247 Embryonic Renal Progenitor Cells Activate the γ Catenin/TCF Signaling Pathway during Integration into Damaged Adult Renal Tubules Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © T[.]
FETAL AND ADULT STEM CELLS MicroRNA Card to analyze a focused set of genes and miRNA expression patterns in iPSC clones generated using CytoTuneTM iPS Sendai Reprogramming Kit and isolated using AP Live stain Comprehensive analysis of the resulting can be utilized not only to derive similaritites between the iPSC clones and control hESC lines but also dissect subtle differences further evaluate their impact on functionality and long term stability In addition, TaqMan® assays can be extended to carry out additional analysis for the confirmation of cellular karyotype 245 An ‘Open-Source’ Approach to Simple and Precise Genome Editing with rAAV Rob Howes,1 Alex Rieck,1 Eric Rhodes.1 Horizon Discovery, Cambridge, United Kingdom The ability to edit the human genome in a targeted and specific way in cell lines to introduce specific mutations relevant to human diseases is essential in understanding disease biology Horizon Discovery’s GENESIS gene-editing platform uses the unique ability of targeted rAAV vectors to naturally stimulate and take advantage of the high fidelity homologous recombination DNA repair pathway in human cells to precisely engineer any DNA variation or tag in any endogenous protein of choice rAAV has been used in multiple cell types to introduce disease-associated mutations to allow understanding of their phenotype in an isogenic background It has also been used to correct existing mutations in cell lines derived from patient samples, for example correction of a mutation causing osteogenesis imperfecta in patient-derived iPSC’s Horizon Discovery established its Centre of Excellence (COE) program in 2011 to allow rapid adoption of this technology within academia The aim of this program is to give the global academic community free access to Horizon’s GENESIS genome editing technology to facilitate new discoveries in life science research using our novel vector improvements and the depth and breadth of knowledge about AAV There are many benefits to joining the COE program including expert support, access to in-house optimised protocols and produced reagents and technical improvements in rAAV gene targeting Members also become part of the rAAV gene editing community allowing collaboration and interaction with world-leaders in this technology The program has developed rapidly since its launch with over 30 centres and 120 projects, across a range of therapeutic areas including oncology and regenerative medicine, and using over 25 different cell lines in various tissue types To demonstrate the success of the program, we will describe in detail the generation and characterisation of human isogenic cell lines involved in oncology and cardiovascular disease We will present data detailing how the use of these cell lines has led to a better understanding of these diseases We will also describe how academic groups can access this precise and flexible technology through the COE program at no cost 246 UV Light Modulates Transcriptional Activity of Egr-1 Promoter on Human Primary Tenocytes Francisco Martinez-F,1,2 Araceli Barrera-Lopez,1 Hugo SandovalZamora,1 David T Curiel,3 Juan A Madinaveitia-V,1 Rebecca E Franco-Bourland.4 Molecular Biotherapeutic Program, Skin & Tissue Bank, National Institute of Rehabilitation Ministry of Health, Mexico City, DF, Mexico; 2Department of Pharmacology, School of Medicine, National Univeristy of Mexico, Mexico City, DF, Mexico; 3Department of Radiation Oncology, School of Medicine, University of Washington, St Louis, MO; 4Department of Biochemistry, National Institute of Rehabilitation, Mexico Introduction: Tenocytes are the principal cell population in human tendon with high metabolic activity and synthesis of matrix protein Egr-1 expression has been implicated in tendon differentiation, S94 synthesis and increase of collagen expression during tendon cell differentiation However, transcriptional regulation on the egr-1 promoter has not been described in adult human tenocytes Hereby, we evaluate the effect of UV light on egr-1 promoter in human primary tenocytes transduced with adenoviral vector Adegr1-Luc Materials and methods: Cells and adenoviral vectors: No replicative recombinant adenovirus (AdEgr1-Luc) were packaged at large scale in HEK-293 cells and purified according to the current protocol of the Core Facility of PBM-INR, based on cesium chloride gradient protocol for in vivo application Viral Stock was titled by plaque assay and OD Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol cells were cultured in DMEM/F12 media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for week and stored for experimental procedure 5x104 tenocytes were seeded/well After 12 hrs, cells were infected with AdEgr1-Luc at 50 MOI´s during two hrs in serum reduced media After infection, cells were keeped in 1% of FBS for 24 hours at environment standard conditions for culture until exposition to UV light (15, 30, 60, 90 and 120 seg) Protein extraction was performed at 2, and hrs based on Cell Glo Lysis Buffer (Promega corp.) and luciferasa activity was quantified using a multidetector DTX-880 Results: Luciferase assays from proteins obtained of not transduced cells not shows luciferase activity (0.33-0.5 LC/s) UV exposition of human tenocytes transduced with Adegr1-Luc induces positive activity of egr-1 promoter Luminescent activity was observed at two, and hrs (1,490 LC/S; 2,904.66 LC/s and 29,511.33 LC/s, respectively) Transcriptional activity induced by EGF (29,511.33 LC/s) was over the positive control with 10% of FBS (23,915.33 LC/s.) Conclusion UV light activates transcriptional activity of egr-1 promoter in human tenocytes However, this activity could be a final event of no genomic pathway, and could be discerned by other studies using proteomic tools This research project is granted by the National Council of Science and Technology of México Grant FOSIS/CONACYT-Salud-2011-1-161624 Fetal and Adult Stem Cells 247 Embryonic Renal Progenitor Cells Activate the β-Catenin/TCF Signaling Pathway during Integration into Damaged Adult Renal Tubules Paul Goodyer,1 Zhao Zhang,1 Diana Iglesias.1 Pediatrics, McGill University, Montreal, QC, Canada The therapeutic potential of exogenously infused mesenchymal stem cells is limited to their salutary paracrine effects, since they are unable to differentiate toward an epithelial phenotype and are rarely integrated into damaged adult tissue In contrast, renal progenitor cells (RPC) isolated from the metanephric mesenchyme of embryonic kidney are rapidly integrated into damaged proximal tubules following acute glycerol-induced kidney injury During nephrogenesis, CD24(+) RPC in the cap mesenchyme are induced to differentiate by Wnt9b signals arising from the ureteric bud (UB) We reasoned that activation of the canonical WNT/ -catenin signaling might be essential for successful integration and differentiation of exogenous stem cells into the damaged adult kidney To address this hypothesis, we first isolated CD24(+) cells from embryonic E15 mouse kidney, labeled them with PKH26 Red and infused them into adult mice and days after glycerol-induced renal proximal tubular injury We observed wide CD24(+) cell integration into damaged tubules The red-labeled cells also showed epithelial polarization and staining for the proximal tubular cell marker lotus tetragonolobus agglutinin We then isolated embryonic CD24(+) cells from a mouse bearing a -catenin/TCF reporter transgene (RPCTCF) and showed that they activate the canonical WNT pathway in response to co-culture with E15 UB cells or L-cells expressing WNT3a We next infused the CD24(+) RPCTCF into adult mice at the peak of Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy FETAL AND ADULT STEM CELLS glycerol-induced proximal tubular injury and tracked their WNT signalling pathway activity with X-gal staining The exogenous RPCTCF showed robust reporter activity at sites within the urinary pole of Bowman’s capsule along the proximal tubule Cells with robust reporter activity co-stained for both WNT4 and PCNA To examine WNT/ -catenin pathway activation in endogenous tubular cells, we also induced injury in adult mice bearing the -catenin/ TCF reporter and noted patchy but widespread reporter activity and WNT4 expression among endogenous tubular cells Based on these observations, we propose that infused embryonic RPC activate the WNT -catenin/TCF pathway as they integrate, proliferate and differentiate within the damaged tubule; this recapitulates the events which occur during primary nephrogenesis and may be essential for renal tubular regeneration 248 Preclinical Investigations of Therapeutic Benefits and Biodistribution from Stress Erythropoiesis on Minimal Dose of Lysosomal Enzyme Required for Phenotypic Correction in Mice with Hurler Syndrome Jingfen Han,1 Mei Dai,2 Salim S Elamouri,1 Phuong Cao,1 Dao Pan.1,2 Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Department of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH The benefits of reprogramming erythroid cells for high-level lysosomal enzyme production with less risk of activating oncogenes in hematopoietic stems cells (HSC) and their progeny have been recently reported by our group However, it remains unclear what is the minimal dose of alpha-L-iduronidase (IDUA), a lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I), required for phenotypic correction in organs and if increasing erythropoiesis can provide further therapeutic benefits We sought to evaluate IDUA expression and potential therapeutic improvement following stress erythropoiesis induced by repeated phlebotomy, and determine the minimal transgene doses required for metabolic correction in major organs, including liver, spleen, kidney, heart and brain, in MPS I mice after HSC-mediated gene transfer with a lentiviral vector expressing IDUA from an erythroid/megakaryocytic promoter Based on transgene frequency determined by qPCR in bone marrow, treated MPS I animals were divided into four groups as MPS/ GT 200%, MPS/GT 10%, MPS/GT 2%, and MPS/GT