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690. Antioxidant Treatment of Muscle Stem Cells for Transplantation Increases Cardiac Repair
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 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 1 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, 1 to 2 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 1 and 25-30% of human CD34+ cord blood cells expressed GFP on day 2 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 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 1 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; 3 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
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 1 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, 1 to 2 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 1 and 25-30% of human CD34+ cord blood cells expressed GFP on day 2 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 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 1 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; 3 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