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1.  Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes 
Nature methods  2014;11(8):855-860.
Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.
PMCID: PMC4169698  PMID: 24930130
Human induced pluripotent stem cell; differentiation; cardiomyocyte; heart; chemically defined medium; small molecule
2.  Short Hairpin RNA Gene Silencing of Prolyl Hydroxylase-2 with a Minicircle Vector Improves Neovascularization of Hindlimb Ischemia 
Human Gene Therapy  2013;25(1):41-49.
In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×105±3.3×105 (MC-Luc) versus 0.4×105±0.3×105 (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31+ expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.
PMCID: PMC3900020  PMID: 24090375
3.  Transplanted Terminally Differentiated Induced Pluripotent Stem Cells Are Accepted By Immune Mechanisms Similar To Self-Tolerance 
Nature communications  2014;5:3903.
The exact nature of the immune response elicited by autologous induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic contexture of intra-graft characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B, and perforin intra-graft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.
PMCID: PMC4075468  PMID: 24875164
Induced pluripotent stem cells; differentiated derivatives; immunogenicity; T cell receptor (TCR) sequencing
4.  Costimulation-Adhesion Blockade is Superior to Cyclosporine A and Prednisone Immunosuppressive Therapy for Preventing Rejection of Differentiated Human Embryonic Stem Cells Following Transplantation 
Stem cells (Dayton, Ohio)  2013;31(11):2354-2363.
Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.
To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.
Methods and Results
We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP), and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging (BLI). Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hindlimb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte (hESC-CM) survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging (MRI). Mechanistically, costimulation-adhesion therapy is associated with systemic and intra-graft upregulation of T cell immunoglobulin and mucin domain 3 (TIM3) and a reduced pro-inflammatory cytokine profile.
Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.
PMCID: PMC3938393  PMID: 24038578
immune tolerance; costimulation blockade; immunosuppressive drugs; embryonic stem cells; endothelial cells; myocardial infarction
5.  Noninvasive Evaluation of Immunosuppressive Drug Efficacy on Acute Donor Cell Survival 
The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death.
Procedures and Results
Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3 × 106) were injected into thigh muscles of Sprague–Dawley rats (N = 30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day −3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0 ± 12.1% (dexamethasone), 30.5 ± 12.5% (tacrolimus), and 21.5 ± 3.5% (mycophenolate) vs. 12.0 ± 5.0% (control) and 8.3 ± 5.0% (cyclosporine) at day 4 (P < 0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.
This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.
PMCID: PMC4161130  PMID: 16555032
Molecular imaging; Cell transplant; Cyclosporine; Tacrolimus; Mycophenolate
6.  Screening Adverse Drug-Induced Arrhythmia Events Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Low-Impedance Microelectrode Arrays 
Circulation  2013;128(11 0 1):10.1161/CIRCULATIONAHA.112.000570.
Drug-induced arrhythmia is the most common cause of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve upon industry-standard, preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. Human iPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.
Methods and Results
Pharmacological responses of beating embryoid bodies (EBs) exposed to a comprehensive panel of drugs at 65 to 95 days post-induction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers such as sotalol and quinidine produced statistically and physiologically significant effects, consistent with patch-clamp studies on human embryonic stem cell-derived cardiomyocytes (hESC-CMs). False negative and false positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations (EADs) and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared to negligible EADs and ectopic beats in untreated controls.
We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. We believe that this system holds great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
PMCID: PMC3855862  PMID: 24030418
stem cell; cardiomyocyte; pharmacology; arrhythmia; genomics; pharmacogenomics
7.  Development of Poly (β-amino esters)-Based Biodegradable Nanoparticles for Non-Viral Delivery of Minicircle DNA 
ACS nano  2013;7(8):7241-7250.
Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements, and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly (β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by an Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising non-viral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.
PMCID: PMC3789527  PMID: 23837668
Non-viral; minicircle; gene delivery; polymer synthesis; biodegradable nanoparticles
8.  Cortical Bone-Derived Stem Cells: A Novel Class of Cells for Myocardial Protection 
Circulation research  2013;113(5):10.1161/CIRCRESAHA.113.302083.
PMCID: PMC3855861  PMID: 23948579
cardiovascular disease; ischemic heart disease; stem cells
9.  Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular Diseases 
Circulation research  2013;112(12):10.1161/CIRCRESAHA.113.300939.
High throughput sequencing technologies have become essential in studies on genomics, epigenomics, and transcriptomics. While sequencing information has traditionally been elucidated using a low throughput technique called Sanger sequencing, high throughput sequencing (HTS) technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows HTS to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within HTS technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing (RNA-seq) can be used to analyze how the transcriptome changes. Chromatin immunoprecipitation sequencing (ChIP-seq) and methylation sequencing (Methyl-seq) can be used to identify epigenetic changes whereas ribosome sequencing (Ribo-seq) can be used to determine which mRNA transcripts are actively being translated. In this review, we will outline the differences in various sequencing modalities and examine the main sequencing platforms on the market in terms of their relative read depths, speeds, and costs. Lastly, we will discuss the development of future sequencing platforms and how these new technologies may improve upon current sequencing platforms. Ultimately, these sequencing technologies will be instrumental in further delineating how the cardiovascular system develops and how perturbations in DNA and RNA can lead to cardiovascular disease.
PMCID: PMC3831009  PMID: 23743227
DNA sequencing; RNA sequencing; chiP sequencing; cardiac disease; transcriptome; genomics; genetics
10.  Patient-Specific Stem Cells and Cardiovascular Drug Discovery 
PMCID: PMC4033311  PMID: 24240927
induced pluripotent stem cells; cardiovascular disease; toxicity screening; drug discovery; drug repurposing
11.  Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease Specific Patterns of Cardiotoxicity 
Circulation  2013;127(16):10.1161/CIRCULATIONAHA.113.001883.
Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.
Methods and Results
Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs).
We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays.
PMCID: PMC3870148  PMID: 23519760
Induced pluripotent stem cells; stem cells; cardiotoxicity; arrhythmia
13.  A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards 
Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.
PMCID: PMC3556463  PMID: 23229562
pluripotent stem cells; cardiovascular disease; toxicity screening; drug discovery
14.  Advances in Nanotechnology for the Management of Coronary Artery Disease 
Nanotechnology holds tremendous potential to advance the current treatment of coronary artery disease. Nanotechnology may assist medical therapies by providing a safe and efficacious delivery platform for a variety of drugs aimed at modulating lipid disorders, decreasing inflammation and angiogenesis within atherosclerotic plaques, and preventing plaque thrombosis. Nanotechnology may improve coronary stent applications by promoting endothelial recovery on a stent surface utilizing bio-mimetic nanofibrous scaffolds, and also by preventing in-stent restenosis using nanoparticle-based delivery of drugs that are decoupled from stents. Additionally, nanotechnology may enhance tissue-engineered graft materials for application in coronary artery bypass grafting by facilitating cellular infiltration and remodeling of a graft matrix.
PMCID: PMC3566293  PMID: 23245913
Nanotechnology; coronary artery disease; atherosclerosis; percutaneous coronary intervention; coronary artery bypass graft
15.  MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2 
Stem cells (Dayton, Ohio)  2013;31(2):259-268.
MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay, and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we use global bioinformatics analysis of miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of the miR-302, which we experimentally confirm by reporter luciferase assays and real-time PCR. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and the miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells (hASCs) into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as “KMOS3”) when compared to using four factors (“KMOS”). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.
PMCID: PMC3572288  PMID: 23136034
microRNA; reprogramming; induced pluripotent stem cells; OCT4
16.  Immunogenicity of Pluripotent Stem Cells and Their Derivatives 
Circulation research  2013;112(3):549-561.
The ability of pluripotent stem cells to self-renew and differentiate into all somatic cell types brings great prospects to regenerative medicine and human health. However, prior to clinical applications, much translational research is required to ensure that their therapeutic progenies are functional and non-tumorigenic, that they are stable and do not de-differentiate, and that they do not elicit immune responses that could threaten their survival in vivo. For this, an in-depth understanding of their biology, genetic and epigenetic makeup, and their antigenic repertoire is critical for predicting their immunogenicity and for developing strategies needed to assure successful long-term engraftment. More recently, the expectation that reprogrammed somatic cells would provide an autologous cell therapy for personalized medicine has been questioned. Induced pluripotent stem (iPS) cells display several genetic and epigenetic abnormalities that could promote tumorigenicity and immunogenicity in vivo. Understanding the persistence and effects of these abnormalities in iPS cell derivatives is critical to allow clinicians to predict graft fate following transplantation, and to take requisite measures to prevent immune rejection. With clinical trials of pluripotent stem cell therapy on the horizon, the importance of understanding immunological barriers and devising safe, effective strategies to bypass them is further underscored. This approach to overcome immunological barriers to stem cell therapy can take advantage of the validated knowledge acquired from decades of hematopoietic stem cell transplantation.
PMCID: PMC3638957  PMID: 23371903
immunogenicity; embryonic stem cells; induced pluripotent stem cells; transplantation; tolerance; patient-specific therapy; stem cell therapeutics
17.  Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nuclease for Cellular Imaging 
Circulation research  2012;111(12):1494-1503.
Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied upon random integration, a method that is associated with unwanted insertional mutagenesis and position effects on transgene expression.
To address this barrier, we used genome editing with zinc finger nuclease technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) for molecular imaging.
Methods and Results
We employed ZFN technology to integrate a construct containing monomeric red fluorescent protein (mRFP), firefly luciferase (Fluc), and herpes simplex virus thymidine kinase (HSVtk) reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for bioluminescence imaging (BLI) and positron emission tomography (PET) imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both hESCs and iPSCs. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited hESCs, iPSCs, and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the utility of ZFN-edited cells for preclinical studies in regenerative medicine.
Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells (PSCs) and their progeny for molecular imaging in vitro and in vivo.
PMCID: PMC3518748  PMID: 22967807
Induced pluripotent stem cells; zinc finger nuclease; homologous recombination; reporter gene; molecular imaging; stem cells; imaging
19.  Induced Pluripotent Stem Cells as a Disease Modeling and Drug Screening Platform 
Induced pluripotent stem cells (iPSCs) hold great hopes for therapeutic application in various diseases. While ongoing research is dedicated to achieving clinical translation of iPSCs, further understanding of the mechanisms that underlie complex pathogenic conditions is required. Compared to other classical models for studying diseases, iPSCs provide considerable advantages. A newly emerging application of iPSCs is in vitro disease modeling, which can significantly improve the never-ending search for new pharmacological cures. Here, we will discuss current efforts to create iPSC-dependent, patient-specific disease models. Furthermore, we will review the use of iPSCs for development and testing of new therapeutic agents, and the implications for high-throughput drug screening.
PMCID: PMC3343213  PMID: 22240913
Induced pluripotent stem cells; Disease modeling; Cardiovascular disease; Drug screening; High-throughput screening
20.  Microfluidic Single Cell Analysis Show Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation 
Circulation research  2012;111(7):882-893.
Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large animal iPSC models.
To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.
Methods and Results
Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction (MI). Compared to control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% vs. 22.3±1.1%; P<0.001) and magnetic resonance imaging (ejection fraction at week 4: 45.8±1.3% vs. 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single cell PCR profiling showed that piPSC-ECs released pro-angiogenic and anti-apoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.
In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function following MI via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.
PMCID: PMC3473073  PMID: 22821929
Induced pluripotent stem cells; large animal models; paracrine activation; myocardial infarction; molecular imaging; ischemic heart disease; vascular biology
21.  Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique 
Circulation  2012;126(11 Suppl 1):S20-S28.
Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.
Methods and Results
We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging.
The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.
PMCID: PMC3481839  PMID: 22965984
cell therapy; stem cells; imaging; cardiovascular disease
24.  Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency 
Scientific Reports  2015;5:8081.
The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.
PMCID: PMC4308704  PMID: 25628230
25.  Early Stem Cell Engraftment Predicts Late Cardiac Functional Recovery: Pre-Clinical Insights from Molecular Imaging 
Human cardiac progenitor cells have demonstrated great potential for myocardial repair in small and large animals, but robust methods for longitudinal assessment of their engraftment in humans is not yet readily available. In this study, we sought to optimize and evaluate the use of positron emission tomography (PET) reporter gene imaging for monitoring human cardiac progenitor cell (hCPC) transplantation in a mouse model of myocardial infarction.
Methods & Results
hCPCs were isolated and expanded from human myocardial samples and stably transduced with variations of the thymidine kinase (TK) PET reporter gene. TK-expressing hCPCs were characterized in vitro and transplanted into murine myocardial infarction models (n=60). Cardiac echocardiographic, magnetic resonance imaging (MRI), and pressure-volume (PV) loop analyses revealed improvement in left ventricular contractile function two weeks after transplant (hCPC vs. PBS, P<0.03). Noninvasive PET imaging was used to track hCPC fate over a four week time period, demonstrating a substantial decline in surviving cells. Importantly, early cell engraftment as assessed by PET was found to predict subsequent functional improvement, implying a “dose-effect” relationship. We isolated the transplanted cells from recipient myocardium by laser capture microdissection for in vivo transcriptome analysis. Our results provide direct evidence that hCPCs augment cardiac function after their transplantation into ischemic myocardium through paracrine secretion of growth factors.
PET reporter gene imaging can provide important diagnostic and prognostic information regarding the ultimate success of human cardiac progenitor cell treatment for myocardial infarction.
PMCID: PMC3400712  PMID: 22565608
cell therapy; stem cells; imaging; positron emission tomography

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