
- NakagawaMKoyanagiMTanabeKGeneration of induced pluripotent stem cells without Myc from mouse and human fibroblastsNat Biotechnol20082610110618059259
- ParkIHZhaoRWestJAReprogramming of human somatic cells to pluripotency with defined factorsNature200845114114618157115
- WaldmanSATerzicATherapeutic targeting: a crucible for individualized medicineClin Pharmacol Ther20088365165418425084
- ZhaoXYLiWLvZiPS cells produce viable mice through tetraploid complementationNature2009461869019672241
- BolandMJHazenJLNazorKLAdult mice generated from induced pluripotent stem cellsNature2009461919419672243
- HannaJMarkoulakiSSchorderetPDirect reprogramming of terminally differentiated mature B lymphocytes to pluripotencyCell200813325026418423197
- UtikalJPoloJMStadtfeldMImmortalization eliminates a roadblock during cellular reprogramming into iPS cellsNature20094601145114819668190
- OkitaKIchisakaTYamanakaSGeneration of germline-competent induced pluripotent stem cellsNature200744831331717554338
- AoiTYaeKNakagawaMGeneration of pluripotent stem cells from adult mouse liver and stomach cellsScience200832169970218276851
- HuangfuDOsafuneKMaehrRInduction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2Nat Biotechnol2008261269127518849973
- EminliSUtikalJArnoldKReprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expressionStem Cells2008262467247418635867
- KimJBZaehresHWuGPluripotent stem cells induced from adult neural stem cells by reprogramming with two factorsNature200845464665018594515
- FengBJiangJKrausPReprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor EsrrbNat Cell Biol20091119720319136965
- StadtfeldMNagayaMUtikalJInduced pluripotent stem cells generated without viral integrationScience200832294594918818365
- OkitaKNakagawaMHyenjongHGeneration of mouse induced pluripotent stem cells without viral vectorsScience200832294995318845712
- KajiKNorrbyKPacaAVirus-free induction of pluripotency and subsequent excision of reprogramming factorsNature200945877177519252477
- WoltjenKMichaelIPMohseniPpiggyBac transposition reprograms fibroblasts to induced pluripotent stem cellsNature200945876677019252478
- NelsonTJTerzicAInduced pluripotent stem cells: reprogrammed without a traceRegen Med2009433335519438303
- YuJHuKSmuga-OttoKHuman induced pluripotent stem cells free of vector and transgene sequencesScience200932479780119325077
- ZhouHWuSJooJYGeneration of induced pluripotent stem cells using recombinant proteinsCell Stem Cell2009438138419398399
- KimDKimCHMoonJIGeneration of human induced pluripotent stem cells by direct delivery of reprogramming proteinsCell Stem Cell2009647247619481515
- ShiYDespontsCDoJTHahmHSSchölerHRDingSInduction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compoundsCell Stem Cell2008356857418983970
- BanitoARashidSTAcostaJCSenescence impairs successful reprogramming to pluripotent stem cellsGenes Dev2009232134213919696146
- HongHTakahashiKIchisakaTSuppression of induced pluripotent stem cell generation by the p53-p21 pathwayNature20094601132113519668191
- MariónRMStratiKLiHA p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrityNature20094601149115319668189
- LiHColladoMVillasanteAThe Ink4/Arf locus is a barrier for iPS cell reprogrammingNature20094601136113919668188
- KawamuraTSuzukiJWangYVLinking the p53 tumor suppressor pathway to somatic cell reprogrammingNature20094601140114419668186
- NelsonTJBehfarATerzicAStem cells: biologics for regenerationClin Pharmacol Ther20088462062318701884
- WaldmanSATerzicMRTerzicAMolecular medicine hones therapeutic arts to scienceClin Pharmacol Ther20078234334717851568
- SegersVLeeRTStem-cell therapy for cardiac diseaseNature200845193794218288183
- NelsonTJBehfarATerzicAStrategies for therapeutic repair: The “R3” regenerative medicine paradigmClin Transl Sci2008116817119756244
- DaleyGQScaddenDTProspects for stem cell-based therapyCell200813254454818295571
- RosenthalNPrometheus’s vulture and the stem-cell promiseN Engl J Med200334926727412867611
- LeriAKajsturaJAnversaPFrishmanWHMyocardial regeneration and stem cell repairCurr Probl Cardiol2008339115318243902
- DimmelerSZeiherAMSchneiderMDUnchain my heart: the scientific foundations of cardiac repairJ Clin Invest200511557258315765139
- ChienKRDomianIJParkerKKCardiogenesis and the complex biology of regenerative cardiovascular medicineScience20083221494149719056974
- MenaschePCell-based therapy for heart disease: a clinically oriented perspectiveMol Ther20091775876619277020
- BehfarAPerez-TerzicCFaustinoRSCardiopoietic programming of embryonic stem cells for tumor-free heart repairJ Exp Med200720440542017283208
- NelsonTJFaustinoRSChiriacACrespo-DiazRBehfarATerzicACXCR4+/FLK-1+ biomarkers select a cardiopoietic lineage from embryonic stem cellsStem Cells2008261464147318369102
- HannaJWernigMMarkoulakiSTreatment of sickle cell anemia mouse model with iPS cells generated from autologous skinScience20073181920192318063756
- WernigMZhaoJPPruszakJNeurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s diseaseProc Natl Acad Sci U S A20081055856586118391196
- XuDAlipioZFinkLMPhenotypic correction of murine hemophilia A using an iPS cell-based therapyProc Natl Acad Sci U S A200910680881319139414
- NelsonTJMartinez-FernandezAYamadaSRepair of acute myocardial infarction with human stemness factors induced pluripotent stem cellsCirculation200912040841619620500
- Martinez-FernandezANelsonTJYamadaSiPS Programmed without c-MYC yield proficient cardiogenesis for functional heart chimerismCirc Res200910564865619696409
- BartunekJShermanWVanderheydenMDelivery of biologics in cardiovascular regenerative medicineClin Pharmacol Ther20098554855219212313
- BelmonteJCEllisJHochedlingerKYamanakaSInduced pluripotent stem cells and reprogramming: seeing the science through the hypeNat Rev Genet20091087888319859062
- ByrneJANguyenHNReijo PeraRAEnhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblastsPLoS ONE200949e711819774082