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693 Effect of IL 3 on Transduction Efficiency in CD34+ Cell Derived Erythroid Cultures with Lentiviral Vectors Expressing the Human beta Flobin Gene Molecular Therapy Volume 17, Supplement 1, May 2009[.]
STEM CELL THERAPIES II 690 Antioxidant Treatment of Muscle Stem Cells for Transplantation Increases Cardiac Repair Lauren Drowley,1 Masaho Okada,1 Bradley Keller,2 Kimimasa Tobita,2 Johnny Huard.1 University of Pittsburgh, Pittsburgh, PA; 2Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA One major issue with cellular transplantation for cardiac repair has been the poor survival of the transplanted cells, which is related to the limited functional recovery seen clinically This is in part due to the local environment at the site of transplantation, with oxidative stress, inflammation, and ischemia all playing key roles The ability of cells to survive in this harsh environment could be critical in the repair process, and cells that have increased survival could have a greater impact on functional repair We have previously shown that muscle-derived stem cells (MDSCs) significantly ameliorate the remodeling process in the ischemic heart compared to myoblasts after myocardial infarction MDSCs also have lower levels of apoptosis than myoblasts when oxidative stress is induced with hydrogen peroxide Based on these results, we hypothesized that one major characteristic of stem cells was an enhanced ability to survive in stressful environments and that by up or down-regulating antioxidants, we could influence repair To examine this, we reduced levels of glutathione, a prevalent antioxidant, in MDSCs with diethyl maleate (DEM), and also treated cells with N-acetylcysteine (NAC), a molecule that has direct antioxidant effects as well as increasing glutathione synthesis We examined in vitro characteristics including cell survival after stress, proliferation, differentiation, and VEGF secretion and found that antioxidant levels played a role in each In a mouse model of myocardial infarction, we found that NAC-treated MDSCs significantly increased cardiac function, have decreased formation of scar tissue, and increased levels of angiogenesis compared to untreated and DEM-treated MDSCs These results show that antioxidant treatment prior to cell transplantation can have a significant beneficial effect 691 Sleeping Beauty-Mediated Gene Transfer into Hematopoietic Stem Cells Kendra A Hyland,1 Erik R Olson,1 Ron T McElmurry,2 Bruce R Blazar,2 R Scott McIvor,1 Jakub Tolar.2 Discovery Genomics, Inc., Minneapolis, MN; 2Pediatrics, Medical School, University of Minnesota, Minneapolis, MN The Sleeping Beauty (SB) transposon system can transpose sequences into mammalian chromosomes, supporting long term expression of both reporter and therapeutic genes Advantages of the SB system include ease of manufacture, the lack of genotoxic effects after transposition and the absence of immunological complications due to repeated administration Hematopoietic stem cells (HSC) are an ideal target cell as they are used in therapy for a variety of hematologic and other conditions Optimization of electroporation conditions, assessed with a green fluorescent protein (GFP) reporter plasmid and a CytoPulse electroporator, included varying voltage, pulse width, and pulse number to improve the loading of purified cell populations containing HSC, such as mouse lineage negative cells and human umbilical cord blood CD34+ cells Cell viability was reduced by 10-30% compared to untreated cells, to days after electroporation alone We demonstrate successful in vitro electroporation of transposon and reporter plasmid DNA into mouse HSC, such that 5-10% of lineage negative cKit+ Sca-1+ cells (SKL) expressed GFP on day and 25-30% of human CD34+ cord blood cells expressed GFP on day post electroporation SB-mediated transposition of HSC with a transposon containing the L22Y mutated dihydrofolate reductase (DHFR) gene conferred methotrexate resistance after electroporation as assessed in hematopoietic progenitor cells by in vitro colony forming cell assays Studies are in progress to confirm transposition-mediated integration of individual transgenes by S264 sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR Our data provide a platform to establish a system for SB-mediated transposition of HSC and subsequent application to the treatment of human patients 692 Mechanical Loading of Stem Cells for Improvement of Cellular Cardiomyoplasty Lauren Drowley,1 Theresa Cassino,1 Masaho Okada,1 Bradley Keller,2 Kimimasa Tobita,2 Philip LeDuc,3 Johnny Huard.1 University of Pittsburgh, Pittsburgh; 2Children’s Hospital of Pittsburgh, Pittsburgh; 3Carnegie Mellon University, Pittsburgh Although stem cell therapy for tissue repair is a rapidly developing field, the factors which dictate the physiological responsiveness of stem cells remain under investigation In this study we tested the hypothesis that controlling the loading history of muscle derived stem cells (MDSCs) with cyclic mechanical stimulation prior to transplantation significantly improves MDSC survival and angiogenic effects on injured recipient myocardium Murine MDSCs were first isolated using a preplating technique and then underwent 24 hours of cyclic mechanical stretch To test their efficacy for this loading history based approach, an acute myocardial infarction was created by permanently ligating the left coronary artery, and both unstimulated and mechanically stretched MDSCs were transplanted into the injured left ventricular myocardium Hearts implanted with mechanically preconditioned cells attenuated post-infarcted dilatation and sustained cardiac contractility for 12 weeks after myocardial infarction, which was significantly better than hearts transplanted with non-stretched MDSCs Myocardium transplanted with stretched MDSCs also displayed significantly higher angiogenesis in comparison to the non-stretched MDSC transplanted myocardium, which was further supported by a significant increase in vascular endothelial growth factor secretion after mechanical preconditioning Results suggest that transplantation of mechanically stretched MDSCs into acute infarcted myocardium ameliorates post-infarcted remodeling better than non-stretched MDSC transplantation by promoting higher levels of angiogenesis through paracrine factor secretion These findings highlight the importance of loading history for increasing the efficacy of stem cell transplantation 693 Effect of IL-3 on Transduction Efficiency in CD34+ Cell-Derived-Erythroid Cultures with Lentiviral Vectors Expressing the Human betaFlobin Gene Leda Ferro,1 Clare Taylor,1 Daniel Hollyman,1 Michel Sadelain,1,2,3,4 Isabelle Riviere.1,2,3,4 Gene Transfer and Somatic Cell Engineering Facility, Memorial Sloan-Kettering Cancer Center, New York; 2Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York; Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York; 4Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York Gene transfer approaches for β-thalassemia using recombinant LVs requires efficient gene transfer into HSCs and high level, stable expression of the b-globin gene in the erythroid lineage Although LVs are able to transduce non-proliferating cells, HSCs display low permissiveness to LVs and require cytokine prestimulation and high vector doses for high transduction efficiency Standard CD34+ prestimulation protocols use a combination of three earlyacting cytokines, Stem Cell Factor (SCF), Thrombopoeitin (TPO) and Flt3 ligand (Flt3-L) IL-3 alone or in combination with other hematopoietic cytokines has been shown to support multilineage colony formation, improve lentiviral and retroviral transduction and expansion of different lineages in liquid culture However its effect on the engraftment potential of cultured CD34+ cells is controversial Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy STEM CELL THERAPIES II We have developed a method to investigate the effects of different prestimulation conditions on transduction efficiency in vitro in short term culture of GCS-F mobilized human peripheral blood (PB) CD34+ cells Selected CD34+ cells are prestimulated, transduced and differentiated into either myeloid (Dendritic cell) or erythroid (Red blood cell) lineages in order to quantify vector copy number (VCN) and transgene expression in the different lineages Using this system we sought to investigate the effects of IL-3 prestimulation using TNS9.3, a LV carrying the human β-globin gene and associated regulatory sequences imparting erythroid-restricted transgene expression, as well as the enhanced green fluorescent protein (eGFP) reporter gene driven by the human PGK promoter We demonstrate that addition of IL-3 to the regular prestimulation cocktail (SCF, TPO and Flt3-L) increases the percentage of eGFP positive cells from 10% to 35% and the average VCN from 0.5 to After normalization of the VCN to the percentage of GFP+ cells, the VCN per cell was approximately 2.5 in the groups in which IL-3 was added and with the conventional cytokines prestimulation alone The VCN of single erythroid and myeloid methylcellulose colonies was 1-3 (average 1.6) for the groups in which the IL-3 was added and in the range of 1-5 (average 2.3) in the absence of IL-3 These data suggest that addition of IL-3 increases the number of CD34+ progenitor cells permissive to LV transduction Where as the absolute number of transduced cells increases upon treatment with IL-3, we observed an accelerated loss of the CD34 marker and an earlier onset of glycophorin A receptor expression and a skewing of colony formation manifested as a reduction in large immature colonies (BFU-E, CFU-GM) and an increase of BFU-E, CFU-M and CFU-G We are currently performing LTC-IC assays to further investigate the effect of IL-3 prestimulation on self-renewal and multilineage potential of CD34+ cells 694 Optimization of Vector Design for Lentiviral Gene Transfer into Human Embryonic Stem Cells Emi Aizawa,1 Kaoru Mitsui,1 Keiichiro Suzuki,1 Hirofumi Suemori,2 Norio Nakatsuji,3,4 Ko Mitani.1 Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan; 2Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Kyoto University, Kyoto, Japan; 3Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; 4Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan Lentiviral vectors (LVs) have become a valuable tool for gene delivery because they can stably transduce a variety of cells, including primate embryonic stem (ES) cells, in high efficiency An important characteristic of LVs is that they form a number of pseudotypes with different tropisms by incorporating envelopes derived from other viruses into the vector particles We compared ten pseudotypes to optimize stable gene delivery into 293 cells, mouse ES (mES) cells, cynomolgus monkey ES (cES) cells, and human ES (hES) cells The envelope genes used for this experiment were those from vesicular stomatitis virus (VSV-G), amphortopic retrovirus 4070A, amphortopic retrovirus 10A1, gibbon ape leukemia virus (GALV), RD114 feline endogenous virus, baculovirus (gp64), rabies virus, Mokola virus, lymphocytic choriomeningitis virus (LCMV)-WE, and LCMV-Arm536 Titers on each cell line were determined by counting the number of G418-resistant colonies after infection with a LV encoding the PGK-neo marker gene The VSV-G was the most effective for stable gene transfer into 293 cells, mES cells, and cES cells For hES cells, the gp64, the 4070A and the 10A1 have been effective, as well as the VSV-G However, at multiplicities of infection (MOIs) of higher than 100 vector particles/cell, the transfer efficiency with the gp64 was the highest Next, we have compared the relative strengths as well as the extent of suppression of the five ubiquitous promoters as an internal promoter for LV in hES Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy cells They are the cytomegalovirus (CMV) enhancer/promoter, the human phosphoglycerate kinase (PGK) promoter, the mutant murine leukemia virus LTR (MNDU3c) enhancer/promoter, the human translation elongation factor alpha (EF-1α) promoter, and the CMV enhancer/chicken β-actin (CAG) promoter They were tested to drive the downstream enhanced green fluorescent protein (EGFP) gene expression and the mean fluorescence intensity was compared Because the titer of LV with CAG promoter was consistently lower than that with CMV by ∼10-fold, LV with an internal CAG was not investigated further While CMV was the strongest in 293 cells, EF-1α promoter showed the activity, which was higher than other three by approximately 3-fold, in hES cells Interestingly, for all four promoters, only 2-4% of the integrated vector expressed the marker gene, suggesting that high degree of suppression observed in human ES cells might be caused not by an internal promoter but by vector backbone sequences By using the EF-1α promoter in a gp64pseudotyped LV, as high as 75% and 45% of the infected KhES-1cells and KhES-3 cells became GFP(+) at an MOI of 300, respectively Our results indicate that LVs are highly efficient and versatile methods for gene transfer into hES cells and potentially other types of human stem cells, such as induced pluripotent stem cells 695 Application of a “Double-Feature” Promoter To Identify Cardiomyocytes Differentiated from human Embryonic Stem Cells Tamas I Orban,1 Agota Apati,1 Andrea Nemeth,1 Nora Varga,1 Virag Krizsik,1 Anita Schamberger,1 Gyorgy Varady,1 Katalin Nemet,1 Zsuzsanna Izsvak,2 Balazs Sarkadi.1 Membrane Research Group of the HAS, Semmelweis University and National Blood Center, Budapest, Hungary; 2Mobile DNA Group, Max-Delbruck Center for Molecular Medicine, Berlin, Germany Human embryonic stem (HuES) cells represent a great potential for cell- and gene-therapy applications Apart from technical and experimental difficulties, however, serious ethical concerns must be resolved before they are actually used in clinical studies Major obstacles of HuES-based applications include the development of efficient and stable gene delivery systems, as well as the efficient control of directed tissue differentiations Nowadays, viral and nonviral applications are both used for gene transfer experiments and they are combined with various methods to obtain the tissue(s) of interest, including antibiotic selection with tissue-specific promoters or applying artificial drug cocktails Here we describe a novel approach to identify tissues differentiated from HuES cells using a “doublefeature” behavior of one version of the CAG promoter We generated GFP-expressing HuES clones under the expression of different promoters using the Sleeping Beauty non-viral transposon system or the SEW lentiviral system Neither of the gene delivery methods modified the subsequent differentiation of these HuES clones, and all kinds of tissue types could be identified after a spontaneous differentiation via the embryoid body formation method By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, EF1α, or PGK), an exceptionally high GFP expression was observed in differentiated cardiomyocytes This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites Such “double-feature” promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling To provide evidence that this promoter behavior manifests itself in tissue-dependent transcriptional profiles, we collected and studied cell populations with different GFP signal intensities by fluorescence activated cell sorting after differentiating the CAG-GFP or EF1α-GFP expressing HuES clones Using realtime quantitative PCR, we could confirm that GFP mRNA levels S265 ... methylcellulose colonies was 1 -3 (average 1.6) for the groups in which the IL- 3 was added and in the range of 1-5 (average 2 .3) in the absence of IL- 3 These data suggest that addition of IL- 3 increases... the percentage of GFP+ cells, the VCN per cell was approximately 2.5 in the groups in which IL- 3 was added and with the conventional cytokines prestimulation alone The VCN of single erythroid and... (VCN) and transgene expression in the different lineages Using this system we sought to investigate the effects of IL- 3 prestimulation using TNS9 .3, a LV carrying the human β-globin gene and associated