ISSCR 2019 Poster Abstracts

600POSTER ABSTRACTSF-3164MODELLING NON-ALCOHOLIC FATTY LIVER DISEASE USING HUMAN INDUCED PLURIPOTENT STEM CELLSLo, Peggy Cho Kiu - Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, UK Grove, Jane - Faculty of Medicine and Health Sciences, Nottingham Digestive Diseases Centre, Nottingham, UK Aithal, Guruprasad - Faculty of Medicine and Health Sciences, Faculty of Medicine and Health Sciences, Nottingham, UK Hannan, Nicholas - Faculty of Medicine and Health Sciences, The University of Nottingham, UKNon-alcoholic fatty liver disease (NAFLD) refers to a spectrum of disorders caused by accumulation of excess dietary lipids within the liver. NAFLD is the fastest growing chronic liver disease and without intervention can progress to liver cancer and liver failure. Poor prognosis and limited therapeutic options for patients is hindered by the absence of robust, human specific platforms for modelling NALFD. To address this need we have created an in-vitro human model of NAFLD using hIPSCs derived from patients with familial NAFLD. In this study, we have improved our differentiation protocol to differentiate hIPSCs into hepatocytes that are more similar to primary human hepatocytes than foetal hepatocytes. We demonstrate higher expression of cytochrome P450 enzymes as well as many genes involved in a broad range of metabolic processes including lipid biosynthesis and metabolism. We have then applied this more advanced platform to model NAFLD by inducing lipid accumulation in healthy and familial-NAFLD-hepatocytes using varying concentrations of palmitic acid. Palmitic acid replicates a high fat, western diet and we observed both control and NAFLD-hepatocytes demonstrate key features of hepatic steatosis such as accumulation of lipids within the cytoplasmic space. Familial-NAFLD-hepatocytes when compared to healthy hepatocytes treated with palmitic acid show unique differences in their inflammatory and fibrotic gene expression profiles that may represent biomarkers for people with genetic predisposition to NAFLD. Familial-NAFLD-hepatocytes also show unique activation of signalling kinases that may represent novel interventions for NALFD. Finally, we also observe significant differences in metabolic profiles and mitochondrial functionality between healthy and NAFLD-hepatocytes that may represent new mechanisms that drive the spectrum of clinical observations in the pathogenesis of NALFD. Our study demonstrates the potential of NAFLD-hepatocytes to provide mechanistic insight and discovery of novel biomarkers and drug targets for NAFLD.Funding Source: Rosetrees Trust and the Nottingham University Digestive Diseases CentreF-3166REPERTAXIN ATTENUATES THE COLITIC PHENOTYPE IN HUMAN-DERIVED ULCERATIVE COLITIS ORGANOIDSKamali, Samaneh - Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Yeu, Yunku - Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Fisher, Robert - Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Cruise, Michael - Department of Pathology, Cleveland Clinic, Cleveland, OH, USA Spence, Jason - Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Huang, Emina - Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USAUlcerative colitis (UC) is a relapsing inflammatory colonic disorder characterized by bloody diarrhea associated with sloughing of the colonic mucosa. Current ex-vivo models of UC inadequately recapitulate the complexity of the pathogenesis. We established an ex-vivo human derived organoid model (iHCOs) to compare the developmental process of UC to normal colon (NL). This model has both epithelial and mesenchymal components reflecting major phenotypic characteristics of UC. We hypothesize that the inflammatory phenotype of UC iHCOs affects their developmental pattern, specifically the aberrant formation of the adherens junction (AJ) vs. tight junction (TJ) structure in the epithelial barrier. Yamanaka factors were applied to reprogram NL and UC isolated fibroblasts into induced pluripotent stem cells (iPSCs) followed by directed differentiation to iHCOs. Downstream effects of the pro-inflammatory phenotype in UC iHCOs originating from the interaction of CXCL8-CXCR1/2 axis were studied in the context of development. Furthermore, the effect of repertaxin, a CXCR1/2 antagonist (3 weeks of exposure) on the development of UC iHCOs was investigated. We established robust conditions to differentiate iPSCs to iHCOs reflecting the major phenotypes in the tissue of origin including an elevated secretion of CXCL8, a pro-inflammatory chemokine, in the mesenchyme compartment of UC iHCOs (7.167x NL; p < 0.005). The transcriptome of UC vs. NL iHCOs revealed an inflammatory signature consisting of the AJ vs.TJ structure in the epithelial barrier. In response to repertaxin, UC iHCOs revealed more NL phenotype including morphology, cell junction and a decrease in the aberrant regeneration pattern of the epithelium (average of 20 μm decrease in epithelium thickness and a 65% decrease in the number of organoids vs. control; p < 0.0001). UC iHCOs recapitulate the phenotype of their primary tissues and are functionally responsive to CXCL8-CXCR1/2 axis inhibition. The creation of human derived organoids model will permit investigation not only of the developmental, pharmacologic, and genetic aspects of UC, but also, the role of inflammatory mediators in the microenvironment.Funding Source: This work is sponsored, in part, by NIH CA157663, U01 CA214300, and ASCRS RF LPG 100.

601POSTER ABSTRACTSF-3168ESTABLISHMENT OF A DRUG SCREENING PLATFORM FOR FAMILIAL HYPERTROPHIC CARDIOMYOPATHY IN HIPSCS USING CRISPR/CAS9Park, Sang-wook - New Drug Development Center, DGMIF, Daegu, Korea Min, Sang-hyun - New Drug Development Center, DGMIF, Daegu, KoreaProgrammable nucleases such as CRISPR/Cas9, ZFN, TALEN, and Cpf1 are versatile tools for editing specific genomic sites in hiPSCs. In particular, CRISPR/Cas9 efficiently enable the introduction of genetic changes into hPSCs in a site-specific manner, including correction of pathogenic mutations in patient-derived hPSCs and introduction of specific mutations into normal hPSCs. These approaches enable the generation of genetically matched, isogenic iPSC lines. In our study, we established a Familial Hypertrophic Cardiomyopathy (FHC) model from normal hiPSCs using a CRISPR/Cas9 genome editing tool. About 80% of for FHC is caused by genetic mutations in MYH7 and MYBPC3. In particular, an intronic 25bp deletion in MYBPC3 gene is a prevalent genetic mutation in Southeast Asia. It is estimated that 4% of the Southeast Asians carries this pathogenic mutation. To better understand and develop drugs for FHC, we generated FHC-hiPSC harboring an 25bp deletion in intron 32 of MYBPC3 gene by introducing carefully designed dual sgRNAs, Cas9 protein, and 195mer ssODN in normal hiPSCs. About 4% of CRISPR/Cas9 transfected hiPSCs showed bialleclic 25bp deletion in MYBPC3 gene by targeted deep sequencing and Sanger sequencing. These gene-edited hiPSCs exhibited normal pluripotency and stemness compared with genetically matched normal hiPSCs. Based on these results, we are going to find effective drugs from various clinically available drug libraries by differentiating MYBPC325bp -hiPSCs into cardiomyocytes.ΔF-3170TARGETED PRECISION THERAPIES FOR INFANTILE PARKINSONISMBarral, Serena - ICH, University College London, UK Ng, Joanne - Institute for Women’s Health, University College of London, UK De La Fuente Barrigon, Carmen - GOS-Institute of Child Health, University College of London, UK Lignani, Gabriele - Institute of Neurology, University College of London, UK Erdem, Fatma - Centre of Physiology and Pharmacology, Medical University of Vienna, Austria Wallings, Rebecca - , Department of Physiology, Anatomy and Genetics, University of Oxford, UK Privolizzi, Riccardo - Institute for Women’s Health, University College of London, UK Meyer, Esther - GOS-Institute of Child Health, University College of London, UK Alrashidi, Haya - GOS-Institute of Child Health, University College of London, UK Ngoh, Adeline - GOS-Institute of Child Health, University College of London, UK Pope, Simon - Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK Karda, Rajvinder - Institute for Women’s Health, University College of London, LUK Perocheau, Dany - Institute for Women’s Health, University College of London, UK Baruteau, Julian - Institute for Women’s Health, University College of London, UK Antinao Diaz, Juan - Institute for Women’s Health, University College of London, UK Schorge, Stephanie - Institute of Neurology, University College of London, UK Cowley, Sally - Sir William Dunn School of Pathology, University of Oxford, UK Freissmuth, Michael - Centre of Physiology and Pharmacology, Medical University of Vienna, Austria Counsell, John - GOS-Institute of Child Health, University College of London, UK Wade-Martins, Richard - Department of Physiology, Anatomy and Genetics, University of Oxford, UK Heales, Simon - Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK Rahim, Ahad - School of Pharmacy, University College of London, UK Bencze, Maximilien - GOS-Institute of Child Health, University College of London, UK Waddington, Simon - Institute for Women’s Health, University College of London, UK Kurian, Manju - GOS-Institute of Child Health, University College of London, UKMost inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in Dopamine Transporter Deficiency Syndrome (DTDS), caused by bi-allelic mutations in SLC6A3, which encodes the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuronal model of DTDS. Patient-derived neurons exhibited impaired DAT activity and apoptotic neurodegeneration associated with TNF -mediated inflammation and dopamine toxicity. Whilst DAT αactivity was ameliorated with the pharmacochaperone pifithrin-μ, the effect was mutation-specific. In contrast, Lentiviral gene transfer restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress towards clinical translation, we utilized the knockout (KO) mouse model of DTDS, which recapitulates human disease, with reduced survival and parkinsonism features, including tremor and bradykinesia.

602POSTER ABSTRACTSNeonatal intracerebroventricular injection of adeno-associated virus (AAV) vector provided neuronal expression of human DAT which rescued motor phenotype, lifespan and neuronal survival in the substantia nigra (SNc) and striatum.F-3172GENERATION AND FUNCTIONAL CHARACTERIZATION OF HUMAN MICROGLIA FROM INDUCED PLURIPOTENT STEM CELLSBurke, Tom - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USA Burton, Sarah - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USA Hancock, Michael - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USA Hilcove, Simon - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USA Kim, Kwi Hye - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USA Rajesh, Deepika - Fujifilm Cellular Dynamics, Fujifilm Cellular Dynamics, Madison, WI, USAMicroglia are immune-competent cells residing within the brain that play a critical role in maintaining immunological balance for normal brain function. As phagocytic cells, they are activated following clearance of pathogens and secreted proteins and respond by either exacerbating or dampening a pro-inflammatory environment. Primary human microglia are difficult to acquire and stably culture in vitro. We generated and characterized functional human induced pluripotent stem cell-derived microglia (iCell® Microglia) from episomally reprogrammed iCell Hematopoietic Progenitor Cells (proprietary technology) under defined conditions. iCell Microglia express CD45, CD11b and CD33, and consistent with a microglial phenotype, they also express PU.1, CX3CR1, IBA, TREM-2 and P2RY12. The purity for all these markers is greater than 80% and the retain the expression of all these markers post cryopreservation. iCell Microglia were able to phagocytose opsonized bacteria and fibrillar Aβ within a span of 2-60hrs and reveal a ramified morphology when treated with 5ÂμM Thiazovivin. Additionally, iCell Microglia secrete cytokines and chemokines including TNFα, IL-8, IL-10, CCL2, CCL4, CCL3, CCL4, CXCL10, CXCL11, CXCl1, CXCl2 and CXCL10 when stimulated with LPS and interferon gamma. iCell Microglia will serve as a great tool for disease modeling and drug testing for neuroscience research.F-3174USING HIPSC DERIVED ASTROCYTES TO INVESTIGATE APOE EFFECTS IN ALZHEIMER’S DISEASERaman, Sreedevi - School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA Brafman, David - School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USAAlzheimer’s disease (AD) is the sixth leading cause of death in the United States and poses an increasing burden on the healthcare system. Following the first report of AD in 1907, numerous clinical trials addressing the classic amyloid hypothesis have failed to identify a cure. It is essential to understand the role of genetic risk factors such as the cholesterol transport protein apolipoprotein E (ApoE) in the more prevalent sporadic form of the disease. ApoE is highly expressed by astrocytes in the human brain, where it is found as the E2, E3 and E4 isoforms, with ApoE3/3 being the most common genotype. The less common E4 allele greatly increases the risk of developing sporadic AD and reduces the mean age of onset from 84 to 68 years, whereas the E2 allele has a protective effect. The mechanism of this effect remains unknown and ApoE has been implicated in amyloid peptide aggregation and clearance defects in animal models. Although the mouse model of AD has provided invaluable mechanistic insight and led to the identification of early biomarkers of the disease, translation into human therapy has been unsuccessful. With the advent of iPSC technology, it is possible to model AD using reprogrammed patient cells and study disease progression as it relates to the complex human genetic landscape. We differentiated six (two non-demented control, familial and sporadic AD) patient derived iPSC lines into neural progenitor cells (NPC) and subsequently into astrocytes on laminin and VDP, a synthetic substrate that supports the maintenance of cell types of the CNS. Our robust protocol generated mature astrocytes that secreted ApoE, exhibited calcium transience and were responsive to inflammatory stimuli. These astrocytes took up fluorescently labeled A -42, indicative of their role in βA peptide clearance. They retained their astrocytic identity βand functionality following cryopreservation. Additionally, the differentiation protocol was scaled up to a bioreactor system to generate mature functional astrocytes. We are currently studying the isoform specific effects of ApoE in astrocytes and neurons generated using isogenic lines derived from a familial AD patient. We plan to study the mechanism of ApoE isoform specific effects in co-culture systems of astrocytes and neurons derived from patient iPSCs.F-3176ALTERED NEURAL PROGENITOR PROLIFERATION ASSOCIATED WITH COPY NUMBER VARIANTS IN PSYCHIATRIC DISORDERSBrickler, Thomas - Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, CA, USA Li, Jingling - Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, CA, USA Banuelos, Allison - Stem Cell Institute, Stanford University, Palo Alto, CA, USA Marjon, Kristopher - Stem Cell Institute, Stanford University, Palo Alto, CA, USA Bian, Jing - Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, CA, USA Chetty, Sundari - Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, CA, USA

603POSTER ABSTRACTSCopy number variations (CNVs) of certain chromosomal regions are closely associated with neurodevelopmental and neuropsychiatric disorders such as autism spectrum disorder (ASD) and schizophrenia. Recent evidence suggests that alterations in neural progenitor cell (NPC) proliferation associated with particular CNVs may underlie the cause of abnormal brain development. However, we still have limited knowledge of the cellular and molecular profiles of NPCs during neurodevelopment that adds to the complexity of these diseases, which can affect multiple cell types in different regions of the brain. In order to investigate the cellular and molecular mechanisms underlying neuropsychiatric disorders, we have used NPCs and neurons derived from human induced pluripotent stem cells (hiPSCs). Here, we generated NPCs and neurons from patients diagnosed with a psychiatric disorder and particular CNVs to better model these disorders and identify molecular targets for intervention. We have found that proliferation rates of NPCs can be closely tied to specific CNVs, allowing us to group patients by particular disease phenotypes to help gain insight into the disruption of cellular and molecular pathways during development. Interestingly, these differences in NPC proliferation pathways mechanistically converge with neuroimmune mechanisms. To identify how these signaling roles play in cell survival and clearance, we have co-cultured our derived NPCs and neurons with immune-derived cells and assessed differences in cellular elimination. Many forms of psychiatric disorders such as ASD and schizophrenia are associated with abnormal synapse connections which could allude to improper elimination of NPCs that lay the groundwork for the brain architecture. Our study will be the first to systematically investigate novel neuroimmune-related mechanism(s) tied to ASD and schizophrenia as well as correlate a clinical phenotype to a cellular phenotype.F-3178A HUMAN IPS CELL FUNCTIONAL MYOGENIC MATURATION SYSTEM ENABLING ACUTE AND CHRONIC DISEASE MODELING OF DUCHENNE MUSCULAR DYSTROPHYUchimura, Tomoya - Center for iPS Cell Research and Application/CiRA, Kyoto University, Fujisawa, Japan Sakurai, Hidetoshi - Clinical Application, Kyoto University/CiRA, Kyoto, JapanDuchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and degeneration. There are currently no available treatments for the disease. Although the lack of dystrophin protein causes the disease, the mechanisms underlying its pathogenesis still remain unclear. While induced pluripotent stem cells (iPSCs) are a powerful tool for understanding the pathogenesis of intractable diseases, currently iPSCs-based disease modeling of muscular dystrophy is critically lacked. Such a model is essential to understand the cellular and molecular events during muscle weakness and degeneration. However, in vitro culture system enabling skeletal muscle cells to be mature enough to function is quite limited. In this study, we developed a MyoD expression-induced myogenic culture system combined with replating technique and electrical-field stimulating (EFS)-mediated muscle training on a 12kPa soft gel. Myotubes trained under this condition showed progressed myogenic maturation characterized by increased cell fusions and expression of slow and fast muscle markers. In addition, cells started contracting (excitation-contraction coupling) at day 14, and the sarcomere formation was organized by day 16. Furthermore, we developed an acute and chronic disease model of DMD using patient-derived iPSCs. In the acute model, dystrophic cells showed a significant reduction of muscle performance as well as induction of inflammatory responses and apoptosis. In the chronic model, dystrophic cells showed comparable muscle performance until day 20 compared to normal cells. However, the muscle performance of dystrophic cells started to drop down after day 22, indicating progressive muscle weakness and fatigue. Finally, we stablished a new model for culturing contracting skeletal muscle cells and recapitulated dystrophic phenotypes using patient-derived iPSCs in vitro. Further investigations will identify how muscle weakness and degeneration are initiated and progressed under the dystrophic condition, and design a strategy to develop a novel drug to treat the disease.F-3180STRUCTURE-FUNCTION STUDY OF NEUROGENIN3 DISEASE CAUSING ALLELES DURING HUMAN PANCREAS AND INTESTINAL DEVELOPMENTZhang, Xinghao - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA McGrath, Patrick - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Salomone, Joseph - Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA Rahal, Mohamed - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA McCauley, Heather - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA Schweitzer, Jamie - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA Gebelein, Brian - Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USA Wells, James - Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cinicinnati, OH, USAThe transcription factor Neurogenin3 (NEUROG3) is required for formation of all endocrine lineages of the pancreas and intestine. Patients with homozygous or compound heterozygous NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells (EECs), whereas the impact on endocrine pancreas development varies in an allele-specific manner, ranging from neonatal diabetes to

604POSTER ABSTRACTSlater onset in life. These findings suggest a context dependent requirement for NEUROG3 in pancreas versus intestine that can only be accurately studied in an endogenous context. We utilized a NEUROG3-/- human pluripotent stem cell line to functionally analyze NEUROG3 mutants in the context of developing human pancreatic and intestinal tissue. NEUROG3-/- cultures failed to form any pancreatic or intestinal endocrine cells, demonstrating its requirement for human endocrine development. Endocrine specification was fully rescued by physiologic expression of wild type NEUROG3, and we used endocrine cell rescue to test the activity of mutants’ effect on pancreatic and intestinal endocrine development. Most disease-associated NEUROG3 alleles had similar hypomorphic or null phenotypes in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but was largely null in the intestine. Molecular and biochemical studies revealed NEUROG3 variants have distinct molecular defects that affect protein stability, phosphorylation, heterodimer formation with E-proteins, and/or DNA binding. Moreover, we found that the NEUROG3 protein was highly unstable in the intestinal epithelium relative to the pancreas, possibly explaining the enhanced sensitivity of intestinal endocrine cell defects relative to the pancreas in patients with disease associated NEUROG3 alleles. In addition to revealing the molecular and developmental defects in patient-derived NEUROG3 mutations, these studies emphasize that studies of human mutations in the endogenous tissue context may be required to accurately assess structure-function relationships.Funding Source: NIH grants R01NS044080, R01DK092456, U19 AI116491, P01 HD093363-01, UG3 DK119982 and the Digestive Disease Research Center (P30 DK078392)F-3182HUMAN INDUCED PLURIPOTENT STEM CELL DERVIED CARDIOMYOCYTES AS DRUG SCREENING PLATFORM OF CARDIAC LAMINOPATHYlEE, Yee Ki, Carol - Medicine, The University Of Hong Kong, Pokfulam, Hong Kong Lau, Yee Man - Medicine, The University of Hong Kong, Pokfulam, Hong Kong Ran, Xinru - Medicine, The University of Hong Kong, Pokfulam, Hong Kong Cai, Zhu Jun - Medicine, The University of Hong Kong, Pokfulam, Hong Kong Lai, Wing Hon - Medicine, The University of Hong Kong, Pokfulam, Hong Kong Siu, Chung wah - Medicine, The University of Hong Kong, Pokfulam, Hong Kong Tse, Hung Fat - Medicine, The University of Hong Kong, Pokfulam, Hong KongLamin A (LMNA) is an essential component in nuclear matrix served to maintain chromosome and genome integrity. We hypothesize that premature heart block and DCM phenotype of lamnopathy is due to retarded mechano-sensitivity of lamin in nuclear structure via actin-polymerization pathway. We aim to investigate the arrhythmogenic effects of mechanosensitive ion channels in cardiac laminopathy by human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) bearing LMNA mutations. hiPSC of a patient bearing LmnaR225X/WT, an isogenic corrected, LmnaWT/WT and mutated lines, LmnaR225X/R225X were created by CRISPR-Cas9. To investigate mechanical-related regulation, the hiPSC-CMs were treated with either F-actin depolymerization small molecules, latrunculin B (0.5 μM), cytochalasin D (10 μM), or myofilament desensitizer, blebbistatin (250 nM). To study subsequent force-frequency relationship (FFR) of electrical-contraction (EC) coupling, calcium transients and contractility were recorded under electrical field stimulation or stretching condition. Apart from action potential (AP) recording to study proarrhythmic risk related to APD prolongation, we further extrapolate our study to develop mechanism-based drug interventions using multielectric array (MEA) and optical mapping of cell monolayers in a high throughput way. Disorganization of F-actin in LmnaR225X/WT-hiPSC-CMs, interconnecting nuclear lamina to sarcomere and cell surface, reduced nuclear integrity and disrupted uniform force generation making the cells being overstretched. Latrunculin B and blebbistatin could resume the compromised EC coupling in the LmnaR225X/R225X cells with upstroke velocity significantly boosted by ~75% (n=5). Furthermore, electrical instability as reflected by loss in rate dependency (1-2 Hz) of the mutated group could be rescued by another small molecule for actin depolymerization, cytochalasin D (n=10). The ranolazine-sensitive APD30 and APD50 shortening by 57.5% and 27.4% (n=5) respectively in LMNA-mutated cells indicated the overactivated forward mode of the actin-dependent ion channels, sodium-calcium exchanger (NCX1). The channel might be overstimulated by signal from nuclear lamina leading to sodium influx, which triggers premature heart beats.F-3184THE EFFECTS OF HYPOXIA ON DIFFERENTIATION AND MATURATION OF HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED NEURAL PROGENITORSOkada, Yohei - Department of Neurology, Aichi Medical University, Nagakute, Japan Okada, Rina - Department of Neurology, Aichi Medical University, Nagakute, Japan Li, Jiawei - Department of Neurology, Aichi Medical University, Nagakute, Japan Onodera, Kazunari - Department of Neurology, Aichi Medical University, Nagakute, Japan Ito, Takuji - Department of Neurology, Aichi Medical University, Nagakute, Japan Okano, Hirotaka - Division of Regenerative Medicine, Jikei University, Tokyo, Japan Doyu, Manabu - Department of Neurology, Aichi Medical University, Nagakute, Japan

605POSTER ABSTRACTSDisease specific human induced pluripotent stem cells (hiPSCs) have been expected as useful disease models for pathophysiological analysis and drug discovery. However, hiPSC-derived neural cells are often immature for the detection of phenotypes of adult onset neurological diseases. In this study, to obtain iPSC-derived mature neural cells, we examined the effects of hypoxia on differentiation and maturation of hiPSC-derived neural progenitors. First, hiPSCs (201B7) were differentiated into motor neuron (MN) progenitors under hypoxia (2% or 5%) or normoxia (20%), and were examined for neural induction. As a result, hiPSCs showed massive cell death under hypoxia and did not show any positive effects of hypoxia on neural induction. Then, we differentiated hiPSC-derived MN progenitors under hypoxia, and examined their differentiation and maturation. Hypoxia induced significant downregulation of the markers of immature neural progenitors, Nestin and Sox1, and upregulation of the markers of mature MNs, ChAT, Synaptophysin and PSD95, in an oxygen concentration dependent manner. We also examined functional maturation of iPSC-derived MNs by multielectrode array (MEA). Motor neurons differentiated under hypoxia showed significantly more mature electrical activity compared with those differentiated under normoxia. Finally, we investigated whether the hypoxia facilitate the detection of the phenotypes of disease specific iPSCs of adult onset neurdegenerative diseases. iPSCs established from patient of spinal bulbar muscular atrophy (SBMA) and control iPSCs were induced to MN progenitors, and differentiated under hypoxia or normoxia for up to four weeks. In comparison with control iPSC-derived MNs, SBMA iPSC-derived MNs cultured under hypoxia showed significant increase in the expression of CALCA and in the phosphorylation of c-Jun, known phenotypes in SBMA model mice, while those cultured under normoxia showed less or insignificant differences. Together, it is suggested that hypoxia is effective for promoting differentaition and maturation of iPSC-derived neural cells, and detecting phenotypes of disease specific iPSCs of adult-onset neurological disorders. Thus, hypoxia is applicable to varieties of pathophysiological analysis and drug screening using disease specific iPSCs.Funding Source: AMED 18ek0109243h0002 KAKENHI 17H05707 KAKENHI 17K19465F-3186LOSS-OF-FUNCTION DUE TO MODY1/HNF4A MUTATION ABROGATES HUMAN PANCREAS AND LIVER DIFFERENTIATION FROM MODY1-IPSCSLau, Hwee Hui - Stem Cells and Diabetes Lab/IMCB, Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore, Singapore Ng, Natasha Hui Jin - Stem Cells and Diabetes Lab, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore Jasmen, Joanita - A*STAR, A*STAR, Singapore, Singapore Lim, Chang Siang - Institute of Molecular and Cell Biology, A*STAR, Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore Gomathi Krishnan, Vidhya - Molecular Engineering Lab, A*STAR, A*STAR, Singapore, Singapore Kadiwala, Juned - Anne McLaren Laboratory, Wellcome Trust-Medical Research Council Stem Cell Institute, United Kingdom, UK Kulkarni, Rohit - Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA Raeder, Helge - Department of Clinical Science, Jebsen Center for Diabetes Research, Bergen, Norway Vallier, Ludovic - Anne McLaren Laboratory, Wellcome Trust-Medical Research Council Stem Cell Institute, United Kingdom, UK Hoon, Shawn - Molecular Engineering Lab, A*STAR, A*STAR, Singapore, Singapore Teo, Adrian - Stem Cells and Diabetes Lab, Institute of Molecular and Cell Biology, A*STAR, Singapore, SingaporeMaturity onset diabetes of the young 1 (MODY1) is caused by autosomal dominant mutations in the HNF4A gene. HNF4A is a key determinant dictating foregut endoderm development which eventually forms the liver and pancreas. Based on knowledge from rodent development, we hypothesised that mutations in human HNF4A gene implicate foregut endoderm development that subsequently leads to the eventual loss of insulin secretory ability of the pancreatic -cells in MODY1 βpatients. To understand the pathogenesis of MODY1 in humans, we differentiated patient-derived iPSCs into foregut endoderm/hepato-pancreatic progenitors (HPP) and hepatic or pancreatic β-like cells in a stepwise manner. We observed that mutant HNF4A protein is sequestered in the cytoplasm in MODY1-HPP. The downregulated gene expression and the inability of mutant HNF4A protein to enter the nucleus suggest that these MODY1 patients could have a reduced HNF4A gene dosage. Genome-wide transcriptomic analysis revealed a diversion towards hindgut lineage evident by upregulation of numerous HOX genes in MODY1-HPP, while HNF4A and foregut genes were downregulated. HNF4A haploinsufficiency also led to the downregulation of several key hepatic and pancreatic markers. We then demonstrated that MODY1/HNF4A mutation resulted in a loss of transcriptional activation on selected hepatic and pancreatic gene promoters. Collectively, our patient-derived iPSC MODY1 model revealed that HNF4A haploinsufficiency results in downstream gene dysregulation and impairs foregut development and its derivatives. These effects may propagate to long-term pathological consequences observed in MODY1 patients.Funding Source: IMCB A*STAR, NHG-KTPH SIG/14033, NUHS-CG Metabolic In-Vitro Core Seed Funding, JCO Career Development Award A*STAR, NMRC OF-YIRGF-3188PATIENT IPS CELL DERIVED NEURONAL CELLS AS DISEASE MODELS FOR POMPE DISEASECheng, Yu-Shan - NCATS, nCATS/NIH, Rockville, MD, USA Li, Rong - National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA

606POSTER ABSTRACTSBaskfield, Amanda - National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA Beers, Jeanette - National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Zou, Jizhong - National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Liu, Chengyu - National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Zheng, Wei - National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USAPompe disease is an autosomal recessive disease caused by a deficiency of acid alpha-glucosidase, a lysosomal enzyme involved in the breakdown and recycling of glycogen. Although the clinical manifestations largely reflect on the skeletal and cardiac disorders, glycogen accumulation in the brain, brainstem nuclei and anterior horn cells has also been observed in the autopsy studies. To facilitate drug discovery and model disease pathophysiology, we generated neural stem cells (NSCs) and neuronal cells from Pompe patient-derived induced pluripotent stem cells (iPSCs). The NSCs exhibited characteristic disease phenotypes with deficiency of acid alpha-glucosidase, accumulation of glycogen and neutral lipid within cells. Using this NSCs disease model, we observed the reduction of lipid accumulation by the treatment of two small molecular compounds: hydroxypropyl- -cyclodextrin (HP CD) ββand -tocopherol. Our data demonstrate that the patient-δderived NSCs can be used as a cell-based disease model to study disease pathology and perform compound screening for drug development.REPROGRAMMINGF-3190A TRANSIENT SWITCH IN THE SWI/SNF SUBUNITS BCL11A AND BCL11B ORCHESTRATES REPROGRAMMING TOWARD PLURIPOTENCY AND MALIGNANCYFurlan, Giacomo - Cell Plasticity, Cancer Research Centre of Lyon (CRCL), Lyon, France Huyghe, Aurélia - Cell Plasticity, Cancer Research Center of Lyon, France Schroeder, Jan - Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia Yu, Yong - Cambridge, Wellcome Trust Sanger Institute, Cambridge, UK Wang, Juexuan - Cambridge, Wellcome Trust Sanger Institute, Cambridge, UK Lainé, Alexandra - Immunology, Cancer Research Center of Lyon, France Wajda, Pauline - Cell Plasticity, Cancer Research Center of Lyon, France Gadot, Nicolas - Cell Plasticity, Cancer Research Center of Lyon, France Goddard, Isabelle - Cell Plasticity, Cancer Research Center of Lyon, France Marie, Julien - Immunology, Cancer Research Center of Lyon, France Liu, Pentao - Cambridge, Wellcome Trust Sanger Institute, Cambridge, UK Polo, Jose - Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia Lavial, Fabrice - Cell Plasticity, Cancer Research Center of Lyon, FranceA key challenge for developing organisms is to establish cell identities by restricting plasticity. In contrast, such plasticity is regained during the conversion of somatic cells into induced pluripotent stem cells (iPS) by Oct4, Sox2, Klf4 and c-Myc (OSKM). Malignant transformation also implies cellular identity loss and the acquisition of embryonic features, leading to the hypothesis that oncogenes such as c-Myc and K-Ras induce a direct reprogramming process that accompanies the acquisition of the tumorigenic phenotype. For these reasons, a better characterization of cellular identity loss occurring during iPS cells generation and malignant transformation might have profound implications for both regenerative medicine and cancer biology. The aim of this study is to perform a comparative and comprehensive analysis of the early steps of pluripotent reprogramming, induced by OSKM, and malignant transformation triggered by c-Myc and Kras in MEF. RNA-seq analysis of FACS sorted reprogramming intermediates led us to demonstrate that, in the early days of both iPS and malignant cells emergence, loss of cellular identity is correlated with a switch in the expression of the transcription factors Bcl11b and Bcl11a, components of the SWI/SNF complex. Gain- and loss-of-function approaches revealed that this balance controls cellular identity loss and the emergence of pluripotent and malignant reprogrammed derivatives. Moreover, the use of Bcl11-reporter mice led us to (i) define a molecular roadmap of reprogramming and (ii) characterize novel and rare “intermediate states” emerging in the early days of both processes at the transcriptomic and epigenomic levels. Collectively, this project shed light on molecular mechanisms that constraining reprogramming and therefore iPS cells generation and tumorigenesis.F-3192MANIPULATING CELL FATE TO IMPROVE TISSUE REGENERATION DURING AGINGAlle, Quentin - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France Bechir, Nelly - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France Gabanou, Melissa - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France Lemey, Camille - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France

607POSTER ABSTRACTSLe Borgne, Enora - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France Lemaitre, Jean-Marc - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, France Milhavet, Ollivier - IRMB - INSERM U1183, French National Institute of Health and Medical Research (Inserm), Montpellier, FranceAging is a complex process modulated by genetic and epigenetic factors and marked by progressive appearance of age-related pathologies and decrease of cell and tissue regenerative capacity. Our objective is to delay these effects by inducing global rejuvenation or increasing the organismal regenerative capacity. In 2007, Dr. Yamanaka showed that human fibroblasts can be converted into pluripotent stem cells by inducing the expression of four transcription factors. Ourselves, we showed in 2011 that senescent cells which are accumulating in aging organisms can be reprogrammed and regained a rejuvenated physiology and metabolism. Our hypothesis is that in vivo induction of a transient reprogramming process could erase the marks of cellular aging and improve tissue regeneration to restore altered cell physiology and delay tissue aging and deleterious consequences. For this, we use a transgenic mouse model to control the induction of the expression of the reprogramming factors in order to reprogram the cells of the whole organism. These mice also recapitulate the human phenotype of Hutchinson-Gilford progeria syndrome, mimicking accelerated physiological aging. Our results showed a significant increase in life expectancy and improvement in tissue integrity related to activation of tissue regeneration. The impact of controlled transient reprogramming on age-related pathologies has also been monitored and a protective role in osteoarthritis and osteoporosis has been observed in these animals at eight months of age. Also, a two-week low-dose treatment, at two months of age, allows for increased life expectancy in later ages while decreasing phenotypic markers associated with aging suggesting a memory effect of transient reprogramming maintained along the entire life. Cellular and molecular mechanisms were examined in vitro. We highlighted, in the context of transient reprogramming, the implication of Foxo3a, an actor of the stress response, the involvement of DNA repair mechanisms and the activation of autophagy. We also initiated a global analysis of gene expression on cells isolated from our murine models, and experiments to determine the role of endogenous stem cells. This work demonstrates how transient cellular reprogramming reduces the negative impacts of aging at the organismal level.F-3194INFERRING INDIVIDUAL CELL TYPE CHIP-SEQ PROFILES FROM POPULATION CHIP-SEQ AND SINGLE CELL RNA-SEQ DATASabri, Shan - Biological Chemistry, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Ernst, Jason - Biological Chemistry, University of California, Los Angeles, CA, USA Langerman, Justin - Biological Chemistry, University of California, Los Angeles, CA, USA Plath, Kathrin - Biological Chemistry, University of California, Los Angeles, CA, USAHistone modification profiles are informative for understanding gene regulation for a given cell type. However, current histone modification profiling technologies yield averaged profiles of a mixture of cells that mask individual cell type-specific profiles. This is a major shortcoming given that cellular variability is inherent in most cell populations. The current method for single cell ChIP-seq has low per-cell sequencing coverage and exhibits a signal-to-noise ratio too high for meaningful interpretation. Here, we present a method that predicts chromatin maps at the single cell type level by jointly modeling population epigenetic data with single cell transcriptomics. We train a machine learning model to learn relationships between gene expression and chromatin features using a compendium of data containing histone modification maps and gene expression profiles. To deconvolve the histone profiles of a complex mixture of cells we integrate two types of models that account for within- and across-cell type relationships. The within-cell type model leverages information about expression of nearby genes, and the across-cell type model predicts ChIP-seq profiles from the expression of all genes. Using these relationships, we can infer histone modification profiles at the individual cell type level using single cell expression data. This allows us to annotate difference in cis-regulatory sites, such as enhancers, between cell types from complex population of cells. We apply this framework to the context of reprogramming somatic cells into induced pluripotent stem cells (iPSCs), a process limited by low conversion efficiency potentially due to epigenetic barriers that are difficult to overcome. It is unclear which chromatin changes allow some cells to progress towards pluripotency. Our preliminary results suggest our model can predict cell type-specific enhancers within somatic and iPSC populations solely from single cell gene expression data. Utilizing these different data types will provide a foundation for understanding the regulatory program at the single cell or subpopulation level.Funding Source: Rose Hills Foundation Pre-doctoral Training Award and UCLA Broad Stem Cell Research Center (BSCRC) FellowshipF-3196ENHANCED CARDIAC REPROGRAMMING BY ELECTRICAL STIMULATION USING MICROPILLAR ARRAYMin, Sungjin - Department of Biotechnology, Yonsei University, Seoul, Korea Lee, Hyo Jung - Department of Materials Science and Engineering, Yonsei University, Seoul, Korea Jin, Yoonhee - Department of Biotechnology, Yonsei University, Seoul, Korea Kim, Yu Heun - Department of Biotechnology, Yonsei

608POSTER ABSTRACTSUniversity, Seoul, Korea Choi, Heon-Jin - Department of Materials Science and Engineering, Yonsei University, Seoul, Korea Cho, Seung-Woo - Department of Biotechnology, Yonsei University, Seoul, KoreaCardiac diseases including cardiomyopathy, arrhythmias, and myocardial infarction are life-threatening. Cellular reprogramming technology has been attracting great attention for patient-specific cell therapy and drug screening. In particular, direct reprogramming can readily turn somatic cells into other lineage cells in a short period without using stem cells. However, there is a need to be improved in terms of conversion efficiency and maturation. In this study, we applied electrical cues to promote cardiac reprogramming and generation of induced cardiac spheroids. We confirmed upregulated cardiac gene expressions in cardiac reprogramming as 3D spheroids, compared with reprogramming under 2D condition. Induced cardiac spheroids also showed autonomous beating, which could be facilitated by isoproterenol. Direct electrical stimulation using micropillar array to induced cardiac spheroids could further promote differentiation and maturation of cardiac lineage cells in the spheroids. Our study provides a new engineering platform for electrical stimulation to improve direct reprogramming, which would be applied for understanding disease mechanism and testing potential drugs.Funding Source: This research was supported by a grant (19172mfds168) from Ministry of Food and Drug Safety in 2019.F-3198INDUCED SENSORY HAIR CELLS: UNDERSTANDING AND ENHANCING REPROGRAMMING EFFICIENCY, SPECIFICITY AND MATURITYTrecek, Talon - Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Menendez, Louise - Neuroscience, University of Southern California, Los Angeles, CA, USADeafness affects 360 million people worldwide, the leading cause of which is loss of sensory hair cells in the cochlea. Hair cells are scarce and fragile, making research studies difficult. Here we used direct cellular reprogramming to generate induced sensory hair cells (iHCs) in vitro. Direct reprogramming is an indispensible technique for studying rare and inaccessible cell types but comes with its own limitations of efficiency and heterogeneity. We used a combinatorial analysis of hair cell specific transcription factors (TF), and identified an optimal cocktail of four TFs for reprogramming mouse fibroblasts towards a hair cell fate. RNA sequencing of iHCs indicates a robust recapitulation of the transcriptional profile of primary hair cells. Further bioinformatics analysis revealed that iHCs bypass a progenitor stage, yet can still up regulate 72% of primary hair cell genes. Interestingly the genes that fail to activate fall into known developmental pathways of hair cell differentiation (ie. Notch signaling). To understand if these unique differences are attributable to failure to activate primary hair cell enhancer networks we performed ATAC sequencing. Despite bypassing normal developmental pathways, the iHCs open >90% of the primary hair cell enhancers. This correlated with significant up regulation of the putative gene targets of these enhancers. Taken together these results indicate a need to examine whether the failed developmental pathways are limiting the reprogramming efficiency, specificity, and maturity of iHCs. To address this question we are using an inducible polycistronic vector for reprogramming to ensure proper stoichiometry of the TFs, single cell sequencing to better illustrate the temporal changes and maturity achieved in reprogramming, and ChIP sequencing on the fibroblasts that fail to reprogram in order to profile histone marks that may be acting as barriers to reprogramming. Ultimately as we push towards a bona fide iHC model the goal is to provide the biological material necessary to perform high throughput studies for protective and regenerative initiatives of sensory hair cells.F-3200EPIGENETIC MECHANISM OF DIRECT CONVERTING ADULT HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS INTO IMSCS USING A NOVEL REPROGRAMMING SYSTEMChen, Wanqiu - Center for Genomics, Loma Linda University, Loma Linda, CA, USA Wang, Chenguang - Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China Choi, Hannah - Center for Genomics, Loma Linda University, Loma Linda, CA, USA Mi, Xianqiang - Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China Zhang, Xiao-Bing - Department of Medicine, Loma Linda University, Loma Linda, CA, USA Wang, Charles - Center for Genomics, Loma Linda University, Loma Linda, CA, USABone marrow and adipose tissue are the major sources in obtaining mesenchymal stem cells (MSCs); unfortunately, the acquisition process requires invasive surgery. Previously we reported that human cord blood hematopoietic progenitors (CB-CD34+ cells) could be directly converted into iMSCs with a single factor, OCT4. To expand this finding, we attempted to generate patient-specific iMSCs from adult peripheral blood mononuclear cells (PBMCs) using a modified oriP/EBNA1-based episomal vector (EV) system. After screening multiple combinations of factors, we identified a cocktail containing five factors (including OCT4 and SOX9) that was highly efficient in reprogramming adult human PBMCs into iMSCs with trilineage differentiation potential in vitro. More importantly, SOX9, unlike SOX2, restricts cell fate to MSC without going through the pluripotent stage, evidenced by 0% TRA-1-60+ cells during the reprograming. Inclusion of SOX2 led to 1-2% TRA-1-60+ cells, even when cultured in the MSC medium. Factor omission studies showed that without OCT4, MSC-like colonies were observed and the reprogramming efficiency was reduced by

609POSTER ABSTRACTSonly 20-30%. However, four factors (without OCT4) generated iMSCs showed significantly impaired multilineage differentiation potential. In particular, the expression of osteogenic related genes like GBLAP, SP7, and ALP was reduced by 40-80%. To understand the epigenetic mechanism of iMSC reprogramming involving OCT4, we analyzed chromatin accessibility by ATAC-seq, DNA methylome by RRBS and transcriptome by RNA-seq and single-cell RNA-seq. We found that without OCT4, the 4F-derived iMSCs were hypermethylated (P<0.0001) at the transcription start sites, suggesting that OCT4 is critical for reshaping the DNA methylation pattern during reprogramming. ATAC-seq results confirmed a distinctive chromatin accessibility pattern when omitting OCT4. Overall, we developed an efficient EV-based reprogramming system containing the factors for generating integration-free patient-specific iMSCs from PBMCs, and there was an epigenomic reprogramming orchestrating with transcriptomic changes during the cellular reprogramming. Our data also suggest that transient expression of OCT4 may play a critical role in promoting the direct reprogramming by reshaping the global epigenome.Funding Source: This work was supported by AHA grants 18IPA34170301, an innovative project award of the American Heart Association to C.W.F-3202BI-POTENT PROGENITOR CELLS GENERATION THROUGH SMALL MOLECULE-MEDIATED HUMAN HEPATOCYTES REPROGRAMMINGKim, Yohan - College of Medicine, Hanyang University, Seoul, Korea Kang, Kyojin - College of Medicine, Hanyang University, Seoul, Korea Yoon, Sangtae - College of Medicine, Hanyang University, Seoul, Korea Buisson, Elina - College of Medicine, Hanyang University, Seoul, Korea Lee, Chang Hee - College of Medicine, Hanyang University, Seoul, Korea Yim, Ji-Hye - College of Medicine, Hanyang University, Seoul, Korea Jeong, Jaemin - College of Medicine, Hanyang University, Seoul, Korea Choi, Dongho - College of Medicine, Hanyang University, Seoul, KoreaGiven the shortage of organ donors, cell-based regenerative medicine can prove useful and ground-breaking in terms of gene and/or stem cell therapy for patients suffering from end stage liver disease. Unlike mouse bipotent progenitor cells that have been proved to provide ease of isolation and long-term expansion from terminally differentiated mouse hepatocytes, the challenge remains with adult human hepatocytes. Using hepatocytes from diseased and healthy livers in combination with two small molecules and growth factors, we report a new technique to generate patient-specific hepatic progenitor cells from human hepatocytes. Three days after the treatment of hepatocytes with small molecules and growth factors, a key driver of hepatic progenitor cell activity, generated small polyglonal cells with expansion properties which co-expressed hepatic progenitor cells and lineage specific marker genes. These chemically derived human hepatic progenitor cells (hCdHs) showed a retention in normal phenotype and karyotype when passaged to at least 10 passages. They were also able to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Furthermore, molecular similarity between hCdHs and human hepatoblasts was seen through a next-generation sequencing analysis. Finally, the transplantation of hCdHs into immunocompromised mice with diseased liver showed effective restoration and repopulation capacity of hCdHs. In conclusion, hCdHs provide a safe novel tool that permits expansion and genetic manipulation of patient-specific hepatic progenitor cells to study regeneration and repair of diseased liver.Funding Source: This work was carried out with the support of the ‘‘Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01100202)” Rural Development Administration, Republic of Korea.F-3204REPROGRAMMING HUMAN SOMATIC CELLS DIRECTLY TO NAIVE IPSCS USING NON-MODIFIED MRNAS AND MIRNASRen, Yongming Luke - REPROCELL USA, Beltsville, MD, USA Guo, Ge - Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, UK Yang, Jian - Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, Guangzhou, China Bredenkamp, Nicholas - Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, UK Clarke, James - Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, UK Baker, Duncan - Sheffield Children’s NHS Foundation Trust, Sheffield, UK Eminli-Meissner, Sarah - REPROCELL USA, Beltsville, MD, USA Smith, Austin - Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, UKPluripotent stem cells (PSCs), including induced PSCs (iPSCs), hold great potential for both basic research and biomedical applications due to their unlimited self-renewal and differentiation potential into multiple somatic cell lineages. Human epiblasts in the embryo, the founder of the fetus and the origin of PSCs, develop continuously from inner cell mass to a single-layered epithelial disc, which is primed for gastrulation. Human PSCs representing the more primed post-implantation epiblast have been well studied. We have recently reported generation of human naïve stem cells from dissociated inner cell masses and primed human PSC lines. These cells have molecular signatures related to emerging naïve epiblast. Here we report establishment of transgene-free human naïve iPSCs by direct somatic cell reprogramming using an RNA-based reprogramming method

610POSTER ABSTRACTSthat combines a novel cocktail of synthetic, non-modified reprogramming [OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (OSKMNL)] and immune evasion mRNAs [E3, K3, B18-R] with reprogramming-enhancing mature, double-stranded microRNAs. This unique combination of different RNAs results in a fast, highly efficient and robust reprogramming protocol that can generate not only iPSCs directly from somatic cells but also naïve iPSCs. mRNA is the most favorable technology for reprogramming of somatic cells into naïve iPSCs since it does not integrate into the genome and therefore overcomes many limitations for the translational of the overall iPSC technology for clinical applications. We validated this technology on multiple adult human fibroblasts and endothelial progenitor cells (EPCs) derived from adult blood. Clonal naïve RNA-iPSC lines can be expanded in culture for more than ten passages and retain a normal karyotype. They express a panel of naïve specific transcription factors and surface markers. Direct production of transgene-free naïve iPSCs offers a new platform to evaluate differentiation propensity of naïve versus primed pluripotency.Funding Source: This research is funded by the Medical Research Council.F-3206BETA-HYDROXYBUTYRATE PREVENTS VASCULAR SENESCENCE THROUGH HNRNP A1-MEDIATED UPREGULATION OF OCT4 IN VASCULAR CELLSHan, Young-min - CMTM, Georgia State University, Buford, GA, USAHere we report that -HB promotes cellular quiescence in βvascular cells, which significantly inhibits both stress-induced premature senescence and replicative senescence through p53-independent mechanisms. Further, by using a ligand fishing pulldown approach, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a direct binding target of β-HB. This binding of -HB to hnRNP A1 markedly enhanced βhnRNP A1 binding with Octamer-binding transcriptional factor (Oct) 4 mRNA. The binding of hnRNP A1 with Oct4 mRNA stabilizes Oct 4 mRNA and Oct4 expression. Finally, we found that both fasting and intraperitoneal injection of -HB in vivo βupregulates Oct4 and Lamin B1 in both vascular smooth muscle and endothelial cells in mice. We conclude that the ketone body β-HB exerts anti-aging effects in vascular cells by upregulating an hnRNP A1-induced Oct4-mediated Lamin B1 pathway.TECHNOLOGIES FOR STEM CELL RESEARCHF-3208THE PROGENIES OF HUMAN OR CHIMP PLURIPOTENT STEM CELLS DISTURB INTERSPECIES CHIMERA DEVELOPMENTMasaki, Hideki - Cell Therapy, The University of Tokyo, Tokyo, Japan Nakauchi, Hiromitsu - Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, JapanWe have been working on generating human organ in animal body by injecting pluripotent stem cells (PSCs) to organ deficient animal embryos. Previously, we generated mouse pancreas in mouse-rat chimera by injecting mouse embryonic stem cells into apancreatic rat embryos. Diabetic mice transplanted with generated mouse pancreatic islets showed normal blood glucose level throughout their life. Despite the various advantages of this approach, difficulty to create human-animal chimera prevent human application. To solve this problem, we used chimp iPS cells, as an alternative to human PSCs, and screened chimera forming cells with xenogeneic animals. Chimp-mouse and chimp-pig chimeric fetus were obtained by preventing apoptosis in chimp iPS cells, however, all of developed interspecies chimeric fetus showed low chimerism compared to rat-mouse chimeras. Consistent to our results, several reports succeeded to generate human-animal chimeric fetus also showed low chimerism. The reason why chimp (or human) PSC derived chimeras showed poor chimerism is remain unknown. Here we created chimp-mouse chimeras by forced expressing BCL2 in conventional chimp iPSCs and analyzed at various developmental stages. Highly chimeric embryos disappear before E8.5 and only poorly chimeric embryos could develop further, which is unlikely to rat-mouse chimeras. Chimp-mouse highly chimeric embryos showed severe malformation even from egg cylinder-stage, and then resulted in degeneration or abortion at later stages. Consistently, chimp-pig chimeric fetus developed to later stage showed lower chimerism than degenerated fetus. It was also consistent with human iPSC-derived chimeras with mouse embryos. These observations suggested that the reason only poorly chimeric fetus were observed was that human (or chimp) iPSC-derived progenies disturb host animal’s development and highly chimeric embryos could not survive. Some sort of modification would be required to harmonize human PSC-progenies in mouse or pig embryo development and obtain highly chimeric animals, which maximize the chance to generate whole human organ in animal body.Funding Source: This work was supported by grants from AMED LEAP (grant number JP18gm0010002), research grant for type 1 diabetes, Japan IDDM network and California Institute for Regenerative Medicine Grant Number LA1-06917.F-3210MULTIPLEXED AUTOMATED IMAGING ASSAYS FOR COMPOUND TESTING USING INDUCED PLURIPOTENT STEM CELL-DERIVED CELL MODELSSirenko, Oksana - R&D, Molecular Devices, San Jose, CA, USA Spira, Felix - R&D, Molecular Devices, San Jose, CA, USA

611POSTER ABSTRACTSThere is an increased need for expanding variety and complexity of cell-based assays for biologic research and drug discovery. Stem cell-derived cells and tissues become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. In this study, we used human iPSC-derived cardiomyocyte and neuronal cell models to develop functional and morphological readouts for testing effects of different compounds in a multi-parametric assay format. The effects of selected compounds on cardiac physiology were monitored by measuring spontaneous contractions of stem cell derived cardiomyocytes. Kinetic patterns were characterized by measurements of calcium ion flux using time-lapse recording of the fluorescence intensity of calcium ion sensitive dyes. In addition, the effects on cell viability and mitochondria integrity were monitored at the end-point using additional readouts. We used automated cell imaging and analysis with the ImageXpress Pico Imaging System to simultaneously determine calcium oscillation frequency, cell viability, cytoskeletal integrity, apoptosis, and mitochondrial function. We demonstrated the effects of several cardio-active and cardio-toxic compounds on amplitude and the frequency of calcium oscillations and determined EC50 values for the effects on calcium oscillations, as well as EC50 values for cell viability and mitochondria potential. For neuro-spheroids automated imaging was used to evaluate the number of live cells and monitor calcium oscillations using time-lapse imaging. Multiplexed assessment of different readouts provides additional insight of the mechanisms of action of various compounds. The methods were characterized using a set of cardio-active drugs, neurotransmitters, and selected neurotoxic or cardiotoxic compounds. Overall, our results demonstrate how a variety of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and neuronal models and enable rapid and cost-efficient multidimensional biological profiling.F-3212DEVELOPMENT OF NOVEL QUALITATIVE AND QUANTITATIVE METHODS TO ASSESS MATURATION STATUS OF CARDIOMYOCYTE DERIVED FROM MOUSE PLURIPOTENT STEM CELLSChanthra, Nawin - Regenerative Medicine, Jichi Medical University, Tochigi, Japan Hanazono, Yutaka - Regenerative Medicine, Jichi Medical University, Tochigi, Japan Uosaki, Hideki - Regenerative Medicine, Jichi Medical University, Tochigi, JapanPluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a promising cell source for research and medical applications. Although PSC-CMs are efficiently obtained from cardiac differentiation using conventional protocols, but these cells solely show fetal-like phenotype and arrest at embryonic state of maturation instead of developing to adult cardiomyocytes (CMs). Presently, generation of fully matured CMs in a reasonable time is still a challenge. A primary limitation in the production of fully mature CMs is a lack of applicable high-throughput technologies providing distinguished parameters for determining how CMs mature and measuring the maturity of CMs. To this end, we aimed to develop novel qualitative and quantitative methods to assess the effects of hormone agonists on CM maturation. For a qualitative method, we generated a maturation reporter line, that is a PSC-line with the RFP gene knocked-in so that it is fused to a sarcomere gene and upregulated postnatally. Unless cells differentiated to mature CMs, no RFP could be detectable. After 2 weeks of CM differentiation, weak RFP became visible in approximately 40% of PSC-CMs. In this research, we first examined if hormone agonists could enhance CM maturation using the reporter line. We found that hydrocortisone added to the culture increased Myom2-RFP intensity, whereas the RFP signal was significantly reduced in triiodothyronine (T3)-treated condition. These data indicated that these agonists affected the maturation of PSC-CMs. For a quantitative method, we collected a reference transcriptome dataset from embryos to adults with a cost-effective transcriptome method with next generation sequencing. Next, we compared transcriptome of the treated PSC-CMs to that of in vivo counterparts and quantitatively assessed the maturation status according to microarray-based method as we demonstrated in our previous paper (Uosaki, Cell Rep, 2015). We found that T3 enhanced CM maturation to the maturity equivalent to that of postnatal day 7 to 14 and promoted isoform switches of sarcomere proteins, as well as increased expressions of CM maturation markers. In this study, we highlight T3 as a hormone that potentially enhances CM maturation.Funding Source: This study is sponsored by grants from AMED, Novartis Pharma Research Grant, Japan Research Promotion Society for Cardiovascular Diseases, Takeda Science Foundation, and Uehara Memorial Foundation.F-3214HARNESSING HIERARCHICAL NANOTOPOGRAPHIES TO DIRECT STEM CELL FATEWang, Peng-Yuan - Center for Human Tissues and Organs Degeneration, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaManipulation of cell fate is a critical process in regenerative medicine and cell-based therapies. Strategies and methods to maintain stemness of stem cells and direct them into specific cell types are ongoing challenges in cell biology. To date, a number of studies have reported that biophysical stimulation in the form of surface nanotopographies can influence stem cell attachment, proliferation, and differentiation. In addition, specific surface nanotopographies can enhance efficiency of cell reprogramming and maintain stemness of stem cells. While biochemical cues are generally effective, biophysical cues have other advantages such as scalability, cost effective, longer lifetime, and easily to be defined. In our group, we dedicated

612POSTER ABSTRACTSto fabrication of various surface nanotopographies including nanogrooves, nanopillars, nanopores, and binary colloidal layers using different nanotechnologies including electron beam lithography, reactive ion etching, soft lithography, and self-assembly. Having these cell culture tools we were able to direct stem cell fate into desired outcome. We believe that combining biochemical and biophysical stimulation has the greatest potential to generate functionally mature cells at a scalable and inexpensive way for diverse applications in regenerative medicine and cell therapy.Funding Source: P.-Y. Wang acknowledges the National Natural and Science Foundation of China (grant number 31870988) and the Australia Research Council for providing a Discovery Early Career Researcher Award (DECRA).F-3216EXPANSION OF MESENCHYMAL STEM CELLS ON MICROCARRIERS WITH OPTIMIZED PHYSICAL AND CHEMICAL PROPERTIESRogers, Robert E - College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA Haskell, Andrew - College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA Leong, Tiffany - Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA Dai, Jing - Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA Han, Arum - Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA Gregory, Carl - College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA Kaunas, Roland - Department of Biomedical Engineering, Texas A&M University, College Station, TX, USAMesenchymal stem cells (MSCs) are attractive avenues for cell therapeutics due to their immunomodulatory capabilities and ability to secrete trophic factors. However, current culture techniques inhibit their translation into clinical medicine in part due to the cost of growth media. Serum supplementation of cell culture media can be significantly reduced with three-dimensional bioreactor culture, easing MSC culture into clinical medicine by reducing cost. Previous work has shown that gelatin-derived materials can be generated with tunable mechanical properties and the capability to release growth factors in a time-dependent manner. Therefore, the purpose of these experiments is to optimize expansion of induced pluripotent stem cell-derived MSCs (iPS-MSC) on gelatin-methacrylamide (GelMA) microcarriers. Monodisperse GelMA microcarriers with tunable stiffness are mass-produced using a custom, inexpensive microfluidic device that operates under biocompatible conditions and can be run in parallel for scale-up. Using various bioreactor configurations, future experiments on GelMA microcarriers include coupling and encapsulating bioactive factors for release, and quantifying expansion and downstream therapeutic properties of iPS-MSCs on microcarriers with various parameters. Finally, a parametric computer model will be prepared to optimize the expansion of iPS-MSCs while minimizing the amount of media supplementation and volume requirements. Collectively, this proposed method aims to reduce cell culture costs by limiting the amount of required supplements and materials without sacrificing cell quality.Funding Source: Grants from the Texas A&M X-Grant Presidential Excellence Fund, NIAMS R01AR066033 and the Cancer Prevention and Research Institute of Texas.F-3218DISCOVERING THE ROLE OF LONG NON-CODING RNAS DURING PANCREATIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS USING DCAS9 TECHNOLOGYWestmoreland, Shania D - San Diego State University, California Institute for Regenerative Medicine (CIRM), Oceanside, CA, USA Touboul, Thomas - Reproductive Medicine, University of California San Diego, San Diego, CA, USA Foster, Mikelle - Biological Sciences, Cal Poly San Luis Obispo, San Luis Obispo, CA, USA DeHoff, Peter - Reproductive Medicine, University of California San Diego, San Diego, CA, USA Gobbur, Tanuja - Reproductive Medicine, University of California San Diego, San Diego, CA, USA Laurent, Louise - Reproductive Medicine, University of California San Diego, San Diego, CA, USAFrom ultra-deep RNA sequencing studies, non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs), have been identified throughout the human genome. lncRNAs are thought to play roles in embryonic development, dosage compensation, disease regulation, immune response and cell regulation. Through a small number of studies on specific lncRNAs, it has been demonstrated that lncRNAs can operate through diverse mechanisms. However, only a small fraction of known lncRNAs have been thoroughly studied and characterized. Recent advances in CRISPR technology has enabled programmable control of gene expression for studying gene function. The nuclease dead CRISPR-Cas9 (dCAS9) enzyme can be directed to the promoter of a specific target genes to either block the initiation of the transcription (CRISPRi), or, when fused to a transactivator domain (e.g. VPR), to promote transcriptional initiation (CRISPRa). However, many of the existing CRISPRi/CRISPRa systems do not work well in hESCs due to gene silencing. This study aims to optimize these systems in hESCs to determine the functions of lncRNAs during pancreatic differentiation. Using a TALEN assisted gene-trap, doxycycline-inducible dCAS9 and dCAS9/VPR constructs were integrated into the “safe harbor” AAVS1 locus. hESC clones carrying the correct integration of the construct into the AASV1 safe harbor without random integration events were identified. Induction of dCas9 mRNA and protein expression in undifferentiated and differentiated hESCs was used to select optimal clones. Constructs for constitutive expression of guide RNAs (gRNAs)

613POSTER ABSTRACTStargeting the promoters of lncRNAs and transcription factors (TFs) differentially expressed during pancreatic differentiation were introduced into these clones. Using this system, we are able to overexpression and knock-down selected target transcripts in undifferentiated hESCs. Ongoing studies are designed to disrupt or promote gene expression at specific time points during differentiation and study the effects on the differentiation process. In addition to delineating the functions of the specific lncRNAs and TFs targeted in these experiments, this study aims to develop a more robust system for programmable manipulation of gene expression in hESCs, particularly during directed differentiation.Funding Source: California Initiative for Regenerative MedicineF-3220HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELL EXPANSION IN XENO-FREE CONDITIONS FOR CLINICAL-GRADE APPLICATIONSBecerra-Bayona, Silvia M - Facultad de Ciencias de la salud, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia Solarte-David, Victor - Facultad de Ciencias de la Salud, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia Alviar-Rueda, Juan - Cirugía Plástica, Fundación Oftalmológica de Santander, Clínica Carlos Ardila Lulle (FOSCAL), Floridablanca, Colombia Sossa, Claudia - Facultad de Ciencias de la Salud, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia Serrano, Sergio - Facultad de Ciencias de la Salud, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia Arango-Rodríguez, Martha - Banco Multitejidos y Centro de Terapias Avanzadas, Fundación Oftalmológica de Santander, Clínica Carlos Ardila Lulle (FOSCAL), Floridablanca, ColombiaThe ability to rationally propose xeno-free culture environments to expand human multipotent mesenchymal stromal cells (hMSCs) for clinical-grade applications would represent a significant progress in stem cell-based therapies. Nevertheless, rational selection of the appropriate source of nutrients and growth factors for cell maintenance and proliferation requires a deeper understanding of hMSC response to the molecules present in supplemented media. In particular, fetal bovine serum (FBS) has been broadly accepted as a growth factor source for hMSCs. However, its use is problematic because of its xenogeneic origin and biosafety concerns, especially, for cell-based therapies. A number of alternatives to FBS have been explored, including human platelet-derived growth factors (PDGF). Although several studies have evaluated the impact of PDGF on hMSC proliferation and differentiation, few studies have assessed their influence on relevant hMSC processes, such as metabolism and gene expression. Thus, we began to address this challenge by culturing human adipose-derived MSCs (hAD-MSCs) in media supplemented with 10% PDGF or FBS in order to characterize them and evaluate its effect on cell metabolism and gene expression of associated regenerative factors, during cell expansion. Following confirmation of the expression of stem cell surface markers, hAD-MSCs were differentiated into osteogenic and adipogenic lineages. We found a significant osteogenic differentiation in the presence of PDGF without using differentiation medium (p<0.05). Likewise, compared to FBS, PDGF induced a significant two-fold increase in the specific growth rate (p<0.02). Nevertheless, in terms of cell metabolism profile, no significant differences were found between both culture conditions. Furthermore, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions (p<0.01). The present results demonstrate that PDGF influence hAD-MSC behavior; in particular, their metabolic profile suggested that the required carbon source for cell growth could be different from glucose that may be present in the media. Our results advance our understanding of the processes associated with MSC responses for clinical applications.Funding Source: We gratefully acknowledge funding from UNAB and FOSCAL.F-3222ANALYZING HUMAN PLURIPOTENT STEM CELL FATE DYNAMICS BY LIVE, MULTI-REPORTER HIGH CONTENT MICROSCOPYKim, Sungmin - School of Cellular and Molecular Medicine, University of Bristol, UK Casanova, Paola - Pharmacology, University of Cambridge, UK Samacoits, Aubin - School of Cellular and Molecular Medicine, University of Bristol, UK Ren, Edward - School of Cellular and Molecular Medicine, University of Bristol, UK Piddini, Eugenia - School of Cellular and Molecular Medicine, University of Bristol, UK Carazo Salas, Rafael - School of Cellular and Molecular Medicine, University of Bristol, UKStem cells integrate a variety of dynamical inputs – the activity of multiple transcription factors, morphological and proliferative properties, cell-cell interactions and others – in order to allow the formation of correctly differentiated, organized and stratified tissues. Dissecting how those various inputs together impact on the fate of individual cells has been challenging, due to the limited number of reporters allowing simultaneous monitoring of those inputs within live cells. To overcome this limitation we have developed ORACLE, a novel and versatile fluorescent tool allowing live observation of multiple cell fate reporters at the single-cell level. ORACLE is a genetically-encoded two-colour fluorescent reporter - whose expression can be controlled by transcription factor promoters of choice - that localizes to the nuclear envelope, making it distinguishable from and compatible with conventional nuclear-localized reporters. We validate the tool with constitutive and differentially regulated promoters, and show that ORACLE can be used with to monitor the dynamical changes of multiple transcription factors within individual human ESCs, as they undergo differentiation. We further demonstrate

614POSTER ABSTRACTSthat co-expression of ORACLE with fluorescently-tagged histone H2B or the FUCCI cell cycle reporter allows to directly observe how transcription factors and dynamical proliferative features co-vary in individual cells through time, which has been impracticable until now. Thus, ORACLE provides an important new tool to improve live interrogation and dissection of the dynamics of cell fate establishment with single cell resolution.F-3224RAPID EXPANSION OF MESENCHYMAL STEM CELLS USING OPTIMIZED MEDIA SUPPLEMENTED WITH HUMAN PLATELET LYSATE PLTMAX® OR PLTGOLD®, SUITABLE FOR CGMP EXPANSION AT LARGE SCALEAlonso-Camino, Vanesa - Laboratory R&D, Mill Creek Life Sciences, Rochester, NY, USA Mirsch, William - Management, Mill Creek Life Sciences, Rochester, MN, USAMesenchymal Stem Cells (MSCs) are a promising candidate for tissue engineering and regenerative medicine applications. They are currently being used in hundreds of clinical trials and have been safely administered to thousands of patients with an expanding body of evidence of therapeutic efficacy. Mill Creek Life Sciences’ PLTMax®, derived from normal human donor platelets, arose as an alternative to FBS to be used as a supplement for in vitro expansion of human cells used in clinical applications. It is the only media supplement to date demonstrating clinical cultures of MSCs with long term genetic fidelity, rapid expansion and potent clinical activity. PLTMax® is being used worldwide in over 30 clinical trials including Phase I, II and III. Our latest product PLTGold®, is a second-generation human platelet lysate that does not require the addition of heparin to remain clot free, thus providing a fully xenogeneic free alternative supplement. In traditional monolayer expansion systems, we have demonstrated growth of adipose derived and bone marrow derived MSCs using an optimized, fully cGMP compliant culture system. We have found that MSC NutriStem® XF Basal Medium (Biological Industries USA, Inc, Cromwell, CT) supplemented with PLTMax® or PLTGold® exceeds the performance of other conventional media products, obtaining up to 5x108 cells (5 times more cells than with other commercially available products) in only 5 passages. Furthermore, when we use MSC NutriStem® XF Complete Medium supplemented with PLTMax® or PLTGold®, we obtained up to 2x1010 cells (200 times more cells than with other commercially available products) in only 5 passages. Cells grown in these media maintained MSC phenotype and capacity to undergo differentiation. The effective transfer into the clinic of allogenic cell therapies using MSCs will depend predominantly on the development of large scale and cost effective manufacturing platforms that allow production of functional cells at the scale required to meet clinical demand. We will also present the extension of these studies using this optimized method in to evaluate 9 different microcarriers. Together, we will present an optimized protocol for the establishment of large scale expansion of MSCs in bioreactors.F-3226DEVELOPING SAFE AND IMMUNE-TOLERATED CELL THERAPY FOR TREATING NEUROLOGICAL DISEASESMa, Xiaoxue - Australian Regenerative Medicine Institute, Monash University, Melbourne, Australia Payne, Natalie - Australian Regenerative Medicine Institute, Monash University, Melbourne, Australia Nagy, Kristina - Lunenfeld Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada Harding, Jeffery - Lunenfeld Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada Liang, Qin - Lunenfeld Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada Monetti, Claudio - Lunenfeld Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada Nagy, Andras - Lunenfeld Tanenbaum Research Institute, Sinai Health System, Toronto, ON, CanadaEmploying genome-editing strategies, we addressed the safety concerns associated with cell-based regenerative medicine through the development of the FailSafe (FS) Cell System. This proprietary technology inserts a suicide gene into a cell division essential locus, allowing selective elimination of proliferative cells through the administration of a pro-drug, whilst also protecting the suicide gene from inactivation. To develop economically feasible ‘off-the-shelf’ cell products, we developed a strategy for induced allogeneic cell tolerance (iACT). In this system, expression of eight transgenes involved in immune tolerance and rejection allows FS mESCs to form teratomas in MHC-mismatched recipient mice. In the current study, we sought to demonstrate the application of our technology for treatment of neurological diseases. By incorporating expression of firefly luciferase, we used bioluminescence imaging (BLI) to monitor the survival and proliferation of FS mESCs after stereotaxic injection into the mouse brain. Small numbers (1x10^4) of FS mESCs injected into the lateral ventricles were detectable by BLI. Weekly monitoring of live animals and changes in the BL signal over time identified those recipients in which the FS mESCs grafted and proliferated. Histological analysis of brain tissue confirmed that the grafted cells developed into teratomas containing structures derived from the three primary germ layers. To assess whether proliferating cells could be eliminated from the brain, we administered the pro-drug either at the time of transplantation to prevent teratoma formation, or at a delayed time point to allow for terminal differentiation of grafted cells. Longitudinal BLI studies and histological analyses confirmed that our suicide system was effective in eliminating proliferating FS cells from the brain, leaving the non-proliferating component intact. Moreover, proliferating FS mESCs engineered with iACT could be eliminated from the brain by administration of the pro-drug. We are now testing this approach in an allogeneic transplant setting. Ultimately, our FS and iACT technologies will contribute to the development of safe and immune tolerated cell therapies for treating neurological disease.

615POSTER ABSTRACTSFunding Source: This work is supported by a grant form The CASS Foundation.F-3228TWEEN-80 IS RESPONSIBLE FOR THE DELETERIOUS IMPACT OF COMMERCIAL CHEMICALLY DEFINED LIPID CONCENTRATE ON HUMAN PLURIPOTENT STEM CELL CULTUREFaxiang, Xu - Faculty of Healthy Sciences, University of Macau, Macau Meng, Ya - Zhuhai Precision Medical Center, Zhuhai People’s Hospital, Zhuhai, China Liu, Weiwei - University of Macau, Faculty of Health Sciences, Macau, Macau Chen, Guokai - University of Macau, Faculty of Health Sciences, Macau, MacauChemically defined lipid concentrate has been widely used as lipid supplement to culture numerous cell types such as CHO, hybridoma, endothelial cells, cardiomyocytes and various stem cells. Here, we report that the addition of lipid concentrate leads to the cell death of human pluripotent stem cells during passaging and proliferation in chemically defined E8 medium. Systematic analysis of individual components in the lipid concentrate reveals that surfactant Tween-80 is the main contributor to the cell death. Tween-80 permeabilizes cell membrane and disrupt the cellular integrity. However, its toxicity can be suppressed by such as albumin, but not by other polymers such as Pluronic F-68. Tween-80 significantly promotes trophoblast differentiation. We further demonstrate that we could increase the concentration of lipids to support cell growth by removing Tween-80.FundingSource:MYRG2018-00135-FHS,CellFateDetermination by Pyruvate in Human Pluripotent Stem CellsF-3230INTEGRATED COLLECTION OF STEM CELL BANK DATA BY MIACARMChen, Ying - Deptartment of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Fujibuchi, Wataru - Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA) Kyoto University, Kyoto, JapanLast decade has witnessed the fast growth in scale and number of cell lines in stem cell banks in all over the world. Although the worldwide stem cell banks may provide us with proper cell lines for cell therapies, the reproducibility and data exchange among stem cell banks are constrained by the lack of a standardized format. To solve this problem, MIACARM (Minimum Information About a Cellular Assay for Regenerative Medicine) was first developed and released in 2016 as a guideline for standardizing items and formatting cellular assay data produced from stem cell banks all over the world. Based on the MIACARM data model, a user-friendly integrated collection database was developed for cross search among 6 databases, UK Stem Cell Bank, RUCDR, hPSCreg, SKIP, Riken, and eagle-i. Presently, a total of 14,314 registered stem cell lines are available by the online search of http://icscb.stemcellinformatics.org/, where researchers can easily approach the standardized information of stem cells or stem cell lines from cell banks throughout the world. Advantages by adopting MIACARM include the deposition of minimum information and assay metadata to reduce missing and unnecessary information about the cells, the progress toward reproducible experiments, and the retrieval of stem cell lines by omnibus search across all cell banks and registries in the world. Also, MIACARM based integrated database will be promising and flexible to incorporate with newly developed cell banks with increasing number of cell lines in future. Currently, basic structure of MIACARM includes two levels (I and II) with five module structure (project, source cell, assay, experimental technology and data). MIACARM-I contains the guideline for basic cell research, which includes omics assays using human cells. MIACARM-II contains the guideline for stem cell based regenerative medicine, which includes terms related to stem cell and source cell quality checks. We further plan to extend MIACARM to be able to characterize cells differentiated from the stem cell lines (MIACARM-III).F-3232INVESTIGATION OF MURINE DENTAL PULP STEM CELLS WITH 3D IMAGING OF TOOTH AFTER TISSUE CLEARINGKim, Young Hwan - Department of Oral Biochemistry, Pusan National University School of Dentistry, Yangsan, Korea Lee, Jeong Sang - Department of Pediatric Dentistry, Pusan National University Dental Hospital, Yangsan-si, Korea Seo, Eun Jin - Department of Oral Biochemistry, Pusan National University School of Dentistry, Yangsan-si, Korea Park, Jae Kyung - Department of Oral Biochemistry, Pusan National University School of Dentistry, Yangsan-si, Korea Ha, Chang Man - Brain Research Resource Center, Korea Brain Research Institute, Daegu, Korea Ryu, Youngjae - Brain Research Resource Center, Korea Brain Research Institute, Daegu, Korea Jung, Tae-Sung - Department of Pediatric Dentistry, Pusan National University Dental Hospital, Yangsan-si, Korea Jang, Il Ho - Department of Oral Biochemistry, Pusan National University School of Dentistry, Yangsan-si, KoreaTo achieve a comprehensive understanding of tissue-resident dental stem cells and regeneration mechanism, we develop 3D imaging protocol for tooth using tissue clearing and light sheet microscopy. Mouse molars were partially decalcified in 10% EDTA (pH8.0) for 2 weeks with or without formic acid, followed by CUBIC or bone CLARITY tissue clearing protocol using X-CLARITY. Cleared molars were subjected to the primary antibody staining for 10 days and the secondary antibody staining for 10 days. After antibody staining, molars were stained with the hoechst 33342 for overnight and finally immersed in

616POSTER ABSTRACTSthe refractive index matching solution (RIMS). Samples were imaged with ZEISS Lightsheet Z.1 microscope and 3D images were rendered using Arivis software. Addition of formic acid produced opaque area along dental pulp and tissue clearing with CUBIC protocol was not efficient, thus either protocol was not adopted. After 10% EDTA and bone CLARITY process, molars were divided half with razor and subjected to staining with antibodies of BMI1 or SOX2 among stem cell markers and b-actin for counterstaining. Followed by the corresponding secondary antibody staining and RIMS immersion, molars were imaged with light sheet microscope. The result showed the presence of dentin layer and adequate staining of beta-actin in dental pulp area, which suggests the successful penetration of antibody and light sources for imaging. Interestingly, both BMI1 and SOX2 staining were localized to root area with increasing signal to root apex and decreasing signal toward pulp chamber. These results suggest the higher stem cell activity at root canal in dental pulp in comparison with crown chamber. We established 3D imaging protocol for tooth using bone CLARITY tissue clearing and lightsheet microscope, which provides a platform for the analysis of cellular and molecular mechanism of dentin and pulp regeneration.Funding Source: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (NRF-2018R1D1A1A02048916).F-3234A SMALL MOLECULE COCKTAIL PROMOTES CELL SURVIVAL AND TRANSLATIONAL APPLICATIONS OF HUMAN PLURITOTENT STEM CELLSChen, Yu - Stem Cell Translational Laboratory, National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Tristan, Carlos -National Center for Advancing Translational Sciences , NIH, Rockville, MD, USA Chen, Lu -National Center for Advancing Translational Sciences , NIH, Rockville, MD, USA Jovanovic, Vukasin -National Center for Advancing Translational Sciences , NIH, Rockville, MD, USA Malley, Claire -National Center for Advancing Translational Sciences , NIH, Rockville, MD, USA Chu, Pei-Hsuan -National Center for Advancing Translational Sciences , NIH, Rockville, MD, USA Ormanoglu, Pinar - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Austin, Christopher - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Simeonov, Anton - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Singec, Ilyas - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USAAccess to human induced pluripotent stem cells (iPSCs) has created novel paradigms for drug discovery and regenerative medicine. However, the poor survival of iPSCs during routine passaging and after cryopreservation poses major challenges to the establishment of well-controlled workflows to produce and store iPSCs on a large scale, and to the development of efficient genome editing protocols based on optimized single-cell cloning procedures. The ROCK inhibitor Y-27632 has been widely used to improve cell survival, but significant amounts of cell death remain evident in many iPSC applications. Here, we developed a four-component small-molecule cocktail named “CEPT” that dramatically improves iPSC viability. Testing 12, 744 compounds in quantitative high-throughput screening (qHTS), we first identified 114 hits that improved iPSC survival. Advancing 29 hits to combination screening based on their diverse modes of action, we discovered compound C and compound E as a synergistic pair (CE) that improved iPSC survival during routine passaging by approximately 50% as compared to Y-27632. Despite the dramatic effect of CE during routine passaging, its benefit was only modest when iPSCs were seeded at a low density (25 cells/cm2) or in a 1 cell/well condition. Therefore, we designed another combination screening assay to search for additional compounds that, when applied together with CE, can further enhance iPSC survival at low cell density conditions. Screening 8,011 compounds using the new assay, we found that CE together with two additional compounds (compounds P and T) dramatically improved iPSC survival at low cell density conditions, increasing single-cell cloning efficiency to ~65% as compared to the ~10% with Y-27632. We then extensively tested CEPT and demonstrated that this cocktail was highly efficient in improving cell survival during routine cell passaging, embryoid body formation, single-cell cloning following genome editing using CRISPR/Cas9, the establishment of new iPSC lines, as well as cell recovery after cryopreservation. Hence, the versatility of CEPT provides a powerful chemical platform for establishing efficient protocols and may become a widely used approach in drug development and regenerative medicine.F-3236MECHANOBIOLOGICAL REGULATION OF PLURIPOTENCY IN MOUSE EMBRYONIC FIBROBLASTSLee, Jason - Biomedical Engineering, The University of Texas at Austin, TX, USA Armenta-Ochoa, Miguel - Biomedical Engineering, University of Texas at Austin, TX, USA Maceda, Pablo - Biomedical Engineering, University of Texas at Austin, TX, USA Yoon, Eun - Biomedical Engineering, University of Texas at Austin, TX, USA Samarneh, Lara - Biomedical Engineering, University of Texas at Austin, , TX, USA Wong, Mitchell - Biomedical Engineering, University of Texas at Austin, TX, USA Baker, Aaron - Biomedical Engineering, University of Texas at Austin, TX, USAProducing induced pluripotent stem cells (iPSCs) from mature somatic cells is a promising strategy for creating new therapies for many diseases. However, most strategies for creating iPSCs often require genetic manipulation, creating concern for their

617POSTER ABSTRACTSuse as therapeutic cell lines. In this study, we examined whether mechanical force could play a role in enhancing the generation of iPSCs without genetic modification. We recently developed a high throughput biaxial stretching device that is capable of applying dynamic and complex mechanical strain on 576 cell cultures wells simultaneously to perform mechanobiological screening assays. Using this device, we applied mechanical stretch to mouse embryonic fibroblasts expressing an Oct-4 reporter construct. We first performed a dose response to mechanical load by applying a range of mechanical loads from 2.5-17.5% strain using the system. All mechanical strains induced some increase in Oct-4 and we observed a maximum increase of 2.5-fold in cells treated with 17.5% strain at 0.1 Hz. MEFs stretched under these conditions also expressed five-fold increase in Sox2 expression and three-fold increase in SSEA1. We also performed a drug screen in combination with mechanical loading and found several compounds that synergistically increased Oct-4 expression in MEFs with mechanical load over seven days of treatment. Further gene expression analysis through RT-PCR confirmed increase in several pluripotency markers such as Sox2, SSEA1, Nanog, and related genes. Overall, we demonstrated a new non-viral methodology to prime MEFs by efficiently identifying optimal mechanical and pharmacological treatments.Funding Source: The authors gratefully acknowledge funding through the American Heart Association, the DOD CDMRP, and the National Institutes of Health to ABB.F-3238UNCOATED AND STARCH-COATED SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES FOR EFFECTIVE STEM CELL LABELING AND TRACKINGEl-Badri, Nagwa - Biomedical Sciences, Zewail City Of Science And Technology, Giza, Egypt Elkhenany, Hoda - Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt Abd Elkodous, M. - Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt Ghoneim, Nehal - Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt Ahmed, Toka - Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt Ahmed, Sara - Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, EgyptExperimental stem cell therapy protocols require cell tracking and monitoring in vivo.to determine cell differentiation, proliferation and migration. Superparamagnetic Iron Oxide (SPIO) nanoparticles have emerged as one of the contrast agents that can be detected by magnetic resonance imaging (MRI). We hypothesized that starch-coated SPIOs could be effectively used as bio-compactible labeling nanoparticle for adipose tissue-derived stem cells (ASCs). ASCs were labelled with the following forms of SIPOs: ferric oxide (Fe3O4), ferrous oxide (Fe2O3), or cobalt-nickel ferrite (CoxNi1-x Fe2O4) nanoparticles. The particles were either coated or uncoated with starch. Proliferation, viability, migration and vasculogenic differentiation of labeled cells were evaluated. Our results showed that uncoated and starch-coated Fe2O3 nanoparticles were more efficient in labeling ADSC as confirmed by transmission electron microscopy (TEM). Both uncoated Fe2O3 and starch-coated Fe3O4 nanoparticles were biocompatible as determined by lack of cell toxicity to ASCs and their significant proliferation. Both uncoated and starch-coated Fe2O3 showed a protective effect against apoptosis. Migration scratch-healing assay revealed that cells labeled with starch-coated Fe2O3 and Fe3O4 exhibited a significantly higher migration rate after 48 hrs. The angiogenic potential of ASCs was more robust in cells labeled with starch coated Fe2O3 and starch coated Fe3O4 than the CoxNi1-x Fe2O4-loaded cells. Our data show that starch-coated Fe2O3 and Fe3O4 can be safely used to track stem cells, as shown by their biocompatibility, and enhancing cell proliferation, migration and angiogenic potential.Funding Source: This study was funded by the Egyptian Science and Technology Development Fund (STDF; Grant ID: 5300).F-3240H-Y- INCOMPATIBLE MOUSE EMBRYONIC STEM CELL TRANSPLANTATION CAUSES REJECTION OF NUCLEUS MATCHED CELLSHu, Xiaomeng - Department of Surgery/ TSI Lab, University of California, San Francisco, CA, USA Kueppers, Simon - Heart Center, UKE, Hamburg, Germany Kooreman, Nigel - Stanford Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA Gravina, Alessia - Surgery, University of California, San Frascsico, CA, USA Wang, Dong - Surgery, University of California, San Franscico, CA, USA Tediashvili, Grigol - Surgery, University of California, San Franscico, CA, USA Marcus, Sivan - Surgery, University of California, San Franscico, CA, USA Deuse, Tobias - Surgery, University of California, San Franscico, CA, USA Schrepfer, Sonja - Surgery, University of California, San Franscico, CA, USAPluripotent stem cells (PSCs) are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, gene engineering approaches, immunosuppression, or cell banks are discussed. In this context, reducing the immunogenicity of the donor cell is crucial, but little is known about the contribution of minor histocompatibility antigens

618POSTER ABSTRACTS(mHA) mismatched grafts in stem cell immunobiology. For whole organ transplantation, donor H-Y antigens, a class of mHAs encoded by the Y chromosome, has revealed worse graft outcomes for male to female transplants of heart, liver, and kidney. In addition, male recipients of female hemotopoietic stem cell grafts are more likely to develop graft versus host disease (GvHD). In this study, we investigated the immunogenicity of the H-Y antigen in a murine nucleus matched, male-to-female embryonic stem cell (ESC) transplant setting. We demonstrate in vitro and in vivo immune responses in a H-Y mismatched model, that is mainly T-cell mediated, resulting in limited survival of H-Y mismatched murine ESCs in syngeneic female mice. In addition, using the acquired tolerance concept, we show that tolerance towards H-Y can be induced in the neonatal phase. Furthermore, our data indicate that the H-Y immunogenicity is preserved upon differentiation of ESCs into endothelial cells. The findings presented in our study are therefore of significant clinical importance since they reveal the need to match beyond HLA towards mHA mismatches and define optimal donor cells with caution.Funding Source: NAF-3242GENOME-SCALE CRISPR-CAS9 KNOCKOUT SCREENING FOR GENES REGULATING GLUCOSE UPTAKE IN HESC-DERIVED VASCULAR SMOOTH MUSCLE CELLSChen, Zhifen - Cardiovascular Institute, BIDMC, Harvard Medical School, Boston, MA, USA Cowan, Chad - Cardiovascular Institute, BIDMC, Harvard Medical School, Boston, MA, USABy causing damage on micro- and macrovascular system, hyperglycemia is an independent risk predictor for cardiovascular morbidity and mortality, as well as for total mortality, independent of the subsequent development of overt diabetes mellitus. However, paucity is known about genes responsive to the high glucose stimulus in vascular system. Here, we used a genetically encoded glucose sensor in the genome-scale Crispr Cas9 knockout (GeCKO) screening for genes regulating glucose uptake in human embryonic stem cell derived vascular smooth muscle cells (hESC-VSMC). The gene candidates identified by the GeCKO screening were used in the second pooled screening to prioritize the top ones critical for regulating glucose uptake in hESC-VSMC. Through a detailed mechanistic study of the top genes for glucose uptake, we intend to gain insight about how high blood glucose level or hyperglycemia affects the VSMC function and contributes to macrovascular complications.LATE-BREAKING ABSTRACTSF-4002SUBCELLULAR DYNAMICS OF RNAS AND MICRORNAS IN MOUSE EMBRYONIC AND TROPHOBLAST STEM CELLSPark, Brian J - Department of Physiology, University of Toronto, , ON, Canada Cox, Brian - Department of Physiology, University of Toronto, ON, Canada Magony, Alex - Department of Physiology, University of Toronto, ON, Canada Nosi, Ursula - Department of Physiology, University of Toronto, ON, Canada Yang, David - Department of Physiology, University of Toronto, ON, CanadaSubcellular trafficking of RNAs plays a pertinent role in regulation of key biological processes, such as maintenance and progression of cell identity and function. Mouse embryonic stem cells (ESCs) and trophoblast stem cells (TSCs), as representatives of early cell fate decision – the inner cell mass (ICM) and the trophoectoderm (TE), respectively – rely on reciprocal molecular signaling between coding RNAs and regulatory RNAs for self-maintenance and proliferation. Understanding the spatial dynamics of RNA localization and interaction in ESCs and TSCs gives insight on pertinent processes involved in cell fate determination. Here, a biochemical fractionation of cytosolic and nuclear compartments of mouse ESCs and TSCs is coupled to gene expression profiling by high-throughput RNA- and smallRNA-sequencing. To ensure proper quantitative analysis between cytosolic RNA (cytRNA) and nuclear RNA (nucRNA), a mass-balance scaling factor is applied to normalize against RNA concentration and cell equivalence. Next, bioinformatics analysis of coding and noncoding RNAs shows asymmetric expression of pluripotency genes across ESCs and TSCs, suggesting our methodology of cell fractionation and data normalization is suitable for comparative analysis. Differential expression analysis of compartments revealed that nucRNA in both ESCs and TSCs show enrichment of terms associated with RNA processing, transport, and splicing. Uniquely, TSCs nucRNA was enriched in regulatory noncoding RNA. Furthermore, genes related to RNA and DNA processing show higher retention of introns relative other genes; a phenomenon conserved across cytRNAs and nucRNAs in both cell lines. A parallel pipeline for microRNAs (miRNAs) show differentially expressed miRs in both the cytosol and the nucleus of ESCs and TSCs, suggesting a nuclear role of mature miRs associated with ICM and TE phenotype. This high-throughput, compartment-specific method of surveying differentially expressed RNAs – including introns – allows a lead to a deeper understanding of regulatory dynamics governing ICM and TE fate.

619POSTER ABSTRACTSF-4004ACID-EXTRUDING AND -LOADING MECHANISM IN HUMAN STEM CELLS AND CANCER CELLSChao, Chih-Chi - Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan Chen, Hsuan-Yu - Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan Lin, Yu-Shan - Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan Dai, Niann-Tzyy - Division of Plastic and Reconstructive Surgery, Tri-Service General Hospital, Taipei, Taiwan Lee, Shiao-Pieng - Division of Oral and Maxillofacial Surgery, Tri-Service General Hospital, Taipei, Taiwan Wu, Gwo-Jang - Department of Obstetrics and Gynecology, Tri-Service General Hospital, Taipei, Taiwan Loh, Shih-Hurng - Department of Pharmacology, National Defense Medical Center, Taipei, TaiwanPluripotent stem cells (PSCs) were high proliferation and rely on glycolysis. In tumor, the up-regulation of acid-extruders, such as Na+-H+ exchanger (NHE), was to adapt the higher production of metabolic acid from glycolysis, and its further created a reverse pH gradient: microenvironment acidification and intracellular alkalization. Thus, it’s easy to associate that PSCs have the higher activity of acid-extruders. Some studies had shown that acidic culture medium retained the pluripotency during differentiation. However, acidic environment also reduced the quality of PSCs during the cultured process, through inhibited the growth and increased the apoptosis. Until now, pHi regulating mechanism in human stem cells is still unknown. The aims of this study were to investigated the role of pHi regulation in the different stem and cancer cells. The hiPSCs (HPS0077), human adipose-derived stem cells (hADSCs) and OEC-M1 was kindly provided from Dr. Dai and Dr. Lee. The change of pHi was detected either by microspectrofluorimetry method. Weak acid-base pre-pulse were used to induce the intracellular acidification and alkalization. Our present results showed that the buffering capacity were increased with the intracellular alkalization in hiPSCs. NHE, Na+-HCO3- cotransporter (NBC), Cl-/ OH- exchanger (CHE) and Anion exchanger (AE) were activated for the pHi regulating mechanism in all cell lines, while V-ATPase was only activated for the acid-extruding in HeLa and A549. Interestingly, the unknown Na+-independent acid extruder(s) and unknown Cl--independent acid-loader(s) could only detected in hiPSCs. The resting pHi value was found to be 7.7 in hiPSCs, which was much higher than that of other cells (7.46, 7.13, 7.49, 7.4 and 7.39 in hDPSCs, hADSCs, OEC-M1, HeLa and A549). The strengthening of buffering capacity and the weakening of acid extruding mechanism, including intracellular acidification and decreasing of activity of acid-extruders, was observed during the loss of pluripotency. Summary, our study provided a model and the dynamic changes of pHi regulating mechanism at different pluripotent state in hiPSCs. Moreover, the pHi in hiPSCs was much higher than the others cancer and adult stem cells. Key words: hiPSCs; hMSCs; cancer cells; acid-extruder; acid-loader; buffering capacity.F-4006USING ACTIVITY-BASED CHEMICAL PROTEOMICS TO UNCOVER ESSENTIAL REGULATORS OF PLURIPOTENCYTu, William B - David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Cao, Jian - Biological Chemistry, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Backus, Keriann - Biological Chemistry, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Plath, Kathrin - Biological Chemistry, University of California - Los Angeles (UCLA), Los Angeles, CA, USAPluripotent stem cells exist in a spectrum of states with distinct cellular and molecular features, including differential signaling, transcriptomic and chromatin landscapes, X-inactivation state and developmental properties. For instance, mouse embryonic stem cells (ESCs) represent a naïve pluripotent state of the pre-implantation embryo, while mouse epiblastic-derived stem cells (EpiSCs) resemble the developmentally more advanced primed state of the post-implantation embryo. Better understanding these pluripotent states will yield insights into the mechanisms underlying earliest embryonic development. Functional genomic studies, such as genome-wide RNAi screens, have identified transcription factors and chromatin complexes as regulators of pluripotent states, but may not capture regulatory events at the protein-level, including regulation by enzymatic activities and post-translational modifications. Activity-based protein profiling (ABPP) in conjunction with chemical proteomics has emerged as an unbiased proteome-wide approach to identify functional and small molecule-targetable proteins in disease states. We apply the quantitative isotopic Tandem Orthogonal Proteolysis (isoTOP)-ABPP method to compare the cysteine (Cys) reactivity profiles of ESCs and epiblast-like cells (EpiLCs) to capture the molecularly different naïve and primed states, as Cys residues are important for protein stability, enzymatic activity and cellular redox reactions. Overall, we identify 3872 reactive Cys from 2192 proteins. Of these Cys, 170 show elevated labeling specifically in ESCs and 160 in EpiLCs. The Cys preferentially labeled in ESCs are enriched on proteins involved in carbon and amino acid metabolism and fatty acid degradation, while EpiLC-specific Cys labeling occurs in proteins involved in ribosomes and lipid biosynthesis. We also identify proteins with reactive Cys of unknown functions in the regulation of pluripotency. Using electrophilic compounds that target Cys in specific proteins, we observe that compound treatment inhibited the maintenance of pluripotency in ESCs. Together, these results suggest that activity-based chemical proteomics identifies novel classes of pluripotency regulators, which can be targeted by small molecules to manipulate the pluripotent state.Funding Source: This research is supported by UCLA Broad Stem Cell Research Center.

620POSTER ABSTRACTSF-4008L-PROLINE INDUCES MESC DIFFERENTIATION TO A SPATIALLY DISTINCT EPL-CELL POPULATION THROUGH A COMPLEX SIGNALLING REGULATORY NETWORK TO PROMOTE NEURAL DIFFERENTIATIONGlover, Hannah - Bosch Institute, Discipline of Physiology, University of Sydney, Camperdown, Australia Morris, Michael - Bosch Institute, Discipline of Physiology, School of Medical Sciences, University of Sydney, Camperdown, AustraliaThe amino acid L-proline has novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesis. Addition of 400 μM L-proline to self-renewal medium drives mouse embryonic stem cells (mESCs) to a transcriptionally distinct pluripotent cell population - early primitive ectoderm-like (EPL) cells - which lies between the naïve and primed states. Both EPL cells and EpiSCs express ‘primed pluripotency’ genes such as Fgf5, Dnmt3b and Otx2. EPL cells have high expression of neurogenesis genes, and map to the anterior aspect of the ~7.0 dpc embryo – the site of the prospective neurectoderm. Conversely, EpiSCs have a propensity to differentiate to mesendoderm, upregulate expression of cardiovascular development genes, and more closely map to the distal-posterior aspect of the embryo. While both these cell types retain pluripotency, these gene expression changes indicate EPL cells and EpiSCs are predisposed to different lineage commitments. Here, we use EPL cells to understand the molecular mechanisms underpinning pluripotency and neural induction. In mESCs, L-proline acutely increases phosphorylation of MAPK, mTOR and PI3K signalling-pathway intermediates. Inhibition of these signalling pathways individually had little effect on cell fate. When these inhibitors were used in combination, more robust changes were seen in gene expression and emergent properties including colony morphology, cell number, proliferation and apoptosis. This data was analysed using statistical modelling to identify synergistic effects between inhibitors – where two pathways converge to produce a change larger than either individually, or interaction effects - where two pathways converge and produce an effect equal to either pathway alone. Further analysis using linear regression was used to determine which signalling pathways make the largest contribution to changes in gene expression and emergent properties. This statistical model showed that a decrease in expression of the naïve pluripotency marker Rex1 or an increase in the primed pluripotency marker Fgf5 could be explained by a single pathway (MAPK and mTOR, respectively). Changes in the other 12 parameters were attributed to signalling pathways acting together - indicating a complex, self-regulating signalling network.F-4010GENERATION OF CLINICAL-GRADE FUNCTIONAL HEPATOCYTES FROM HUMAN EMBRYONIC STEM CELLS IN XENO-FREE CONDITIONSLi, Zhongwen - Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Wu, Jun - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Wang, Lei - Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Yu, Juan - Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Wang, Yukai - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Feng, Guihai - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Li, Wei - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Gu, Qi - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Hu, Baoyang - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Wang, Liu - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Zhou, Qi - Institute of Zoology, Chinese Academy of Sciences, Beijing, China Hao, Jie - Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaAs one of the most important organs in the body, the liver is susceptible to many major diseases, which are the main causes of morbidity and mortality. Patients with acute liver injuries or end-stage liver diseases have no choice but to receive a liver transplant. However, the source of donor livers is restricted. In recent years, hepatocyte transplantation has been attempted to treat liver failure clinically. However, the use of primary or fetal hepatocytes has been restricted due to the lack of available healthy donors as well as limited cell proliferation, functional deficits, the risk of immune rejection and the concern of ethical issues. Hepatocytes have been successfully generated from human pluripotent stem cells (hPSCs). However, the cost-effective and clinical-grade generation of hepatocytes from hPSCs still need to be improved. In this study, we reported the production of functional clinical-grade hepatocytes from clinical-grade human embryonic stem cells (hESCs) under GMP requirements. During hepatoblast differentiation, dimethylsulfoxide (DMSO) and four small molecules (transferrin, Vc-Mg, insulin and sodium selenite) were used instead of cytokines and FBS/KOSR. Then, hepatoblasts were differentiated into hepatocyte-like cells (HLCs) that had a typical hepatocyte morphology and possessed characteristics of mature hepatocytes, such as metabolic-related gene expression, albumin secretion, fat accumulation, glycogen storage and inducible cytochrome P450 activity in vitro. HLCs integrated into the livers of Tet-uPA Rag2-/- Il2rg-/- (URG) mice, which partially recovered after transplantation.

621POSTER ABSTRACTSFurthermore, a series of biosafety-related experiments were performed to ensure future clinical applications. This efficient platform could facilitate the treatment of liver diseases using hESC-derived HLCs transplantation.Funding Source: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030400), the National Key Research and Development Program (2017YFA0104403, 2016YFA0101502, 2017YFA0105001).F-4012THE TCA CYCLE METABOLITES REGULATE HUMAN EMBRYONIC STEM CELL SURVIVAL AND MITOCHONDRIAL DYNAMICS DURING NAÏVE PLURIPOTENCYChien, Yu - Department of Obstetrics and Gynaecology, National Taiwan University Hospital, Kaohsiung, Taiwan Sytwu, Huey-Kang - National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan Chen, Mei-Jou - Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan Ho, Hong-Nerng - Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, TaiwanDuring early embryonic development, embryonic stem cells (ESCs) shift naïve pluripotency into primed epiblast stem cells (EpiSCs). The conversion of the metabolic demand between oxidative phosphorylation and glycolysis can impact on the transition from naïve to primed pluripotency. The TCA cycle metabolite -ketoglutarate ( KG) can maintain naïve pluripotency, ααwhereas aerobic glycolysis-derived cytosolic acetyl-CoA contributes primed pluripotency through epigenome remodeling. However, whether other TCA cycle metabolites can affect the pluripotent status have not been comprehensively characterized yet. Here we identify citrate, malate, oxaloacetate and succinyl CoA of the eight intermediates of the Krebs cycle led to naïve human ESC death. The cellular death attributed to the formation of autophagosomes and mitochondria with degenerate cristae manifested by transmission electron microscopy. Isocitrate but not KG promoted hESC differentiation and rescued the cellular αdeath caused by oxaloacetate both in naïve hESC and mouse early embryos. In vitro manipulation of the pluripotency from naïve hESC into primed hEpiSC, the mitochondria changed from punctate into the filamentous network. The primed hEpiSC counteracted the oxaloacetate-caused cell death by restoring cristae structure and elongating mitochondrial configuration. These results suggest that mitochondrial membrane structures and dynamics correlate with pluripotent status and TCA cycle metabolites have distinct functions in regulating naïve hESC pluripotency and survival.F-4014THE REGULATION OF HEDGEHOG SIGNALING PATHWAY BY PUM1 IN MOUSE EMBRYONIC STEM CELLSLu, Ting - Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China Li, Xiajun - School of Life Science and Technology, ShanghaiTech University, Shanghai, China Shi, Shuo - Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China Qi, Hongying - Yale Stem Cell Center, Yale School of Medicine, New Haven, USA Lin, Haifan - Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaThe PUF (Pumilio and FBF) family of RNA-binding proteins are evolutionally conserved post-transcriptional regulators. Through binding to recognition sites in target mRNAs, they can regulate gene expression by promoting mRNA degradation or inhibiting translation. PUFs have been shown to be required for germline stem cell maintenance in D. melanogaster, C. elegans, and the planarian S. mediterranea, but little is known about their function in mammalian stem cells. Pumilio1 (PUM1) and Pumilio2 (PUM2) are two murine members of the PUF family proteins. Our previous study showed that Pum1 deficiency leads to smaller body size and multiple developmental defects, indicating Pum1 may play important roles in mouse embryogenesis. Here we report that knockdown of Pum1 in ESCs does not affect their self-renewal, but results in defects in embryoid body differentiation, especially in the mesodermal lineage. Furthermore, PUM1 can negatively regulate Gli1 expression and Hedgehog (HH) signaling pathway activity both in mouse embryos and in ESCs. Thus, we hypothesize that PUM1 affects ESC differentiation and mouse embryonic development at least in part by influencing the HH signaling pathway. By investigating the regulatory mechanism of PUM1 on the HH signaling pathway, our study will shed new light on how PUF proteins and the HH signaling pathway regulate ESCs and embryogenesis in mammals.F-4016SEARCHING FOR A PREDICTIVE BIOMARKER TO SELECT HUMAN INDUCED PLURIPOTENT STEM CELLS WITH HIGH CARDIAC DIFFERENTIATION POTENTIALOhashi, Fumiya - Research and Development Center, Terumo Corporation, Kanagawa, Japan Miyagawa, Shigeru - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Yasuda, Satoshi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, Japan Miura, Takumi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, Japan Kuroda, Takuya - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, Japan

622POSTER ABSTRACTSItoh, Masayoshi - Preventive Medicine and Diagnosis Innovation Program, RIKEN Center, Kanagawa, Japan Kawaji, Hideya - Preventive Medicine and Diagnosis Innovation Program, RIKEN Center, Kanagawa, Japan Ito, Emiko - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Yoshida, Shohei - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Saito, Atsuhiro - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Kanagawa, Japan Oyama, Kenji - Research and Development Center, Terumo Corporation, Kanagawa, Japan Matsuda, Isamu - Research and Development Center, Terumo Corporation, Kanagawa, Japan Sameshima, Tadashi - Research and Development Center, Terumo Corporation, Kanagawa, Japan Kawai, Jun - Preventive Medicine and Diagnosis Innovation Program, RIKEN Center, Kanagawa, Japan Sawa, Yoshiki - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Sato, Yoji - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, JapanSelection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening, but current pluripotency markers such as OCT-4, LIN28, and NANOG cannot be used to distinguish the direction of differentiation. The purpose of the present study was to identify a biomarker for predicting efficient cardiac differentiation that can be used for selecting individual hiPSC lines by comparing the gene expression profiles of undifferentiated hiPSC lines with varying cardiac differentiation potential. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA array, and microRNA array to efficiently screen biomarkers related to cardiac differentiation of hiPSCs. Subsequently, we subjected all hiPSC lines to cardiac differentiation. Differentiation capacity was quantified based on the percentage of cardiac troponin T positive cell populations using flow cytometry and cardiac related genes using qPCR. As a result, we observed that a difference in the purity of cardiac troponin T among hiPSC lines ranging from 7.7%±2.6% in RIKEN-12A to 92.3%±1.2% in 409 B2. Differential global gene expression within the undifferentiated hiPSCs state revealed 22 distinguishing candidate genes among hiPSC lines with caridiac propensity. Of the candidate genes, PF4 was validated as a biomarker expressed in undifferentiated hiPSCs with high potential for cardiac differentiation in 13 additional hiPSC lines. In addition, we found that pretreatment with PF4 enhanced the cardiac differentiation potential of hiPSCs, suggesting that PF4 could promote cardiac differentiation. These observations suggest that PF4 gene expression, which could vary not only between hiPSC lines but also between pharmacological conditions, reflect the potential of hiPSCs to differentiate into cardiomyocytes and can thus be used as a quality control marker of hiPSCs. In conclusion, differential global gene expression of hiPSC lines with high and low differentiation capacity showed that cardiac differentiation potential may be predicted by measuring the expression of PF4, suggesting that these genes may be helpful in selecting hiPSCs lines for regenerative therapy and drug screening.F-4018NAÏVE VERSUS PRIME HUMAN STEM CELL FOR LIVER ORGANOID DIFFERENTIATIONThompson, Wendy - Developmental Biology, Cincinnati Children’s Hospital and Medical Center, Cincinnati OH, USA Zhang, RanRan - Developmental Biology, Cincinnati Children’s, Cincinnati, OH, USA Cai, Yuqi - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, USA Kimura, Masaki - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, USA Takebe, Takanori - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, USAOrganoid based approaches hold promise for advancing human health and disease biology, drug development, and regenerative medicine. For example, we have differentiated human induced pluripotent stem cells (hiPSCs) to make multi-lineage human liver organoids (HLOs) that are capable of modeling steatohepatitis in vitro. However, there is a considerable variability in deriving these HLOs in terms of cell type differentiation ability as well as organoid forming efficiency across hiPSC lines, even among clones derived from the same individual. We reasoned this at least in part is associated with epigenetic variabilities between lines. To overcome these challenges, we have developed a novel method to produce putative “naïve” human cells from “primed” hiPSCs. Naïve stem cells normalizes many of the epigenetic changes that occur during natural development and that can alter gene expression. After culturing hiPSCs with naïve mouse embryonic stem cells (mESCs) for 5 days and sorting out the mouse cells the hiPSCs undergo a morphological change from 2D flat shaped colonies to round, domed shaped colonies that become more stable with increased passaging. These human putative “naïve” cells express naïve markers by qPCR and immunostaining such as DPPA3, KLF17, TFCP2L1, and TFAP2C, and have downregulated primed markers such as DUSP6. This method is consistent across several hiPSC lines, and is accompanied by diverse chromatin accessibility changes. We are currently working to 1) re-prime these putative “naïve” cells and differentiate these cells into HLOs or 2) use embryoid body differentiation, to compare the HLO efficiency to the same cell line that has not been previously converted into a putative “naïve” status. To compare HLO efficiency between naïve and primed iPSC, we will quantify organoid number, levels of liver specific markers, hepatic production and drug metabolism enzyme activity. This work will help to provide valuable insights into how epigenetic changes can affect differentiation and development in HLOs and beyond.

623POSTER ABSTRACTSF-4020AN INTEGRATED GENOMIC APPROACH TO STUDY ASTROCYTE-INDUCED MATURATION IN HUMAN INDUCED GLUTAMATERGIC NEURONSKotter, Mark R - Department of Clinical Neurosciences and Wellcome Trust MRC Cambridge Stem Cell Institute, University of Cambridge, UK Abdul Karim, Muhammad Kaiser - Department of Clinical Neurosciences and Wellcome Trust MRC Cambridge Stem Cell Institute, University of Cambridge, UK Baranes, Koby - Department of Clinical Neurosciences and Wellcome Trust MRC Cambridge Stem Cell Institute, University of Cambridge, UK Patikas, Nikolaos - UK Dementia Research Institute, University of Cambridge, UK Cooper, Sarah - Wellcome Sanger Institute, Cambridge, UK Bello, Erica - UK Dementia Research Institute, University of Cambridge, UK Tourigny, David S. -, MRC Laboratory of Molecular Biology, Cambridge, UK Metzakopian, Emmanouil - UK Dementia Research Institute, University of Cambridge, UK Bassett, Andrew - Wellcome Sanger Institute, Cambridge, UK O’Neil, John S. - MRC Laboratory of Molecular Biology, Cambridge, UKAstrocytes are known to modulate many aspects of neuronal development, including their maturation, axon outgrowth and synapse formation. Whilst most studies of neurons are based on non-human models, the development of rapid neuronal reprogramming protocols has provided a unique opportunity to study the biology of human neurons. We have previously demonstrated that gene targeting the components of a genetic switch into genomic safe harbour sites enables optimised expression of NGN2 in hiPSC (OptiOx) and yields homogenous cultures of pure cortical glutamatergic neurons (hiNeuron) in less than four days. Here we aimed to gain a wholistic insight into how astrocytes modulate the function of human excitatory neurons. To assess electrophysiological activity, on the 3rd day of reprogramming, primary rat astrocytes were added to the hiNeuron cultures maintained on multi-array-electrodes (MEAs). Serial MEA recordings demonstrated spontaneous electrophysiological activity of hiNeurons as early as 11 days after induction. At 21 days post induction, synchronised burst patterns were detected across the network. In the absence of astrocytes hiNeurons cell bodies tended to cluster together and limited spontaneous electrophysiologic activity was recorded. The presence of astrocytes therefore profoundly affected neuronal function. In order to study genome-wide effects of astrocytes on hiNeurons, three independent biological samples were collected of hiNeurons cultured in the presence and absence of astrocytes at day 4, 14, and 21 following induction and submitted to bulk RNA-seq, bulk ATAC-seq, and single cell RNA-seq. Bulk sequencing data was de-convoluted according to species to separate human neuronal reads from those of rat astrocytes. The presence of astrocytes induced profound transcriptional and epigenetic changes in hiNeurons. Sc-RNA seq demonstrated that hiNeurons in the presence of astrocytes are distinct from hiNeurons in the absence of glia. In conclusion, mixed-species in vitro cultures of astrocytes and hiNeurons are a useful tool to study human neuronal biology. The integration of RNA-Seq, ATAC-Seq, and scRNA-Seq provides unique and wholistic insights how the presence of astrocytes affects the function of human neurons.F-4022TRANSIENT AND STABLE OVER-EXPRESSION OF MIR690 ACCELERATES OSTEOGENIC DIFFERENTIATION IN EMBRYONIC STEM CELLSKarmach, Omran - Molecular, Cell, and Systems Biology, University of California, Riverside, Moreno Valley, CA, USA zur Nieden, Nicole - Molecular, Cell, and Systems Biology, UC Riverside, Riverside, CA, USAOsteogenesis is a complex and critical process for the proper development of vertebrates. The complexity of osteogenesis resides in the multiple pathways that embryonic stem cells (ESCs) can take to differentiate into osteoblasts and the intricate gene regulatory network, which controls this lineage development. Specifically, osteogenic differentiation can be induced from mesoderm or neural crest cells; both of which can differentiate into mesenchymal stem cells and then into osteoblasts. This overlap of differentiation into mesenchymal cells creates a convenient point at which to study osteogenesis, but reduces the focus on the intermediate stages of differentiation. Additionally, the role of microRNAs (miRNAs) in these stages also appears to be crucial for the fate of specific progenitors, making them an important piece of the puzzle. Our lab has shown that overexpression of a specific miRNA, mir690, upregulates osteogenesis through the direct targeting of -catenin. Additionally, the increased βosteogenesis achieved from miR690 overexpression during differentiation is a time dependent process, best achieved with a transfection during days 5-7 of differentiation. Furthermore, both the stable and transient transfection of mir690 result in morphological differences as well as a shift in the calcification time line possibly stemming from a shift in the overall cell population origin. Additionally, we have analyzed the role of mir690 in DGCR8 KO cells, which have been shown to maintain a higher level of pluripotency even during standard differentiation cultures, and we have shown this cell line to have a reduced ossification potential. However, the transfection of mir690 into DGCR8 KO cells also showed an increase in calcification when transfected during the same differentiation window as the wildtype cells. Lastly, increased osteogenesis was accompanied by an earlier increase in alkaline phosphatase activity as well as specific osteoblast genes. Together, our data indicate that miR690 supports osteogenesis both when stably and transiently transfected.Funding Source: National Institute of Health (NIH) Translational Centre for Regenerative Medicine (TRM Leipzig)

624POSTER ABSTRACTSF-4024REDUCED LEVELS OF MIR28 AND MIR377 EFFECT OSTEOGENESIS IN HYPERGLYCEMIC CONDITIONS IN MOUSE EMBRYONIC STEM CELLSHardy, Ariana R - Cell, Molecular, and Developmental Biology, University of California, Riverside, CA, USA Kamrach, Omran - Biochemistry and Molecular Biology, University of California, Riverside, CA, USAThe International Diabetes Federation stated in 2017 that approximately 425 million adults were living with diabetes and by 2045 this will rise to 629 million. Diabetes is a metabolic disease that is characterized by the presence of elevated glucose levels, and sadly the rates of diagnoses will continue to increase worldwide. Diabetes has been shown to cause numerous health problems, one being improper bone development, which could lead to other bone diseases such as osteoporosis or even bone fractures. Our lab previously showed that two miRNAs, miR28 and 377, were significantly regulated during osteogenesis of mouse embryonic stem cells (mESCs) and directly target AMP kinase (AMPK), which is typically activated by a hyperglycemic stress response. To uncover the downstream effectors of miR28, miR377 and AMPK that control lineage fate early on during specification, mRNA expression of neural crest, paraxial mesoderm and lateral plate mesoderm, which all are potential precursor to bone cells, were examined in both glucose concentrations of 5mM (low) or 25mM (high). Furthermore, miR28 was found to target FOXO1, a transcription factor downstream of AMPK, which may transcriptionally mediate the metabolic response. Ultimately, these results indicate that osteogenesis is reduced in the presence of high glucose and that manipulation of candidate miRNAs may represent a feasible treatment option to prevent developmental consequences of hyperglycemia.F-4026UNEXPRESSED PROTEIN TAGGING AS A TOOL TO OPTIMIZE CHARACTERIZATION IN HUMAN STEM CELLS USING CRISPR/CAS9Nair, Esther - Cell Biology, California State University, San Marcos, San Marcos, USA Chesnut, Jonathan - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA Potter, Jason - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA Liang, Xiquan - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USAMethods to confirm iPSC cell differentiation to a desired lineage are often complex and time-consuming. To streamline this process, we are developing methodology to fluorescently tag genes that are unexpressed in the iPSC state, but expressed in the desired differentiated cell line. The expression of the marker after differentiation allows for rapid visual screening, which will minimize cost and time associated with clonal isolation of properly differentiated cells, and optimization of factors relevant to characterization. As a proof of concept, we have currently tagged two genes with Emerald GFP (EmGFP) as the differentiation reporter using the CRISPR/Cas9 ribonucleoprotein (RNP) system, donor DNA, and Neon electroporation. We targeted genes indicating differentiation of iPSC cells to a neuronal lineage using beta III tubulin (TUBB3), and an astrocyte lineage using glial fibrillary acidic protein (GFAP). The donors were linear dsDNA with short homology arms coding for EmGFP and puromycin driven by the EF1 promoter. Proper integration of the donor DNA was confirmed via junction PCR and sequencing. Next, edited cells were enriched using a puromycin selection. Surviving cells were expanded in Essential 8 media for 5 days, when individual colonies harboring the knock-in had grown large enough to be picked and isolated for clonal selection. At this point, no EmGFP expression was observed, as expected. Junction PCR and sequencing was then repeated to confirm the presence of the tags in the clonal samples. The resulting TUBB3_EmGFP iPSC cell line was induced to differentiate into neurons, whereas the GFAP_EmGFP iPSC cell line was induced to differentiate into astrocytes. Since TUBB3 and GFAP only express in mature neurons and astrocytes respectively, upon differentiation the cells visibly expressed the EmGFP, demonstrating that cellular fate can be successfully and easily tracked via visualization of EmGFP expression. Further improvements can be made to this cell differentiation tracking system by tagging multiple proteins with different colored reporters to potentially visualize different stages of differentiation of lineages.F-4028ROBUST AND EFFICIENT GENERATION OF HUMAN MACROPHAGES USING INDUCED PLURIPOTENT STEM CELLS FOR DRUG DISCOVERYArmesilla-Diaz, Alejandro - Functional Genomics, GSK, GlaxoSmithKline, Stevenage, UK Martufi, Matteo - Functional Genomics, GSK, GlaxoSmithKline, Stevenage, UK Ashby, Charlotte - SPMB, GSK, GlaxoSmithKline, Stevenage, UK Escudero-Ibarz, Leire - Functional Genomics, GSK, GlaxoSmithKline, Stevenage, UK Schmidt, Sara - Functional Genomics, GSK, GlaxoSmithKline, Stevenage, UK Santivanez-Perez, Jessica - SPMB, GSK, GlaxoSmithKline, Stevenage, UK Faelth-savitski, Maria - Functional Genomics, GSK-CellZome, Stevenage, UK Vlachou, Denise - SPMB, GSK, GlaxoSmithKline, Stevenage, UK Eberl, Chris - Functional Genomics, GSK-CellZome, Stevenage, Germany Mohamet, Lisa - Functional Genomics, GSK, GlaxoSmithKline, Stevenage, UK

625POSTER ABSTRACTSRobust and efficient generation of human macrophages using induced pluripotent stem cells (iPSCs) for drug discovery. After decades of research there are still persistent failures to translate preclinical drug candidates into clinical success, highlighting the limited effectiveness of disease models currently used in drug discovery. iPSC technology has provided new tools to improve drug discovery efforts from efficacy and toxicity testing to novel target identification and understanding disease mechanisms. Macrophages perform key functions in regulating homeostasis, immune response and tissue repair. Equally, their dysfunction may drive pathogenesis of inflammatory and degenerative diseases, making them a key therapeutic target. Currently, macrophages are differentiated from peripheral blood monocytes (PBMCs), which is costly at scale and results in poor reproducibility due to donor variability. Moreover, advances in gene editing of human macrophages have been hampered due to viral delivery systems and their limited proliferative capacity. We have developed a robust cellular platform to generate human macrophages using innovative iPSC technology. This provides a cost-effective, reproducible and biologically relevant large-scale source of human macrophages for use in drug discovery. We have identified a simple three-step method which enabled the generation of a continuous source of human macrophages within twenty days of initial differentiation. Careful biochemical, functional and cellular assays were performed for comparative analysis with blood-derived macrophages and other commercially available iPSC-derived macrophages. In addition, we have successfully implemented highly efficient precision gene editing in both iPSCs and iPSC-macrophages by using CRISPR RNP. This ambitious methodology demonstrates comparable results to current primary cell-based models and adds value to existing strategies by reducing donor variability, reducing timelines and ultimately decreasing costs. Moreover, the use of engineered iPSC-macrophages provides great opportunity to accelerate early-stage drug development pipelines by generating physiologically and clinically relevant models.F-4030INTERROGATING THE THERAPEUTIC POTENTIAL OF TRPC6 FOR ALZHEIMERS DISEASE IN NEURONS FROM PATIENT SPECIFIC IPSCSTao, Ran - State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Shanghai, China Lu, Rui - Laboratory of Neural Signal Transduction, Institute of Neuroscience, Shanghai, China Wang, Junfeng - Laboratory of Neural Signal Transduction, Institute of Neuroscience, Shanghai, China Zeng, Shujun - Department of Neurology, Ruijin Hospital Affiliated with the School of Medicine, Shanghai Jiao Tong University, Shanghai, China Zhang, Ting - State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China Guo, Wenke - State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China Zhang, Xiaobing - Department of Medicine, Loma Linda University, Loma Linda, CA, USA Cheng, Qi - Department of Neurology, Ruijin Hospital Affiliated with the School of Medicine, Shanghai, China Yue, Chunmei - State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China Wang, Yizheng - National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China Jing, Naihe - State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, ChinaThe patient-specific induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer’s disease (AD) in live neurons. Then, the key question in the field is subsequent applications of iPSC-derived cellular models. In this study, the possible role of transient receptor potential canonical 6 (TRPC6) in AD pathogenesis was interrogated in iPSC-derived live AD neurons. The iPSCs generated from peripheral blood cells of sporadic AD patients efficiently differentiated into mature cortical neurons that displayed higher levels of AD pathological markers A and phospho-tau, but lower levels of TRPC6, than βthose of control neurons. Treatment of AD neurons with TRPC6 peptides or agonist inhibited the elevation of A and phospho-βtau. Our results in AD neurons confirm that the compromised expression of TRPC6 substantially contributed to A pathology βof sporadic AD, which fit well with our previous findings in mouse models. The inhibition of A elevation suggested that targeting βTRPC6 could help to develop novel therapeutic strategies for the treatments of AD.F-4032A HUMAN EMBRYONIC STEM CELL REPORTER LINE FOR CARDIOTOXICITY PLATFORMTsai, Su-Yi - Department of Life Science, National Taiwan University, Taipei, Taiwan Hsu, Zi-Ting - Life Science, National Taiwan University, Taipei, Taiwan Wang, Hou-Jun - Life Science, National Taiwan University, Taipei, TaiwanHuman embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes for studying cardiac biology, disease modeling, drug screens, and potentially for regenerative therapies. Directed differentiation protocols for cardiomyocytes using hESCs are well established, but methods to isolate highly pure population of cardiomyocytes are limited. Reporter cell lines can be valuable for purification and visualization of cells for such applications. We used CRISPR/Cas9 in hESCs to place an mCherry reporter gene into the MYH6 locus, facilitating a simple method to purify cardiomyocytes. MYH6:mCherry positive cells express atrial and ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human cardiomyocytes and can be used successfully to monitor drug-

626POSTER ABSTRACTSinduced cardiotoxicity and oleic acid-induced cardiomyocyte arrhythmia. The MYH6:mCherry hESC reporter line should serve as a useful tool for disease modeling and drug development relevant to cardiomyocyte biology.F-4034IN VITRO DISEASE MODELING OF THE FTDP-17 TAU R406W MUTATION USING PATIENT-DERIVED IPSCSNakamura, Mari - Department of Physiology, Keio University, Tokyo, Japan Shiozawa, Seiji - Department of Physiology, Keio University, Shinanomachi, Japan Tsuboi, Daisuke - Department of Cell Pharmacology, Nagoya University, Nagoya, Japan Amano, Mutsuki - Department of Cell Pharmacology, Nagoya University, Nagoya, Japan Watanabe, Hirotaka - Department of Physiology, Keio University, Shinanomachi, Japan Maeda, Sumihiro - Department of Physiology, Keio University, Shinanomachi, Japan Kimura, Taeko - Department of Functional Brain Imaging Research, National Institute of Radiological Sciences, Inage, Japan Yoshimatsu, Sho - Department of Physiology, Keio University, Shinanomachi, Japan Kisa, Fumihiko - Department of Physiology, Keio University, Shinanomachi, Japan Karch, Celeste - Department of Psychiatry, Washington University in St. Louis, St. Louis, MO, USA Miyasaka, Tomohiro - Department of Neuropathology, Doshisha University, Kyoto, Japan Takashima, Akihiko - Department of Life Science, Gakushuin University, Mejiro, Japan Sahara, Naruhiko - Department of Functional Brain Imaging Research, National Institute of Radiological Sciences, Inage, Japan Hisanaga, Shinichi - Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Japan Ikeuchi, Takeshi - Department of Molecular Genetics, Niigata University, Niigata, Japan Kaibuchi, Kozo - Department of Cell Pharmacology, Nagoya University, Nagoya, Japan Okano, Hideyuki - Department of Physiology, Keio University, Shinanomachi, JapanFrontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative disease caused by mutations in the microtubule-associated protein tau (MAPT) gene, which encodes the tau protein. Among the MAPT mutations, the R406W mutation is a unique missense mutation whose patients have been reported to exhibit Alzheimer’s Disease (AD)-like clinical phenotypes independent of A accumulation. To date, there is no treatment known to βbe effective for FTDP-17 patients, including those with the R406W mutation. The objective of this study is to establish a suitable model for studying the abnormalities induced by R406W mutant tau and elucidating the pathological role of tau in neurodegenerative diseases, as a basis for drug screening. iPSC lines were established from patients heterozygous for the MAPT R406W mutation (MAPT R406W/ ). These lines were ＋gene-edited using CRISPR/Cas-9 to establish WT isogenic lines (MAPT+/+), or homozygous mutants (MAPT R406W/R406W). iPSCs were then induced into a homogenous population of cortical neurons with more than 85% neuronal purity by dissociating cerebral organoids. In these neurons, R406W tau were less phosphorylated than WT tau by several kinases and were increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized to the dendrites and induced axonal phenotypes, including morphological changes and mitochondrial transport defects, which could be rescued with microtubule stabilization. In summary, we elucidated the abnormalities induced by the R406W mutant tau using iPSC-derived neurons. These findings provide new mechanistic insight into tau pathology and a potential for therapeutic intervention.F-4036DUCHENNE MUSCULAR DYSTROPHY: A DEVELOPMENTAL DISEASEMournetas, Virginie - Muscular Disease, IStem, Corbeil Essonnes, France Massouridès, Emmanuelle - Muscular Disease, IStem, Corbeil-Essonnes, France Kornobis, Etienne - Bioinformatics and Biostatistics Hub, Institut Pasteur, Paris, France Jarrige, Margot - Sequencing Platform, IStem, Corbeil-Essonnes, France Polvèche, Hélène - Sequencing Platform, Istem, Corbeil-Essonnes, France Dupont, Jean-Baptiste - Muscular Disease, IStem, Corbeil-Essonnes, France Górecki, Dariusz C. - School of Pharmacy and Biomedical Sciences, Portsmouth University, Portsmouth, UK Pinset, Christian - Muscular Disease, IStem, Corbeil-Essonnes, FranceDMD boys are currently diagnosed around 4, an age at which muscles have already suffered from the pathology. Meanwhile, no treatment is available yet to stop this degenerative disease. Efficacy of developing therapies aiming at restoring the expression of dystrophin in muscle stays too low to really be beneficial. All of this stresses the need of better defining the first steps of DMD in Human to be able to 1) find earlier and more specific biomarkers to increase diagnosis sensitivity; and 2) develop alternative therapeutic approaches by finding targets that compensate the lack of dystrophin in addition to restoring it. To identify theses biomarkers and therapeutic targets, we have modelled DMD in vitro using both human primary adult myoblasts and human pluripotent stem cells differentiating into myotubes. In our previous study, we identified Dp412e, an embryonic isoform of the dystrophin leading us to investigate further DMD onset during development. Comparisons at 7 cell differentiation time points of 3 healthy and 3 DMD human cell

627POSTER ABSTRACTSlines by omics (bulk transcriptomes, single-cell transcriptomes, miRnomes as well as proteomes) demonstrate that 1) we have access to both embryonic/foetal and adult myotubes ; 2) embryonic/foetal myotubes exhibit described DMD phenotypes (eg. dystrophin-associated protein complex destabilization, disrupted Ca2+ homeostasis and reorganisation of extracellular matrix); 3) our in vitro model properly recapitulates myogenesis, with a mesoderm entry at D3 followed by a specification toward skeletal muscle, up to myotube formation by D25; 4) DMD cells show marked dysregulations from D10, before the expression of skeletal muscle transcription factors is even detected at D17, with a clear downregulation of mitochondrial genes and 5) our cell model is well adapted for identifying drugs using high-throughput screening. Altogether, our data identify human pluripotent stem cells as a suitable cell model to study muscle development in the context of genetic diseases, and use it as a tool for therapy development. They strongly argue for an early developmental manifestation of DMD and lead us to rethink dystrophin functions during development.Funding Source: AFM-Telethon Fondation Maladies RaresF-4038IDENTIFICATION OF KCC2 EXPRESSION ENHANCING COMPOUNDS AS A BASIS FOR TREATMENT OF RETT SYNDROMETang, Xin - Whitehead Institute for Biomedical Research, Cambridge, MA, USA Drotar, Jesse - Whitehead Institute for Biomedical Research, Cambridge, MA, USA Li, Keji - Picower Institute for Learning and Memory, MIT, Cambridge, USA Clairmont, Cullen - Whitehead Institute, MIT, Cambridge, MA, USA Brumm, Sophie - University of Heidelberg, Germany Sullins, Austin - Picower Institute for Learning and Memory, MIT, Cambridge, USA Wu, Hao - Whitehead Institute, MIT, Cambridge, MA, USA Liu, Shawn - Whitehead Institute, MIT, Cambridge, USA Wang, Jinhua - Dana Farber Institute, Harvard University, Cambridge, MA, USA Gray, Nathanael - Dana Farber Institute, Harvard University, Boston, USA Sur, Mriganka - Picower Institute for Learning and Memory, MIT, Cambridge, USA Jaenisch, Rudolf - Whitehead Institute, MIT, Cambridge, USAA critical need in the drug development process is the ability to identify chemicals that regulate specific molecular targets in disease-relevant cell types in order to rescue functional deficits caused by faulty gene expression. The neuron-specific protein K+/Cl- co-transporter 2 (KCC2) has emerged as a promising therapeutic target for treatment of a number of human brain disorders including epilepsy, schizophrenia, spinal cord injury, and Rett syndrome (RTT), a severe neurodevelopmental disorder. Due to the lack of a neuron-based high-throughput screening (HTS) assay, it has been challenging to discover chemical compounds that enhance the expression of the KCC2 gene. In this study, we report the development of a robust high-throughput drug-screening platform through genome editing in human embryonic stem cells, which allows for the rapid assessment of KCC2 gene expression in human reporter neurons. We have identified a group of compounds from an unbiased screen of over 900 small molecule chemicals that enhance KCC2 expression termed KCC2 expression-enhancer compounds (KEECs). The identified KEECs include FDA-approved drugs that are inhibitors of the FLT3 or GSK3 kinase βpathways, and activators of the SIRT1 or TRPV1 pathways. We demonstrate that treatment with these hit compounds increases KCC2 expression in human WT and isogenic Methyl CpG binding Protein 2 (MECP2) mutant RTT neurons, and rescues the deficits in GABA reversal potential, excitatory synaptic transmission, and morphological development of RTT neurons to levels equivalent to WT neurons. Moreover, injection of KEECs KW-2449 or Piperine into a Mecp2 mutant mouse model of RTT ameliorates disease-associated respiratory and locomotion phenotypes. The small molecule compounds described in our study could potentially benefit various brain diseases through a novel mechanism of enhancing KCC2 expression.Funding Source: NIH grants HD 045022, R37-CA084198, NS088538, MH104610, MH085802-06, SCSB award# 2389069, RSRT grant 50-1873-0201, IRSF postdoctoral fellowship. DRCF postdoctoral fellowship. NCI P30-CA14051.F-4040NOVEL IMPRINTED SINGLE CPG SITES FOUND BY GLOBAL DNA METHYLATION ANALYSIS IN HUMAN PARTHENOGENETIC INDUCED PLURIPOTENT STEM CELLSChoi, Na Young - Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea Bang, Jin Seok - Stem Cell Biology, Konkuk University, Seoul, Korea Lee, Minseong - Stem Cell Biology, Konkuk University, Seoul, Korea Ko, Kinarm - Stem Cell Biology, Konkuk University, Seoul, KoreaGenomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, ovarian mature cystic teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium Human Methylation 450K array. This analysis identified novel single imprinted CpG sites.

628POSTER ABSTRACTSWe further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.Funding Source: This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) [grant number 2018R1A2B6001072].F-4042AN EFFICIENT PLATFORM TO EDIT COMPLEX PLURIPOTENT STEM CELLS FOR DISEASE MODELLINGVyas, Sapna - Cell Line Engineering, Horizon Discovery Group Plc, Waterbeach, Cambridge, UK Augereau, Cecile - Cell Line Engineering, Horizon Discovery, Waterbeach, UK Hardy, Emily - Cell Line Engineering, Horizon Discovery, Waterbeach, UK Trenchard, Elizabeth - Cell Line Engineering, Horizon Discovery, Cambridge, UK Chartier, Solene - Cell Line Engineering, Horizon Discovery, Cambridge, UK Anderson, David - Cell Line Engineering, Horizon Discovery, Cambridge, UK Santos, Rodrigo - Cell Line Engineering, Horizon Discovery, Cambridge, UK Escudero-Ibarz, leire - Cell Line Engineering, Horizon Discovery, Cambridge, UK Armesilla-Diaz, Alejandro - Cell Line Engineering, Horizon Discovery, Cambridge, UK Laurent, Thibault - Cell Line Engineering, Horizon Discovery, Cambridge, UK Collin, Philippe - Cell Line Engineering, Horizon Discovery, Cambridge, UKHuman Pluripotent Stem Cells (hPSCs) including human Embryonic Stem Cells (hESCs) and human induced pluripotent stem cells (hiPSCs) offer a unique in vitro platform for the generation of large quantities of cells for disease modelling, drug screening and ultimately cell based therapy. Development of efficient targeted genome editing/alteration technologies is essential to explore the full potential of stem cells. The CRISPR-Cas9 system has emerged to be a powerful tool in this field due to its effectiveness and robustness in genome engineering at low cost. The system has been used to modify genomes of various species including microbial, plant, animal, and human cells. Genome editing/correction in healthy or patient-derived iPSCs offers one of the most reproducible and relevant approaches for personalized therapy in regenerative medicine. hPSCs are suitable for gene editing as they can be easily engineered and expanded from a single cell state without losing their pluripotency and unique ability to differentiate into various lineages and specialized cells, however this can be challenging due to the variability between different hESCs and hiPSCs. A common and efficient method to develop an hiPSC disease model is to generate a CRISPR mediated knockout of disease relevant genes through non-homologous end joining (NHEJ) and explore the pathogenic mechanism in the derived cells. Isogenic hiPSCs can also be generated using Homology Directed Repair (HDR) in order to correct a genotype, introduce disease relevant mutation or endogenously tag a protein and monitor its expression through differentiation. Here we show, through our iPSC platform created in 2015, how we adapt and characterise commercially available and patient derived PSCs to chemically defined media conditions, select optimal targeting strategies and apply the CRISPR-Cas9 technology to generate disease relevant models with a large range of editing. Moreover, we have developed a good understanding of our customer needs, thanks to our collaboration with biotech companies specialised in hiPSC reprogramming, allowing us to generate a panel of unique gene-engineered cell types, which can then generate functional tissue for disease modelling as well as drug discovery and screening.F-4044INVESTIGATION OF TDP RISK OF AZITHROMYCINE USING CARDIOMYOCYTE DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLLee, Jin-Moo - Pharmacological Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, Korea Ahn, Sun-Young - Pharmacological Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, Korea Cha, Hye Jin - Pharmacological Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, Korea Kim, Young-Hoon - Pharmacological Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, Korea Suh, Soo Kyung - Pharmacological Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, KoreaIn the field of cardiovascular safety pharmacology, hERG assay and telemetry are used according to the International Council for Harmonisation (ICH) guideline. However, there are some medications, which were discontinued due to adverse reactions to the heart in clinical application. Therefore, to address this issue the Comprehensive in Vitro Proarrhythmia Assay (CiPA) project has been launched to develop a new method using cardiomyocytes derived from human induced pluripotent stem cell (hiPSC-CM). In this study, we established a new method that can predict the cardiovascular adverse effects of drugs in human using hiPSC-CM with reference to the CiPA. Azimilide, terfenadine, and verapamil were selected as reference drugs which were known to cause high, intermediate and very low TdP risk, respectively. Azithromycin and diphenhydramine were selected as test group because of their cardiac adverse events. To evaluate the functions of heart after administration of these drugs, the field potential duration (FPD) and spike amplitude

629POSTER ABSTRACTSwere investigated by using microelectrode array (MEA). Azimilide and terfenadine increased FPD by 32.7%, 18%, and verapamil decreased FPD by 15%, while azithromycin and diphenhydramine decreased FPD by 74% and 7%, respectively. Spike amplitude was decreased by 73% for azimilide, 55% for terfenadine, 11% for verapamil, 39% for azithromycin and 11% for diphenhydramine. In addition, we analyzed proteins such as Troponin, ROS, and ion channels as biomarkers to predict cardiac function. Azimilide, azithromycin, and diphenhydramine showed about two fold increases in ROS. Troponin was increased in a dose-dependent manner in all groups. These results showed that azithromycine has a cardiac arrhythmia potential at a moderate level and diphenhydramine has a low level. Taken together, it was possible to evaluate a potential of cardiac adverse events of new molecules using hiPSC-CM in new drug development.Funding Source: This study was supported by grants (18181MFDS381, 19181MFDS405) funded by the Ministry of Food and Drug Safety, Republic of Korea.F-4046HIGH-THROUGHPUT MICROFLUIDIC PLATFORM FOR DRUG SCREENING OF VASCULARIZED 3D TISSUESVulto, Paul - Mimetas, Leiden, Netherlands Previdi, Sara - Department of Internal Medicine - Nephrology, Leiden University Medical Center, Leiden, Netherlands Kurek, Dorota - Model Development, Mimetas, Leiden, Netherlands Nicolas, Arnaud - Hardware Research and Development, Mimetas, Leiden, Netherlands Schavemaker, Frederik - Hardware Research and Develo, Mimetas, Leiden, Netherlands Trietsch, Sebastiaan - Hardware Research and Develo, Mimetas, Leiden, Netherlands Lanz, Henriette - Model Development, Mimetas, Leiden, Netherlands3D tissues such as spheroids or organoids derived from human pluripotent stem cells (PSCs) represent a new type of three-dimensional in vitro model for understanding organ development, disease mechanism, and drug testing. Despite the success in generating 3D cultures resembling different tissue types (brain, heart, intestine, liver, lung, and kidney), these mini-organs show limited growth potential and an immature phenotype due to lack of vascularization. Several groups have attempted to improve vascularization of organoids by transplanting them into a host (i.e. mouse, chick). However, the low predictivity of animal models, boost the development of in vitro alternative strategies. In this regard, microfluidic techniques are increasingly recognized as important toolbox able to add physiologically relevant cues to traditional cell culture models. We recently described the use of the Organoplate® for generating 3D perusable angiogenic vessels. Here, we present the use of a high-throughput ‘grafting’ platform which allows vessels co-culture with 3D tissue aggregates and tissue vascularization. One unit of the Mimetas Organoplate® Graft is made of two microfluidic channels in which endothelial cells can be patterned against ECM through the use of the PhaseGuide® technology. Gradient of pro-angiogenic factors (VEGF, PMA, S1P, and FGF-b) allows the formation of a perfused vascular bed on top of which tissue fragments (i.e. organoids or spheroids) can be added to enable vascularization. Tissue dependent vessels remodeling and stabilization can be monitored overtime by real time imaging and barrier integrity. When liver spheroids are used, vessels became leak-tight to dextran 150 kDa after 14 days of co-culture. Moreover, expression of CD31+ cells around and in within the spheroids proves that endothelial cells migration and tissue envelopment occurred during co-culture. The high number of units (up to 64 chips in 384 well format) enables functionality studies and compound screening in a robust and automated way. We propose the use of the Organoplate® Graft as a vessels grafting platform for multiple 3D tissues allowing drug screening and disease modeling in a more physiological environment.F-4048ENHANCED OSTEOGENIC CONVERSION OF HUVECS WITH OCT4-30KC19 CELL PENETRATING PEPTIDE AND BMP4 GROWTH FACTORKim, Seung Hyun - Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea Hwang, Nathaniel - School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea Kwon, Janet - Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea Lee, Seunghun - Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea Lee, Jaeyoung - Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon-si, Korea Park, Ju Hyun - Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon-si, Korea Lee, Hwajin - School of Dentistry, Seoul National University, Seoul, KoreaOsteoblasts mineralizes bone matrix thereby play an essential role in bone remodeling. Recently, functional osteoblasts were successfully transdifferentiated from dermal fibroblasts through ectopic expression of RUNX2, OSTERIX, OCT4 and L-MYC. However, this method viral transduction of cells which provides risks such as unwanted genetic change and teratoma formation. Herein, we developed protein-based strategy to induce transdifferenatiaton of endothelial cells into osteoblasts via nuclear delivery of OCT4 protein combined with BMP4 treatment. For the nuclear delivery of OCT4 protein, we created OCT4 recombinant protein fused with 30Kc19, which is derived from silkworm hemolymph of Bombyx mori that has cell-penetrating, protein stabilizing, non-cytotoxic and anti-apoptotic properties. Our results show enhanced osteogenic induction of HUVECs. Cells treated with OCT4-30Kc19 protein combined with BMP4

630POSTER ABSTRACTSshowed increased gene markers, BSP, OPN and COL I, and strong alizarin red s staining in in vitro. In addition, results from in vivo mouse cranial defect experiment demonstrate successful bone regeneration of HUVECs treated with both OCT4-30Kc19 and BMP4. Ultimately, using protein-based transdifferentiation method allows bypassing cell delivery of genetically modified cells which may induce unintended genetic change and remains as challenge to be used in clinical applications.Funding Source: National Research Foundation of Korea (NRF) - NRF-2017M3A9C6031786F-4050CELL RESPONSE TO MECHANO CUE AT EARLY PHASE OF CELL REPROGRAMMING VIA SELF-MODULUS SUDDEN CHANGESong, Yang - Department of Bioengineering, University of California,), Los Angeles, CA, USA Soto, Jennifer - Department of Bioengineering, University of California, Los Angeles, CA, USA Chen, Binru - Department of Bioengineering, University of California, Los Angeles, CA, USA Yang, Li - Department of Bioengineering, Chongqing University, Chongqing, China Li, Song - Department of Bioengineering, University of California Los Angeles, CA, USAThis study is to report a deterministic sudden change of cell modulus and an F-actin re-polymerization at the early phase of cell reprogramming. The study has been carried out through investigating the adult mice fibroblasts direct reprogramming into neurons via the forced expression of three transcription factors: Ascl1, Brn2, and Myt1l (ABM). After active above exogenous transcription factors via Dox, cell modulus was measured by atomic force microscopy (AFM) and quantitative deformability cytometry (q-DC) every 24 hr, and F-actin was stained by FITC-Phalloidin. Results showed that cell modulus significantly decreased in 24 hr after Dox initiate cell reprogramming and F-actin re-polymerized to a bird’s nest structure at the same time. Additionally, it is believed that the process could affect the reprogramming efficiency when somatic cells response to mechano cue. Then, ABM-infected adult mice fibroblasts cultured on PA gels with different stiffness (1kpa, 20kpa, and 40kpa) before and after cell modulus changes, and reprogramming efficiency was quantified by neuron-specific Class III -tubulin β(Tuj1) staining. It is found that 20 kPa and 1kPa PA gels increased the reprogramming efficiency when ABM-infected cells cultured on gels before and after cell modulus change, respectively. This study implies that a maximum mechanical interaction between somatic cell and matrix can occur to efficaciously regulate cell reprogramming efficiency at the early phase of reprogramming when cell modulus is comparable to the matrix stiffness.Funding Source: National Institute of Health (HL121450), National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH (T32AR059033), National Nature Science Foundation of China (11532004).F-4052ROLE OF INTRACELLULAR STRUCTURES IN THE DIRECT CONVERSION OF FIBROBLASTS INTO NEURONSSoto, Jennifer - Bioengineering, -University of California, Los Angeles, CA, USA Wong, SzeYue - Bioengineering, University of California, Berkeley, , CA, USA Chu, Julia - Bioengineering, University of California, Berkeley, USA Li, Song - Bioengineering, University of California, Los Angeles, USADirect reprogramming is the process of converting from one cell type into a very distantly related cell type without proceeding through an intermediate proliferative stem-cell like stage. Previous studies have shown that somatic cells can be directly reprogrammed into specific neuronal subtypes using different combinations of transcription factors and microRNAs and in addition, biochemical factors can aid in reprogramming these cells into induced neuronal (iN) cells. However, how intracellular structures may regulate iN reprogramming through mechanotransductive pathways is not well understood. Here we show, for the first time, that disruption of actin-myosin contractility via treatment with blebbistatin can enhance the efficiency of iN conversion. The derived iN cells displayed a typical neuronal morphology, expressed neuronal markers and exhibited functional neuronal properties. The involved mechanism relied on the modulation of gene expression patterns by blebbistatin. We found that blebbistatin downregulated fibroblast markers while concurrently upregulated neuronal genes. Furthermore, our findings suggest that focal adhesions and the nuclear lamina play a critical role in iN conversion. In essence, our findings highlight that the disruption of intracellular mechano-structures can regulate the direct reprogramming of fibroblasts into iN cells. By investigating signaling pathways, these novel findings can provide insights into the mechanisms that determine cell fate with potential applications in neurological disease modeling and drug discovery.F-4054DIRECT REPROGRAMMING OF HEPATOCYTES INTO PANCREATIC BETA CELLS BY CRISPR-DCAS9 MEDIATED MULTIPLEX ENDOGENOUS GENES ACTIVATIONYang, Xiaofei - Translational Medicine Collaborative Innovation Center, Shenzhen People’s Hospital, Shenzhen, China Wang, Hanyue - Department of Pathology and Pathophysiology, Ji’nan University, Guangzhou, China Cheng, Albert Wu - Jackson Laboratory for Genomic Medicine, The Jackson Laboratory, Farmington, USA Li, Furong - Translational Medicine Collaborative Innovation Center, Shenzhen People’s Hospital, Shenzhen, China

631POSTER ABSTRACTSDirect reprogramming of autologous cells from diabetes patients to insulin producing cells is a new method for pancreatic cell replacement therapy. At present, trandifferentiation among mature cells is achieved mainly by introducing foreign genes into the starting cells. However, since the chromatin of the endogenous transcription factor is in an inhibitory state, the exogenously introduced transcription factor is transiently expressed, resulting in inefficient and poor maturation of the directly reprogrammed beta cells. Here, we constructed a CRISPR/dCas9 based new Casilio system that targeted Tgif2, Pdx1, Ngn3 and Mafa, which can continuously activate these four endogenous specific transcription factors. We found that when activating single gene by Casilio with targeted gRNA pools, endogenous gene expression levels can increase 10 times. When activating all four factors simultaneously, gene expression increased substantially and can maintain higher levels until 7 days. When these four factors were activated simultaneously in Ins1-EGFP reporter HepG2 cells, Ins1-EGFP+ cells can be found at 24 hours, and peaked at 72hrs. The expression of various islet beta cell specific genes in these cells was detected at the mRNA level and protein level. In conclusion, we provide a new approach for the treatment of diabetes by direct reprogramming of liver cells to insulin producing cells through CRISPR-dCas9.Funding Source: Project 81800686 supported by National Natural Science Foundation of ChinaF-4056CONVERSION OF MOUSE FIBROBLASTS INTO OLIGODENDROCYTE PROGENITOR LIKE CELLS THROUGH A CHEMICAL APPROACHZhang, Mingliang - Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University, School of Medicine, Shanghai, ChinaTransplantation of oligodendrocyte progenitor cells (OPCs) is a promising way for treating demyelinating diseases. However, generation of scalable and autologous sources of OPCs has proven difficult. Here we found that chemical condition M9 could specifically initiate neural program in mouse embryonic fibroblasts, and induce the generation of colonies that underwent mesenchymal-to-epithelial transition at the early stage of reprogramming, which represent unstable and neural lineage-restricted intermediates that have not established a neural stem cell identity. By modulating the culture signaling recapitulating the principle of OPC development, these intermediate cells could be reprogrammed towards OPC fate. The chemical-induced OPC-like cells (ciOPLCs) resemble primary neural stem cell-derived OPCs in terms of their morphology, gene expression, and the ability of self-renewal. Upon differentiation, ciOPLCs could produce functional oligodendrocytes and myelinate the neuron axons in vitro, validating their OPC identity molecularly and functionally. Our study provides a non-integrating approach to OPC reprogramming that may ultimately provide an avenue to patient-specific cell-based or in situ regenerative therapy, and conceptually extends our previously established Cell Activation and Signaling Directed-reprogramming paradigm to a chemical-based lineage-specific manner.Funding Source: This work was supported by Shanghai Jiao Tong University, National Natural Science Foundation of China (31771643), Shanghai Pujiang Program (17PJ1405200), The Eastern Scholar at Shanghai Institutions of Higher Learning.F-4058CYTOSINE BASE EDITOR GENERATES SUBSTANTIAL OFF-TARGET SINGLE NUCLEOTIDE VARIANTS IN MOUSE EMBRYOSYang, Hui - Institute of Neuroscience, Chinese Academy of Science (CAS), Shanghai, China Zuo, Erwei - Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China Sun, Yidi - CAS-MPG partner Institute for Computational Biology, University of Chinese Academy of Sciences, Shanghai, China Wu, Wei - Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA Yuan, Tanglong - Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, ChinaGenome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.F-4060OPTIMIZATION OF HOMOLOGY-DIRECTED REPAIR USING CRISPR/CAS9 SYSTEMSKim, Sungtae - Glaxosmithkline, Collegeville, USARepertoire of genomic alterations via CRISPR/Cas9 genome editing depends on the efficiency of dsDNA breaks and the subsequent DNA repair events, which consist of two distinct pathways: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). HDR enables precise genome modifications which can be used to mimic disease relevant genotypes or to integrate functional domains of interest into the genome. However, the efficiency of HDR via genome editing remains quite low, mostly ranging from 0.1 to10%. Although several strategies have been claimed to enhance

632POSTER ABSTRACTSHDR efficiency, there are still concerns about the variability and reproducibility. We surveyed factors influencing HDR efficiency and explored the possibility of optimizing conditions by combinatorially modulating them. Highly efficient NHEJ without a donor is pre-requite for the success. Chemical modifications of the gRNAs and the donor template seem to have significant roles in increasing HDR by stabilizing the molecules. Some small molecule NHEJ antagonists successfully increased HDR in multiple cell lines. Notably, the choice of CRISPR system delivery appears to have the most profound effect on the HDR efficiency.F-4062COST-EFFECTIVE DIFFERENTIATION OF HUMAN IPS CELLS TO PANCREATIC BETA-CELLS BASED ON AGGREGATE SUSPENSION CULTURE WITH DIALYSIS OPERATIONSChoi, Hyunjin - Department of Bioengineering, The University of Tokyo, Japan Shinohara, Marie - Department of Chemical System Engineering, University of Tokyo, , Japan Sakai, Yasuyuki - Department of Chemical System Engineering, University of Tokyo, JapanThe production of insulin-secreting pancreatic -cells from human βinduced pluripotent stem cells (hiPSCs) is a promising strategy to solve the donor shortage problem in cell transplantation therapy for diabetes. However, the production cost of pancreatic β-cells is extremely high, because the high-priced cytokines are necessary for induction of hiPSCs’ differentiation to pancreatic β-cells. Furthermore, the cytokines remaining in the used culture medium and possible autocrine factors secreted by cells are removed along with toxic metabolites when the culture medium is exchanged daily. In this study, in order to reduce the production cost of pancreatic -cells, we investigated the βfeasibility of dialysis suspension culture system in differentiation induction of hiPSCs into pancreatic β-cells. This culture system enables to minimize the use of growth factors by their retention and full utilization of autocrine factors in the cell culture compartment. As the first step, we examined the feasibility in the most expensive Stages 1 and 6 among the 6 stages of the differentiation protocol. Stage 1 is the induction Stage for definitive endoderm and Stage 6 is the maturation stage of endocrine progenitor cells to pancreatic -cells. For the dialysis βsuspension culture, we developed a simple culture device consists of flat-type dialysis membrane-loaded culture insert for the cell culture compartment and a deep well for the dialysate compartment. Such a dialysis culture remarkably improved the cultural environment with continuous glucose supply and lactate removal. Furthermore, it was possible to produce cells without lowering the gene expression level with a smaller amount of growth factors, possibly through by retaining and reusing the growth factors and the autocrine factors in the culture medium, Particularly in Stage 6, the gene expression levels of insulin were increased. These findings demonstrates that dialysis suspension culture system is a very promising method for cost-effective and scalable production of pancreatic -cells for future implantation-βbased treatments of diabetic patients.Funding Source: This research was partially funded by AMED-Kyoten B (Japan).F-4064SIMPLIFYING DEPOSIT OF IPSC LINES INTO THE EUROPEAN BANK FOR INDUCED PLURIPOTENT STEM CELLSSteeg, Rachel - EBiSC, Fraunhofer Research UK Ltd, Glasgow, UK Bruce, Kevin - COO, Censo Biotechnologies, Edinburgh, UK Seltmann, Stefanie - BCRT - Berlin Institute of Health Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany Dewender, Johannes - BCRT - Berlin Institute of Health Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany Mah, Nancy - BCRT - Berlin Institute of Health Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany Bultjer, Nils - BCRT - Berlin Institute of Health Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany Mueller, Sabine - Biomedical Data Science, Fraunhofer Institute for Biomedical Engineering (IBMT), Würzburg, Germany Kurtz, Andreas - BCRT - Berlin Institute of Health Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany Neubauer, Julia - Project Centre for Stem Cell Process, Fraunhofer Institute for Biomedical Engineering (IBMT), Würzburg, Germany Zimmerman, Heiko - Head of Institute, Fraunhofer Institute for Biomedical Engineering (IBMT), Sulzbach, Germany Ebneth, Andreas - Neuroscience, Janssen Research and Development, Beerse, BelgiumThe European Bank for induced Pluripotent Stem Cells (EBiSC, cells.ebisc.org) is a centralised, non-profit repository and distribution hub ensuring long term storage of deposited hiPSC lines and their distribution to academic and commercial researchers worldwide. In a second project phase launched in March 2019 (EBiSC2), EBiSC supports researchers through provision of high quality iPSC lines by allowing researchers to deposit iPSC lines into EBiSC and distribute the lines on their behalf. EBiSC has now established simplified procedures to ease deposition to increase deposit of further existing iPSC lines and thus ensure researchers can access these vital resources long term. In early cases, Depositors occasionally used consent templates which limited downstream use of donated Biosamples, leading to re-consenting or inability to proceed with iPSC line deposit. Reliance on manual data collection was also time-consuming and error prone, especially for large cohorts of iPSC lines. Lastly, institutions needed support to

633POSTER ABSTRACTSfinalise EBiSC deposit agreements. Using input from ethical and scientific leaders, EBiSC developed consent templates and a flexible yet robust review process that maximises utility of donated Biosamples whilst providing suitable research and ethical provenance to meet regulatory standards. Use of the human Pluripotent Stem Cell Registry (hPSCreg.eu) for direct data input by depositors bypasses reliance on burdensome methods for manual data collection and enables mass data uploads through direct database data transfer. Direct guidance for legal representatives is established to progress completion of deposit agreements. Using these experiences, EBiSC is now guiding hiPSC research projects from the outset in establishing ethical and legal frameworks compliant with deposition into a public repository. With these developments and collaborations over the past 5 years, EBiSC has successfully safeguarded iPSC lines generated by projects such as StemBANCC and HipSci, created mutual benefits across consortia, promoted scientific excellence and reduced duplication of efforts and public funds. A second project phase, EBiSC2, strives to supply iPSC related services such as cell line generation and gene editing, Quality Control, automated data input and streamlined governance processes.Funding Source: The EBiSC and EBiSC2 projects have received funding from the Innovative Medicines Initiative Joint Undertaking (JU) and EFPIA under grant agreement No 115582 and No 821362 respectively.F-4066GENE EDITED INDUCED PLURIPOTENT STEM CELL-BASED THERAPIES IN PATANT APPLICATIONSMorita, Yasushi - International Center for Cell and Gene Therapy, Fujita Health University, Osaka, Japan Maekawa, Hiromi - Regenerative Medicine and Stem Cell Biology, School of Medicine, Fujita Health University, Osaka, Japan Bessho, Kanako - Regenerative Medicine and Stem Cell Biology, School of Medicine, Fujita Health University, Osaka, Japan Okura, Hanayuki - International Center for Cell and Gene Therapy, Fujita Health University, Osaka, Japan Matsuyama, Akifumi - Regenerative Medicine and Stem Cell Biology, School of Medicine, Fujita Health University, Toyoake, JapanPatent application tends were investigated for induced pluripotent stem cell (iPSC)-based therapies in PCT applications from 2006 to 2018. The number of patent applications for iPSC technologies was 1966 with 3% of applications for gene edited iPSC-based therapies. Patent applications for iPSC-based therapies described zinc-finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system in claims were extracted. The first patent application for gene edited iPSC-based therapy using CRISPR/Cas system was filed in 2013, then the number of applications has been increasing dramatically. In contrast, the first patent applications using ZFN and TALEN systems were filed in 2010 and 2012, respectively, however, the patent applications using these systems after 2015 also included CRISPR/Cas system. The patent applications for gene edited iPSC-based therapies were categorized into 14 disorder groups: neurodegenerative/neurodevelopmental,blood,metabolic,immune, bone/cartilage, ophthalmological, skin disorders, trisomy syndrome, pain, and infertility. Most patent applications were using CRISPR/Cas system for 10 disorder groups, which mostlyincludedneurodegenerative/neurodevelopmental,blood, and metabolic disorders. The patent applications for gene edited iPSC-based therapies were filed by applicants in the United States, Switzerland, Belgium, Spain, France, Germany, and Republic of Korea. Most patent applications were filed by Switzerland applicant and US applicants. CRISPR Therapeutics, AG was filed the patent applications for gene edited iPSCs or iPSC-derived cells used CRISPR/Cas system for treatment of neurodegenerative, metabolic, blood, immune, skin disorders, and pain. Sangamo Biosciences, Inc. was filed the patent applications for gene edited iPSC used ZFN system for treatment of immune, neurodegenerative, blood, digestive, ophthalmological disorders, cancer, and infection.Funding Source: AMED under Grant Number JP17bm0504009F-4068PLANT-DERIVED CELLULOSE HYDROGEL AS A MATRIX FOR 3D HUMAN STEM CELL PROLIFERATION AND DIFFERENTIATIONSpencer-Fry, Jane - UPM Biomedicals, UPM-Kymmene Corporation, Helsinki, Finland Paasonen, Lauri - UPM Biomedicals, UPM-Kymmene Corporation, Helsinki, FinlandIn the pursuit of in vitro cell models that are biological relevant with improved functionality, new materials and methods for creating three-dimensional (3D) cell culture systems are a key requirement. GrowDex®, nanofibrillar cellulose (NFC) hydrogel which is derived from the Birch tree, has been shown to provide an effective support matrix for culturing various cell types in 3D. 1) hESC (WA07) and hiPSC (iPS(IMR90)-4): Cell colonies were embedded in 0.5% GrowDex (UPM) in mTeSR1 (STEMCELL Technologies) media and cultured on 96-well plate up to 26 days. hPSCs pluripotency was analysed with OCT4 and SSEA-4 marker expression, in vitro EB-mediated differentiation, and teratoma assay. 2) hESC derived neuronal cells: Pre-differentiated neuronal cells were embedded in 1.5 and 1.0% GrowDex. Formation of the neuronal networks was evaluated by immunocytochemical staining against neuronal markers MAP-2 and -Tubulin III and confocal microscopy. 3) Adipose tissue βderived hMSCs: Cells were embedded in 0.2% GrowDex in DMEM media and transferred on 24-well tissue culture inserts. Adiopogenic differentiation and osteogenic differentiation were induced with StemProTM differentiation kits (ThermoFisher) for 21 days, and analysed with Oil Red O and Alizarin Red S stainings. 1) hESC and hiPSC proliferated in GrowDex without

634POSTER ABSTRACTSfeeder cells, formed spheroids with 100-200 μm diameter, and the cells remained their pluripotency throughout the 26 day study. 2) GrowDex supported the 3D growth of hESC derived neuronal cells. The formation of neurospheres and neuronal networks by neurite outgrowth were observed. 3) Adipose tissue derived hMSCs were able to differentiate to both adipogenic, and osteogenic direction in GrowDex 3D culture. GrowDex offers a well-defined, tunable 3D culture matrix for various regenerative medicine applications. Authors would like to thank Yan-Ru Lou, Tiina Joki and Jonathan Sheard for performing the experiments.

635PRESENTER INDEXAbo, KristineT-3173Adhikarla, Vikram F-3225Adileh, Lana T-2122Adler, Andrew F W-3082Agarwal, Trisha F-2080Agarwala, Sobhika F-2054Aguiari, Paola F-2013Ahern, DarcyW-3177Ahn, Eun Hyun T-2130Ahsan, SanjanaF-2019Ahuja, Christopher S W-3029Aihara, Yuki F-3151Akatsuka, Kyoko T-3077Akiyama, Kentaro W-3071Akiyama, Tetsuya F-3026Alani, BanaW-2072Albert, Silvia F-3059Ali, Noelle J.A. T-2023Alizadeh, Hamed W-2114Alle, QuentinF-3192Allison, Tom F-3137Alonso-Camino, Vanesa F-3224Alsanie, Walaa F-3143Amartuvshin, Oyundari F-3089AmmothumKandy, Aswathy W-3022Ampuja, Minna T-4048An, Ju-HyunF-3152An, Ju-HyunT-3221An, Ju-HyunW-3158An, Ju-HyunW-3211An, QinT-2103Andersen, Carsten W-3025Andreadis, Stelios T F-2105Andreadis, Stelios T T-2012Andrews, Madeline G W-1011Anna, JanzT-2029Appleton, Evan M W-3142Arata, Claire E W-2017Arduini, Brigitte L W-3077Arjun, ArpanaW-3006Armesilla-Diaz, Alejandro F-4028Aros, Cody J W-2110Aryal A C, Smriti F-3060Ashmore-Harris, Candice W-2074Aspegren, Anders T-4056Attico, Eustachio W-2079Aung, Kyaw Thu W-3072Autio, Matias I T-3238Avior, YishaiF-3112Aydin, BegumW-3128Azzouni, Karima W-3155Babos, Kimberley N T-3196Bachiller, Daniel T-3225Bachiller, Daniel W-3209Bachman, John F T-2015Badner, Anna W-3005Baer, MeghanF-3081Bagger, Frederik O W-3144Bai, YunfeiF-3207Bairapura Manjappa, Akshay W-2008Bang, Jin Seok W-4022Banno, KimihikoT-2044Bar, ShiranF-3100Barabino, Andrea W-2093Baranov, Petr T-2102Barish, Michael T-3055Barral, Serena F-3170Barrett, Robert F-3046Barretto, Tanya F-3025Bartoccetti, Michela T-3206Bassal, Mahmoud A T-2056Batalla-Covello, Jennifer T-2118

636PRESENTER INDEXBatista, Luis T-3177Becerra-Bayona, Silvia M F-3220Belair-Hickey, Justin J T-3006Belling, Jason N W-3239Beltran, Adriana S W-3213Bernardo, Andreia S W-2029Bernotiene, Eiva W-2020Bertero, Alessandro W-2027Bian, JingT-3025Biscola, Natalia P W-4057Blackford, Samuel J F-3125Blenkinsop, Timothy F-2099Bliley, Jacqueline T-2025Block, Travis J F-2111Bo, WangW-4038Boers, RubenF-4059Booker, Cori N W-2018Borcherding, Dana C W-3051Borkowska, Paulina F-4009Bosworth, Colleen W-3228Bozon, KayleighW-1001Braam, MitchellT-3241Brickler, Thomas F-3176Brown, RobertW-4055Brumbaugh, Justin T-3100Budinger, Dimitri W-3179Burberry, Aaron W-3159Burke, ConnorW-2122Burke, TomF-3172Burkeen, Gregory A W-2125Burns, TerryF-2110Burridge, Paul W T-2040Busskamp, Volker F-2100Cagavi, Esra W-2038Cai, YuqiF-3057Calvanese, Vincenzo W-1005Calza, LauraT-3120Calza, LauraW-3001Cao, XuF-3147Carabana Garcia, Claudia W-2086Carey, BryceF-2064Carico, Zachary M F-3102Carless, Melanie A T-3032Carmel Gross, Ilana T-3108Caron, LeslieT-3028Carromeu, Cassiano W-3027Castaldi, Alessandra W-2082Castro, Nadia P T-3242Catala, Pere T-2101Celen, CemreW-2075Chan, Chun Ho T-3066Chan, Chun Ho W-2024Chang, Hsiang-TsunT-2104Chang, VivianF-2059Chanthra, Nawin F-3212Chao, Chih-ChiF-4004Chao, Hsiao-MeiT-2089Charney, Rebekah M T-3155Chau, AnthonyT-4065Chechik, Lyuba F-3095Chen, BoT-2053Chen, BoW-2049Chen, Chu-YenW-3009Chen, ChuanW-3109Chen, DiT-1027Chen, GuokaiW-3146Chen, HaoF-1029Chen, JiayuT-3088Chen, JiekaiW-3207Chen, Kevin G F-4005Chen, Show-LiF-3021Chen, WanqiuF-3200

637PRESENTER INDEXChen, Wei-JuF-3110Chen, XiuqingF-3146Chen, Yi-FanF-2086Chen, Yi-HuiT-3090Chen, Yi-HuiW-2079Chen, YichangF-3056Chen, YingF-3230Chen, YuF-3234Chen, YuejunF-3016Chen, ZhifenF-3242Cheng, XiuyuanF-2125Cheng, Yu-CheF-3071Cheng, Yu-ShanF-3188Chern, JeffyW-3241Chi, FangtaoW-3093Chiba, MayumiT-3138Chien, Peggie J F-2014Chien, YuF-4012Chilin Vidal, Brian W-3007Chiou, Shih-HwaF-2095Chirshev, Evgeny W-3143Cho, Ssang-GooF-3134Cho, Sung-RaeW-3016Cho, Yong-HeeF-2128Choi, AlexanderW-3217Choi, BogyuW-3224Choi, EunjungW-3234Choi, HannahW-3148Choi, Hong Seo T-3106Choi, HyunjinF-4062Choi, Kyung-DalT-3012Choi, Na Young F-4040Choi, Seon-AF-3128Chondronasiou, Dafni W-3201Choo, HyojungT-4046Chow, MelissaT-2097Christoffersson, Jonas T-3041Chu, Edward Po-Fan W-3230Chu, JianhuaT-3210Chu, Pei-HsuanT-3148Chu, VirginiaF-3140Chung, Yun Shin W-4027Clark, Elisa C W-1004Clark, Elisa C W-2035Clark, Kaitlin C W-2002Cober, Nicholas D T-2115Coles, Brenda L W-2096Colin, MargauxT-3116Collier, Amanda J W-3097Conder, Ryan K F-3047Conti, AnastasiaW-2055Contreras-Trujillo, Humberto W-2127Cooper, Fay T-4043Cortes, Daniel W-3011Cortes-Servin, Alan W-3195Corveloni, Amanda C W-3105Cox, AndyW-3088Crane, AndrewW-3076Cruz, AngelicaW-2069Cuesta - Gomez, Nerea T-2061Cunningham, Amy W-3196Dabbah, Mahmoud T-2124Dai, ZhiyuT-4042Darmani, Homa T-2004David, LaurentT-3093Davis, JeffreyT-2068Davis, Richard P F-2026de Caires Junior, Luiz Carlos T-3060De Castro, Mateus V W-4016de Luzy, Isabelle R T-3004de Peppo, Giuseppe Maria W-3236De Rosa, Maria Caterina T-3179

638PRESENTER INDEXDe Souza Santos, Roberta F-2068De Soysa, Yvanka F-2041Delsing, Louise T-2047Deng, ChunhaoW-3131Deng, TaoF-3136Desai, DivyaW-3136Deshpande, Aditi W-3160Detera-Wadleigh, Sevilla F-3171Dexter, Dwayne F-4045Dhahri, Wahiba F-2037Di Domenico, Francesca T-4057Dias, Marlon L T-1021Diette, Nicole F-2007Dillman, Robert O F-2119Ding, QianqianT-3150Dirkx, NinaF-3162Dizon, JordanT-3213Dobrovolny, Robert F-2091Dobson, Samori F-2018Dohi, HiromiT-3082Doi, DaisukeW-3133Dong, ZhenF-3154Dos Santos, Aurelie W-2095Drakhlis, Lika T-3047Drela, KatarzynaT-4041Dror, IrisT-3096Drouin-Ouellet, Janelle T-3022Dunn, AndrewT-4031Dziedzicka, Dominika W-3138D’Antonio-Chronowska, Agnieszka W-2026D’Ignazio, Laura T-3174D’Sousa, Saritha S F-2050Eastman, Anna E W-3222Ebisu, FumiW-3235Ederer, Maxwell T-2018Eguchi, Asuka W-2031El-Badri, Nagwa F-3238El-Badri, Nagwa T-2117El-Badri, Nagwa W-2105Elcheva, Irina T-2042Eleuteri, Boris F-3219Elsayed, Ahmed F-4035Emara, AlaaW-3214Emperumal, ChitraPriya F-2089Enoki, TatsujiF-2044Enzo, ElenaW-1008Eppler, Felix W-4048Eshwara Swamy, Vinutha F-2083Evseenko, Denis W-2019Eze, UgommaT-1014Fajardo, Viviana F-2036Fan, YipingF-3035Fang, FangW-3092Fang, JunW-4001Faxiang, Xu F-3228Faynus, Mohamed T-2099Feaster, Tromondae K T-2041Fedorova, Veronika F-3139Felkner, Daniel W-3111Feng, QiangT-2091Feng, QiangW-2050Fernandez, Nestor T-2062Fieldes, Mathieu T-3137Fiorenzano, Alessandro W-3045Fiscella, Michele T-4022Fitzgerald, Michael Z T-4055Flynn, Kevin C W-2089Fonoudi, Hananeh F-1038Foong, Chee (Patrick) k W-3079Foster, Mikelle T-3142Frank, AaronW-3122Franzen, Julia T-3097

639PRESENTER INDEXFrieman, Amy L W-3197Friesen, Max T-3184Fu, XuebinF-2038Furlan, Giacomo F-3190Furuta, Asuka W-3089Gaafar, Ameera W-2052Gahyang, Cho W-2076Galat, YekaterinaW-4013Galloway, Kate E T-3195Gan, PeihengF-2024Gao, JinghuiT-3111Gao, LinW-4010Gao, NiF-4047Gao, ZhonghuaT-4016Garcia, Gustavo T-3159Garg, VidurF-3199Gasparini, Sylvia J F-2098Gaudreault, Nathalie F-3211Geens, MiekeT-3207Geisse, Nicholas A F-3213Geisse, Nicholas A W-4037Gell, Joanna J T-3089Genolet, Oriana T-3147George, Aman W-3162George, Benson M W-2062Georgieva, Daniela W-3202Gerami, Amir W-3004Geusz, RyanF-2069Gevorgian, Melinda T-3015Ghazizadeh, Soosan T-2087Ghazvini, Mehrnaz T-3205Ghosheh, Nidal T-3146Gifford, Casey W-3198Gill, Stanley P F-3023Glackin, Carlotta A W-2119Glaeser, Juliane D T-2016Glover, Hannah F-4008Glykofrydis, Fokion W-4028Go, YounghyunF-3233Godoy-Parejo, Carlos W-3107Goetjen, Alexandra M W-3096Goetzke, Roman F-3121Goldenberg, Regina F-2066Gomez, EmildaW-3205Gomila Pelegri, Neus F-3003Gonzalez Teran, Barbara W-2032Gonzalez, Sheyla F-2094Goto, TakasumiF-2034Goulart, Ernesto T-1024Grajcarek, Janin T-3164Grezella, Clara W-3175Griffin, Casey W-3026Grise, Kenneth N T-2094Gu, MingxiaF-1035Gunawardane, Ruwanthi T-3122Gunawardane, Ruwanthi W-3212Guo, PeipeiT-4036Guo, QiuyuF-2104Guo, WentingF-3029Guo, XiaolinF-2060Gupta, AditiT-4064Gupta, Ashwani K W-2071Gupta, SandeepF-3008Gupta, SuchiF-3221Gutierrez, Maria L F-3074Gutova, Margarita T-3234Ha, SeungyeonW-4002Haddadderafshi, Bahareh W-3099Haferkamp, Undine F-3028Hahn, SoojungT-4011Han, Ho Jae W-4007Han, Ho Jae W-4058

640PRESENTER INDEXHan, Ji-youT-2032Han, Min-JoonF-3203Han, Min-JoonT-3200Han, Young-minF-3206Hansen, Marten T-2057Hao, YiT-4061Hardy, Ariana R F-4024Harris, Violaine K F-3086Hashem, Sherin F-2031Haskell, Andrew W W-3219Hatanaka, Emily A W-3032Hatou, ShinF-2096Hayashi, Yohei F-3150He, AliceW-3218He, JiangpingT-3110He, Xi (CiCi)F-2084Hedenskog, Mona F-3209Hegab, Ahmed E W-2081Hemmati, Pouya W-2025Henderson, Kayla T-2035Hendricks, Eric W-4060Hendrickson, Michael T-3016Henn, AliciaF-2115Henn, AliciaT-3208Heo, June Seok T-2009Herbert, Franklin J W-1012Hernandez, Juan Carlos T-2120Heydarkhan-Hagvall, Sepideh W-3063Hicks, Michael R W-2115Hidalgo San Jose, Lorena T-4037Higgins, Will T-4013Higuchi, Yuichiro F-3037Hiler, Daniel J W-3134Hirano, Minoru T-4019Hirano, Sugi F-3063Hirasaki, Masataka T-3135Hirst, Adam J W-3104Ho, MirabelleF-1036Ho, MirabelleT-3187Ho, MirabelleW-3120Ho, MirielT-3121Ho, MirielW-3121Ho, RitchieF-3165Hoban, Deirdre B W-3013Holder, Daniel W-2099Hollands, Jennifer W-4052Hoover, Malachia Y T-2113Hor, Jin Hui T-3024Hor, PoojaT-3149Hrstka, Sybil W-4035Hsi, ChangW-2015Hsieh, Chen-ChanW-2016Hsu, Yi-chaoT-3040Hsu, Yi-chao F-3040Hu, JiangnanW-3058Hu, ShijunF-2032Hu, XiaomengF-3240Hu, XiaomengT-3224Huang, BoxianT-2001Huang, Chun-ChungW-4011Huang, DantongW-3065Huang, GuanyiW-3190Huang, JijunT-2031Huang, LungYungT-3232Huang, PengyuW-4018Huebsch, Nathaniel F-3066Huebsch, Nathaniel T-2026Hultmark, Simon F-2053Hung, Shu-TingF-3022Hussein, Abdiasis W-3087Hwang, Dong-YounW-3014Hwang, Ji Yoon T-3209

641PRESENTER INDEXIbrayeva, Albina W-3019Iefremova, Vira F-3053Iida, TsuyoshiT-1015Ikeo, SatoshiF-1031Iniguez, Karen T-3226Inoue, MakotoT-4008Ishibashi, Nobuyuki F-3004Ito, TakujiF-3175Iwamori, Naoki T-4060Iwasawa, Kentaro W-4031Iworima, Diepiriye G W-3135Izrael, Michal T-3086Izrael, Michal W-3002Jabali, Ammar A T-1018Jacob, Sheela P F-2052Jacobs, Elizabeth H W-3053Jacques-Smith, Krystal T-2072Jakimo, Alan L W-3215Jamieson Morris, Isabella C W-2128Janas, Justyna A T-3194Jang, SujeongF-3195Jang, Woong Bi F-2045Jang, Young-JooF-2087Janiszewski, Adrian W-3192Jee, JooHyunT-3042Jeon, KilsooT-3125Jeong, Kyu-ShikT-4045Jeong, Yun-MiF-2040Jervis, Eric W-3232Jha, RajneeshT-3153Ji, GuangzhenT-3105Jiang, DanW-2098Jiang, DuW-2064Jiang, XueyingF-3032Jin, YoonheeF-3070Jo, Hye-YeongF-4025Johannesson, Bjarki W-3129Johansson, Markus T-3183Jonlin, Erica C T-3076Jordan, Zachary S F-3038Josephson, Richard T-4032Joshi, PiyushF-3108Journot, Laurent T-3175Jovanovic, Vukasin M F-3012Ju, HyeinT-3218Ju, HyeinW-2001Ju, YoungheeF-3185Judd, JustinT-3044Jung, Kyong-JinT-2084Jung, Seung Eun F-2030Jung, SookyungF-3133Kageyama, Tatsuto F-3064Kamali, Samaneh F-3166Kambli, Netra K T-2100Kanber, Deniz W-2101Kang, EunjinW-2111Kannan, Nagarajan F-2116Kapyla, Elli W-3066Karaca, Esra W-2047Kardel, Melanie D T-3139Kargaran, Parisa F-3163Karmach, Omran F-4022Kato, MidoriW-4026Kausar, Rukhsana F-3006Kawabori, Masahito T-3031Kawakami, Eri T-4007Kawashima, Akihiro W-2006Keller, Alexander W-3124Khazaei, Mohamad F-3002Khedr, MoustafaF-3159Kheur, Supriya M F-2124Khoury, Hanane T-2050

642PRESENTER INDEXKikuchi, Tetsuhiro F-3085Kim, Albert D W-2068Kim, Byung Woo W-3024Kim, ChoonghyoW-3028Kim, Eun Young W-4008Kim, EunhyeW-2021Kim, Jean J W-3240Kim, Jong-TaeT-2105Kim, JonghunT-4003Kim, JunyeopT-3192Kim, Kyung-MinT-3127Kim, MinYoungF-3082Kim, Pyung-HwanF-2118Kim, Sang Woo F-2029Kim, Seung Hyun F-4048Kim, SuminF-3024Kim, SungminF-3222Kim, SungtaeF-4060Kim, YejiF-3069Kim, Yeon Ju F-2046Kim, YeseulF-3149Kim, YohanF-3202Kim, Young Hwan F-3232Kim, Young-KyuF-3187Kim, YubinT-4006Kim, YunaF-2129Kimura, Azuma W-1006Kimura, Masaki T-3236Kishimoto, Keiko T-3092Kishore, Siddharth F-1039Kiskinis, Evangelos W-3033Kitada, Kohei T-3171Kitahara, Takahiro F-3051Klim, JosephT-3027Knoebel, Sebastian F-3239Knoebel, Sebastian T-2027Knox, RonaldF-3217Kobayashi, Yuki F-4017Koh Belic, Naomi F-2006Kohyama, Jun T-3099Kojima, Yoji T-4058Komarovsky Gulman, Nelly F-3160Koning, Marije T-3052Korsakova, Elena W-3031Kosanke, Maike F-4049Kotian, Shweta T-4027Kotter, Mark R F-4019Kotter, Mark R F-4020Koui, YutaF-2078Kousi, MariaT-3181Kramer, Philipp M F-3043Kramerov, Andrei W-2094Krefft, Olivia F-3042Kroeger, Heike T-2092Kuirsaki, Akira W-2007Kumar, SanjayF-2002Kumar, VivekF-3141Kuninger, David W-3229Kuo, Hung-ChihT-3003Kurdian, Arinnae W-3048Kuroda, Takuya F-4011Kurowski, Agata W-3095Kuzenna, Olga M T-4001Kwon, DaekeeW-4068Kwon, Yoo-wookF-3113Laan, LooraT-3176Lacham-Kaplan, Orly F-2012Laha, KurtT-3011Lai, DongmeiW-3084Lai, Jesse D T-3048Lai, Jesse D W-3017Landon, Mark F-2130

643PRESENTER INDEXLandon, Mark T-2125Langerman, Justin T-3193Lara, JacquelineW-2129Lau, CynthiaW-3050Lau, Hwee Hui F-3186Lau, Hwee Hui T-3119Lavoie, Jessie R W-3242Lawson, Erica J T-4068Lazure, Felicia W-2022Le, Khoa T T-3113Le, Minh N T-3143Lech, WiolettaF-2005Lee, Chang Hyun W-2034Lee, Choon-SooF-1030Lee, EmilyW-3182Lee, HanbyeolT-2081Lee, Hyun Jung W-4033Lee, JasonF-3236Lee, JasonT-3219Lee, Jia-JungF-4029Lee, Jin-MooF-4044Lee, Jin-WooT-3165Lee, JiyunT-2014Lee, JoohyungT-2008Lee, JooyeonT-2083Lee, MingyunF-3107Lee, MinjiW-4039Lee, Vivian M F-3153Lee, Vivian M T-3123Lee, WeiF-2004lEE, Yee Ki, Carol F-3182Lee, YukyeongW-4051Leitner, Lucia M T-2036Leitoguinho, Ana Rita W-2054Leushacke, Marc T-4034Levy, ShiriF-3101Levy, ShiriT-3103Lew, HelenF-2101Lew, HelenT-2003Lewis, KyleT-4039Lezmi, ElyadW-3141Li, Bo W-2084Li, CuiW-3030Li, Ian M W-3150Li, JinglingT-3186Li, JinglingW-3119Li, JunjunT-2034Li, LeeAnnW-4017Li, Lu W-4032Li, MeiT-2059Li, RongF-3017Li, Wan-JuF-2020Li, XiajunF-3092Li, YichenF-3183Li, YingchunF-3201Li, YuanyuanF-4037Li, ZhongwenF-4010Liang, XiquanF-3227Libby, AshleyW-3062Lim, Jae-YolW-3069Limone, Francesco W-3036Lin, HangW-4025Lin, Kun-YangT-2111Lin, LihuiT-3201Lin, Phyo Nay T-4025Lin, Po-YuF-2015Lin, XiaolinF-3031Linares, Gabriel R W-3037Linda, KatrinT-3168Lindqvist, Maria T-3204Lis, RaphaelT-2054Lis, RaphaelW-2043

644PRESENTER INDEXLiu, CambrianW-1007Liu, HongjunT-2093Liu, HuiW-3056Liu, JiadongW-3206Liu, JingT-3026Liu, LipingT-3130Liu, XinyuanF-3145Liu, YingT-3069Liu, YingW-3003Liu, YongF-3015Lo Cascio, Costanza W-2123Lo Nigro, Antonio W-2070Lo, Peggy Cho Kiu F-3164Lohmussaar, Kadi T-1019Long, JenniferT-2013Lopez Davila, Victor F-3065Lopez, George A W-3187Lovelace, Michael D F-3001Lovelace, Michael D W-4044Low, Blaise Su Jun W-3168Low, Walter C F-3078Lowe, MatthewF-3087Lowman, John W-4023Lozito, Thomas W-4056Lu, Huai-EnW-3237Lu, JiafengT-2005Lu, KarolT-3078Lu, KarolW-3075Lu, MinT-3056Lu, TingF-4014Lu, VivianF-3142Lucich, Katherine L T-3189Lui, Nga Chu F-3161Luo, ZhengliangF-2021Luthra-Guptasarma, Manni T-3203Luttrell, Shawn F-3129Lynch, Cian J W-3110Ma, XiaoxueF-3226Ma, XunF-3093MacArthur, Chad C W-3199Machado, Leo T-3230Maddah, Mahnaz T-3222Maekawa, Hiromi W-4063Mafreshi, Maryam T-3235Maguire, Colin T T-4029Mah, NancyW-4006Mahlke, Megan F-3106Malley, Claire W-3132Mallon, Barbara F-4001Mana, MiyekoT-2129Mandalay, Prasanthi T-3037Mangala, Melissa M T-2037Manstein, Felix W-3115Mantripragada, Venkata R T-3223Mantripragada, Venkata R W-2012March, Alexander R W-3060Marczenke, Maike W-4015Mariga, Abigail F-4033Marques, Marcelo R T-3233Martin, Heather M T-2090Martinez Becerra, Francisco J F-4069Martins, Manuella M T-3133Martins, Soraia F-3173Marzorati, Elisa W-3140Masaki, Hideki F-3208Masters, Haley T-3001Masumoto, Kanako W-3154Matsuo, Junichi T-4033Matsuoka, Akihiro J W-2106Maxwell, Kristina G. T-3172Mayner, Jaimie T-2028McCarrey, John R T-3091

645PRESENTER INDEXMcGarr, Tracy F-3223McGrath, Patrick S F-3191Mcintire, Erik W-3233McLelland, Bryce W-3049McMullen, James W-4014Mcvicar, Rachael N T-3152Meer, ElliottW-3231Mei, YangF-2062Meissner, Torsten B F-2057Meitz, Lance E T-3128MeloEscobar, Maria Isabel T-3061Memon, BushraF-1034Mendez, Gilberto W-3188Meng, LingjunT-2123Meng, ShulinF-2043Mengarelli, Isabella T-3163Milagre, Ines T-3095Miller, Duncan C W-3181Millette, Katelyn T-2069Milliex, Julia W-4069Min, Sang Hyun F-2121Min, SungjinF-3196Minocha, Ekta W-2077Mir, Yasir A W-4036Mitchell, Jana T-3021Mithal, Aditya T-1020Miyagishima, Kiyoharu J T-2095Miyaoka, Yuichiro F-2039Miyauchi, Masashi T-1022Mochizuki, Mai F-3080Mogen, Austin Blake W-3223Mohanty, Sujata F-3068Mohanty, Sujata T-3068Mohanty, Sujata W-2067Molakandov, Kfir T-2070Montel-Hagen, Amelie W-2053Monville, Christelle W-2097Moon, Byoung San W-2118Moon, HyeJiT-3107Moore, Jennifer C T-3023Moran, Deborah J W-2087Morey, RobertT-3156Morita, Yasushi F-4066Moriyama, Hiroyuki W-2010Moriyama, Mariko T-3182Moser, V. Alexandra W-4059Mournetas, Virginie F-4036Mu, LiliT-2112Muckom, Riya J F-3119Mueller, Sabine C F-4065Muffat, Julien W-4067Mulay, ApoorvaW-2083Mun, Chin Hee T-4038Murphy, Matthew P T-2017Murray, John T-2007M’Callum, Marie-Agnes T-3134Na, JieT-2043Nagaishi, Kanna T-3216Nagwa, El-BadriW-3203Nair, EstherF-4026Nair, Vani Manoharan F-2076Nakai, KentoT-3170Nakajima, Taiki T-1023Nakamura, Mari F-4034Nakano, Tokushige F-3052Nakata-Arimitsu, Nagisa W-4053Nakielski, Pawel T-3065Nath, AneeshaW-2057Natsume, Yusuke F-3229Neely, M Diana T-3178Nefzger, Christian W-3200Negoro, Takaharu F-3076

646PRESENTER INDEXNegoro, Takaharu W-4034Negoro, Takaharu W-4064Neiman, Gabriel W-3145Nekanti, Usha F-3011Nell, PatrickW-4019Nella, Kevin T T-2109Nelli, Rahul K F-3044Newby, Steven D F-3062Ngai, Hoi Wa W-2126Ngan, EllyW-2037Nguyen, Linh T T-4002Nicoleau, Camille W-3170Nicolini, Anthony T-4014Nicolini, Anthony T-4047Nii, TakenobuF-2056Nio, YasunoriT-4009Nishikawa, Masaki T-3054Nishinaka-Arai, Yoko W-1010Nishino, Taito F-2074Nissenbaum, Yonatan (Jonathan) F-2127Nistor, Gabriel W-2113Niu, WeiF-3179Niwa, AkiraW-2116Niwa, SatoshiF-3010Noggle, Scott F-3049Nolbrant, Sara F-3009Nold, PhilippF-3215Ock, Sun A F-2072Ogawa, MinaF-3158Ogawa, ShimpeiF-3130Ogoke, OgechiT-2078Oh, DeniseW-2100Oh, Il-HoanT-4044Oh, JonghyunW-3059Oh, Steve K F-3135Ohashi, Fumiya F-4016Ohlemacher, Sarah K F-3020Okada, RyuF-3048Okada, YoheiF-3184Okuno, HironobuW-3102Oliva, JoanF-2082Oliva, JoanT-2080Olivarria, Gema M W-3038Oliveira, Karina G T-3167Onen, SelinF-3090Ongstad, Emily F-2025Oommen, Saji F-2028Ortega, Juan Alberto T-3214Ortmann, Daniel W-3112Osnato, Anna W-3106Osorio, Maria Joana T-3180Otero, ChristopherW-3174Otomo, JunW-3152Ouyang, John F T-3191Paganelli, Massimiliano T-2076Pajcini, Kostandin V T-2110Paluh, Janet L T-3085Pan, ShaohuiF-2093Pandya-Jones, Amy W-3098Pang, JeremyT-2030Panopoulos, Athanasia D W-2117Papadimitriou, Elsa W-3204Papalmprou, Angela W-2013Papandreou, Apostolos W-3021Paquin, Karissa W-4029Paris, MarylineT-3062Park, ArumT-4066Park, Brian J F-4002Park, Hyun Jung T-3017Park, Jae Kyung T-3083Park, Ju Hyun F-4067Park, JuchanT-3215

647PRESENTER INDEXPark, KijeongT-3229Park, Kwang-SookT-3198Park, Sang In W-4024Park, Sang-wookF-3168Park, So Young F-3061Park, Tea Soon T-3050Park, Tea Soon W-2090Park, ZewonF-4043Parvez, Riana K F-2067Passanha, Fiona W-4045Patel, SamF-2017Pathak, Varun F-2058Pawlowski, Matthias T-2058Pearse, Yewande F-3033Pearson, Caroline A W-3008Pecori, Federico W-3114Pei, PeiF-3099Peng, GuangdunT-4059Peng, YiF-2114Perez-Bermejo, Juan T-2033Pernstich, Christian T-4040Perry, John M T-2119Petrosyan, Astgik T-2116Petrosyan, Astgik W-2065Petrova, Ralitsa W-1013Pezhouman, Arash W-3126Pham, KristenT-3045Pijuan-Galito, Sara F-2107Pires, Cristiana F F-4053Pirozzi, Filomena W-3035Platero-Luengo, Aida W-3194Plaza Reyes, Alvaro W-2092Poe, Adam J W-2091Pollante, Michael Vincent V T-3033Poole, AleksandraW-2078Poon, EllenW-2036Popova, Semiramis F-2051Porterfi eld, Veronica W-4065Pouraghaei Sevari, Sevda W-4005Pouyanfard, Somayeh F-3132Pranke, Patricia F-3067Pranke, Patricia T-3067Pranke, Patricia W-2066Prasad, Maneeshi S T-3132Previdi, Sara T-4010Prieto Gonzalez Albo, Isabel W-4043Prowse, Andrew W-3220Prutton, Kendra F-4039Pulecio, Julian T-3098Qi, ZhenF-3073Qian, LiT-3199Qian, TongchengF-2035Qian, XuyuF-3055Quadrato, Giorgia W-3054Queckbörner, Suzanna T-4052Questa, Maria T-4067Quijano, Janine W-3057Quiroz, Erik J W-2088Quiskamp, Nina F-3041Radecki, Daniel W-3012Radwan, Ahmed W-3193Rajagopal, Jayaraj T-2082Ramamoorthy, Mahesh F-3096Raman, SreedeviF-3174Ramos Calcada, Raquel M T-3154Ramos, Michael Edison P T-2077Randolph, Lauren N T-3129Rapino, Francesca W-3185Rauch, John N T-3212Ravindran, Geeta W-3186Regier, Mary T-4028Reinhardt, Anika W-3221

648PRESENTER INDEXReinholdt, Laura T-3114Ren, HaoW-4054Ren, Yongming Luke F-3204Ressler, Andrew W-3164Richey, Alexandra T-4050Rizal, GandhiT-3104Rockne, Russell F-2120Rogers, Robert E F-3216Roh, SanghoT-2086Roh, Seung Ryul F-3109Romagnuolo, Rocco F-2027Rosa, AlessandroW-3046Rosen, Siera A T-4054Rothmiller, Simone F-2109Rowland, Teisha W-4021Rukstalis, Michael T-3141Ruoss, SeverinF-3084Ryan, SeanF-3169Ryu, SeungmiF-3050Sa-Ngiamsuntorn, Khanit T-2071Sabri, ShanF-3194Sagal, JonathanT-3057Sagi, IdoW-3103Saha, DebapriyaW-3100Sahabian, Anais W-3125Saito, MikakoT-2128Sakaguchi, Hideya W-3055Sako, MisatoT-3035Salameti, Vasiliki W-4020Salehin, Nazmus F-4027Salgado, Bianca M W-2073Salie, MuneebF-3098Samarasinghe, Ranmal F-3045Samsonraj, Rebekah Margaret W-4046Sances, Samuel F-3167Sanders, Bret W-3169Sandoval, Aaron Gabriel W-4047Sandoval, Kadellyn T-3007Sankar, Aditya T-1025Santiago, Isaac E T-3018Santin Velazque, Natalia L T-2114Santos, Jerran T-2006Santosa, Munirah T-1017Saremi, Shahin F-2122Sari, Siska Y T-4049Sawada, Rumi F-3122Sawai, TsutomuF-3077Saxton, Sarah T-3073Scaramozza, Annarita W-2014Schaefers, Catherine W-3130Schaukowitch, Katie M T-3008Schell, John P T-3112Schiffmacher, Andrew T W-2107Schruf, Eva W-3184Schröter, Manuel W-4062Schumacher, Anika W-3047Schwartz, Joshua T-3166Secreto, Frank J W-2040Selich, Anton W-2108Seol, Dong-WonW-3139Serowoky, Maxwell T-2019Shablii, Volodymyr F-2003Shah, RuchiF-2092Shaharuddin, Syairah Hanan Binti F-2073Sharma, Arun W-1003Sharma, Arun W-2030Sharma, Maryada T-3053Sharma, Ruchi T-3072Sharma, Yogita F-3014Shayler, Dominic W-2102Sheldon, Michael T-3188Shen, Chia-NingW-3108

649PRESENTER INDEXShevade, Kaivalya T-3102Sheyn, DmitriyW-3137Shi, Chung-ShengW-2121Shibata, Shun F-2097Shibuya, Yohei F-3036Shichinohe, Hideo T-4051Shiga, TakahiroT-1016Shih Min, Wang W-4012Shimba, Kenta T-3010Shimbo, Takashi T-2088Shin, YoungchulF-2047Shineha, Ryuma F-3075Shingo, Miyawaki F-3097Shrestha, Rupendra F-3123Sidhu, Harpreet K F-4063Signer, Robert A F-2049Silva, JoseF-4055Sim, Zixuan Erinn F-3155Sinha, DivyaW-3180Sinnaeve, Justine T-2121Sirenko, Oksana F-3210Sirenko, Oksana W-2033Sirenko, Oksana W-3042Skiles, Matthew L W-4040Skowronska, Marta F-3104Slamecka, Jaroslav W-3151Sligar, Andrew D W-2045Smith, AlecW-3216Smith, CambrayW-3078So, Kyoung-HaT-3046Soares Da Silva, Francisca W-2112Soares, Eduardo W-2103Soleimani, Vahab F-2112Solis, Mairim A W-2004Soncin, Francesca T-2002Song, Byeong-WookW-3225Song, JihwanT-3019Song, YangF-4050Songsaad, Anupong W-4049Sosa, EnriqueT-1026Soto, JenniferF-4052Soufi, AbdenourW-3191Sozen, BernaW-3090Spencer-Fry, Jane F-4068Sperandeo, Alessandra W-3161Sporrij, Audrey W-2060Sproul, Andrew W-3020Sridhar, Akshayalakshmi W-3044Srinivasan, Gayathri W-3067Stan, RodicaF-3079Steeg, RachelF-4064Steiner, Embla W-3015Steininger, Holly M F-2022Streckfuss-Boemeke, Katrin T-3169Strunk, Dirk O T-4005Sturgeon, Christopher M W-2063Su, Emily Y T-4023Su, KevinT-3058Su, XinyunF-2085Subramanyam, Deepa W-3113Sucov, HenryT-2024Sugawara, Tohru F-4003Sugimori, Michiya T-2127Sui, LinaT-2073Sultana, Zeba F-3115Sun, ChichengW-3173Sun, DaweiW-3052Sun, LuluT-2075Sundberg, Maria K T-3030Sung, Jin Jea T-3220Sung, Jin Jea W-3210Sung, Li-YingT-3131