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Stem Cell Therapy for Vascular Regeneration

2010; Lippincott Williams & Wilkins; Volume: 122; Issue: 5 Linguagem: Inglês

10.1161/circulationaha.109.881441

1524-4539

Nicholas J. Leeper, Arwen L. Hunter, John P. Cooke,

Electrospun Nanofibers in Biomedical Applications

HomeCirculationVol. 122, No. 5Stem Cell Therapy for Vascular Regeneration Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBStem Cell Therapy for Vascular RegenerationAdult, Embryonic, and Induced Pluripotent Stem Cells Nicholas J. Leeper, MD, Arwen L. Hunter, PhD and John P. Cooke, MD, PhD Nicholas J. LeeperNicholas J. Leeper From the Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, Calif. , Arwen L. HunterArwen L. Hunter From the Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, Calif. and John P. CookeJohn P. Cooke From the Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, Calif. Originally published3 Aug 2010https://doi.org/10.1161/CIRCULATIONAHA.109.881441Circulation. 2010;122:517–526Broadly speaking, vascular regeneration includes the restoration of normal vascular function and structure, the reversal of vascular senescence, and the growth of new blood vessels. Therapeutic applications of vascular regeneration for coronary or peripheral arterial diseases are directed to relieving symptoms of ischemia; preventing target-organ damage due to hypoxia, reperfusion, or capillary leak; and avoiding cardiovascular catastrophes due to acute thrombosis, embolism, plaque rupture, or dissection.Clinicians have sought methods to harness the potential of therapeutic vascular regeneration, but studies focused on gene therapy or small molecular approaches have largely failed thus far. Recently, efforts have shifted to stem cell–based approaches given their theoretical capacity to replicate, differentiate, and form new blood vessels in a directed fashion. Initial preclinical studies evaluated the pluripotent embryonic stem cell (ESC) and the more lineage-committed "adult" stem cells, which include the endothelial progenitor cells (EPCs) found within the bone marrow. Early clinical trials indicate some benefit of EPC therapy in patients with ischemic or cardiomyopathic disease. In the meantime, scientific interest has shifted to a newly described class of stem cell, the induced pluripotent stem cell (iPSC). This fascinating cell is derived from terminally differentiated adult somatic cells that are "reprogrammed" to an embryonic-like state with transcription factors that govern cell differentiation. Interest in iPSCs is high because these cells are autologous (do not require immunosuppression when delivered), have pluripotential (can differentiate into tissue from each of the 3 germline lineages), are noncontroversial (are derived from adult tissue), and come from a plentiful source (are derived from any adult cell [eg, skin fibroblasts]).The focus of this review is on the use of stem cell therapies for the growth of new blood vessels (ie, angiogenesis, vasculogenesis, and arteriogenesis). In particular, we will focus on the promise of iPSCs for cell-based vascular regeneration compared with other stem cell approaches.General Properties of Stem CellsA stem cell is defined by its capacity for both self-renewal and directed differentiation. Historically, investigators have recognized 2 broad categories of stem cells, the ESC and the so-called adult stem cell. The ESC is derived from the inner cell mass of the fetal blastula and is pluripotent, ie, it has the ability to differentiate into any cell type found in the adult body. ESCs can replicate via mitotic division while retaining their undifferentiated state (self-renewal) or differentiate into lineage-specific cells under the appropriate stimuli.In contrast to ESCs, adult stem cells are partially lineage-committed and therefore have the capacity to give rise only to cells of a given germ layer. In other words, they are multipotent rather than pluripotent. As an example, the adult hematopoietic stem cell can repopulate the bone marrow of the leukemia patient after transplantation, generating all blood cell lineages; however, this multipotent adult stem cell cannot produce cells of endodermal or ectodermal lineage. Another form of multipotent stem cell, the EPC, is described in detail below. In addition to multipotent adult stem cells, unipotent stem cells have been described. Such cells have increased replicative capacity but can only differentiate into 1 cell lineage. Compared with adult differentiated cells, adult stem cells have greater capacity for proliferation and ability to repopulate or repair tissue.1 Although adult differentiated cells typically give rise only to cells of identical lineage, there is rare evidence for transdifferentiation between lineages. For example, Barrett's metaplasia is due to transdifferentiation of esophageal epithelial cells into cells that resemble intestinal mucin-secreting goblet cells.A third form of stem cell that has great potential for regenerative medicine is the iPSC. In 2006, Yamanaka and colleagues2 reported that mouse fibroblasts could be reprogrammed into iPSCs by viral transduction of 4 transcription factors. That a small set of genes can induce "nuclear reprogramming" of adult differentiated cells into cells with many of the same characteristics as pluripotent ESCs was soon confirmed by others.3–6 In 2007, human fibroblasts were reprogrammed into iPSCs by viral transduction of octamer-binding transcription factor-3/4 (Oct 3/4) and SRY-related high-mobility-group (HMG)-box protein-2 (Sox2), in combination with Krüppel-like factor 4 (Klf4) and c-Myc or in combination with Nanog and Lin28.7,8 The iPSCs resemble ESCs in that they have the potential to differentiate into any adult cell. Ultimately, iPSCs may represent the most attractive cellular approach for regenerative medicine. In the following sections, each of these 3 cell types will be discussed in turn, with an emphasis on the translation to therapeutic application in patients with vascular disease (Figure 1). Download figureDownload PowerPointFigure 1. ESCs, adult stem cells (SCs), and iPSCs. The 3 progenitor cell types available for vascular regenerative therapy: their promise and peril.Adult Stem Cells in Vascular RegenerationThe therapeutic application of adult stem cells is farther along in clinical development than any of the other stem cell approaches. Adult stem cells exist in the bone marrow and the circulation or as residents within a specific tissue.9 Therapies aimed at neovascularization to date have included bone marrow–derived and circulating stem cell approaches. The use of adult stem cells for vascular regeneration was presaged by Asahara's discovery of the vasculogenic EPC subpopulation in 1997 and has culminated in recent clinical trials.13Parenthetically, the widely used term "EPC" is a misnomer. The surface markers that are commonly used for identification of human EPCs include markers that are not specific for endothelial lineage, such as CD133 and KDR (kinase insert domain receptor).10 In addition, methods for harvesting, purifying, and culturing EPCs are not standardized. Thus, semantic confusion is compounded by methodological variation. Casual readers of the literature may not recognize that EPCs are a mixed population of progenitor cells of different lineages. Within this population of cells, there are true endothelial progenitors that can incorporate into the vascular network, whereas hematopoietic progenitors may contribute by secreting angiogenic cytokines. In cell culture, EPCs may form early-outgrowth and late-outgrowth colonies. Cells derived from the former colonies are clearly not of endothelial origin, expressing markers of hematopoietic lineage, and are morphologically distinct from late-outgrowth cells, which grow in a cobblestone pattern reminiscent of endothelial cells (ECs). At present, there are no surface markers that clearly distinguish early endothelial progenitors; the best approach currently is to define endothelial lineage morphologically (ie, with tubulogenesis assays). The formation of a tubular network in Matrigel is a defining feature of endothelial progenitors, which can also incorporate into existing tubular networks formed by differentiated ECs.EPCs are postulated to arise from an earlier progenitor, termed the "hemangioblast," which also generates hematopoietic stem cells.11 Preclinical studies indicate that EPCs reside in the marrow (adhering to supporting stromal cells in a "stem cell niche") and circulate in the blood at very low levels (<0.01% of circulating white cells).12 Their prevalence in the blood can change in response to various stimuli. Ischemia increases VEGF expression, which in turn activates matrix metalloproteinases, which releases the EPCs (CD34+/cKit+ cells) from the vascular niche by cleaving Kit ligand. The mobilized EPCs enter the circulation and home to the site of ischemia.11,13,14 Bone marrow–derived cells mobilized by ischemia have been shown to incorporate into the vasculature, differentiating into ECs, pericytes, or smooth muscle cells. Alternatively or in addition, they may potentiate local angiogenesis via the elaboration of paracrine factors.15–17 Numerous animal studies in both hindlimb ischemia and coronary ischemia models have indicated that EPCs can be harvested, expanded ex vivo, and administered to augment capillary density, perfusion, and organ function.11,18–22To avoid semantic confusion, it may be best to eschew the term "EPC" and simply describe the isolated cells by their morphology, surface markers, and method of isolation. Thus, in humans, vascular regeneration has been attempted with autologous bone marrow mononuclear cells (BMNCs) or granulocyte colony-stimulating factor–expanded peripheral blood mononuclear cells in patients with coronary and peripheral arterial disease. Phase I and II trials have been completed in patients with acute myocardial infarction,9 as well as in subjects with chronic ischemic heart disease.23–25 Thus far, the majority of these early trials have suggested modest but consistent improvements in cardiac end points such as global and regional contractility and little risk for these cell-based therapies.9Strauer and colleagues26 found that the intracoronary injection of Ficoll gradient–isolated autologous BMNCs 5 to 9 days after revascularization was associated with a small improvement in ejection fraction and infarct size (as assessed by left ventriculography) and improved myocardial perfusion by nuclear scintigraphy 3 months after delivery. This work was quickly followed by several other small nonblinded trials, including TOPCARE-AMI (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction; BMNCs or peripheral blood mononuclear cells) and BOOST (BOne MarrOw transfer to enhance ST-elevation infarct regeneration; sedimentation-protocol isolated BMNCs), both of which confirmed improved pump function globally and in the infarct zone at 4 to 6 months after intracoronary cell therapy, again with no observed serious adverse events.27–29 Enthusiasm was briefly tempered by negative findings reported by Janssens et al30 and in the Autologous Stem Cell Transplantation in Acute Myocardial Infarction (ASTAMI) trial of autologous BMNCs in patients with acute ST-segment elevation myocardial infarction.31 In addition, the 18-month BOOST follow-up data failed to show a persistent benefit of cell therapy on contractility compared with standard care.32 It is possible that a single injection of stem cells may not be sufficient to produce long-term differences in pump function and that the methods used to isolate and process the BMNCs in the ASTAMI trial may have affected their performance adversely.9 Also, although Janssens et al30 did not show global improvements in the ejection fraction of the patients in their randomized trial, those who received cell therapy did in fact have smaller infarct sizes and greater recovery of regional cardiac function.By contrast, the largest and most rigorously designed trial to date, the multicenter, randomized, double-blind, placebo-controlled REPAIR-AMI trial (Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction), revealed a modest improvement in global left ventricular ejection fraction at 4 months after the delivery of BMNCs 3 to 7 days after infarct.33 Although not powered to do so, this study also revealed a significant reduction in death, recurrent myocardial infarction, and revascularization at 1 year. The majority of trials completed thus far have not attempted to uncover the mechanism of observed benefit with cell transplantation. Furthermore, most studies do not control for the possible effects of paracrine factors secreted by the administered cells (which could be addressed in part by use of a conditioned medium control). Nevertheless, a subset of patients in the REPAIR-AMI trial who underwent coronary flow reserve testing did manifest a marked improvement in microvascular function and vasomotor responsiveness at 4 months, which suggests that the administered BMNCs may in fact enhance vascular repair.34 Overall, the studies of adult stem cell therapy for myocardial ischemia suggest that cell therapy is feasible, safe, and associated with modest improvements in contractility; however, the mechanisms of this modest benefit remain unknown.There have also been a number of small pilot trials of adult stem cells in peripheral arterial disease. Clinical benefit has been observed in small, uncontrolled studies of BMNC therapy for subjects with claudication.14,35–37 Pathological specimens of amputation specimens have confirmed the presence of therapeutic angiogenesis in patients who had undergone bone marrow transplantation relative to matched control subjects.22 The only controlled trial (Therapeutic Angiogenesis using Cell Transplantation [TACT]) revealed improvements in transcutaneous oxygen pressure, rest pain, and pain-free walking time after intramuscular injections of BMNCs compared with vehicle control.38 Several international phase II trials are now under way to confirm these promising early safety and efficacy findings in peripheral vascular disease (clinicaltrials.gov).Mechanisms of Benefit of Adult Stem Cells in Vascular RegenerationIt appears that only a small subset of EPCs are of true endothelial lineage in humans. These endothelial colony-forming cells can form vascular structures in vivo but are rare, only 1 to 2 per 100 million mononuclear cells.39 Another subset of EPCs, which are more common, are of hematopoietic lineage. These EPCs share the same surface markers (CD31, CD105, CD144, CD146, von Willebrand factor [VWF], KDR, and UEA-1 [ulex europaeus agglutinin 1 lectin]) and incorporate acetylated low-density lipoprotein, but they express the myeloid surface markers CD45 and CD14 and have other features of the monocyte/macrophage phenotype. Some of these cells may contribute to angiogenesis not by incorporating into the vasculature but by secreting angiogenic cytokines and matrix metalloproteinases.40,41 Still other bone marrow–derived stem cells can form pericytes, which associate with and stabilize endothelial networks.42 To conclude, circulating adult stem cells mobilized from the bone marrow, termed "EPCs," are a heterogenous population of cells that may promote EC survival and proliferation, contribute to network formation, and/or stabilize newly formed vessels.The horizon for adult stem cell therapies is broadening rapidly. Treatments composed of 2 or more cellular components, such as those with both mononuclear and mesenchymal populations, are currently under investigation in patients with coronary and peripheral vascular disease (the MESENDO trials). Approaches that move beyond the injection of a single progenitor cell type are intuitively appealing in that they may enhance paracrine cross talk between intimal and medial constituents or reinforce the scaffolding of the developing vascular tree.43 Investigations are also evolving past the bone marrow–derived and circulating cells studied thus far to include resident tissue stem cells such as the adipocyte-derived and cardiac progenitor cell populations (eg, the PRECISE [Randomized clinical trial of adiPose-deRived stEm and regenerative Cells In the treatment of patients with nonrevaScularizable ischEmic myocardium], APOLLO [Randomized clinical trial of AdiPOse-derived stem CeLLs in the treatment of patients with ST-elevation myOcardial infarction), and Caduceus [CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction] trials).44–48 Cardiospheres expanded after endomyocardial biopsy may have a particular role in repopulating areas of infarcted myocardium,49 whereas mesenchymal adipose-derived stem cells have demonstrated the capacity to differentiate into endothelial, pericytic, and smooth muscle cells, which indicates less lineage restriction and more plasticity than a comparable EPC.50–52 Finally, the future of adult stem cell therapy may lie in even more innovative areas, as highlighted by examples such as the so-called EPC-capture stent, which is coated with immobilized anti-CD34 antibodies that aim to guide circulating stem cells to the site of vessel injury (eg, the Endothelial Progenitor Cells-Capture Stents in Acute Coronary Syndromes [JACK-EPC] trial).53 Novel approaches that enhance progenitor homing, cell-cell interaction, and cell integration or that incorporate progenitor cells with vascular matrices and synthetic conduits are likely to define the future of adult stem cell therapies for vascular disease.54,55Strengths and Weaknesses of Adult Stem CellsAll adult stem cells, whether harvested from the marrow, blood, or tissue, share a number of traits that make them appealing as candidates to lead the field of cell-based regeneration. The first is that these cells are harvested from the patient in whom they are ultimately to be used and do not need to overcome an immunologic barrier. Also, this approach is not burdened by the ethical concerns that surround the use of human embryos. Adult stem cells, and EPCs in particular, have undergone the most translational and human studies of all stem cell approaches. The trials done to date show little increased risk associated with their therapeutic use in humans.On the other hand, there are deficiencies of adult stem cell therapy. Autologous delivery of cells inevitably means that there is a delay in treatment due to the time needed to collect the cells and isolate and then propagate progenitors ex vivo so as to obtain adequate numbers before injection. Adverse effects of their delivery could include microvascular embolism, as well as unintended acceleration of pathological neovascularization, as in the case of an occult malignancy. Furthermore, most investigators use a small set of surface markers to define "EPCs," which is problematic.56 As discussed above, the typical surface markers used to isolate EPCs generate a mixed population of cells that have not been completely characterized.11,14,57,58 It is likely that a greater effect size and/or more persistent benefit of cell therapy could be realized with a more precisely defined cell population. It is not known what combination of progenitor cells might be most therapeutic in humans, eg, purified EPCs or some combination with smooth muscle precursors and/or subsets of hematopoietic progenitors.59–61 Of importance, the mechanisms by which these adult stem cells interact to confer benefit must be further defined so that they can be manipulated therapeutically.62 Reproducible and efficient methods to isolate, expand, and deliver angiogenic cells are needed. Finally, in the patients who most need EPC therapy, these cells are rare, have limited replicative capacity, and are often dysfunctional. For example, EPCs derived from older individuals demonstrate reduced ability to proliferate, incorporate into existing capillary networks, and enhance limb perfusion in preclinical models. Conditions typically associated with vascular disease, such as age and diabetes mellitus, impair angiogenic functionality.11,63 The limitations of adult stem cell therapy for vascular disease have increased the interest in other sources for stem cell therapy, such as ESCs.ESCs as a Source of Vascular RegenerationESCs are derived from the inner cell mass of the blastocyst and, unlike adult stem cells, can differentiate into any cell type or any organ of endodermal, mesodermal, or ectodermal lineage.64 The ESC can be directed to differentiate into vascular endothelial and smooth muscle cells and cardiomyocytes.65–67 ESC-derived ECs (ESC-ECs) manifest endothelial surface markers (such as CD31), express endothelial proteins (such as VWF and PECAM [platelet and endothelial cell adhesion molecule]), and manifest endothelial functions (such as uptake of acetylated low-density lipoprotein and formation of capillary tubes in Matrigel). Animal studies have shown that ESC-derived ECs and mural cells incorporate into the vasculature of the ischemic limb or myocardium.68–70 We tracked the fate and function of transplanted ESC-ECs in the ischemic murine myocardium.71a Murine ESCs were first transduced with a construct that encoded luciferase (for bioluminescence imaging) and red fluorescent protein (for histological tracking). After the ESCs differentiated into ECs, the ESC-ECs or vehicle was injected into the ischemic zone of the left ventricle after ligation of the left anterior descending coronary artery. Bioluminescence imaging showed persistence of the ESC-ECs up to 8 weeks later, and echocardiography revealed improved systolic function in hearts injected with ESC-ECs compared with vehicle. Histological studies revealed increased myocardial capillary density in the hearts treated with cell therapy. Preclinical studies of ESC-ECs for vascular regeneration are at an early stage. Although the ESC has not yet transitioned from bench to bedside for patients with cardiovascular diseases, the world's first trial of human ESC therapy was recently granted clearance by the US Food and Drug Administration. This trial of ESC-derived neuronal cells for use in acute spinal cord injury is an important milestone in ESC therapy, and the results are likely to have a dramatic impact on the future of ESCs in regenerative medicine, including vascular regeneration.Strengths and Weaknesses of ESCsCompared with adult stem cells, ESCs have the advantage of pluripotentiality and greater proliferative capacity. On the other hand, their clinical use remains conflicted by the ethical debate surrounding the use of human embryos and is challenged by an immunologic barrier. Cells derived from ESCs will be allogeneic, and their use is likely to require the coadministration of immunosuppressive agents, which carry their own substantial risks. Additionally, the great regenerative potential of these cells has raised fears about the inadvertent administration of even a single pluripotent ESC. The undirected growth and pathological differentiation after transplantation of an ESC creates a risk of teratoma, a complication that can occur late after administration.71b Much more robust differentiation and purification protocols are required, and studies proving the long-term safety profile of embryonic progenitors are needed before widespread use in humans will be possible.iPSCs for Vascular RegenerationiPSC BackgroundAn exciting new milestone in the field of regenerative medicine is the development of the iPSC, which has opened a new avenue for cardiac and vascular regeneration.72 Previous work had revealed that the differentiation state of adult somatic cells can be fluid. It was known that the nuclear DNA of differentiated cells could be "reprogrammed" to express stem cell genes by fusion with ESCs or after nuclear transfer into oocytes.73,74 More recently, it has been shown that a handful of reprogramming factors are sufficient to reprogram adult differentiated cells into pluripotential stem cells.2 These iPSCs are capable of giving rise to all 3 germ layers (endoderm, ectoderm, and mesoderm) and subsequently differentiating into adult cells.3–6The set of reprogramming factors used by Yamanaka and colleagues2 included Oct 3/4, Sox2, Klf4, and c-Myc. In parallel studies, Thomson and colleagues8 focused on Oct 3/4 and Sox2 together with Nanog and Lin28. Both groups initially used viral vectors to overexpress the genes encoding the reprogramming factors in human fibroblasts, so as to generate iPSCs. Although the exact mechanism by which these genes induce dedifferentiation remains incompletely understood,75 putative pathways have been theorized.2,74 The proto-oncogene c-Myc likely promotes histone acetylation and chromatin remodeling to facilitate access of Oct 3/4 and Sox 2 to their binding sites and to accelerate cellular proliferation. Tumor suppressor–induced apoptosis, which should naturally follow, is avoided by a second oncogene, Klf4, which inhibits cell death by its suppression of p53 and upregulation of the renewal gene Nanog. The pluripotency-related factors Oct3/4 and Sox2 activate other critical embryonic genes and/or recruit chromatin remodeling complexes to promote reprogramming. Lin28 is an RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing. The creation of iPSCs does not require antibiotic selection5,76 nor the insertion of the retroviruses in specific genomic locations.77 Furthermore, these transcription factors are required only for the induction of, and not the maintenance of, pluripotency.78 Indeed, the persistent expression of these exogenously introduced genes (a particular problem with the lentiviral constructs) appears to impair the ability to differentiate the iPSCs into the desired lineage.Although fibroblasts were the first target of reprogramming efforts, other cell types have been induced to form pluripotential cells, with differences in the ease of reprogramming noted.77 For example, murine neural progenitor cells can be reprogrammed with overexpression of a single factor, Oct3/4. Compared with human fibroblasts, induction efficiencies of human scalp keratinocytes are 100-fold higher.79 The difference in the ease of reprogramming probably relates to differences in the basal epigenetic state and transcriptional activation of cells. Other advances have been made to streamline the reprogramming process.6,7 Exogenous introduction of c-Myc and Klf4 is dispensable, although the differentiation efficiency is reduced without ectopic Myc.73,80 The addition of small molecules that enhance chromatin remodeling can enhance the reprogramming progress. For example, with the use of valproic acid (which is a histone deacetylase inhibitor at high concentrations), only overexpression of Sox2 and Oct3/4 is required for reprogramming.73The Promise and Perils of iPSCsThe therapeutic potential of iPSCs is considerable, because they are patient-specific stem cells that do not face the immunologic barrier that confront cells derived from ESCs. Furthermore, these cells can be derived from plentiful and easily accessible sources of tissue, such as the donor's skin, fat, or hair. Thus, immediate advantages over other progenitors include their immune-privileged status as autologous tissue (compared with allogeneic ESCs) and their potential abundance (relative to the rare EPC). Furthermore, these cells are "almost indistinguishable from [embryonic stem] cells"66 and have "almost completely identical" differentiation properties,81 epigenetic states, surface markers, and gene expression profiles as the pluripotential ESC73 (Figure 2). iPSCs are germline competent and form trophoblastic tissue, which actually makes them totipotent cells.82 The generation and use of iPSCs are not encumbered by the ethical concerns and political barriers faced by those using ESCs.83 Additionally, because they can be derived from subjects with genetic diseases and easily propagated in vitro, they represent an ideal vehicle for the investigation of heritable disorders and for screening of novel therapeutics.84,85Download figureDownload PowerPointFigure 2. Characterization of iPSCs. iPSCs derived from human fibroblast reprogrammed with lentiviral vectors expressing Oct-3/4, Sox2, and Klf4 maintain colony morphology characteristic of ESCs (A, magnification ×4; B, magnification ×20), demonstrate alkaline phosphatase activity (C), and express pluripotency markers as detected by immunocytochemistry including Nanog (D), TRA-1-81 (E), and SSEA-4 (stage-specific embryonic antigen 4; F). For panels D through F, pluripotency markers were detected with antibodies tagged by Alexa-594 (red), and nuclei are marked by DAPI staining (blue). Notably, control plates of differentiated fibroblasts that were mock transfected failed to give rise to any human ESC–like colonies.A number of hurdles remain for the clinical development of these cells. The combination of gene manipulation with cell therapy increases safety concerns and regulatory barriers. For example, the use of retroviruses or lentiviruses leads to integration of viral DNA into the chromosome, which raises the risk of silencing indispensable genes or inducing oncogenesis.78 These concerns are overcome in part with the use of adenoviruses or plasmid constructs,86–88 but even these episomal vectors carry a risk of DNA integration. Accordingly, any iPSCs created with DNA-based strategies will still need to be screened carefully to exclude any DNA integration. Most recently, Cre/LoxP and piggyBAC transposable elements have been used to generate iPSCs.89,90 These strategies offer certain advantages in that the elements can be silenced or excised, which decreases the possibility of reactivation. However, in