ISSCR 2019 Poster Abstracts_clone

500POSTER ABSTRACTSNADH/NAD+ ratio compared to a clone exhibiting no detectable heteroplasmy. This study demonstrates genetically encoded SoNar as an efficient platform based metabolic screening could serve as a valuable approach for drug discovery.F-3165SINGLE-CELL RECONSTRUCTION OF HUMAN HINDBRAIN AND SPINAL CORD IN IPSC MODELS OF AMYOTROPHIC LATERAL SCLEROSIS REVEALS CELL TYPE-SPECIFIC TRANSCRIPTIONAL SIGNATURES OF DISEASEHo, Ritchie - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Workman, Michael - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Kellogg, Mariko - Product Development, Illumina, San Diego, CA, USA Montel, Valerie - Product Development, Illumina, San Diego, CA, USA Mathkar, Pranav - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Oheb, Daniel - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Banuelos, Maria - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Huang, Steven - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Khrebtukova, Irena - Product Development, Illumina, San Diego, CA, USA Watson, Lisa - Product Development, Illumina, San Diego, CA, USA Taylor, Kevin - Product Development, Illumina, San Diego, CA, USA Svendsen, Clive - Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USAAmyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder defined by cortical and spinal motor neuron (MN) death, typically presented in adulthood as a spreading paralysis of various motor circuits throughout the hindbrain and spinal cord, ultimately leading to asphyxiation. ALS patients have an average life expectancy of three years after diagnosis. Of ALS cases, ten percent are attributed to genetic mutations, among which hexanucleotide repeat expansions in C9orf72 are the most common, and 90 percent are of unknown genetic cause (sporadic ALS). While several pathways and cell types have been associated with ALS etiology, there are currently no cures available due to the lack of definitive mechanisms and thus therapeutic targets. Given the heterogeneous genotypes and clinical and molecular phenotypes associated with ALS, iPSC models, which can capture the genetic architecture of ALS patients in differentiated MNs as well as accessory cell types, are amenable to study the underlying causes of ALS in search of early biomarkers or candidate drug targets. Here we expression profile iPSC-differentiated neuronal cultures from C9orf72 ALS, sporadic ALS, and unaffected patients at the single cell level to characterize the developmental, anatomical, and functional identity of each cell. Using a rostro-caudal developmental HOX gene expression code, we observe that iPSC-differentiated neural cultures resemble hindbrain and rostral rather than caudal spinal cord segments. Using a combination of developmental marker genes with global clustering algorithms, we define ventral MN, V1, and V2 interneuron populations in cultures. We subsequently detect transcriptional dysregulation by ALS within each cell type. Despite resembling immature fetal tissue, iPSC-derived MNs specifically exhibit transcriptional changes that are detectable in and concordant with post-mortem, laser captured MNs from sporadic ALS patients. Overall, our analysis enables the resolution of cell identities (e.g. rostral vs. caudal, hindbrain vs. spinal cord, dorsal vs. ventral regions, and progenitors vs. postmitotic neurons) and disrupted physiologies relevant to individual ALS patients. Furthermore, our results illustrate that early signatures and potential therapeutic targets of ALS can be observed in iPSC models.Funding Source: This work was funded by the Neuro Collaborative by the ALS Association, Answer ALS, NeuroLINCS, and a National Institute on Aging Pathway to Independence Award.F-3167IPSC DERIVED BRAIN CHIP SYSTEMS TO STUDY HUMAN NEURODEGENERATIVE DISEASESances, Samuel - Regenerative Medicine Institute, Cedars Sinai Medical Center, Santa Monica, CA, USA West, Dylan - Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Woodbury, Amanda - Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Ondatje, Briana - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA El-Ghazawi, Kareem - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Laperle, Alexander - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Ho, Ritchie - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Meyer, Amanda - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Dardov, Victoria - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Shu, Zhan - Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA, USA Spivia, Weston - Advanced Clinical Biosystems Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Maidment, Nigel - Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA, USA Van Eyk, Jennifer - Advanced Clinical Biosystems Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Svendsen, Clive - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

501POSTER ABSTRACTSThe physiological, molecular and cellular changes that underlie amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are complex. Rare monogenetic forms of these diseases, while informative, do not reflect the approximately 90% of ALS and PD cases with no known genetic mutations (termed sporadic). The greater prevalence of sporadic ALS and PD cases prompts the immediate need to develop sporadic disease models. Working with the Cedars-Sinai’s Induced Pluripotent Stem Cell (iPSC) Core to generate large cohorts of ALS and PD lines, we have developed advanced differentiation techniques for cell types relevant to central nervous system function. Among these are robust methods to derive spinal motor neurons (spMNs) and dopaminergic neurons (DANs) that are uniquely susceptible in ALS and PD, respectively. From iPSCs, we can derive other cell types that are also affected in disease, including astrocytes and microglia, as well as brain microvascular endothelial cells (BMECs) that replicate blood brain barrier (BBB) function. Here we have combined these iPSC-derived cells with scalable microphysiological systems (MPS), also known as Organ-Chips (Emulate Inc.). Through co-culture of either spMNs or DANs with additional supportive cell types, we have developed highly physiological models of ALS and PD on Organ-Chips referred to as ALS-Chip and PD-Chip. These neurodegenerative Chip models are perfuseable culture systems capable of sustained flow of media and even human blood. To uncover disease-specific pathophysiology in the ALS-Chip or PD-Chip and to generate novel biomarkers for both diseases, we have developed a comprehensive array of genomic, proteome, metabolomic, and electrophysiological assays. The primary outcome of this project is the establishment of reproducible disease-specific phenotypes of both sporadic ALS and PD that will then be used as physiological biomarkers to screen for novel pathological-mitigating drugs.Funding Source: ALS Association, National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS), National Center for the Advancing Translational Science (NCATS), Grant ID 1UG3NS105703-01F-3169INFLAMMATION AND PHAGOCYTIC DYSREGULATION IN A MODEL OF HIV NEUROPATHOGENESISRyan, Sean - Pathology, University of Pennsylvania, Philadelphia, PA, USAHIV-Associated Neurocognitive Disorders (HAND) affect 55% of HIV-infected individuals worldwide. While antiretroviral treatments have reduced the severity of HAND, the prevalence has increased due to increased life expectancy. In addition, little progress has been made in developing therapeutics to reduce the prevalence of HAND. While the major pathological manifestation of HAND is synaptodendritic damage, the full, underlying mechanism is unknown partly since there is no in vitro model to study the direct interactions between HIV-infected macrophages/microglia and neurons. In order to address this problem, we have developed a human-induced pluripotent stem cell (HiPSC) based model; whereby, we separately differentiate HiPSCs into forebrain, glutamatergic-like neurons, astrocytes, and microglia and create a co-culture of the three cell types with or without HIV-infection. As expected, we found increased production of inflammatory cytokines by HIV-infected microglia. However, we discovered a reduction in synaptic phagocytosis by the infected microglia, suggesting phagocytosis is not the mechanism behind synaptodendritic damage in HAND.F-3171AN INDUCED PLURIPOTENT STEM CELL RESOURCE FROM A POPULATION ISOLATE INTEGRATES GENETICS WITH BIOLOGY FOR STUDIES OF BIPOLAR AND RELATED NEUROPSYCHIATRIC DISORDERSDetera-Wadleigh, Sevilla - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Ahn, Kwangmi - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Akula, Nirmala - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Besancon, Emily - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Blattner, Megan - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Corona, Winston - Human Generic Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Cross, Joanna - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Dumont, Cassandra - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Gordovez, Francis - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Jiang, Xueying - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Kassem, Layla - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Lopes, Fabiana - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA McMahon, Francis - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USA Schulze, Thomas - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD,

502POSTER ABSTRACTSUSA Sheridan, Laura - Human Genetics Branch, National Institute of Mental Health / National Institutes of Health, Bethesda, MD, USAMultiple risk-associated variants revealed through genome-wide association studies (GWAS) highlight the polygenic architecture of bipolar disorder (BD), major depressive disorder (MDD) and schizophrenia (SCZ). These findings have fueled intensive investigations on the biological impact of common alleles on the pathogenesis of these complex neuropsychiatric disorders. Common low-risk alleles may be complemented by variants of higher impact that are enriched in genetically isolated populations. To identify such variants, we are carrying out a genetic study of Amish and Mennonite individuals whose common ancestors originated from central and northern Europe. These communities convey the advantages of large families, relatively homogeneous lifestyles, well-documented genealogies, and increased frequencies of certain otherwise rare alleles. Cases of BD and their relatives undergo a mental health evaluation, neurocognitive testing, and provide blood samples. Whole genome SNP array analysis has revealed rare copy number variants, such as a 16p11.2 duplication previously associated with schizophrenia and bipolar disorder. Exome sequencing in the first 600 participants has detected numerous otherwise rare variants, some of which are predicted to be damaging, within genes implicated in BD or SCZ by GWAS. Probands and one unaffected relative per family have provided skin biopsies, over 50 of which have been reprogrammed to iPSC. Neural derivatives of these clones constitute cellular reagents to model biological impact of risk alleles and to screen for novel therapeutics. We have performed initial characterization of selected clones, which are available to collaborating laboratories at no cost. Expansion of the resource continues in earnest. Comprehensive genetic and phenotype analyses on iPSC-derived neural cells may help explain how polygenic variants and rare alleles contribute to the neurobiological changes that put carriers at increased risk for debilitating mental disorders.Funding Source: NIMH Intramural Research ProgramF-3173THE TREM2 R47H RARE VARIANT AND ALZHEIMER DISEASE: AN IPSC-BASED PLATFORM FOR DISEASE MODELINGMartins, Soraia - ISRM - Institute For Stem Cell Research And Regenerative Medicine, UKD - University Hospital Düsseldorf, Germany Müller-Schiffmann, Andreas - Department Neuropathology, Heinrich-Heine University, Duesseldorf, Germany Bohndorf, Martina - Institute for Stem Cell Research and Regenerative Medicine, UKD - University Hospital Düsseldorf, Germany Wruck, Wasco - Institute for Stem Cell Research and Regenerative Medicine, UKD - University Hospital Düsseldorf, Germany Sleegers, Kristel - Neurodegenerative Brain Diseases group, VIB-UAntwerp Center for Molecular Neurology, Antwerp, Belgium Van Broeckhoven, Christine - Neurodegenerative Brain Diseases group, VIB-UAntwerp Center for Molecular Neurology, Antwerp, Belgium Korth, Carsten - Department Neuropathology, Heinrich-Heine University, Duesseldorf, Germany Adjaye, James - Institute for Stem Cell Research and Regenerative Medicine, UKD - University Hospital Düsseldorf, GermanyRecently, genes associated with immune response and inflammation have been identified as genetic risk factors for late-onset Alzheimer disease (LOAD). One of them is the rare p.Arg47His (R47H) variant within the gene encoding triggering receptor expressed on myeloid cells 2 (TREM2), which has been shown to increase the risk of developing AD by 2-3-fold. TREM2 is a cell surface receptor of the immunoglobulin superfamily that initiates a signalling cascade modulating cell proliferation and differentiation, survival, chemotaxis and inflammation. Importantly, TREM2 is required for microglial phagocytosis of a variety of substrates, including apoptotic neurons and A and thus plays a prominent role in driving microgliosis. βAlthough the role of TREM2 in AD has been a focus of study using post-mortem brains, mouse models and heterologous cell lines, no concluding agreement has been made probably due to the inability to model this complex disease. Here, we report the generation and characterization of a model of LOAD using lymphoblast-derived iPSCs from patients harbouring the R47H mutation in TREM2, as well as from control individuals without dementia. iPSCs efficiently differentiated into mature neuronal networks composed of neurons and glia cells. Comparative global transcriptome analysis identified a distinct gene expression profile in AD TREM2 neuronal networks suggesting that these lines exhibit alteration in key signaling pathways related to metabolism and immune system in comparison to control. Although the neuronal networks derived from AD TREM2 lines secreted A with a similar A 42 ratio compared ββto controls, manipulation with an A 42-S8C peptide dimer βrevealed metabolic dysregulation, impaired phagocytosis-related pathway and failed to induce an inflammatory response. In conclusion, our study has shown that our AD-iPSCs based model can be used for indepth studies to reveal putative molecular mechanisms underlying the onset of Alzheimer disease and for screening of potential therapeutic targets.Funding Source: Medical faculty of Heinrich Heine University DüsseldorfF-3175ELUCIDATION OF NON-CELL AUTONOMOUS NEURODEGENERATION IN SPINAL AND BULBAR MUSCULAR ATROPHY USING IPSC-DERIVED NEUROMUSCULAR MODELSIto, Takuji - Neurology, Aichi Medical University, Nagakute, Japan Tanaka, Satoshi - Department of Orthopedics, School of

503POSTER ABSTRACTSMedicine, Nagoya University, Nagoya, Japan Shimojo, Daisuke - Department of Neurology, School of Medicine, Aichi Medical University, Nagoya, Japan Doyu, Manabu - Department of Neurology, School of Medicine, Aichi Medical University, Nagoya, Japan Okano, Hideyuki - Department of Physiology, School of Medicine, Keio University, Tokyo, Japan Okada, Yohei - Department of Neurology, School of Medicine, Aichi Medical University, Nagoya, JapanSpinal bulbar muscular atrophy (SBMA) is an adult onset lower motor neuron disease caused by the abnormal expansion of polyglutamine tract (CAG repeat) in Androgen receptor (AR). So far, SBMA have been considered to be caused by cell-autonomous motor neuron degeneration. However, recent analyses have shown non-cell autonomous neurodegeneration by skeletal muscles, though detailed molecular mechanisms have not been fully elucidated. In this study, we established a human induced pluripotent stem cells (iPSCs) based neuro-muscular co-culture system to elucidate neuro-muscular pathology of SBMA. We first established human myoblast cell lines (Hu5/E18) stably expressing wild-type (Hu5/E18-AR24Q) or mutant human AR (Hu5/E18-AR55Q, and 97Q), and found that mutant AR expressing myoblasts exhibited poor differentiation into myotubes compared with those expressing wild type AR. Then, to examine the roles of mutant AR in neuromuscular interaction, motor neurons derived from control iPSCs (201B7) were co-cultured with myotubes expressing wild-type or mutant AR. As expected, iPSC-derived motor neurons formed significantly less numbers of BTX-positive neuromuscular αjunctions (NMJs) with myotubes expressing mutant AR than with myotubes expressing wild-type AR, suggesting that mutant AR expressed in myotubes suppresses NMJ formation even with healthy motor neurons. Moreover, motor neurons co-cultured with mutant AR expressing myotubes exhibited significantly higher numbers of cleaved Caspase-3 positive apoptotic cells than those co-cultured with myotubes expressing wild-type AR. These results suggest that mutant AR expressed in skeletal muscles may induce neuronal cell death through NMJs. To further elucidate actual pathogenesis of non-cell autonomous neurodegeneration caused by mutant AR in myotubes, we are currently undergoing co-cultures of patient or control iPSC-derived skeletal muscles and motor neurons in various combinations. In our preliminary data, co-culture of iPSC-derived motor neurons (Control) and skeletal muscles (SBMA) showed dying back-like neurodegeneration by time-lapse imaging. In the future, we would investigate underlying mechanisms of non-cell autonomous motor neuron degeneration to explore common or unique therapeutic targets for motor neuron disease.Funding Source: AMED 18ek0109243h0002 KAKENHI 17H05707 KAKENHI 18K15470 GSK Japan Research Grant RIKAKEN Research Grant SONPO Research GrantF-3177HEPATITIS B VIRUS INFECTION IN METABOLICALLY IMPROVED STEM CELL DERIVED HEPATOCYTE-LIKE CELLSTricot, Tine - Stem Cell Institute Leuven, KU Leuven, Belgium Thibaut, Hendrik Jan - Rega Institute, Katholieke Universiteit Leuven, Belgium Abbasi, Kayvan - Rega Institute, Katholieke Universiteit Leuven, Belgium Boon, Ruben - Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Kumar, Manoj - Stem Cell Institute Leuven, Katholieke Universiteit Leuven, Belgium Neyts, Johan - Rega Institute, Katholieke Universiteit Leuven, Belgium Verfaillie, Catherine - Stem Cell Institute Leuven, Katholieke Universiteit Leuven, BelgiumWorldwide, an estimated 260 million people are chronically infected with the hepatitis B virus (HBV) and at high risk of developing liver cirrhosis and hepatocellular carcinoma. Although the currently available nucleoside viral polymerase inhibitors are highly efficient in lowering viral load, they are unable to eradicate the virus. Consequently, life-long treatment is mostly necessary as the covalently closed circular DNA (cccDNA) is retained in the hepatocyte nucleus. Hence, there is an urgent need for therapeutics that allow to cure patients from their infection. Primary human hepatocytes (PHH) are currently the standard for HBV studies. However, because of shortage of donor organs and the labour-intensive work with PHH, alternatives are needed. Therefore, the use of human pluripotent stem cells (hPSC) derived hepatocyte-like cells (HLCs) in HBV infection models is being explored, which also offer the advantage of diverse genetic backgrounds. Although protocols have been developed to generate HLCs from hPSC, all hPSC-HLCs remain immature, lacking major drug metabolizing enzymes such as CYP3A4. To address this maturation problem, the Verfaillie lab has generated a hPSC line that overexpresses three liver-specific transcription factors (termed HC3x) in a doxycycline inducible manner as well as optimised medium conditions for these HC3x-HLCs. Differentiation of HC3x-HLCs in the optimised medium resulted in a more mature hepatocyte progeny, with increased production of albumin and functional CYP3A4 in comparison to control hPSC-HLCs. HC3x-HLCs could efficiently be infected with HBV, as demonstrated by staining for HBV core and surface antigen (infection efficiency between 9 and 20%). We are now further validating the culture system with various known anti-HBV antiviral drugs. The culture system can also be downscaled to 96 and 384-well format culture plates, which will enable us to perform large scale small molecule screens to identify novel and selective anti-HBV drugs to improve and expand the existing therapeutic regimens.Funding Source: Research Foundation-Flanders (FWO) (1185918N)

504POSTER ABSTRACTSF-3179MODELING PCDH19-RELATED EPILEPSY IN HUMAN EMBRYONIC STEM CELL DERIVED NEURONS AND CEREBRAL ORGANOIDSNiu, Wei - Neurology, University of Michigan, Ann Arbor, MI, USA Deng, Lu - Neurology, University of Michigan, Ann Arbor, MI, USA Du, Xixi - Neurology, University of Michigan, Ann Arbor, MI, USA Jalilian, Elmira - Neurology, University of Michigan, Ann Arbor, MI, USA Mojica-Perez, Sandra - Neurology, University of Michigan, Ann Arbor, MI, USA Tidball, Andrew - Neurology, University of Michigan, Ann Arbor, MI, USA Parent, Jack - Neurology, University of Michigan, Ann Arbor, MI, USAPCDH19-Related Epilepsy (PRE) is caused by mutations of the PCDH19 gene on the X-chromosome and exclusively affects females and mosaic males while male carriers are spared. Mosaic expression of PCDH19 due to random X-inactivation is thought to cause impaired cell-cell interactions between mutant and wild type PCDH19-expressing cell populations to produce the disease phenotype. However the precise function of PCDH19 during human cortical development and how the mosaic expression of PCDH19 leads to PRE remain unclear. Our goal is to use genome editing and stem cell approaches, including human cerebral organoids (hCOs), to interrogate the function of PCDH19 and how its mutations lead to seizure-like activity in developing human brain in vitro. To this end, we used CRIPSR/Cas9 to generate in-frame epitope tagged PCDH19 H9 female hESCs that allow us to use a standard antibody against the epitope to detect PCDH19 expression as most antibodies to date have shown non-specific labeling. We found that PCDH19, along with N-Cadherin, is localized to the apical lumens of neural rosettes in both 2D adherent cultures and 3D hCOs that are derived from the tagged hESCs, consistent with its high mRNA expression level at this stage. Using CRISPR/Cas9, we also established homozygous PCDH19 knockout H9 hESCs and generated a “virtual PRE patient” model in which isogenic H9 lines with a HA-FLAG-tagged PCDH19 allele are mixed with knockout cells, providing a reliable system to model the mosaic expression of PCDH19 that occurs in vivo due to random X-inactivation. Lastly, we cultured hCOs to enable a detailed examination of early human cortical development, preserving structural aspects and temporal-spatial relations critical for understanding epileptogenesis. We observed altered N-Cadherin+ apical lumens and abnormal cell segregation in hCOs derived from a “virtual PRE patient” model. Our results suggest that PCDH19 acts as a critical cell-cell adhesion molecule through the interactions with other cadherin proteins in the developing human brain. Further analyses including gene expression and electrophysiology in hCOs will help to identify PRE-related abnormalities in human brain development and seizure mechanisms that should lead to novel therapies.Funding Source: This work is founded by the PCDH19 AllianceF-3181ROLE OF TBX3 IN HUMAN STEM CELL-DERIVED HYPOTHALAMIC NEURONSXu, Yanjun - Pediatrics, Columbia University, New York, NY, USA De Rosa, Maria Caterina - Columbia Stem Cell Initiative, Naomi Berrie Diabetes Center, Department of Pediatrics, Columbia University, New York, NY, USA Quarta, Carmelo - Neurocentre Magendie, INSERM, Bordeaux, France Fisette, Alexandre - Helmholtz Diabetes Center, Helmholtz Center Munich, Munich, Germany Rausch, Richard - Columbia Stem Cell Initiative, Naomi Berrie Diabetes Center, Department of Pediatrics, Columbia University, New York, NY, USA Tschoep, Matthias - Helmholtz Diabetes Center, Helmholtz Center Munich, Germany Thaker, Vidhu - Naomi Berrie Diabetes Center, Division of Molecular Genetics, Department of Pediatrics, Columbia University, New York, NY, USA Garcia-Caceres, Cristina - Helmholtz Diabetes Center, Helmholtz Center Munich, Germany Doege, Claudia - Columbia Stem Cell Initiative, Naomi Berrie Diabetes Center, Department of Pathology and Cell Biology, Columbia University, New York, NY, USAThe leptin-melanocortin pathway of the hypothalamus is a master regulator of body weight. Thus, it is no surprise that mutations in genes expressed in hypothalamic pro-opiomelanocortin (POMC) neurons have been identified as monogenic causes of human obesity. So far, the functional relevance of novel variants identified in sequencing studies of obese humans has been tested mostly in non-human models and human non-neuronal cell lines. Here, we use human stem cell-derived hypothalamic neurons as a model to dissect the role of novel variants associated with human obesity. Mutations in the transcription factor T-box 3 (TBX3) have been associated with obesity in humans, but the molecular mechanism underlying this association remains unknown. We have recently shown that loss-of-function of TBX3 homolog in Drosophila causes excessive fat accumulation, while hypothalamic loss-of-function of Tbx3 in mice disrupts peptidergic identity and maturation of POMC-expressing neurons. Based on these findings from non-human models, we further investigated the role of TBX3 in the differentiation of human stem cells into hypothalamic neurons to establish a functional and causal link between TBX3 mutations and the development of obesity in humans. Loss-of-function models were created using CRISPR/Cas9 and their cellular and molecular phenotypes were obtained at several time points during the course of differentiation from stem cells into

505POSTER ABSTRACTShypothalamic neurons. These studies revealed the critical role of TBX3 in the maturation of hypothalamic progenitors into POMC-expressing neurons. These results also suggest that the role of TBX3 in the regulation of energy homeostasis is conserved across species.Funding Source: NIH (R01 DK52431, R01 DK110113 , P30 DK26687) Columbia Stem Cell Initiative Seed Fund Program CIHR (152588) NIH 5K23DK110539, BHCMG NHGRI 5U54HG006542 ICEMED, Helmholtz Initiative on Personalized Medicine iMed ERC (695054)F-3183DISSECTING THE MECHANISMS OF SCHIZOPHRENIA USING PATIENT-DERIVED STEM CELLSLi, Yichen - Stem Cell Biology and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USA Focking, Melanie - Psychiatry, Royal College of Surgeons in Ireland, Dublin, Ireland Ragoussis, Vassilis - Wellcome Trust Centre for Human Genetics, University of Oxford, UK Haenseler, Walther - Sir William Dunn School of Pathology, University of Oxford, UK Pagnamenta, Alistair - Wellcome Trust Centre for Human Genetics, University of Oxford, UK Devesa, Pablo - Ear, Nose and Throat, John Radcliffe Hospital, Oxford, UK Taylor, Jenny - Wellcome Trust Centre for Human Genetics, University of Oxford, UK Cowley, Sally - Sir William Dunn School of Pathology, University of Oxford, UK Cristino, Alex - Diamantina Institute, The University of Queensland, Brisbane, Australia Cotter, David - Psychiatry, Royal College of Surgeons in Ireland, Dublin, Ireland Mackay-Sim, Alan - Griffith Institute for Drug Discovery (GRIDD), Griffith University, Brisbane, Australia James, Anthony - Department of Psychiatry, University of Oxford, UK Szele, Francis - Department of Physiology, Anatomy and Genetics, University of Oxford, UK Ichida, Justin - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USASchizophrenia affects about 1% of the world’s general population. Although widely accepted as having a strong genetic basis and a root in neurodevelopment, the exact mechanisms of the disease remain unknown. We recruited a group of patients (n=17) with adolescent-onset schizophrenia who suffer from severe symptoms and were free from prolonged medications. Whole exome sequencing using DNA from patients and their parents revealed both inherited and de novo candidate variants. Olfactory stem cells, as a unique source of adult stem cells in the nasal cavity, when isolated from patients showed a significantly reduced cell-to-matrix adhesion. This was coupled with significantly disturbed global protein synthesis, stress responses and actin dynamics in patient olfactory stem cells based on proteomics analysis. These results highly agreed with previous studies addressing adult-onset schizophrenia, suggesting that the adolescent-onset group and adult-onset group share very similar mechanisms. Induced pluripotent stem cells were derived from three patients with family histories of schizophrenia, as well as age- and gender-matched controls. Organoids that resemble the fetal forebrains, and microglia that were incorporated into the organoids, were generated and the key components in neurodevelopment are currently being checked longitudinally. Induced neurons resembling glutamatergic layer 2/3 neurons in frontal lobes are being derived from a patient with variants in SYNGAP1 and SHANK2 –two important members of the post-synaptic density. Electrophysiological studies will be performed on induced neurons from this patient and isogenic controls. Together, this study used a group of patients enriched for clinically severity, and aims to understand the possible mechanisms of schizophrenia from the cellular and molecular perspectives.F-3185SHP2 MUTATIONS CAUSE NEURODEVELOPMENTAL ABNORMALITIES IN NOONAN SYNDROME DERIVED IPSCSJu, Younghee - Department of Biological Sciences, KAIST, Daejeon, Korea Park, Jun Sung - Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea Kim, Daejeong - Department of Bio and Brain Engineering, KAIST, Daejeon, Korea Kim, Bumsoo - Department of Biological Sciences, KAIST, Daejeon, Korea Lee, Jeong Ho - Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea Nam, Yoonkey - Department of Bio and Brain Engineering, KAIST, Daejeon, Korea Choi, Jin-Ho - Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea Lee, Beom Hee - Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea Yoo, Han-Wook - Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea Han, Yong-Mahn - Department of Biological Sciences, KAIST, Daejeon, KoreaNoonan syndrome (NS) is a genetic disorder caused by gain-of-function mutations in SHP2 (Src homology 2 domain-containing protein tyrosine phosphatase 2). Although approximately 30-50% of NS patients have the cognitive deficits including lower intelligence and neuropsychological complications, how SHP2 mutations are associated with neural development in NS patients remains elusive. In this study, induced pluripotent stem cells generated from NS-patient dermal fibroblasts (NS-iPSCs)

506POSTER ABSTRACTSdifferentiated into embryoid bodies (EBs), neural rosettes (NRs), neural precursor cells (NPCs), and neural cells in vitro. NS-EBs showed abnormal morphologies and defective development to NRs. Inhibition of both BMP and TGF- signaling pathways βrescued impaired early neuroectodermal development of NS-iPSCs. Recued NS-EBs normally differentiated into NRs and NPCs. NS-neural cells developed from NS-NPCs exhibited phenotypic abnormalities such as increment of glial cells and shortened neurites of neuronal cells as compared with wild-type (WT)-neural cells. They also decreased in extracellular spontaneous firing in NS-neural cells at 12 weeks during neural differentiation of NS-NPCs. SHP2 inhibition helps partially restore defective phenotypes and dysfunctional electrophysiology of NS-neural cells. Recently, cerebral organoids developed from NS-iPSCs recapitulated biased differentiation into glial cells. Our results provide a possibility that imbalanced neural development may contribute to cognitive deficits in NS patients.Funding Source: This research was supported by the NRF Stem Cell Program Grant (2011-0019509) funded by the Ministry of Science and ICT, Republic of Korea.F-3187DEVELOPMENT OF DRUG SCREENING PLATFORM THE ALPHA-1 ANTITRYPSIN DEFICIENCY IN PATIENT DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLKim, Young-Kyu - New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Taegu, Korea Park, sang-wook - New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundaiton (DGMIF), Teagu, Korea Min, Sang-Hyun - New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundaiton (DGMIF), Teagu, KoreaThe liver genetic diseases associated with alpha-1 antitrypsin deficiency (A1ATD) is a gain-of-toxic function mechanism. The misfolded insoluble globular proteins accumulate in the endoplasmic reticulum, leading to hepatic fibrosis and even hepatocellular carcinoma. However, efforts to identify pharmaceuticals to treat heritable liver diseases have been hamper by the lack of human model systems. Recently, the rapidly development of human induced pluripotent stem cells (iPSCs) technology has ushered in new era for the fields of disease modelling and drug discovery. The advantages of iPSCs used drug development are their expandability, easy accessibility, human origin, avoidance of ethical concerns associated with human embryonic stem cells (ESCs) and the potential to develop personalized medicine using patient-specific iPSCs. In this study, we reprogrammed the A1ATD patient fibroblasts into iPSCs using Yamanaka factor (Oct3/4, Sox2, Klf4, c-Myc). From these patient-specific hiPSCs, we have successfully differentiated into hepatocyte-like cells (HLCs) using various growth factors and chemicals. The percentage of hepatocytes differentiated from iPSCs using FACS analysis and immunocytochemistry (ICC) was confirmed more than 90% of hepatocyte specific marker albumin and HNF4 positive cells. The liver function of glycogen αstorage was also observed in A1ATD-iPSC-derived HCLs by the periodic acid Schiff (PAS) staining. Furthermore, these HCLs showed the aggregation of alpha-1 antitrypsin (A1AT) similar to the hepatocytes of the A1ATD patient. Based on these results, we are going to establish drug screening platform targeting A1ATD in liver genetic diseases.REPROGRAMMINGF-3191COMBINED RNA-BASED GENE EDITING AND REPROGRAMMING OF HUMAN IPSCS: A CLINICALLY RELEVANT APPROACHMcGrath, Patrick S - Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Butterfield, Kiel - Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Pavlova, Maryna - Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Roop, Dennis - Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Bilousova, Ganna - Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Kogut, Igor - Dermatology, University of Colorado Anschutz Medical Campus, CO, USAInduced pluripotent stem cells (iPSCs) hold great promise as a therapeutic for many currently incurable genetic diseases. The successful development of autologous therapeutics derived from iPSCs depends on several critical steps: (1) reprogramming patient cells into iPSCs, (2) correction or replacement of a disease-causing gene in iPSCs, and (3) differentiation of the corrected iPSCs into functional replacement tissues. Usually these steps are performed sequentially, leading to a lengthy, complicated, and expensive manufacturing process. To reduce the number of steps associated with the generation of genetically corrected iPSCs, we combined our previously reported high-efficiency RNA-based reprogramming protocol together with CRISPR/Cas9-mediated correction into a one-step procedure which can be performed within 5-6 weeks. As proof-of-principle for our one-step correction/reprogramming approach, we generated corrected iPSCs from multiple patients with recessive dystrophyic epidermolysis bullosa (RDEB) resulting from various COL7A1 mutations (c.7485+5G>A, IVS26-3T>G, c.6781C>T). We observed correction efficiencies as high as 5% in reprogrammed iPSCs which were recoverable in clonally expanded colonies. Keratinocytes differentiated from corrected iPSCs express Col7 protein detected by immuno-staining, indicating functional recovery of the corrected gene. Due to its high efficiency and reproducibility, our combined RNA-based gene editing and reprogramming approach

507POSTER ABSTRACTSprovides an opportunity to shorten the time between patient biopsy and the generation of gene edited iPSC lines, simplifying the manufacturing process of iPSC-based therapies for clinical applications.Funding Source: This study was supported by the US Department of Defense (W81XWH-18-1-0706), the Epidermolysis Bullosa (EB) Research Partnership, the EB Medical Research Foundation, and the Cure EB Charity.F-3193MOUSE ADULT NEURAL CREST-DERIVED STEM CELL REPROGRAMMING TO PLURIPOTENT STATE AND THE SEROTONIN SYSTEMVasyliev, Roman - Cell and Tissue Technologies, State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Kiev, Ukraine Rodnichenko, Anzhela - Cell and Tissue Technologies, State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Kiev, Ukraine Gubar, Olga - Functional Genomics, Institute of Molecular Biology and Genetics NASU, Kiev, Ukraine Zlatska, Alona - Cell and Tissue Technologies, State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Kiev, Ukraine Gordiienko, Inna - Molecular and Cellular Pathobiology, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NASU, Kiev, Ukraine Bader, Michael - Molecular Biology of Peptide Hormones, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Alenina, Natalia - Molecular Biology of Peptide Hormones, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Tomilin, Alexey - Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology RAS, St. Petersburg, Russia Zubov, Dmytro - Cell and Tissue Technologies, State Institute of Genetic and Regenerative Medicine, Kiev, Ukraine Novikova, Svitlana - Cell and Tissue Technologies, State Institute of Genetic and Regenerative Medicine, Kiev, UkraineThe discovery of the phenomenon of somatic cell reprogramming into a pluripotent state was a scientific breakthrough. In addition to new fundamental data, it enables obtaining any cell types for use in regenerative medicine. Despite intensive studies of the reprogramming and the pluripotent states, many issues remain unresolved: the choice of the starting cell type, the appropriate cocktail of transcription factors, the type of transfer vector, the use of small molecules etc. Adult neural crest-derived stem cells (NCSCs) are a promising cell type, both for studying the reprogramming and for potential clinical applications. The NCSCs express a number of genes associated with pluripotency: Sox2, Oct3/4, Nanog, Klf4, Lin28, cMyc (albeit at a lower level than ESCs and iPSCs). Thus, probably fewer factors may be used to reprogram adult NCSCs to iPSCs than the classic “magic of four” of Yamanaka. Small molecules are widely used in reprogramming to increase efficiency. There is evidence that serotonin, or molecules that affect the serotonin system (agonists and antagonists of 5-HT receptors, or inhibitors of the Slc6a4 transporter or the two rate-limiting enzymes TPH 1 and 2 may be used for this purpose. Accordingly, it was recently discovered that the 5-HT3 agonist, 2-methyl-serotonin, can replace the Oct3/4 in this process. In our study, we compared the reprogramming efficiency of mouse adult NCSCs using: the classic combination of single lentiviruses with the four Yamanaka’s factors (Oct3/4, Sox2, Klf4 and cMyc); a combination of three single lentiviruses without Sox2 due to its high expression at the protein level in NCSCs, as well as two polycistronic vectors expressing all four factors, OSKM and OKSM. The reprogramming efficiency of NCSCs from WT and 2KO mice, lacking both TPH isoforms (Tph1/Tph2), was also compared. When using the classic combination of Yamanaka’s factors as single vectors, the reprogramming efficiency was 0.071 ± 0.013 %. When using a cocktail of three single vectors (without Sox2): 0.011 ± 0.003 %. In case the OSKM vector, adult NCSCs were not reprogrammed unlike MEFs. When using the OKSM vector, the reprogramming efficiency was 0.015 ± 0.006 % for WT and 0.087 ± 0.016 % for 2KO. NCSCs. The NCSCs are a promising tool for the study of reprogramming and the association of this process with the serotonin system.Funding Source: This research was supported by grant from the Volkswagen Foundation (VolkswagenStiftung), Germany under initiative Trilateral Partnerships – Cooperation Projects between Scholars and Scientists from Ukraine, Russia and GermanyF-3195A NOBLE FINDING OF MIRNAS IN NEUROGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLSJang, Sujeong - Physiology/ Chonnam National University Medical School, Chonnam National University Medical School, Gwangju, Korea Jeong, Han-Seong - Physiology, Chonnam National University Medical School, Gwangju, Korea Park, Jong-Seong - Physiology, Chonnam National University Medical School, Gwangju, Korea Park, Sah-Hoon - Physiology, Chonnam National University Medical School, Gwangju, KoreaMicroRNAs (miRNAs) are small noncoding RNAs that emerge as regulators of stem cell lineage such as proliferation, development, differentiation, and apoptosis. We hypothesized that miRNA was involved in the neurogenic differentiation of mesenchymal stem cells. Here, the role of miRNAs in neurogenic differentiation of human mesenchymal stem cells (MSCs) is investigated. By performing a miRNA-mRNA paired microarray screening, we identified miR-4650-5p and miR-3146 among the most upregulated miRNAs during neurogenic differentiation. After selection of the miRNAs, we investigated the ability of neurogenic differentiation of miRNAs in human adipose tissue-derived MSCs (hADSCs). We found that miR-4650-5p or miR-3146 was increased the most of neuronal gene expressions by a quantitative PCR. Using bioinformatics and functional assay, we confirmed that miR-4650-5p and miR-3146 potentially targeted

508POSTER ABSTRACTSon JNK and GSK3 to regulate Wnt signaling pathway. Overall βcomparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role in neurogenic differentiation of hADSCs. These findings suggest that the miR-4650-5p and miR-3146 expression contributes the neurogenic differentiation of MSCs by increasing the neuronal genes and Wnt signaling pathway. The miRNAs regulation and downstream pathway network suggested the important role of miRNAs and Wnt signaling in the neurogenic differentiation of MSCs.Funding Source: 1.Basic Science Research Program through the NRF of Korea funded by the Ministry of Education (2016R1A6A3A11936076,2018R1D1A1B07050883)2.Chonnam National University Hospital Biomedical Research Institute (BCRI19044)F-3197ABCG2-MEDIATED TRANSPORTATION OF N-METHYL-PROTOPORPHYRIN IX TRIGGERED HEPATOCYTE REPROGRAMMING IN LIVER INJURYWu, Sung-Yu - Genomics Research Center, Academia Sinica, Nankang, Taiwan Chang, Hsiao-Min - Genomics Research Center, Academia Sinica, Taipei, Taiwan Kuo, Tzu-Chien - Genomics Research Center, Academia Sinica, Taipei, Taiwan Chen, Tai-Lin - Institution of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan Chien, Chiao-Yun - Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Lee, Hsuan-Shu - Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Mao, Wan-Yu - Genomics Research Center, Academia Sinica, Taipei, Taiwan Shen, Chia-Ning - Genomics Research Center, Academia Sinica, Taipei, TaiwanLiver transplantation can aid to improve survival of patients with liver failure. However, the shortage of donor liver and poor liver graft survival limit the potential use of liver transplantation. Since the liver has been renowned for its remarkable regenerative capacity, we tried to address the potential of reprogramming mature hepatocytes to bipotential progenitors for the purpose of repairing liver injury. Initial efforts had demonstrated that periportal hepatocytes could be reprogrammed into Sox9-expressing progenitor cells in mice treated 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In order to reveal insight into the cellular and molecular mechanism of the liver regeneration and to develop strategies to trigger hepatocyte reprogramming toward bipotential progenitor fate via activating the genetic routes that mimicking natural regeneration machineries, we generated mice that were knockout of ATP binding cassette transporter ABCG2 and discovered ABCG2 deficiency affected reprogramming-mediated liver repairing. The further analysis revealed, in DDC-treated mice, N-methyl-protoporphyrin IX(N-methyl-PPIX) produced from the breakdown of Cytochrome P450 and transported through ABCG2 could trigger induction of Sox-9 expression in hepatocyte reprogramming. We found depletion ABCG2 possibly restricted N-methyl-PPIX transportation thus inhibiting Sox9 induction and hepatocyte reprogramming. In contrast, ductular reaction and hepatocyte reprogramming toward Sox9-expressing progenitor cells could be stored either by p53 knockout or by rescuing ABCG2 expression via hydrodynamic injection. Immunohistochemical staining further identified induction of SOX9 was found to associated hepatocyte dedifferentiation together with upregulation of Yap1 and NFATc1. Importantly, we found that treatment of human and mouse primary hepatocytes with N-methyl-PPIX was sufficient to be converted into Sox9-expressing cells. And transplantation of reprogrammed Sox9-expressing cells into to the mice treated with carbon tetrachloride (CCL4) could replenish damaged hepatocytes. Hopefully, the findings from the work can lead to understanding the insight of liver regeneration and to development of cell therapeutic strategies for patients suffering from liver injury.F-3199SWITCHING FATES TO UNDERSTAND LINEAGE DECISIONS AND THE RISE OF PLURIPOTENCYGarg, Vidur - Weill Cornell Graduate School, Memorial Sloan Kettering Cancer Center, New York, NY, USA Apostolou, Effie - Medicine, Weill Cornell Medicine, New York, NY, USA Hadjantonakis, Kat - Developmental Biology, Sloan-Kettering Institute, New York, NY, USAThe blastocyst, a developmental stage shared among all mammals, is the source of a bona fide in vivo pluripotent population, the epiblast (EPI). Three lineages comprise the mature blastocyst – trophectoderm (TE), primitive (extra-embryonic) endoderm (PrE), and epiblast – which can also be captured ex vivo as trophoblast (TS), eXtraembryonic ENdoderm (XEN) and embryonic (ES) stem cells, respectively. A progenitor population, the inner cell mass (ICM), makes a bi-lineage choice to generate the EPI versus PrE. The EPI and ES cells give rise to all somatic tissues of adult mammals. Thus, determining how pluripotency is established in vivo holds far-reaching implications for stem cell biology, and regenerative medicine. While genetic and ex utero experiments have helped explain how these two lineages are segregated, we do not yet understand the mechanisms underlying individual cell-fate decisions, and factors maintaining a developmental barrier between the sister lineages. Much has been gleaned about the establishment of pluripotency by transcription factor-based reprogramming of somatic cells. Ectopic expression of Oct3/4, Klf4 and Sox2 induces pluripotency in somatic and TS cells, but XEN cells have never been tested for their potential to acquire a pluripotent identity despite being developmentally closer than TS cells. Conversely, ES cells acquire a XEN-like state with Gata4 misexpression. We are probing the mechanisms underlying the EPI-vs-PrE fate decision by inducing pluripotency within the PrE in vivo and in XEN cells in vitro. This in parallel with a Gata4-induced ES-to-XEN conversion will serve as an ex vivo model of ICM lineage specification.

509POSTER ABSTRACTSF-3201GENERATION, GENETIC MANIPULATION, AND AIRWAY DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PRIMARY HUMAN AIRWAY EPITHELIAL CELLSLi, Yingchun - Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA Goldfarbmuren, Katherine - Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA Morris, Carolyn - Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA Rios, Cydney - Center for Genes, Environment and Health, National Jewish Health, Denver, CO, USA Montgomery, Michael - Center for Genes, Environment and Health, National Jewish Health, Denver, CO, USA Ren, Luke - R&D, REPROCELL USA, Beltsville, MD, USA Eminli-Meissner, Sarah – Research &Development, REPROCELL USA, Beltsville, MD, USA Seibold, Max - Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USAAir-liquid interface (ALI) differentiation of human airway epithelial cells (AEC) is a well-established model to study the airway epithelium in vitro. The replicative airway basal epithelial cell used for ALI culture lacks the ability to be passaged indefinitely and is not easily genetically manipulated. Therefore, alternative approaches for epithelial basal cell regeneration and gene editing are needed to further model airway development and disease, as well as for regenerative therapeutics. Recently, various human cell types have been reprogrammed into induced pluripotent stem cells (iPSCs) using integration-free methods like mRNAs. Here we demonstrate for the first time the successful generation of iPSCs using readily accessible epithelial cells from the upper airways as a source, with a clinically-relevant non-modified RNA based reprogramming technology that combines a cocktail of synthetic reprogramming and immune evasion mRNAs with reprogramming-enhancing mature, double-stranded microRNAs. We collected human primary AECs from nasal and bronchial brushings, and generated iPSCs with reprogramming efficiencies of up to 0.17%. These iPSCs expressed pluripotency markers (OCT4, NANOG, SSEA4, and ALP) and effectively formed tissues representing all three germ layers under both targeted and spontaneous differentiation conditions. We successfully differentiated these iPSCs to Keratin 5-expressing basal-like airway epithelial cells through stage specific culture conditions. Further, we successfully expanded and maintained these iPSC-derived basal-like cells for multiple passages (>3) without losing the basal cell markers Keratin 5 and TP63. Moreover, using a CRISPR/Cas9 gene editing approach, we knocked out the epithelial cell marker Epithelial Cell Adhesion Molecule (EPCAM) in these iPSCs with >66% efficiency as measured by flow cytometry. We were also able to utilize CRISPR/Cas9 homology-directed repair (HDR) to incorporate full-length eGFP-tagged B-actin protein with 1.17% HDR efficiency. In conclusion, we found airway brushings can serve as a minimally invasive source of cells capable of reprogramming to iPSCs, gene manipulation, and redifferentiation to airway cells. This work highlights a potential strategy for gene correction and regeneration of the human airway.F-3203MITOCHONDRIAL ACTIVE NAIVE-LIKE PLUIPOTENT STEM CELL INDUCED BY PRIMITIVE GROWTH FACTOR, NME7ABHan, Min-Joon - Hematology/St. Jude, St. Jude, Memphis, TN, USANaïve pluripotent stem cells (PSCs) display a distinctive phenotype when compared to their “primed” counterparts. This phenotype includes, but is not limited to, increased potency to differentiate and more robust mitochondrial respiration. The cultivation and maintenance of naïve PSCs has been notoriously challenging, requiring the use of complex cytokine cocktails. NME7AB is a newly discovered embryonic stem cell growth factor that is expressed exclusively in the first few days of human blastocyst development. It has been previously reported that growing primed state iPSCs in bFGF depleted medium with NME7AB as the only added growth factor facilitates the regression of these cells to the naïve state. Here, we confirmed this regression by demonstrating the reactivation of mitochondrial function in the naïve state PSCs and their increased ATP production, as compared to that in primed state iPSCs.Funding Source: This work was supported by St. Jude institutional funds (to M-J.H.).F-3205INTERCELLULAR RNA TRANSFER-DRIVEN EPIGENETIC REPROGRAMMING OF HUMAN PLURIPOTENT STEM CELLS INTO NAÏVE-LIKE STATEYoneyama, Yosuke - Institute of Research, Tokyo Medical and Dental University, Bunkyo-ku, Japan Zhang, Ran-Ran - Division of Gastroenterology, Hepatology and Nutrition, Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Kimura, Masaki - Division of Gastroenterology, Hepatology and Nutrition, Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Takebe, Takanori - Division of Gastroenterology, Hepatology and Nutrition, Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USAUnderstanding the molecular basis of intercellular coordination will facilitate our ability to control collective cell behaviors. Herein we found the unexpectedly high incidence of bidirectional mRNA exchanges between human and mouse pluripotent stem cells in the xenogeneic coculture. Transferred mRNA accounts for

510POSTER ABSTRACTS0.5-3.0% of the transcripts in the cells, that can be inhibited by the toll-like receptor 4 (TLR4) pathway. The mRNA transfer was primarily driven by direct cell-cell contact rather than by secretion mechanisms. Remarkably, human primed induced pluripotent stem cells (iPSCs) were converted from flat into domed colony morphology when cocultured with mouse naïve embryonic stem cells (ESCs). These human iPSCs expressed the naïve-specific markers including KLF4, TFCP2L1, and CD130, indicating the conversion from primed into naïve-like pluripotency in human iPSCs. The naïve-like conversion of human iPSCs was inhibited by the TLR4 ligand treatment. Furthermore, transcriptome and epigenome analyses revealed that during the conversion some mRNAs coding transcription factors were transferred from mouse ESCs into human iPSCs accompanied by naïve-specific enhancer openings in hiPSCs, strongly suggesting the potential of the intercellular mRNA transfer in cell fate conversion. Taking these results together, we propose that this intercellular mRNA transfer-based reprogramming phenomenon is a potential strategy to fine-tune a variable intercellular epigenomic identity, that can be leveraged for a novel reprogramming method for human cells without the use of conventional reprogramming factors.F-3207INDUCING HEPATOCYTE-LIKE CELLS FROM FIBROBLASTS BY PURE CHEMICAL APPROACHBai, Yunfei - Institute of Molecular Medicine, Peking University, Beijing, China Yang, Zhenghao - Institute of Molecular Medicine, Peking University, Beijing, China Zhao, Yang - State Key Laboratory of Natural and Biomimetic Drugs, the MOE Key Laboratory of Cell Proliferation and Differentiation, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, ChinaRecently, pure chemical methods are reported to reprogram fibroblasts into a series of cell types, such as pluripotent stem cells, functional neurons, neural progenitor cells, astrocytes, skeletal muscle cells and cardiomyocytes, which may provide a promising approach to obtain stem/progenitor cells or functional cell types for tissue engineering and regenerative medicine, and bypass the concerns of potential genetic alterations by transgenic approach. Here in our study, we find that the expression of hepatocyte-associated transcription factors (TFs) is stimulated in the first 4 days of 40-day chemical reprogramming process from mouse embryonic fibroblasts (MEFs) into chemically-induced pluripotent stem cells (CiPSCs), suggesting that these cells may be primed to hepatic lineage. Thus, we optimize the combination of chemical cocktails after day 4 of chemical reprogramming while using hepatocyte culture medium (HCM), and we find pure chemically-induced hepatocyte-like cells (CiHeps) emerged 12 days later. The CiHeps are epithelioid and colony-forming, co-expressing hepatic markers Albumin and Hnf4a, detected by immunostaining. The upregulated mRNA levels of other hepatic genes, such as Hnf1a, Foxa2, Ttr, Tdo2, Cps1, and Cyp2d10 are also detected by real-time PCR. Moreover, we find few CiHeps are obtained without the chemical treatment of the stage I medium of CiPSC induction for the first 4 days, supporting our hypothesis that an early cell plasticization process mediates chemical reprogramming into multiple cell lineages. Furthermore, we find the cells induced during hepatic reprogramming process do not express Sall4, a master gene of the extraembryonic endoderm (XEN), suggesting that the generation of those hepatocyte-like cells are dispensable of the generation of XEN-like cells, an intermediate state during the process of CiPSC induction. Overall, our findings not only pave a new way for obtaining hepatocytes with pure chemicals, but also suggest a general approach in developing chemical reprogramming methods through a multiple-lineage priming state.Funding Source: This study was supported by the National Natural Science Foundation of China (Grant No. 31771645) and Boehringer-ingelheim pharmaceutical international GmBH.TECHNOLOGIES FOR STEM CELL RESEARCHF-3209ESTABLISHING A CLINICALLY COMPLIANT GMP HUMAN EMBRYONIC CELL LINE DERIVED UNDER XENO-FREE AND DEFINED CONDITIONS IN A GMP FACILITYHedenskog, Mona - CLINTEC, Karolinska Institutet, Huddinge, Sweden Efstathopoulos, Paschalis - CLINTEC, Karolinska Institutet, Stockholm, Sweden Lanner, Fredrik - CLINTEC, Karolinska Institutet, Stockholm, Sweden Main, Heather - CLINTEC, Karolinska Institutet, Stockholm, Sweden Padrell Sanchez, Sara - CLINTEC, Karolinska Institutet, Stockholm, Sweden Peutrus-Reurer, Sandra - CLINTEC, Karolinska Institutet, Stockholm, Sweden Plaza Reyes, Alvaro - CLINTEC, Karolinska Institutet, Stockholm, SwedenHuman embryonic stem cells (hESCs), are a promising cell source for therapies in regenerative medicine since they can differentiate in vitro to many different cell types. Consequently cell transplantation has a great potential as treatment for many diseases. Crucial for clinical translation is high-quality hESC derived under GMP conditions. We have established a clinically compliant GMP hESC line derived under these conditions and we now present the protocol. We have previously published xeno-free and defined protocols for derivation and culture but now all the components in the original protocol are reevaluated and this resulted in a new media formulation and a GMP compatible laminin 521 culture matrix. All reagents have also been functionally tested and the protocol showed good derivation efficiency. The derivations are done using frozen surplus human

511POSTER ABSTRACTSembryos, donated with informed consent and with ethical approval from the Regional Ethics Board in Stockholm. We are now expanding the first lines at Vecura, a GMP facility within Karolinska University hospital. The first line has been analyzed for pluripotency by immunostaining and FACS, has been karyotyped, analyzed by iCS digital pluritest, and tested for the absence of human viruses. Our facility will produce cell lines and provide clinical grade GMP hESCs for experimental research and for clinical applications in regenerative medicine.F-3211AUTOMATED PASSAGING, FEEDING AND PLATING ON GLASS OF GENE-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS FOR HIGH-THROUGHPUT 3D LIVE CELL MICROSCOPY PIPELINEGaudreault, Nathalie - Microscopy, Allen Institute for Cell Science, Seattle, WA, USA Allen Institute for Cell Science, Team - Microscopy, Allen Institute for Cell Science, Seattle, WA, USA Gregor, Ben - Microscopy, Allen Institute for Cell Science, Seattle, WA, USAThe Allen Institute for Cell Science combines genomics, gene editing, and 3D live cell imaging of cell organization to understand what defines different stem cell states and to unlock the underlying mechanisms for their production. We use human induced pluripotent stem cell (hiPSC) lines expressing green fluorescent protein tagged to proteins identifying specific cellular organelles and structures. To produce large numbers of standardized images, we have developed an automated hiPSC culture procedure. We use the Hamilton Star robotic platform to generate imaging plates with uniform and reproducible cellular confluency and morphology. Here we provide specific values on parameters such as the movements of the plates across the deck, the angle and speed of the aspiration and dispensing of media, the seeding strategies and the timing of every step that we optimized. This approach was developed and applied to coating, seeding, passaging and feeding procedures for cell expansion in 6-well plastic plates and 96-well glass bottom imaging plates. The overall optimization procedure aimed to preserve the undifferentiated state, maintain pluripotency, and prevent karyotyping abnormality of our gene-edited hiPSC lines for up to 10 passages. We present a side by side comparison of quality control results obtained from manual and automated operations. We also developed an automated image-based colony segmentation pipeline to measure and track colony growth characteristics over time. The colony feature measurements (number, size, textures, etc.) are used to rank 96-well plates and ensure consistent and adequate quality control. We have also implemented cell passaging based on image-based confluency calculations to eliminate the need for manual cell counting steps. In addition to providing higher uniformity, reproducibility, and overall cell quality for imaging samples, the standardized automation protocol ensures consistency by removing operator-to-operator variability and potential error or bias introduced by manual repetitive tasks and fatigue of the operators.F-3213HIGH-PRECISION AND SCALABLE FABRICATION OF BIOMIMETIC CULTURE ENVIRONMENTS FOR ENHANCING STEM CELL MATURATIONGeisse, Nicholas A - Research and Development, NanoSurface Biomedical, Seattle, WA, USA Fisher, Elliot - Research and Development, NanoSurface Biomedical, Seattle, WA, USA Gray, Kevin - Research and Development, NanoSurface Biomedical, Seattle, WA, USA Ghazizadeh, Hamed - Research and Development, NanoSurface Biomedical, Seattle, WA, USA Smith, Alec S.T. - Bioengineering, University of Washington, Seattle, WA, USA Kim, Deok-Ho - Bioengineering, University of Washington, Seattle, WA, USADifferentiated stem cells when kept in culture can lose cell-type specific phenotypes or fail to express many mature phenotypes found in vivo. Traditional cell culture environments—typically composed of glass or plastic—are partially responsible for this effect. Considerable effort has been directed at generating biomimetic cell culture environments to maintain or promote mature in vivo phenotypes. However, fabrication of biomimetic substrates capable of mimicking different aspects of the extracellular matrix (ECM) in vivo typically involves costly or hard-to-reproduce techniques that are often incompatible with many standard assays. Here, we will present a novel method of generating surfaces that mimic mechanical, nanoscale shape and structural cues of the collagen ECM. The fabrication scheme described is highly reproducible, scalable, and amenable to integration with most industry-standard endpoint assays, including high-NA optical microscopy. We present techniques to fabricate biomimetic culture surfaces out of elastomers that can be stretched in order to reproduce mechanical cues that are critical in the development and function of certain tissues. Various cell types were tested and were amenable to this approach. For example, hiPSC-derived cardiomyocytes (CMs) showed more in vivo-like myofibril alignment, sarcomere spacing and width, and expression of CM-specific proteins that are present in mature myocytes. Furthermore, higher-ordered 2D anisotropic myocyte tissues also showed adult-like structure and electrophysiological responses to drugs in vitro when compared to traditional unordered 2D isotropic constructs. Examples of phenotype enhancement of other mammalian cell types will be presented, further demonstrating the utility of the approach for fabrication of highly scalable and precise biomimetic surfaces.

512POSTER ABSTRACTSF-3215ROLE OF IMPELLER DESIGN FOR THE CULTIVATION OF STEM CELLS IN STIRRED-TANK BIOREACTORSNold, Philipp - Eppendorf AG Bioprocess Center, DASGIP GmbH, Jülich, GermanyThe routine use of stem cells and stem cell-derived cells in drug research and regenerative medicine requires the constant supply of high cell numbers in consistent quality. Stirred-tank bioreactors have emerged as promising cultivation systems, which facilitate close control of critical process parameters and have proven their value for efficient process scaling. Cell cultivation on microcarriers or as cell aggregates are two widely used methods for expanding anchorage-dependent cells in stirred-tank bioreactors. Suitable agitation conditions have to be established to keep cell aggregates or microcarriers in suspension while avoiding damaging shear forces.We have developed a novel 8-blade impeller to improve the cultivation of stem cells as aggregates and on microcarriers in the DASbox Mini Bioreactor System. Use of this impeller supported the formation and growth of stem cell aggregates in the bioreactor. Cell aggregate formation was more efficient than when using a conventional pitched-blade or Rushton-type impeller. Furthermore, microcarriers could be efficiently brought to and kept in suspension. Our results suggest that the impeller shape is an important parameter to consider when optimizing the agitation conditions in stirred-tank bioreactors.F-3217NOVEL SOLUTIONS FOR CARDIAC DRUG SAFETY AND TOXICOLOGY RESEARCHKnox, Ronald - Nanion Technologies GmbH, Germany Costantin, James - Nanion Technologies Inc. Dragicevic, Elena - Nanion Technologies GmbH, Nanion Technologies GmbH, Munich, Germany Juhasz, Krisztina - Nanion Technologies GmbH, Nanion Technologies GmbH, Munich, Germany Reinhardt, Oliver - Translational Molecular Imaging Group, University OF Toyama, Göttingen, Germany Okeyo, George - Nanion Technologies Inc., Nanion Technologies Inc., Livingston, NJ, USA Stölzle-Feix, Sonja - Nanion Technologies GmbH, Nanion Technologies GmbH, Munich, Germany Alves, Frauke - Translational Molecular Imaging Group, MPI of Experimental Medicine, Göttingen, Germany Haedo, Rodolfo - Nanion Technologies Inc., Nanion Technologies Inc., Livingston, NJ, USA Fertig, Niels - Nanion Technologies GmbH, Nanion Technologies GmbH, Munich, GermanyHuman induced pluripotent stem cells (hiPSCs) have been proven instrumental for cardiac safety and toxicology testing due to their validated predictivity (e.g. CiPA study). We combined impedance and extracellular field potential (EFP) measurements with solid-supported membrane (SSM) based electrophysiology to study drug safety and toxicology in hiPSC-CMs. We have performed dual impedance and EFP-recordings to monitor cell proliferation and contractility, over prolonged time periods, contrary to standard mostly endpoint cytotoxicity assays. As the emerging field of cardio-oncology aims to find a balance between oncologic efficacy and reducing adverse cardiovascular effects, we tested the same treatment used in breast cancer chemotherapy, on hiPSC derived cardiomyocytes (iPSC-CMs). One of the standard clinical regimens for breast cancer is a combination of cyclophosphamide, adriamycin (doxorubicin) and 5-fluorouracil (CAF) administered for 4 months. We investigated putative cardiovascular side effects of CAF mix and paclitaxel and their long-and short-term implications on iPSC-CMs viability. We show a dose dependent negative effect of paclitaxel on iPSC-CMs viability (base impedance reduction). This was also observed for doxorubicin alone, but not the rest of the CAF compound mix. Paclitaxel and CAF also induced negative changes in cell contraction properties. We also investigated the role of the Na+/Ca2+- exchanger (NCX) in cellular Ca2+ homeostasis of iPSC-CMs. NCX is important for Ca2+ homeostasis, and it can contribute to cell damage by Ca2+ overloading or induce an anti-arrhythmic effect, when inhibited or when direction reversed. By developing a suitable sensor-based method (SSM based electrophysiology) we recorded specific NCX current responses of high amplitude. These currents showed similar Ca2+ affinity compared to NCX1 expressed in HEK cells and were sensitive to nickel, KB-R7943 and SEA0400. Using the impedance-based system, we also observed a significant increase in beating rate, as a long-term effect, when inhibiting NCX with SEA0400, in contrast to previous studies using non-human systems and focusing on short-term effects. In summary, we demonstrate the importance of new combination of tools, which help generate new insights into the pharmacology and toxicology of iPSC-CMs.F-3219LAMININ-FUNCTIONALIZED 3D SILK SCAFFOLD MAINTAINS EXPANSION, STEMNESS AND DIFFERENTIATION POTENTIAL OF HUMAN PLURIPOTENT STEM CELLSEleuteri, Boris - Research and Development, BioLamina, Stockholm, Sweden Xiao, Zhijie - Research and Development, BioLamina AB, Stockholm, Sweden Kallur, Therese - Business Development, BioLamina AB, Stockholm, SwedenTissues are 3D formations of cells integrated in an extracellular matrix (ECM) with specific sites for cell anchorage offering positional and instructive information, regulating cell behavior. A vast number of publications in the last decades have proven the reciprocal interaction between the ECM, cytoskeleton and nuclear matrix, showing that these structures exert a physical and chemical influence over gene expression. Thus, to culture authentic cells from which real biological questions

513POSTER ABSTRACTScan be answered, environmental context is pivotal. Laminins comprise a family of 16 unique heterotrimeric glycoproteins naturally present in the body. Laminins are tissue specific, supply a natural environment for all cell types, and influence cell adhesion, differentiation, migration, phenotypic stability, and cell functionality. As such, recombinant laminins are biologically relevant ECM protein substrates that can be used to mimic the in vivo cell niche in vitro. The laminin 521 isoform is a critical factor supporting pluripotent stem cells in culture, and given the right context, this isoform also provides support for other differentiated cell types. Silk 521 is a biomaterial made of recombinant silk, which is functionalized with human recombinant laminin 521 protein (Biolamininâ„¢ 521). This material supports integration and proliferation of human pluripotent stem cells (PSCs) in vitro and serves as a viable base for the development of subsequent lineage-specific differentiation in a 3D format. Human PSCs seeded in the Silk 521 scaffold integrate, migrate, and form small colonies at day 1 post seeding and retain pluripotency thereafter. Human PSCs in the Silk 521 scaffold could be cultured using different types of medium, expressed pluripotent markers, spontaneously differentiated to all three tissues germ layers, and directly to neural fates. On the contrary, cells seeded in silk only (control), could hardly integrate into the foam and the cells did not amplify at all, regardless of media or cell line used, and regardless of the presence of ROCKi. Together these data highlight the biological effects of the laminin substrate within the Silk 521 biomaterial. In sum, Silk 521 is a biorelevant 3D system that is biodegradable and non-immunogenic, and thus ideal for cell culturing and many other biomedical applications.F-3221ASSOCIATION OF HIF-1 AND ALIX IN ENHANCED ΑBIOGENESIS AND SECRETION OF EXOSOMES FROM HUMAN MESENCHYMAL STEM CELLS UNDER HYPOXIAGupta, Suchi - Stem Cell Facility, All India Institute of Medical Sciences Delhi (AIIMS), New Delhi, India Mann, Zoya - Stem Cell Facility, AIIMS, New Delhi, India Mohanty, Sujata - Stem Cell Facility, AIIMS, New Delhi, India Nayak, Baibaswata - Departement of Gastroenterology, AIIMS, New Delhi, India Sharma, Harshita - Stem Cell Facility, AIIMS, New Delhi, IndiaWith the advancements in research, it is now well-known that Mesenchymal Stem Cells (MSCs) exhibit their therapeutic effect via a paracrine mechanism where MSCs produce membrane-bound nanovesicles called exosomes. These exosomes contain biologically active proteins and miRNAs. Due to their role in cell free therapy, they are considered as ideal candidates for regenerative medicine. However, the yield of secreted exosomes is a limiting factor for obtaining sufficient amounts of exosomes for cell-free therapies. To this end, we cultured MSCs isolated from adipose tissue (ADSC) and bone marrow (BMSC) in different culture condition (hypoxia -2.5% Oxygen and normoxia-21% oxygen) for different time points, i.e., 24h and 48h. The cells were assessed for HIF-1 expression αthrough qPCR, and conditioned media was used for exosome secretion using Nanoparticle Tracking Analysis (NTA).We further studied differential expression of genes involved in exosomes biogenesis and secretion with primary focus on ESCRT dependent pathway genes (ALIX, Tsg101) and secretion genes (Rab27a and Rab27b). It was found that tissue specific MSCs showed differential response to hypoxia treatment with 2 fold up regulation for HIF-1 expression 48h in ADSC while it was at α24h in BMSC. Also, NTA showed that secreted exosomes size range from 30nm to 150nm. Exosome secretion for ADSC was 1.4 X 106 at 48h in hypoxia while it was 1.6 X 106 for BMSC at 24h. This suggested that there is a direct co-relation between HIF-1 and exosome secretion. Also, with respect to exosome αbiogenesis and secretion gene expression across these tissue specific MSCs in hypoxia, there was specifically up regulation of ALIX expression. BMSC exhibits 2 fold increase in ALIX expression at 24h whereas it was a 6 fold increase in ADSC at 48h. Hence, we found that the expression of ALIX was directly related to HIF-1 expression. This study suggests that in MSCs αthere is ALIX dependent release of exosomes. Further studies need to be done for understanding the role of ALIX in MSCs derived exosome secretion.ALIX role in other cell type like Hela, DC cells is well known, however, it is still in its preliminary stage for MSCs.Funding Source: The work done in this study was generously supported by Department of Biotechnology, IndiaF-3223REFERENCE CELL LINES FOR THE ANALYSIS OF ONCOGENIC TRANSFORMATION OF HUMAN PLURIPOTENT STEM CELLSMcGarr, Tracy - Mammalian Genetics, The Jackson Laboratory, Bar Harbor, ME, USA Carney, Lisa - Mammalian Genetics, The Jackson Laboratory, Bar Harbor, ME, USA Shultz, Leeonard - Mammalian Genetics, The Jackson Laboratory, Bar Harbor, ME, USA Pera, Martin - Mammalian Genetics, The Jackson Laboratory, Bar Harbor, ME, USAThe potential for tumor formation is a major safety concern for the use of cellular therapeutics derived from human pluripotent stem cells (hPSC). Low level product contamination with genetically abnormal cells with oncogenic potential poses particular challenges to safety assessment. At present there are no in vitro surrogate assays for oncogenic transformation of hPSC. While a few reports suggest that teratoma assays in mice can identify abnormal hPSC with malignant potential, there is limited evidence to validate this approach. Embryonal carcinoma (EC) cell lines provide a positive control for tumor formation, but often differ markedly from hPSC in their limited differentiation capacity and growth requirements in vitro. We describe two clonal cell lines derived from a primary teratocarcinoma of the testis which encompass a broad range of biological behavior. Both cell lines express pluripotency-associated cell surface markers and transcription factors, and both show karyotypic abnormalities

514POSTER ABSTRACTScommon to germ cell tumors and genetically abnormal hPSC. Cell line GCT27C4 shows high cloning efficiency in standard culture medium without feeder cell support, but its sibling GCT27D1, like normal hPSC, clones poorly even in the presence of a mouse embryo fibroblast feeder cell layer (cloning efficiency <0.1%). GCT27C4 shows little capacity for differentiation in vitro, but GCT27D1 can differentiate spontaneously into a range of cell types. The two cell lines form xenograft tumors in immunodeprived mice with similar efficiency. Tumors derived from GCT27C4 consist of undifferentiated EC cells only. Tumors derived from GCT27X1 contain derivatives of all three embryonic germ layers, but unlike tumors derived from normal hPSC, they also contain undifferentiated EC cells, sometimes accompanied by large blocks of primitive neuroectodermal tissue and yolk sac elements, features seen in malignant pediatric germ cell tumors. RNA-seq analysis reveals significant differences in gene expression between each of the two EC cell clones and normal hPSC cell lines. These two EC cell lines should prove useful in the refinement of teratoma assays and for the development of in vitro assays for oncogenic transformation of hPSC. We will distribute these cell lines through a central cell bank.F-3225COMPUTATIONAL MODELING OF NEURAL STEM CELL MIGRATION ROUTES IN THE BRAINAdhikarla, Vikram - Department of Computational and Quantitative Medicine, Division of Mathematical Oncology, City of Hope National Medical Center, Duarte, CA, USA Tsaturyan, Lusine - Department of Developmental and Stem Cell Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Gutova, Margarita - Department of Developmental and Stem Cell Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Rockne, Russell - Department of Computational and Quantitative Medicine, Division of Mathematical Oncology, Beckman Research Institute, City of Hope, Duarte, CA, USANeural stem cells (NSCs) are inherently patho-tropic and have been shown to be effective for tissue regeneration and delivery of therapeutics in cases of brain injuries and tumors. The delivery of NSCs in the brain can be either via intracranial, intraventricular or intranasal routes. The therapeutic efficiency of NSCs depends both on the distance between the injection site and target, as well as the route characteristics. Prediction of NSC delivery efficiency and routes of migration based on the injection site is instrumental for optimizing NSC dosage. We have recently shown the migration of NSCs along white matter in naïve mice brain. Here, we present a computational model for simulation of NSC migration within mice and human brain. Fractional anisotropy atlas of the mouse brain was obtained using Diffusion Tensor Magnetic Resonance Imaging (DT-MRI). The Fiber Assignment with Continuous Tracking (FACT) algorithm which is typically used in tractography, is used to predict migration routes from potential injection sites. The FACT algorithm generates the migration direction based on the direction of dominant eigenvector (from DT-MRI atlas) at the tissue voxel. An injection site was simulated as a specific region from which a number of paths would be stochastically simulated to originate from. Migration paths in mice brain are simulated with potential injection sites in the corpus callosum (CC injection) and olfactory bulb (OB injection). Results predicted NSC migration routes to be along the corpus callosum and towards the frontal cortex for the CC injection and towards the optic chiasm for the OB injection. The model is applied to human brain DT-MRI atlas to simulate NSC migration paths with putamen as the potential injection site. The model predicts the migration of NSCs to the frontal cortex and along the subventricular zones. The model is well poised to incorporate directed cues for simulation of NSC migration in the presence of tumors or injuries. A successful modeling framework verified against experimental data will be useful for optimizing stem cell therapeutic doses and modes of delivery.Funding Source: The project is supported by NIH NSC grant R03CA216142.F-3227A ROBUST METHOD FOR TAGGING ENDOGENOUS GENES THROUGH PROMOTER TRAPPING AND SHORT HOMOLOGY ARMSLiang, Xiquan - Cell Biology, Thermo Fisher Scientific, San Diego, CA, USA Potter, Jason - Cell Biology, Life Technologies, Carlsbad, CA, USAPrecise genome editing via homology-directed repair (HDR) pathway holds great promise for gene and stem cell therapy. However, the efficiency of integrating large DNA molecules into mammalian genome via HDR is inherently low. Recently, we showed that the use of short homology arms (~35nt) was sufficient to introduce small changes in mammalian genome. Now, we take a step further and develop a novel method for tagging endogenous genes through promoter trapping and short homology arms, which dramatically increases the efficiency and specificity of integration. The efficiency of tagging endogenous genes with a 1.4 kb promoterless GFP reporter ranges from 50% to 100% upon antibiotic selection with higher level of specificity occuring at the C-terminus than at the N-terminus. The method has been validated using multiple targets in many different cell lines, including human induced pluripotent stem cells and hematopoietic stem cells. The basal expression levels of various fluorescent fusion proteins and their subcellular locations could be visualized by fluorescence microscopy or detected by western blotting. This method has broad applications in general genome engineering, DNA cloning, protein production and immune cell therapy.Funding Source: Thermo Fisher scientific

515POSTER ABSTRACTSF-3229AN IMAGE PROCESSING METHOD FOR CREATING HIGH QUALITY IPS CELL STOCKSNatsume, Yusuke - Ajinomoto Co., Inc., Kawasaki, Japan Yusuke, Yasuko - Ajinomoto Co., Inc. Yokoyama, Mizuho - Research Institute For Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc., Kawasaki-shi, Japan Aritomi, Shizuka - Research Institute For Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc., Kawasaki-shi, Japan Okamoto, Satoru - Research Institute For Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc., Kawasaki-shi, JapanCells used in research are generally stored as a certain amount of stockpile for maintaining cell performance. Regarding induced pluripotent stem (iPS) cells, we have previously reported that it is important to set an appropriate timing of cryopreservation after cell seeding for increasing the cell stock quality. In fact, iPS cells cryopreserved in the late stage of cell growth had less ability to attach to culture dishes despite high cell yield. We also assumed that morphological features such as colony size, colony-colony contact, and colony circularity before freezing also affect the iPS cell stock quality. This study aims to reveal which morphological parameters of iPS colonies would be effective to estimate cell stock quality and quantity prior to freeze-thawing. In the first step, we cultured iPS cells (line 201B7 and 1210B2) under multiple culture conditions (multiple seeding density, culture period, and the passage number), and obtained colony images by a common phase contrast microscope. We next freeze-thawed and cultured the iPS cells to confirm the stock conditions. In the second step, we applied an image analysis technique to evaluate multiple colony morphological parameters by a computational software, and then statistically analyzed the data. The result indicates that a proper combination of the threshold values for each parameter is effective for preventing cell death and greater stability in long-term cell culture after thawing. In summary, this method allows us to evaluate cells non-invasively and quantitatively for cryopreservation, and establish valuable iPS cell resources.F-3231A SAFEGUARD SYSTEM FOR HUMAN PLURIPOTENT STEM CELL-BASED THERAPIESWu, Youjun - Department of Translational Research and Cellular Therapeutics, City of Hope, Duarte, CA, USA Chang, Tammy - Department of Translational Research and Cellular Therapeutics, City of Hope, Duarte, CA, USA Huang, He - Bone Marrow Transplantation Center, Zhejiang University, Hangzhou, China Long, Yan - Bone Marrow Transplantation Center, Zhejiang University, Hangzhou, China Yee, Jiing-Kuan - Department of Translational Research and Cellular Therapeutics, City of Hope, Duarte, CA, USAHuman pluripotent stem cells (hPSCs) hold enormous promise for cell-based therapy. However, the risk of generating malignancy from contaminating undifferentiated hPSCs in the final cell product for therapeutics is still one major concern for clinical applications. A suicide protein, iC9, containing a fusion between human Caspase 9 and FK506-binding protein, was developed previously. Dimerization of iC9 with AP1903, a small molecule which is well tolerated in culture cells and in clinical studies, activates one of the last steps in the apoptotic cascade to induce rapid cell death. To selectively eradicate undifferentiated hPSCs, we inserted a suicide gene in-frame into the stem cell-specific SOX2 locus in human embryonic stem cell line H1 (H1-iC9) by Crispr-Cas9. The inserted iC9 gene was under the direct transcription control of SOX2 while the endogenous SOX2 expression was not disrupted. This strategy restricted iC9 expression to undifferentiated cells. Administration of AP1903 dimerized iC9 and induced apoptosis of undifferentiated H1-iC9 cells, while the differentiation products derived from H1-iC9 cells, including hematopoietic cells, beta-like cells and neurons, were not affected. Our results showed the feasibility of using suicide gene to remove undifferentiated stem cells without affecting the viability of differentiated cell products. This strategy therefore provides a layer of safety control to reduce the risk of teratoma when applying hPSC-derived cell products in therapies.Funding Source: Wanek Innovation ProgramF-3233STRUCTURE-ACTIVITY RELATIONSHIP ANALYSIS OF YM155 FOR INDUCING SELECTIVE CELL DEATH OF HUMAN PLURIPOTENT STEM CELLSGo, Younghyun - Life Science, Sogang University, Seoul, Korea Lim, Chang-Jin - Chemistry, CHA University, Seoul, Korea Jeong, Ho-Chang - Life Science, Sogang University, Seoul, Korea Kwon, Ok-Seon - Pharmacy, Seoul National University, Seoul, Korea Lee, Mi-Ok - Life Science, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Cha, Hyuk-Jin - Pharmacy, Seoul National University, Seoul, Korea Kim, Seok-Ho - Chemistry, CHA University, Seoul, KoreaDespite great potential for regenerative medicine, the high tumorigenic potential of human pluripotent stem cells (hPSCs) to form undesirable teratoma is an important technical hurdle preventing safe cell therapy. Various small molecules that induce the complete elimination of undifferentiated hPSCs, referred to as ‘stem-toxics’, have been developed to facilitate tumor-free cell therapy, including the Survivin inhibitor YM155. In the present work, based on the chemical structure of YM155, total 26 analogs were synthesized and tested for stem-toxic activity toward human embryonic stem cells (hESCs) and induced PSCs (iPSCs). We found that a hydrogen bond acceptor in the pyrazine ring of YM155 derivatives is critical for stem-toxic activity, which is completely lost in hESCs lacking SLC35F2 encoding a solute

516POSTER ABSTRACTScarrier protein. These results suggest that hydrogen bonding interactions between the nitrogens of the pyrazine ring and the SLC35F2 protein are critical for entry of YM155 into hPSCs, and hence stem-toxic activity.Funding Source: This research was funded by the National Research Foundation of Korea, NRF-2017M3A9B3061843 (bioassay) and NRF-2017R1D1A1B03034612 (synthesis of small molecules)and Seoul National University, 370C-20180086 (bioassay).F-3235LEVERAGING ADVANCES IN BIOMEDICINE TOWARD DEVELOPMENT OF THE CELL-BASED MEAT INDUSTRYSwartz, Elliot - Science and Technology, The Good Food Institute, Los Angeles, CA, USAThe utilization of animal stem cells to grow muscle and fat tissues in vitro for consumption, dubbed “cell-based meat,” offers an unprecedented opportunity to transform animal agriculture and produce meat in a humane and sustainable way. Here, we provide an industry snapshot and highlight advances in regenerative medicine, genetic engineering, bioengineering, and large-scale bioprocessing methods for their applicability to this fast-growing nascent industry. We discuss the current challenges in scaling cell-based meat production and opportunities for development of new tools, resources, or optimizations required to reach price parity with traditional animal meat.Funding Source: The Good Food Institute is a 501(c)3 nonprofit funded entirely by philanthropic donors.F-3239EFFICIENT GMP-COMPLIANT EXPANSION OF MESENCHYMAL STROMAL CELLS (MSCS) IN A CLOSED CULTIVATION SYSTEM USING XENO-FREE MSC-BREW GMP MEDIUMKnoebel, Sebastian - R&D, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Godthardt, Kathrin - R&D, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Heifer, Conny - R&D, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Bosio, Andreas - R&D, Miltenyi Biotec GmbH, Bergisch Gladbach, GermanyHuman mesenchymal stem cells (MSCs) hold great promise for clinical use and cell therapy applications and can be isolated from multiple tissue, e.g. bone marrow (BM), umbilical cord (UC) or adipose tissue (AT). As there is a high number of clinical trials and applications using MSCs, quality and safety of the resulting cellular products are indispensable. The xeno-free MSC-Brew GMP Medium developed and manufactured following the recommendations of USP <1043> on ancillary materials enables a standardized expansion and culture of MSC from different tissue sources. To increase the level of process standardization and product safety we developed the CliniMACS Prodigy® Adherent Cell Culture System for GMP compliant isolation and cultivation of adherent cells using a closed single used tubing set. The process includes the flexible combination of the following modules: 1. Density Gradient Centrifugation (DGC) 2. Surface coating 3. Inoculation 4. Culture 5. Media Change 6. Harvest. Here we show that MSCs from different tissue sources (AT, BM, UC) can be expanded and passaged using the CliniMACS Prodigy® Adherent Cell Culture System from primary tissue or single-cell suspensions combining the process modules in a flexible way. BM-MSCs were isolated via DGC and subsequent adherence to plastic using semi-automatic feeding, harvesting and reseeding procedures. AT-MSCs as well as UC-MSCs were isolated and expanded from single-cell suspensions obtained after enzymatic digestion. UC was digested using an optimized and automated procedure using the gentle MACSTM Dissociator. Subsequently, MSCs were isolated from single cell suspension using the flexible cultivation procedure of the CliniMACS Prodigy® Adherent Cell Culture System. Resulting cells displayed an MSC specific phenotype as defined by the ISCT consortium. Furthermore MSCs revealed their immunomodulatory potential as assessed by in vitro assay and multi-color flow cytometric analysis. With the CliniMACS Prodigy®, MSCs can be expanded in large scales, thus providing high numbers of cells for consecutive clinical applications.F-3241HUMAN IPS CELL-DERIVED PERIPHERAL NEURONS MODULATE TARGET ORGAN TISSUE FUNCTIONS BY CO-CULTURING IN MICROTUNNEL-EMBEDDED MICROFABRICATED DEVICETakayama, Yuzo - Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Kida, Yasuyuki - Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, JapanThe peripheral nervous system (PNS) is connected to the central nervous system (CNS) in our body, and the PNS governs the homeostasis of various tissues and organs. Understanding the molecular mechanism of peripheral neuropathy progression and the interaction of the PNS with target organs might contribute to the development of novel therapeutic methods for a complete cure. Thus, induction of peripheral neurons in vitro and construction of co-culture model system of peripheral neurons and target organ tissues are useful for clarifying the progressive mechanism of PNS-related diseases. In this study, we have constructed co-culture networks using human PNS and target organs cells. First, we fabricated a poly(dimethylsiloxane) (PDMS) -based co-culture chamber, which consisted of two culture compartments connected with 20 microtunnels, and we cultured induced PNS and CNS neurons differentiated from human iPS (induced pluripotent stem) cells. Development of their functional

517POSTER ABSTRACTSconnections was evaluated with microscopic observations, immunochemical analysis, and calcium imaging. In particular, we confirmed that CNS neurons showed an increase in calcium signals during electrical stimulation of networked bundles from PNS neurons, which demonstrated the formation of functional cell-cell interactions. Furthermore, we prepared a co-culture system using PNS neurons and cardiomyocytes, both derived from human iPS cells, or insulin-secreting cells, to confirm that our microfabricated device can be used with various cell types. These co-culture systems would be a promising tool to form networks of PNS neurons and target organs, and might help to understand functional mechanisms under normal and pathological conditions.Funding Source: Funding was provided by a Grant-in-Aid for Young Scientists (A) (#26702015) from Japan Society for the Promotion of Science (JSPS) and by AMED under Grant Number JP18be0304324.LATE-BREAKING ABSTRACTSF-4001HUMAN NAÏVE PLURIPOTENCY: VARIATIONS ON A THEMEMallon, Barbara - NIH Stem Cell Unit / NINDS, NIH, Bethesda, MD, USA Chen, Kevin - NINDS, NIH, Bethesda, USA Johnson, Kory - NINDS, NIH, Bethesda, USA Park, Kyeyoon - NINDS, NIH, Bethesda, USA Robey, Pamela - NIDCR, NIH, Bethesda, USA Shi, Yijun - NINDS, NIH, Bethesda, USA Yang, Forest - NINDS, NIH, Bethesda, MD, USAAlthough naïve mouse embryonic stem cells (mESC) are generally derived and maintained at normoxia (O2 > 19%), many protocols for the derivation or reversion of human pluripotent cells to a naïve state are performed under hypoxia (< 5% O2). We previously used RSeT medium under normoxia to evaluate differences between the naïve and primed pluripotent states described in the literature. We observed some results contrary to those published but it was unclear if these discrepancies were due to cells used, normoxic conditions or lab-to-lab variation. We have now compared 3 hESC lines in primed and ‘naïve’ culture conditions under normoxia and hypoxia with and without the use of the ROCK inhibitor, Y27632. Morphologically, naïve cells should be domed and exhibit rapid cell division, but we found that all 3 cell lines behaved differently in RSeT medium with H9 performing best and H1 performing worst. With the removal of Y27632 after passage 4, H1 cells exhibited extremely poor growth, which was exacerbated by hypoxia (3% O2). Hypoxia appeared to reduce the mitotic index of all lines under both culture conditions but did not otherwise affect morphology. H9 also outperformed H1 and H7 with respect to other indicators of the naive pluripotent state. For example, a reduction in global methylation was only observed in ‘naïve’ H9 cells under both hypoxia and normoxia, although ‘naïve’ H7 cells did show decreased methylation levels under hypoxia. In addition, when immunostained for H3K27me, only primed H9 cells demonstrated a punctate nuclear pattern, indicative of an inactive X chromosome, which was lacking in the ‘naïve’ cells. These data suggest that cell line choice strongly affects how successful naïve culture will be. In contrast, we found that discrepancies between our previous data and the published literature were not due to normoxia. We found agreement in upregulated RNA expression of several genes such as TFCP2L1, KLF2 and KLF4 under ‘naïve’ conditions but pluripotency markers such as POU5F1 and NANOG were still downregulated in our hands in both hypoxia and normoxia. Interestingly, these differences appeared more pronounced in cultures grown without Y27632. We will present further gene expression data regarding changes specific to hypoxia/normoxia, naïve/primed and +/- Y27632 as well as protein levels and metabolic analyses.Funding Source: This research was supported by the Intramural Research Program of the NIH, NINDSF-4003HSA-MIR-302S ARE ESSENTIAL FOR SELF-RENEW OF HUMAN PLURIPOTENT STEM CELLSSugawara, Tohru - Center for Regenerative Medicine, National Center for Child Health and Development, Setagaya-Ku, Japan Miura, Takumi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, Japan Kawasaki, Tomoyuki - Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan Umezawa, Akihiro - Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan Akutsu, Hidenori - Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, JapanRecent studies have revealed that human pluripotent stem cell (PSC)-specific microRNAs (miRs), including hsa-miR-302/367 cluster, are important for self-renewal, differentiation, and cellular reprogramming, however, their functional roles are largely unknown. Here, we showed that hsa-miR-302s are essential for self-renew of hPSCs by targeting hsa-miR-302/367 region via Cas9 nuclease complex with guide RNA and replacement that region to fluorescent protein. Using homologous donor with green or red florescence protein, we confirmed the deletion of the hsa-miR-302/367 region under microscope, but no homozygous knockout colony was detected, suggesting that hsa-miR-302/367 homozygous knock out cells were negatively selected. Next we analyzed the comprehensive expression dynamics of both miRs and mRNAs and identify 22 candidate targets of human PSC-specific miRs that were moderately expressed in undifferentiated hPSCs and more than 2-fold up-regulated during differentiation. The deleted in azoospermia-associated protein 2 (DAZAP2), one of those targets, was directly repressed by hsa-miR-302a/b/c/d, but not by hsa-miR-367. We found moderate expression of DAZAP2 mRNAs but hardly found expression of DAZAP2 proteins in undifferentiated iPSCs,

518POSTER ABSTRACTSindicating that their expression was strictly regulated by hsa-miR-302s. Overexpression of dazap2 caused cell proliferation decreased in undifferentiated hPSCs, although morphology and undifferentiated marker gene expressions was not affected. In addition, neural differentiation was suppressed in DAZAP2 overexpressing hPSCs. Taken together, our study revealed that hsa-miR-302s control self-renew and also differentiation via repressing DAZAP2, suggest that overexpression of target genes could be a solution for dissecting functions of miR.F-4005CONCERNING THE UNCERTAINTY OF HUMAN NAÏVE PLURIPOTENCYChen, Kevin G - National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA Chen, Kevin - National Institute of Neurological Disorders and Stroke”, NIH, Bethesda, MD, USA Johnson, Kory - National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA Park, Kyeyoon - National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA Yang, Forest - National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA Shi, Yijun - National Institute of Neurological Disorders and Stroke”, NIH, Bethesda, MD, USA Fann, Yang - National Institute of Neurological Disorders and Stroke”, NIH, Bethesda, MD, USA Robey, Pamela - National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA Mallon, Barbara - National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USATo achieve precision use of desired human pluripotent stem cells (hPSCs) for medical and pharmaceutical applications, it is essential to have a thorough understanding of all fundamental properties of these starting cell sources. One of the most important properties is related to the ground or naïve pluripotent state that is primarily established in mouse embryonic stem cells (mESCs). Thus far, more than five well-established research groups have reported the existence of human naïve pluripotency, which is believed to have major benefits for hPSC growth, genetic engineering, disease modeling, and drug discovery. In this study, we have derived and characterized naïve-like hPSCs (NLPs) using five initially reported human naïve protocols under normoxic growth conditions. Our data indicated that there is a significant heterogeneity regarding various pluripotent states in NLPs using these protocols. In general, current derived NLPs lack some hallmarks of naïve pluripotency as described in mESCs. Briefly, these NLPs exhibit much lower single-cell plating efficiency and growth rates, commonly lack unique mouse naïve cell surface marker (e.g., SSEA-1) expression, and have altered or abrogated cell growth under normoxia. Moreover, some examined NLPs are independent of BMP4 signaling and sensitive to the inhibitor of the Janus kinase (JAK). The genome-wide meta-analysis also revealed various fundamental inconsistencies between NLPs and their human and mouse counterparts. Thus, our data suggest the existence of dynamic pluripotent states in these derived NLPs, arguing about the rationale of genuine naïve pluripotency in these cells under normoxic growth conditions. To further recapitulate physiologically relevant human naïve pluripotency in hPSCs under hypoxic growth conditions would warrant their future broad applications.Funding Source: This work was supported by the Intramural Research Program of the NIH at the National Institute of Neurological Disorders and Stroke and in part by the National Institute of Dental and Craniofacial Research.F-4009OVEREXPRESSION OF BCL-2 AND BDNF AS EFFECTIVE PROTECTORS AGAINST INDUCED CELL DEATH AND EFFICIENT INDUCERS OF THE DIFFERENTIATION OF HUMAN WHARTONS JELLY MESENCHYMAL STEM CELLS INTO DOPAMINERGIC NEURONSBorkowska, Paulina - Department of Medical Genetics, Medical University of Silesia, Katowice, Poland Kowalski, Jan - Department of Medical Genetics, Medical University of Silesia, Katowice, PolandDopamine (DA) neurons, which are derived from MSCs, are a valuable source for cell replacement therapy in Parkinson’s disease. Although there have been numerous studies to develop a protocol for differentiating MSCs into DAergic neurons in recent years, there is still no efficient method that enables a relatively homogeneous population of functional DAergic neurons with an increased resistance to cell-death factors that could be used for transplantation to be obtained. We think that cells that have all of the above-mentioned features will survive after transplantation and will create a network of functional neurons. We hypothesize that this can be done because of the synergistic overexpression of Bcl-2 and BDNF. A stable overexpression of Bcl-2 in MSCs protects cells from apoptotic factors and a stable overexpression of BDNF enables MSCs to differentiate into functional DAergic neurons. We determined that the synergistic overexpression of Bcl-2 and BDNF in MSCs is stable and lasts at least 60 days. As a result, the level of Bcl-2 and BDNF proteins significantly exaggerated the level that was observed in MSCs without an overexperssion. These results suggest that the Bcl-2 and BDNF proteins that are produced are functional because the transfected MSCs are more resistant to the apoptotic factors that induce cell death via intrinsic and extrinsic pathways. Overexpressed MSCs with a high efficiency differentiated into DAergic cells (thyrosine hydroxylase possitive). The trace expression of the genes that are typical for neurons with different phenotypes was also observed. The new cells produced dopamine, and after depolarization, their quantity increased sharply. These results suggest that the new population of dopaminergic neurons was homogeneous and functional. The results, which are based on the synergistic overexpression of genes, may indicate a new pathway for experiments to obtain homogeneous populations of functional neurons with an increased survival capacity. In this model, the overexpressed genes can be changed to obtain

519POSTER ABSTRACTSa homogenous population of different neurons that can be investigated. In the near future, I plan to study the functionality of the obtained DAergic neuron population in a rat model of Parkinson’s disease.F-4011SALL3 EXPRESSION BALANCE UNDERLIES LINEAGE BIASES IN HUMAN IPS CELL DIFFERENTIATIONKuroda, Takuya - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kanagawa, Japan Yasuda, Satoshi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Tachi, Shiori - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Matsuyama, Satoko - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Kusakawa, Shinji - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Tano, Keiko - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Miura, Takumi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Matsuyama, Akifumi - Department of Regenerative Medicine, Fujita Health University, Toyoake, Japan Sato, Yoji - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, JapanHuman induced pluripotent stem cells (hiPSCs) have the ability to differentiate into a variety of cells and to self-renew in vitro. Because of these two characteristics, hiPSCs have been expected to provide new applications for regenerative medicine/cell therapy. Although various in vitro differentiation protocols have been developed for efficient derivation of specific cell types, hiPSC lines vary in their ability to differentiate into specific lineages. For efficient selection of hiPSC lines suitable for differentiation into desired cell lineages, we tried identifying the marker genes that potentially predict the differentiation propensity of hiPSCs into three germ layers. Our approach for identifying differentiation propensity markers is essentially based on the statistical comparison of the geneexpression profiles of undifferentiated hiPSCs with each cell line’s in vitro differentiation potential using the rank correlation method. In this study, we identified the SALL3 gene as predicting differentiation propensity using the rank correlation method and analysis of 10 hiPSC lines. We show that SALL3 expression correlates positively with ectoderm differentiation and negatively with mesoderm/endoderm differentiation during embryoid body (EB) formation and that SALL3 inversely regulates the capacities of cardiac and neural differentiation in hiPSCs. Mechanistically, SALL3 is found to repress gene body methylation in hiPSCs, leading to their epigenetic changes. Our findings provide a practical method for selecting appropriate hPSC lines in clinical-grade cell banks, allowing for the prediction of differentiation capacity toward a desired cell lineage.F-4013EFFICIENT GENERATION OF CLINICAL-GRADE MIDBRAIN DOPAMINERGIC NEURAL PROGENITORS FROM HUMAN EMBRYONIC STEM CELLSWang, Yukai - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Feng, Lin - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Liang, Lingmin - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Hao, Jie - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Wang, Liu - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Hu, Baoyang - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Zhou, Qi - Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, ChinaDifferentiation of human pluripotent stem cells (hPSCs) into midbrain dopaminergic neural progenitors (mDA) promises a cell-based therapy for Parkinson’s disease. Here, we report the generation of clinical-grade mDA from embryonic stem cells (ESCs) under defined xeno-free conditions. By optimizing the treatment time of SHH and the concentration of CHIR99021, we could efficiently generate EN1+ mDA and developed a chemical recipe and a streamlined protocol. Using this protocol, the percentage of EN1 positive cells increased from 20% to about 50%. The cells also expressed appropriate mDA markers, such as FOXA2, LMX1A and NURR1. Upon transplantation into brains of rat model of Parkinson’s disease, they exhibited sufficient safety and reasonable efficacy. Therefore, this protocol enables advancement of stem cell-based therapies towards neural regeneration.Funding Source: This work was supported by grants from the Program of National Key Research and Development and the National Basic Research Program of China.F-4015NFIA IS A GLIOGENIC SWITCH ENABLING RAPID DERIVATION OF FUNCTIONAL HUMAN ASTROCYTES FROM PLURIPOTENT STEM CELLSTchieu, Jason - Developmental Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

520POSTER ABSTRACTSAromolaran, Kelly - Anesthesiology, Weill Cornell Medicine, New York, NY, USA Calder, Elizabeth - Developmental Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Goldstein, Peter - Anesthesiology, Weill Cornell Medicine, New York, NY, USA Guttikonda, Sudha - Developmental Biology, Weill Cornell Medicine, New York, NY, USA Gutzwiller, Eveline - Developmental Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Steinbeck, Julius - Developmental Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Studer, Lorenz - Developmental Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USAAstrocytes are the most abundant glial cell type in the human brain, and their dysfunction is a driver in the pathogenesis of both neurodevelopmental and neurodegenerative disorders. During early development, neural stem cells (NSCs) are fate-restricted to exclusively produce neurons, while at later stages, they undergo a switch from neurogenic to gliogenic competency resulting in progressive production of astrocytes and oligodendrocytes. The molecular nature of the gliogenic switch has remained elusive, and its timing varies dramatically across species from 7 days in the mouse to 6-9 months during human development. Those species-specific timing differences similarly apply to NSCs derived from human pluripotent stem cells (hPSCs). The highly protracted timing of acquiring glial competency in hPSCs presents a major roadblock in the quest for deriving human astrocytes for basic and translational applications. Here, we identify Nuclear Factor IA (NFIA) as the molecular switch for inducing human glial competency. Transient expression of NFIA for 5 days, in the presence of factors promoting glial differentiation, is sufficient to trigger glial competency and generate hPSC-derived astrocytes as compared to 3-6 months of differentiation using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli, and engraft in the adult brain. Finally, NFIA-induced astrocytes can be induced to express features of region-specific and of reactive astrocytes. The underlying mechanism of NFIA-induced glial competency involves rapid but reversible chromatin remodeling, GFAP promoter demethylation, and a striking lengthening of the G1 phase in the cell cycle. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function in glial competency. Our study addresses a significant roadblock in hPSC and glial biology by defining key mechanistic features of the gliogenic switch and by enabling the rapid production of human astrocytes for disease modeling and regenerative medicine.Funding Source: J.T. was supported by the Tri-I Starr Stem Cell Scholars postdoctoral training fellowship. The work was supported by R21 NS084334 (LS) and the core grant P30CA008748.F-4017WNT INHIBITION AND BMP4 REGULATE OCULAR SURFACE EPITHELIAL FATE IN HUMAN PLURIPOTENT STEM CELLSKobayashi, Yuki - Department of Ophthalmology, Osaka University Medical School, Suita, Japan Hayashi, Ryuhei - Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Japan Quantock, Andrew - School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK Nishida, Kohji - Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, JapanThe corneal epithelium is derived from the ocular surface ectoderm (OSE) via the non-neural ectoderm. How and when the OSE commits to presumptive corneal epithelial cells, however, is not well defined. What is well-known, is that BMP and WNT signalling pathways have crucial roles within cells during various stages of development. BMP4 is expressed during the beginning of spontaneous human induced pluripotent stem cells (hiPSC) differentiation, as well as gastrulation, and cells exposed to BMP4 are directed towards an ectodermal fate. Simultaneously, WNT signalling is precisely controlled for optic cup formation in eye development. Standard hiPSC differentiation in vitro, involves a cultivation period of 10 to 12 weeks, before isolation of presumptive corneal epithelial cells that express p63 and PAX6, markers for epithelial stem cells and OSE, respectively. Interestingly, we found that p63 and PAX6 double positive (p63+/PAX6+) cells were detected by immunofluorescence at 10 days of differentiation, and this finding is mimicked in vivo by p63+/PAX6+ cells within mouse embryonic eye at E8.5. Based on these findings, hiPSCs were treated with a combination of WNT inhibitor (IWP2) and BMP4, during the initial 4 days of differentiation, to observe effects of OSE formation at 10 days and 6 weeks of differentiation. A p63 knock-in hiPSC-line was used in order to trace epithelial stem cells via EGFP expression. To determine the OSE differentiation efficiency, p63-EGFP+ cells were isolated by FACS. p63-EGFP+ cells were then stained with immunofluorescent-PAX6, in order to quantify the p63+/PAX6+ cell ratio. Results showed 1.66-fold increase on 10 days of differentiation and 3.24-fold increase on 6 weeks of differentiation, of p63+/PAX6+ cells compared to non-treated cells. As such, combined treatment of IWP2 and exogenous BMP4, greatly enhanced OSE differentiation efficiency. Furthermore, the data indicates simultaneous WNT inhibition and BMP4 were promoted not only p63+ cells, but considerably maintained p63+/PAX6+ cells within the OSE. Therefore, surface ectodermal cell lineage is likely determined during early eye development. This study has great future potential for research to clarify the intricate mechanisms of epithelial development, as well as OSE cell lineage determination.Funding Source: This work was supported in part by the Project for the Realization of Regenerative Medicine of The Japan Agency for Medical Research and Development (AMED).

521POSTER ABSTRACTSF-4019REPROGRAMMING IPSCS INTO GLUTAMATERGIC NEURONS - RECONSTRUCTION OF TRANSCRIPTIONAL EVENTS AND CELLULAR STATES USING AN INTEGRATED GENOMIC APPROACHKotter, 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 Cooper, Sarah - Wellcome Sanger Institute, Wellcome Sanger Institute, Cambridge, UK Bello, Erica - Wellcome Sanger Institute, Wellcome Sanger Institute, Cambridge, UK Patikas, Nikolaos - UK Dementia Research Institute, University of Cambridge, UK Metzakopian, Emmanouil - UK Dementia Research Institute, University of Cambridge, UK Bassett, Andrew - Wellcome Sanger Institute, Wellcome Sanger Institute, Cambridge, UK Kotter, Mark - Department of Clinical Neurosciences and Wellcome Trust MRC Cambridge Stem Cell Institute, University of Cambridge, UKDirect cell reprogramming is a rapidly growing field that challenges traditional concepts of cellular identity. Expression of Neurogenin 2 (NGN2) has been used to efficiently reprogram human pluripotent stem cells (PSCs) into functional cortical glutamatergic neurons. This approach combines the advantage of the highly proliferative and epigenetically malleable PSC stage with the efficiency of direct reprogramming. We have previously demonstrated that gene targeting the components of a Tet-On system into two separate safe harbour sites overcomes gene silencing, results in optimised transgene expression in hiPSCs (OptiOx), and yields highly homogenous cultures of pure glutamatergic neurons within less than four days. The mechanisms that mediate this remarkable cellular metamorphosis remain poorly understood. To study the transcriptional events occurring as a result of NGN2 expression, three independent biological replicates were harvested at the iPSC stage (day 0), and 6h, 12h, 24h, 36h, day 2, day 3, day 4, day 14, and day 21 post induction. Cell extracts were subsequently processed for bulk RNA sequencing. This demonstrated distinct and highly reproducible transcriptional changes across each of the individual time points. To gain a more detailed insight into the cellular states during reprogramming, we complemented our bulk-RNA-Seq approach with single cell RNA sequencing of cells harvested at the iPSC stage (day 0), as well as 12h, 24h, day 2, day 3, day 4, day 14, and day 21 post induction. Louvain network analysis demonstrated distinct transient populations through which cells synchronously progressed with little heterogeneity until attaining a distinct neuronal phenotype. In order to differentiate direct from indirect NGN2 down-stream effectors, an inducible hiPSC cell line in which NGN2 tagged with HA was constructed. CHiP-Seq data acquired on day 2 after induction was subsequently overlaid with bulk seq data and single cell seq data. This enabled identification of direct and indirect effectors of NGN2 with regards to gene regulatory networks as well as peripheral genes. In conclusion, we provide a detailed analysis of transcriptional events that govern the transition of iPSCs into mature glutamatergic neurons following NGN2 expression.F-4021MEDIAL INDUCTION ACCELERATES NEUROEPITHELIAL CONVERSION OF HUMAN PLURIPOTENT STEM CELLSWalsh, Patrick - Division for Stem Cell Engineering, Anatomi Corp, Minneapolis, MN, USA Truong, Vincent - Division of Stem Cell Engineering, Anatomi Corp, Minneapolis, MN, USA Dutton, James - Stem Cell Institute, University of Minnesota, Minneapolis, MN, USADifferentiation of human pluripotent stem cells (hPSCs) into ectoderm provides neurons and glia useful for research, disease modeling, drug discovery, and potential cell therapies. In current protocols, hPSCs are traditionally differentiated into ectoderm after 6 to 10 days in vitro when protected from mesendoderm inducers. This protracted timing has made ectoderm a difficult germ layer to access and manipulate, hindering development of efficient differentiation protocols for ectoderm-derived cell types. Here we report efficient and serum-free differentiation of hPSCs into ectoderm within 24 hours using a novel combination of chemical inhibitors. This method is greater than 70% efficient, is broadly applicable to a panel of five independent hPSC lines, and accelerates the emergence of downstream intermediate and terminal neurodevelopmental landmarks. Given its rapid and flexible nature, we expect this method to democratize the development and execution of significantly more efficient protocols for the differentiation of ectoderm-derived cell types from hPSCs.F-4023DEVELOPMENT OF COMMERCIALLY VIABLE MANUFACTURING PROCESSES FOR CELL THERAPY PRODUCTS DERIVED FROM IPSCSYang, Fan - Cell Therapy Development Services, Lonza, Walkersville, USA Ahmadian Baghbaderani, Behnam - Development, Lonza, Walkersville, MD, USA Menendez, Laura - PD, Lonza, Walkersville, MD, USA Neo, Boon Hwa - PD, Lonza, Walkersville, MD, USA Tian, Xinghui - BAS, Lonza, Walkersville, MD, USA Panchalingam, krishna - PD, Lonza, Walkersville, MD, USA Shafa, Mehdi - PD, Lonza, Walkersville, MD, USA

522POSTER ABSTRACTSHuman induced pluripotent stem cells (iPSCs) reprogrammed from different type of starting materials are able to renew indefinitely and differentiate into specialized cell types of the body. These characteristics give iPSCs very attractive potential as unlimited source of cells for a wide range of cell therapy applications. While current processes for derivation, expansion, and directed differentiation of iPSCs are significantly improved and demonstrate potentials of these cells, they face significant manufacturing challenges because they are open, manual, and carried out under uncontrolled 2D environment. To address these challenges and allow manufacturing of iPSC derived CT products under commercial setting, it is important to develop robust and reproducible processes by implementing appropriate process optimization and process control strategies along with incorporating innovative technologies during the development phase. We previously reported manufacturing of human iPSC master cell banks (MCB) under current Good Manufacturing Practices (cGMP). We have also shown that these cGMP compliant iPSCs can be differentiated into specialized cell types from all three embryonic lineages with morphological and cellular characteristics of cardiomyocytes, definitive endoderm (DE), and neural stem cells (NSCs) in 2D. Here we report our approach for development of a robust and reproducible expansion and directed differentiation of iPSCs under controlled condition in 3D in computer controlled bioreactors. In particular, we demonstrate that these fully characterized human iPSC lines can readily differentiate into cardiomyocytes, and the directed differentiation process can be further optimized to establish a robust and reproducible process. This is a major step forward in the development of a salable and commercially viable cGMP-compliant manufacturing possess to generate clinical quantities of iPSC derived cell therapy products.F-4025INTENSIVE GENETIC QUALITY TESTS OF GMP COMPLIANT IPSC AND THEIR DERIVATIVESJo, Hye-Yeong - Division of Intractable Diseases, Korea National Institute of Health, CheongJu, Korea Han, Hyo-Won - Division of Intractable Diseases, Korea National Institute of Health, Cheong Ju, Korea Jung, Inuk - Bioinformatics Institute, Seoul National University, Seoul, Korea Ju, Ji Hyeon - Division of Rheumatology, The Catholic University of Korea, Seoul, Korea Park, Soon-Jung - Department of Medical Science, Konkuk University School of Medicine, Seoul, Korea Moon, Sung-Hwan - Department of Medical Science, Konkuk University School of Medicine, Seoul, Korea Geum, Dongho - Department of Medical Science, Korea University, Seoul, Korea Kim, Hyemin - Department of Predictive Toxicology, Korea Institute of Toxicology, DaeJeon, Korea Park, Han-Jin - Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Korea Kim, Sun - Interdisciplinary program in Bioinformatics, Seoul National University, Seoul, Korea Koo, Soo Kyung - Division of Intractable Diseases, Korea National Institute of Health, Cheongju, Korea Stacey, Glyn - International Stem Cell Banking Initiative, International Stem Cell Banking Initiative, Hertfordshire, UK Park, Mi-Hyun - Division of Intractable Diseases, Korea National Institute of Health, CheongJu, Korea Kim, Jung-Hyun - Division of Intractable Diseases, Korea National Institute of Health, CheongJu, KoreaMost hiPSC lines are karyotypically normal during long-term maintenance however, still possess mutations in risk, therefore, it is imperative to perform intensive genetic quality controls in clinical grade iPSC, better in cost-effective manner. As a conceptual approach, we performed an intensive genetic quality test with a clinical grade homozygous HLA iPSC and their derivatives in post-distributing condition using a single RNA sequencing data. We found that degree of accumulated SNPs by prolonged culture and differentiation were differ by institutes. However, CNVs and mutations in HLA molecular types were not detected in any of the samples. The RNA expression levels of cancerous and immunogenic genes were not different between the early and late passaged iPSC and their derivatives. Advanced time-course analysis techniques identified 5 clusters showing different patterns in prolonged -culture condition and 40 clusters remaining in similar pattern. Importantly, we were able to access theses genetic quality testing results using a single RNA sequencing data. We believe the benefit of theses analysis approaches and the post-distribution monitoring would facilitate identification of a right iPSC seed stock for the clinical application.Funding Source: This work was supported by the Korea National Institute of Health Intramural Research Program 4800-4861-312-210-13 (grant nos. 2017-NG61003-00 and 2017-NG61004-00).F-4027TIMED INDUCTION OF MIXL1 PLAYS A ROLE IN ENDODERM BIAS IN MOUSE EPIBLAST STEM CELLSSalehin, Nazmus - Embryology Unit, Children’s Medical Research Institute, Westmead, Australia Osteil, Pierre - Embryology Unit, Children’s Medical Research Institute, Westmead, New South Wales, Australia Santucci, Nicole - Embryology Unit, Children’s Medical Research Institute, Westmead, New South Wales, Australia Knowles, Hilary - Embryology Unit, Children’s Medical Research Institute, Westmead, Australia Cai, Simon - Bioinformatics Group, Children’s Medical Research Institute, Westmead, Australia Studdert, Joshua - Children’s Medical Research Institute, Westmead, Australia Teber, Erdahl - Bioinformatics Group, Children’s Medical Research Institute, Westmead, Australia Tam, Patrick PL - Embryology Unit, Children’s Medical Research Institute, Westmead, Australia

523POSTER ABSTRACTSIn early mouse embryos, gastrulation forms the three germ layers from which all cells of the mouse will derive. The signalling factors and gene networks involved in the formation of the mouse endoderm have not been fully elucidated. We have focused on Mix1 homeobox-like 1, encoded by Mixl1, which is expressed in the primitive streak during gastrulation and is vital for the formation of the endoderm. Previous in vitro studies have shown that Mixl1 expression is necessary for both haematopoietic and endoderm differentiation. Furthermore, a previous study within the lab found a correlation between the timing of Mixl1 expression and germ layer specification. However, the role of Mixl1 expression timing as well as the direct targets of Mixl1 had not been fully explored. In attempting to determine how Mixl1 could be responsible for both mesoderm and endoderm differentiation, we used epiblast stem cells (EpiSC) that are inducible for Mixl1. Using timed doxycycline inductions of Mixl1, we have performed the first ChIP-seq analysis of Mixl1 in a proper gastrulation model. We have built a comprehensive gene regulatory network centred on Mixl1 by combining ATAC-seq and RNA-seq in the same cell conditions and adding on published data on promoter capture Hi-C in EpiSC and histone methylation marker ChIP-seq in human embryonic stem cells. We found that the early induction of Mixl1 during gastrulation results in the expression of the anterior primitive streak markers, Gsc and Lhx1, from where the endoderm progenitors emerge. Mixl1 also activates WNT signalling pathway genes and results in reduced accessibility in genes involved in stem cell maintenance. Moreover, we have found Mixl1 timing to be instrumental in endoderm differentiation. Together, these results suggest that early Mixl1 activation pushes cells towards endodermal progenitors within the anterior primitive streak.F-4029MODELING THE CARDIAC ELECTRICAL PHENOTYPE OF AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE USING PATIENT IPS CELL-DERIVED CARDIOMYOCYTESLee, Jia-Jung - Faculty of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Chen, Hung-Chun - Faculty of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Hsieh, Patrick CH - Institute of Biomedical Sciences, Academia Sinica, Taipei, TaiwanMutations in PKD1 or PKD2 gene lead to autosomal dominant polycystic kidney disease (ADPKD). The mechanism of ADPKD progression and its link to increased cardiovascular mortality is still elusive. In this study, we applied patient-specific induced pluripotent stem cells (iPSCs) to establish a human cell-based, in vitro model for studying ADPKD-associated cardiac manifestations. We efficiently generated iPSC-derived cardiomyocytes (CMs) with predominantly ventricular-like cells that exhibited PKD1 and PKD2 expression. The ADPKD patient-specific iPSC-CMs had decreased sarcoplasmic reticulum calcium content compared with control-CMs. Spontaneous action potential of the PKD2 mutation line-derived CMs demonstrated lower beating rate and longer action potential duration. The PKD1 mutation line-derived CMs showed a comparable dose-dependent shortening of phase II repolarization with the control-CMs, but a significant increase in beating frequency in response to L-type calcium channel blocker. The PKD1-mutant iPSC-CMs also showed a relatively unstable baseline as a greater percentage of cells exhibited delayed afterdepolarizations (DADs). Both the ADPKD patient-specific iPSC-CMs showed more -adrenergic agonist-elicited DADs βcompared with control-CMs suggesting a possible common proarrythmogenic phenotype of ADPKD. The close mimicry of the electrophysiological characteristics of patient-specific iPSC-CMs with their clinical cardiovascular phenotype demonstrated that iPSC-CMs may be invaluable in disease modeling and as a drug screening platform for ADPKD-associated cardiovascular complications.Funding Source: Ministry of Science and Technology, National Health Research Institutes, Academia Sinica, and Kaohsiung Medical University Hospital, Taiwan.F-4031HOMOLOGY-MEDIATED END JOINING (HMEJ)-BASED STRATEGY IN GENOME EDITING AND GENE THERAPIES IN MOUSE AND MONKEY EMBRYOWang, Xing - Institute of Neuroscience, Chinese Academy of Science (CAS), Shanghai, China Yao, Xuan - Institute of Neuroscience, Chinese Academy of Science, Shanghai, China Liu, Zhen - Institute of Neuroscience, Chinese Academy of Science, Shanghai, China Hu, Xinde - Institute of Neuroscience, Chinese Academy of Science, Shanghai, China Shi, Linyu - Institute of Neuroscience, Chinese Academy of Science, Shanghai, China Sun, Qiang - Institute of Neuroscience, Chinese Academy of Science, Shanghai, China Yang, Hui - Institute of Neuroscience, Chinese Academy of Science, Shanghai, ChinaAs a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse and monkey zygotes,as well as in hepatocytes and neurons in vivo. Moreover, this HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency

524POSTER ABSTRACTSinduced liver failure mice.Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.F-4033DEVELOPING STEM CELL-BASED ASSAYS FOR NEUROLOGICAL DISEASE DRUG DISCOVERYMariga, Abigail - Bioassays and High Throughput Screening, Biogen, Cambridge, USA Dragan, Sofya - Bioassays and High Throughput Screening, Biogen, Cambridge, MA, USA Engle, Sandra - Translational Cellular Sciences, Biogen, Cambridge, MA, USA Faloon, Patrick - Bioassays and High Throughput Screening, Biogen, Cambridge, MA, USA Hurt, Jessica - Translational Genome Science, Biogen, Cambridge, MA, USA Little, Kevin - Bioassays and High Throughput Screening, Biogen, Cambridge, MA, USA Micozzi, Jack - Translational Cellular Sciences, Biogen, Cambridge, MA, USA O’Malley, Tiernan - Translational Sciences Biomakers, Biogen, Cambridge, MA, USA Smith, Robert - Translational Cellular Sciences, Biogen, Cambridge, MA, USA Swalley, Susanne - Chemical Biology and Proteomics, Biogen, Cambridge, MA, USAA major challenge in neurological drug discovery has been establishing translatability between use of heterologous cell lines in preclinical studies and predictive responses in patients. The application of human induced pluripotent stem cells (iPSCs) represents a promising avenue for drug discovery where patient-specific cellular phenotypes that model aspects of neurological diseases can translate into clinically relevant outcomes. Our goal was to develop iPSC-based assays using patient-derived iPSCs and iPSC-derived cells to support neurological drug discovery efforts. We are interested in evaluating assay performance in self renewing iPSC vs iPSC-motor neurons as the latter is the most relevant to phenotypes that manifest in patients. Here, we present iPSC-based assays for two phenotypic discovery projects. The assays are evaluating protein readouts in response to treatment with well characterized tool compounds and antisense oligonucleotides (ASO). The first assay measures downregulation of a protein in response to treatment with small molecules or knockdown ASO in wild type iPSCs and iPSC-motor neurons using electrochemiluminescence. The second assay uses undifferentiated patient-derived iPSCs and patient iPSC-motor neurons to determine an increase in expression of a protein by Homogenous Time-Resolved Fluorescence (HTRF). For the second assay, we are using two patient-derived iPSC lines and their respective iPSC-motor neurons to assess assay performance across different iPSC lines. We will present assay performance characteristics including signal window, assay sensitivity, specificity and consistency across runs. Preliminary data for the second assay indicate a robust performance with a high signal to background ratio and low inter-assay variability. Differences between iPSC lines were subtle. This assay was very scalable for HTS and was adapted to automation without compromising assay metrics. We ran pilot studies using informer compound sets to thoroughly assess these assays prior to HTS. The HTS and follow up experiments using iPSC-derived motor neurons are in progress and will be presented.F-4035PATIENT-SPECIFIC IPSC-DERIVED KERATINOCYTES RECAPITULATE THE GENETIC DEFECTS IN PSORIASIS DISEASEElsayed, Ahmed - Qatar Biomedical Research Institute-Qatar Foundation, Doha, Qatar Ali, Gowher - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Nandakumar, Manjula - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Bashir, Mohamed - Hamad Medical Corporation, Doha, Qatar Abu Aqel, Yasmin - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Aghadi, Maryam - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Memon, Bushra - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Temanni, Ramzi - Biomedical Informatics Division, SIDRA, Doha, Qatar Karam, Manale - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar Taheri, Shahrad - Weill Cornell Medicine-Qatar, Weill Cornell Medicine-Qatar, Doha, Qatar Abdelalim, Essam - Diabetes Research Center, Qatar Biomedical Research Institute, Doha, QatarPsoriasis is a chronic inflammatory disease of the skin characterized by keratinocytes hyperproliferation, affecting 2-3% of world population. The etiology of psoriasis is complex and is supposed to result from the interplay between genetic predisposition and environmental factors. A major limitation in understanding the pathogenesis of psoriasis and subsequent development of novel therapies is the lack of suitable in vitro models mimicking the disease. Recent progress in the generation of induced pluripotent stem cells (iPSCs) has provided unique opportunities for obtaining patient specific cells for modeling skin diseases and future clinical applications. In the present work, we showed the successful generation of iPSCs from patients having familial history of psoriasis as well as from healthy controls. The generated iPSC lines were characterized for the pluripotency and self-renewal properties. All iPSCs lines showed normal karyotype, expressed pluripotency markers and differentiated into all three germ layers upon spontaneous and direct differentiation. The pluripotent ability of the generated iPSC lines was further validated by the TaqMan hPSC Scorecard assay in vitro. Patient-specific iPSCs and healthy controls were efficiently differentiated into keratinocyte progenitors and mature keratinocytes, which expressed the key markers, including p63,

525POSTER ABSTRACTSKRT18, KRT14, LORICRIN, KRT1, INVOLCURIN, and LAMININ. High throughput sequencing analysis showed dysregulated expression of psoriasis-associated genes in patient specific iPSC-derived keratinocytes. Those genes are mainly involved in epithelial proliferation, keratinocyte differentiation, and in the immune system. These include members of the solute carriers family, interleukins, Human leukocytes antigen (HLA) and Keratins (KRT) as well as filaggrin (FLG), involucrin (IVL), TGM5, PSORSICI genes shown to be implicated in the pathogenesis of psoriasis. Our results demonstrate successful modeling of psoriasis from patient derived iPSCs that could be used to understand the pathogenesis of psoriasis and screening of novel therapeutics.Funding Source: This work was supported by a grant from Qatar National Research Fund (QNRF), Qatar Foundation (NPRP 9-283-3-056)F-4037OPTIMIZED PROTOCOL FOR GENERATING SAFE SPINAL CORD NEURAL STEM CELLS FROM HUMAN EMBRYONIC STEM CELLSLi, Yuanyuan - Department of Neurosciences, University of California San Diego, La Jolla, USA Brock, John - Department of Neurosciences, University of California San Diego, La Jolla, USA Kumamaru, Hiromi - Department of Neurosciencess, University of California San Diego, La Jolla, USA Rosenzweig, Ephron - Department of Neurosciences, University of California San Diego, La Jolla, USA Lu, Paul - Department of Neurosciences, University of California San Diego, La Jolla, USA Tuszynski, Mark - Department of Neurosciences, University of California San Diego, La Jolla, USARecently, we reported the generation of spinal cord neural stem cells(NSCs) from human pluripotent stem cells (hPSCs) (Kumamaru et al., 2018, Nat Methods). However, the prolonged culture of NSCs with high doses of small molecules, such as a SMAD inhibitor, SB431542 and GSK-3 inhibitor, CHIR99021, may cause chromosomal instability. To reduce the potential negative effect of small molecules used in the neural induction and maintenance of spinal cord neural stem cells, we reduced the concentration of CHIR99021 and SB431542. In addition, we carefully assessed the effects of the concentration of FGF proteins and reduced their concentration as well. We successfully developed a modified protocol with reduced doses of small molecules and FGF proteins while retaining high efficiency in generating and maintaining spinal cord NSCs. In optimum culture conditions, the spinal cord NSCs show an equivalent identity to the original protocol and can be maintained for prolonged time periods, up to twenty passages. Cluster analysis of RNAseq data shows that hPSC-derived spinal cord NSCs are closely grouped with human fetal spinal cord tissue, and karyotype analysis shows that 8 out of 10 batches exhibited normal karyotype. Two-three months after transplantation into injured spinal cords of rats and monkeys, the grafted human NSCs survive and integrated well without evident expansion. In addition, grafted human spinal cord NSCs differentiate into both neurons and glia and extended very large numbers of axons into the host spinal cord. Long-term studies are underway to assess the safety of human spinal cord NSCs for potential human translation.F-4039EFFECT OF TRISOMY 21 ON THE DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS TO NEURAL PROGENITOR CELLSPrutton, Kendra - Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, CO, USA Jain, Abhilasha - Pharmaceutical Sciences, University of Colorado Denver, CO, USA Marentette, John - Pharmaceutical Sciences, University of Colorado Denver, CO, USA Roede, James - Pharmaceutical Sciences, University of Colorado Denver, CO, USADown syndrome (DS) is characterized by a complex phenotype, including intellectual disability, developmental complications, and chronic health conditions. The ubiquity of cognitive deficits in DS has made structural and cellular changes in the brain the focus of much research effort. However, no prior studies have utilized DS induced pluripotent stem cells (iPSC) to assess the effect of trisomy 21 on neurogenesis. The main aim of the current study was to define, evaluate, and compare neural differentiation in DS and euploid iPSCs through the formation and maturation of embryoid bodies (EB) to neural progenitor cells (NPC). Embryoid body formation is an important initial step and common platform in directed differentiation protocols that is often overlooked and under-researched. Here we show that DS iPSCs produce larger and more EBs compared to their euploid counterparts. We found that several neural differentiation markers, such as TuJ1 (neurons) and GFAP (glial cells), are upregulated in DS EBs. Furthermore, DS EBs showed increased expression of several genes involved in the TGF- βpathway – a critical regulator of pluripotency in embryonic stem cells and lineage determination in progenitor cells. In particular, DS EBs showed an increased gene expression of several bone morphogenetic proteins (BMP) which have been shown to switch progenitors to an astrocytic fate. These observations and the lack of proper neural rosette formation in DS iPSCs suggest accelerated neural and glial differentiation and reduced progenitor cell development. Irregular cell fate specification in DS iPSCs is consistent with the fact that DS individuals show an increased number of astrocytes. Our results demonstrate an increased number of differentiated neurons and glial cells, and dysregulation in cell lineage specification early in development of the DS brain, which may lead to altered synapse formation, function, and elimination, decreasing the efficiency of neuronal

526POSTER ABSTRACTStransmission. Due to the lack of previous research on this topic, this experimental data provides evidence that trisomy 21 affects EB formation and NPC cell fate specification during neurogenesis.Funding Source: NIH/NIEHS R01ES027593F-4041A MUTATION IN SCN1A SELECTIVELY IMPAIRS IPSCS-DERIVED INHIBITORY NEURONS DERIVED FROM A PATIENT WITH DRAVET SYNDROMETanaka, Yasuyoshi - Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan Ishikawa, Mitsuru - School of Medicine, Keio University, Shinjuku-Ku, Japan Higurashi, Norimichi - School of Medicine, Jikei University, Minato-Ku, Japan Okano, Hideyuki - School of Medicine, Keio University, Shinjuku-Ku, Japan Hirose, Shinichi - Department of Pediatrics School of Medicine, Fukuoka University, Fukuoka, JapanDravet syndrome (DS), a devastating type of infantile-onset epilepsy that presents with cognitive deficits and autistic traits, is caused by a mutation in SCN1A, which encodes the -subunit of the voltage-gated sodium channel, Nav1.1. αSeveral types of mutations, including nonsense, frame-shift, and missense mutations, located at different sites in SCN1A have been identified in patients with DS. Excitatory/inhibitory (E/I) imbalance in the cerebral cortex can cause central nervous system disorders, such as DS (or epilepsy). However, the underlying cellular disturbance remains ill-defined owing to the reliance of available knowledge on animal models that are not readily transferable to the syndrome in humans. Recently, we generated induced pluripotent stem cells (iPSCs) derived from a DS patient (D1) with a c.4933C>T substitution in SCN1A predicted to cause truncation in the fourth homologous domain of the protein (p.R1645*). Moreover, to elucidate the mechanism of neurodegeneration in DS caused by c.4933C>T mutation, we performed gene correction in D1 iPSCs using TALEN (transcription-activator-like effector nuclease)-mediated genome editing, generating D1 TALEN iPSCs. In this study, we generated excitatory or inhibitory neurons by employing direct in vitro conversion of iPSCs through the overexpression of specific transcription factor cocktails as a novel approach for neuronal differentiation. These cells were seeded on multi-electrode array (MEA) systems, which is a measuring device with multiple electrodes integrated in a cell culture dish, and the spontaneous neuronal activity was recorded. We present data comparing excitatory or inhibitory neurons derived from healthy (WT), DS (D1), and isogenic control (D1 TALEN) iPSCs that were measured using MEA systems. We found differences in physiological activity between WT and D1 inhibitory neurons. The spontaneous firing of D1 inhibitory neurons was significantly impaired compared to that in WT neurons. Inhibitory neurons derived from iPSCs with the c.4933C>T mutation in SCN1A (D1) showed fewer spontaneous spikes in the burst. This phenotype was rescued in the isogenic control inhibitory neurons (D1 TALEN) to a level equal to that in the WT neurons.F-4043PHYSIOLOGICAL ANALYSIS OF PATIENT SPECIFIC IPSC DERIVED DOPAMINERGIC NEURONS; PARKINSON’S DISEASE MODELINGPark, Zewon - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju-Si, Korea Bang, Yunsu - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Choi, Juhyun - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Lee, Jonggu - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Yi, Jung-Yeon - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Kim, Kisoon - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Oh, woo Yong - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Korea Jung, Jehyuk - Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Cheongju, KoreaNeurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and other age related dementias are incurable diseases. Parkinson’s disease (PD) is the second most common neurodegenerative disease which is a slowly progressive disease and is the result of degeneration of neuromelanin(NM)-containing dopamine neurons(DA) in the substantia nigra par compacta. There are many PD disease models, but in vitro models are limited which have difficulties in mimicking PD. Here, we observed clinical potentials of iPSC derived dopaminergic neurons from PD patients. First data showed distinct differentiation markers were expressed each steps of differentiation, such as progenitor, development and maturation. Dopamine secretion was confirmed by LC-MS method. Next step, Drug test was performed using Patch-Clamp detection. Action-potential is detected at –57mV. The frequency decreased at 10 uM of L-Dopa but two action potentials appeared simultaneously, 30 μM L-Dopa increased the frequency compared to the action potential of the control in the current clamp mode. In the voltage clamp mode, the frequency of dopaminergic neurons were increased and the amplitude of L-Dopa increased at 30uM. However, the 60uM L-dopa increased the frequency compare to 10uM L-dopa but decreased level than 30uM L-dopa. Dose dependent dopamine releasing result using high sensitive ELISA showed similar releasing patterns as patch clamp data. As a result, dopaminergic neurons differentiated from PD patient iPSCs were showed recovery results as L-dopa treatment, so patient specific iPSCs can be useful tools for modeling Parkinson’s disease.

527POSTER ABSTRACTSFunding Source: This research was supported by a grant (17181MFDS432) from Ministry of Food and Drug Safety in 2018F-40453D MODEL OF PARKINSONS DISEASE SPECIFIC-DOPAMINERGIC NEURONS FOR HIGH-THROUGHPUT PHENOTYPING AND DRUG SCREENINGDexter, Dwayne - Director, Mimetas, Gaithersburg, MD, USA Chiang, Chiwan - Model Development, Mimetas, Leiden, Netherlands Wilschut, Karlijn - Model Development, Mimetas, Leiden, Netherlands Lanz, Henriette - Model Development, Mimetas, Leiden, Netherlands Trietsch, Sebastiaan - Hardware R&D, Mimetas, Leiden, Netherlands Joore, Jos - Management, Mimetas, Leiden, Netherlands Vulto, Paul - Management, Mimetas, Leiden, NetherlandsParkinson’s disease (PD) is a neurodegenerative disease which is characterized by the progressive loss of dopaminergic neurons in the substantia nigra which leads to motor dysfunction. The high heterogeneity of the disease and the lack of preclinical models that recapitulate the features of PD prohibiting successful development of neuroprotective therapies. By using induced pluripotent stem cell (iPSC) technology we have a powerful tool in hand to develop in vitro PD disease models applicable in the field of regenerative medicine. Here, we describe the development of a human 3D Parkinson’s disease model of iPSC-derived dopaminergic neurons in Mimetas’ OrganoPlate® to discover new drug candidates. The culture platform contains 96 tissue chips with 3D microfluidic channels that support the development of neuronal cultures of patient-derived iPSCs towards an in vitro phenotype resembling that of dopaminergic neurons in vivo. Here, intermediate human neuroepithelial stem cells were seeded pre-mixed in Matrigel into the OrganoPlate® and differentiated for 4 weeks towards functional dopaminergic neurons. Immunostaining confirmed the presence of dopaminergic neurons in a 3D environment. Imaging of calcium fluctuations showed the spontaneously electrophysiological activity of the neurons, which is a characteristic feature of mature neurons in vivo. Mito-functionality assay has been developed to screen potential mitochondrial dysfunction. We developed an industrial quality microfluidic PD model containing, mature, differentiated, midbrain-like dopaminergic neurons. This PD model will be applied in high-throughput screening to identify mitochondrial dysfunction in PD patients and finally used to develop new candidate neuroprotective agents for PD patients.F-4047IN VIVO SIMULTANEOUS TRANSCRIPTIONAL ACTIVATION OF MLTIPLE GENES IN THE BRAIN USING CRISPR-DCAS9-ACTIVATOR TRANSGENIC MICEGao, Ni - Institute of Neuroscience, Chinese Academy of Science (CAS), Shanghai, ChinaDespite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.F-4049REPROGRAMMING ENRICHES FOR SOMATIC CELL CLONES WITH SMALL SCALE MUTATIONS IN CANCER-RELATED GENESKosanke, Maike - Hannover Medical School, Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover, Germany Osetek, Katarzyna - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Haase, Alexandra - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Wiehlmann, Lutz - Hannover Medical School, Department of Paediatric Pneumology and Neonatology, Hannover, Germany Davenport, Colin - Hannover Medical School, Department of Paediatric Pneumology and Neonatology, Hannover, Germany Chouvarine, Philippe - Hannover Medical School, Department of Paediatric Pneumology and Neonatology, Hannover, Germany Merkert, Sylvia - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Wunderlich, Stephanie - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Opel, Ulrike - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Menke, Sandra - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany Dorda, Marie - Hannover Medical School, Department of Paediatric Pneumology and Neonatology, Hannover, Germany

528POSTER ABSTRACTSMielke, Samira - Hannover Medical School, Department of Paediatric Pneumology and Neonatology, Hannover, Germany Steinemann, Doris - Hannover Medical School, Institute of Human Genetics, Hannover, Germany Schambach, Axel - Hannover Medical School, Institute of Experimental Haematology, Hannover, Germany Martin, Ulrich - Hannover Medical School, Research Laboratories for Biotechnology and Artificial Organs, Hannover, GermanyRecent studies observed high mutational load in iPSCs, which is largely derived from their parental cells, but as yet it is unknown whether reprogramming may enrich for individual mutations that pre-exist in the parental cell population. We have derived 30 human iPSC clones from neonatal and aged individuals under comparable conditions. High accuracy exome and amplicon sequencing showed that all analyzed small scale variants pre-existed in their parental cell population. We demonstrate that individual mutations present in small subpopulations of parental cells become enriched among iPSC clones during reprogramming. Evaluation of the variant impact and gene function in cellular processes and in cancer development imply a potential role of some of those mutations as putatively actionable driver mutations. Especially as some of the enriched, putatively actionable mutations affect genes of cell death / survival, cell cycle control, and pluripotency, somatic cells carrying such a mutation might experience a selective advantage during reprogramming. In view of the various common characteristics of (pluripotent) stem cells and cancer stem cells, the same mutations are likely to account for an increased tumor risk. Notable, on average, iPSCs of aged donors carry a higher number of these putatively actionable mutations. The reprogramming-associated selection for individual potentially pathogenic or carcinogenic mutations that have been acquired during lifetime may impact the clinical value of patient-derived iPSCs.F-4051SAFER AND MORE STABLE IPSC GENERATED USING DOGGYBONE DNA VECTORSThornton, Christopher - Centre for Bioscience, Manchester Metropolitan University, Manchester, UK Caproni, Lisa - R&D, Touchlight Genetics, London, UK Karbowinczek, Kinga - R&D, Touchlight Genetics, London, UK Tite, John - R&D, Touchlight Genetics, London, UK McKay, Tristan - Centre for Bioscience, Manchester Metropolitan University, Manchester, UKThe application of induced pluripotent stem cell (iPSC)-derived cells in clinical trials is in its infancy but the potential is vast. A key asset of iPSCs is the ability to apply autologous cell therapies, but to date most current or approved clinical trials are using fully characterised allogeneic or non-allogeneic cell banks alongside immunosuppressive drugs. Until now, all current or approved clinical trials worldwide utilise iPSC generated using EBNA1 expressing plasmids containing the OriP sequence to maintain a self-replicating episome. These vectors are amplified in bacterial hosts and contain bacterial DNA motifs recognised by the transfected cell’s innate and intrinsic interferon host defense responses. Moreover, the continued forced expression of the Epstein-Barr virus EBNA1 protein is known to cause widespread alterations in gene expression as well as elevated oxidative stress and DNA damage. All of these have potentially significant implications for the safe clinical use of iPSC generated using OriP/EBNA1 plasmid episomes. We describe efficient iPSC reprogramming by applying equivalent gene sequences transiently expressed from Doggybone DNA (dbDNA) vectors free of OriP/EBNA1 sequences, bacterial motifs and produced in a chemically defined, low endotoxin, cGMP compliant manufacture. In direct comparator experiments with the current state-of-the-art OriP/EBNA1 episomes, dbDNA vectors produced iPSC colonies with the same efficiency but dbDNA-iPSC displayed evidence of greater stability in terms of maintenance of pluripotency. Mechanistic evaluations showed that the persistence of OriP/EBNA1 episomes resulted in elevated STAT1/IFN signalling in iPSC when compared to dbDNA generated iPSC. This in turn resulted in increased spontaneous differentiation and slowing of the cell cycle in OriP/EBNA1-iPSC. We propose a potential that utilising dbDNA vectors presents a safer and more stable approach to iPSC production and development.F-4053INDUCING ANTI-TUMOR IMMUNITY WITH DENDRITIC CELL REPROGRAMMINGPires, Cristiana F - Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden Rosa, Fabio - Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden Kurochkin, Ilia - Skolkovo Institute of Science and Technology, Skolkovo Institute of Science and Technology, Moscow, Russia Humbert, Marion - Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland Ferreira, Alexandra - Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden Reis e Sousa, Caetano - Immunobiology Laboratory, The Francis Crick Institute, London, UK Hugues, Stephanie - Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland Pereira, Carlos-Filipe - Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, SwedenCell fate reprogramming towards pluripotency or unrelated somatic cell-types have highlighted the plasticity of adult somatic cells, providing new technologies to generate any desired cell type for regenerative medicine. Dendritic cells (DCs) are professional antigen presenting cells specialized in the recognition, processing and presentation of antigens to T-cells. In particular, the mouse conventional DC type 1 (cDC1) subset excels on the ability to perform antigen cross-presentation on MHC-I, a critical step for inducing cytotoxic responses. We hypothesized that the unique properties of cDC1s could be induced in unrelated cell-types, allowing the direct control

529POSTER ABSTRACTSof immune responses with cell reprogramming. Here, the requirements to induce cDC1s were investigated using a combinatorial overexpression of transcription factors (TFs) in Clec9a-tdTomato mouse fibroblasts. In the hematopoietic system, this reporter system specifically marks the DC lineage, including all cDC1 cells. We have identified Pu.1, Irf8 and Batf3 as sufficient and necessary to induce Clec9a reporter activation, establish DC morphology and activate a cDC1 transcriptional program in mouse fibroblasts. The overexpression of the three factors ignites the expression of cDC1 markers, major histocompatibility complex (MHC) class I and II molecules and co-stimulatory receptors. Functionally, induced DCs (iDCs) secrete inflammatory cytokines and engulf, process and present antigens to CD4+ T cells. Remarkably, iDCs have established the competence for cross-presentation, a hallmark of cDC1s, eliciting antigen-specific CD8+ T-cell responses. We have shown that intra-tumoral vaccination of B16-OVA and EG7-OVA syngeneic tumor mouse models with iDCs conferred protection against tumor growth. Moreover, injection of iDCs increased infiltration of antigen-specific CD8+ T cells in the tumor, as well as promoted cytotoxic profile of T-cells in the draining lymph nodes. Hence, we have shown for the first time that antigen presentation can be programmed by a small combination of TFs providing evidence that mouse immune cells and triggered immune responses can be modulated through cell reprogramming. Collectively our work represents a platform for future development of cancer immunotherapies based on DC reprogramming.Funding Source: This project was co-funded by the Knut and Alice Wallenberg Foundation, Cancerfonden, Crafoord Foundation, VR and FCT, Portugal (SFRH/BPD/121445/2016, SFRH/BD/130845/2017, CENTRO-01-0145-FEDER-030013).F-4055DISTINCT ROUTES CONVERGE ON A UNIFYING TRANSITION LOGIC TO ESTABLISH NAÏVE PLURIPOTENCYSilva, Jose - Welcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, UK Stuart, Hannah - Welcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, UKTo decipher how cellular identity is instructed by interplay between transcription factors and signals, we employ defined reprogramming systems in which genetic and signalling parameters can be independently varied and successfully transitioning cells isolated. We show that naïve pluripotency can be induced from EpiSCs along transcriptionally and mechanistically distinct routes. Relative to development, one route moves forward, with productive cells acquiring mesodermal signature prior to naïve pluripotency induction. In contrast, another route goes backwards, transcriptionally resembling the earlier embryo and gaining its greater developmental potency. Nevertheless, these distinct trajectories reach the same endpoint, demonstrating surprising flexibility for the establishment of a single identity from a single origin. We reconcile route differences, revealing precise Oct4 expression as a unifying, essential and sufficient feature. We propose that fine-tuned regulation of this “transition” factor underpins multidimensional access to the naïve identity. This offers a conceptual framework for the understanding of cell identity transitions.Funding Source: Wellcome Trust (WT101861)F-4057RHINO INDUCED PLURIPOTENT STEMCELLS TO RESCUE THE NORTHERN WHITE RHINOTelugu, Narasimha - Stemcell Core Facility, Max-Delbrück Center for Molecular Medicine (MDC), Berlin, Germany DIecke, Sebastian - Stemcell Core Facility, Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany Drukker, Micha - Induced Pluripotent Stem Cell Unit, Helmholtz Zentrum München, Munich, Germany Hildebrandt, Thomas - Reproduktionsmanagement/Reproduction Management, Leibniz-Institut für Zoo- und Wildtierforschung (IZW) im Forschungsverbund Berlin e.V., Berlin, Germany Hayashi, Katsuhiko - Developmental Stem Cell Biology, Kyushu University, Fukuhoka, Japan Galli, Cesare - Laboratorio di Tecnologie della Riproduzione, AVANTEA srl, Cremona, Italy Lazzari, Giovanna - Laboratorio di Tecnologie della Riproduzione, AVANTEA srl, Cremona, ItalyThe northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal at present. Only two females have survived and even before the death of the last male, both were incapable of natural reproduction. The only way to create a self-sustaining population of the NWR is the use of assisted reproduction technologies combined with emerging stem cell technologies. A crucial step in this process is gaining knowledge of the regulation of pluripotency and differentiation. Here, we show that pluripotency states of northern white rhino (NWR) and the human are comparable in terms of growth conditions and certain marker gene expression. We derived intrinsically renewing NWR iPSCs and show that these cells can differentiate to progeny of the germ layers when applying protocols optimized for human iPSCs. Finally, we ectopically express Bcl2 and show that ground state cells can integrate in mouse epiblast-stage chimeras. Moreover, we were able to establish ESC from southern white rhinos which also can be differentiated into three germ layers. We hope by using these “gold standard“ pluripotent stem cells to establish a germ cell differentiation protocol. Taken together, we have generated NWR iPSCs and ESCs, as well as validated both pluripotent maintenance as well as germ layer differentiation in this critically endangered mammal, with a view to optimizing germ lineage differentiation. This could one day provide a tool to help restore the NWR using cryopreserved somatic cells.

530POSTER ABSTRACTSF-4059DCM-TIME-MACHINE: A NOVEL METHOD TO DETECT TRANSCRIPTION RETROSPECTIVELYBoers, Ruben - Developmental Biology, Erasmus Medical Center, Rotterdam, Netherlands Boers, Joachim - Developmental Biology, Erasmus Medical Center, Rotterdam, NetherlandsA doxycyclin inducible DCM-RpolIIb fusion gene is able to methylate actively transcribed genes. Bacterial DCM methylation introduced in the murine epigenome is detected using MeD-seq together with the endogenous CpG methylation and does not affect gene expression. DCM methylation is propagated to daughter cells enabling retrospective analysis of gene expression. Using the murine small intestine as a validation model we were able to trace stem cell expression. DCM methylation changes during pulse labeling generated distinct DCM methylation profiles corresponding to specific intestinal cell types.F-4061A SCALABLE AND PHYSIOLOGICALLY RELEVANT SYSTEM FOR HUMAN INDUCED PLURIPOTENT STEM CELL EXPANSION AND DIFFERENTIATIONWang, Ou - Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE, USA Lei, Yuguo - Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE, USA Li, Qiang - Bioengineering, Harvard University, Boston, MA, USA Lin, Haishuang - Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, USAHuman induced pluripotent stem cells (iPSCs) and their derivatives are needed in large. However, scalable and cost-effective manufacturing of high quality iPSCs and their derivatives remains a challenge. In vivo, cells reside in a 3D microenvironment that has cell-cell and cell-extracellular matrix interactions, sufficient nutrients and oxygen, and minimal hydrodynamic stress. The current iPSC culturing methods provide highly-stressed microenvironments, leading to low efficiency. For instance, iPSCs typically expanded 4-fold/4 days to yield ~2.0x10^6 cells/mL with current 3D suspension culturing. These cells occupy ~0.4% of the bioreactor volume. To our best knowledge, the largest culture volume for iPSCs is less than 10 liters. There is a critical need to develop new culture technologies to achieve the iPSCs’ potential.We report a novel technology that can overcome all limitations of current methods and provide a physiologically-relevant culture microenvironment. iPSCs are processed into and cultured in microscale alginate hydrogel tubes termed stress-free intratubular cell culture (SFIT) that are suspended in the cell culture medium (Figs. 1A and B). The hydrogel tubes create free microspaces that allow cells to interact with each other and expand. Meanwhile, they protect cells from hydrodynamic stresses in the culture vessel and confine the cell mass 10 passages) of iPSCs without uncontrolled differentiation and chromosomal abnormalities. Cultures between batches and cell lines were very consistent. iPSCs in SFIT had high viability, growth rate (1000-fold/10 days/passage in general) and yield (~5x10^8 cells/mL microspace). The expansion per passage (e.g. up to 4200-fold/passage) and volumetric yield are much higher than current methods, which significantly reduce the culture volume and time, and the production cost, making large-scale cell production technically and commercially feasible. iPSCs could be efficiently differentiated into various cells in SFIT. Additionally, other human cells, such as T cells, could also be efficiently cultured in this technology. Two SFIT-based automated bioreactors for producing autologous and allogenic iPSCs and their derivatives are under developing. This technology has high potential to address the cell manufacturing challenge.F-4063CHARACTERIZATION OF HUMAN ADIPOSE STEM CELLS GROWING IN OPTIMIZED SERUM-FREE XENO-FREE MEDIASidhu, Harpreet K - Biopharma, Rinati Skin LLC, Beverly Hills, CA, USA Newman, Nathan - Biopharma, Rinati Skin LLC, Beverly Hills, CA, USA Talavera-Adame, Dodanim - Biopharma, Rinati Skin LLC, Beverly Hills, CA, USAAdipose stem/stromal cells (ASCs) are a type of adult mesenchymal stem cells (MSCs) that can be easily isolated from adipose tissue. ASCs can be cultured in vitro over several passages, but conventional culture conditions involve the use of human or animal serum to enhance cell attachment and promote cell survival and proliferation. Since these cells or their conditioned media, which contains many beneficial secretory factors, are increasingly being used in regenerative medicine, it is crucial to eliminate serum supplements to ensure scalability and prevent adverse immune reactions. We aim to formulate a unique serum-free media (SFM) that supports the growth and expansion of human ASCs in vitro, and, to characterize their cell viability, proliferation, differentiation potential and secretory factor profiles. ASCs were isolated from adipose tissue obtained from human donors through liposuction. These cells were then cultured in vitro using either media supplemented with 2% human serum or SFM developed in our laboratory. Morphology, viability, proliferation rates, differentiation potentials, and cytokine profiles were evaluated in both groups of cells. Our SFM supported the growth and proliferation of ASCs up to passage 14 in vitro while maintaining their mesenchymal stem cell characteristics and stemness. These cells were successfully cultured in monolayers and three-dimensional scaffolds, and, were able to differentiate into adipocytes, chondrocytes and osteocytes. In addition, they secreted less pro-inflammatory chemokines, unlike ASCs cultured in conventional media supplemented with 2% human serum. These ASCs or their

531POSTER ABSTRACTSconditioned media, which exhibits biological activity, may thus be safely used for downstream cosmetological or regenerative applications without the risk of viral transmission or adverse immunological reactions.F-4065HIGH THROUGHPUT BANKING AND CARDIAC DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS IN A SUSPENSION BIOREACTORMueller, Sabine C - Biomedical Data Science, Fraunhofer-Institute for Biomedical Engineering (IBMT), Würzburg, Germany Steeg, Rachel - EBiSC, Fraunhofer Research UK Ltd, Glasgow, UK Meiser, Ina - Cryobiotechnology, Fraunhofer Institute for Biomedical Engineering (IBMT), Sulzbach, Germany Bur, Stephanie - Cryobiotechnology, Fraunhofer Institute for Biomedical Engineering (IBMT), Sulzbach, Germany Fischer, Benjamin - TBC, Fraunhofer Institute for Biomedical Engineering (IBMT), Würzburg, Germany Neubauer, Julia - Project Centre for Stem Cell Process, Fraunhofer Institute for Biomedical Engineering (IBMT), Würzburg, Germany Ebneth, Andreas - Neuroscience, Janssen Research and Development, Beerse, Belgium Zimmerman, Heiko - IBMT, Fraunhofer Institute for Biomedical Engineering (IBMT), Sulzbach, GermanyHuman induced pluripotent stem cells (hiPSCs) are one of the most promising options for regenerative medicine, disease modeling, toxicity testing and drug discovery. However, the current reliance on ‘traditional’ culture systems for propagation of hiPSCs and differentiated progenitors are intensive in time, labour and reagent costs, restricting investments into actual research using these tools. To provide, at low cost, the needed quantity of high-quality hiPSCs and hiPSC derived differentiated cells, technologies and methodologies for cell culture have to fundamentally evolve. To address this problem, in conjunction with the European Bank for induced Pluripotent Stem Cells (EBiSC), Fraunhofer-IBMT have established a novel system for culturing and differentiating hiPSCs in suspension. Here, the bioreactor system BioLevitatorTM (now CERO from OLS OMNI Life Science) was used in combination with innovative impeller-free vessels to avoid high shear forces and culture multiple hiPSC lines in parallel, simultaneously. Disease-unaffected EBiSC hiPSC lines were thawed and directly inoculated with alginate microcarriers and after only one passage between thaw and harvest, at least 54 vials with 1x106 cells per vial were banked for each line. Media volume, matrix volume, staff time and overall culture time were significantly reduced in comparison to traditional culture methods. All banked lines showed viability, recovery, adhesion rate, expression of pluripotency markers and karyology comparable to banking using traditional two-dimensional culture systems. Additionally, using this bioreactor system, bulk production of spheroids and subsequent differentiation of two hiPSC lines into autonomously contracting cardiac clusters was achieved. This generated 7500 cardiac clusters in just 10 ml of differentiation medium for each hiPSC line. Cardiac clusters successfully expressed lineage-specific markers Cx43 and -Actinin and at least 93% αwere spontaneously contracting. In conclusion, we present a multifunctional system for expansion and banking, as well as cardiac differentiation of hiPSCs in suspension. EBiSC2 aims to integrate such valuable tools to overcome current limitations in generating large numbers of high-quality cells from many donors in parallel.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-4067EFFICIENT OSTEOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS BY DIRECT DELIVERY OF LINEAGE-SPECIFIC TRANSCRIPTION FACTORSPark, Ju Hyun - Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon-si, Korea Lee, Jaeyoung - Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon-si, KoreaMesenchymal stem cells (MSCs) have a great potential for regenerative medicine due to their multi-lineage differentiation potency. Especially, many studies have revealed that the osteogenic differentiation of MSCs can contribute to the treatment of bone diseases such as osteoporosis. However, the precise control of their lineage commitments has been regarded as a key issue in the therapeutic applications because MSCs have multipotency and many factors are involved in MSC differentiation to each lineage. Although some groups have reported the methods to control MSC differentiation by delivering lineage-specific genes, these approaches have limitations such as clinical incompatibility or low efficiency. We hypothesized that direct delivery of osteoblast-specific transcription factors (TFs) can induce osteogenesis of MSCs without any genetic manipulations. In this study, we produced Osterix, a typical osteogenic TF, as recombinant protein using E.coli expression system. To intracellularly deliver the recombinant protein, a silkworm-derived cell-penetrating protein, 30Kc19 was fused to the Osterix. The fusion protein was efficiently delivered into the cells without any significant cytotoxicity and some of the protein was located at nucleic space. By alizarin red S and alkaline phosphatase staining, we determined that osteoblast differentiation of MSCs was significantly augmented when the cells cultured in osteogenic medium were repeatedly treated with Osterix-30Kc19. Quantitative real-time PCR analysis also demonstrated the expression of osteoblast-specific markers such as Runx2 and Osteocalcin was increased by Osterix-

532POSTER ABSTRACTS30Kc19. Consequently, our strategy to directly deliver Osterix using cell-permeable 30Kc19 protein is anticipated to hold great potential for treating bone disorders in stem cell-based therapies.Funding Source: This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (Ministry of Science and ICT) (2017M3A9C6031798).F-4069CAR-T MEDIA DEVELOPMENT: NOVEL FORMULATIONS FOCUSED ON IMPROVED TRANSDUCTION EFFICIENCY AND PRESERVATION OF RELEVANT T CELL SUBPOPULATIONS.Martinez Becerra, Francisco J - Research and Development, Nucleus Biologics, San Diego, USA Ghassemi, Saba - Perelman School of Medicine, University of Pennsilvania, Philadelphia, PA, USA Heo, David - Perelman School of Medicine, University of Pennsilvania, Philadelphia, PA, USA Master, Alyssa - Research and Development, Nucleus Biologics, San Diego, CA, USA O'Connor, Roddy - Perelman School of Medicine, University of Pennsilvania, Philadelphia, PA, USAAdoptive immunotherapy involves patient T cell expansion in optimal conditions for the purpose of re-infusing their progeny as therapy. This calls for the generation and characterization of novel media supplements and formulations to boost the progress of the cell therapy market by providing physiologically relevant protein sources in concentrations suitable for biological activity. PhysiologixTM XF Human Growth Factor Concentrate (hGFC) is a cGMP, xeno-free serum replacement that replaces supplements such as fetal bovine serum (FBS) or human serum (HS). In this study, T cell media such as OpTmizer and X-VIVO 15 were supplemented with 5% human serum or 2% PhysiologixTM XF. In addition, RPMI 1640 with 10% FBS was compared due to its prevalent use in non-clinical settings. T cells from three healthy donors were activated with CD3/CD28 Dynabeads and then cultured for 14 days in varying media formulations. Proliferative capacity was assessed with flow cytometry. PhysiologixTM XF showed increased potency relative to human serum when used along X-VIVO 15 or OpTmizer media. Differentiation status was assessed using CCR7 and CD45RO to distinguish T cell subpopulations. We detected an increase in naïve and central memory populations when cells were cultured in PhysiologixTM XF compared to human serum. These phenotypes are highly relevant for the outcome of patients undergoing CAR-T cell therapy. To further define the advantage of our supplement in generating therapy ready products, we compared the transduction efficiency of a lentiviral-GFP in OpTmizer, X-VIVO 15 or RPMI supplemented with 2% PhysiologixTM XF or 5% HS at different multiplicities of infection (MOIs). A higher transduction efficiency was consistently observed when using PhysiologixTM XF at a high MOI of 4 down to a MOI of < 1. These results were also observed for anti-GD-2 virus. This increased transduction efficiency and preservation of T cell subpopulations can translate into a higher number of transfusable CAR+ cells with relevant phenotypes which allow for reduced overall costs and better clinical outcomes. The ability of anti-GD-2 CAR-T cells expanded in medium supplemented with PhysiologixTM XF to directly kill target cells and control tumor burden in an aggressive leukemia xenograft model is discussed.POSTER III - EVEN 19:00 – 20:00PLACENTA AND UMBILICAL CORD DERIVED CELLSF-2002A QUEST FOR OPTIMIZING IN VITRO HMSCS EXPANSION PRECONDITIONS: A COMPARATIVE PROTEOMIC ANALYSIS EMPHASIZES 3D-SPECIFICATIONS WITH 1% OXYGENKumar, Sanjay - Center for Stem Cell Research, Christian Medical College, Vellore, India Sundaram, Balasubramanian - Centre For Stem Cell Research, Christian Medical College, Vellore, India Balasankar, Ananthi - Department of Proteomics, Clinbiocare Technology, Chennai, IndiaHuman Mesenchymal Stem/Stromal Cells (hMSCs) has the potential for numerous regenerative clinical applications. But so far there is no defined culture condition, as a standard protocol for the in vitro expansion of hMSCs. We hypothesized that the common norm of two-dimensional (2D) culture expansion with atmospheric oxygen levels (21% O2) could alter the properties of the de novo hMSCs. Thus, we attempted to provide natural homely 3D-microenvironmental space with 1% physiological oxygen. In this study, we have done the comparative proteomic analysis of human Wharton’s Jelly-derived MSCs (hWJMSCs) cultured in four different culture conditions (2D Normoxia (21% O2), 2D Hypoxia (1% O2), 3D Normoxia (21% O2) and 3D Hypoxia (1% O2) by two-dimensional gel electrophoresis and mass spectrophotometry. The comparative proteomic data revealed that among the culture groups of 2D Normoxia Vs. 2D Hypoxia 12 protein spots; 2D Normoxia Vs. 3D Normoxia 20 protein spots; 2D Normoxia Vs. 3D Hypoxia 23 protein spots; 2D Hypoxia Vs. 3D Hypoxia 16 protein spots and between 3D Normoxia Vs. 3D Hypoxia 5 protein spots were differentially expressed. Further identification of 20 spots by LC-MS/MS revealed that several filaments network proteins including plectin, vimentin, keratin, tropomyosin, and myosin isoforms differentially expressed between 2D and 3D culture conditions; insisting on the role of the microenvironment in cytoskeleton reorganization, cell shape, cell binding, focal adhesion and migration of hMSCs. Interestingly, 3D Hypoxia (1% O2) cultured hWJMSCs showed enhanced expression of galectin-1 than

533POSTER ABSTRACTSother culture conditions. Galectin-1 involved in the inhibition of immune effector cells in an inflammatory setting. These findings along with relatively superior multilineage-differentiation capability, better in vivo performing exosomes packed with key therapeutic molecules, elevated pluripotency-associated gene expression, greater levels of immunomodulatory cytokine expression, higher expression of genes related to cellular migration is implicating that 3D Hypoxia (1% O2) culture condition maintains better intrinsic properties of hMSCs and also primes them with enhanced immunomodulatory potential, in turn providing better culture condition for expansion of hWJMSCs for clinical applications.Funding Source: We would like to thank UGC for SRF to Balasubramanian. DBT, Govt of India for Ramalingaswami Fellowship to Sanjay and Research grants. Also appreciate Core support grants from CSCR. Technical help from CSCR core is acknowledged.F-2004TISSUE-SPECIFIC INTERACTIONS OF HUMAN MESENCHYMAL STROMAL CELLS (HMSCS) IN MODULATING PERIPHERAL B LYMPHOCYTES MATURATIONLee, Wei - Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Wang, Li-Tzu - Departments of Obstetrics/Gynecology, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan Hsu, Pei-Ju - Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan Lee, Yu-Wei - Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan Liu, Ko-Jiunn - National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan Yen, B. Linju - Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, TaiwanB lymphocytes, also called B cells, are a type of white blood cell within the immune system. Circulating in the blood and activated by pathogens/antigens, peripheral B cells play an important role in both physiological and pathological conditions, being responsible for the humoral immunity of the adaptive immune system as well as acting as professional antigen-presenting cells (APCs) to critically orchestrate T cell responses. Despite the importance of B cells, surprisingly little is known about their interactions with mesenchymal stromal cells (MSCs), a type of multilineage somatic progenitor cell with strong immunomodulatory properties. First isolated from the bone marrow (BM), MSCs have since been found to exist in numerous adult as well as fetal-derived tissues/organs. Given that the BM is the site for B lymphocyte development, interactions between B cells and MSCs from this source are likely to be different from MSCs derived from other sources. We therefore were interested in the interactions of B cells to BM-MSCs and term placenta-derived MSCs (P-MSCs). We found that P-MSCs but not BM-MSCs affect the maturation of activated B cell at several stages. Moreover, similar findings were seen in an in vivo mouse model of B cell activation. Our preliminary findings demonstrate that B cell interactions with MSCs can differ significantly depending on the source of MSCs. Studies are ongoing to elucidate the mechanisms mediating tissue-specific MSCs interactions with this important population of the adaptive immune system.ADIPOSE AND CONNECTIVE TISSUEF-2006HUMAN ADIPOSE-DERIVED STROMAL CELL PROTEOMEKoh Belic, Naomi - School of Life Sciences, University of Technology Sydney, Australia Bicknell, Fiona - School of Life Sciences, University of Technology Sydney, Australia Santos, Jerran - School of Life Sciences, University of Technology Sydney, AustraliaHuman adipose-derived stromal cells are often marketed as stem cell treatments despite the fact that there is little to no scientific evidence of their safety let alone efficacy. The number of predatory clinics offering these unproven stem cell treatments is rising, with clinics across the globe in countries such as Australia, Japan, Canada and the United States. Despite their wide use, surprisingly little is known about these cells. This study is the first to characterise the proteome of human adipose-derived stromal cells and compares both fresh and cryogenically preserved samples. 10 patients underwent liposuction and adipose-derived stromal cells were extracted and expanded. Whole cell lysates, membrane bound fractions and extracellular vesicles were analysed on Q Exactive Plus Orbitrap mass spectrometer, resulting in the detection of more than 5000 proteins per patient sample. Whole cell lysates provided vital insight into cellular function, while analysis of membrane bound proteins provided an extensive catalogue of cell surface markers that are useful for antibody-based assay development. Extracellular vesicles were investigated as adipose-derived stromal cells secrete them in substantial quantities and they are known to play a significant role in cancer, injury healing and immune suppression. As expected, there was variation in the detectable proteome across patients, however almost half of the detected proteins were conserved across all 10 patient samples, providing unique insight into the phenotype and functionality of these cells. 27 secreted cytokines were also investigated through the utilisation of a Bioplex multiplex immunoassay as cytokines facilitate cellular communication of immune signals. With adipose-derived stromal cell use rising in unproven stem cell treatments it is crucial that more research is conducted to identify exactly how these cells function. This novel study provided fundamental insight by identifying and comparing the proteome of fresh and cryogenically preserved human adipose-derived stromal cells.

534POSTER ABSTRACTSF-2008CHARACTERIZATION OF HUMAN BONE MARROW- AND ADIPOSE TISSUE DERIVED- MESENCHYMAL STROMAL CELLS IN AN IMPROVED ANIMAL COMPONENT-FREE CULTURE MEDIUMWagey, Ravenska - Research and Development, STEMCELL Technologies Inc, Vancouver, BC, Canada Bertram, Karri – Research and Development, STEMCELL Technologies Inc, Vancouver, Canada Elliott, Melissa – Research and Development, STEMCELL Technologies Inc, Vancouver, Canada Christie, Jennifer – Research and Development, STEMCELL Technologies Inc, Vancouver, Canada Thomas, Terry – Research and Development, STEMCELL Technologies Inc, Vancouver, Canada Eaves, Allen - STEMCELL Technologies Inc, Vancouver, Canada Szilvassy, Stephen - Research and Development, STEMCELL Technologies Inc, Vancouver, Canada Louis, Sharon - Research and Development, STEMCELL Technologies Inc, Vancouver, CanadaWe characterized Mesenchymal Stromal Cells (MSCs) derived from human bone marrow (BM) and adipose (AD) tissues in an improved animal component-free (ACF) culture medium (MesenCult™-ACF Plus) and in medium containing fetal bovine serum (FBS). Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs). MSCs from both tissue sources were plated at 1.5–3x10³ cells/cm² in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MesenCult™-ACF Plus (MACF-P) was evaluated by co-culture of BM-MSCs with Peripheral Blood Mononuclear Cells (PBMCs). The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days. Total CFU-F per 10^6 AD-SCs and per 10^6 BM-MNCs was comparable in MACF-P and FBS containing medium (AD-SCs: 236 ± 127 vs. 268 ± 120 [mean ± SEM; n=3]; BM-MNCs: 37 ± 7 vs. 32 ± 5 [mean ± SEM; n=6] in MACF-P and FBS-containing media, respectively). However, the average fold-expansion of AD-MSCs at each subculture over 9 passages was significantly higher in MACF-P medium (12.2 ± 0.9; mean ± SEM; n=5) than in FBS-containing medium (3.8 ± 0.3; mean ± SEM; n=5; p<0.05). Similarly, the average fold-expansion of BM-MSCs at each subculture over 6 passages was also significantly higher in MACF-P (11.6 ± 1.2; mean ± SEM; n=5) than in FBS-containing medium (3.9 ± 0.4; mean ± SEM; n=5; p<0.05). Both AD-MSCs and BM-MSCs cultured in MACF-P differentiated more robustly in vitro under the appropriate conditions into adipogenic, osteogenic and chondrogenic cells compared to the same cells cultured in FBS-containing medium. Co-culture of BM-MSCs with PBMCs in MACF-P indicated that proliferation of CD4+ T cells is suppressed in a cell concentration-dependent manner when BM-MSCs were added in vitro. These data demonstrate that both BM-MSCs and AD-MSCs can be efficiently derived and expanded in MACF-P medium under complete ACF conditions and that BM-MSCs cultured in MACF-P medium exhibit immunosuppressive activity in vitroF-2010SUV39H1 AND CITED2 ARE NEGATIVE REGULATORS OF HUMAN ADIPOGENESISZhao, Yuanxiang - Biological Sciences, California State Polytechnic University at Pomona, CA, USA Tan, Lun - Research and Development, Inheritor Cell Technology, Arcadia, CA, USA Trujillo, Amparo - Biological Sciences, California State Polytechnic University at Pomona, CA, USA Ibili, Esra - Research and Development, Inheritor Cell Technology, Arcadia, CA, USAHuman adipogenesis is the process through which uncommitted human mesenchymal stem cells (hMSCs) differentiate into adipocytes. Understanding the molecular and cellular regulation of human adipogenesis may provide a way to prevent and/or treat obesity and obesity related diseases. Based on a siRNA high throughput screen, a list of genes whose expression knock-down by its siRNAs led to enhanced adipogenic differentiation of hMSCs were uncovered, including SUV39H1 and CITED2. SUV39H1 encodes a histone methyltrasferase that catalyze H3K9Me3, while CITED2 encodes a CBP/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2. The role of SUV39H1 and CITED2 during human stem cell fate commitment has never been studied. Here we report that both SUV39H1 and CITED2 are down regulated during adipogenic differentiation of hMSCs. Single knockdown of either SUV39H1 or CITED2 by siRNA significantly promoted adipogenic differentiation of hMSCs by both accelerating fat accumulation in individual adipocytes and increasing the number of adipocytes, despite slight reduction in total cell numbers as compared to control treatment. In addition, both siSUV39H1 and siCITED2 were able to promote hMSCs to commit to adipogenic lineage even in the presence of osteogenic inducing media, which normally only induces osteogenic differentiation of hMSCs in the absence of siSUV39H1 or siCITED2. Furthermore, siSUV39H1 significantly enhanced adipogenic differentiation of human dermal fibroblasts, which was further improved by siCITED2. Concomitantly, expression of CEBP and PPAR , two master αγregulators of adipogenesis, were both markedly increased in siSUV39H1 and siCITED2 individually transfected cells as compared to siControl cells. Finally, simultaneous knockdown of both SUV39H1 and CITED2 demonstrated an accumulative effect on promoting both adipogenesis and the expression of CEBP and PPAR . Taken together, this study demonstrates αγthat SUV39H1 and CITED2 are both negative regulators of human adipogenesis, whose expression knockdown exerts

535POSTER ABSTRACTSaccumulative effect in promoting adipogenic differentiation efficiency through both accelerated adipogenic maturation and enhanced adipogenic fate commitment, and such effect was mediated by promoting the expression of CEBP and PPAR .αγFunding Source: NIH grant (1SC3GM116720) 2016 - 2020, PI Yuanxiang ZhaoMUSCULOSKELETAL TISSUEF-2012IN VITRO REGULATION OF MOUSE MUSCLE STEM CELL DIFFERENTIATION BY ESTROGEN (E2) AND EICOSAPENTAENOIC ACID (EPA)Lacham-Kaplan, Orly - Mary MacKillop Institute of Health Research, Australian Catholic University, Melbourne, Australia Camera, Donny - Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, Australia Hawley, John - Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, AustraliaMyogenesis involves interactions between signal transduction pathways and transcription factors resulting in the expression of myogenic regulatory factors (Mrfs). In-vivo, skeletal muscle mass is maintained by E2 and polyunsaturated fatty acids (ie: EPA) due to their anti-inflammatory characteristics and ability to modify the expression of Mrfs suggesting that they should be incorporated into in-vitro models of skeletal muscle differentiation. However, inconsistent reported effects of E2 and EPA on in-vitro myogenesis discourage their use in skeletal muscle regenerative studies. Using C2C12 mouse myoblasts to reiterate myogenesis in-vitro, the present study provides (a) time-dependent (0-120hr) immunofluorescence imaging and targeted gene expression (qPCR) analyses during differentiation in 10nM E2, 50μM EPA and 10nM E2/50μM EPA treatments, (b) a bench mark global transcriptome (Illumina HiSeq 2500 RNA-seq) at 48hr differentiation in E2 or EPA. We show that E2 augments myotube formation and fusion index at 48 and 120hr, while EPA and E2/EPA treatments significantly inhibit tube formation and reduce fusion index (P<0.001). E2 increases the expression of ER and the signal transduction pathway genes MAPK and αAkt (P<0.01) within 1hr from treatment leading to increased expression of Mrfs genes (MyoD, myogenine, Myh1) and the membrane fusion gene Tmeme8c (P<0.01) at 48hr or 120hr. EPA and E2/EPA treatments have no effect on E2 receptor expression and significantly reduce the expression of genes associated with the myogenic pathway and membrane fusion (P<0.01). Cell number and viability are not affected by treatment. We also show that a whole transcriptome profile following E2 treatment is similar to control profile but significantly different to EPA treatment with 4,000/15,000 transcripts expressed differently (P<0.05-0.001). The latter are associated primarily with the myogenic, muscle contraction, E2 metabolism, fat metabolism and signal transduction pathways. Results from the present study suggest that in-vitro regulation of myogenesis is significantly different between E2 and EPA. What directs the cellular response to augment or attenuate in-vitro myogenesis by E2 and EPA or which cellular mechanism(s) they use to regulate myogenesis remains equivocal and requires further research.Funding Source: ACURF project grant 2016 to Orly Lacham-KaplanF-2014THE IDENTIFICATION OF SMALL MOLECULES REGULATING PAX7 EXPRESSION IN SKELETAL MUSCLE PROGENITOR CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLSChien, Peggie J - Molecular Biology Interdepartmental Program, University of California, Los Angeles, CA, USA Xi, Haibin - Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA, USA Pyle, April - Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA, USAEmbryonic and fetal skeletal muscle progenitor cells (SMPCs) and adult skeletal muscle stem cells (satellite cells, SCs), identifiable by their expression of transcription factor PAX7, participate in developmental myogenesis and postnatal muscle regeneration, respectively. The lack of ability to maintain pure SMPC and SC populations in vitro poses great challenges for analyzing these populations in detail, both for better understanding of the basic biology of human skeletal muscle development as well as for potential translational utilization in cell replacement therapies including for muscle disorders such as Duchenne Muscular Dystrophy. Human pluripotent stem cells (hPSCs) present a source for modeling human skeletal myogenesis and generating SMPCs and SCs for regenerative medicine. However, current hPSC directed myogenic differentiation protocols always generate heterogenous cultures comprised of both myogenic and other cell types. Moreover, different myogenic commitment states coexist within the myogenic subpopulations themselves, and PAX7 expression gets lost over continuous passaging in culture. To address these concerns, we developed a high-throughput small molecule screen and identified small molecule candidates that maintain PAX7 expression in hPSC-derived SMPCs. We hypothesize that these novel small molecules modulate survival or self-renewal pathways in SMPCs and enable expansion while maintaining stemness. In conjunction with developing strategies to expand SMPC populations, we have utilized single-cell RNA-sequencing to identify cell surface markers that not only enable isolation of myogenic cells from other cell types, but also enrich for PAX7-expressing SMPCs while excluding those of more differentiated MYOD- or MYOG-positive myogenic states. This is crucial considering that previous studies in mice and human clinical trials have shown that only cells of high stemness with the ability to self-renew are capable of conferring long-term regeneration in vivo. In summary, our combined approaches to purify and sustain

536POSTER ABSTRACTShPSC-derived SMPCs will enable detailed mechanistic studies of SMPCs and potentially SCs derived from hPSCs and human development and facilitate the evaluation of potential myogenic populations for cell-based therapies to treat muscle diseases.Funding Source: Support from the UCLA Center for Duchenne Muscular Dystrophy Ruth L. Kirschstein National Research Service Award T32AR065972, CIRM Grant DISC2-10695, and NIH/NIAMS Grant R01 AR064327.F-2016G-RESIST: A DEEPLY QUIESCENT STATE ADOPTED BY STEM CELLS IN RESPONSE TO DISTANT LIVER INJURYTiwari, Rajiv L - Stem Cell and Regenerative Medicine, University of Southern California, Alhambra, CA, USA Cheng, Pin-Chung - Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Rodgers, Joseph - Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USASkeletal muscle stem cells (MuSCs), also known as satellite cells, are the central factors responsible for muscle repair and regeneration. In normal conditions, these cells reside in host muscle in a quiescent state. An injury to their host muscle induces these cells to enter the cell cycle and divide, a process known as activation. Previous work has shown that following muscle injury, or isolation-induced activation, MuSCs from adult mice require ~50 hours to complete cell division, MuSCs from humans require ~70 hours. In our previous work, we found that speed of MuSC activation was plastic and could be regulated. We identified that the HGFA-HGF-cMet-mTORC1 pathway could induce MuSCs into a primed state of quiescence in which, following muscle injury, they are able to activate much more quickly than normal, in ~35 hours. We termed this primed quiescent state “GAlert”. Because the systemic factor responsible for stimulating MuSCs into GAlert, Hepatocyte Growth Factor Activator (HGFA), is primarily produced and regulated by the liver, we decided to test how MuSCs could response to changes in liver function. We performed liver injury by partial hepatectomy (PHX) and, to our surprise, found that 2 days after PHX, MuSCs required significantly longer time, ~70 hours, to activate compared to the MuSC from animals that received a sham surgery. Furthermore, PHX had a significant impact on muscle regeneration. Animals that were subject to muscle injuries several days after PHX displayed profound impairments and delays in muscle regeneration. Interestingly, these PHX-induce defects in MuSC function and muscle regeneration were transient, 30 days after PHX MuSC function and muscle regeneration returned to normal. Using mouse genetic models, we have identified that these PHX-induce MuSCs functional changes are independent of HGFA levels or activity. We propose that we have identified a novel functional response by MuSCs in which they enter a, reversible, deeply quiescent state and have low muscle regenerative potential. We putatively term this state “GResist”. It is well known that patients with liver disease and impaired liver function have a potently impaired ability to heal wounds and injuries, anywhere on their body, we suspect that this systemic, stem cell, response to liver injury is a mechanism that underlies this connection.Funding Source: The Donald E. and Delia B. Baxter Foundation to J.T.R. and by grants from the Glenn Foundation for Medical Research, the NIH (P01 AG036695 and R01 AR062185F-2018PLURIPOTENT STEM CELL DERIVED MUSCLE FUNCTION ASSAY FOR DUCHENNE AND BECKERS MUSCULAR DYSTROPHYDobson, Samori - California State University, San Marcos, California State University, San Marcos, CA, USADuchenne’s Muscular Dystrophy (DMD) and Becker’s muscular dystrophy (BMD) are X-linked diseases that are caused by a mutation in the DMD gene that will eventually progress to fatality. Recent evidence suggests that SkM cells die in DMD due increased mechanical stress, which activates NADPH-oxidase (NOX2) and reactive oxygen species (ROS) production. This in turn activates calcium influx via TRPC channels, causing calcium overload and inhibition of mitochondrial function ultimately leading to SkM lost, reducing ambulatory and respiratory function, and a reduced life expectancy to 35 years. Currently, there is no effective cure for either DMD or BMD and current therapies at best, only delay progression of symptoms. Our lab has recently developed a platform, kinetic image cytometer (KIC), that will accelerate drug discovery for skeletal muscles disorders by creating a more precise/reproducible translational assay utilizing hPSC- disease-derived cells to quantify physiological functions of DMD. For this study, we devised aims to develop normal, DMD, and BMD hPSC-SkMs, develop methods to quantify physiological function of hPSC-SkM, and develop methods to stress the hPSC-SkMs cultures. Both healthy and DMD-derived hPSCs will be differentiated into mature skeletal muscle myotubes and examined for DMD-specific defects. Furthermore, we have identified a variety of clinically relevant DMD phenotypes that are amenable to high-throughput utilizing automated digital microscopy, including elevated levels of creatine kinase, calcium transient, contractile motion and ROS. While our data supports that calcium reuptake is disrupted more in BMD and DMD lines further investigation is needed to determine the correlation of electrode stimulation strength with overall cell fatigue, shock and degradation. Additionally, data suggest that the organization of the myotubes, regardless of cultivation densities, could be a key contributing factor to the rapid degradation in DMD. This platform is a proof-of concept for DMD but could be applicable to many other myopathic disorders.

537POSTER ABSTRACTSF-2020CELLULAR REPROGRAMMING REJUVENATES HUMAN SYNOVIAL FLUID-DERIVED STEM CELLS FOR ENHANCING PROPERTIES AND FUNCTIONSLi, Wan-Ju - Orthopedics and Rehabilitation, University of Wisconsin-Madison, WI, USA Jiao, Hongli - Orthopedics, University of Wisconsin-Madison, WI, USA Walczak, Brian - Orthopedics, University of Wisconsin-Madison, WI, USA Lee, Ming-Song - Orthopedics, University of Wisconsin-Madison, WI, USACellular senescence is one of the major concerns associated with the use of mesenchymal stem cells (MSCs) for regenerative applications. Among different methods that have been developed to address the issue, rejuvenation of MSCs through cellular reprogramming has been shown particularly promising. In this study, we reprogrammed human synovial fluid-derived MSCs (SF-MSCs) into induced pluripotent stem cells (iPSCs), which were then induced to differentiate into MSCs to establish iPSC-MSC lines. Activities of iPSC-MSCs and their parental SF-MSCs were compared to determine if reprogramming is able to alter their age-associated properties and functions. Our results showed that compared to SF-MSCs, iPSC-MSCs exhibited similarity in the morphology and immunophenotype but an increase in cell proliferation, osteogenesis, and chondrogenesis. Functional enrichment analysis indicated that senescence-related hallmark gene sets, including those associated with reactive oxygen species, the p53 pathway, and senescence-associated secretory phenotype-related inflammatory cytokines and chemokines were down-regulated in reprogrammed MSCs compared to non-reprogrammed ones. We also demonstrated that senescence-associated -galactosidase activity and the βexpression of p53 and p21CIP1 were attenuated in iPSC-MSCs compared to those in SF-MSCs. Cellular reprogramming resulted in an increase in the expression of telomerase reverse transcriptase and telomerase activity, in turn lengthening telomere and a decrease in the expression of p53 and p21CIP1 through regulation of -catenin in iPSCs and their derived-βMSCs compared to non-reprogrammed cells. Taken together, this study demonstrates that cellular reprogramming is able to rejuvenate MSCs through -catenin-mediated telomere βlengthening and p53/p21CIP1 attenuation, providing a potential solution to the concern of MSC senescence.Funding Source: Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01 AR064803.F-2022HUMAN SKELETAL STEM CELLS FROM ACUTE FRACTURES MAINTAIN SELF-RENEWAL AND DIFFERENTIATION INDEPENDENT OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAID)Steininger, Holly M - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Mountain View, CA, USA Ambrosi, Thomas - Surgery, Stanford University, Stanford, CA, USA Goodnough, Henry - Surgery, Stanford University, Stanford, CA, USA Hoover, Malachia - Surgery, Stanford University, Stanford, CA, USA Bellino, Michael - Surgery, Stanford University, Stanford, CA, USA Bishop, Julius - Surgery, Stanford University, Stanford, CA, USA Gardner, Michael - Surgery, Stanford University, Stanford, CA, USA Longaker, Michael - Surgery, Stanford University, Stanford, CA, USA Chan, Charles - Surgery, Stanford University, Stanford, CA, USAWhether NSAIDS hinder human fracture healing by direct action on osteochondrogenic differentiation remains unknown. While animal studies suggest NSAIDs are deleterious to osteoblast differentiation in vitro and in vivo, human clinical trials have yet to demonstrate an effect of NSAIDs on fracture healing. We have recently identified a purified bona fide skeletal stem cells (SSCs) as the source for osteogenic and chondrogenic cell populations, which can be isolated from intact bones and fracture sites and sought to investigate functional changes by NSAID administration. Human SSCs (Podoplanin+, CD146- CD73+ CD164+) and osteoprogenitors (hOPs (MSCs); Podoplanin- CD146+) were FACS-isolated from human fractures. Purified SSCs were cultured in the presence or absence of three common NSAIDs and analyzed subsequently for colony-forming units (CFU-F). After osteogenic and chondrogenic differentiation, cell avidity for Alizarin Red and Alcian Blue was quantified by spectrophotometry. Experiments were performed in triplicate on n =4 healthy adults (age. 44-85 years). Physiologic and supra-physiologic concentrations, as well as initial pulsed and continuous NSAID administration, failed to inihibit hSSC (and hOPs) differentiation into osteoblasts and chondrocytes. NSAID administration did not affect clonogenicity of hSSCs. In contrast and as expected from results of previous studies, mouse SSCs showed impaired osteochondrogenic differentiation potential when treated with physiological concentrations of NSAIDs. Microarray gene expression data of mouse and human SSCs suggests this could be due to the lack of Cox-2 expression in hSSCs. We demonstrate that hSSCs prospectively isolated

538POSTER ABSTRACTSfrom human fracture sites maintain functionality independent of NSAID application. Our results add evidence to emerging clinical data suggesting NSAID administration for post-operative analgesia is safe for fracture healing.CARDIAC TISSUE AND DISEASEF-2024THE ROLE OF TNNI3K IN ADULT MAMMALIAN HEART REGENERATIONGan, Peiheng - USC Stem Cell, University of Southern California (USC), Charleston, SC, USA Patterson, Michaela - Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA Sucov, Henry - Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, USAAdult mammalian heart regeneration capacity is thought to be extremely limited. All fetal and newborn mouse cardiomyocytes are mononuclear and diploid, and most become polyploid during the first postnatal week. Our lab showed that mononucleated diploid cardiomyocytes (MNDCMs) retain regenerative capacity, and that the percentage of this subpopulation in the adult mouse heart is variable and can be surprisingly high. We reported that cardiac troponin I-interacting kinase (TNNI3K) is a key regulator of MNDCM frequency. Nonetheless, a detailed mechanism of how Tnni3k regulates MNDCM frequency is still lacking. Studies from another group showed that Tnni3k mediates adult cardiomyocyte oxidative stress response. We therefore hypothesize that similar pathways might explain how Tnni3k affects cardiomyocyte ploidy. We found that reducing postnatal oxidative stress by transgenic expression of mitochondrial catalase increased MNDCM frequency. MNDCM frequency was further increased with Tnni3k deletion. We also found that several common human TNNI3K polymorphisms compromise kinase activity, which may influence variation in MNDCM frequency and heart regenerative capacity in the human population.F-2026CARDIOMYOCYTES FROM A SET OF ISOGENIC HUMAN IPSC LINES HARBORING MUTATIONS IN KCNH2 DISPLAY DIFFERING RESPONSES TO A TORSADOGENIC COMPOUNDDavis, Richard P - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Brandao, Karina - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands van den Brink, Lettine - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Miller, Duncan - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Sala, Luca - Laboratory of Cardiovascular Genetics, Istituto Auxologico Italiano, Milan, Italy van Meer, Berend - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Grandela, Catarina - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Mol, Mervyn - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Mummery, Christine - Anatomy and Embryology, Leiden University Medical Center, Leiden, Netherlands Verkerk, Arie - Department of Medical Biology, Academic Medical Center, Amsterdam, NetherlandsCongenital long QT syndrome type 2 (LQT2) is one of the most common genetic cardiac channelopathies leading to life-threatening arrhythmias and sudden cardiac death. A broad range of phenotypes are associated with this cardiac channelopathy and nearly 500 variants identified in the gene KCNH2, encoding the ion channel hERG, have been associated with this disease. Studies have shown that the location of the mutation within KCNH2 is an important determinant of arrhythmic risk in LQT2 patients, with patients harboring mutations in the pore-loop region at higher risk of cardiac events than those with mutations located in other regions of the channel. An understanding of how the mutation type and location contributes to phenotypic variability could lead to improved intragenotype risk stratification of patients. Patient-derived human induced pluripotent stem cells (hiPSCs) can be used to investigate the pathogenicity of mutations identified in LQT2 patients. However, as these lines are from different individuals, each harbours additional genetic variants that may functionally influence the disease phenotype observed and complicate their use for broad intragenotype risk stratification. To overcome this issue, we have generated a set of isogenic hiPSC lines by introducing KCNH2 mutations into a wild-type hiPSC line using Crispr/Cas9 technology. Analysis of cardiomyocytes derived from these isogenic lines (hiPSC-CMs) show a prolongation of both action potential and field potential duration when compared to wild type, consistent with the cardiomyocyte phenotype observed in the LQT2 patient iPSC lines. Treating the cardiomyocytes from this isogenic set of KCNH2-variant hiPSC lines to a hERG channel-blocking compound revealed different sensitivities between the lines, with the hiPSC-CMs containing a mutation in the pore-loop region more susceptible to the proarrhythmic effects of the drug. These findings demonstrate the potential of hiPSC-CMs to correlate the functional effects of KCNH2 mutations with patient risk when using genetically-matched lines, and offers new opportunities for predicting risk in patients as well as in the development of patient-specific pharmacotherapy.Funding Source: This work was supported by a European Research Council (ERC) Starting Grant (STEMCARDIORISK; #638030) and a VIDI fellowship from the Netherlands Organisation for Scientific Research (ILLUMINATE; #91715303).

539POSTER ABSTRACTSF-2028INTRA-MYOCARDIAL TRANSPLANTATION OF HUMAN PLURIPOTENT STEM CELL-DERIVED CARDIAC LINEAGE CELLS INTO NOD SCID GAMMA (NSG) MICE: PRECLINICAL ENGRAFTMENT AND SAFETY EVALUATIONOommen, Saji - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Emrich, Tobin - Regenerative Medicine, Mayo Clinic, Rochester, NY, USA Cantero Peral, Susana - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Secreto, Frank - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Theobald, Genevieve - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Moore, Timothy - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Deng, Wei - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Rasmussen, Boyd - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Wobig, Joan - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Hajyusuf, Aliya - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Nelson, Timothy - Regenerative Medicine, Mayo Clinic, Rochester, MN, USAHeart disease is a major cause of death and morbidity in the western world. Transplantation remains the only viable long-term treatment for patients with progressed cardiac failure. For this reason, stem cell-derived cardiomyocyte-based therapies have emerged as a potential therapeutic approach to initiate cardiac remodeling and delay transplantation. Cardiac stem cell therapy challenges include the ability of cells to engraft into myocardium, mitigation of stem cell-induced tumor formation, and life threatening arrhythmias. A population of Day 20 human iPSC-derived cardiac lineage cells (iPSC-CL) was produced though 3D culture and injected into the myocardium of NSG mice. Mice were divided into 4 groups that received either the placebo (cardiomyocyte media) or increasing doses of Day 20 iPSC-CL; placebo group (n=10), and treatment groups receiving iPSC-CL at 0.3x106/animal (n=25), 1.0x106/animal (n=25), and 3.0x106/animal (n=25). A telemetry device was implanted into 6 mice from each group to detect ventricular ectopic activity. Each subject was assessed for iPSC-CL engraftment, tumor formation, and cardiac side effects. No test article-related effect was observed on the heart rate or PR, QRS, or QT intervals at any dose level. Ventricular premature complexes were observed in mid and high dose groups early in the study; however, these events were not detected at week 14. All surviving mice were sacrificed 14 weeks post-treatment. Hematology, biochemistry, and troponin data collected at that time indicated no significant abnormalities or variability between groups. Histologically, no evidence of tumor in the myocardium or other ectopic sites was observed and immunohistochemistry indicated mature grafts of viable transplanted iPSC-CL present within the myocardium. Results demonstrate 3D culture production of Day 20 iPSC-CL achieved engraftment within the myocardium, the cells maintained a high survival rate in a dose-dependent manner, induced no teratomas, and incurred no significant risk factors. Results from our study suggest that grafting high-purity iPSC-CL at a dose of 3.0x106 does not form tumors and persistent ventricular arrhythmias are not observed.Funding Source: Todd and Karen, Wanek Family Program for Hypoplastic Left Heart SyndromeF-2030EFFECT OF CONNEXIN 43 AND SGSM3 THROUGH HIF-1 -MEDIATED MODULATION IN RAT BONE ΑMARROW-DERIVED MESENCHYMAL STEM CELLSJung, Seung Eun - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Kang, Misun - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Lee, Jiyun - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Park, Jun-Hee - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Song, Byeong-Wook - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Choi, Jung-Won - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Lim, Soyeon - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Kim, Sang Woo - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Kim, Il-Kwon - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Lee, Seahyoung - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, Korea Hwang, Ki-Chul - Institute for Bio-Medical Convergence, Catholic Kwandong University, Incheon, KoreaConnexin 43 (Cx43) contributes gap junction-mediated communication as a gap junction protein but has shown channel independent functions. Many reports have suggested that Cx43 regulates other cellular mechanisms, including cell cycles, differentiation, and proliferation. Recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. In the previous study, we found that Cx43-interaction protein, small G protein signaling modulator 3 (SGSM3) plays a critical role in stress cells. Moreover, their interaction plays a key role in Cx43 internalization for connexin turnover in cardiomyocytes of infarcted hearts. Here, we investigated for SGSM3, a potential partner of Cx43, in an attempt to identify for enhancing survival markers in bone marrow-derived mesenchymal stem cells (MSCs). Cx43 co-immunoprecipitated analysis identifying two proteins, and gap junction proteins were predicted that SGSM3 was highly

540POSTER ABSTRACTScorrelated with Cx43 in GeneMANIA network analysis. Results of Hif1a and Sgsm3 siRNA knockdown experiments suggest that SGSM3 possibly plays a role in the cellular response to stress or ischemia with Cx43 dependently on Hif1 . In conclusion, αthese data demonstrate a role for SGSM3 in Cx43 endocytic trafficking and further substantiate its role in Cx43 turnover. This knowledge of SGSM3-mediated regulation of Cx43 may help to identify a novel therapeutic target to counteract the loss of Cx43 or impairment of Cx43-GJIC that disrupt normal cell functions and are associated with many human diseases.FundingSource:Thisstudywasfundedby2018R1D1A1B07049416 and NRF-2015M3A9E6029519.F-2032MIR148A FAMILY REGULATES CARDIAC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS BY INHIBITING THE DLL1-MEDIATED NOTCH SIGNALING PATHWAYHu, Shijun - Institute for Cardiovascular Science, Soochow University, Suzhou, China Miao, Shumei - Institute for Cardiovascular Science, Soochow University, Suzhou, China Fang, Xing - Institute for Cardiovascular Science, Soochow University, Suzhou, China Yu, You - Institute for Cardiovascular Science, Soochow University, Suzhou, China Han, Xinglong - Institute for Cardiovascular Science, Soochow University, Suzhou, China Wu, Hongchun - Institute for Cardiovascular Science, Soochow University, Suzhou, China Zhao, Zhen-Ao - Institute for Cardiovascular Science, Soochow University, Suzhou, China Wang, Yongming - School of Life Sciences, Fudan University, Shanghai, China Lei, Wei - Institute for Cardiovascular Science, Soochow University, Suzhou, ChinaMicroRNAs (miRNAs), as a type of naturally occurring RNAs, play important roles in cardiac physiology and pathology. There are abundant miRNAs showing multifarious expression patterns during the cardiomyocyte genesis. Here, we focused on the MIR148A family, which is composed of MIR148A, MIR148B and MIR152, three highly conserved miRNAs sharing same seed sequences. The expression levels of all MIR148A family members were progressively increased during the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes. The deletion of MIR148A family (MIR148A-TKO) resulted in decreased cardiomyocyte proportion after cardiac induction, which could be restored by the ectopic expression of MIR148A family members. The transcriptome analysis indicated that MIR148A family was the potential repressor of paraxial mesoderm after primitive streak formation. These miRNAs, in turn, promoted the differentiation of lateral mesoderm and cardiomyocytes. Furthermore, the NOTCH ligand Delta-like1 (DLL1) was validated as the target gene of MIR148A family, and the knockdown of DLL1 could promote cardiac differentiation of MIR148A-TKO hESCs. Thus, our results demonstrate MIR148A family promotes cardiomyocyte differentiation as a novel paraxial mesoderm repressor, which provides a new insight into heart development and cardiac differentiation.Funding Source: National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81770257, 81600218), Natural Science Foundation of Jiangsu Province (BK20170002)F-2034MESENCHYMAL STEM CELLS MIGHT BE MOBILIZED FROM BONE MARROW TO THE PERI-INFARCTION AREA BY SYSTEMIC HMGB1 ADMINISTRATION VIA SDF1-CXCR4 SIGNALING COMPLEX IN RAT INFARCTION MODELGoto, Takasumi - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Toyonaka, Japan Miyagawa, Shigeru - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Tamai, Katsuto - Department of Stem Cell Therapy Science, Osaka University Graduate School of Medicine, Osaka, Japan Matsuura, Ryohei - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Harada, Akima - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Ueno, Takayoshi - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Toda, Koichi - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Kuratani, Toru - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan Sawa, Yoshiki - Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, JapanHigh-mobility group box 1 (HMGB1) is reportedly a regenerative factor to mobilize CXCR4 positive bone marrow mesenchymal stem cells (BM-MSC) to the damaged tissue to promote tissue regeneration. However, actual mechanism of BM-MSCs recruitment remains uncertain. Whereas, SDF-1, which is a ligand of CXCR4, plays an important role in various stem cells migration, including BM-MSCs. So, we hypothesized that, in rat myocardial infarction (MI) model, systemic administration of HMGB1 could mobilize BM-MSCs to the damaged myocardium via SDF-1/CXCR4 signaling, leading to inhibition of left ventricular (LV) adverse remodeling. HMGB1 (3 mg/kg) or PBS (3 ml/kg) was administered intravenously for 4 days to 26 MI model rats, and cardiac function was evaluated by ultrasonography; antifibrotic action, by immunostaining. Using GFP-bone marrow transplantation (GFP-BMT) rat, BM-MSC recruitment was evaluated. Furthermore, in MI model rat, SDF-1 expression was evaluated by QT-PCR and histology. Four weeks after each injection, the LVEF was significantly improved in the HMGB1 group than in the control (HMGB1 vs. control; 48.6% ± 5.5% vs. 33.6% ± 5.4%; p < 0.01). LV remodeling exhibiting interstitial fibrosis, cardiomyocyte hypertrophy and

541POSTER ABSTRACTSdecrease of capillary density were significantly attenuated in the HMGB1 group compared with the control. On QT-PCR analysis, VEGF mRNA level was significantly higher in the HMGB1 group than in the control (border zone; 1.6 ± 0.6 vs. 1.1 ± 0.2; p = 0.02). In GFP-BMT rat MI model, confocal microscopy image showed that there were more GFP+/PDGFR + cells in the HMGB1 αgroup compared with the control. Some of those cells were present at vessel constituent cells in the peri-infarction area. On PCR analysis, SDF-1 expression significantly increased in MI model rat than in normal rat, with SDF-1 level of peri-infarction area was the highest in all area (MI rat vs. normal; peri-infarction area; 2.1 ± 0.4 vs 0.9 ± 0.1; p < 0.01). Histological analysis also showed SDF-1 expression along the border zone. Based on these findings, systemic administration of HMGB1 could inhibit LV adverse remodeling by enhancing BM-MSCs recruitment to the damaged myocardium, leading to LV functional recovery. This study also suggests that those BM-MSCs may be mobilized to the peri-infarction area via SDF-1/CXCR4 signaling complex.Funding Source: NoneF-2036THE ROLE OF GLUCOSE AS A PROMOTER FOR CARDIAC REGENERATIONFajardo, Viviana - Pediatrics/Division of Neonatology, University of California, Los Angeles, CA, USA Nakano, Haruko - Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA Shigeta, Ayako - Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA Nakano, Austin - Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USAHeart failure is the leading cause of death worldwide. Our focus is on non-genetic mechanisms by which cardiac regeneration can be lengthened or enhanced. Specifically, we are interested in the cyto-protective effects of glucose in cardiomyocyte growth, differentiation and proliferation and how this knowledge can be applied to regeneration therapies. Our preliminary data showed that glucose induces cardiomyocyte proliferation and inhibits cardiomyocyte maturation in human embryonic stem cells derived cardiomyocytes (hESC-CM) via the Pentose Phosphate Pathway in a dose dependent manner. Whether this pathway can be a therapeutic target for heart regeneration is unknown. Our hypothesis is that glucose promotes neonatal heart regeneration in a murine model. Non-Transmural cryoinjury was performed to the apex of the left ventricle in wild-type pups and cardiac specific overexpression of Glucose Transporter 1 Transgenic pups. In the acute phase (P1-P7), the level of cardiomyocyte cell proliferation was measured via flow cytometry analysis and immunostaining with PH3 and cTnnt. Glucose uptake by cardiomyocytes was measured by 18F-FDG assay and Glut1 immunostaining. In the chronic phase (P14, P21, P40), we quantified the level of fibrosis by histology (H&E and Picrosirius Red) and neovascularization by immunostaining with PECAM. Increased cardiomyocyte proliferation was observed in the Transgenic Glut1 pups. Myocardial glucose uptake declines from the muscular layer towards the trabecular layer, corresponding with maturation of the heart. We observed that Glut1 cardiomyocyte-specific overexpression resulted in improved cardiac repair compared to wild type (WT) mice at 21 days postnatally. Compared to wild-type, Glut1 hearts showed increased angiogenesis around the site of injury. We believe that increased in blood vessel formation is secondary to an increase in cardiomyocyte proliferation. This study would be the first to demonstrate the potential role of glucose as a promoter for cardiac regeneration and reveal a potential mechanism for congenital cardiomyopathy associated with diabetic pregnancy.Funding Source: UCLA CDI Junior Faculty Career Development GrantF-2038INVESTIGATION PATHOGENIC MECHANISMS OF PEDIATRIC DILATED CARDIOMYOPATHY BY USING PATIENT SPECIFIC INDUCED PLURIPOTENT STEM CELL MODELFu, Xuebin - Department of Surgery, University of Maryland, Baltimore, MD, USA Arfart, Mir Yasir - Department of Surgery, University of Maryland, Baltimore, MD, USA Sharma, Sudhish - Department of Surgery, University of Maryland, Baltimore, MD, USA Mishra, Rachana - Department of Surgery, University of Maryland, Baltimore, MD, USA Li, Deqaing - Department of Surgery, University of Maryland, Baltimore, MD, USA Kaushal, Sunjay - Department of Surgery, University of Maryland, Baltimore, MD, USADisease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-CMs could eliminate confounding species-specific and inter-personal variations, and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. Unlike in adult dilated cardiomyopathy (DCM) patients, demonstrated benefits of RAAS inhibition and -adrenergic receptor blockade are not observed in DCM children. Independent of classic G-protein coupled receptor signaling pathway, the pediatric DCM pathogenic progression maybe related to other causes such as microRNAs. Compared to current animal models, we applied DCM iPSCs which are reprogramed from pediatric patient blood as our DCM patient-specific model. Compared to healthy control iPSC-CM, the DCM iPSC-CM are intensively characterized. The potential microRNAs have been identified by using patient specific iPSC-CM model. Overexpression of the specific microRNA

542POSTER ABSTRACTSaggregated the DCM progression. On the other hand, the inhibition of the microRNA is reverse the disease progression. Our results enhanced current understanding of DCM pathogenic progression and provide novel therapeutic targets.F-2040ELEVATED EXPRESSION OF MITF IN FAR-INFRARED-PRECONDITIONED RAT BONE MARROW-DERIVED STEM CELLS PROTECTS CELL SURVIVAL AGAINST METABOLIC STRESSJeong, Yun-Mi - Division of Cardiology, Department of Internal Medicine, Kyung Hee University Medical Center, Seoul, Korea Kim, Weon - Division of Cardiology, Department of Internal Medicine, Kyung Hee University Hospital, Kyung Hee University, Seoul, KoreaBone marrow-derived stem cells (BMSCs) have been broadly investigated for treatment of ischemic heart diseases. However, there are many uncertainties that are prevailing against bench-to-bedside research related to BMSC-based therapy: like the optimal rout of cell transplantation, appropriate dosage, duration, safety, and efficacy of applications. Herein, we focus on a strategy for improving the low survival rate of BMSC after transplantation. To achieve this, we extend our previous study about the effects of preconditioning with far-infrared irradiation (FIR) on survival of BMSCs under condition of metabolic stress, such as oxidative stress, low temperature, and ischemic hypoxia condition. BMSCs were isolated and harvested from femur bone marrow of 6-week-old male Sprague-Dawley rat. To determine the effects of a FIR generator with an energy flux of 0.13 mW/cm2 on viability of rat BMSCs following metabolic stress, survival of BMSCs was measured by crystal violet staining and PI staining. FIR preconditioning was observed to significantly increase BMSC survival against H2O2, low temperature, and ischemic hypoxia condition. Of note, qRT-PCR and Western blot analysis demonstrated that FIR induced microphthalmia-associated transcription factor (MITF), BCL2, HIF-1a, mTOR and CD63 at mRNA and protein levels. It is well known that the mTOR or MITF is a master regulator of various important cellular responses, including protein synthesis, cellular growth, proliferation, autophagy, lysosomal function, and cell metabolism. In agreement with these observations, MITF-depleted BMSCs or rapamycin-treated BMSCs is decreased proliferation and survival of preconditioned BMSC by FIR-associated with up-regulation of MITF and mTOR. Overall, our results demonstrated for the first time that preconditioning with FIR can open new insights to help therapeutic efficacy of BMSCs, and the expression of MITF and mTOR-mediated cellular response is a key to understanding its role in survival or death of BMSCs after transplantation.Funding Source: NRF-2016R1A6A3A119334448ENDOTHELIAL CELLS AND HEMANGIOBLASTSF-2042ENDOTHELIAL CELLS DERIVED FROM HEMOPHILIA PATIENT-SPECIFIC IPSCS FOR SUSTAINED FVIII DELIVERY AND THE TREATMENT OF HEMOPHILIA AZhou, Ping - Internal Medicine, University of California Davis, Sacramento, CA, USA Rose, Melanie - Internal Medicine, University of California Davis, Sacramento, CA, USA Gao, Kewa - Department of Internal Medicine, University of California Davis, Sacramento, CA, USA Cortez-Toledo, Elizabeth - Department of Internal Medicine, University of California Davis, Sacramento, CA, USA Agu, Emmanuel - Department of Internal Medicine, University of California Davis, Sacramento, CA, USA Pan, Guangjin - Department of Burns and Plastic Surgery, The Third Xiangya Hospital of Central South University, Changsha, China Nolta, Jan - Department of Internal Medicine, University of California Davis, Sacramento, CA, USA Wang, Aijun - Department of Surgery, University of California Davis, Sacramento, CA, USAHemophilia A is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells in a non-diseased human being, endothelial cells hold great potential for development as a cell therapy for hemophilia A. We showed that hemophilia A patient-specific induced pluripotent stem cells (HA-iPSCs) could provide a renewable supply of endothelial cells. The HA-iPSC-derived endothelial cells were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA-iPSC-derived endothelial cells were retained in the animals for at least 10-16 weeks and maintained their expression of FVIII, GFP and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into neonatal hemophila A mice, these transduced HA-iPSC-derived endothelial cells significantly reduced blood loss in a tail-clip bleeding test. Thus, our studies provide proof-of-concept that HA-iPSC-derived endothelial cells can serve as a relatively long-term cell factory to deliver FVIII for the treatment of hemophilia A in not only adults but also newborns.Funding Source: This work is supported by the Milstein Medical Asian American Partnership Foundation, University of California Davis, NIH and California Institute for Regenerative Medicine training grant.

543POSTER ABSTRACTSF-2044ROBUST DIFFERENTIATION PROCEDURE INTO HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS AND THEIR PROPERTIESEnoki, Tatsuji - CDM Center, Takara Bio Inc., Kusatsu, Japan Tosaka, Yasuhiro - CDM Center, Takara Bio Inc., Kusatsu, Japan Kudo, Yoko - CDM Center, Takara Bio Inc., Kusatsu, Japan Kubo, Kaori - CDM Center, Takara Bio Inc., Kusatsu, Japan Okamoto, Sachiko - CDM Center, Takara Bio Inc., Kusatsu, Japan Yamashita, Jun - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Mineno, Junichi - CDM Center, Takara Bio Inc., Kusatsu, JapanPrimary human endothelial cells (ECs) such as HUVECs are widely used for vascularization study, various organoids formation or safety/toxicological test. However, primary ECs sometimes show uncontrollable lot-to-lot variances in these researches due to genetic diversity of derived donors, and thereby may lead to the difficulty of obtaining the reproducibility of the experiment. Human induced pluripotent stem cell-derived ECs (iPS-ECs) are expected to use for these research field as cell source having stable properties among manufacturing batches from single donor. Nevertheless, iPS-ECs have not been widely utilized in those experiments, since current iPS-ECs have some problems such as unstable differentiation from iPS cells or poor growth capacity. To overcome this, we have developed robust differentiation procedure into iPS-ECs in which continuous proliferation has been observed. We tested their ability of the procedure to differentiate into ECs from various iPS cell clones. All iPS-ECs from tested iPS cell clones showed more than 90% of both CD31 and CD144 positive population, which are typical endothelial cell surface markers. Then, these iPS-ECs were evaluated for their growth capacity for one month. Although all iPS-ECs exhibited almost similar endothelial cell-related genes and markers expression, there were big differences in the proliferation rate. The most proliferated iPS-ECs showed about one thousand fold expansion, even though one out of 6 iPS-ECs could little proliferate after differentiation (about a few fold expansion). In spite of the differences in growth capacity, all iPS-ECs retained both CD31 and CD144 positive population during observation period. We also confirmed whether iPS-ECs could be used for angiogenesis inhibition assay by observing the tube formation by measuring the fluorescence of calcein-labeled ECs. As a result, the inhibition of tube formation was observed in the addition of Wortmannin in a dose-dependent manner. Overall we have successfully developed iPS-ECs differentiation system. It can be overcome the lot variation problems of primary endothelial cells, making them suitable for various kinds of vascularization researches.F-2046LONG-TERM PRIMING BY THREE SMALL MOLECULES IS A PROMISING STRATEGY FOR ENHANCING LATE ENDOTHELIAL PROGENITOR CELL BIOACTIVITIESKim, Yeon Ju - Physiology/Medical Research, Pusan National University, Yangsan, Korea Kwon, Sang-Mo - Physiology, Pusan National University, Yangsan, KoreaEndothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs) play a pivotal role in vascular regeneration in ischemic tissues; however, their therapeutic application in clinical settings is limited due to the low quality and quantity of patient-derived circulating EPCs. To solve this problem, we evaluated whether three priming small molecules (tauroursodeoxycholic acid, fucoidan, oleuropein) could enhance the angiogenic potential of EPCs. Such enhancement would promote the cellular bioactivities and help to develop functionally improved EPC therapeutics for ischemic diseases by accelerating the priming effect of the defined physiological molecules. We found that preconditioning of each of the three small molecules significantly induced the differentiation potential of CD34+ stem cells into EPC lineage cells. Notably, long-term priming of OECs with the three chemical cocktail (OEC-3C) increased the proliferation potential of EPCs via ERK activation. The migration, invasion, and tube-forming capacities were also significantly enhanced in OEC-3Cs compared with unprimed OECs. Further, the cell survival ratio was dramatically increased in OEC-3Cs against H2O2-induced oxidative stress via the augmented expression of Bcl-2, a prosurvival protein. In conclusion, we identified three small molecules for enhancing the bioactivities of ex vivo-expanded OECs for vascular repair. Long-term 3C priming might be a promising methodology for EPC-based therapy against ischemic diseases.Funding Source: National Research Foundation (NRF- 2015M3A9B4066493).HEMATOPOIESIS/IMMUNOLOGYF-2050EFFICIENT MODIFICATION OF CCR5 LOCUS IN NONHUMAN PRIMATE INDUCED PLURIPOTENT STEM CELLS AND THEIR USE FOR PRODUCTION OF LYMPHOID AND MYELOID CELLSD’Sousa, Saritha S - Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA Park, Miae - Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA Kumar, Akhilesh - Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA Weinfurter, Jason - Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin,

544POSTER ABSTRACTSMadison, WI, USA Tao, Lihong - Wisconsin National Primate Research Center,, University of Wisconsin, Madison, WI, USA Kang, HyunJun - Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA Reynolds, Matt - Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA Slukvin, Igor - Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USAThe discovery that CCR5 serves as an R5-HIV-1 receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on targeting of CCR5. Among them are technologies based on iPSCs with edited CCR5 locus which have emerged as promising option for providing unlimited source of HIV-resistant blood cells for cellular therapies. To advance preclinical models for assessing iPSC-based therapies for AIDS, we established method for efficient CCR5 gene disruption in nonhuman primate (NHP) iPSCs using the CRISPR/Cas9 system. NHP T-iPSC lines were generated from peripheral blood T cells from Mauritian cynomolgus macaque (MCM) using Sendai Reprogramming kit. To successfully disrupt CCR5, we designed two CCR5 gRNAs to target sequences within exon 2, including 24-bp deletion region which was found to prevent functional CCR5 expression in NHPs. Following CCR5 editing in T-iPSCs using these gRNAs, we were able to achieve CCR5 disruption in 50% of colonies, 33% of which showed biallelic editing. NHP T-iPSCs with biallelic CCR5-disruption (CCR5mut-T-iPSC ) showed normal traits of pluripotent stem cells and efficiently differentiated into CD34+CD45+ multipotent hematopoietic progenitors (MHPs). MHP from CCR5mut-T-iPSCs were differentiated into CD45+CD14+MHCclassII+ macrophages in presence M-CSF and IL-1b for a week to yield about 6x106 macrophages from 1x10^6 MHPs. For T cell differentiation MHP were cultured with OP9-DLL4 in presence of IL7 and SCF. Resistance of immune cells from wild and CCR5mut NHP T-iPSCs to SIV infection was evaluated in vitro following challenge with SIV virus. Overall these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in NHP preclinical model.F-2052PRODUCTION OF ASSAY-READY HUMAN IPSC-DERIVED CD34+ CELLS, MONOCYTES, AND MESENCHYMAL STEM CELLSJacob, Sheela P - Stem Cell/ Primary Cell Dept, American Type Culture Collection, Gaithersburg, MD, USA Spencer, Michelle - Stem Cell/ Primary Cell Department, , American Type Culture Collection, Gaithersburg, MD, USA Desai, Himanshi - Stem Cell/ Primary Cell Department, , American Type Culture Collection, Gaithersburg, MD, USA Yin, Dezhong - Stem Cell/Primary Cell Department, , American Type Culture Collection, Gaithersburg, MD, USAThere is an unmet need for highly characterized and reproducible cell models for a wide range of applications including mechanisms of action, drug development and toxicity testing. Human induced pluripotent stem cells (iPSCs) have the capability to differentiate into all somatic cell types and therefore hold great promise for development into cell models for a wide range of applications. We have developed processes for the scalable generation of functional CD34+ cells, monocytes, and mesenchymal stem cells (MSCs) derived from iPSC lines. It is well documented that starting cell types and donor background play an important role in the efficiency of iPSCs to terminally differentiate. We screened four iPSC lines, three from bone marrow CD34+ cells and one foreskin fibroblast-derived iPSC line, for differentiation into CD34+, monocytes, or MSCs. All CD34+ cells-derived iPSC lines exhibited high efficiency for differentiation into CD34+ cells and MSCs, while the fibroblast-derived iPSC line favored differentiation into monocytes. To assess the differentiation potential of the iPSCs-derived cells, we demonstrated that CD34+ cells could be differentiated into erythroid cells, myeloid cells, and megakaryocytes while monocytes were capable of differentiating into dendritic cells and functionally active macrophages. Compared to primary MSCs, iPSCs-derived MSCs exhibited similar immunophenotypes and T-cell suppression activity as well as the ability to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, these iPSC-derived cells exhibited very stable phenotypes across multiple lots. The ability to obtain sufficiently large quantities of certain cell types, including CD34+ cells, for high throughput assays for drug and toxicity screening can be an issue. To address this issue, in this study we show that we have developed processes for the scalable and reproducible generation of high purity bio functionally active assay-ready iPSCs-derived CD34+ cells, monocytes, and MSCs. In addition, these stable phenotype cells remove the inherent variability of donor-derived material. The availability of these highly characterized cells for screening applications will serve as a catalyst for developmental studies, drug screening, and toxicity testing.F-2054A CORRELATIVE LIGHTSHEET AND ELECTRON MICROSCOPY APPROACH TO CHARACTERISE THE ADULT HEMATOPOIETIC STEM CELL NICHE IN ZEBRAFISHAgarwala, Sobhika - Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA Kim, Keun-Young - Centre for Research in Biological Systems, University of California at San Diego, San Diego, CA, USA Bushong, Eric - Center for Research in Biological Systems, University of California at San Diego, San Diego, CA, USA Ellisman, Mark - Center for Research in Biological Systems, University of California at San Diego, San Diego, CA, USA Drummond, Iain - Nephrology Division, Massachusetts General Hospital, Charlestown, MA, USA Tamplin, Owen - Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA

545POSTER ABSTRACTSHematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium in the dorsal aorta, then migrate to colonize the fetal liver, before finally homing and engrafting within the fetal bone marrow (BM). Within the BM, HSPCs are retained in a quiescent state in a complex microenvironment and divide occasionally to self-renew and to repopulate the blood lineages. While it is known that HSPCs are maintained by surrounding niche cells such as megakaryocytes, peripheral nerves, endothelial and mesenchymal stromal cells, the ultrastructure of these HSPCs within the niche is not well defined, as current imaging technology does not allow direct visualization of the fetal BM niche. Zebrafish have a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To track HSPCs during niche colonization, we used our previously validated HSPC-specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We could visualize the presumptive adult niche, kidney marrow (KM) in fixed larvae at 5 days post fertilization using a tissue clearing technique and detected ~100 HSPCs/larva. To precisely locate these rare HSPCs within the larger dense KM, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high resolution serial block-face scanning electron microscopy (SBEM) (XY=10.8 nm/pixel, Z=70 nm/pixel). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in a SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as a novel perivascular HSPC niche with a defined cellular assembly. In this perivascular site, a single HSPC was seen to simultaneously contact one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell, and other hematopoietic cells. This shows that within a distinct anatomical niche, multiple cell types can directly contact and regulate HSPCs. Our technique can be used as a general approach to identify the ultrastructure of single rare cells within dense tissues by using multiple imaging techniques. Further, we can now identify novel intercellular structures that form between an unperturbed HSPC in its endogenous perivascular niche.F-2056BIOACTIVE PEPTIDE SL-13R EXPANDS HUMAN UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLSNii, Takenobu - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Konno, Katsuhiro - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Shigeto, Mami - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Kaneyuki, Ayako - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Sumasu, Motoko - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Owaki, Toshiyuki - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, Japan Sugiyama, Daisuke - Incubation Center for Advanced Medical Science, Kyushu University, Fukuoka, JapanHematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized in clinical setting. Although umbilical cord blood (UCB) is a promising source of HSCs, insufficient number of HSCs in UCB limits their use, prompting need for ex vivo HSC amplification method. HSCs emerge from hemogenic endothelium in the aorta-gonads-mesonephros (AGM) region, and then expand in liver. We previously showed that Delta-like 1 (Dlk1) positive hepatoblasts are the niche-like cells of HSCs in fetal liver. Dlk1 is a transmembrane protein and also secreted as soluble protein known as fetal antigen 1 (FA1). We hypothesized that hepatoblasts regulate HSC self-renewal via DLK1/FA1. Several bioactive peptides were generated from extracellular domain of human DLK1 and a bioactive peptide, SL-13R, which has ability to expand HSCs was identified. UCB CD34+ cells were cultured with the SL-13R in xeno- and serum-free medium containing a cytokine cocktail for 9 and 14 days. The number of CD34+CD38- cells were increased by SL-13R compared to control (9 days: 1.5 fold, 14 days: 2.5 fold). Transplantation of CD34+ cells cultured with SL-13R into immunodeficient NOG mice confirmed that the cultured cells possess long-term reconstitution ability. Moreover, we performed second transplantation and showed the self-renewal ability of the cultured cells, but not untreated control cells. These results suggest that SL-13R has ability to expand HSCs and maintain its self-renewal capacity. To understand the mechanisms of HSC expansion by SL-13R, we investigated peptide binding proteins using biotin-conjugated SL-13R and identified PLEC and ERLIN2 proteins as interactors of SL-13R by LC-MS/MS and MASCOT analysis. PLEC knockdown UCB CD34+ cells cultured with bioactive peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of SL-13R. These results suggest that PLEC functions in HSC expansion promoted by SL-13R. In summary, we have identified a novel bioactive peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs.F-2058PENTRAXIN 3 PLAYS A KEY ROLE IN HUMAN MYELOID ANGIOGENIC CELLS PHAGOCYTIC FUNCTIONPathak, Varun - Centre for Experimental Medicine, Queens University Belfast, UK Peixoto, Elisa - Centre for Experimental Medicine, Queens University Belfast, UK Pedrini, Edoardo - Centre for Experimental Medicine, Queens University Belfast, UK McLoughlin, Kiran - Centre for Experimental Medicine, Queens University Belfast, UK Chambers, Sarah - Centre for Experimental Medicine, Queens University Belfast, UK

546POSTER ABSTRACTSAllen, Lynsey-Dawn - Centre for Experimental Medicine, Queens University Belfast, UK Johnston, Louise - Centre for Experimental Medicine, Queens University Belfast, UK Stitt, Alan - Centre for Experimental Medicine, Queens University Belfast, UK Medina, Reinhold - Centre for Experimental Medicine, Queens University Belfast, UKMyeloid angiogenic cells (MACs) were originally described as endothelial progenitors, but we and others have demonstrated that their revascularisation properties are explained by release of soluble factors. Furthermore, MACs do not differentiate into endothelial cells, but exhibit a defined myeloid phenotype CD14+CD163+CD204+CD54- related to alternative activated macrophages. Their therapeutic potential has been tested in preclinical models for ischaemic disease in heart, retina, and lower limbs. Besides their paracrine pro-angiogenic effect, MACs may also clear cellular debris from ischaemic tissues to reduce inflammation. Pentraxin 3 (PTX3) is a pattern recognition receptor expressed in myeloid cells with reported functions in inflammation, angiogenesis, and phagocytosis. Therefore, here we studied PTX3 role in MACs function. First, MACs were exposed to 25 mM D-glucose to model diabetes or to 1% O2 to mimic tissue hypoxia. After 7 days exposure to high glucose, PTX3 mRNA expression was significantly (P <0.01) downregulated, while IL1- mRNA was significantly (P <0.05) βincreased when compared to untreated controls. Similarly, there was a significant reduction in PTX3 mRNA expression after 2, 4, and 6 hours exposure to hypoxia. This was associated with a significant decrease in MACs phagocytic index using pHrodo™ Bioparticles™ and GFP-positive apoptotic bodies, when exposed to a high glucose milieu for 7 days (P<0.05). Second, we tested whether treatment with recombinant (r) PTX3 rescue MACs from high glucose-induced decrease in phagocytosis. Pre-treatment with 100 ng/ml rPTX3 for 24 hours restored the phagocytosis of pHrodo bioparticles and GFP-apoptotic bodies (P<0.05) in high glucose-treated MACs. Finally, we isolated MACs from PTX3 knockout (KO) and wild type (WT) mice. MACs derived from PTX3 KO mice showed less uptake of pHrodo bioparticles as compared to MACs from WT mice (P<0.05). This evidence demonstrates that PTX3 promotes phagocytosis in MACs. Furthermore, treatment of MACs with rPTX3 significantly reduced the expression of IL1- mRNA (P <0.05) with an increase βin CD163 expression (P <0.05), suggesting an anti-inflammatory role for PTX3. These findings support our hypothesis that MACs act as phagocytic cells and their dysfunction under diabetic conditions can be corrected by PTX3.Funding Source: This project is funded by Novo Nordisk.F-2060IDENTIFICATION OF THE SPATIO-TEMPROAL VULNERABILITIES TO GENOTOXIC STRESS IN FETAL HEMATOPOIETIC STEM AND PROGENITOR CELLSGuo, Xiaolin - Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, China Wu, Renyan - Shanghai Jiao-Tong University School of Medicine, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai, ChinaChildhood leukemia demonstrate being prenatal in origin and associated with chromatin alterations. However, the developmental vulnerabilities to genotoxic stress and underlying mechanism in fetal hematopoietic stem and progenitor cells (HSPCs) remain much unknown. Here we report genotoxic insults of Etoposide or UV, both in vivo and ex vivo, induced more DNA breaks in mouse HSPCs in 12.5~14.5 dpf (days post fertilization) fetal livers (FLs) than that in 12.5dpf placentas and in 15.5~18.5 dpf FLs. Consistently, lineage-tracing observation showed that relatively retarded hepatocyte development in early FLs exposed HSPCs in an unprotected environment to genotoxic stress. ATAC-seq and RNA-seq showed that the chromatin in 12.5dpf FLs HSPCs was more accessible, more enriched in CTCF occupancy, but less in YY1 binding, which might determine the chromatin fragility in early FLs HSPCs. These findings will be important in further studies of the evolutionary “trade-off” of development and etiology of childhood malignancies.F-2062DIAPHANOUS-RELATED FORMINS MODULATE BETA 2 INTEGRINS THROUGH SERUM RESPONSE FACTOR TO REGULATE THE ENGRAFTMENT OF MURINE HEMATOPOIETIC STEM AND PROGENITOR CELLSMei, Yang - Pathology, Northwestern University, Chicago, IL, USA Mei, Yang - Pathology, Northwestern University, Chicago, IL, USA Han, Xu - Pathology, Northwestern University, Chicago, IL, USA Liu, Yijie - Pathology, Northwestern University, Chicago, IL, USA Yang, Jing - Pathology, Northwestern University, Chicago, IL, USA Sumagin, Ronen - Pathology, Northwestern University, Chicago, IL, USA Ji, Peng - Pathology, Northwestern University, Chicago, IL, USA

547POSTER ABSTRACTSMature blood cells are derived from hematopoietic stem and progenitor cells (HSPC) that possess self-renewal and engraftment ability. The diaphanous-related formins are effectors of Rho GTPases and involved in membrane and cytoskeleton physiology. How they are involved in the regulation of HSPC remains unknown. Here we show that loss of formins mDia1 or mDia2 impaired the competitive repopulation capacity of HSPC, in which mDia2 deficiency exhibited a more prominent phenotype. mDia1 partially compensates the defects in mDia2 deficient HSPC since mice with double knockout of mDia1 and mDia2 exhibited rapid lethality after birth with severely reduced HSPC populations. The major role of mDia2 in HSPC was further implicated in that mice transplanted with mDia2 deficient bone marrow mononuclear cells showed decreased HSPC with loss of stem cell quiescence and rapid lethality under serial transplantation. Unexpectedly, loss of mDia2 did not affect HSPC localization to the bone marrow vasculatures during competitive transplantation. However, these HSPC failed to undergo trans-endothelial migration to the bone marrow niche. Mechanistically, loss of mDia2 reduced the linear actin filaments in HSPC, which led to a repression of the transcriptional activity of serum response factor (SRF). We identified that the expression levels of beta 2 integrins were significantly attenuated in mDia2 deficient HSPC, which could explain the trans-endothelial migration defects in these cells. We further revealed both Itgam and Itgb2 as new downstream targets of SRF with intronic binding in their genomic loci. Ectopic expression of SRF or a naturally occurred SRF variant rescued dysregulated gene expression profile as well as engraftment defects of mDia2 deficient HSPC. Consistently, mice with hematopoietic specific ItgaM and Itgb2 knock out through CRISPR-Cas9 exhibited serve defects in HSPC engraftment and long-term self-renewal. In addition, exogenously expressing human ItgaM is sufficient to rescue the engraftment defects in mDia2 deficient HSPC. Collectively, our data revealed a novel mDia-SRF-beat 2 integrin axis that regulates HSPC engraftment in mice.F-2064OPTIMIZED POST-CRYOPRESERVATION RECOVERY OF HUMAN STEM-CELL DERIVED BETA-CELLS FOR TREATMENT OF TYPE 1 DIABETESCarey, Bryce - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Thompson, Evrett - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Hsiung, Michael - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Gomez, Ander - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA McPartlin, Lori - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Chinn, Rebecca - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Lucich, Katherine - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Kalenjian, Lena - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Yasin, Jay - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Kaplan, Jonah - Bio Process Development, Semma Therapeutics, Cambridge, MA, USA Pagliuca, Felicia - Cell Biology, Semma Therapeutics, Cambridge, MA, USA Thiel, Austin - Bio Process Development, Semma Therapeutics, Cambridge, MA, USASemma Therapeutics is developing a novel therapy for Type 1 diabetes using human pluripotent stem cells (hPSCs) to derive functional insulin-producing islets (SC-Islets). These SC-Islets can be cryopreserved and thawed, and are glucose-responsive in vitro and in vivo with the capacity to ameliorate hyperglycemia in diabetic mice. To optimize the post-cryopreservation SC-Islet yield and function, we have characterized the contribution of signaling pathways that improve CHGA+ endocrine cell re-aggregation. We identify novel signaling requirements that enhance re-aggregation efficiency and cluster size. Further, these conditions improve SC-islet composition and SC-beta function in-vitro. These findings will contribute to the development of a scalable process suitable for Phase 1 clinical trials and future commercial manufacturing.PANCREAS, LIVER, KIDNEYF-2066REDUCTION OF HEPATIC DAMAGE INDUCED BY RADIATION IN MICE TREATED WITH G-CSFGoldenberg, Regina - Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Brazil Ramos, Isalira - National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, Brazil Andrade, Cherley - Translational Endocrinology Laboratory, Federal University of Rio de Janeiro, Brazil Moraes, Alan - Laboratory of Cellular and Molecular Cardiology, Federal University of Rio de Janeiro, Brazil Almeida, Thays - Laboratory of Cellular and Molecular Cardiology, Federal University of Rio de Janeiro, Brazil Azevedo, Rafaella - Laboratory of Cellular and Molecular Cardiology, Federal University of Rio de Janeiro, Brazil Batista, Cintia - Laboratory of Cellular and Molecular Cardiology, Federal University of Rio de Janeiro, Brazil Meireles, Fernanda - D’Or Institute for Research and Education, Rio de Janeiro, Brazil Lima, Carolina - D’Or Institute for Research and Education, Rio de Janeiro, Brazil Kasai- Bunswski, Tais - National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, BrazilAlthough liver cancer cells are sensitive to radiation, this treatment cannot be used at very high doses because normal liver tissue is also easily damaged by radiation. To overcome this problem, we investigated the role of Granulocyte Colony Stimulating Factor (G-CSF) in a model of alcohol and ionizing

548POSTER ABSTRACTSradiation induced liver injury. C57BL/6 mice (n=45) were equally divided into 3 groups: irradiated (IR) receiving vehicle; irradiated and G-CSF (IR+G-CSF) and non-irradiated control (CT). The IR and IR+G-CSF groups were subjected to local irradiation with a single dose of 18Gy. Three days prior irradiation, animals from the IR+G-CSF group received 50 μg/kg of G-CSF subcutaneously every 24 hours. To confirm the mobilization of bone marrow hematopoietic precursors, a set of CD45+low, CD117+, CD90.2 and CD34+ markers were analyzed by flow cytometry. The animals were submitted to euthanasia 7, 30 and 60 days after the irradiation date, serum was collected for biochemical analysis of albumin (ALB) and alanine amino transferase (ALT) and liver fragments processed for H&E analysis. Flow cytometry showed that animals receiving G-CSF presented 0.3 ± 0.1% of hematopoietic precursors, whereas those receiving vehicle showed 0.1 ± 0.02%. The animals submitted to IR showed ALB reduction on days 7 (1.4 ± 0.15g/ dl), 30 (1.5 ± 0.09g/dl) and 60 (1.2 ± 0) compared to CT group: 7 (2.2 ± 0.1g/dl), 30 (2.5 ± 0.3g/dl) and 60 (2.5 ± 0.5g/dl), however, the IR+G-CSF group showed that albumin level were similar to control: 7 (2.1 ± 0.14g/dl), 30 (2.2 ± 0.11g/dl) and 60 (2.5 ± 0.19g/dl). IR and IR+G-CSF groups showed a significant increase in ALT-IR: 7 (50 ± 7U/L), 30 (100 ± 10U/L) and 60 (50 ± 8 U/L) and IR + G-CSF: 7 (45 ± 5U/L), 30 (55 ± 3U/L) and 60 (62 ± 1 U/L) - when compared to CT: 7 (37 ± 5U/L), 30 (40 ± 7U/L) and 60 (41 ± 5U/L). H&E staining showed the presence of inflammatory infiltrate in the IR group; however, this change was not observed in both IR+ G-CSF and control groups. The data suggest that irradiation caused hepatic dysfunction (ALB reduction, ALT increase and presence of inflammatory infiltrate). In the G-CSF group, there was no inflammatory infiltrate and albumin levels were similar to those in the control group. Therefore, mobilization of hematopoietic progenitors, by administration of G-CSF prior to irradiation showed hepatoprotective effects.Funding Source: CAPES, CNPq, INCT-REGENERA and FAPERJF-2068AUTOLOGOUS CO-CULTURE HUMAN IPSC MODEL FOR PANCREATIC ENDOCRINE AND VASCULAR ENDOTHELIAL CELLSDe Souza Santos, Roberta - RMI, Cedars-Sinai Health System, West Hollywood, CA, USA Ramos, Michael - RMI, Cedars-Sinai, Los Angeles, CA, USA Shaharuddin, Hanan - RMI, Cedars-Sinai, Los Angeles, CA, USA Gross, Andrew - RMI, Cedars-Sinai, Los Angeles, CA, USA Sareen, Dhruv - RMI, Cedars-Sinai, Los Angeles, CA, USADiabetes is a clinical condition that affects millions of people, and its major causes are the death or dysfunction of insulin-producing beta-cells within the pancreatic islets, resulting in improper insulin secretion and failure to maintain normal glycemia. Most of the patients are reliant on multiple exogenous insulin injections as treatment, and some of them are recipients of cadaveric islet transplantations; however, the source of cadaveric islets is scarce, and these cells are not long-term functional. Thus, new strategies to create scalable and compatible pancreatic islets containing insulin-producing beta cells are necessary. Differentiation of human induced pluripotent stem cells (iPSCs) into pancreatic beta cells appears to be a promising alternative cell source for diabetic patients, as well as for other applications such as disease-modeling and to study pancreas development. Several protocols to generate iPSC-derived beta cells have been developed over the past years; however, improvements are still necessary since many immature polyhormonal cells remain and cannot attain a monohormonal and functional state. During human development, pancreas co-develops with endothelium and shares signals, allowing for better maturation of beta cells, and these are not currently included in the current differentiation protocols. Thus, here we describe a novel autologous co-culture model using iPSC-derived pancreatic endocrine and endothelial cells from the same patient to obtain more functional and monohormonal pancreatic beta-cells from iPSCs. We have established an efficient “in-house” protocol to differentiate iPSCs into pancreatic endocrine cells, with high expression of beta-cell markers such as C-PEPTIDE, NKX6.1 and NGN3. However, these cells have limited glucose stimulated insulin secretion response. To improve functionality of the iPSC-derived pancreatic endocrine cells, we have co-cultured them with iPSC-derived endothelial cells, as well as with primary HUVECs, in trans-wells and Organ-Chip devices. Preliminary data indicate that iPSC-derived pancreatic endocrine and endothelial cells can be adapted to trans-wells and Organ-Chip devices, and co-culture improves endothelial cell markers.F-2070CHARACTERISTICS AND DIFFERENCES OF HEPATIC PROGENITOR CELLS ACCORDING TO DONOR AGE IN MICE AND HUMANSYoon, Sangtae - Department of Surgery, Hanyang University, Seoul, Korea Kang, Kyojin - Department of Surgery, Hanyang University, Wangsip-ri, Korea Kim, Yohan - Department of Surgery, Hanyang University, Wangsip-ri, Korea Buisson, Elina Maria - Department of Surgery, Hanyang University, Wangsip-ri, Korea Lee, Changhee - Department of Surgery, Hanyang University, Wangsip-ri, Korea Yim, Ji-hye - Department of Surgery, Hanyang University, Wangsip-ri, Korea Jeong, Jaemin - Department of Surgery, Hanyang University, Wangsip-ri, Korea Choi, Dongho - Department of Surgery, Hanyang University, Wangsip-ri, KoreaDue to a shortage of organ donors, liver transplantation being the only choice of treatment for end-stage liver disease is problematic. Therefore, researchers are now leaning towards stem cells as a solution. We recently reported human hepatocytes can convert into human chemically derived hepatic progenitors

549POSTER ABSTRACTS(hCdHs) through a cocktail of three small molecules (A83-01, CHIR99021, HGF). Interestingly, clinical feature analysis revealed that age is to be the most important factor in CdH generation. To analyze features of intergenerational mCdHs, we used E16.5 hepatoblast, young mice (6-8 weeks old) and old mice (72-96 weeks old) hepatocytes. Mouse hepatocytes of different ages were stably reprogrammed. In particular, the generation efficiency of mCdHs was different in hepatocytes of different ages. The best of them were liver precursor cells (EmCdHs) made from E16.5 hepatoblasts. These various age-matched mCdHs expressed precursor cell markers, and they also had the ability to differentiate into functional hepatocytes. Afterwards, mCdHs generated from the hepatocytes of various age groups were transplanted into FRG - / - mice and the cell viability was analyzed. Furthermore, significant survival results were seen in the mCdH transplantation group compared to non-transplanted mice. Hepatocytes of various ages were reprogrammed as hepatic progenitor cells. The generation, differentiation, and hepatic function efficiency of the youngest E16.5 hepatoblast-derived mCdHs (EmCdHs) showed a significant difference when compared to others. Also, we obtained the same results from human samples in the process of studying mice. Specifically, human hepatoblasts (14.2 weeks old) showed the same generation efficiency as old hepatocytes (75 years old) similarly to the mice model. The efficiency of hepatic differentiation and function showed adequate results which are better correlated with age. In conclusion, our study has showed that age is an important factor in the generation of CdH which can open the door and provide opportunities for future specific liver disease treatments.F-2072REPRESSING OF -CELL FUNCTIONS IN INDUCE ΑPORCINE MONOHORMONAL PANCREATIC BETA-CELLS GENERATED BY INSERTION OF TRANSCRIPT FACTORS, PDX1/MAFA/PAX4Ock, Sun A - Animal Biotechnology, National Institute of Animal Science (NIAS), Wanju-gun, Korea Ullah, Imran - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, Korea Lee, Ran - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, Korea Kim, Youngim - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, Korea Woo, Jae-Seok - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, Korea Oh, Keon Bong - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, Korea Hwang, Seongsoo - Animal Biotechnology Division, National Institute of Animal Science, Wanju-gun, KoreaTranscription factors, such as Pdx1 and the intrinsic signal pathways involved in pancreatic organogenesis developmental pathways have been revealed recently. As an alternative source for pancreas or islet transplant in the treatment of type 1 diabetes, studies have been carried out on stem cells or somatic cells to generate functional insulin-producing -cells via a combination βof small molecules and/or transcription factors. Therefore, we tested whether pig fibroblasts treated with specific transcription factors (Pdx1/MafA/Pax4) and a small molecule (A83-01) could be differentiated into direct monohormonal cells ( -cells). The βgeneration of insulin-producing -cells was carried out through βthe transfection (MO1=1~2) of a lentivirus vector inserting 3 transcription factors (Pdx1/MafA/Pax4) into pig ear fibroblasts for 1 day. The cells were cultured in N2B27 medium supplemented with A83-01/bFGF (basic culture medium, BCM) + activin-A for the first 6 days, BCM + retinoic acid for 2 days, and BCM + ITS for 3 weeks. In induced -cell-like cells (i -cells), we tested the ββexpression of specific islet proteins (Pdx1/c-peptide/glucagon/somastatin/pancreatic polypeptide) and performed whole-transcriptome analysis and DTZ. Morphologically, i -cells were βobserved 1 week after transfection. They formed many clusters of > 100 mm diameter after 1 month and maintained their cluster formation ability until 15 passages. They even formed spherical structures like isolated pancreatic islets and showed strong Pdx1/c-peptide but no Pdx1/glucagon co-expression, and a DTZ-positive reaction. Whole-transcriptome analysis confirmed the activation of endocrine cell differentiation and ²-cell maturation pathway-related genes, such as FOXA3, NKX2-3, NKX6-1, PAX6, ISL1, NEUROD1, in the i²-cells. Overall, i²-cells shared characteristics with real pancreatic ²-cells. Repressing ±-cells thus induces characteristic monohormonal cell (²-cell) functions and could be used to develop cellular therapy for T1D.Funding Source: This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (Project no. PJ01094404).F-2074DISCOVERY OF SMALL-MOLECULE CELL GROWTH ACTIVATORS THAT PROMOTE LIVER REGENERATIONNishino, Taito - Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka, Japan Aihara, Ayako - Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka-shi, Japan Nakajima, Hiroyuki - Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka-shi, Japan Otsuka, Keiichiro - Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka-shi, Japan Abe-Fukasawa, Natsuki - Biological Research Laboratories, Nissan Chemical Corporation, Shiraoka-shi, JapanThe liver is the only internal organ that has a remarkable capacity for repair following injury. The ability for the liver to regenerate is critical for patients of liver diseases when the partial removal of the liver is an indispensable treatment process. Recently, regenerative medicine of the liver is expected as a new method to cope with a shortage of living donors for liver transplantation. However, the mechanism of liver regeneration has not been fully elucidated, and further improved therapeutic approaches are needed to accomplish the realization of liver regenerative medicine. In this study, we have performed a phenotypic high