660 bowelbladder sensation and control in patients with spinal cord injury treated with human embryonic stem cell therapy

660 Bowel/Bladder Sensation and Control in Patients with Spinal Cord Injury Treated with Human Embryonic Stem Cell Therapy Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American[.]

Stem Cell Engineering and Therapies II 657 Delivery of Genetically Engineered Adipose-Derived Cell Sheets for Treatment of Ischemic Disorders - Development of Application in Animal Models Pavel I Makarevich,1,3 Konstantin V Dergilev,1 Zoya I Tsokolaeva,1 Anastasia Yu Efimenko,1,2 Evgeny V Gluhanuk,2 Julia O Gallinger,2 Polina A Rodina,2 Stepan S Sarkisyan,2 Ksenia V Kerova,2 Yu-Chen Hu,4 Yelena V Parfyonova.2,3 Laboratory of Regenerative Medicine, Lomonosov Moscow State University, Moscow, Russian Federation; 2Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russian Federation; 3Laboratory of Angiogenesis, Russian Cardiology Research and Production Complex, Moscow, Russian Federation; Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan Cell sheet (CS) approach is extensively developed in recent years for a number of applications in regenerative medicine including lesion treatment, tissue-engineered constructs and organoplastics In this study we have targeted ischemic diseases - myocardial infarction (MI) and peripheral artery disease (PAD) using CS from differetnt sources transplanted as CS to the impaired region We established a protocol for generation of CS from c-kit+ cardiac primitive cells and cardiosphere cells for delivery to rats with induced MI using clinical-grade fibrin glue for CS adhesion subepicardially We found CS to be vascularized after transplantation and used ultrasound assessment to detect improvement of left ventricle function and significant positive changes of tissue status in histology studies: capillary density and reduction of fibrosis) Moreover, we conducted a comparative analysis of this approach vs CS from adipose-derived stomal cells (ADSC) and VEGF-expressing ADSC The latter were obtained via baculovirus transduction of CS, which enabled significant production of human VEGF165 by murine cells and, thus, enhanced their therapeutic potential Our previous application of ADSC for therapeutic angiogenesis and established methods for genetic modification of cells allowed us to develop a protocol in limb ischemia model We have demonstrated, that subcutaneous implantation of CS or VEGF-expressing CS from mouse ADSC resulted in significant improvement of limb perfusion with modified cells to be superior to mock-transduced Increased blood flow was supported by higher CD31+ capillary density, reduction of necrosis and detection of viable CS subcutaneously with focal proliferation of mADSC and minimal (app 10%) prevalence of apoptotic cells at 14 days post transplantation Paracrine mechanism for CS mode of action is suggested due to the fact, that ww found vessel growth within CS mass, which indicates possibility of cells’ nutrition and uptake of secreted proteins from the site of transplantation besides diffusion Thus, our findings indicate, that CS-based delivery for treatment of MI or PAD is a potential method in regenerative medicine in this field and furthermore genetic modification of cells used for CS generation may be a way to “tune up” the cells’ paracrine activity and efficacy of their application 658 Increasing Fetal Hemoglobin Expression With TALEN Induced Small Deletions in Peripheral Blood Stem Cells Christopher T Lux,1 Andrew M Scharenberg,1 Brandon K Hadland.2 Seattle Children’s Research Institute, Seattle Children’s Hospital, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA Hemoglobinopathies are common and morbid inherited conditions caused by mutations in hemoglobin genes that generate pathologic S262 globin molecules or reduced expression of wild type hemoglobin A subset of individuals with hemoglobinopathy-associated beta globin mutations have a mild clinical phenotype and are found to carry additional disease modifying mutations including small deletions or point mutations that induce fetal hemoglobin expression This condition is referred to as hereditary persistence of fetal hemoglobin Recent progress in the arenas of gene editing and hematopoietic stem cell (HSC) expansion has raised the possibility of treating hemoglobinopathies through direct modification of genes in HSCs to increase the expression of fetal hemoglobin in the erythroid lineage To date, most attempts to induce fetal hemoglobin have targeted the expression of transcription factors implicated in regulation of fetal hemoglobin production, such as Bcl11a, an approach which could lead to unintended and potentially deleterious effects on cellular phenotype Here, we are evaluating the application of mRNA transfection of TALENs to directly modify transcription factor binding sites within the hemoglobin locus in HSCs to achieve efficient, stable expression of fetal hemoglobin We have designed TALENs to induce small deletions in the beta hemoglobin promoter at the binding site of KLF1, postulated to play a role in localizing the active chromatin hub to and preferentially expressing adult beta hemoglobin, as well as other globin locus target regions implicated in the regulation of fetal hemoglobin production By combining TALEN-mediated gene modification with novel HSC expansion approaches involving the recently described small molecule UM729, we hope to realize both efficient HSC modification and expansion with a sufficient level of fetal hemoglobin production in erythrocytes to achieve a functional cure for a wide range of beta hemoglobinopathies 659 Precision Genome Engineering in Human Induced Pluripotent Stem Cells Using CRISPR/ CAS9 Jon P Connelly,1 Amber M Neilson,1 Sam T Peters,1 Rita Martinez,1 Jeffrey D Milbrandt,1 Shondra M Miller.1 Genetics, Washington University, St Louis, MO Site-specific nucleases are engineered to create a double-stranded DNA break at a defined genomic locus Subsequent repair of these DNA breaks by the cellular endogenous DNA repair pathways can then be exploited to create genetically modified cell lines Genes can be knocked out, disease relevant mutations introduced, or proteins can be tagged for subsequent monitoring in differentiation assays and high throughput small molecule screens Human induced pluripotent stem cells (iPSCs) have the potential to differentiate into any somatic cell in the body Importantly, they can be derived from both normal individuals, as well as from patients The combination of these two technologies allows investigators to create custom modified cell lines harboring site specific genetic changes of interest relevant to their field of study Here, we present two examples of precise and efficient genome editing without the use of antibiotic selection using the CRISPR/Cas9 system in iPSCs First we demonstrate the steps involved in the creation of a knockout (SARM1) iPSC line Second, we outline the workflow for creating a custom iPSC line that has been tagged with a fluorescent protein at the endogenous locus (CHAT) 660 Bowel/Bladder Sensation and Control in Patients with Spinal Cord Injury Treated with Human Embryonic Stem Cell Therapy Geeta Shroff Nutech Mediworld, New Delhi, India Introduction: Spinal cord injury (SCI) involves a complex series of pathological events resulting in long-lasting locomotor and sensory neuron degeneration The present study evaluated the bowel/bladder sensation and control in patients with SCI after human embryonic stem cell (hESC) therapy Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Stem Cell Engineering and Therapies II Methods: This is a retrospective analysis (May 2005-Aug 2012) of 226 SCI patients (mean age-28 years) treated with hESC therapy Therapy schedule had four treatment phases (T1, T2, T3, T4) lasting 4-6 weeks separated with gap phases (4-8 months) hESCs in an injectable form were administered at a dose of 0.25 ml (