ISSCR 2019 Poster Abstracts

400POSTER ABSTRACTST-3156SINGLE-CELL TRANSCRIPTOME ANALYSIS OF HESC PANCREATIC DIFFERENTIATION REVEALS PRODUCTIVE AND NON-PRODUCTIVE PATHS OF DIFFERENTIATIONMorey, Robert - Department of Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Khater, Marwa - Department of Reproductive Medicine, University of California, San Diego, CA, USA Laurent, Louise - Department of Reproductive Medicine, University of California, San Diego, CA, USA Mora-Castilla, Sergio - Department of Reproductive Medicine, University of California, San Diego, CA, USA To, Cuong - Department of Reproductive Medicine, University of California, San Diego, CA, USA Touboul, Thomas - Department of Reproductive Medicine, University of California, San Diego, CA, USAHESCs differentiated to specific cell types have tremendous potential for use in cell replacement therapy for a variety of conditions. Among the most sought after target cell types are pancreatic beta cells for treatment of Type 1 diabetes mellitus. However, the efficacy and safety of such therapies remain suboptimal. We performed single-cell RNAseq on hESCs at sequential stages of pancreatic differentiation, as well as human fetal pancreas tissue and adult islets, to determine the heterogeneity at each stage of differentiation. We applied a network permutation analysis to the key transcriptional networks characterizing component cell subpopulations. Our results indicate that the overall trajectory of differentiation for the bulk of the population of hESCs travels smoothly from the undifferentiated state toward the developmentally relevant states occupied by fetal pancreas and adult islet cells, with the exception of the pancreatic progenitor stage of differentiation, which deviates from the expected direct path between the preceding posterior foregut and following endocrine pancreas stages. In addition, our approach provides evidence of some individual cells transitioning between cell fates. We found a subpopulation of cells positive for both POU5F1 and the SOX17 mediator CER1. Consistent with other studies, these results suggest that POU5F1 might be required during early differentiation to transition out of the pluripotent state, and may partner with SOX17 for endodermal specification. This subpopulation of POU5F1/CER1 double positive cells may be evidence of cells that have not yet achieved a definitive endoderm-like state. These results point to complex transcriptional networks that may be activated or repressed during the in vitro differentiation process, and will be applied to future studies aimed at optimizing this process to enable production of mature functional pancreatic beta cells derived from hESCs or other types of human pluripotent stem cells.PLURIPOTENT STEM CELL: DISEASE MODELINGT-3160MRI TRACKING OF IPS CELLS-INDUCED NEURAL STEM CELLS IN TRAUMATIC BRAIN INJURY RATSTang, Hailiang - Neurosurgery Department, Huashan Hospital, Fudan University, Shanghai, China Zhu, Jianhong - Neurosurgery Department, Huashan Hospital, Fudan University, Shanghai, ChinaInduced pluripotent stem cells (iPS cells) are promising cell source for stem cell replacement strategy applied to brain injury caused by traumatic brain injury (TBI) or stroke. Neural stem cell (NSCs), derived from iPS cells could aid the reconstruction of brain tissue and the restoration of brain function. However, how to trace the fate of iPS cells in host brain is still a challenge. In our study, iPS cells were derived from skin fibroblasts using the four classic factors Oct4, Sox2, Myc and Klf4. Then these iPS cells were induced to differentiate into NSCs, which were incubated with superparamagnetic iron oxides (SPIOs) in vitro. Next, 30 TBI rat models were prepared and divided into 3 groups (n=10). One week after brain injury, group A&B rats received NSCs (labeled with SPIOs) implantation, while group C rats received non-labeled NSCs implantation. After cell implantation, all the rats were performed T2*-weighted magnetic resonance imaging (MRI) scan at day 1, and 1 week to 4 weeks, to track NSCs distribution in rats’ brains. One month after cell implantation, all the rats were performed manganese-enhanced MRI (ME-MRI) scan. In group B, diltiazem was infused during the ME-MRI scan period. Thus, (1) iPS cells were successfully derived from skin fibroblasts using the four classic factors Oct4, Sox2, Myc and Klf4, expressing the typical antigens including SSEA4, Oct4, Sox2 and Nanog. (2) iPS cells were induced to differentiate into NSCs, which could express Nestin and differentiate into neural cells and glia cells. (3) NSCs were incubated with SPIOs overnight, and prussian blue staining showed intra-cellular particles. (4) After cell implantation, T2*-weighted MRI scan showed these implanted NSCs could migrate to the injury area in chronological order. (5) The subsequent ME-MRI scan detected NSCs function, which could be blocked by diltiazem. In conclusion, using in vivo MRI tracking technique to trace the fate of iPS cells-induced NSCs in host brain is feasible.T-3162EXOSOMES DERIVED FROM MESENCHYMAL STEM CELLS ALLEVIATED ILC2-DOMINANTE ALLERGIC AIRWAY INFLAMMATIONZhang, Hongyu - Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Fang, ShuBin - Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Xu, ZhiBin - Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

401POSTER ABSTRACTSWu, ZhangJin - Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Fu, qingLing - Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, ChinaType 2 innate lymphoid cells (ILC2s) were reported to be involved in the pathogenesis of allergic diseases. The cost of allergic airway inflammation is a significant burden to the society, promoting us to look for better treatment. We previously reported the immunomodulation of mesenchymal stem cells (MSCs) on suppressing airway inflammation. Extracellular vesicles (EVs) or exosomes are one of the key secretory products of MSCs involved in their immunomodulation. In this study, we aimed to investigate the effects of exosomes from MSCs on allergic airway inflammation and the possible mechanisms. We isolated exosomes from human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) media, and determined their characteristics. We induced mouse ILC2 dominant airway inflammation model by intratracheal instillation of IL-33. Compared with the model group, the intravenous injection with MSC-derived exosomes (MSCexo) significantly suppressed the level of IL-5/13 Th2 cytokines in bronchoalveolar lavage fluid (BALF) and reduced the proportion of ILC2s in lung. Additionally, we co-cultured the peripheral blood mononuclear cells (PBMCs) from patients with allergic rhinitis with MSCexo, and MSCexo significantly inhibited the IL-5 production in response to IL-33. However, fibroblast-derived exosomes (Fbexo) did not exhibit the similar effects. We further identified that MSCexo had higher levels of miR-146a-5p compared to Fbexo. After inhibiting the expression of miR-146a-5p in MSCs, the inhibitory effect of exosomes on inflammation was significantly reversed both in mouse model and in vitro. Our data suggest that exosomes from MSCs suppress the ILC2 dominant allergic airway inflammation, which is at least partly mediated via transfer of miR-146a.T-3164GENOME-WIDE MICROHOMOLOGIES ENABLE PRECISE TEMPLATE-FREE EDITING OF PATHOGENIC DELETION MUTATIONSGrajcarek, Janin - Department of Life Science Frontiers / Center For IPS Cell Research And Application (CiRA), Graduate School of Medicine / Kyoto University, Kyoto, Japan Bourque, Guillaume - Department of Human Genetics, McGill University, Montréal, QC, Canada Lougheed, David - Computer Science and Biology, McGill University, Montréal, QC, Canada Matsuo, Shiori - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Monlong, Jean - UC Santa Cruz Genomics Institute, University of California, Santa Cruz, CA, USA Nagai, Miki - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Nakamura, Michiko - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Nishinaka-Arai, Yoko - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Saito, Megumu - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Sakurai, Hidetoshi - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Woltjen, Knut - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, JapanTo investigate the function of a gene or to create disease models, researchers often aim to generate gene “knockouts” using designer nucleases such as CRISPR/Cas9. This type of gene editing faces two challenges, the predictability of mutations created at the target loci and of their phenotypic outcome. Recently published manuscripts report predictable mutations using CRISPR/Cas9-induced template-free non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) DNA repair at random genetic loci or in artificial sequence libraries. However, these published tools give limited insight into probable phenotypic outcomes. In order to combine predictable gene editing outcomes with existing knowledge on phenotypes, we devised a methodology relying on precise deletions generated by MMEJ to re-create already known mutations. MMEJ is mediated by microhomologies (μHs) flanking the DNA double strand break. Here we introduce a tool called MHcut that identified μHs flanking the majority of all naturally occurring deletion mutations across the human genome. In total, 11 million deletions are flanked by μHs, covering 88% of protein-coding genes. Notably, over 99% of these mutations are as of yet unexplored. Using CRISPR/Cas9 in human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs), we precisely recreated target μH-flanked pathogenic deletion mutations by MMEJ. In addition, we demonstrated both gain- and loss-of-function phenotypes in three hiPSC disease models. We anticipate this precise gene editing methodology and the data set of over 11 million μH-flanked deletions to enable not only functional genetic studies and drug screening, but to also yield potential targets for gene therapy.Funding Source: Grant to K.W. from the Cell Science Foundation (Japan), to G.B. from Fonds de Recherche Santé Québec (FRSQ-25348) and to H.S from AMED (17bm0804005h0001). K.W. is a Hakubi Center Special Project Researcher.T-3166GENERATION OF A SERIES OF ISOGENIC HUMAN PLURIPOTENT STEM CELL LINES AS MODELS OF AUTISM SPECTRUM DISORDERSchwartz, Joshua - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, Songstad, Allison - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA Krach, Florian - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA Chu, Josephine - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA Roberts, Elizabeth - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

402POSTER ABSTRACTSAigner, Stefan - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA Goldstein, Lawrence - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA Yeo, Gene - Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USAAutism spectrum disorder (ASD) is a severe neurodevelopmental disorder that affects 1 in 59 children, but lacks effective medical treatment. Although several highly penetrant genetic variants have been linked to ASD, researchers have a limited understanding of the causal gene networks, disease-relevant cell types, druggable targets, and biological signatures associated with ASD. Induced pluripotent stem cells (iPSCs) preserve the patient’s genome and serve as valuable tools to dissect the cellular basis of disease pathology, but their comparisons to unaffected controls can be hampered by differences in genetic background, reprogramming approaches, cell culture methods, or quality standards. To overcome these limitations, we used CRISPR-mediated genome engineering to introduce ASD-linked genetic variation into a well-characterized iPSC line from a healthy neurotypical adult. Specifically, we prioritized modeling 13 syndromic forms of ASD: five are monogenic- MECP2 y/- (Rett), SHANK3 +/- (Phelan-McDermid), TSC2 +/- (tuberous sclerosis), FMR1 y/- (fragile X), and SLC9A6/NHE6 y/- (Christianson), and eight are copy number variants- reciprocal deletions and duplications at 7q11.23 (Williams-Beuren), 15q11-13 (Angelman/Prader-Willi), 17p11.2 (Smith-Magenis) and 16p11.2. All lines are derived from single-cell clonal isolates and have been rigorously validated by digital and G-banding karyotyping. While the monogenic models are currently available through the NIMH Stem Cell Center at RUCDR, additional characterization of copy number variation lines is underway prior to widespread distribution to the research community. We anticipate that our comprehensive resource of monogenic and copy number variant ASD stem cell models built on the same genetic background will be invaluable for uncovering convergent and divergent molecular and cellular mechanisms in ASD.Funding Source: NIMH NCRCRG U19: MH107367T-3168ROS DEPENDENT INCREASED AUTOPHAGOSOME FORMATION MEDIATES SYNAPTIC DYSFUNCTION IN A PATIENT DERIVED MODEL FOR KOOLEN-DE VRIES SYNDROMELinda, Katrin - Human Genetics, Radboud UMC, Nijmegen, Netherlands Lewerissa, Elly - Human Genetics, Radboud UMC, Nijmegen, Netherlands Devilee, Lynn - Human Genetics, Radboud UMC, Nijmegen, Netherlands Frega, Monica - Human Genetics, Radboud UMC, Nijmegen, Netherlands Verboven, Anouk - Human Genetics, Radboud UMC, Nijmegen, Netherlands KleinGunnewiek, Teun - Anatomy, Radboud UMC, Nijmegen, Netherlands Koolen, David - Human Genetics, Radboud UMC, Nijmegen, Netherlands de Vries, Bert - Human Genetics, Radboud UMC, Nijmegen, Netherlands Nadif Kasri, Nael - Human Genetics, Radboud UMC, Nijmegen, NetherlandsKoolen-de Vries syndrome (KdVS) is a heterogeneous multisystem disorder characterized by developmental delay, intellectual disability, facial dysmorphisms, epilepsy, and congenital malformations in multiple organ systems. It is caused by heterozygous loss of KANSL1. KANSL1 is a scaffold protein of the nonspecific lethal complex that contains the histone acetyltransferase MOF, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Our recent studies in mice have shown that heterozygous loss of Kansl1 leads to changes in gene expression related to synaptic transmission and to a decrease in basal synaptic transmission and plasticity, but the underlying cellular mechanisms remain unknown. H4K16ac is known to be essential for the regulation of autophagy, a process controlling degradation and recycling of proteins and shown to play a role in synapse development and function. Here, we made use of KdVs patient- and control-derived induced pluripotent stem cells (iPSCs) and derived neurons thereof (iNeurons) to link heterozygous loss of KANSL1 to deregulated autophagy and subsequent synaptic dysfunction. Using several KdVS patient cell lines we found that, under basal conditions, the number of autophagosomes is increased in both, KANSL1 deficient iPSCs and iNeurons. By investigating gene expression profiles that are associated with H4K16 acetylation, we found that SOD1 gene expression was down-regulated. Reduced expression of this antioxidant enzyme is leading to a consecutive increase in oxidative stress and autophagy. Furthermore, in maturing KdVS iNeurons, increased autophagosome formation at the synapse resulted in reduced synaptic density and network activity. We could partially rescue the observed autophagy phenotype as well as the synaptic deficits by treating KANSL1 deficient neurons with antioxidants to lower the amount of reactive oxygen species (ROS). Taken together these results identify increased oxidative stress as a cause for increased autophagosome formation and subsequent synapse loss in KdVs, providing a promising starting point for the development of a more targeted therapy.T-3170ELECTROPHYSIOLOGICAL RECORDINGS OF NEURONS DERIVED FROM SLEEP BRUXISM PATIENT-SPECIFIC IPSCSNakai, Kento - Department of Prosthodontics, Showa University School of Dentistry, Ota-ku, Japan Shiga, Takahiro - Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, Bunkyo-ku, Japan Abe, Yuka - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, Japan

403POSTER ABSTRACTSHoashi, Yurie - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, Japan Nakamura, Shiro - Department of Oral Physiology, Showa University School of Dentistry, Shinagawa-ku, Japan Yasuhara, Rika - Division of Pathology, Department of Oral Diagnostic Sciences, Showa University School of Dentistry, Shinagawa-ku, Japan Matsumoto, Takashi - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, Japan Avijite, Sarker - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, Japan Kotani, Keisuke - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, Japan Inoue, Tomio - Department of Oral Physiology, Showa University School of Dentistry, Shinagawa-ku, Japan Mishima, Kenji - Division of Pathology, Department of Oral Diagnostic Sciences, Showa University School of Dentistry, Shinagawa-ku, Japan Akamatsu, Wado - Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, Bunkyo-ku, Japan Baba, Kazuyoshi - Department of Prosthodontics, Showa University School of Dentistry, Shinagawa-ku, JapanSleep bruxism (SB) is classified as a sleep-related movement disorder characterized by involuntary jaw-closing muscle activity. Mechanical stress of SB is shown to be responsible for poor prognosis of dental treatment and seriously compromises patients’ quality of life. However, little is known about the etiology of SB. We previously found that a single nucleotide polymorphism (SNP) in serotonin 2A receptor (5-HT2A) gene, rs6313 C>T, increases the risk of SB. 5-HT2A receptors are widely expressed in central nervous system including GABAergic premotor neurons projecting to the trigeminal motor nucleus, which play a central role in inhibitory regulation of masticatory muscle activity during sleep. The aim of this study is to elucidate the functional difference of these GABAergic neurons between C and T allele carriers using electrophysiological recording of iPSC-derived neurons. First of all, human iPSCs from SB and healthy controls were differentiated into neurons with the characteristics of the ventral hindbrain, where 5-HT2A positive neurons are enriched. Regional identity of neurospheres were confirmed by qRT-PCR with 5-HT2A and VGAT gene-specific primers. Neurons derived from neurospheres were immunostained with anti-5-HT2A receptor and anti-GABA antibodies. Whole-cell patch-clamp recordings were performed on these neurons with current-clamp method. Then, voltage-clamp method was utilized to record currents following the administration of selective 5-HT2A receptor agonist (TCB-2). We confirmed that iPSCs were successfully differentiated into GABAergic and 5-HT2A receptor positive neurons by using qRT-PCR and immunostaining. Furthermore, these differentiated neurons generated repetitive action potentials in response to current injections and TCB-2 evoked inward currents. These functional observations suggested that neurons differentiated from patient-specific iPSCs contained both GABAergic and 5-HT2A receptor positive neurons. In conclusion, the successful electrophysiological recording of iPSC-derived neurons allows us to examine the effects of SB-associated genetic variation, which might elucidate the etiology and underlying mechanism of SB.T-3172RAPID REVERSAL OF DIABETES AFTER TRANSPLANTATION OF HUMAN BETA CELLS FROM PATIENT IPS CELLS WITH CRISPR CORRECTIONMaxwell, Kristina G. - Department of Biomedical Engineering, Washington University, Saint Louis, MO, USA Millman, Jeffrey - Department of Medicine, Washington University in St. Louis, Saint Louis, MO, USAStem cell-derived (SC- ) cells are a powerful tool for disease ββmodeling, drug screening, and cell therapy. Patient induced pluripotent stem (iPS) cells allows for the study of diabetes by providing an unlimited cell source and has potential in autologous cell therapy, removing the need for immunosuppression. Monogenetic forms of diabetes, including Wolfram Syndrome (WS), are an immediate application of SC- cell technology, βas cells from WS patients are rarely available for study and βmouse models do not faithfully recapitulate the disease. WS is caused by a pathogenic variance in the WFS1 gene, inducing endoplasmic reticulum (ER) stress. To enable disease modeling and determine the cell therapy potential of autologous SC- βcells, we reprogrammed WS patient fibroblasts into iPS cells and corrected the disease-causing sequence with CRISPR/Cas9. After differentiation of patient-matched unedited and corrected WS iPS cells with our 6-stage protocol to produce SC- cells, βsingle-cell RNA sequencing was performed on approximately 10,000 cells. Unsupervised clustering using Seurat identified -, αβ-, and -like cell populations, with corrected -like cells having δβincreased cell and decreased stress markers. Surprisingly, βcluster analysis identified multiple off-target non-pancreatic cell types produced with the unedited iPS line, including neural, epithelial, and muscle tissues, while the corrected line produced predominately pancreatic endocrine. Corrected WS SC- cells βachieved robust dynamic glucose-stimulated insulin secretion (GSIS) similar to primary islets and to SC- cells generated from βunrelated non-patient donors, demonstrating their maturity and projected cell therapy success in vivo, while unedited WS SC- cells had greatly reduced GSIS and insulin content. βTransplantation of corrected WS SC- cells rapidly alleviated βdiabetes within 10 days in mice with pre-existing diabetes that had been treated with streptozotocin. However, the unedited WS SC- cells were unable to regulate blood glucose in mice even βafter 10 weeks. Human insulin was detected in all transplanted mice although was significantly less for unedited WS SC- cell βtransplants. This technological platform allows for the study of human diabetes pathology and is enabling for drug screening and cell replacement therapy.FundingSource:NIH(1R01DK114233-01and5T32DK10842-02), JDRF (Career Development Award), WashU Center of Regenerative Medicine, Diabetes Research Center

404POSTER ABSTRACTST-3174NOVEL INSIGHTS INTO THE MOLECULAR MECHANISMS UNDERLYING X-LINKED DYSTONIA-PARKINSONISM BY HARNESSING X-CHROMOSOME INACTIVATIOND’Ignazio, Laura - Department of Neurology / Johns Hopkins School of Medicine, Lieber Institute for Brain Development, Baltimore, MD, USA Benjamin, Jade - Lieber Institute for Brain Development, Baltimore, MD, USA Feltrin, Arthur - Lieber Institute for Brain Development, Baltimore, MD, USA Katipalli, Tarun - Lieber Institute for Brain Development, Baltimore, MD, USA Pankonin, Aimee - Salk Stem Cell Core Facility, Salk Institute for Biological Studies, La Jolla, CA, USA Diffenderfer, Kenneth - Salk Stem Cell Core Facility, Salk Institute for Biological Studies, La Jolla, CA, USA Hendriks, William - Massachusetts General Hospital, Boston, MA, USA Bragg, Cristopher - Massachusetts General Hospital, Boston, MA, USA Paquola, Apua - Lieber Institute for Brain Development, Baltimore, MD, USA Erwin, Jennifer - Lieber Institute for Brain Development, Baltimore, MD, USAX-linked Dystonia-Parkinsonism (XDP) is an adult-onset Mendelian neurodegenerative disease endemic to the island of Panay, Philippines. Conventional genetics analysis identified a founder haplotype consisting of: five single-nucleotide variants (disease-specific single-nucleotide changes, DSC-1, -2, -3, -10, and -12), a 48-bp deletion, and a 2627 bp SINE-VNTR-Alu (SVA)-type retrotransposon insertion. XDP haplotype boundaries mapped on Xq13.1, with the main disease-associated mutations lying within the non-coding region of TAF1 gene. TAF1 (TATA-binding-protein (TBP)-associated factor 1), being a subunit of the TFIID complex, mediates the transcription by RNA polymerase II (RNAP II). Therefore, it has been proposed that XDP pathogenesis might be dependent on TAF1 dosage insufficiency or aberrant transcription around the SVA region. For X-linked human diseases, such as XDP, the natural process of X-chromosome inactivation (XCI) provides an efficient way to generate genetically matched isogenic female induced pluripotent stem cell (iPSC) lines. However, XCI status in human iPS cell models is highly variable. In this study, we characterize the status of XCI in iPSC lines derived from multiple XDP female carriers. In particular, we use these iPS cell lines to uncover the mechanisms underlying XDP etiology. As such, cellular and molecular phenotypes associated with XDP are here identified by delineating the transcriptome profiles of a pair of isogenic female iPSC lines where either the wild-type or the mutant X chromosome are active. Overall, this initial analysis allowed us to harness the X-chromosome inactivation to understand the molecular features underlying XDP pathology, opening further possibilities to deepen into other XDP etiology-associated factors, such as brain regions, TAF1 isoform, as well as retrotransposon-derived transcripts and their regulators.Funding Source: We would like to thank the Collaborative Center for X-linked Dystonia Parkinsonism and the Lieber Institute for Brain Development for funding this research project.T-3176DOWN SYNDROME-IPSC NEUROGENESIS CONNECTS DIFFERENTIAL METHYLATION TO DYSREGULATED GENESLaan, Loora - Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Klar, Joakim - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Sobol, Maria - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Hoeber, Jan - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Zakaria, Muhammad - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Anneren, Goran - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Falk, Anna - Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Schuster, Jens - Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Dahl, Niklas - Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenDown syndrome (DS) is caused by trisomy 21 (T21) in humans and affects approximately one in 700 live births. Impaired cognition is a major disabling feature in DS and despite major efforts the molecular mechanisms leading to the morphological and functional brain abnormalities associated with T21 remain unknown. To clarify the role of differential methylation on transcriptional dysregulation and neurodevelopment in T21, we established an induced pluripotent stem cell (iPSC) derived neural cell model showing a transcriptional profile comparable to the early-mid gestational period. The DNA methylation pattern was analysed using Illumina HumanMethylation 450k BeadChip and paralleled by RNA sequencing. We first assessed the genome wide methylation pattern in T21 and euploid iPSC neural derivatives and identified 500 differentially methylated positions (DMPs). The DMPs in T21 neural lines were distributed across the genome with enrichment of hypomethylated sites on chromosomes 2, 8, 19, 21 and 22. Approximately half of DMPs (281 of 500) could be annotated to a total of 202 genes. Gene Ontology (GO) analysis of these 202 genes revealed enrichment of neurotransmitter transporters (GO:0006836). Integrated analysis of methylation and transcriptome data sets revealed altered expression in 77 out of the 202 genes in T21 neural lines. Furthermore, the most profound methylation changes (>9DMPs/gene) associated with differential expression in T21 neural lines was observed for a cluster of genes (ZNF441,

405POSTER ABSTRACTSZNF69, ZNF700 and ZNF763) on chromosome 19 encoding zink finger transcription factors. These ZNF genes with yet unknown functions are highly expressed during normal embryonic brain development. Taken together, our data suggest that differential methylation contributes to transcriptional dysregulation in T21 neural cells and our data highlights a set of candidate ZNF transcription factor genes that now require further investigations.Funding Source: This work was funded by the Stiftelsen Sävstaholm and the Swedish Research Council.T-3178PATIENT-SPECIFIC TISSUE CHIP TECHNOLOGY TO STUDY RARE DISEASES OF THE BRAINNeely, M Diana - Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA Brown, Jacquelyn - Physics and Astronomy, Vanderbilt University, Nashville, TN, USA Carson, Robert - Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA Lippmann, Ethan - Biomedical Engineering, Vanderbilt University, Nashville, TN, USA Bowman, Aaron - School of Health Sciences, Purdue University, West Lafayette, IN, USA Ess, Kevin - Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA Wikswo, John - Physics and Astronomy, Vanderbilt University, Nashville, TN, USAHere we describe patient-specific brain-on-a chip technology to model Tuberous Sclerosis Complex (TSC), a rare neurodevelopmental disorder manifesting itself with cortical malformations (“tubers”), subependymal giant cell astrocytomas, and epilepsy, but also affecting other organs. TSC is caused by loss-of-function mutations in the TSC1 or TSC2 gene that result in aberrant mTOR signaling. Our patient-specific brain-on-a-chip model, the NeuroVascular Unit (NVU), consists of a vascular chamber populated with hiPSC-derived brain microvascular endothelial cells (BMEC) seeded on a supporting membrane, with astrocytes seeded on the opposite side of this membrane that defines a neuronal compartment containing hiPSC-derived neurons and astrocytes embedded into hydrogel. We compared cell viability in the neuronal chamber using a live/dead stain and found twice as much cell death in TSC- than control neuronal compartments (p=0.01, N=10). In addition, we found that TSC vascular compartments are significantly more leaky than their control counterparts as assessed by measuring FITC-dextran diffusion across the BMEC cell layer (p=.001, N=10). Interestingly, the replacement of TSC astrocytes seeded onto the opposing side of the BMEC-containing vascular chamber with control astrocytes in otherwise TSC-cell populated NVUs restored viability in the neuronal compartment and BMEC barrier function in the vascular compartment to control levels. The present model for TSC pathogenesis suggests a key role for increased mTORC1 signaling. Rapamycin, which is therapeutically used in some TSC patients, inhibits mTORC1 (but not mTORC2) activity. Continuous perfusion of rapamycin (0.2 nM) through the vascular chamber significantly improved cell viability in the neuronal compartment and barrier function of the vascular chamber (p=0.02, N=10). We are presently assessing mTOR signaling by quantifying the phosphorylation of downstream targets such as S6-Kinase, S6 ribosomal protein and 4E-BP1 in the neuronal and vascular compartments of control and TSC NVUs. In summary, we have developed a patient-specific brain-on-a-chip model to study TSC pathogenesis by determining TSC cellular and biochemical phenotypes and to assess effects of presently used and prospective therapeutics.Funding Source: NIH/NCATS UG3 TR002097T-3180INTRINSINC HUMAN GLIAL PROGENITOR CELLS TRANSCRIPTOME DYSREGULATION IN VANISHING WHITE MATTER DISEASEOsorio, Maria Joana - Center for Translational Neuromedicine, University of Copenhagen, Denmark Goldman, Steve - Center for Translational Neuromedicine, University of Copenhagen, Denmark Lassen, Mette - Center for Translational Neuromedicine, University of Copenhagen, DenmarkVanishing White Matter (VWM) disease is one of the most common congenital disorders of myelin. It is caused by mutations in the eukaryotic translation initiation factor subunit genes (EIF2B1-4), the pathogenic effects of which manifest almost exclusively in the CNS. VWM is unique in its susceptibility to stress events, such as fever or head trauma, which typically precede a rapidly progressive demyelination and clinical deterioration. Despite its association with white matter loss, the cellular basis of VWM has remained unclear. Since both astrocytes and oligodendrocytes have been implicated in this disorder, we asked if bipotential human glial progenitor cells (hGPCs) might manifest pathology in the setting of EIF2B1-4 mutations. To that end, we engineered a human embryonic stem cell line (GENEA19) with homozygous single point missense mutations associated with distinct VWM phenotypes (a severe form, EIF2B5 cG584A>R195H; a classic form, EIF2B4 cC728T>P243L; and a milder form, EIF2B5 cG338A>R113H). Cells were instructed to hGPCs fate and isolated by CD140a-based FACS for RNA-sequence analysis. Functional analysis of VWM GPCs expressed higher levels of pro-apoptotic genes (TP53I3, BAK1, BAX, FAS), and exhibited dysregulation of genes involved in cell proliferation (CCNA1, CDKN2D, CDKN1B, SIX3). VWM-derived GPCs upregulated genes involved in inflammatory response (ANXA1, ANXA2. HLA-A, HLA-B, HLA-C, IFITM2, IFITM3, TRIM5, TRIM22) as well as in extracellular matrix (ECM) remodeling (CD44, MMP2; the integrins ITGB1, ITGA3, ITGA7; and the laminins LAMA5, LAMB1). Although the most severe line manifested the larger number of dysregulated genes, a subset of genes commonly dysregulated were identified as possible key denominators of VWM pathology, including CCNA1, SIX3, AQP4, TP53I3, BRINP1, ANXA1, NELL2 and ARHGEF28. VWM-derived hGPCs thus manifest intrinsic pathology at a stage preceding their differentiation as astrocytes or oligodendrocytes. They express

406POSTER ABSTRACTSpro-apoptotic transcripts at baseline, suggesting vulnerability to cell stress, and upregulate genes involved in inflammation and ECM remodeling, suggesting the potential for aberrant intercellular signaling patterns among glial cells within VWM white matter.Funding Source: Lundbeck Foundation (Denmark) Child Neurology Foundation (USA)T-3182GENERATION OF KERATINOCYTES FROM INDUCED PLURIPOTENT STEM CELLS DERIVED FROM A KINDLER SYNDROME PATIENTMoriyama, Mariko - Pharmaceutical Research and Technology Institute, Kindai University, Higashi-Osaka, Japan Ozawa, Toshiyuki - Graduate School of Medicine, Osaka City University, Osaka, Japan Hayakawa, Takao - Pharmaceutical Research and Technology Institute, Kindai University, Higashi-Osaka, Japan Moriyama, Hiroyuki - Pharmaceutical Research and Technology Institute, Kindai University, Higashi-Osaka, JapanKindler syndrome is an autosomal recessive disorder caused by the mutations in the kindlin-1 gene, which is known to bind to integrins and regulate integrin activation at cell adhesions. Kindler syndrome is characterized by skin blistering, erosion and photosensitivity. Radical treatments, such as regenerative therapies using stem cells are strongly desired because of its difficulties of complete cure. Therefore, we decided to establish induced pluripotent stem cells (iPSCs) from human adipose tissue-derived stem cells (hADSCs) or keratinocytes isolated from patient with Kindler syndrome, and differentiate them into keratinocytes in order to discover the pathogenic mechanism. In this study, we have established patient-specific, transgene-free iPSCs through electroporation of episomal vectors and growth under 5% O2 or 20% O2 condition. Consistent with previous report, 5% O2 significantly increased the iPSCs-like colony formation. The resulting iPSC lines were verified by the expression of pluripotent stem cell markers through immunofluorescent staining, quantitative PCR analysis, and flow cytometry analysis. Pluripotency of the iPSC lines were also confirmed by differentiation capacity into three germ layers. Then, the patient-specific iPSCs were differentiated into keratinocyte lineage through sequential applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen IV-coated plates. Keratinocytes differentiated from iPSCs displayed similar expression profiles with normal epidermal keratinocytes. We also found that iPSCs derived from patient’s keratinocytes possessed a more pronounced ability to differente into keratinocyte lineage than those from patient’s hADSCs. In addition, we have established Keratin 14-EGFP reporter iPSCs using Crispr/Cas9 system in order to monitor the differentiation status into keratinocytes. This study is expected to be a first step in the investigation of the underlying mechanism and a novel therapeutic development of Kindler syndrome.T-3184INSULIN RESPONSE IN HUMAN STEM CELL-DERIVED METABOLIC TISSUESFriesen, Max - Jaenisch Laboratory, Whitehead Institute for Biomedical Research, Cambridge, MA, USA Jeppesen, Jacob - Global Research, Novo Nordisk, Cambridge, MA, USA Jaenisch, Rudolf - MIT Department of Biology, Whitehead Institute for Biomedical Research, Cambridge, MA, USAWe are in the midst of a worldwide epidemic of type 2 diabetes (T2D) and associated obesity. These disorders represent a complex interaction between genes and environment. In T2D there is a well-defined progressive pathogenesis, beginning with insulin resistance in peripheral tissues such as muscle, fat and liver. This is initially compensated for by increased insulin secretion, but eventually beta cells exhaust and insulin level gradually declines leading to clinical hyperglycemia. While some alterations in insulin action leading to insulin resistance have been defined and genome wide association studies have identified many genes associated with risk of T2D, the primary defect(s) in peripheral tissues leading to insulin resistance remains unclear. To elucidate the molecular basis of insulin resistance, we first need to accurately define the acute insulin response. Omics data on human insulin response is sparse, especially in a time-lapse fashion, as there are severe practical restrictions on performing these experiments. We show transcriptional response to insulin stimulation in a human in vitro model system, and we are able to characterize this acute response in several metabolic tissues derived from human pluripotent stem cells (hPSCs). The tissues we have investigated are adipocytes, hepatocytes, skeletal muscle, endothelial cells and vascular smooth muscle. We see a response in gene expression as early as 5 minutes after stimulation and can monitor this signal over time. These types of experiments are impossible to conduct in humans, demonstrating a clear advantage of our system. Several crucial transcription factors, such as MLXIPL and SREBF1, have a conserved response in multiple tissues. Both of these proteins are well known players in glucose and lipid metabolism. These early responders will turn on consecutive waves of transcription, from which we can gain an understanding of the cell’s exact reaction to insulin stimulation. In this setting we then induce insulin resistance, through genome editing or chemical means, and comprehensively survey the changes in insulin response. Ultimately, we aim to illuminate novel and druggable targets in the insulin signaling pathway that would be of value in understanding and treating insulin resistance in the setting of human metabolic disease.T-3186USING PATIENT-DERIVED HIPSCS TO MODEL THE ROLE OF GLIA IN AUTISM SPECTRUM DISORDERLi, Jingling - Department of Psychiatry, Stanford University School of Medicine, Stanford, CA, USA

407POSTER ABSTRACTSChetty, Sundari - Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USAAutism Spectrum Disorder (ASD) is defined as a group of neurodevelopmental disorders associated with impaired social communication, repetitive behaviors, and intellectual deficits. However, early diagnosis of ASD remains challenging due to its complexity and heterogeneity. In some subtypes of ASD, an increase in brain size precedes the first clinical signs, suggesting that understanding the mechanisms leading to brain overgrowth could provide important insights in disease onset. Here, we use human induced pluripotent stem cell (hiPSC) technology to model ASD associated with disproportionate megalencephaly (ASD-DM) and investigate the cellular and molecular mechanisms involved. While an overall enlargement in brain size has been shown through brain imaging of autistic children with megalencephaly, the iPSC models to date have primarily focused on modeling neurons in ASD. In this study, we investigate changes in glial cells by differentiating hiPSCs to brain-derived glia from control subjects and ASD-DM subjects. Changes in morphology, including the cell body size and the number of processes, and proliferation rates were compared across control and patient-derived hiPSCs. Preliminary data shows that the ASD-DM-derived glia have more processes. Also, increases in proliferation could be observed in glia from ASD-DM compared to the controls. We also investigate and compare changes in gene expression associated with the cell cycle. Overall, these data indicate that ASD with megalencephaly is possibly a result of enhanced proliferation. While prior studies have focused on changes in neurons, this study is the first to systematically investigate changes in glial cells in ASD-DM.T-3188THE NIMH REPOSITORY AND GENOMICS RESOURCE (NRGR): A GLOBAL RESOURCE FOR THE STUDY OF THE GENETICS OF PSYCHIATRIC DISEASESheldon, Michael - Genetics/Rutgers University, RUCDR Infinite Biologics, Piscataway, NJ, USA Moore, Jennifer - Genetics, Rutgers University, Piscataway, NJ, USA Chu, Jianhua - Genetics, Rutgers University, Piscataway, NJ, USA Tischfield, Jay - Genetics, Rutgers University, Piscataway, NJ, USA Brzustowicz, Linda - Genetics, Rutgers University, Piscataway, NJ, USAEstablished in 1998, RUCDR Infinite Biologics (RUCDR, www.rucdr.org) is the world’s largest university-based integrated cell and DNA repository, assisting researchers throughout the world by providing the highest quality biomaterials, technical consultation, and logistical support. Its services include sample collection and bioprocessing (i.e., blood fractionation, nucleic-acid extraction, cell-line creation, etc.) and analytical services such as gene expression, sequencing, and genotyping. RUCDR offers comprehensive stem cell culture services that include the reprogramming of source cells such as skin fibroblasts and blood cells to yield induced pluripotent cells (iPSC) and genome editing using CRISPR/CAS technology. In addition, RUCDR performs a complete range of assays to characterize iPSCs to assess their quality, pluripotency, germline potential and genomic stability, and distributes a cGMP grade iPSC line. RUCDR has been awarded a cooperative grant from NIMH to establish stem cell repositories that provide high quality patient and control iPSCs and somatic cells from a wide range of disorders. The NIMH Stem Cell Center has 376 fibroblast and 325 induced pluripotent stem cell (iPSC) lines in support of investigators engaged in stem cell-based research relevant to mental disorders, including but not limited to anxiety disorders, attention deficit hyperactivity disorder, autism spectrum disorders, bipolar disorder , depression, eating disorders, obsessive-compulsive disorder, post-traumatic stress disorder, and schizophrenia. The NIMH cell line collection administered through the Stem Cell Center can be accessed through the menus at https://www.nimhgenetics.org/.Funding Source: Funded by a grant from NIMH (6U24MH068457-16)REPROGRAMMINGT-3192EFFICIENT DIRECT LINEAGE REPROGRAMMING OF FIBROBLASTS INTO INDUCED CARDIOMYOCYTES USING NANOTOPOGRAPHICAL CUESKim, Junyeop - Lab of Stem Cells and Cell Reprogramming, Dongguk University, Seoul, Korea Kim, Jongpil - Lab of Stem Cells and Cell Reprogramming, Dongguk University, Seoul, KoreaInduced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show the efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Direct conversion into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes compared with that on flat substrates. Additionally, the enhanced reprogramming by the substrate nanotopography was derived from changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.T-3194CRITICAL ROLE OF THE CHROMATIN REGULATOR TIP60 IN NEURONAL FATE SPECIFICATIONJanas, Justyna A - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA

408POSTER ABSTRACTSZhang, Lichao - Chemical and Systems Biology, Stanford University, Stanford, CA, USA Mall, Moritz - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA Elias, Joshua - Chemical and Systems Biology, Stanford University, Stanford, CA, USA Jackson, Peter - Baxter Laboratory, Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA Wernig, Marius - Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University, Stanford, CA, USADirect lineage reprogramming of a somatic cell type into another has emerged as an attractive approach for generating patient-specific cells for cell replacement and tissue repair without the complications of immune rejection and teratogenicity. The direct conversion of non-neuronal cells into functional neurons is particularly promising in the context of developing treatments and modeling of neurological disorders given the lack of access to human neurons. We have previously shown that fibroblasts can be reprogrammed directly into functional induced neuronal (iN) cells by ectopic expression of three transcription factors, Ascl1, Brn2 and Myt1l (BAM). We have also found that Ascl1 alone is sufficient to initiate the iN reprogramming process and acts as a ‘pioneer transcription factor’ by binding and activating inaccessible chromatin regions. However, the precise mechanism by which Ascl1 activates host chromatin and neuronal gene expression in fibroblasts remains elusive. Here we used unbiased proteomics approach to identify chromatin regulators that mediate Ascl1-driven reprogramming in MEFs and identified Tip60 acetyltransferase complex as a key player in iN cell reprogramming. We found that suppression of Tip60 via shRNA mediated knockdown prevented iN generation induced by Ascl1. This reprogramming block could be rescued by re-expression of wild type Tip60, but not catalytically deficient mutant of Tip60. We further demonstrate that the acetyltransferase activity of Tip60 is essential for proper regulation of a subset of genes during reprograming and for establishing of neuronal cell identity. By elucidating detailed mechanisms of the function of Tip60 during iN induction our study provides insights into molecular mechanisms of iN cell reprogramming and basis for future strategies of iN cell generation for biomedical and therapeutic applications.T-3196MITIGATING ANTAGONISM BETWEEN TRANSCRIPTION AND PROLIFERATION ALLOWS NEAR-DETERMINISTIC CELLULAR REPROGRAMMINGBabos, Kimberley N - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Galloway, Kate - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Kisler, Kassandra - Physiology and Biophysics, University of Southern California, Los Angeles, CA, USA Zitting, Madison - Physiology and Biophysics, University of Southern California, Los Angeles, CA, USA Li, Yichen - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Shi, Yingxiao - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Quintino, Brooke - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Chow, Robert - Physiology and Biophysics, University of Southern California, Los Angeles, CA, USA Zlokovic, Berislav - Physiology and Biophysics, University of Southern California, Los Angeles, CA, USA Ichida, Justin - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAAlthough cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining the reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription, and that maintaining hypertranscription early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and restricts cell proliferation, which also promotes reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation in part through activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes epigenetic barriers to cellular reprogramming.T-3198RAPID GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS USING A BETA-DEFENSIN3-DERIVED PEPTIDE AND INTEGRATION-FREE NUCLEOFECTIONPark, Kwang-Sook - School of Dentistry, Seoul National University, Seoul, Korea Lee, Dongwoo - School of Dentistry, Seoul National University, Seoul, Korea Lee, Jue-Yeon - Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul, Korea Chung, Chong Pyoung - Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul, Korea Park, Yoon Jeong - School of Dentistry, Seoul National University, Seoul, Korea

409POSTER ABSTRACTSSomatic reprogramming from matured cells to induced pluripotent stem cells (iPSCs) is one of the best model to represent a mesenchymal-to-epithelial transition (MET). The low efficiency of iPSC generation has been improved by enhancing MET using microRNA, small molecules, and surface stiffness. However, they have some limitations like stability and toxicity. Here, we discovered a synthetic peptide from human beta-defensin-3 (hBD3) with anti-inflammatory functions and applied to iPSC generation. The generation of iPSCs was induced by nucleofection of four transcription factors (Oct4, Sox-2, KLF4 and c-Myc) and the treatment of the hBD3-derived peptide. We found that the peptide significantly increases endogenous expression of stemness genes (Oct4 and Nanog) and epitheial genes (E-cadherin and Ep-CAM) compared to non-treated group. Also the peptide reduces the time of iPSC generation compared to non-treated group. The colonies produced using nucleofection and peptide treatment has in vitro and in vivo differentiation potentials into three germ layers. Taken together, our peptide eliminates potential risks associated with the use of chemical small molecules, providing the promising technique for patient-derived iPSCs.T-3200CHANGES IN ENERGY METABOLISM AND SUBCELLULAR ORGANELLES IN PLURIPOTENT STEM CELLS DUE TO METABOLIC SWITCHINGHan, Min-Joon - Hematology/St. Jude, St. Jude, Memphis, TN, USABecause of their distinctive character, human pluripotent stem cells (hPSCs) undergo significant changes not only in their gene expression but also in the production and/or expenditure of energy in the form of ATP via switching of their metabolic signature from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. This phenomenon is referred to as the Warburg effect and is a hallmark of hPSCs. The translation of functional proteins from mRNA in the cytoplasm is a major cause of energy consumption in the cell. We performed a comparative proteomics study of induced hPSCs and the fibroblasts from which they were derived, and the results suggest that mRNA translation is reduced in hPSCs. In addition, compared with somatic cells, hPSCs have large nuclei and scanty cytoplasm, which signify changes in their subcellular organelles. Our investigation of organelle function in hPSCs revealed significant changes in the mitochondrial and lysosomal functions that lead to the metabolic switch in these cells.Funding Source: This work was supported by St. Jude institutional funds (to M-J Han)T-3202INDUCTION OF PLURIPOTENCY BY ALTERNATIVE FACTORSWu, Linlin - Guangzhou Institute of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, China Academy of Sciences, Guangzhou, ChinaReprogramming somatic cells to pluripotency represents a paradigm for cell fate determination. A binary logic of closing and opening chromatin provides a simple way to understand iPSC reprogramming driven by both Yamanaka factors or chemicals. Here we apply this logic to the design a seven factors combination, Jdp2, Jhdm1b, Mkk6, Glis1, Nanog, Essrb and Sall4 (7F), that reprogram MEFs to chimera competent iPSCs efficiently. RNA-and ATAC-seq reveal differences between 7F and Yamanaka Factors induced pluripotency, 7IP and YIP, in transcriptomic and chromatin accessibility dynamics(CAD). Sall4 emerges as a dominant force that can close and open chromatin with the help of Jdp2 and Glis1 in resetting somatic chromatin to a pluripotent state. These results reveal a previously unknown path between somatic and pluripotent states, open a door for cell fate control.T-3204VANGL2 RECEPTOR OVEREXPRESSION INCREASES APOPTOSIS AND EFFECTS INFLAMMATION AS WELL AS MIGRATION AND DIFFERENTIATIONS IN HUMAN HEK293 CELLSLindqvist, Maria - Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, SwedenWe had earlier shown that Wnt7a which is a ligand for Vangl2 receptor effects apoptosis and migration of epithelial cells.The purpose of current study is to investigate if Vangl2 is effected in wound healing and if this signaling pathway is effected when anesthetics are used. 1.Analyzing closer which molecules mediate Vangl2 signalings effect on apoptosis by investigating caspase-8 singling using live cell imaging and investigate if this signaling pathway is intervened when anesthetics are applied. 2.Analyzing closer which molecules mediate Vangl2 signalings effect on migration and see if its mediated via ezrin,paladin and FGF7 and look closer into whether this signaling pathway is intervened when anesthetics are applied. We conclude that Vangl2 overexpression/silencing effects apoptosis and hypothezise that this is done via caspase-8 signaling. We also suggest that the rearrangement of p53 and Th17 expression obtained in our preliminary experiments is a result of actin translocation from the cytoplasm into the nucleus and is probably mediated by exosomes/micro-RNAs and possibly interfered when anesthetics are applied.We further suggest that the delay in differentiation is a result of Vangl2 signalings effect on the expression of Cx43.Funding Source: The experiments were funded by her royal highness Crownprincess Lovisa foundation for the care of sick children

410POSTER ABSTRACTST-3206TEMPORAL RESOLUTION OF GLOBAL GENE EXPRESSION AND DNA METHYLATION CHANGES REVEALS UNIQUE FEATURES OF EPIGENETIC REMODELLING IN THE FINAL PHASES OF INDUCED PLURIPOTENCYBartoccetti, Michela - Stem Cell Institute Leuven, KU Leuven, Belgium Luo, Xinlong - Stem Cell Institute Leuven, KU Leuven, Belgium van der Veer, Ben - Stem Cell Institute Leuven, KU Leuven, Belgium Koh, Kian Peng - Stem Cell Institute Leuven, KU Leuven, BelgiumTranscription factor-mediated reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) progresses via sequential events to clear epigenetic roadblocks in the path of full pluripotency acquisition. A better understanding of the global epigenome resetting involved is not only relevant to improving the quality of iPSCs, but also in understanding disease. The use of markers to characterize intermediate stages is essential to explore the underlying mechanisms, but existing markers do not provide sufficient temporal resolution of global transcriptomic and DNA methylation changes that occur during the late maturation stage of iPSC generation. The 5-methylcytosine dioxygenase Tet1 is part of the DNA demethylation machinery and is highly elevated in endogenous expression along with the master pluripotency factor Oct4 late in reprogramming. Here, we generated murine transgenic lines harboring dual fluorescent reporters reflecting cell-state specific expression of Oct4 and Tet1. By assessing reprogramming intermediates based on dual reporter patterns, we identified a sequential order of Tet1 and Oct4 gene activation at proximal and distal regulatory elements following pluripotency entry. Full induction of Tet1 marks a pivotal late intermediate stage occurring after a phase of global gene repression, and preceding full activation of Oct4 along with late naive pluripotency and germline-specific genes. Sequential activation of Tet1 further distinguishes two waves of global DNA demethylation, targeting distinct genomic features and largely uncoupled from transcriptional changes. Absence of Tet1 is compatible with reprogramming towards full Oct4 gene activation, but generates iPSCs with aberrant DNA methylation, chromosomal instability during lineage priming and defective differentiation potential. Our study captured the dynamics of global epigenome reprogramming with unprecedented precision, revealing molecular parallels and distinction from physiological reprogramming that may account for observed epigenetic differences between embryo-derived and experimentally induced pluripotent cells. Furthermore, the transcriptional logic of Tet1 expression signals a deterministic epigenetic roadmap towards generation of high-quality iPSCs.Funding Source: Fonds voor Wetenschappelijk Onderzoek (FWO) Research Foundation – Flanders Odysseus Program grants G.0C56.13N, Research Project grant G.0632.13 and KU Leuven Internal Funds C14/16/077.T-3208PHYSIOXIC CONDITIONS WITHOUT SUBOPTIMAL TRANSIENTS DURING CELL HANDLING INCREASES HUMAN BONE MARROW MSC YIELDSHenn, Alicia - BioSpherix Medical, Parish, NY, USA He, Yan - Scientific, BioSpherix, Parish, NY, USA Darou, Shannon - Scientific, BioSpherix, Parish, NY, USA Yerden, Randy - Scientific, BioSpherix, Parish, NY, USAIsolated from tissues that normally are at low oxygen levels like the bone marrow stem cell niche, Mesenchymal Stromal Cells (MSC) and the exosomes derived from them are of intense clinical interest. Many cell culturists still handle MSC in conventional room air biological safety cabinets (BSC) even though MSC are isolated from and returned to tissues with profoundly low oxygen levels. This causes stress for MSC. We have shown previously that subjecting cells to room air conditions (suboptimal transients) during cell handling negative impacts MSC yields. Here, we sought to determine what unbroken O2 level produced the best yields of human bone marrow MSC, testing 0.5%, 1%, 3%, or 18% O2, each with 5%CO2. Using the Xvivo System, all temperature and gas levels were controlled continuously, making it possible to maintain optimal low O2 conditions for the cells during all cell handling steps as well as incubation. Our null hypothesis was there would be no difference in MSC growth between the conditions. The cell handling chamber conditions were set to match incubation conditions, so each culture was in constant conditions and media were pre-equilibrated to the matching O2 levels overnight before use. Cells were counted at each passage and cells were seeded into 96-well plates and stained with crystal violet to visualize cell growth. Vessel headspace and pericellular O2 were measured before each passage. Differences were seen in cell growth with higher cumulative cell yields and faster cell growth were seen when cells were maintained continuously at 1% and 3% O2, than at 0.5% or 18%O2. Pericellular O2 levels were lower than chamber O2 levels. Cells maintained at 18% O2 senesced earlier than the other cultures. We conclude that MSC cultures can be grown to higher yields when conditions are controlled for cell handling at the appropriate tissue O2 levels.TECHNOLOGIES FOR STEM CELL RESEARCHT-3210CRISPR/CAS9 GENE EDITING FOR GENERATING IPSC MODELS OF HUMAN DISEASES AND DEVELOPMENTChu, Jianhua - RUCDR Infinite Biologics, Piscataway, NJ, USA Moore, Jennifer - Department of Genetics, Rutgers University, Piscataway, NJ, USA Sheldon, Michael - Department of Genetics, Rutgers University, Piscataway, NJ, USA

411POSTER ABSTRACTSSutherland, Margaret - The National Institute Neurological Disorders and Stroke, Rockville, MD, USA Swanson-Fischer, Christine - The National Institute Neurological Disorders and Stroke, Rockville, MD, USA Tischfield, Jay - Department of Genetics, Rutgers University, Piscataway, NJ, USASince its inception in 1998, RUCDR Infinite Biologics (RUCDR, www.rucdr.org) has provided the scientific community with the highest quality biomaterials, technical consultation, and logistical support. The stem cell lab of RUCDR provides stem cell services to researchers, the NIH and non-profit foundations. These services include the banking and distribution of source cells and induced pluripotent stem cells (iPSC) and the generation of iPSC from human somatic cells. Most recently, we have begun offering CRISPR gene editing service in human iPSCs through the use of CRISPR (clustered regularly-interspaced short palindromic repeats)/Cas9 technology. The diverse genetic background of the human subjects has hampered the usefulness of iPSCs for modeling human diseases and development. The use of the CRISPR/Cas9 system can create isogenic cell lines that will serve as better controls and help eliminate effects that are due to genetic variance rather than a biological mechanism. At RUCDR/Infinite Biologics we have developed a high throughput, cost efficient workflow for using CRISPR/Cas9 to genetically modify iPSC from affected or unaffected subjects. Using this strategy, we have generated footprint-free and precisely edited isogenic iPSC pairs harboring mutations involved in neurological disorders such as Alzheimer’s Disease, Amyotrophic Lateral Sclerosis (ALS) and Parkinson’s Disease. All edited iPSC lines are tested rigorously for off-target effects, homogeneity, pluripotency and genetic integrity. Data of our quality control process is included in the COA for each iPSC line we distribute.T-3212QUANTITATIVE LIVE-CELL ANALYSIS CHARACTERIZING MORPHOLOGY AND FUNCTION OF IPSC-DERIVED NEURONS AND SUPPORT CELLSRauch, John N - Essen BioScience, Ann Arbor, MI, USA Alcantara, Susana - Research and Development, Essen Bioscience, Welwyn Garden City, UK Appledorn, Daniel - Research and Development, Essen Bioscience, Ann Arbor, MI, USA Dale, Tim - Research and Development, Essen Bioscience, Welwyn Garden City, UK Lovell, Gillian - Research and Development, Essen Bioscience, Welwyn Garden City, UK Oupicka, Libuse - Research and Development, Essen Bioscience, Ann Arbor, MI, USA Overland, Aaron - Research and Development, Essen Bioscience, Ann Arbor, MI, USA Schramm, Cicely - Research and Development, Essen Bioscience, Ann Arbor, MI, USARecent advances in stem cell technologies offer an exciting alternative to rodent models for investigating the human nervous system and neurological disorders. The ability to simultaneously monitor morphology and functional readouts over time is critical for thorough characterization of human induced pluripotent stem cell (hiPSC)-derived models. Typically endpoint assays of subtype-specific surface marker expression and morphological features are employed. These techniques have proven valuable, but they fail to couple insight into the ability of these cells to exhibit neuronal activity and develop mature networks. In the case of hiPSC-derived microglia, it is important to ensure that these cells are capable of critical neuroimmune functions such as phagocytosis and chemotaxis. Here we present live-cell imaging data used to quantify functional readouts of hiPSC-derived neurons and microglia and qualitatively assess morphology using the IncuCyte® for Neuroscience. Monitoring of differentiating neuro-progenitor cells (Axol Bioscience) enabled the maturation to iPSC-derived neurons to be visualized over >80 days, with marked morphological and functional development. Activity of hiPSC-derived glutamatergic and GABAergic neurons (CDI, iCell Neurons) was visualized and analyzed using the Incucyte® NeuroBurst reagent, a genetically encoded calcium indicator. While both neuronal types exhibited spontaneous activity, only glutamatergic neurons developed coordinated synaptic network activity over time in culture. To exemplify the benefit of long term monitoring of these models, the CNS.4U® (NCardia) co-culture model of hiPSC-derived neurons and astrocytes were evaluated for over 40 days. While spontaneous activity was detected after one week, correlated network bursting was only observed after 30 days in culture. Finally, hiPSC-derived microglia (Axol Bioscience) were monitored for morphological changes following differentiation and evaluated for phagocytic potential by use of a pH sensitive dye. Collectively, these data illustrate the ability to monitor and characterize hiPSC-derived neuronal cultures and support cells over time using live-cell analysis techniques.T-3214ENHANCING FUNCTIONAL MATURATION OF HUMAN IPSC-DERIVED NEURONS WITH NOVEL PEPTIDE AMPHIPHILE NANOSTRUCTURESOrtega, Juan Alberto - Neurology / Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Alvarez, Zaida - Simpson Querrey Institute of BioNanotechnology, Northwestern University, Chicago, IL, USA Sato, Kohei - Simpson Querrey Insitute of BioNanotechnology, Northwestern University, Chicago, IL, USA Sasseli, Ivan - Simpson Querrey Insitute of BioNanotechnology, Northwestern University, Chicago, IL, USA Quinlan, Katharina - George and Anne Ryan Institute for Neuroscience, University of Rhode Island, Kingston, RI, USA Edelbrock, Alexandra - Simpson Querrey Insitute of BioNanotechnology, Northwestern University, Chicago, IL, USA Kiskinis, Evangelos - Neurology / Feinberg School of Medicine,

412POSTER ABSTRACTSNorthwestern University, Chicago, IL, USA Stupp, Samuel - Simpson Querrey Insitute of BioNanotechnology, Northwestern University, Chicago, IL, USAHuman induced pluripotent stem cell (iPSC)-based technologies offer a unique resource for modeling disease and regenerating complex tissues such as the central nervous system (CNS). However, iPSC models are still fraught with significant technical limitations including inefficient maturation, abnormal aggregation and reduced long-term viability of neurons. We reasoned that the lack of physiological extracellular matrix (ECM) conditions contributes to these problems and establishing a stable and bioactive ECM environment would facilitate the functional maturation of iPSC-derived neurons. To test this, we utilized peptide amphiphiles (PAs), a class of biomaterials that have the ability to assemble into supramolecular nanofibers capable of morphologically and chemically mimicking the ECM. We designed a series of PAs scaffolds containing a short bioactive peptide (IKVAV) found in Laminin-1, which plays a major role in neuronal behavior in the CNS. The newly design PAs have an identical chemical bioactivity but the supramolecular fibers that they form drastically differ in terms of mobility. Here, we have specifically explored how the molecular mobility of ECM signals impacts the maturation of different classes of iPSC-derived human neurons. We identified an IKVAV-PA that dramatically enhanced the maturation of iPSC-derived cortical and motor neurons in vitro. The beneficial activity of this IKVAV-PA strongly correlated with its intermolecular hydrogen-bonding domain, which facilitated high molecular mobility, mimicking the dynamic ECM-neuronal interactions. Neurons assembled within the high dynamic IKVAV-PA exhibited reduced aggregation, enhanced arborization, and a mature pattern of electrical activity closely resembling neurons co-cultured with primary glial cells. Using global proteomic analysis and high-resolution microscopy, we traced these effects to specific and relevant molecular pathways that were downstream of laminin-1/Integrin beta-1 signaling pathway. Our PA technology highlights the importance of the ECM in recapitulating in vivo conditions and offers a more physiological and translational platform to study the development, function and dysfunction of the CNS in disease or injury using iPSC-based approaches.FundingSource:SimpsonQuerreyInstituteofBioNanotechnology and US National Institutes of Health (NIH) / National Institute on Neurological Disorders and Stroke (NINDS) and National Institute on Aging (NIA) R01NS104219.T-3216CORRELATION BETWEEN CELL MORPHOLOGICAL INFORMATION OBTAINED FROM DIGITAL IN-LINE HOLOGRAPHIC MICROSCOPY (D-IHM) AND FUNCTIONAL CHARACTERISTICS OF HUMAN MESENCHYMAL STEM CELLSNagaishi, Kanna - Second Department of Anatomy, Sapporo Medical University, Sapporo, Japan Yamamoto, Shuhei - Cell Business Development Section, Analytical and Measurements Division, Shimadzu Corporation, Kyoto, Japan Sawada, Ryuji - Cell Business Development Section, Analytical and Measurements Division, Shimadzu Corporation, Kyoto, JapanIn this study, we focused on the morphological information of human mesenchymal stem cells (MSCs) obtained from digital in-line holographic microscopic images (D-IHM) acquired by CultureScanner CS-1. MSCs are considered the most attractive cell source for regenerative medicine. MSCs have been highlighted because of their multi-potentialities, ease of culture and expansion in vitro. MSCs have already been utilized in clinical trials, including for GVHD, autoimmune diseases, inflammatory bowel disease and spinal cord injury, as therapeutic applications for tissue regeneration and in immune regulation. The therapeutic effect of MSCs is highly affected by the characteristic of cultured cells. Characteristics of MSCs are mainly evaluated by cell function, such as gene expression, protein expression, proliferation, differentiation and cellular energy metabolism analysis. While these have advantages in evaluating various factors, there are risks of destruction and invasion of cells during non-sterile operations. Furthermore, it is difficult to directly evaluate the cells themselves used for therapeutic administration. In contrast, the morphological evaluation of cultured cells is mainly performed by phase contrast microscopy. Morphological characteristics, such as cytoplasmic and nuclear shapes in individual cells, cell density and cell luminance are obtained through this method, but it is difficult to estimate these features objectively or further predict cell function. Therefore, we focused on the morphological analysis using D-IHM, which provide information of optical thickness and transmittance of cultured cells non-invasively and over time in a wide field of view. We have previously shown the significance of measuring the lengths of the minor axis (LMA) of cultured MSCs in phase contrast images. In smaller LMA, there is higher proliferation, migration and the secretion of regenerating factors. However, it is difficult to quantify LMA objectively in large numbers of cells. We investigated how D-IHM tended to correlate with LMA and functional characteristics of human umbilical cord-derived MSCs. We will report on the utility of morphological information obtained from D-IHM as a novel non-invasive method for predicting functional properties of MSCs.Funding Source: Shimadzu CorporationT-3218LONGITUDINAL INTRAVITAL IMAGING OF TRANSPLANTED MESENCHYMAL STEM CELLS ELUCIDATES THEIR FUNCTIONAL INTEGRATION AND THERAPEUTIC POTENCY IN AN ANIMAL MODEL OF INTERSTITIAL CYSTITISJu, Hyein - Department of Biomedical Sciences, University of Ulsan College of Medicine, Gyeong, Korea Lim, Jisun - Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Korea Ryu, Chae-Min - Department of Biomedical Sciences,

413POSTER ABSTRACTSUniversity of Ulsan College of Medicine, Seoul, Korea Yu, Hwan Yeul - Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Korea Heo, Jinbeom - Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Korea Shin, Jung-Hyun - Department of Biomedical Sciences, Asan Medical Center, Seoul, Korea Kim, Jun Ki - Biomedical Engineering Center, Asan Medical Center, Seoul, Korea Choo, Myung-Soo - Department of Urology, Asan Medical Center, Seoul, Korea Shin, Dong-Myung - Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, KoreaMesenchymal stem cell (MSC) therapy may be a novel approach to improve interstitial cystitis/bladder pain syndrome (IC/BPS), an intractable disease characterized by severe pelvic pain and urinary frequency. Unfortunately, the properties of transplanted stem cells have not been directly analyzed in vivo, which hampers elucidation of the therapeutic mechanisms of these cells and optimization of transplantation protocols. Here, we monitored the behaviors of multipotent stem cells (M-MSCs) derived from human embryonic stem cells (hESCs) in real time using a novel combination of in vivo confocal endoscopic and microscopic imaging and demonstrated their improved therapeutic potency in a chronic IC/BPS animal model. Ten-week-old female Sprague-Dawley rats were instilled with 10 mg of protamine sulfate followed by 750 μg of lipopolysaccharide weekly for 5 weeks. The sham group was instilled with phosphate-buffered saline (PBS). Thereafter, the indicated dose (0.1, 0.25, 0.5, and 1×106 cells) of M-MSCs or PBS was injected once into the outer layer of the bladder. The distribution, perivascular integration, and therapeutic effects of M-MSCs were monitored by in vivo endoscopic and confocal microscopic imaging, awake cystometry, and histological and gene expression analyses. A novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, together with immunofluorescence analysis of bladder tissues, demonstrated that transplanted M-MSCs engrafted following differentiation into multiple cell types and gradually integrated into a perivascular-like structure until 30 days after transplantation. The beneficial effects of transplanted M-MSCs on bladder voiding function and the pathological characteristics of the bladder were efficient and long-lasting due to the stable engraftment of these cells. This longitudinal bioimaging study of transplanted hESC-derived M-MSCs in living animals reveals their long-term functional integration, which underlies the improved therapeutic effects of these cells on IC/BPS.T-3220UNIVERSAL CORRECTION OF BLOOD COAGULATION FACTOR VIII IN PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS USING CRISPR/CAS9Sung, Jin Jea - Department of Physiology, Yonsei Medical School, Seoul, Korea Park, Chul-Yong - Department of Physiology, Yonsei University College of Medicine, Seoul, Korea Cho, Sung-Rae - Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea Kim, Dong-Wook - Department of Physiology, Yonsei University College of Medicine, Seoul, KoreaHemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII). Genome editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correcting various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein in vitro and functionally rescued the disease phenotype in vivo. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.Funding Source: Ministry of Science, ICT and Future Planning (2016R1C1B1008742)(CYP) and National Research Foundation (2017M3A9B4042580), Ministry of Health and Welfare (HI15C0916)(DWK).T-3222ARTIFICIAL INTELLIGENCE CAPTURES STRUCTURAL TOXICITY IN HUMAN IN VITRO CELL MODELSMaddah, Mahnaz - Dana Solutions, Palo Alto, CA, USA Loewke, Kevin - Dana Solutions, Palo Alto, CA, USA Mandegar, Mohammad - Tenaya Therapeutics, South San Francisco, CA, USA Ribeiro, Alexandre - Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USAPredicting drug toxicity is an important part of the drug development process as adverse side effects are the main causes for drug attrition. In vitro human cell models, such as cells differentiated from induced pluripotent stem cells (iPSCs), can reflect human-specific physiology and pharmacology and have been increasingly investigated to evaluate toxicity early in drug development. Most cell-based imaging assays focus

414POSTER ABSTRACTSon cell damage through mitochondrial deficiency or nuclear count, or functional defects such as arrhythmia or prolonged contraction time in cardiomyocytes. There is a need for new assays that can capture structural changes in cells, which may complement existing assays, improve sensitivity, and better predict clinical toxicity. We propose a novel method, PhenoTox, which uses artificial intelligence (deep neural networks) to capture subtle structural changes in cell cultures that relate to toxic drug effects. The input to PhenoTox is a collection of microscopy images captured and grouped at multiple doses and time-points for the drug of interest and a control set for each time point where no drug is applied. PhenoTox performs a series of 2-class neural network trainings comparing controls to the test conditions and generates a classification accuracy for each training. The final output is a heatmap of the z-factors across all test conditions, depicting the doses and timepoints at which structural changes have happened and how strongly they differ from controls. As part of a large ongoing study, we applied PhenoTox to characterize the effects of drugs with known toxicity profiles on both iPSC-derived hepatocytes and cardiomyocytes, including Tamoxifen, Doxorubicin, and Aspirin as a negative control. The cells were fixed and stained, and fluorescence microscopy images were collected from multiple wells with multiple locations per well. For Tamoxifen-treated hepatocytes, PhenoTox detected structural changes that correlated with loss of Cytochrome P450 3A4 activity. For Doxorubicin-treated cardiomyocytes, PhenoTox detected structural changes that correlated strongly with decreased contraction displacement. No structural change was detected for Aspirin. These results indicate that PhenoTox captures structural changes across multiple cell types that correlate with functional results.T-3224MURINE SCNT-DERIVED ESCS AND IPSCS WITH MISMATCHED MITOCHONDRIA TRIGGER AN IMMUNE RESPONSE IN VIVOHu, Xiaomeng - Department of Surgery/ TSI Lab, UCSF, San Francisco, CA, USA Deuse, tobias - Surgery, UCSF, San Francsico, CA, USA Agbor-Enoh, Sean - NHLBI, NIH, Bethesda, MD, USA Gravina, Alessia - Surgery, UCSF, San Francsico, CA, USA Alawi, Malik - Heinrich Pette Institute, UKE, Hamburg, Germany Marishta, Argit - NHLBI, NIH, Bethesda, MD, USA Wang, Dong - Surgery, UCSF, San Francsico, CA, USA Valantine, Hannah - NHLBI, NIH, Bethesda, MD, USA Weissman, Irving - Stanford Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA Schrepfer, Sonja - Surgery, UCSF, San Francsico, CA, USAThe generation of human pluripotent stem cells by somatic cell nuclear transfer (SCNT) has sparked new interest in this technology since this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer derived mouse embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies. In this context, despite their autologous nature, rejection of induced pluripotent stem cell (iPSC)-derived cells has been reported, although mechanistic details remain largely unknown. We hypothesized that new mutations in the mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than the chromosomal DNA, can give rise to neoantigens capable of eliciting an allogeneic immune response and causing rejection. Here we show that in mice, mutagenic nonsynonymous mtDNA single nucleotide polymorphisms (SNPs) generate relevant neoantigens, which initiate a highly specific immune response. This neo-antigenicity of SNPs can diminish the survival of iPSC grafts and their derivatives in autologous recipients. Thus, autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and therefore techniques need to be developed to monitor mtDNA mutations and SNP enrichments during the manufacturing of autologous iPSC products.T-3226GENE EDITING AND MODULATION TOOLS FOR LONG NON-CODING RNA APPLICATIONSIniguez, Karen - Biology, California State University, San Marcos, San Marcos, CA, USA Yang, Jian-Ping - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Braun, Julia - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Magnon, Veronica - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Jacobsen, Natasha - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Zou, Yanfei - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Chesnut, Jonathan - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USA Ravinder, Namritha - Cell Biology Gene Editing, Thermo Fisher Scientific, Carlsbad, CA, USAApproximately 98% of the human genomic sequence is characterized as non-coding RNA. A large subgroup of RNA, called long non-coding RNAs (lncRNAs), are characterized as having sequences of 200 nucleotides long or more and mediate numerous biological processes, including stem cell differentiation and maintenance. Although more and more novel lncRNAs have been identified through advancement of sequencing technologies, the biological purpose of the majority of lncRNAs remain unclear due to their low and cell specific expression

415POSTER ABSTRACTSlevels, varied cellular localization, shared genomic sequences with coding transcripts, and lack of effective investigating technologies. Here we evaluated several gene editing and modulation technologies that can be applied for lncRNA study: (1) RNA interference: transfection of siRNA enabled efficient repression of targeted mRNA level; (2) Cas9/RNP: Co-delivery of two synthetic gRNA with Cas9 protein into the cells resulted in gene knockouts through specific gene deletion; (3) Lentiviral based CRISPR system: we further established a dual gRNA lentiviral system that allows expression of two gRNAs for deleting user defined genomic DNA sequences. We will discuss advantages and limitations of each technology and provide useful guidance for lncRNA applications in relevant cell models, including, but not limited to, stem cells.Funding Source: California Institute for Regenerative Medicine Thermo Fisher ScientificT-3228BOVINE PLATELET LYSATE-DERIVED SERUM NEOSERA IS SAFE, LESS ETHICAL AND POWERFUL ALTERNATIVE TO FETAL BOVINE SERUM FOR THE CULTURE OF MESENCHYMAL STEM CELLSYamahara, Kenichi - Laboratory of Medical Innovation, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan Hamada, Akiko - Laboratory of Medical Innovation, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan Umezawa, Ko - General Manager, Japan Biomedical Co., Ltd, Hokkaido, Japan Sudo, Toshita - Representative Director, Japan Biomedical Co., Ltd, Hokkaido, Japan Yoshihara, Kyoko - Department of Transfusion Medicine and Cell Therapy, Hyogo College of Medicine, Hyogo, Japan Yoshihara, Satoshi - Department of Transfusion Medicine and Cell Therapy, Hyogo College of Medicine, Hyogo, Japan Okada, Masaya - Department of Transfusion Medicine and Cell Therapy, Hyogo College of Medicine, Hyogo, Japan Soma, Toshihiro - Department of Transfusion Medicine and Cell Therapy, Hyogo College of Medicine, Hyogo, Japan Ohnishi, Shunsuke - Department of Gastroenterology and Hepatology, Hokkaido University, Hokkaido, Japan Fujimori, Yoshihiro - Department of Transfusion Medicine and Cell Therapy, Hyogo College of Medicine, Hyogo, JapanFetal bovine serum (FBS) is a common component of culture media and usually used for cellular research, as well as recent cell-based medical products. However, due to the high risk of contaminations and the variation from batch to batch, FBS might influence the outcome of research or cellular manufacturing. FBS also contains moral concerns because it harvested from bovine fetuses taken from pregnant cows. In addition, FBS is most expensive part of cell culture. To overcome these problems, we developed a new serum, adult bovine platelet lysate-derived serum “NeoSERA” .Using apheresis medical devices with closed disposable kits, sterile bovine serum NeoSERA is collected from healthy bovine receiving a regular veterinary check. After stimulation and removal of coagulated fibrin by centrifugation, NeoSERA is collected in a completely closed system. To meet the scope of directives that apply to produce medicinal products from the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/743/00) and the United States Department of Agriculture (9CFR§113.450), NeoSERA is finally gamma-irradiated at a dose of more than 30 kGy. NeoSERA® completely meets the standard for biological ingredients in Japan (Ministry of Health, Labour and Welfare Notification No.375 2014) and also obtained the certificate of eligibility for the raw material of regenerative medicine from Pharmaceuticals and Medical Devices Agency (PMDA, No.0417002, 2017. 4.17). Similar to blood donation, NeoSERA can repeatedly obtain from few adult bovine without sacrifice, indicating less moral problem and lot-to-lot variation. To test whether NeoSERA is useful for the expansion of mesenchymal stem cells (MSCs), a cell culture experiment was performed. 3 to 5 days after NeoSERA treatment, the proliferation of human bone marrow-, adipose tissue-, umbilical cord- and amnion-derived MSCs was significantly increased (p < 0.05) compared to FBS. Our results confirm that safe, less ethial and powerful adult bovine platelet-lysate-derived serum NeoSERA profoundly enhances MSC proliferation.T-3230UBIQUITOUS AND CELL-SPECIFIC RESPONSES TO DISSOCIATION: IN SITU FIXATION AS A TOOL TO INVESTIGATE DYNAMIC TRANSCRIPTIONAL EVENTSMachado, Leo - Biology of the Neuromuscular System, INSERM IMRB U955-E10, UPEC, ENVA, EFS, IMRB, Crosne, France Camps, Jordi - Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, Belgium Van Herck, Jens - Laboratory of Reproductive Genomics, Department of Human Genetics, KU Leuven, Belgium Legendre, Rachel - Institut Pasteur, Plate-forme Transcriptome and Epigenome, Biomics, Centre d’Innovation et Recherche Technologique (Citech), Pasteur Institute, Paris, France Proux, Caroline - Institut Pasteur, Plate-forme Transcriptome and Epigenome, Biomics, Centre d’Innovation et Recherche Technologique (Citech), Pasteur Institute, Paris, France Varet, Hugo - Institut Pasteur, Plate-forme Transcriptome and Epigenome, Biomics, Centre d’Innovation et Recherche Technologique (Citech), Pasteur Institute, Paris, France Sampaolesi, Maurilio - Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, Belgium Voet, Thierry - Laboratory of Reproductive Genomics, Department of Human Genetics, KU Leuven, Belgium Relaix, Frederic - Biology of the Neuromuscular System, INSERM IMRB U955-E10, UPEC, ENVA, EFS, IMRB, Créteil, France

416POSTER ABSTRACTSMourikis, Philippos - Biology of the Neuromuscular System, INSERM IMRB U955-E10, UPEC, ENVA, EFS, IMRB, Créteil, FranceThe microenvironment plays a critical role in the specification, maintenance and alteration of cell identity. It is hard to conceive a more radical niche alteration than disconnecting a cell from its tissue of origin. Nevertheless, dissociating a cell from a tissue to study its in vivo properties is a standard practice in almost every study. As most cell dissociation protocols are an order of magnitude (hours) longer than a cell’s transcriptional response to stimuli (minutes) it is likely that the profiles of freshly isolated cells no longer reflect that of their in vivo counterparts. To this end, our team developed In situ Fixation, an aldehyde-based protocol that preserves a cell’s transcriptome during isolation. We uncovered that standard dissociation of skeletal muscle stem cells (MuSCs) induces significant alterations on over half of the detected transcripts, shifting MuSCs from a quiescent to an activated state (Machado et al. 2017). In our most recent work, we have applied In situ Fixation to dissect the molecular events leading to MuSCs quiescence exit in a temporally resolved manner. Comprehensive time-course experiments coupled to in vivo single cell RNA-seq (scRNA-seq) on resting and injured muscle has identified early response factors involved in the transition of quiescent MuSC to activation. The work presented at this meeting, describes the first precise transcriptional cartography of MuSC quiescence and activation, featuring modules of coexpressed genes with dynamic and complex transcriptional behavior. Moreover, by scrutinizing published scRNA-seq atlases of various cell types from diverse tissues, we found signatures strikingly akin to the dissociation-induced artefact we detected in freshly isolated MuSCs. To investigate global and cell-specific responses to dissociation, methods to produce single-cell tissue atlases coupled to In situ Fixation in a high-throughput format, will be discussed. To conclude, our work aims to raise awareness about the universality and magnitude of dissociation-induced artefacts and call for a paradigm shift in how we think of freshly isolated cells. Finally, we propose In situ Fixation as an affordable, accessible and versatile method to produce artefact-free transcriptomic data for an increasingly accurate profile of cells in multicellular organisms.Funding Source: Funded by: TRANSLAMUSCLE PROJECT 19507 FRM grants FDT20130928236 and DEQ20130326526 ANR-10-LABX-73 ANR 11 BSV2 017 02 ANR-13-BSV1-0011-02 ANR-12-BSV1-0038-04ANR-15-CE13-0011-01ANR-15-CE13-0012-02 ANR-15-RHUS-0003T-3232DEVELOPING A HUMAN MESENCHYMAL STEM CELL CULTURE MEDIUM WITH HIGH GROWTH EFFICIENT, 3D CULTURE SUPPORTED, SERUM-FREE AND XENO-FREE PROPERTIESHuang, LungYung - Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan Su, Hong Lin - Life Sciences, National Chung Hsing University, Taichung, TaiwanHuman mesenchymal stem cell (MSC) therapies have been tested clinically for a variety of disorders, including Crohn’s disease, multiple sclerosis, graft-versus-host disease (GVHD), bone fractures, sepsis, and cartilage defects. Despite of the remarkable clinical advancements in this field, most cell manufacturing processes still rely on the traditional culture medium containing fetal bovine serum (FBS). The highly inconsistence nature and xenogenic infection risks of the bovine serum remain a challenge for the standardization and massive production for clinics. Here, we successfully develop a medium, featured with serum-free, xeno-free components and high efficacy for MSC growth, named as AllPhase. The medium on human adipose tissue-derived mesenchymal stem cells (ADSC) and Wharton’s jelly mesenchymal stem cells (WJ-MSC) enables competitive expansion performance, comparing to traditional culture medium and can maintains tri-lineage mesoderm differentiation potential after fifth passages. Furthermore, most commercial serum-free media need expansive ECM coating, like fibronectin or other commercial coating materials, to support cell adhesion. Especially, AllPhase can support ADSC expansion under 3D culture condition without microcarriers. We hope that our medium can support most bioreactors and cell industrial production for MSC clinical applications.T-3234ENGRAFTMENT AND PERSISTENCE OF L-MYC IMMORTALIZED NEURAL STEM CELLS IN ADULT MOUSE BRAINGutova, Margarita - Developmental and Stem Cell Biology, City of Hope National Cancer Center and Beckman Research Institute, Duarte, CA, USA Tsaturyan, Lusine - Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA Adhikarla, Vikram - Department of Information Sciences, Division of Mathematical Oncology, Beckman Research Institute of City of Hope, Duarte, CA, USA Barish, Michael - Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA Rockne, Russell - Department of Information Sciences, Division of Mathematical Oncology, Beckman Research Institute of City of Hope, Duarte, CA, USAHuman neural stem cells (hNSCs) demonstrate an inherent tropism to sites of damage in the CNS that can in the context of repair can potentially be exploited for delivery of therapeutic agents promoting regeneration, and/or in situ differentiation to achieve overt cell replacement. The effectiveness of any hNSC-mediated therapy will depend on the number and viability of hNSCs reaching the target site, their appropriate engraftment, and the absence of tumorigenicity. We previously demonstrated migration of L-myc immortalized hNSCs (LM-NSC008) to sites of tumor and injury in immunodeficient (NSG) mouse brain (Gutova et al, 2016). Here we have visualized LM-NSC008 cells in optically cleared mouse brains after 2, 4, 8 and 12 months of engraftment without evidence of tumorigenicity, demonstrating their potential for long-term therapeutic deployment. Their distributions over

417POSTER ABSTRACTStime was volumetrically imaged and computationally brought into registration with a Diffusion Tensor Magnetic Resonance Imaging (DT-MRI) mouse brain atlas, and compared favorably with previously predicted migration originating in the corpus callosum. This work shows the feasibility of using 3D optical imaging on cleared tissue to evaluate engraftment, migration, distribution and fate of LM-NSC008 cells in mouse brain models for optimizing NSC-mediated cellular therapies for damaged and diseased brain. This work has also helped to validate prior computational models of NSC migration.Funding Source: R03 CA216142-01A1, NIH-NCIT-3236FORWARD CELLOMICS APPROACH USING MOSAIC HUMAN ORGANOID LIBRARY: FROM PHENOTYPE TO GENOTYPEKimura, Masaki - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Thompson, Wendy - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Cai, Yuqi - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Iwasawa, Kentaro - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Zhang, Ranran - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Takebe, Takanori - Developmental Biology/Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USAEmerging biobanks provide a population-scale human induced pluripotent stem cells (hiPSC) library from genotyped donors, thus enabling an accessible platform to study human gene expressional variation such as gene expression quantitative trait loci (eQTL). Despite the promise of evolving organoid approach for precision analysis, conventional laboratory scale protocols are not equipped to perform large comparative analyses as culturing each organoid separately is heavily variable, costly and labor intensive. To circumvent this challenge, we approached the en masse efficacy screening strategy by developing a mosaic human liver organoid library from iPSC containing up to 20 individuals, followed by in vitro “GWAS” study to determine genotype-phenotype correlation. Specifically, we generated a mosaic human liver organoid by mixing a variety of different donor derived foregut in a single well and evaluated the Non-Alcoholic Steato Hepatitis (NASH) risk of each donor using a lipid accumulation phenotype. As a result of each organoid genotyping with a donor specific barcode, i.e. SNPs, mixed foregut gave rise to form “clonal” liver organoids from a single donor. Furthermore, diagnostic value of this approach was tested by determining sensitivity/specificity of lipid accumulation with the mosaic liver organoid library that includes lysosomal acid lipase deficiency. The area under the ROC curve was 0.888, which indicates the possibility to identify high risk donors in the mosaic organoid library. Next, we stratified them into high / low group with lipid accumulation phenotype, determined the risk SNP haplotypes for steatosis phenotype and compared odds ratio of reported NASH, which was identified through large-scale clinical cohort studies. The NASH risk SNPs PNPLA3 odd ratio was 1.81697(95% confidence interval: 1.16544 - 2.84399). In XKR 6, which should decrease serum triglyceride level, there was inverse correlation with lipid accumulation phenotype (odd rate: 0.566687, 95% confidence interval: 0.334000 - 0.951662). These results suggest the significance of population pooling approach to determine individualized basis for disease precision with relatively small sample due to minimal bias, noise and batch variation, potentially revolutionizing drug development as well as personalized therapy.T-3238CHARACTERISATION OF NOVEL HUMAN GENOMIC SAFE HARBOURS FOR CONTROLLED TRANSGENE EXPRESSIONAutio, Matias I - Human Genetics, GIS, Singapore, Singapore bin Amin, Talal - Computational and Systems Biology, GIS, Singapore, Singapore Efthymios, Motakis - Human Genetics, GIS, Singapore, Singapore Perrin, Arnaud - Human Genetics, GIS, Singapore, Singapore Foo, Roger - Human Genetics, GIS, Singapore, SingaporeControlled expression of transgenes in cells is essential in both therapeutic and research applications. Traditionally, such transgene expression has been accomplished via viral vector integration into the host genome, in a generally random fashion. Genome-engineering technologies, such as CRISPR/Cas9, allow for directed integration of transgenes in the cell of interest. The ideal integration sites for the foreign genetic material are ‘safe harbour’ sites, which allow for controlled expression of a transgene without perturbing endogenous gene expression patterns. To date, only a limited number of targeted integration sites have been reported and characterised in the human genome. These include for example, the AAVS1 site, CCR5 locus and the human homolog of ROSA26. However, none these sites meet stringent requirements for a genomic safe harbour (GHS) locus. We sought to discover putative human GSH, by first identifying loci in the human genome that are located outside DNAse clusters, gene transcription units and ultra-conserved regions, as well as >300kb away from any known oncogenes, miRNAs and lncRNAs. We further refined our list of candidate GSHs by cross referencing against a set of stable housekeeping genes (defined from the GTEx dataset) as well as against highly active chromatin compartments (inferred from published chromatin conformation data of 21 human primary tissues and cell types). Our final list has approximately 2000 putative human safe harbour loci. Using CRISPR/Cas9 targeting in H1 hES cells, we have successfully targeted two shortlisted

418POSTER ABSTRACTSGSH located on chromosomes 1 and 5. Transgenes introduced to the two GSH are expressed stably and at a high level in hES cells and their integration shows no significant alteration in any of the native genes in the chromosomal neighbourhood. The two GSH are currently undergoing further in vitro characterisation via differentiation of targeted hES cells into all germ-lineages, as well as through targeting the GSH in other cell types. The uncovered GSH have the potential to enable truly targeted and controllable transgene expression without interference to the native transcriptome, a desired outcome in both research and gene therapy.Funding Source: BMRC Young Investigator Grant 2016, SingaporeT-3240A STRUCTURE-BASED EXTRACELLULAR MATRIX MODIFICATION PROMOTES PARAXIAL MESODERM DIFFERENTIATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLSZhao, Mingming - Department of Clinical application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Sekiguchi, kiyotoshi - Institute for Protein Research, Osaka University, Osaka, Japan Sakurai, Hidetoshi - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, JapanThe in vitro differentiation of human induced pluripotent stem cells (hiPSCs), which have great therapeutic potentials, was designed by recapitulating embryogenesis. However, more robust methods for lineage-specific differentiation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) should be taken into account as a key factor in concurrence with toning growth factors for lineage-specific differentiation. Here, we describe a system for differentiating hiPSCs on the new generation laminin (NGL) which is a recombinant form of a laminin-421 E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. In this system, HS bind to fibroblast growth factor-2 (FGF2), form a stable high affinity HS-FGF2-FGFR complexes on cell surface, then strongly stimulate FGFR signaling pathway. Recapitulating embryogenesis in hiPSCs differentiation, NGL time-dependently increases the marker genes expression of primitive streak and paraxial mesoderm lineage. These effects depend on the unique structure of NGL, that could not be replaced by treating with high dose of FGF2 or coating the mixture of laminin-421 E8 and perlecan. In addition, using this xeno-free matrix, we established a highly efficient differentiation system for hiPSCs induced paraxial mesoderm lineage, subsequently, paraxial mesoderm derived myocytes and muscle stem cells, thus providing infinite source for disease modeling and regenerative medicine.T-3242DEVELOPMENT OF A BIOPROCESS XENO-FREE HUMAN MSCS CELL CULTURE MEDIACastro, Nadia P - R&D, RoosterBio Inc., Frederick, MD, USA Adlerz, Katrina - R&D, RoosterBio Inc., Frederick, MD, USA Ahsan, Taby - R&D, RoosterBio Inc., Frederick, MD, USA Brennan, James - R&D, RoosterBio Inc., Frederick, MD, USA Hooker, Bill - R&D, RoosterBio Inc., Frederick, MD, USA Lembong, Josephine - R&D, RoosterBio Inc., Frederick, MD, USA Ravishankar, Prarthana - R&D, RoosterBio Inc., Frederick, MD, USA Rowley, Jon - R&D, RoosterBio Inc., Frederick, MD, USA Takacs, Joseph - R&D, RoosterBio Inc., Frederick, MD, USA Walde, Amy - R&D, RoosterBio Inc., Frederick, MD, USAThere are currently more than 1000 clinical trials involving hMSCs registered at clinicaltrials.gov, representing a steady increase of over 20% per year for the last 10 years. With a typical clinical “dose” in the range of 1E7 to 1E8 cells, this proliferation of clinical testing is driving a massive increase in demand for high quality media. In support of these high-volume clinical applications, hMSC culture media needs to be both animal origin-free and straightforward to use in downstream applications. In particular, elimination of substrate coating and media replacement steps can reduce cost and streamline operations. In response to these requirements, we focused on developing a medium formulation that allows batch culture with no media replacement, requires no substrate coating, is xeno-free and still supports high rates of hMSC proliferation. We used high-throughput formulation DOE with multiple and single factorial analysis to identify critical components of the medium, which led to 5 medium formulations. These formulations were then screened for proliferation of MSCs from 5 distinct donors to select a final formulation. Final formulation effects on MSCs were then validated on bone marrow-derived MSCs (4 donors) and umbilical cord-derived MSCs (2 donors). In addition, the final formulation was tested not only in 2D adherent culture but also in 3D suspension culture using microcarriers. We characterized the cells using a panel of assays designed to evaluate cell health and functionality. The panel included expansion kinetics (cell density), cell surface marker expression (flow cytometry analysis for CD14, CD34, CD45, CD73, CD90, CD105, and CD166), cytokine secretion (FGF, HGF, IL-8, TIMP-1, TIMP-2, and VEGF), trilineage differentiation potential (osteogenesis, adipogenesis and chondrogenesis) and immunomodulatory function (response to IFN stimulation). Our results demonstrate γthat our optimized bioprocess xeno-free cell culture medium supports high rates of proliferation across multiple hMSC culture platforms while maintaining cell robustness and functionality.

419POSTER ABSTRACTSLATE-BREAKING ABSTRACTST-4002IMPACT OF METFORMIN ON HUMAN PLURIPOTENT STEM CELL DERIVED NEUROECTODERM AND CEREBRAL ORGANOIDSNguyen, Linh T - Stem Cell and Diabetes Lab, IMCB, A-STAR, Singapore, Singapore Amirruddin, Nur Shabrina - Stem Cell and Diabetes Lab, IMCB, A-STAR, Singapore, Singapore Hoon, Shawn - Molecular Engineering Lab, Biomedical Institute, A-STAR, Singapore, Singapore Shiao-Yng Chan, Shiao-Yng - Department of Obstetric and Gyneacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Teo, Adrian - Stem Cell and Diabetes Lab, IMCB, A-STAR, Singapore, SingaporeMetformin is a biguanide drug and anti-hyperglycemic agent commonly used as treatment for Type 2 diabetes. It is increasingly used before and during pregnancy to treat women with pre-pregnancy diabetes, polycystic ovary syndrome (PCOS) and gestational diabetes (GDM). However, unlike insulin, metformin can easily cross the placenta. Long-term clinical studies on the impact of metformin on offspring health are still very limited. There have been extensive in vitro studies on the anti-hyperglycemic actions of metformin exposure on liver cells, skeletal muscle and adipose tissues. However, there is very little known about the extent of in utero metformin exposure and its effects on fetal development. Using human embryonic stem cells (hESCs) and human pluripotent stem cell (hPSC)-derived germ layers and cerebral organoids, we aimed to study the impact of metformin treatment on the development and function of fetal tissue. We found that metformin treatment resulted in hESCs to exhibit significant, concentration-dependent upregulation of pluripotency factors SOX2 and NANOG. Interestingly, hESC-derived neuroectoderm showed significantly reduced expression of neural markers such as SOX1, despite the upregulation of SOX2 in hESCs. Additionally, further differentiated cerebral organoids demonstrated lowered expression of the forebrain marker FOXG1 at both transcript and protein level. These findings suggest that high concentrations of metformin treatment can perturb fetal neural development. Additional experiments are currently underway to investigate the mechanisms by which metformin elicits these effects on the neural lineage.T-4004AGGREGATION OF HUMAN PLURIPOTENT STEM CELLS ON MICROPATTERNED SURFACE INDUCES TROPHOBLAST-LIKE INTESTINAL EPITHELIUM CELLS FOR MINI-GUT ORGANOID PRODUCTIONTanaka, Yuichi - Research and Development Center, Dai Nippon Printing Co., Ltd. (DNP), Kashiwa, Japan Inoue, Makoto - Research and Development Center, Dai Nippon Printing, Kashiwa, Japan Sugawara, Tohru - Department of Reproductive Biology, National Research Institute for Child Health and Development, Setagaya, Japan Akutsu, Hidenori - Department of Reproductive Biology, National Research Institute for Child Health and Development, Setagaya, JapanThere are variety of methods to obtain intestinal organoids from pluripotent stem cells. We previously reported a new method to induce highly functioned intestinal organoids (Mini-Guts) structure from human pluripotent stem cells using 1500μm diameter micropatterned surface (Uchida et al., JCI Insight 2017). However, detailed mechanism about the differentiation had remained unknown. In this study, we focused on cellular aggregation of pluripotent stem cells, more frequently observed on the micropatterned surface. Since then, we discovered the aggregation contained CDX2+/KRT7+ trophoblast-like intestinal epithelium cells, confirmed by immunostaining and human chorionic gonadotropin (hCG) secretion. This phenomenon might have been caused by decreasing OCT4 expression of the aggregated cells. To support this phenomenon, the epithelium cells of the Mini-Guts co-expressed fetus marker, such as KRT7 and AFP. Next, we drew a comparison between the pattern surface and bare-glass for the cell culture efficiency. We discovered the pattern surface led to rapid intestinal cell differentiation confirmed by villin western blotting and hCG ELISA analysis. Moreover, we also found, using transgenic iPS cells, neuroblast-marker expressing cell in the surrounded aggregated area on 2 or 3weeks from the seeding day, suggesting that the aggregation contains neuronal differentiating cells as well as intestinal differentiating cells. Based on these results, we newly invented ring-pattern shaped cell-culture surface, consisting of cell non-attachable hollow area in the center and cell attachable area. Using this surface, we induced Mini-Guts more efficiently compared to the previous circle pattern. Our results will contribute to produce intestinal organoids more efficiently and to understand the benefits of high-density culture of pluripotent stem cells for the intestinal differentiationT-4006THREE-DIMENSIONAL RECONSTITUTION OF MINIATURE BLADDERS WITH TISSUE STROMA THAT RECAPITULATES IN VIVO TISSUE REGENERATION AND CANCERKim, Yubin - Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Korea Kim, Eunjee - LIfe Sciences, POSTECH, Pohang, Korea Kim, Seungeun - LIfe Sciences, POSTECH, Pohang, Korea Choi, Seoyoung - LIfe Sciences, POSTECH, Pohang, Korea Shin, Kunyoo - LIfe Sciences, POSTECH, Pohang, KoreaCurrent organoid models have limitations because of the missing factors in such platforms, including mature architecture and microenvironment. Here we reconstitute the tissue stem cell-based, multi-layered miniature bladders that structurally and

420POSTER ABSTRACTSfunctionally mimic the mammalian urinary bladders. These mini-bladders recapitulate in vivo tissue dynamics of regenerative response to bacterial infection in that, with the heightened activity of signal feedback between urothelium and the stroma and its associated increase in cell proliferation, the regenerative portions of urothelium arise from single cells through polyclonal expansion. Furthermore, using three-dimensional bioprinting technology, we develop multi-layered tumor organoids with the stroma that recapitulate the in vivo patho-physiology of patient-derived invasive urothelial carcinoma representing tumor-stromal interaction, slower drug response, immune cell infiltration, and muscle invasion. Our study thus provides a conceptual framework for the reconstitution of multi-layered, functional organoids derived from tissue stem cells or tumor cells that mimic the biology of native tissues.T-4008XENOBIOTIC METABOLISM IN HUMAN GUT ORGANOIDS FROM PLURIPOTENT STEM CELLSInoue, Makoto - Converting Products, Dai Nippon Printing Co., Ltd. (DNP), Kashiwa-shi, Japan Sasaki, Kengo - Transplantation Centre, National Centre for Child Health and Development (NCCHD), Setagaya-ku, Japan Kawasaki, Tomoyuki - Center for Regenerative Medicine, National Centre for Child Health and Development (NCCHD), Setagaya-ku, Japan Machida, Masakazu - Center for Regenerative Medicine, National Centre for Child Health and Development (NCCHD), Setagaya-ku, Japan Tanaka, Yuichi - Converting Products, Dai Nippon Printing Co., Kashiwa-shi, Japan Umezawa, Akihiro - Center for Regenerative Medicine, National Centre for Child Health and Development (NCCHD), Setagaya-ku, Japan Akutsu, Hidenori - Center for Regenerative Medicine, National Centre for Child Health and Development (NCCHD), Setagaya-ku, JapanThe absorption, distribution, metabolism, excretion and toxicity (ADMET) processes of drugs are of importance and require preclinical investigation not only in the liver but also in the intestine. Human intestinal absorption is the first gate for an orally taken therapeutic and one of important ADMET properties for drug efficacy. Various models have been developed for prediction of ADMET in the intestine. One of the most available cells is Caco-2 cells, which are colon cancer cell line. Caco-2 based monolayer culture was reported as the first barrier model and has been considered as the standard in studying intestinal disposition of drugs in vitro. However, it is different from the human intestinal cells in the expression pattern of the drug transporters. It is difficult to accurately predict absorption rate in humans, and there is a limit as a model system. In addition, the expression level of metabolizing enzymes such as CYP3A4 is very low, and so it could not be generally used for evaluation of drug metabolism. We have reported to originally generate functional human gut organoids (mini-guts) from pluripotent stem cells (PSCs) under xenogeneic-free culture conditions in vitro. The mini-guts showed the gut tube-like architecture consisted of mucosa and submucosa by histological, immunofluorescence and electron micrograph examinations. Here, we focus on intestinal metabolic activity in the mini-guts. Human drug metabolism PCR array was performed using RNA isolated single mini-gut and showed major intestinal transporters (ABCB1, ABCC2, ABCC1 ABCC3 and SLC15A1) and metabolic enzymes (CYP3A4, CYP3A5, CYP2C9 and CYP2C19) were similar expression level with normal adult intestine as control. Furthermore, induction assay of CYP3A4 and ABCB1 (P-glycoprotein) by 1, 25-dehydroxyvitamin D3 (VD3) demonstrated that markedly induced CYP3A4 and ABCB1 expression in the mini-guts. In contrast, VD3 had little effect on both expressions in Caco-2 cells. Subsequently, CYP3A4 assay with Luciferin-IPA containing a substrate for cytochrome 3A4 showed that CYP3A4 activity in mini-guts. These data suggest that mini-guts from hPSCs have intestinal absorption and metabolism ability in vitro. The mini-guts enable us to evaluate human intestinal ADMET in a dish and thus provide a novel bio-model for pharmacological testing.T-4010HIGH-THROUGHPUT MICROFLUIDIC PLATFORM FOR STUDYING VASCULARIZATION OF IPSC-DERIVED KIDNEY ORGANOIDSPrevidi, Sara - Mimetas B.V., Leiden, Netherlands Kurek, Dorota - Mimetas BV, Leiden, Netherlands Koning, Marije - Department of Internal Medicine, Nephrology, Leiden University Medical Center, Leiden, Netherlands W. van den Berg, Cathelijne - Department of Internal Medicine, Nephrology, Leiden University Medical Center, Leiden, Netherlands Wiersma, Loes - Department of Internal Medicine, Nephrology, Leiden University Medical Center, Leiden, Netherlands Vulto, Paul - Mimetas BV, Leiden, Netherlands Rabelink, Ton - Department of Internal Medicine, Nephrology, Leiden University Medical Center, Leiden, NetherlandsKidney organoids derived from human induced pluripotent stem cells (iPSCs) represent a powerful in vitro model for studying kidney development, disease mechanisms and drug testing. Despite the great level of structural complexity reached in vitro, these kidney organoids are immature possibly due to the lack of a functional vascular system. Transplantation of kidney organoids under the kidney capsule of a mouse can significantly improve their maturation. However, alternative approaches are valuable for studying these processes in vitro. Microfluidic techniques show great potential in bridging the gap between 2D in vitro cultures and animal models. Here, we present the use of a high-throughput in vitro ‘grafting’ platform which allows co-culture of vessels with kidney organoids. One unit of the Mimetas Organoplate® Graft is made of two microfluidic channels in which endothelial cells can be patterned against ECM. Presence of a tissue chamber allows endothelial cell co-culture with 3D tissues. When kidney organoids are used, extensive vascular remodeling occurred with formation of a

421POSTER ABSTRACTScomplex 3D network of angiogenic sprouts growing towards the tissue. Moreover, vessel stabilization can be monitored overtime by real time imaging and perfusion with 150 kDa Dextran. The established kidney organoid-on-a-chip system provides a promising platform for drug testing and disease modeling.T-4012A TUBULAR ORGANOID-DERIVED GUT-ON-A-CHIP MODEL BY PRESERVING THE STEM CELL NICHE OF LGR5+ INTESTINAL EPITHELIAVulto, Paul - Mimetas, Leiden, Netherlands Kosim, Kinga - Model Development, Mimetas, Leiden, Netherlands Puschhof, Jens - Hubrecht Institute, Utrecht, Netherlands Naumovska, Elena - Model Development, Mimetas, Leiden, Netherlands Nicolas, Arnaud - Hardware, Mimetas, Leiden, Netherlands Lanz, Henriëtte - Model Development, Mimetas, Leiden, Netherlands Trietsch, Sebastiaan - Mimetas, Leiden, Netherlands Joore, Jos - Mimetas, Leiden, Netherlands Clevers, Hans - Hubrecht Institute, Utrecht, Netherlands Kurek, Dorota - Model Development, Mimetas, Leiden, NetherlandsMicrofluidic techniques are increasingly recognized as an important toolbox to add physiologically relevant cues to traditional cell culture. These cues include long term gradient stability and continuous perfusion. Microfluidic technology allows patterning of cell layers as stratified co-cultures that are devoid of artificial membranes, in order to capture complex tissue architectures found in vivo. Previously, we have introduced the OrganoPlate® platform for growing human intestinal gut tubules in a membrane-free manner. Although suitable for toxicity studies, this model uses human intestinal cell lines, such as adenocarcinoma line Caco-2, which has limited differentiation capabilities and harbors multiple gene mutations. In contrast, Lgr5+ intestinal organoids can develop crypt-villi morphology and form an epithelial barrier – features associated with gut epithelium. These organoids are usually grown as a polarized ball-like structures embedded in an ECM, with limited apical access. Here we show a human organoid gut-on-a-chip model which is composed of Lgr5+ gut epithelial cells grown inside of the microfluidic channels of the OrganoPlate®. We established a tubular shaped epithelial barrier model of the intestinal tract showing rapid cell polarization, tight junction formation and proper expression of intestinal markers. These gut tubules are suitable for high-throughput screening of compound effects through real time imaging of transport and barrier integrity. Moreover, the OrganoPlate® facilitates development of complex models of gut epithelial tubules co-cultured with endothelial vessels. These complex gut-on-a-chip models allow mimicking disease phenotypes such as inflammatory bowel diseases (IBD) and support screening for potential drug targets. Protocols have been established that allow automated readout of the barrier integrity, followed by image analysis and quantification. The combination of Lgr5+ gut organoids with the OrganoPlate® technology are a powerful combination to study physiology and disease mechanisms in patient specific gut models.T-4014SCALABLE MULTIWELL MICROELECTRODE ARRAY (MEA) TECHNOLOGY FOR THE EVALUATION OF CARDIAC AND NEURAL THREE-DIMENSIONAL CELL CULTURESNicolini, Anthony - Applications, Axion Biosystems, Inc., Atlanta, GA, USA Sullivan, Denise - Applications, Axion Biosystems, Inc., Atlanta, GA, USA Arrowood, Colin - Applications, Axion Biosystems, Inc., Atlanta, GA, USA Millard, Daniel - Applications, Axion Biosystems, Inc., Atlanta, GA, USAThree-dimensional induced pluripotent stem cell (iPSC)-derived in vitro models, commonly referred to as spheroids or organoids, more accurately recreate the multicellular organization and structure of in vivo tissues when compared to traditional monolayer stem cell cultures. However, to effectively characterize 3D iPSC cell cultures, or to extract meaningful and predictive information from these models, new technology is required for evaluating functional cellular and network responses. For electro-active cells, like cardiomyocytes or neurons, measurements of electrophysiological activity across a networked population of cells provide a comprehensive view of function. Microelectrode array (MEA) technology offers such a platform by directly connecting key biological variables, such as gene expression or ion channel distributions, to measures of cellular and network function. Furthermore, the advent of multiwell MEA platforms has enabled scalable throughput capacity for 3D cell culture applications including toxicological and safety screening, disease modeling, and developmental biology. Neural or cardiac electrical activity can be captured simultaneously from each electrode on an MEA multiwell plate and in relation to the position of the spheroid on the array. Here, we present data supporting the use of multiwell MEA technology as an efficient non-invasive approach to capture electrophysiological activity from individual iPSC-derived spheroids. Human iPSC-derived cardiomyocytes and neural spheroids were cultured on 6-well MEA plates and monitored throughout maturation of their network connections. Cardiac electrophysiological activity, including spike amplitude and field potential duration, were recorded in response to compounds to provide information on the depolarization and repolarization of the cardiomyocyte action potential. For neural cultures, functional endpoints, such as network bursting and synchrony, were measured to define cellular activity across neural spheroids within a network. These results support the continued development and use of human

422POSTER ABSTRACTSiPSC-derived cardiomyocyte and neural spheroid assays on multiwell MEA technology for high throughput drug toxicity and safety assessment, evaluation of phenotypic disease-in-a dish models, and cell development.T-4016REGULATION OF NEURONAL DIFFERENTIATION BY AUTS2 IN HUMAN BRAIN ORGANOIDSGao, Zhonghua - Biochemistry and Molecular Biology/Penn State University College of Medicine, Penn State University, Hershey, PA, USA Geng, Zhuangzhuang - Biochemistry and Molecular Biology, Penn State University, Hershey, PA, USA Wang, Qiang - Biochemistry and Molecular Biology, Penn State University, Hershey, PA, USARecent genetic and genomic efforts have identified hundreds of risk genes for neurological disorders. However, the functional impact of most of these disease-associated alleles remains unclear due to the remarkable complexity of the nervous system. There is a tremendous need to define cell type-specific effects and underlying mechanisms in order to better understand disease pathogenesis, improve diagnoses, and ultimately to develop treatments for neurodevelopmental disorders. Our research seeks to address these challenges for a key risk gene, Autism Susceptibility Candidate 2 (AUTS2), and define its role in neural differentiation. AUTS2, initially reported as disrupted in a pair of twins with autism spectrum disorders (ASD), is also linked with other neurodevelopmental disorders, including intellectual disability, attention deficit hyperactivity disorder, and schizophrenia. AUTS2 maps to chromosome 7q11.2 and its expression peaks in neocortex of E13.5 mice, suggesting a role in neurogenesis. Indeed, mouse and zebrafish models of Auts2 deletion show a defect in neurodevelopment, although cellular and molecular mechanisms remain poorly understood. To understand the role of AUTS2 in neural development, we recently generated human embryonic stem cells (hESC) lacking AUTS2 that mimics the disruption in an intellectual disability patient. Using a human brain organoid in vitro culture model, our preliminary results showed that deletion of AUTS2 leads to severe defects in neuronal differentiation. In addition, our initial single-cell RNA-seq (scRNAseq) analysis identified a cell population that is lost upon AUTS2 knockout. Interestingly, these cells are enriched for FOXG1, a master regulator for cortex development through promoting neuronal progenitor cell (NPC) proliferation. Taken together, our studies have provided critical mechanistic insights toward understanding the neuronal differentiation program regulated by AUTS2, a prominent risk factor for neurodevelopmental disorders, which may lead to the development of therapeutic strategies for these diseases.T-4018TRANSCRIPTOMIC STATE OF HUMAN PLURIPOTENT STEM CELLS PREDICTS THE SUCCESS OF CEREBRAL ORGANOID DIFFERENTIATIONTurcios, Felix D - Neurobiology, University of California, Los Angeles (UCLA), Northridge, CA, USA Watanabe, Momoko - Neurobiology, University of California, Los Angeles, CA, USA Malone, Cindy - Biology, California State University, Northridge, CA, USA Novitch, Bennett - Neurobiology, University of California, Los Angeles, CA, USADefects in brain development underlie many neurological diseases; yet, the critical mechanisms are not understood. Efforts to investigate these mechanisms utilize animal models. However, the human brain has distinct features from other species. Increasing evidence suggests that these human-specific features may be impacted, illustrating the need for human-specific models. One such model involves directing human pluripotent stem cells (hPSCs) to form brain-like structures termed organoids. Cerebral organoids recapitulate many aspects of human fetal brains including the formation of neural progenitor regions and cortical layering. Our lab previously developed a reproducible and efficient protocol to generate cerebral organoids. The success of cerebral organoid differentiation is influenced by the maintenance of hPSCs. Variabilities arise due to batch differences in mouse embryonic fibroblast feeders, which are required for organoid formation, and in defined media components. We seek to identify the key factors that impact neural differentiation to improve outcomes. Transcriptional analyses showed that hPSCs yielding the best organoids displayed elevated expression of TGF-beta superfamily signaling molecules and genes associated with naïve pluripotency. We are testing different growth factor combinations to enhance organoid formation under feeder-free conditions. Identifying these molecules will yield effective protocols for optimizing cerebral organoids to elucidate human neurological disease mechanisms and accelerate drug discovery.Funding Source: UCLA-CSUN CIRM-Bridges training program (TB1-00183), UCLA Broad Stem Cell Research Center and the NICHD (K99HD096105), California Institute for Regenerative Medicine (CIRM) (DISC1-08819)T-4020GMP-COMPATIBLE ENGINEERED HUMAN MYOCARDIUM FOR HEART REPAIRTiburcy, Malte - Institute of Pharmacology and Toxicology, University Medical Center Goettingen, Germany Cyganek, Lukas - Cardiology and Pneumology, University Medical Center Göttingen, Germany Riggert, Joachim - Transfusion Medicine, University Medical

423POSTER ABSTRACTSCenter Göttingen, Germany Ullrich, Christian - Quality, Repairon GmbH, Göttingen, Germany Zimmermann, Wolfram-Hubertus - Pharmacology and Toxicology, University Medical Center Göttingen, GermanyHuman pluripotent stem cell-derived cardiomyocytes hold great potential for bona fide remuscularization of the human heart. We have developed a process to generate large heart muscle patches for epicardial implantation. Here, we report on the transfer of our previously established research protocol to a fully GMP-compliant process for heart failure repair in patients. Methods and Results: To obtain sufficient starting material, we generated a GMP working cell bank (WCB) from human iPSC. 200 vials with 1x10E7 cells each were obtained from a GMP master cell bank. The WCB was validated to retain a high pluripotency and stable karyotype. GMP-compliant engineered human myocardium (EHM) is generated from two active substances, cardiomyocytes and stromal cells. Cardiomyocytes were obtained by directed differentiation using small molecules and growth factors. Four test runs were performed to optimize the protocol. In additional validation runs reproducible production of the clinical target dose (8x10E8 cardiomyocytes) with high purity (95±2% sarcomeric actinin-positive cells) was demonstrated per run. By immunostaining and action potential analysis we found that the majority of cardiomyocytes (26 out of 27 measured) displayed a ventricular phenotype with a prominent plateau phase (APD20/APD80>0.3). Similarly, stromal cells (Vim+, CD90+, Collagen I+) were obtained by directed differentiation of the iPSC WCB and cryo-stored until use. To generate GMP-compatible EHM patches iPSC-derived cardiomyocytes were mixed with stromal cells in a collagen hydrogel and cultured under defined, serum-free conditions yielding contracting GMP heart patches in the clinically relevant target size (3.5x3.5 cm). In conclusion,we demonstrate that engineered human myocardium can be prepared in a clinically applicable and scalable manner. The developed process is the basis for the first-in-class BioVAT-HF trial.T-4022ELECTROPHYSIOLOGICAL PHENOTYPE CHARACTERIZATION OF HUMAN IPSC-DERIVED NEURONAL-CELL LINES BY MEANS OF HIGH-RESOLUTION MICROELECTRODE ARRAYSFiscella, Michele - D-BSSE, ETH Zurich, Basel, Switzerland Zorzi, Giulio - DBSSE, ETH Zurich, Basel, Switzerland Ronchi, Silvia - DBSSE, ETH Zurich, Basel, Switzerland Prack, Gustavo - DBSSE, ETH Zurich, Basel, Switzerland Schröter, Manuel - DBSSE, ETH Zurich, Basel, Switzerland Hierlemann, Andreas - DBSSE, ETH Zurich, Basel, SwitzerlandHigh-resolution-microelectrode-array (HD-MEA) technology enables to study neuronal dynamics across different scales, ranging from single-axon physiology to connectivity of large networks (Müller et. al, Lab on a Chip, 2015). We have used this HD-MEA technology (26’400 electrodes at 17.5 m pitch) to compare the electrical phenotypes of 6 human neuronal cell lines (dopaminergic neurons, dopaminergic neurons -synuclein A53T α- Parkinsons disease, glutamatergic neurons, motor neurons, motor neurons SOD1-G93A -ALS, motor neurons TDP43-Q331K -ALS, all from Fujifilm Cellular Dynamics International). All neuronal lines were co-cultured with human astrocytes. Neuronal electrical activity was recorded at DIV7, DIV14 and DIV21. All neuron lines showed robust synchronized/oscillating bursting activity at DIVs 14 and 21. We used the following parameters to characterize the neuron’s electrical activity: Mean Firing Rate (MFR, Hz), Mean Spike Amplitude (MSA, uV), Percentage of Active Electrodes (PAE), Burst Peak Amplitude (BP, Hz) and Interburst Interval (IBI, s). Using HD-MEA technology, we measured the following median coefficients of variation (CVs) for each parameter across developmental time points: MFR = 0.2, MSA = 0.10, PAE = 0.24, BP = 0.27 and IBI = 0.13. Furthermore, we extracted the corresponding CVs for low-resolution MEAs recordings by using the data of 16 electrodes arranged at a pitch of 300 um: MFR = 0.64, MSA = 0.28, PAE = 0.5, BP = 0.42 and IBI = 0.18. We found that the use of HD-MEA technology enables to phenotype the electrical activity of several neuronal lines with significantly lower sample-to-sample variability, as compared to state-of-the art low-resolution MEA technology. Furthermore, we isolated single-neuron electrical activity, (1) quantified network functional connectivity, and (2) characterized subcellular electrical features (e.g., axon propagation velocity along axons). We found differences in network connectivity and subcellular electrical features among the tested lines, and most importantly, between healthy lines and their diseased isogenic counterparts. We conclude that high-resolution MEA systems enable to access novel electrophysiological parameters of iPSC-derived neurons, which can be potentially used as biomarkers for phenotype screening and drug testing.Funding Source: EU, ERC Advanced Grant “neuroXscales” contract number 694829 CH, Project CTI-No. 25933. 2 PFLS-LS “Multi-well electrophysiology platform for high-throughput cell-based assays”T-4024DRUG-INDUCED SEIZURE-LIKE ACTIVITIES IN HUMAN IPSC-DERIVED NEURONSSuzuki, Ikuro - Department of Electronics, Tohoku Institute of Technology, Sendai, Miyagi, Japan Ishibashi, Yuto - Department of Electronics, Tohoku Institute of Technology, Sendai Miyagi, Japan Odawara, Aoi - Department of Electronics, Tohoku Institute of Technology, Sendai Miyagi, JapanHuman iPSC-derived neurons are expected to be applied to toxicity evaluations in nonclinical studies. Microelectrode array (MEA), measurement system of the electrophysiological activity, are suitable to evaluate the seizure liability of drugs. We have previously reported the electrophysiological responses to several convulsive compounds using MEA in cultured hiPSC-derived neurons. However, the identification of analytical parameters to detecting seizure liability remains an important issue. In this study, we identified the parameter sets that can separate the

424POSTER ABSTRACTSresponses between convulsive drugs and negative control, and the responses among the several convulsants with different mechanism of action. Twelve compounds (pentylenetetrazole, picrotoxin, 4-aminopyrdine, linopyridine, amoxapine, strychnine, pilocarpine, amoxicillin, chlorpromazine, enoxacin, phenytoin and acetaminophen) selected by HESI NeuTox MEA subteam were tested at 5 concentrations for each compound. We found that the Total Firing Rate, Inter Burst Interval, Max Firing Rate (MF) and CV of MF are especially effective parameters for classification of drug responses. As a result of using the principal component analysis (PCA) method and the clustering method using four parameters, the convulsive positive compound and the negative compound were separated, and the mechanism of action of the convulsive positive compound was also classified. PCA and clustering method using the selected parameter set are useful for the prediction of seizure liability and mechanism of action of new drugs in cultured hiPSC-derived neuronal networks.Funding Source: This research was supported by AMED under Grant Number JP18bk0104076.T-4026CELLRAFT AIR SYSTEM FOR STEM CELL ANALYSIS: DIFFERENTIATION, CRISPR AND ORGANOID WORKFLOWSTrotta, Nick - Genomics and Gene Editing, Cell Microsystems, Inc., Research Triangle Park, NC, USAContemporary stem cell workflows often rely on isolating single cells for either single cell molecular analysis or clonal colony propagation. Due to stem cell-specific viability properties, isolating single cells remains a bottleneck in analytical workflows. To eliminate these cell biology limitations, Cell Microsystems has developed the CellRaft AIR System. The AIR System allows automated imaging of thousands of individual cells on a single cell culture consumable, the CytoSort Array. CytoSort Arrays come in a range of forms tailored to various laboratory workflows, each comprising thousands of microwells in which cells randomly segregate and settle. Each microwell contains a releasable plastic floor, or CellRaft, which serves as a microscale cell culture substrate. Using the AIR System software, cells on the CytoSort Array can be imaged and sorted based on fluorescence intensity in three fluorescent channels. To isolate single cells from the array, the AIR System mechanically releases the individual microscale CellRafts from each microwell and physically places them into a 96-well plate or collection tube. The CellRaft technology provides a gentle, microwell-based method for imaging, sorting and isolating stem cells for various single cell applications.T-4028PATTERNING HUMAN PLURIPOTENT STEM CELL FATES USING ENGINEERED MORPHOGEN GRADIENTSRegier, Mary - Bioengineering, University of Washington, Seattle, WA, USA Stevens, Kelly - Bioengineering, University of Washington, Seattle, WA, USASpatiotemporal regulation of signaling pathways orchestrates diverse cellular programs, such as cell division, differentiation, and migration throughout development. Perturbing these signaling pathways by applying biomolecules of varying concentrations in cell media is a central tenant in stem cell biology. However, a method to spatially and dynamically regulate biomolecule solute patterns in vitro that is accessible to a wide range of researchers has not previously been achieved. To address this limitation, we developed a method incorporating tunable design, dynamic capabilities, and compatibility with standard 2D culture platforms. We first absorbed biomolecules into spatially-defined agarose-coated surfaces or into device-contained micromolded agarose gels. Gel surfaces loaded with biomolecules were then placed within 100um of cells in standard 2D culture. These predefined patterns of biomolecules were rapidly or sustainably transferred with high fidelity from the device to the underlying cells by diffusion. We demonstrated precise patterning of dissolved species including small molecule cell dyes, macromolecules, and viral particles. We also adapted our devices to produce gradients of morphogen signals (bone morphogenic protein 4 – BMP4, and Wnt agonist – CHIR99021) across human pluripotent stem cells. Immunostaining for lineage-specific transcription factors, Sox2, Brachyury, and CDX2, demonstrated distributions in cell fate specification that correlated with the imposed signal patterns. We further modified these distributions of cell fates by tuning the stimulus gradient geometry and amplitude. Our technology provides added control over the micron-scale spatial location of biomolecule presentation for cell fate patterning. This technology has numerous potential applications across diverse facets of stem cell biology.Funding Source: WRF Postdoctoral Fellowship, NIH grants DP2HL137188 and T32GM008349T-4030AUTOMATED HUMAN INDUCED PLURIPOTENT STEM CELL CULTURE AND DIFFERENTIATION OF RETINAL PIGMENTED EPITHELIUM ON THE TECAN FLUENT WORKSTATION FOR PERSONALIZED DRUG SCREENINGTruong, Vincent - Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA Geng, Zhaohui - Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA Stamper, Mark - Labwerx, Tecan, Morrisville, NC, USA

425POSTER ABSTRACTSGreenough, Scott - Labwerx, Tecan, Morrisville, NC, USA Scheitz, Eric - Labwerx, Tecan, Morrisville, NC, USA Ferrington, Deborah - Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Dutton, James - Stem Cell Institute, University of Minnesota, Minneapolis, MN, USACell types differentiated from human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cells from individual patients and patient populations. This in turn allows drug screening and disease modelling to be carried out using more relevant and phenotypically accurate cells than traditional immortalized cell lines. However, technical complexities including lack of both scale and standardization and the lengthy time periods involved in the standard culture and differentiation of hiPSCs have limited the adoption of these technologies for personalized drug screening. The entry of reproducible end-to-end automated workflows for these processes, demonstrated on commercially available platforms, would provide enhanced accessibility of this technology to commercial pharmaceutical testing. We have utilized a TECAN Fluent automated cell culture workstation to perform hiPSC culture and differentiation in a scalable manner to generate patient-derived retinal pigmented epithelial cells for drug screening. hiPSCs derived from multiple patients with the dry form of age-related macular degeneration (AMD) were introduced into our automated workflow and the patient specific cells were cultured and differentiated into retinal pigmented epithelium (RPE). Each patient’s hiPSC-derived RPE subsequently entered an automated screening workflow with a compound library targeting improved mitochondrial function. Mitochondrial respiration in the treated cells was then measured offline with the Agilent Seahorse XF system. This system performs scalable, robust, reproducible culture and differentiation of multiple individual cell lines from different patients on the TECAN Fluent platform and demonstrates the potential for end-to-end automation of hiPSC-based personalized drug testing.T-40323D MIDBRAIN FLOOR PLATE MODEL FOR DIFFERENTIATION OF HUMAN PSC-DERIVED DOPAMINERGIC NEURONSJosephson, Richard - Cell Biology R&D, Thermo Fisher Scientific, Frederick, MD, USA Sagal, Jonathan - Cell Biology R&D, Thermo Fisher Scientific, Frederick, MD, USA Derr, Michael - Cell Biology R&D, Thermo Fisher Scientific, Frederick, MD, USA Shin, Soojung - Cell Biology R&D, Thermo Fisher Scientific, Frederick, MD, USA Kuninger, David - Cell Biology R&D, Thermo Fisher Scientific, Frederick, MD, USAAccurate in vitro modeling of neurological diseases requires multiple cell types of the brain to interact and develop toward mature functionality. When human pluripotent stem cells (PSC) undergo neural differentiation in 3D, self-organization of progeny cells results in organoids with brain-like structures and functions that are not observed in 2D culture. However, the increased complexity of neural organoids often comes with the costs of low throughput and poor reproducibility. Disease models for drug discovery may therefore have to temper self-organized complexity with inductive specification of desired cell types. To model Parkinsons Disease (PD), we have developed a method for differentiation of human PSC to midbrain dopaminergic (DA) neurons that combines elements of 2D dissociated culture and 3D organoid culture. Cells are efficiently specified as midbrain floor plate (FP) in 2D via an established protocol using a combination of small molecules and growth factors. FP cells are then seeded into suspension culture in defined numbers for spheroid formation and expansion, then maintained in suspension for two to five weeks of differentiation. Early differentiation of the 3D cultures is marked by morphological change and the appearance of NurrI- and tyrosine hydroxylase (TH)-expressing DA neurons at the organoid surface. Single-cell analysis demonstrates that many neurons co-express Sox6+ TH+ and thus resemble midbrain DA neurons of the Substantia Nigra pars compacta (SNc). Replating of spheroids on extracellular matrix results in neurite outgrowth and outward migration of DA neurons. Multielectrode array (MEA) recording of replated spheroids shows spontaneous burst activity within a relatively short time, followed by gradual refinement toward coordinated rhythmic bursting. To test disease modeling in the hybrid 2D/3D system, CRISPR was used to make PD-related mutations (LRRK2 G2019S and SNCA A30P) in a Cas9-expressing iPSC line. These lines were differentiated to generate DA neurons as above, and to demonstrate sensitivity of the mutant SNc-like DA neurons to oxidative stress. In short, a hybrid 2D/3D culture system for iPSC-derived midbrain floor plate improves maturation of DA neurons and makes promising steps toward a reproducible in vitro disease model for Parkinsons.T-4034RSPO AND LGR-MEDIATED WNT SIGNALLING IN STEM CELLS, DEVELOPMENT AND CANCERLeushacke, Marc - Epithelial Regeneration and Cancer, A*STAR Institute of Medical Biology, Singapore, SingaporeStem cells are instrumental during organogenesis, adult tissue homeostasis and promote regeneration in response to injury. Stem cell intrinsic factors as well as extrinsic cues provided by the immediate microenvironment, known as the niche, regulate organ-specific stem cell behaviors. WNT signaling mediates key developmental and homeostatic processes in mammals. During embryonic development, WNT signals induce transcriptional programs that control cell proliferation, cell survival, cell fate determination, and tissue patterning. In adults, the WNT pathway is fundamental in defining stem cell niches in several organs, which in turn maintain tissue homeostasis. Deregulation of the WNT pathway contributes to developmental defects as well as

426POSTER ABSTRACTSto the initiation and progression of human diseases including several types of cancer. The R-spondin family comprises four evolutionary conserved secreted ligands. They function as key WNT enhancers by preventing the degradation of WNT receptor complexes triggered by two E3 ubiquitin ligases RNF43 and ZNRF3. Until recently, RSPOs were believed to exclusively execute this role by engaging their obligate cognate receptors LGR4, LGR5 and LGR6. Our data, however, demonstrates that the LGR4/5/6 receptors are dispensable for RSPO2 and RSPO3 functions during organogenesis. Using transgenic mouse lines, and ex vivo organoid culture techniques we aim to further identify novel WNT-related mechanisms in stem cells, development and cancer. Revealing additional layers of WNT signal modulation will undoubtedly have fundamental implications in cell biology, development and oncogenesis and may uncover novel targets for the development of more effective therapeutics against WNT-associated diseases and cancers.T-4036REPLACEMENT HISTONE VARIANT H3.3 MAINTAINS HSC SELF-RENEWAL AND LINEAGE DIFFERENTIATION BY SAFEGUARDING GENOME INTEGRITYGuo, Peipei - Department of Medicine, Weill Cornell Medicine, New York, NY, USA Ding, Bisen - Medicine, Pulmonary, Critical Care and Sleep Medicine, Mount Sinai School of Medicine, New York, NY, USA Geng, Fuqiang - Medicine, Weill Cornell Medicine, New York, NY, USA Liu, Xiaoyu - School f Life Science and Technology, Tongji University, Shanghai, China Liu, Ying - Medicine, Weill Cornell Medicine, New York, NY, USA Rafii, Shahin - Medicine, Reproductive Medicine, Weill Cornell Medicine, New York, NY, USA Wen, Duancheng - Reproductive Medicine, Weill Cornell Medicine, New York, NY, USA Zhong, Liangwen - Reproductive Medicine, Weill Cornell Medicine, New York, NY, USAEpigenetic signature and chromatin components serve as pivotal regulators of hematopoietic stem cell (HSC) self-renewal and lineage differentiation. Histone variant H3.3 is encoded by two genes, H3.3A and H3.3B. We report the enrichment patterns of H3.3 within adult HSCs. H3.3B is deposited at the regulatory regions and gene bodies of actively transcribed genes, promoter regions of bivalent genes, and constitutive heterochromatin regions. Deletion of H3.3A or H3.3B alone did not alter homeostatic hematopoiesis, demonstrating the compensatory effect of H3.3A and H3.3B. Inducible global deletion of H3f3a on H3.3B -/- background in adult resulted in exhaustion of HSC, imbalanced HSC differentiation towards myeloid lineage, and loss of B and T cells. Competitive transplantation assay demonstrated the cell autonomous requirement of H3.3 in maintaining HSC repopulation and lineage differentiation. Upon deletion of H3.3, HSC manifested reduced lymphoid and myeloid cell differentiation. Using in vitro endothelial-cell based coculture systems, we confirmed that H3.3 is essential for ex vivo expansion, and differentiation of adult HSPCs into B cells or CD11b+Gr1- cells. We performed Chip-seq of constitutive heterochromatin marks H3K9me3 and facultative heterochromatin marks H3K27me3 for HSCs at respective conditions. There are 2299 shared significantly reduced H3K9me3 peaks in HSCs from H3.3A-/- H3.3B-/- double knockout (DKO) mice compared with WT or BKO HSCs, among three biological replicates. Notably, there are H3K9me3 reduced mountains at the telomeric regions, suggesting the requirement of H3.3 in maintaining genome integrity. There are 174 shared significantly reduced H3K27me3 peaks in HSCs from DKO mice compared with WT or BKO HSCs. The commonly reduced H3K9me3 regions reside at LTR, LINE and intron regions, etc. Consequently, deletion of H3.3 resulted in increased death and apoptosis of HSCs, altered cell cycle progression and increased inflammatory signatures both in vitro and in vivo, in line with the premature myeloid priming of HSCs with elevated CD16/32 expression. We thus propose that H3.3 maintains the H3K9me3 levels and genome integrity at telomeric regions and repetitive regions. Loss of H3.3 induces DNA hypersensitivity, increased inflammation and premature myeloid priming of HSCs.Funding Source: NYSTEM National Institute of Diabetes and Digestive and Kidney Diseases (4 R01 DK095039-05)T-4038MESENCHYMAL STEM CELLS-DERIVED SECRETOME SUPPRESSES CHRONIC INFLAMMATION IN COLLAGEN-INDUCED ARTHRITIS MICEMun, Chin Hee - Division of Rheumatology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea Shin, Yong Dae - Division of Rheumatology, Department of Internal Medicine, Yonsei University of College of Medicine, Seoul, Korea Kim, Han-Soo - Department of Biomedical Sciences, Catholic Kwandong University College of Medicine, Gangneung, Korea Park, Yong-Beom - Department of Internal Medicine, Yonsei University College of Medicine, Seoul, KoreaSecretome derived from mesenchymal stem cells (MSCs) contribute to improved recovery against tissue injury. However, the therapeutic effects of secretome are not fully understood. We investigated the potential therapeutic effects of secretome in collagen-induced arthritis (CIA) murine model representing rheumatoid arthritis (RA), and explored the mechanism and regulatory components underlying immune modulation by secretome. Secretome was produced in adipose-derived MSCs. CIA mice were injected intraperitoneally with 200 μg of secretome. Treatment-control animals were injected with 35 mg/kg methotrexate (MTX) twice weekly. Clinical activity in CIA mice, degree of inflammation, cytokine expression in the joint, serum cytokine levels, and regulatory T cells (Tregs) were evaluated.

427POSTER ABSTRACTSCytokine array performed to define immunoregulatory proteins of secetome. Mice treated with secretome showed significant improvement in clinical joint score, comparable to MTX-treated mice. Histologic examination showed greatly reduced joint inflammation and damage in secretome-treated mice compared with untreated mice. Secretome significantly decreased serum tumor necrosis factor (TNF)- , interleukin (IL)-1 , IL-6, and IL-αβ12(p70) and increased IL-10 levels. Helper T cell 1 (Th1) was decreased and anti-inflammatory macrophage was increased in mice treated with secretome compared to untreated or MTX-treated mice. MSCs-derived secretome significantly suppressed joint inflammation in CIA mice and decreased pro-inflammatory cytokines and increased anti-inflammatory cytokines. Therefore, our study suggests that the use of secretome could be an effective therapeutic approach for RA.FundingSource:KoreanHealthTechnologyR&DProject (HI13C1270) Basic Science Research Program (2016R1D1A1B03933603) travel grant by Korean Advanced Institute of Women in Science, Engineering and Technology Support Programs for R&D Activity 2019T-4040IN VIVO COMPLETE REGENERATION OF BONE DEFECTS USING PODS® CRYSTALS, A LOCALIZED AND SUSTAINED RELEASE TECHNOLOGY.Pernstich, Christian - Research & Development, Cell Guidance Systems, Cambridge Jones, Michael - CEO, Cell Guidance Systems, Cambridge, UK Mori, Hajime - Research & Development, Kyoto Institute of Technology, Kyoto, JapanStandard recombinant growth factors are inherently unstable, with short half-lives, which limits their utility in the lab and even more so in the clinic. In contrast, PODS® (POlyhedrin Delivery System) growth factors are a highly durable crystalline protein formulation. These protein crystals are produced in cultured insect cells by co-expressing cargo protein with polyhedrin protein, which self-assembles to encase and protect the protein of interest within each polyhedrin crystal. Thus, PODS® crystals contain intact, native, and functional protein. Resistant to many chemical and physical stresses and therefore extremely stable in storage, PODS® crystals degrade slowly over several weeks and steadily release the active cargo protein. Here, we show the complete healing of a rat calvaria bone defect with a single application of PODS® BMP-2 impregnated in absorbable collagen sponge vastly outperforms the regenerative effect of standard recombinant BMP-2. This proof of principle study demonstrates the use of PODS® crystals for therapeutic protein delivery.T-4042ROLE OF CD133+ PROGENITOR CELLS IN THE PATHOGENESIS OF VASCULAR REMODELING AND PULMONARY HYPERTENSIONDai, Zhiyu - Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago and Northwestern University, Chicago, USA Zhang, Xianming - Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago, IL, USA Zhao, Youyang - Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago, IL, USAPulmonary hypertension (PH) is characterized by progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure and premature death. Owing to the poor understanding of the underlying mechanisms of obliterative vascular remodeling, current therapies result in only modest improvement in morbidity and mortality. The underlying mechanisms of occlusive vascular lesions remain unclear. Recently, we have reported the first mouse model of PH [Tie2Cre-mediated disruption of Egln1, encoding hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2), designated Egln1Tie2Cre] with progressive obliterative vascular remodeling. We identified a subpopulation of lung progenitor cells expressing CD133 which was upregulated in Egln1Tie2Cre mice. Lineage tracing studies (CD133-CreERT2;Rosa26mTmG/+) demonstrated that CD133+ cell give rise to endothelial cells and smooth muscle cells in mice. Genetic depletion of CD133+ cell population in mice (CD133-CreERT2;Rosa26iDTR/+) by diphtheria toxin treatment inhibited chronic hypoxia-induced PH. RNA-seq analysis suggest that there are multiple subpopulations of CD133+ cells including CD133+/CD157+ endothelial progenitor cells, CD133+ /KLF4+ smooth muscle progenitor cells, which showed highly proliferative potential. We also demonstrated that pulmonary endothelial cells release CXCL12, and genetic deletion of CXCL12 receptor CXCR4 in CD133+ cells (CD133-CreERT2;Cxcr4f/f) protected hypoxia-induced PH in mice. Taken together, our studies demonstrated the novel role of CD133+ cells in the pathogenesis of vascular remodeling and PH.Funding Source: NIH grants R01HL123957, R01HL125350, R01HL133951, and P01HL077806 (Project 3), K99HL138278, ATS Foundation Research Program and PHA.T-4044REVERSIBLE SWITCHING OF LEUKEMIC CELLS TO DRUG-RESISTANT, STEM CELL LIKE SUBSETS IN CROSS-TALK WITH MESENCHYMAL STROMAOh, Il-Hoan - Department of Medical Lifescience, Catholic University of Korea, College of Medicine, Seoul, Korea Lee, Ga-Young - Department of Medical Lifescience, Catholic University of Korea, Seoul, Korea Lee, Hae-Ri - Department of Medical Lifescience, Catholic

428POSTER ABSTRACTSUniversity of Korea, Seoul, Korea Humphries, Keith - British Columbia Cancer Agency, Terry Fox Lab, Vancouver, CanadaChemoresistance of leukemic cells has been largely attributed to the clonal evolution secondary to accumulating mutations. However, the possible impact of the bone marrow microenvironment in the development of chemoresistant subsets has not been well explored. Here, we show that subsets of leukemic blasts in contact with mesenchymal stroma undergo functional conversion into a distinct type of blasts that exhibit a stem cell-like phenotype and chemoresistance. These stroma-induced changes occurred in a reversible and stochastic manner independent of cell fusion or mitochondrial transfer. Moreover, the frequency of conversion into new phenotype was maintained at constant levels, being rapidly equilibrated among the equipotent leukemic cell population. The development of distinct leukemic subsets was driven by cross-talk whereby leukemic cells in stromal contact induces IL-4 expression in leukemic cells which in turn targets mesenchymal stroma to facilitate development of new leukemic subsets through up-regulation of VCAM-1 expression in MSCs and tight adherence to leukemic cells. The stroma-dependent generation of resistant leukemic subsets occurred both in murine and human leukemic cells including patients-derived leukemic blasts, but was abrogated on MSCs selectively depleted of self-renewing mesenchymal progenitor populations. Together, our study reveals another class of chemoresistance in leukemic blast created by stromal cross-talk and points to dynamic switching of leukemic cell functionality in concert with the microenvironment.Funding Source: This study is supported by the NRF of Korea and funded by Ministry of Science, ICT, and Future Planning (2017M3A9B3061947) and in part by the NRF (2012R1A5A2047939)T-4046DEVELOPMENT OF CRANIOFACIAL SKELETAL MYOGENIC PROGENITOR CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLSChoo, Hyojung - Cell Biology, Emory University School of Medicine, Atlanta, GA, USA Kim, Eunhye - Cell Biology, Emory University, Atlanta, GA, USA Wu, Fang - Cell Biology, Emory University, Atlanta, GA, USACraniofacial skeletal muscle contains approximately 60 muscles, which have critical functions including eye movements, food uptake, respiration, and facial expressions. While craniofacial and limb muscles are both skeletal muscles, the embryonic origin of these two muscle types differ significantly. Skeletal muscles in the trunk and limb originated from precursor cells in segmented trunk paraxial mesoderm, referred to as somites; craniofacial muscles arise from cranial pharyngeal mesoderm during vertebrate embryogenesis. Most current skeletal muscle differentiation protocols which use induced human pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscles developmental pathways. Since the lack of protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we aim to develop an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human induced pluripotent stem cells (iPSCs) by mimicking key signaling pathways during craniofacial embryonic myogenesis. First, we induced cranial pharyngeal mesoderm from iPSCs by modulating Wnt and bone morphogenetic protein (BMP) pathway signaling with Notch inhibition. Since second heart field also originated from cranial pharyngeal mesoderm, we suppressed cardiac lineages fates using dual inhibitions of the BMP and ROCK signaling. To isolate skeletal myogenic population, cMPCs were sorted using flow cytometry with known surface markers: HNK1-ERBB3+NGFR+. We confirmed that sorted cMPCs were expressed myogenic factor 5 (Myf5), a key marker for early skeletal myogenic precursors (99.0 ± 0.5 %). To facilitate differentiation into mature myotubes, we treated these sorted and enriched cMPCs with transforming growth factor- β(TGF- ) inhibitor and IGF. This work has established a new βprotocol for the generation of iPSC-derived human craniofacial muscles, which could be provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also reliable cellular resources for craniofacial muscle transplantation as part of craniofacial reconstruction surgery.Funding Source: This work was supported by a grant from the NIH grants (R01 AR071397), and the National Research Foundation (NRF) grant funded by the Korea government (NRF-2018R1A6A3A03011703).T-4048HUMAN IPSC-DERIVED ENDOTHELIAL CELLS EXHIBIT A MORE MATURE GENE EXPRESSION PATTERN WHEN EXPOSED TO SHEAR STRESS OR CO-CULTURE WITH IPSC-CARDIOMYOCYTESAmpuja, Minna - Faculty of Medicine, University of Helsinki, Finland Helle, Emmi - Pediatric Cardiology, New Children’s Hospital University of Helsinki and Helsinki University Hospital, Finland Antola, Laura - Faculty of Medicine, University of Helsinki, Finland Kivelä, Riikka - Faculty of Medicine, University of Helsinki, Wihuri Research Institute, Helsinki, FinlandEndothelial cells (EC) are present in almost all tissues in the body. Besides their structural purpose, they are an integral part of the function of organs and tissues. Our aim was to explore if and how culturing human induced pluripotent stem cell (iPSC)-derived ECs in conditions better mimicking the physiology of the body changes the transcriptomic phenotype of these cells. First, ECs in the body are exposed to blood flow and shear stress. Secondly, ECs exhibit constant crosstalk with their neighboring cells, which is important for growth and homeostasis of both cell types. Due to this interaction, it is known that ECs have a different identity in different organ systems. Our hypothesis was that culturing the cells in flow and in co-culture with human iPSC-derived cardiomyocytes (iPS-CMs) would influence EC gene expression and identity to better resemble that of physiological conditions in the body. We first performed single-

429POSTER ABSTRACTScell RNA-seq (scRNA-seq) analysis on iPS-ECs in flow and iPS-ECs in static culture in order to study the effects of shear stress on the cells. We then cultured iPS-ECs together with iPS-CMs, as well as separately, and performed scRNA-seq after two days of co-culture in order to find out how the presence of iPS-CMs effects iPS-EC gene expression. Both iPS-ECs and iPS-CMs were derived from human iPS cells, which gives us access to iPS-CMs that would be difficult to obtain from human patients. Two independent iPS cell lines were used in all scRNA-seq experiments. The results reveal that ECs in flow express higher levels of notable EC markers such as CD34, ANGPT2 and ESM1 compared to ECs grown in static conditions. Endothelial cells in co-culture with CMs start expressing more cardiac endothelial markers, including CCL2. Interestingly, Notch signaling was induced in both co-culture ECs and flow ECs compared to static ECs, evidenced by increased expression of Notch target gene HES4 and Notch ligand DLL4. In addition, an extracellular matrix protein-coding gene HAPLN1 was induced in both flow and co-culture ECs. HAPLN1 has recently been indicated as a cardiac EC-specific gene. In conclusion, iPSC-derived ECs are more mature when grown under flow conditions and more cardiac endothelial cell-like in co-culture with CMs.Funding Source: University of Helsinki, Academy of Finland, Wihuri Research Institute, Finnish Medical Foundation, Finnish Foundation for Pediatric ResearchT-4050THE ROLE OF SCIENTIFIC PAPERS CITED BY DIRECT-TO-CONSUMER STEM CELL BUSINESSES IN THE SOUTHWEST USRichey, Alexandra - School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA Frow, Emma - School for the Future of Innovation in Society and School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USAOur lab has characterized 149 direct-to-consumer stem cell businesses in the Southwest United States. These clinics offer stem-cell-based interventions that have not gone through FDA regulatory approval. They mobilize a variety of forms of evidence on their websites to support their practices, including published research articles. Here we present a detailed analysis of the peer-reviewed scientific articles provided on the websites of stem cell clinics, to ascertain whether they support the treatments being offered by the clinics. Of 149 stem cell businesses operating in the Southwest US, 28 list peer-reviewed scientific papers on their websites. A total of 384 articles are cited, from 224 different scientific journals. The clinics vary widely in the number of citations they present, ranging from 1 over 85. To evaluate whether or not the research findings presented in these papers support the treatments offered by a given clinic, we read the abstract of each paper and extracted information including the condition being investigated, the source of stem cells used, whether the cells were manipulated or cultured before being administered, and details about the study itself (sample size, animal or human study, presence of a control group). The data from each paper was compared to the treatments advertised on the website of the clinic referencing the study. In total, only 2% (6/384) of papers cited were identified as directly supporting the treatments being offered by that clinic. Citing a scientific study is likely a strategy aimed at promoting credibility, but our findings suggest the need to go beyond the surface and evaluate on a case-by-case basis the connection between published findings and a given clinic’s treatments. This work has implications for prospective patients as well as the Federal Trade Commission (FTC) in evaluating the scientific basis for direct-to-consumer stem cell treatments.T-4052HUMAN ENDOMETRIAL STROMAL CELLS EXERT IMMUNOMODULATORY RESPONSES TO INFLAMMATORY STIMULIQueckbörner, Suzanna - Department of Women’s and Children’s Health, Karolinska Institutet, Solna, Sweden Davies, Lindsay - Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden Gemzell-Danielsson, Kristina - Department of Women’s and Children’s Health, Karolinska Institutet, Solna, SwedenInfection and inflammation can lead to Asherman’s Syndrome (intra-uterine adhesions) or thin, unresponsive endometrium. Little is known about the relationship between stromal-immune interactions in the pathogenesis of infertility. The aim of this study was to determine how endometrial stromal cells (eSCs) regulate their immunomodulatory phenotype in response to inflammation. eSCs were isolated from endometrial biopsies (n=6) obtained from healthy, fertile women at cycle day 7-9. eSCs were co-cultured with interferon y (IFNy) and tumour necrosis factor alpha (TNFa) for 7 days, and cell surface expression of CD120a, CD120b, CD119, HLA I and HLA II determined with FACS. HLA II expression and the CIITA regulatory pathway were further evaluated by qPCR. Changes in secretion of interleukin (IL)-6, prostaglandin E2 (PGE2) and indoleamine-2,3 dioxygenase (IDO) activity in response to inflammation were established using ELISA and activity assays respectively. eSCs were co-cultured with allogeneic peripheral blood mononuclear cells in contact and transwell systems, with T cell differentiation and proliferation determined using FACS. eSCs constitutively expressed the IFNyR, CD119 and both TNFRI (CD120a) and II (CD120b). HLA I levels were upregulated in response to inflammation (p=0.0038), while no cell surface expression of HLA II was detected even with prolonged stimulation. Secreted levels of IL6, IDO and PGE2 activity were upregulated in response to inflammation (p<0.0001; p=0.0022; p=0.0159 respectively). eSCs suppressed the proliferation of CD4+ T cells, principally via soluble factors (p<0.0001). The eSC secretome modulated the CD4+ T cell phenotype, significantly suppressing numbers of central memory subsets (p<0.0001). In contrast direct contact cultures induced an effector memory phenotype (p=0.0092). No overall change in naïve or TEMRA fractions were observed. eSCs exert an immunomodulatory phenotype, suppressing the proliferation of activated CD4+ T cells and upregulating the

430POSTER ABSTRACTSsecretion of soluble factors including IL-6, IDO and PGE2. In contrast to different stromal cell subsets, eSCs do not induce cell surface expression of HLA II in response to inflammatory stimulation. The impact of this differential responsiveness may represent a relevant contribution to fetal-maternal tolerance.Funding Source: This study was funded by The Swedish Research Council and Karolinska Institutet.T-4054BIOPROCESS DEVELOPMENT TO MANUFACTURE DOPAMINE NEURON PRODUCT BRT-DA01Rosen, Siera A - Neurology, BlueRock Therapeutics, Marlboro, NJ, USA Mann, Shannon - Neurology, BlueRock Therapeutics, New York, NY, USA Wilkinson, Dan - Neurology, BlueRock Therapeutics, New York, NY, USA Ebel, Mark - Neurology, BlueRock Therapeutics, New York, NY, USA Srinivas, Maya - Neurology, BlueRock Therapeutics, New York, NY, USA Tomishima, Mark - Neurology, BlueRock Therapeutics, New York, NY, USA Lafaille, Fabien - Neurology, BlueRock Therapeutics, New York, NY, USAMany of the clinical symptoms of Parkinson’s disease are caused by the progressive and irreversible loss of midbrain dopamine neurons. At diagnosis, patients have usually lost a majority of their midbrain dopamine neurons which causes a constellation of motor symptoms. L-dopa supplementation can transiently manage motor dysfunction by increasing dopamine levels but has long-term side-effects and does not prevent disease progression. One strategy to provide permanent symptomatic relief is to replace the degenerated cells that are lost in Parkinson’s. Our approach is to manufacture authentic midbrain dopamine neurons from pluripotent stem cells for cell replacement therapy. Here, we report on our development of a closed, scaled system to manufacture billions of our dopamine neural cell product, DA01, that forms the basis of our Phase II manufacturing bioprocess. Raw material and bioprocess refinements over our Phase I bioprocess have further mitigated risk and improved process robustness. Thorough process characterization enabled an in depth understanding of DA01 through conventional quality metrics augmented with machine learning to establish the transcriptomic signature of our cell product and in-process intermediates. The combination of conventional and in silico approaches demonstrated that our scaled system makes a cell product that is nearly identical to our previous Phase I approach.Funding Source: BlueRock TherapeuticsT-4056ESTABLISHING HUMAN EMBRYONIC STEM CELLS MASTER CELL BANKS UNDER GMP CONDITIONSAspegren, Anders - TakaraBio Europe AB, Goteborg, Sweden Andersson, Katarina - TakaraBio Europe AB, Göteborg, Sweden Brandsten, Catharina - TakaraBio Europe AB, Göteborg, Sweden Runeberg, Kristina - TakaraBio Europe AB, Göteborg, SwedenThe increased interest in using human pluripotent stem cells for advanced therapy medicinal products (ATMP) has revealed the urgent need for safe and GMP compliant established hESC Master Cell Banks. The culture conditions for human pluripotent stem cells have evolved since 1998, leaving the use of mouse feeders and animal components behind, and instead one employ defined, feeder free culture conditions. However, the absolute majority of the derived human ES cells are made with feeders and outside the GMP environment, with animal components. Further, the starting material, the blastocysts, are not sourced according to FDA’s guidelines, lacking proper donor testing, and/or sourced in non-prion free regions of the world. In order to derive human embryonic stem cells under GMP and sourcing the starting material according to also FDA’s requirement we have built up the infrastructure required for a successful establishment of human ES cells for clinical applications. Initially we developed a new, xeno-free defined culture medium, that together with a defined coating substrate was successfully evaluated for derivation of hES cells. The xeno-free medium was then produced and released under GMP. We then established appropriate sourcing procedures, e.g. sourcing according to FDA’s requirement and with proper donor consents that allows the future use and needed analyses as well as significant pre-screening of the donor couples before they were approved as eligible donors. In parallel we built the infra structure and processes that rendered us to become a registered tissue establishment and with a manufacturing license for deriving and banking human pluripotent stem cells from the Swedish MPA (according to the Swedish LVFS 20018:12 and LVFS 2004: 7, Eudralex Volume 4 GMP). We will present the workflow as well as data from the ongoing derivation work.T-4058EVOLUTIONARILY DISTINCTIVE GENE REGULATORY NETWORK OF HUMAN GERM CELL SPECIFICATIONKojima, Yoji - Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan Saitou, Mitinori - Graduate School of Medicine, Kyoto University, Kyoto, Japan

431POSTER ABSTRACTSGerm cell specification in human occurs shortly after implantation and therefore unapproachable for experimental procedures. Our recent approach of inducing human iPS cells into primordial germ cell-like cells (PGCLCs) have established a platform to analyze the molecular mechanism behind this process. We have recently clarified the requirement of transcription factors, such as SOX17, TFAP2C and BLIMP1, expressed at proper timing is prerequisite for germ cell specification. However, what factors suffices PGCLC specification has not been elucidated. In this study, we utilized the abovementioned PGCLC differentiation protocol, and tested if an overexpression of a set of transcription factors can induce PGCLC differentiation without the addition of the key cytokine BMP4. We have observed that the overexpression of the combination of SOX17, TFAP2C and BLIMP1 is not sufficient for inducing endogenous germ cell marker genes expression, and thus have concluded that there are other factors necessary for PGCLC specification. From further screening of candidate genes, we have identified a set of transcription factors that induces the expression of endogenous transcripts of the key germ cell genes including SOX17, TFAP2C, BLIMP1 and NANOS3, which suggest the congruence to cytokine-induced PGCLCs. Our findings uncover the downstream target of BMP signaling in human germ cell specification and proved an evolutionarily distinctive molecular cascade from that of mouse.T-4060THE ROLE OF H3K27 DEMETHYLASES IN THE AGING OF SPERMATOGONIAL STEM CELLSIwamori, Naoki - Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan Shima, Sakurako - Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan Iwamori, Tokuko - Graduate School of Medical Sciences, Kyushu Univeristy, Fukuoka, Japan Iida, Hiroshi - Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, JapanSpermatogenesisiscontinuousprocess,becausespermatogonial stem cells (SSCs) can maintain themselves as well as provide differentiated progenies. However, the fertile ability of SSC gradually decreases with age. The molecular mechanisms and characteristics of SSC ageing still remain unclear. We found that histone H3 lysine 27 (H3K27) demethylase, JMJD3 (KDM6B), has some roles in the regulation of SSC aging. Although lack of JMJD3 in germ cells did not affect differentiation of spermatogonia, JMJD3 null mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. However, we also found that UTX (KDM6A), which is another H3K27 demethylase and is not detectable in wildtype undifferentiated spermatogonia, was redundantly expressed in JMJD3 null undifferentiated spermatogonia. These results suggest that not only JMJD3 but also UTX may contribute to control the spermatogonial compartment through the regulation of SSC aging. So far, expression and epigenetic profiles of JMJD3 null SSCs were analyzed to elucidate the role of H3K27 demethylases in the regulation of SSC aging. There were some genes that were downregulated in the aged SSCs but not downregulated in the JMJD3 null aged SSCs. We found a set of genes related to energy metabolism were involved in those genes. The UTX null mice and the UTX-inducible mice were generated and being analyzed. Our findings may be involved in the maintenance of diverse stem cell niches with age.Funding Source: KAKENHI and Takeda Science foundationT-4062GENOMIC PROFILING WITH A NOVEL ANTIBODY REVEALS A ROLE FOR O-GLCNAC IN TRANSCRIPTIONAL REGULATION OF PLURIPOTENCYZhang, Che - Department of Chemical Biology, Peking University, Beijing, China Hong, Weiyao - Department of Chemical Biology, Peking University, Beijing, China Sun, De’en - Department of Chemical Biology, Peking University, Beijing, China Fan, Xinqi - Department of Chemical Biology, Peking University, Beijing, China Wang, Jingyang - Department of Chemical Biology, Peking University, Beijing, China Qin, Ke - Department of Chemical Biology, Peking University, Beijing, China Hao, Yi - Department of Chemical Biology, Peking University, Beijing, China Chen, Xing - Department of Chemical Biology, Peking University, Beijing, ChinaO-GlcNAc is a post-translational modification occurring on serine and threonine residues of intracellular proteins, and is found to participate in various biological processes. As a multi-faceted player in transcriptional regulation, O-GlcNAcylation regulates the function of many transcription and epigenetic factors. At the same time, O-GlcNAc is found to regulate pluripotency maintenance and differentiation in embryonic stem cells, and transcriptional regulation is key to pluripotency. However, the mechanisms of O-GlcNAc regulating transcription and pluripotency are not fully understood. Here, we mapped O-GlcNAc’s occupancy on the mESC genome using ChIP-seq with a novel, nanomolar affinity antibody EPR19847, and identified 21099 loci bearing O-GlcNAc. We found these loci significantly enriched in active promoters marked by RNA Pol II, H3K4me3 and H3K9ac, linking O-GlcNAc to the activation of gene expression. We then profiled the expression of mESC genes under perturbations in global O-GlcNAc level and found 164 genes regulated by O-GlcNAc. These genes participate in differentiation, development, transcription and spermatogenesis,

432POSTER ABSTRACTSindicating that O-GlcNAc regulates pluripotency through these pathways. These results show that O-GlcNAc participates in transcriptional regulation of mESCs, and hopefully will provide more insights into O-GlcNAc’s roles in pluripotency.Funding Source: This project is supported by the National Natural Science Foundation of China (No. 91753206, No.21425204, No. 21672013 and No. 21521003), the National Key Research and Development Projects (No. 2018YFA0507600 and 2016YFA0501500)T-4064REGULATION OF THE CHROMATIN STATE AND CORE PLURIPOTENCY FACTORS IN CANCER STEM CELLSGupta, Aditi - Department of Biochemistry, University of Maryland, Baltimore, USA Portney, Benjiamin - Biochemistry and Molecular Biology, University of Maryland, Baltimore, USA Arad, Michal - Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, USA Zalzman, Michal - Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, USAEmbryonic stem (ES) cells and cancer cells share many properties, including gene expression networks and the unlimited capacity for self-renewal. The naïve state of ES cells is maintained by the core pluripotency factors OCT4, NANOG, and SOX2. Previous studies have demonstrated that, like ES cells, cancer cells can harness the core pluripotency factors for their survival and self-renewal. Furthermore, a small subset of cells, also known as cancer stem cells (CSCs), are marked by these factors and pose a significant challenge to cancer treatment as they remain resistant to radiation and chemotherapeutic drugs. CSCs maintain the ability to self-renew, drive tumor heterogeneity, and contribute to an aggressive phenotype and relapse in many types of cancers. ZSCAN4 (zinc finger and SCAN domain containing 4) is an additional stem cell factor implicated in the preservation of embryonic stem cell potency and genome stability. Although it was suggested to have implications in cancer, the function of human ZSCAN4 in cancer or how it exerts its action remained unknown. In the current study, we show that ZSCAN4 marks the CSC population in head and neck squamous cell carcinoma. Our data suggest that induction of ZSCAN4 promotes the CSC phenotype by activating the core pluripotency factors and altering the CSC epigenetic profile. Consistent with this, ZSCAN4 depletion by gene knockdown leads to loss of the CSC phenotype, including decreased CSC marker expression and the impaired ability to form spheroids. Overall, our study suggests that ZSCAN4 plays a critical role in the maintenance of the undifferentiated state and survival of CSCs, indicating that ZSCAN4 is a potential therapeutic target in HNSCC.T-4066IDENTIFICATION IN CONDITIONED MEDIA OF STEMNESS MARKER FOR HUMAN EMBRYONIC STEM CELL DERIVED MESENCHYMAL STEM CELL USING PROTEOMICSPark, Arum - STDBD, Eulji University, Seongnam-si, Korea Lee, Jiyeong - Department of Biomedical Laboratory Science, Eulji University, Seongnam-si, Korea Kim, Hyo-Jin - Department of Senior Healthcare, BK21 Plus Program, Eulji University, Seongnam-si, Korea Lee, Yoo-Jin - Department of Senior Healthcare, BK21 Plus Program, Eulji University, Seongnam-si, Korea Shin, Miji - Department of Senior Healthcare, BK21 Plus Program, Eulji University, Seongnam-si, Korea Choi, Hyebin - Department of Senior Healthcare, BK21 Plus Program, Eulji University, Seongnam-si, Korea Son, Hyunsong - Department of Senior Healthcare, BK21 Plus Program, Eulji University, Seongnam-si, Korea Kang, Hee-Gyoo - Department of Biomedical Laboratory Science, Eulji University, Seongnam-si, KoreaHuman mesenchymal stem cells (hMSCs) are multipotent adult stem cells that have ability to self-renewal and differentiate into chondrocytes, adipocytes and osteocytes as well as have immune-regulatory, have attracted much attention from researchers due to regenerative medicine implications. Especially, human pluripotent stem cell-derived mesencymal stem cells (hPSC-MSCs) is known that hPSC-MSCs have more benefits which it overcomes cellular senescence during the in vitro expansion and enhanced therapeutic effect, in comparison with hPSC-MSCs isolated from different adult sources such as bone marrow, adipose tissues, fetal liver, peripheral blood and lung and so on. To confirm the stemness in hPSC-MSCs is very important because it presents ability of hPCS-MSCs like self-renewal and multipotency. However, the stemness associated with the mechanism and biological process of hPCS-MSCs remain poorly understand as hPSC-MSCs have the heterogeneity and variation between different sources and culture condition. Therefore, we identified the stemness markers in conditioned media (CM) of hPSC-MSCs compared with CM of chondrocyte cells to improve cell quality and to detect easily than existing methods by assessing quantification of stemness markers using HPLC-MS/MS. We used SWATH-MS which is label free quantitative data-independent acquisition (DIA) to select candidate marker for stemness. In addition, multiplexed liquid chromatography-multiple reaction monitoring spectrometry was used for quantitative analysis. In total 408 proteins were identified in hPSC-MSC and 352 proteins in chondrocyte cells. The eighty-one proteins were identified with more than 1.2-fold change and p-value less than 0.05. The forty-four proteins were up-regulated and thirty-seventy were down-regulated in stroke patients comparison with healthy control. These proteins were classified into cell adhesion, extracellular matrix (ECM) and cell signaling involved in differentiation. We selected several candidate markers for stemness and quantified. These proteins were classified into cell signaling pathway (CHRD

433POSTER ABSTRACTSand FST), ECM (CO1A, COL1A2 and COL1A3) and other (PAI1 and STC2). These results were regarded to be able to support whether hPSC-MSC have stemness ability for in vitro cell culture expansion.T-4068MODELING PICALM ALZHEIMER’S DISEASE VARIANTS IN HUMAN IPSC-DERIVED CELLSLawson, Erica J - Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA Dai, Zhonghua - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Xie, Xiaochen - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Bazzi, Sam - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Nelson, Amy - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Sagare, Abhay - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Zhao, Zhen - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USA Tanzi, Rudolph - Genetics and Aging Research Unit, Harvard Medical School, Charlestown, MA, USA Zlokovic, Berislav - Physiology and Neuroscience, University of Southern California, Los Angeles, CA, USAPICALM plays an essential role in regulating neuronal function and providing a major pathway for amyloid-beta (A ) clearance in βvivo across the blood-brain barrier (BBB). PICALM is abundantly expressed in brain capillaries as well as in neurons in the human brain. Impaired transvascular clearance of A across the BBB βcontributes to A accumulation, accelerating neurovascular βand neuronal dysfunction in the Alzheimer’s disease (AD) brain. Our research has shown that PICALM is required for A βtrafficking across the BBB through endocytotisis via LRP1, and Rab5- and Rab11-mediated vehicular transport resulting in A βexocytosis into circulation. Diminished PICALM levels in the brain endothelium are found in AD and are also associated with increased neuronal vulnerability to A . Using CRISPR/CAS9 βgenome editing technology, we investigated the protective PICALM SNP rs3851179A and the risk rs3851179G variants. By successfully generating iPSC lines homologous for allelic variants of PICALM SNP rs3851179, we further differentiated these iPSCs into the cell types of the neurovascular unit: neurons, astrocytes, microglia, endothelial cells, and pericytes. We confirmed by Western blot analysis higher PICALM expression in iPSC-derived neurons and endothelial cells carrying protective rs3851179A alleles than those carrying non-protective rs3851179G alleles. In a 2D transwell model, we found that endothelial cells carrying the non-protective rs3851179G alleles cleared A much less efficiently across the barrier βcompared to endothelial cells carrying the protective alleles. Neurons carrying protective or non-protective alleles of PICALM can also be functionally compared throughout differentiation in 3D and 2D in vitro models. By generating in vitro models with neurons, astrocytes, microglia, pericytes, and endothelial cells derived from human iPSCs with allelic variants of PICALM, we are able to explore the fundamental characteristics of AD pathology as it relates to PICALM expression.Funding Source: Funding sources: This work is supported by the Cure Alzheimer’s Fund, and National Institute of Health grants AG023084, NS034467 to B.V.Z.FRIDAY, JUNE 28, 2019POSTER III - ODD 18:00 – 19:00PLACENTA AND UMBILICAL CORD DERIVED CELLSF-2001HUMAN AMNIOTIC EPITHELIAL CELLS-DERIVED EXOSOMES RESTORE OVARIAN FUNCTION BY TRANSFERRING MICRORNAS AGAINST APOPTOSISZhang, Qiuwan - International Peace Maternity And Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Sun, Junyan - International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Huang, Yating - International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Lai, Dongmei - International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, Shanghai, ChinaPremature ovarian insufficiency/failure (POI/POF) is one of the most common complications among female tumor patients treated with chemotherapy and requires advanced treatment strategies. Human amniotic epithelial cells (hAECs)-based therapy mediate tissue regeneration in a variety of diseases and increasing evidence suggests that the therapeutic efficacy of hAECs mainly depends on the paracrine action. This study aims to identify exosomes derived from hAECs and explore their therapeutic potential in ovaries damaged by chemotherapy and the underlying molecular mechanism. hAECs-derived exosomes exhibited a cup- or sphere-shaped morphology with a mean diameter of 100 nm and were positive for CD63 and CD9. hAECs-Exosomes transplantation increased the number of follicles and improved ovarian function in POF/POI mice. During the early stage of transplantation, hAECs-Exosomes significantly inhibited granulosa cells apoptosis and protected the ovarian vasculature from damage, and involved in maintaining the number of

434POSTER ABSTRACTSprimordial follicle in the injured ovaries. Enriched microRNAs (miRNAs) existed in hAECs-Exosomes and targeted genes were enriched in phosphatidylinositol signaling and apoptosis pathways. Studies in vitro demonstrated that hAECs-Exosomes inhibited chemotherapy-induced granulosa cells apoptosis via transferring functional miRNAs, such as miR-1246. Our findings demonstrate that hAECs-derived exosomes have the potential to restore ovarian function in chemotherapy-induced POF/POI mice by transferring miRNAs.Funding Source: This study is funded by the National Key Research and Developmental Program of China (2018YFC1004800 and 2018YFC1004802), and the National Natural Science Foundation of China (No.81701397).F-2003DEVELOPING THE CLINICAL-GRADE PROTOCOL OF HUMAN PLACENTAL TISSUE CRYOPRESERVATION FOR HEMATOPOIETIC STEM CELLS AND MESENCHYMAL STROMAL CELLS ISOLATIONShablii, Volodymyr - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Kuchma, Maria - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Nikulina, Viktoriia - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Bukreieva, Tetiana - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Zahanich, Ihor - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Rudenok, Oleksandr - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Kyryk, Vitaliy - Department of Cell and Tissue Technologies, State Institute of Genetics and Regenerative Medicine of Academy of Medicine of Ukraine, Kyiv, Ukraine Boichuk, Iuliia - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, Ukraine Lobintseva, Galyna - Placenta Stem Cell Laboratory, Institute of Cell Therapy, Kyiv, UkrainePlacenta is very attractive source of mesenchymal stromal cells (MSCs) and hematopoietic stem/progenitor cells (HSPCs). Large-scale introduction of placental-derived cells to clinical practice requires the establishing of low-temperature stock of placental tissue. Term placentas were collected after normal delivery or CS provided with women’s written informed consent in the Kyiv city maternity hospital #3. One part of tissue from every specimen was processed as a native control and the other part was frozen by nine variants of cryoprotectants. The combination of different concentration of DMSO (5%, 7.5%, 10%) and sucrose (0.1 M, 0.2 M) were used for cryopreservation. Samples were frozen by special programs in the controlled rate freezer (IceCube, Austria). Colony forming units (CFU) assay was carried out on passages 0, 2, 6 for placental MSCs (pMSCs) isolated from native and thawed tissue. CFUs activity of placenta-derived HSPCs was studied using MethoCult assay (StemCell Technologies, Canada). The content of HSPCs among viable CD45+ cells from native and cryopreserved placental tissue did not significantly differ. The cryopreservation led to the alteration of CD133, CD38, and CD33 expression on HSPCs surface. HSPCs from native tissue gave rise to various types of colonies in vitro and their ratio did not significantly differ compared to ones obtained from cryopreserved samples. 5% DMSO preserved the HSPCs better in compare to 10% (without sucrose). However, HSPCs CFU recovery of samples frozen with 10% of DMSO was higher compare to ones with 5% DMSO in combination with 0.2 M sucrose. The CFU number of pMSCs for nine variants on passages 2 and 6 did not significantly differ. Results of these studies revealed the pattern of interference of two cryoprotectants influence (DMSO and sucrose) on the viability of HSPCs in placental tissue during cryopreservation. The cryoresistance of placenta-derived HSPCs and pMSCs enclosed in tissue fragments differed.F-2005IN VITRO, EX VIVO AND IN VIVO INFLUENCE OF 3D MICROENVIRONMENT ON ENHANCEMENT OF WJ-MSC THERAPEUTIC POTENTIAL IN CNS INJURYLech, Wioletta - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland Figiel-Dabrowska, Anna - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland Zychowicz, Marzena - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland Sarnowska, Anna - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland Domanska-Janik, Krystyna - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland Buzanska, Leonora - Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, PolandMesenchymal stem cells (MSC) exhibit neuroprotective, angiogenic and immunomodulatory properties. Their availability, high plasticity and possibility of expansion have made MSC-based therapy one of the most commonly used in regenerative medicine. In this study we have designed 3D hydrogel scaffolds: human platelet lysate (PL) and fibrinogen (FB) and adjusted culture atmosphere to physiological oxygen concentration in order to provide the optimal microenvironment to in vitro culture and protect cells from adverse host tissue vs. transplant interaction during ex vivo and in vivo experiments. WJ-MSC in vitro cultures in different oxygen conditions (21% O2 vs. 5% O2) were tested for proliferation, viability and gene expression profile. For ex vivo studies the organotypic hippocampal slice culture (OHC) model of oxygen glucose deprivation was used to mimic an ischemic injury of neural tissue. In vivo studies were based on experimental model of brain injury induced by local ouabain injection. The effect of cell transplantation on CNS was tested in isolated animal brains at 7, 14 and 21 days after the grafting. WJ-MSC show higher survivability and increased proliferation rate in FB vs. PL scaffolds and in physiological normoxia (5% O2). 3D in vitro conditions increased expression level of

435POSTER ABSTRACTSselected neurotrophins in both type of the scaffolds. Ex vivo studies indicated strong neuroprotective effect of WJ-MSC on injured hippocampal slices. WJ-MSC growing on the scaffolds and co-cultured with OHC revealed increased expression of several neurotrophins and anti-inflammatory TGF- as well βas decreased expression of pro-inflammatory IL-1 . This βeffect was potentiated by FB (as compared to PL) scaffolds and 5% O2 culture conditions. In vivo studies have shown increased expression of rat cytokines, e.g. NGF, VEGF-A after transplantations of WJ-MSC supported by 3D fibrin scaffolds in the injured brain region. Our results indicate significant influence of microenvironmental conditions, therefore it is necessary to optimize and standardize all aspects of preparation of therapeutically competent cell population. Moreover, the proposed models of scaffolds/cell hybrids transplantation are promising for future use in MSC-based therapy.FundingSource:SponsoredbyNCBRgrantNoSTRATEGMED1/234261/2/NCBR/2014 and statutory funds to MMRC.ADIPOSE AND CONNECTIVE TISSUEF-2007A NOVEL XENOGRAFT MODEL TO VALIDATE CAUSATIVE MUTATIONS IN PATIENTS WITH EHLERS-DANLOS SYNDROMEDiette, Nicole - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Frieman, Amy - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Rozhok, Andrii - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Roop, Dennis - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Kogut, Igor - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Bilousova, Ganna - Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USAThe Ehlers-Danlos Syndrome (EDS) is a connective tissue diseasecharacterizedbyhyperextensibleskin,jointhypermobility, and cutaneous fragility. EDS is primarily caused by mutations in fibrillar collagens or genes involved in collagen protein biosynthesis. However, there are many cases in which the causative genetic defects of EDS have not been identified. To date, there is no cure for EDS with treatment limited to symptomatic care. The lack of appropriate in vitro and in vivo models that faithfully recapitulate the clinical EDS phenotype makes mechanistic studies extremely challenging, thus hindering the development of efficient therapeutic strategies. In order to develop a clinically relevant EDS model, we focused on a cohort of hypermobility type EDS patients who did not harbor known EDS-associated mutations, but exhibited classical symptoms of the disease. We isolated fibroblasts from a skin biopsy of one of these patients, and combined these cells with healthy keratinocytes. Using a silicone chamber, we grafted the cells onto an immunocompromised mouse to recapitulate the EDS skin phenotype in a xenograft model. We discovered that grafts formed with EDS fibroblasts exhibited a disorganized collagen network, a feature previously observed in the skin of EDS patients. We have compared the transcriptional profile of EDS fibroblasts with that of controls using RNA sequencing analysis, and identified a number of promising mutations and transcript variants that may be causing the phenotype in these patients. In order to validate these candidate mutations, we reprogrammed EDS fibroblasts into induced pluripotent stem cells (iPSCs). We are currently correcting these candidate mutations using CRISPR/Cas9. The corrected EDS iPSCs will be differentiated into fibroblasts, and used in our xenografting assay. If the xenografts no longer display a disorganized collagen network, this will confirm that this candidate mutation is indeed the cause of EDS in these patients; thus, confirming the usefulness of our model.F-2009ADIPOCYTES FROM OBESE MICE PRODUCE EGF AND PROMOTE THE SURVIVAL AND GROWTH OF PANCREATIC DUCTAL PROGENITOR CELLS IN ORGANOID CULTUREWu, Xiaoxing - Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA, USA Gao, Dan - Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institude and Beckman Research Institude of City of Hope, Duarte, CA, USA Luo, Angela - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Jin, Liang - Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institude and Beckman Research Institude of City of Hope, Duarte, CA, USA Wedeken, Lena - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Ghazalli, Nadiah - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA LeBon, Jeanne - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Walker, Stephanie - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research

436POSTER ABSTRACTSInstitute and Beckman Research Institute of City of Hope, Duarte, CA, USA Quijano, Janine - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Chan, Yin - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Riggs, Arthur D. - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USA Ku, H. Teresa - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute and Beckman Research Institute of City of Hope, Duarte, CA, USAObesity, a condition of excessive adipose tissue (AT), is a major health problem. AT has been recognized as an endocrine organ that secrets bioactive factors, but the repertoire remains incomplete. Obese AT can affect various differentiated cells, as well as progenitor cells in the endothelium, intestine and bone marrow. However, whether AT may also affect pancreas ductal progenitor cells is unknown. Here we examined the effects of secreted factors, collected in the conditioned medium from AT of obese or lean mice, on lean pancreatic ductal progenitor cell using an organoid culture system. We found that obese compared to lean adipocytes produced and secreted more epidermal growth factor (EGF). Recombinant EGF enhanced the survival and the self-renewal of pancreatic ductal progenitor cells in vitro. Our results uncover EGF as a previously-unrecognized secreted factor from adipocytes that may have implications for obesity-associated diseases.MUSCULOSKELETAL TISSUEF-2013ROLE OF COUP-TFII AND THYROID HORMONE RECEPTOR ALPHA IN MURINE SKELETAL MUSCLE LOSSAguiari, Paola - David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Liu, Yan-Yun - David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Cheng, Sheue-yann - Center for Cancer Research National Cancer Institute, National Cancer Institute, Bethesda, MD, USA Perin, Laura - GOFARR Lab, Children’s Hospital, Los Angeles, CA, USA Brent, Gregory - David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Milanesi, Anna - David Geffen School of Medicine, UCLA, Los Angeles, CA, USAMyopathic changes including muscular dystrophy and weakness are commonly described in hypothyroid and hyperthyroid patients. Impaired skeletal muscle regeneration and sarcopenia with aging have been reported in a mouse model of Resistance to Thyroid Hormone (RTH) carrying a frame-shift mutation in the thyroid hormone receptors (TR ) gene (TR 1PV). As αααwe previously reported, TR 1PV mice present a significantly αsmaller pool of PAX7-positive satellite cells (SCs) in the skeletal muscle and sarcopenia with aging, that was reported to be associated with loss of SC pool. Moreover, SC function during skeletal muscle injury was impaired four days after cardiotoxin (CTX)-induced skeletal muscle injury with a decreased activation of SC and a reduced proliferation of Myf5 expressing cells. Overexpression of the nuclear orphan receptor Chicken Ovalbumin Upstream Promoter-factor II (COUP-TFII, or NR2F2) in murine satellite cells have been shown to induce a similar skeletal muscle phenotype, including skeletal muscle loss with aging, and inhibit myogenesis through modulation of Myf5 and MyoD expression. We detected a higher expression of COUP-TFII in C2C12 (a murine myoblast cell line) during proliferation, and a decline during myogenic differentiation. Moreover, we analyzed the skeletal muscle of mice at different ages and we found a higher expression of COUP-TFII in the first 2 months of life followed by a decline with age, suggesting the important role of COUP-TFII in modulating post-natal myogenesis and onset of sarcopenia with aging. In proliferating C2C12 myoblasts and SCs from wild-type and TR 1PV mice, we demonstrated αvia co-immunoprecipitation that COUP-TFII and TR interact. αIn addition, the skeletal muscle of TR 1PV mice showed αsignificantly higher expression of COUP-TFII compared to their WT siblings, and in TR 1PV mice COUP-TFII expression αremained higher with skeletal muscle aging. These results suggest a COUP-TFII role in skeletal muscle loss with aging and impaired skeletal muscle regeneration in TR PV mice that may αbe mediated by COUP-TFII-TR interaction. These new insights αcan provide a therapeutic target to prevent or treat myopathies, such as sarcopenia and Duchenne-like muscular dystrophy.Funding Source: Sources of Research Support: NIH grants and VA MERIT GRANT.F-2015ENHANCING MESENCHYMAL STEM CELL OSTEOGENESIS AND ADIPOGENESIS BY SMALL MOLECULES COCKTAILSLin, Po-Yu - Genomics Research Center, Academia Sinica, Taipei, Taiwan Lin, Po-Heng - Genomics Research Center, Academia Sinica, Taipei, Taiwan Lai, Pei Lun - Academia Sinica, Genomics Research Center, Taipei, Taiwan Lin, Hsuan - National Taiwan University, Department of Pediatrics, Taipei, Taiwan Lu, Jean - Academia Sinica, Genomics Research Center, Taipei, Taiwan

437POSTER ABSTRACTSHuman mesenchymal stem cells (MSCs) hold great promises for regenerative medicine and cell therapy by their multi-potency, immune-modularity, and risk-free of tumorigenesis. Previously, we have revealed that a cocktail including six chemicals (6C, including p38 inhibitor, JNK inhibitor, PKC inhibitor, ROCK inhibitor, ErK inhibitor and GSK3 inhibitor,) are able to βreprogram human somatic fibroblast to induced mesenchymal stem cells (iMSCs) within six days with a high efficiency ( 38%) . iMSCs fulfill all the criteria of traditional MSCs as determined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT), including cell adhesion, marker expression, and multipotency. The iMSCs have much higher clonogenicity than fibroblasts. Additionally, iMSCs have immunomodulatory that can suppress LPS-mediated acute lung injury as effectively as bone marrow MSCs. After testing 120 different combinations, we further boosted the conversion rate from 38% to 78%. Recently, we tested these chemicals and revealed that 3 chemical- (3C), and 6 chemical-(6C) cocktail can promote cell differentiation in MSCs. Among them, in both young and aging MSCs, we found the osteogenesis markers alkaline phosphatase activity and calcium precipitation increased while we treat the MSCs with 3C. In contrast, the adipogenesis increased upon the treatment of 6C. To sum up, it suggests that the cocktails can enhance the osteogenesis or adipogenesis of MSCs, and has a great potential to treat osteoporosis or alter cell fate in the future.F-2017SMALL-MOLECULE RESTORATION OF DYSTROPHIN PREVENTS DYSFUNCTIONAL GLUTAMATE CLEARANCE IN IPSC MODEL OF DYSTROPHIC ASTROCYTESPatel, Sam - Stem Cell Institute Leuven (SCIL), KU Leuven, Leuven, Belgium Wierda, Keimpe - Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease, Leuven, Belgium Thorrez, Lieven - Development and Regeneration, Kulak Kortrijk Campus, Leuven, Belgium van Putten, Maaike - Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands De Smedt, Jonathan - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium Ribeiro, Luis - Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease, Leuven, Belgium Tricot, Tine - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium Gajjar, Madhavsai - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium Duelen, Robin - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium van Damme, Philip - Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease, Leuven, Belgium De Waele, Liesbeth - Department of Pediatric Child Neurology, University Hospitals Leuven, Belgium Goemans, Nathalie - Department of Pediatric Child Neurology, University Hospitals Leuven, Belgium Tanganyika-de Winter, Christa - Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands Costamagna, Domiziana - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium Aartsma-Rus, Annemieke - Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands van Duyvenvoorde, Hermine - Laboratory for Diagnostic Genome Analysis, Leiden University Medical Center, Leiden, Netherlands Sampaolesi, Maurilio - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, Belgium M Buyse, Gunnar - Department of Pediatric Child Neurology, University Hospitals Leuven, Belgium Verfaillie, Catherine - Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, BelgiumDuchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from DMD and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyper-excitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. Moreover, we feel our results form a key component of increasingly convergent pathways that seem to be involved in multitude of neuropsychological disorders. Akin to findings in Autism iPSC models, and brain MRI studies in mdx mouse model of DMD, involvement of excitatory amino acids (Glutamate in our case) seem to be a common denominator among them all. This is, to our knowledge, the first report to demonstrate the importance of dystrophin protein in a non-myogenic setting at the cellular level in CNS. In summary, we demonstrate that: (1) Loss of dystrophin from astrocytes causes defects in astrocyte glutamate handling, (2) This results in glutamate toxicity towards normal cortical neuronal progeny, and (3) These defects could be reversed when dystrophin levels were restored, as shown here for PTC124, which allows read-through of premature termination codons and which has conditionally been approved for DMD by the EMA in 2014. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development. Lastly, pertinent here is also the subpopulation of patients currently treated with PTC124 for myogenic defects. It will be of great interest to determine effects on cognitive function of this intervention, given that this compound can cross the blood-brain barrier.Funding Source: KU Leuven Rondoufonds voor Duchenne Onderzoek , IWT-iPSCAF grant (no. 150031)

438POSTER ABSTRACTSF-2019MURINE SKELETAL MUSCLE STEM CELLS MAINTAIN BIOENERGETIC HOMEOSTASIS THROUGH MITOCHONDRIAL OXIDATION OF GLUCOSE DURING REGENERATIVE ACTIVATIONAhsan, Sanjana - Department of Stem Cell Biology and Regenerative Medicine/Keck School of Medicine, University of Southern California (USC), Los Angeles, CA, USA Rodgers, Joseph - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAMuscle stem cells (MuSCs) reside in skeletal muscle tissue and are activated by injury to initiate and drive muscle repair. In our previous work, we showed that increasing the speed of MuSC activation (exit cellular quiescence and divide) was sufficient to increase the speed of the entire process of muscle regeneration. While the biologic role of MuSC activation in muscle repair is clear, the mechanisms that regulate MuSC activation remain elusive. We found in our previous work that MuSCs upregulate genes associated with mitochondrial metabolism during injury induced activation. This suggests that MuSCs increase mitochondrial ATP production to meet the energetic demands of activation. To dissect MuSC energy metabolism, we used the Seahorse XFp extracellular flux analyzer to measure extracellular flux in oxygen and proton concentrations and determine the real-time rate of cellular ATP production. In addition, we measured changes in cellular ATP synthesis induced by 2-deoxy-D-glucose, an inhibitor of glycolysis, to determine ATP specifically generated from glucose consumption. Our data show that during the first 48 hours of activation, (1) total cellular ATP production dramatically increases by >10-fold in MuSCs, and (2) cellular consumption of glucose to generate ATP increases by >30-fold. We also found that majority of cellular ATP is produced via mitochondrial metabolism as opposed to glycolysis. Interestingly, our results show that mitochondria in freshly isolated (FI) MuSCs do not metabolize glucose to synthesize ATP, suggesting that FI MuSCs oxidize glucose exclusively through glycolysis. By 48 hours, however, we found that mitochondria carry out majority of glucose consumption (linked to ATP synthesis) in MuSCs. Collectively, these data illustrate the dynamic and dramatic metabolic transitions that occur in MuSCs as they initiate muscle repair, and advance our understanding of how metabolic cues and diet can be used to control endogenous tissue repair or augment the efficacy of regenerative medicine therapies.F-2021TRANSIENT ACTIVATION OF NOTCH SIGNALING ENHANCES STROMAL CELL PROLIFERATION AND SUBSEQUENT OSTEOGENESISLuo, Zhengliang - Orthopaedic Surgery, LSUHSC, Shreveport, LA, USA Zhang, Hao - Orthopaedic Surgery, LSUHSC, Shreveport, LA, USA Shu, Bing - Orthopaedic Surgery, Shanghai Longhua Hospital, Shanghai, China Wang, Yongjun - Orthopaedic Surgery, Shanghai Longhua Hospital, Shanghai, China Barton, Shane - Orthopaedic Surgery, LSUHSC, Shreveport, LA, USA Dong, Yufeng - Orthopaedic Surgery, LSUHSC, Shreveport, LA, USASufficient of mesenchymal stromal cells (MSCs) is crucial for tissue growth and repair. While bone marrow-derived MSCs are an attractive cell source, their availability has been hampered by the low population of MSCs in vivo. We have shown previously that activation of Notch signaling enhances limb bud cell proliferation in vitro and in vivo. Here, we extend this knowledge to examine whether activation Notch signaling by injection of ligand jagged1 (JAG1) influences proliferation and osteogenic differentiation of bone marrow MSCs in a manner consistent with our hypothesis that transient activation of Notch signaling will promote MSC in vivo expansion leading to an enhanced osteogenic differentiation response. 10 week old WT male (n=12) and female (n=12) BL6 mice were intraperitoneal injected daily with 0.5 mg/kg of control lgG or JAG1 for 7 days followed by 6-hour in vivo BrdU labeling before sacrificed for cell and tissue collection. Immunostaining showed that expression of Notch target gene Hes1 in bone marrow cells in JAG1-injected mice was significantly increased compared to the lgG-injected control mice. Similarly, the size of BrdU positive area inside the bone marrow space was also increased in JAG1-injected mice suggesting a greater cell proliferation index than that in lgG-injected control mice. Furthermore, flow cytometry data showed a higher percentage of CD29 and CD44 positive cell population in MSCs was obtained in JAG1-treated mice when compared to lgG-treated mice. When introduced to osteogenic differentiation medium, MSCs from JAG1-treated mice exhibited a significant increased osteogenic differentiation by showing enhanced Alizarin red staining. Finally, our RT-PCR data revealed a significant increase in expression of osteogenic marker alkaline phosphatase (ALP), Runx2, osterix and osteocalicin, as well as osteogenic inducer BMP2 in MSCs from JAG-treated mice. Our data support that transient activation of Notch signaling in vivo increased bone marrow cell proliferation and MSC population. Therefore, the increased ex vivo osteogenesis of MSCs from JAG1-treated mice could be due to the increased number of true stem cells in newly isolated MSCs. Further studies to understand the effects of transient Notch activation on other cell types in multiple organs are ongoing.Funding Source: Orthopedic Research and Education Foundation with funding provided by the Musculoskeletal Transplant Foundation (Grant No.16–004)

439POSTER ABSTRACTSCARDIAC TISSUE AND DISEASEF-2025HUMAN NEONATAL C-KIT+ CARDIAC PROGENITOR CELLS IMPROVE CARDIAC FUNCTION POST-MYOCARDIAL INFARCTION BY ALTERING THE CARDIAC HEALING RESPONSEOngstad, Emily - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USA Bao, Weike - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USA Gaddipati, Ranjitha - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USA Belkhodja, Mehdi - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USA Mishra, Rachana - Division of Cardiac Surgery, School of Medicine, University of Maryland Baltimore, Baltimore, MD, USA Bhagroo, Nicholas - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USA Kaushal, Sunjay - Division of Cardiac Surgery, School of Medicine, University of Maryland Baltimore, Baltimore, MD, USA Karathanasis, Sotirios - Cardiovascular, Renal and Metabolism Research, MedImmune-AstraZeneca, Gaithersburg, MD, USAThe c-kit+ cardiac progenitor cells (CPCs) are a promising therapy for preventing progression to heart failure after myocardial infarction (MI). In clinical trials, cell therapies based on adult CPCs (aCPCs) have demonstrated safety, but minimal efficacy. This is partly due to a heterogeneous, mostly senescent, population of cells derived from adult tissues. Recent research has shown that c-kit+ CPCs from neonatal human hearts (nCPCs) have few senescent cells, display robust proliferative capacity in vitro, and are more effective in improving cardiac function compared to c-kit+ CPCs isolated from adult human hearts in rodent MI models. We examined the ability of nCPCs to increase cardiac function and investigated the potential mechanisms for their effectiveness. nCPCs injected intramyocardially immediately post-MI in male nude rats led to significant improvements in contraction and relaxation in the left ventricle at 4 weeks after injury compared to controls. Heart failure biomarker NT-proANP and proinflammatory cytokine TNF were significantly reduced αby administration of nCPCs. Additionally, nCPC administration led to smaller scar size. As reparative effects of progenitor cells on the heart are exerted by paracrine action, we examined the effect of total conditioned medium (TCM) from several cell types in functional assays relevant to cardiac recovery post-MI. Efferocytosis, an inflammation resolution process, was significantly improved by incubation with neonatal TCM (nTCM), but not adult TCM (aTCM), mesenchymal stem cell (MSC) TCM, or fibroblast TCM. nTCM increased angiogenesis, as measured by the endothelial tube formation assay. In an anti-fibrotic assay, nTCM reduced alpha Smooth Muscle Actin protein levels, and decreased profibrotic genes COL1A1, COL1A2, COL3A1, and fibronectin. In an inflammation suppression assay, nTCM decreased secretion of proinflammatory cytokine IL-8. Overall, these data demonstrated an enhanced ability of c-kit+ nCPCs, but not aCPCs or MSCs, to improve cardiac function post-MI by promoting inflammation resolution and enhancing the cardiac healing response.F-2027REGENERATING THE INFARCTED PIG HEART USING HUMAN EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTESRomagnuolo, Rocco - McEwen Stem Cell Institute, University Health Network (UHN), Toronto, ON, Canada Masoudpour, Hassan - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Porta-Sanchez, Andreu - Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada Qiang, Beiping - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Barry, Jennifer - Schulich Heart Research Program, Sunnybrook Research Institute, Toronto, ON, Canada Laskary, Andrew - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Qi, Xiuling - Schulich Heart Research Program, Sunnybrook Research Institute, Toronto, ON, Canada Masse, Stephane - Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada Magtibay, Karl - Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada Kawajiri, Hiroyuki - Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada Wu, Jun - Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada Valdman Sadikov, Tamilla - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Rothberg, Janet - Centre for Commercialization of Regenerative Medicine, Centre for Commercialization of Regenerative Medicine, Toronto, ON, Canada Titus, Emily - Centre for Commercialization of Regenerative Medicine, Centre for Commercialization of Regenerative Medicine, Toronto, ON, Canada Li, Ren-Ke - Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada Zandstra, Peter - Centre for Commercialization of Regenerative Medicine, Centre for Commercialization of Regenerative Medicine, Toronto, ON, Canada Wright, Graham - Schulich Heart Research Program, Sunnybrook Research Institute, Toronto, ON, Canada Nanthakumar, Kumaraswamy - Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada Ghugre, Nilesh - Schulich Heart Research Program, Sunnybrook Research Institute, Toronto, ON, Canada Keller, Gordon - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Laflamme, Michael - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada

440POSTER ABSTRACTSFollowing a myocardial infarction (MI), the damaged muscle is replaced with non-contractile scar tissue, which often leads to heart failure. In small rodent and macaque models of MI, the transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) mediates the partial remuscularization of the infarct scar leading to improvements in contractile function. However, transplantation of hESC-CMs in small non-human primates results in transient, non-lethal ventricular tachyarrhythmias (VT). We therefore tested the capacity of hESC-CMs to stably engraft in a more translationally relevant preclinical model, the infarcted pig heart. To induce infarction, we used balloon angioplasty to occlude the mid left anterior descending artery. At 3-weeks post-MI, a left lateral thoracotomy was performed and vehicle alone (n=14) or 10^9 hESC-CMs (cardiac purity of 82.3 ± 3.0%; n=13) were directly injected into the infarct zone. Animals were monitored by telemetric ECG recording and serial cardiac MRI for up to 8-weeks post-transplantation, with a small cohort of animals undergoing electroanatomical mapping (10 days post-transplantation; n=4 for vehicle and n=3 for hESC-CMs recipients). Overall, hESC-CM transplantation resulted in substantial myocardial implants within the infarct scar that matured over time, formed vascular networks with the host, and evoked minimal cellular rejection. While arrhythmias were rare in infarcted pigs receiving vehicle alone (n=14), hESC-CM recipients (n=13) experienced frequent monomorphic ventricular tachycardia before reverting back to normal sinus rhythm by approximately 4-weeks post-transplantation. Electroanatomical mapping and pacing studies implicated focal mechanisms for these graft-related tachyarrhythmias as evidenced by an abnormal centrifugal pattern with earliest electrical activation in histologically-confirmed graft tissue. These findings demonstrate the suitability of the pig model for the preclinical development of a hESC-based cardiac therapy and provide new insights into the mechanistic basis of electrical instability following hESC-CM transplantation.Funding Source: OIRM, McEwen Stem Cell Institute, Peter Munk Cardiac Centre, Canadian Foundation for Innovation, Medicine by Design/Canada First Research Excellence Fund initiative and BlueRock Therapeutics.F-2029EFFECT OF SMALL MOLECULE INDUCED BEIGE ADIPOCYTES ON CARDIOMYOCYTES AGAINST HYPOXIA/REOXYGENATIONKim, Sang Woo - 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, 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, KoreaBeige adipocytes gained much attention as an alternative cellular target in regulating metabolic homeostasis. The development of the beige cells that has beneficial metabolic, but how thermogenic stimuli activate and control beige adipocytes is not fully understood. Here, we investigated that the effects and mechanism of isoliquiritigenin (ILG) on adipose-derived stem cells (ASCs) differentiate into functional beige/brown adipocytes. We found that ASCs derived white adipocytes are able to switch to a brown phenotype by expressing both UCP1 and CIDEA according to low dose ILG treatments. Although various pharmacological activities of ILG, the possible role of ILG in white-to beige transdifferentiation of adipose-derived stem cells have never been explored. Moreover, we demonstrated that ILG small molecule dose-response in distinct regulatory mechanisms and functions to induce the white-to-beige trans-differentiation of adipose-derived stem cells. Then, we sought to determine if small molecule induced beige adipocytes into impaired heart tissue affected surround fat in the heart. Therefore, we investigated the expression levels of proteins associated with oxidation, inflammation, and death signals in cardiomyocytes with beige cells under hypoxia/reoxygenation conditions for their roles in many physiological processes in the heart. These studies suggest that the induction of thermogenic adipocytes might offer a new approach to combating human metabolic disorders and ischemic heart diseases.Funding Source: This study was funded by NRF-2018R1A2B6008629, 2016R1D1A1B03935124, and NRF-2015M3A9E6029519.F-2031AUTOPHAGIC FLUX CORRECTION BY HEMATOPOIETIC STEM CELL-DERIVED MACROPHAGES IN A MOUSE MODEL OF DANON DISEASEHashem, Sherin - Pathology, University of California, San Diego, La Jolla, CA, USA Gault, Emily - Medicine, University of California San Diego, La Jolla, CA, USA Sharma, Jay - Pharmacology, University of California San Diego, La Jolla, CA, USA Evans, Sylvia - Medicine, University of California San Diego, La Jolla, CA, USA Cherqui, Stephanie - Pharmacology, University of California San Diego, La Jolla, CA, USA

441POSTER ABSTRACTSAdler, Eric - Medicine, University of California San Diego, La Jolla, CA, USADanon disease is a fatal condition without any specific therapy making heart transplantation the patient’s only choice for survival. Even with heart transplantation, patients continue to suffer from skeletal myopathies. Therefore, the development of a stem cell-based systemic therapy would meet a critical unmet need. Here, we sought to assess the therapeutic potential of using Sca1+ hematopoietic stem and progenitor cell (HSPC) in the context of Danon disease which is caused by the deficiency of the lysosomal associated membrane protein type -2 (LAMP-2), a lysosomal membrane bound protein essential in autophagic flux. First, we co-cultured LAMP-2 knockout (KO) fibroblasts (FBs) with wild-type (WT) macrophages and observed the in vitro transfer of LAMP-2-positive vesicles from WT macrophages to LAMP-2 KO FBs that were near. Importantly, a decrease in the number of autophagic vacuoles (AVs) and a rescue of autophagic flux were noted in co-cultured LAMP-2 KO FBs compared to control, suggesting that fusion of autophagosomes and lysosomes was restored. Next, we transplanted LAMP-2 KO mice with WT-GFP+ mouse derived Sca1+ HSPCs. At 12-months post-transplantation, HSPC-derived GFP+/CD68+ cells were present in the hearts and skeletal muscles of HSPC-transplanted mice, demonstrating that a single infusion of HSPCs had the capacity to migrate to and integrate in the diseased hearts and skeletal muscles, and differentiate into phagocytic cells. Assessment of LAMP-2 expression in the heart and skeletal muscle confirmed that tissue engraftment of the WT-HSPC-derived macrophages resulted in the restoration of LAMP-2 expression in recipient mice. Importantly, immunofluorescence studies showed the diffuse pattern of spatial restoration of vesicular LAMP-2 expression in transplanted mice. The transfer of LAMP-2-positive vesicles from WT-donor-derived macrophages also resulted in a reduction in LC3-II levels and number of AVs in the heart and skeletal muscle tissues of recipient mice, indicating rescue of autophagic flux. Our findings may provide a new paradigm for the treatment of a wide assortment of autophagy-related disorders that collectively have a major impact on public health.F-2033ROLE OF TRPC7 IN REGULATING THE FUNCTIONS OF EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTESTsang, Suk Ying - School of Life Sciences, The Chinese University of Hong Kong, China Liu, Xianji - School of Life Sciences, The Chinese University of Hong Kong, China Yao, Xiaoqiang - School of Biomedical Sciences, The Chinese University of Hong Kong, ChinaMany ion channels serve to maintain the electrophysiological function and calcium homeostasis of cardiomyocytes. Although classical sodium, potassium and calcium channels are intensively studied, many other channels may also contribute to the delicate electrophysiological regulation of cardiomyocytes. Canonical transient receptor potential (TRPC) channels are non-selective cation channel that are activated by G-protein coupled receptors. TRPC channels are widely expressed in different tissues and play important roles in the maintaining cellular homeostasis. The aim of this project is to study the function of TRPC7, the most elusive member in the TRPC family, in cardiomyocytes. Western blotting showed that TRPC7 is expressed in mouse heart. Immunocytochemistry experiments showed that the channel locates at plasma membrane at early differentiation stage of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs), but translocates from membrane to a location closer to the sarcomere during the maturation of mESC-CMs. Translocation of TRPC7 occurs during the whole process of maturation; most channels are located near the M-line in less mature mESC-CMs while they are located near the Z-line in more mature mESC-CMs. Knockdown of TRPC7 led to a decrease in calcium transients in mESC-CMs. In addition, knockdown of TRPC7 caused the disassembly of sarcomere in neonatal ventricular myocytes. Our result suggests that TRPC7 may serve multiple roles in differentiating cardiomyocytes. Further study is needed to elucidate the mechanisms of how TRPC7 regulates the functions of differentiating cardiomyocytes.Funding Source: This work was supported by the General Research Fund (14176817) from the University Grants Committee (UGC) of the Hong Kong SAR. X. L. was supported by the postgraduate studentship from the Chinese University of Hong Kong.F-2035MONITORING STEM CELL-DERIVED CARDIOMYOCYTE MATURATION BY OPTICAL METABOLIC IMAGINGQian, Tongcheng - University of Wisconsin-Madison, Morgridge Institute for Research, Madison, WI, USA Favreau, Peter - Department, University of Wisconsin-Madison, WI, USA Dunn, Kaitlin - Department of Chemical and Biological Engineering, University of Wisconsin-Madison, WI, USA Palecek, Sean - Department of Chemical and Biological Engineering, University of Wisconsin-Madison, WI, USA Skala, Melissa - Morgridge Institute for Research and Department of Chemical and Biological Engineering, University of Wisconsin-Madison, WI, USACardiovascular disease remains the leading cause of death in the world despite advances in treatment. Human pluripotent stem cells (hPSCs) can generate any cell lineage in vitro, including cardiomyocytes. hPSC-derived cardiomyocytes have immense potential to impact clinical care and fundamental research for cardiovascular disease. However, hPSC-derived cardiomyocytes exhibit a relatively immature phenotype. New technologies that can non-invasively quantify the maturation state in live hPSC-cardiomyocytes are needed to effectively optimize a mature phenotype. hPSC-derived cardiomyocytes undergo dramatic metabolic changes during maturation. Here, we monitor these metabolic changes that occur in hPSC-derived

442POSTER ABSTRACTScardiomyocytes during extended time in culture by multiphoton fluorescence lifetime imaging (FLIM) of the metabolic co-enzymes NAD(P)H and FAD. Changes in hPSC-cardiomyocyte metabolism during maturation were non-invasively quantified at a single-cell level with the optical metabolic imaging (OMI) parameters. Our results indicate that the lifetime of NAD(P)H increases and the redox ratio decreases with maturation over a 100-day time-course in hPSC-derived cardiomyocytes. These label-free imaging technologies could be used to test strategies to optimize the maturation of hPSC-derived cardiomyocytes in vitro.F-2037IN VITRO MATURED HESC-DERIVED CARDIOMYOCYTES ELECTRICALLY COUPLE AND FORM IMPROVED GRAFTS IN INJURED HEARTSDhahri, Wahiba - Experimental Therapeutics/McEwen Stem Cell Institute, University Health Network (UHN), Toronto, ON, Canada Sadikov Valdman, Tamilla - Experimental Therapeutics, University Health Network, Toronto, ON, Canada Qiang, Beiping - Experimental Therapeutics, University Health Network, Toronto, ON, Canada Masoudpoor, Hassan - Experimental Therapeutics, University Health Network, Toronto, ON, Canada Ceylan, Eylul - Biomedical Engineering, King’s College, London, UK Wulkan, Fanny - Experimental Therapeutics, University of Toronto, Toronto, ON, Canada Laflamme, Michael A - Experimental Therapeutics, University Health Network, Toronto, ON, CanadaThe ability of pluripotent human embryonic stem cells (hESCs) to yield large quantities of functional cardiomyocytes (CMs) holds tremendous promise for use in cardiac regeneration. However, the immature structural, electrophysiological and contractile phenotype of available hESC-CM populations limits their translational potential. The present study was aimed at testing two hypotheses: 1) previously reported approaches to promote the maturation of hESC-CMs by culture on polydimethylsiloxane (PDMS) membranes can be upscaled for transplantation studies; and 2) PDMS-matured hESC-CMs will efficiently engraft in injured hearts and form graft myocardium with enhanced structural and functional properties. We generated transgenic hESC-CMs that stably expressed the fluorescent voltage-sensitive protein ASAP1, then comprehensively phenotyped these myocytes after 20 and 40 days of in vitro maturation on either tissue culture plastic (TCP) or PDMS. Next, we transplanted day 20 or 40 TCP vs PDMS ASAP1+ hESC-CMs into injured guinea pig hearts (n=6-9 per group). Engrafted hearts were later analyzed by ex vivo optical voltage mapping studies and histology. Relative to TCP controls, hESC-CMs on PDMS at both time-points exhibited increased cardiac gene expression as well as a more mature structural and electrophysiological phenotype in vitro. Although CMs from both substrates showed similar capacity for engraftment (graft area by histomorphometry), graft formed using PDMS-matured myocytes had more mature histological properties, with better-aligned cardiomyocytes and increased sarcomere lengths and gap junction expression. Most importantly, graft formed with PDMS-matured myocytes showed enhanced electrophysiological properties by optical mapping based on ASAP1 fluorescence, including better host-graft electromechanical integration and more rapid and uniform conduction velocity. We demonstrate that hESC-CMs matured on PDMS can be produced in large quantities (scale of 10e8 to 10e9 CMs). Moreover, PDMS-matured myocytes form large intramyocardial grafts with enhanced cardiac structure and greatly improved electrical function, thereby establishing that CM maturation prior to transplantation meaningfully improves outcomes in vivo.Funding Source: McEwen Centre for Regenerative Medicine, the Peter Munk Cardiac Centre and the University of Toronto’s Medicine by Design/Canada First Research Excellence Fund initiative.F-2039GENOME-EDITED IPSC AND PIG CARDIOMYOPATHY MODELS REVEAL MUTANT RBM20 FORMS MISLOCALIZED GRANULES TO DOMINANTLY DISRUPT GLOBAL SPLICINGMiyaoka, Yuichiro - Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Tan, Kenneth - Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA Matsa, Elena - Tenaya Therapeutics, Tenaya Therapeutics, South San Francisco, CA, USA Mayerl, Steven - Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA Chan, Amanda - Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA Herrera, Vanessa - Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA Kulkarni, Aishwarya - Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Venkatasubramanian, Meenakshi - Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Chetal, Kashish - Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Sun, Han - School of Medicine, Stanford University, Stanford, CA, USA Briganti, Francesca - School of Medicine, Stanford University, Stanford, CA, USA Wei, Wu - School of Medicine, Stanford University, Stanford, CA, USA Oommen, Saji - Todd and Karen Wanek Hypoplastic Left Heart Syndrome Program, Mayo Clinic, Rochester, NY, USA Carlson, Daniel - Recombinetics, Inc, Recombinetics, Inc, St. Paul, MN, USA Nelson, Timothy - Todd and Karen Wanek Hypoplastic Left

443POSTER ABSTRACTSHeart Syndrome Program, Mayo Clinic, Rochester, NY, USA Steinmetz, Lars - School of Medicine, Stanford University, Stanford, CA, USA Schneider, Jay - Department of Medicine/Cardiology, UT Southwestern Medical Center, Dallas, TX, USA Conklin, Bruce - Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA Salomonis, Nathan - Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USADilated cardiomyopathy (DCM) is the most common indication for heart transplantation. Among over 50 DCM causal genes identified so far, the gene RBM20 has emerged as a key regulator of cardiac splicing in both early cardiogenesis and inherited dilated cardiomyopathy. The pathogenic RBM20 mutations display a striking recurring pattern of eight tightly clustered single amino acid DCM-associated substitutions in the arginine-serine (RS) domain of RBM20, but the pathogenic mechanism of these clustered mutations is unknown. To understand the role of specific RBM20 alleles, we generated genome-edited human induced pluripotent stem cell (iPSC)-derived cardiomyocytes with heterozygous or homozygous R636S mutation or a functional knockout. In addition to both mutant and loss-of-function alleles mimicking the contractile phenotypes of dilated cardiomyopathy patients, R636S mutation resulted in RBM20 mislocalization from the cardiomyocyte nucleus to the cytoplasm as prominent granules. Global transcriptome analyses revealed distinct subclasses of RBM20 target splicing defects, including pathogenic mutation-specific, dosage dependent and heterozygous mutation-dominant splicing. Providing in vivo context, we independently confirmed global and splice-event specific observations in the hearts of genome- edited pigs with the same R636S mutation. Our results highlight novel pathogenic mechanisms in splicing factor-mediated cardiomyopathy, through both splicing-dependent and independent pathways, that will rationally guide future therapeutic development.Funding Source: This work supported by the National Heart, Lung, and Blood Institute to B.R.C and N.S, JSPS Grant-in-Aid for Young Scientists (A), NOVARTIS, Mochida Memorial, SENSHIN Medical Research, Naito, Uehara Memorial Foundations to Y.M.F-2041SINGLE-CELL TRANSCRIPTOME ANALYSIS DURING CARDIOGENESIS REVEALS BASIS FOR ORGAN LEVEL DEVELOPMENTAL ANOMALIESDe Soysa, Yvanka - Gladstone Institute for Cardiovascular Disease, J. David Gladstone Institutes and UCSF, San Francisco, CA, USA del Sol, Antonio - Computational Biology Group, University of Luxembourg, Luxembourg Gifford, Casey - Gladstone Institute of Cardiovascular Disease, J. David Gladstone Institutes, San Francisco, CA, USA Okawa, Satoshi - Computational Biology Group, University of Luxembourg, Luxembourg Ranade, Sanjeev - Gladstone Institute of Cardiovascular Disease, J. David Gladstone Institutes, San Francisco, CA, USA Ravichandran, Srikanth - Computational Biology Group, University of Luxembourg, Luxembourg Srivastava, Deepak - Gladstone Institute of Cardiovascular Disease, J. David Gladstone Institutes, San Francisco, CA, USAOrganogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. In this work, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. We found dysregulation of retinoic acid signaling, responsible for anterior-posterior cardiac patterning, that was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.ENDOTHELIAL CELLS AND HEMANGIOBLASTSF-2043EFFECTIVE GENERATION OF ENDOTHELIAL CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLSMeng, Shulin - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Gao, Ge - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Zhou, Anyu - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Xia, Houkang - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Liu, Junwei - R&D, IxCell Biotechnology Co., Ltd, Shanghai,

444POSTER ABSTRACTSChina Li, Xin - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Yao, Jian - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Jin, Hongyu - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Hu, Zunlu - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Zhang, Xiaomin - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Hao, Jiali - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Yue, Yan - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Ji, Zhinian - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Yan, Ruyu - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Feng, Jing - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Yang, Chaowen - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Wu, Ying - R&D, IxCell Biotechnology Co., Ltd, Shanghai, China Gao, Jieyu - IxCell Biotechnology Co., Ltd, Shanghai, ChinaCardiovascular disease affects millions of people. Induced pluripotent stem cells (iPSCs) derived functional endothelial cells (ECs) are emerging as unlimited resource for cell and gene therapy for cardiovascular diseases. Therefore, there is an urgent need for developing robust and efficient approaches to differentiate iPSCs into mature ECs. Here we reported an effective protocol for generating mature and functional human endothelial cells from iPSCs for mechanistic studies, drug screening and vascular therapy. Integration-free iPSCs were generated from human peripheral blood mononuclear cells (PBMC). The stemness of the iPSCs were confirmed by staining of the stem cells markers using specific antibodies as well as embryoid body formation. iPSCs were differentiated into endothelial cells using a diversity of combinations of growth factors and small molecules targeting the key signaling pathways of the endothelial lineage development. The expression of the endothelial lineage markers, such as CD31 (PECAM1) ,CD144 (VE-cadherin) and vWF (Von Willebrand factor) was examined by immunofluorescence staining and flow cytometry. In vitro tube formation and Dil fluorescent dye-labeled acetylated low density lipoprotein(Dil-ac-LDL) uptake assay were performed to demonstrate the function of the differentiated cells.iPSCs reprogrammed from PBMCs were positive for pluripotency markers NANOG, OCT4, SOX2 and SSEA4, and exhibited the ability of differentiation to three germ layers. The differentiated cells from iPSCs displayed the phenotypes and function of the primary endothelial cells. CD144 and vWF were positively detected by immunofluorescence microscopy and 98% of the cells expressed the endothelial lineage markers CD31 and CD144 determined by flow cytometry. The differentiated cells also assembled into well-defined vessel-like structures in tube formation assay in vitro. Dil-ac-LDL uptake assay showed that the hiPSCs-derived ECs were able to take Dil-ac-LDL.We established an effective and robust strategy for endothelial cells differentiation from iPS cells. The iPSC-derived endothelial cells demonstrate the phenotype and function of primary human endothelial cells.F-2045ANGIOTENSIN 2 ATTENUATES THE BIOACTIVITIES OF HUMAN ENDOTHELIAL PROGENITOR CELLS VIA DOWNREGULATION OF BETA2-ADRENERGIC RECEPTORJang, Woong Bi - Department of Physiology, Pusan National University, Yangsan, Korea Lee, Seon Jin - Physiology, Pusan National University, Pusan, Korea Kim, Da Yeon - Physiology, Pusan National University, Pusan, Korea Yun, Jisoo - Physiology, Pusan National University, Pusan, Korea Choi, Sung Hyun - Physiology, Pusan National University, Pusan, Korea Jung, Seok Yun - Physiology, Pusan National University, Pusan, Korea Kang, Songhwa - Physiology, Pusan National University, Pusan, Korea Park, Ji Hye - Physiology, Pusan National University, Pusan, Korea Kim, Yeon Ju - Physiology, Pusan National University, Pusan, Korea Ha, Jong Seong - Physiology, Pusan National University, Pusan, Korea Ji, Seung Taek - Physiology, Pusan National University, Pusan, Korea Lee, Dong Hyung - Obstetrics and Gynecology, Pusan National University, Pusan, Korea Lee, Dong Jun - Medical Science, Pusan National University, Pusan, Korea Kwon, Sang-Mo - Physiology, Pusan National University, Pusan, KoreaCross talks between the renin-angiotensin system (RAS), sympathetic nervous system, and vascular homeostasis are tightly coordinated in hypertension. Angiotensin II (Ang II), a key factor in RAS, when abnormally activated, affects the number and bioactivity of circulating human endothelial progenitor cells (hEPCs) in hypertensive patients. In this study, we investigated how the augmentation of Ang II regulates adrenergic receptor-mediated signaling and angiogenic bioactivities of hEPCs. Interestingly, the short-term treatment of hEPCs with Ang II drastically attenuated the expression of beta-2 adrenergic receptor (ADRB2), but did not alter the expression of beta-1 adrenergic receptor (ADRB1) and Ang II type 1 receptor (AT1R). EPC functional assay clearly demonstrated that the treatment with ADRB2 agonists significantly increased EPC bioactivities including cell proliferation, migration, and tube formation abilities. However, EPC bioactivities were decreased dramatically

445POSTER ABSTRACTSwhen treated with Ang II. Importantly, the attenuation of EPC bioactivities by Ang II was restored by treatment with an AT1R antagonist (telmisartan; TERT). We found that AT1R binds to ADRB2 in physiological conditions, but this binding is significantly decreased in the presence of Ang II. Furthermore, TERT, an Ang II-AT1R interaction blocker, restored the interaction between AT1R and ADRB2, suggesting that Ang II might induce the dysfunction of EPCs via downregulation of ADRB2, and an AT1R blocker could prevent Ang II-mediated ADRB2 depletion in EPCs. Taken together, our report provides novel insights into potential therapeutic approaches for hypertension-related cardiovascular diseases.F-2047TRANSDIFFERENTIATION OF HUMAN ADULT FIBROBLASTS INTO AUTHENTIC ENDOTHELIAL CELLSShin, Youngchul - Center of Cell-and Bio-Therapy for Heart, Diabetes, and Cancer, Seoul National University Hospital, Seoul, Korea Han, Jung-Kyu - Center of Cell-and Bio-Therapy for Heart, Diabetes, and Cancer, Seoul National University Hospital, Seoul, Korea Choi, Saet-Byeol - Center of Cell-and Bio-Therapy for Heart, Diabetes, and Cancer, Seoul National University Hospital, Seoul, Korea Sohn, Min-Hwan - Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea Shin, Dasom - Center of Cell- and Bio-Therapy for Heart, Diabetes, and Cancer, Seoul National University Hospital, Seoul, Korea Shin, Jong-Yeon - Precision Medicine Center, Seoul National University Bundang Hospital, Seoul, Korea Seo, Jeong-Sun - Precision Medicine Center, Seoul National University Bundang Hospital, Seoul, Korea Kim, Hyo-Soo - Center of Cell- and Bio-Therapy for Heart, Diabetes, and Cancer, Seoul National University Hospital, Seoul, KoreaPreviously, we reported direct conversion of adult fibroblasts (FBs) into endothelial cells (ECs) using defined factors in mice. Here, we assessed whether this approach can be applied for direct conversion of human adult ECs to authentic ECs. We tested whether 5 defined factors (Foxo1, Er71, Klf2, Tal1, Lmo2) for mouse induced ECs (iECs) could convert human dermal FBs (HDFs) to ECs. 28 days after infection of lentiviruses expressing each gene to HDFs, ECs defined by VE-cadherin expression on FACS were detected (32.1±5.1%). Interestingly, 2 factors were dispensable, and only 3 factors (factor X, Y, Z) were necessary and sufficient to make human iECs (49.4±3.5%). To enhance the efficiency, a lentivirus expressing 3 factors plus GFP altogether were made using 2A system. Unexpectedly, VE-cadherin+/GFP+ cells induced by this virus was integrated with VE-cadherin-/GFP+ and HDF control together in hierarchical analysis in whole transcriptome sequencing, which meant VE-cadherin+ cells were not completely converted yet. The proportion of cells expressing another endothelial specific marker, CD31, together (VE-cadherin/CD31 double positive (DP) cells) was only 5.2±0.6%. To get complete conversion, several means were tried. Among them, rosiglitazone (MET inducer) treatment, prolonged incubation after VE-cadherin sorting, and suppression of some FBs specific transcription factors (TFs) using siRNA worked. However, 2nd stage infection of other endothelial specific TFs, shear stress, treatment of VEGF, SB431542 or Wnt modulators, time dependent expression using Tet-on system did not. Final protocol could convert 19.6±3.0% of HDFs into DP cells 6 weeks after infection. DP cells showed characteristics of authentic human ECs (Matrigel tube formation, Ac-LDL uptake, lectin binding, NO production, IF staining for EC markers, characteristic EC morphology on optical and electron microscope, and whole transcriptome sequencing). Our iEC protocol showed the most efficient EC conversion rate, compared with the protocols suggested by other groups. Furthermore, unlike other studies in which EC-like cells were defined as only one single marker, our study suggests a new perspective that the use of double markers can purify mature iECs which exclude immature iECs that arise during the direct conversion process.Funding Source: This research was supported by SNUH Research Fund (grant number: 03-2018-0450) and Korea Health Technology R&D Project “Strategic Center of Cell and Bio Therapy” (grant number: HI17C2085)HEMATOPOIESIS/IMMUNOLOGYF-2049EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWALSigner, Robert A - Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA Kruta, Miriama - Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA Sunshine, Mary Jean - Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA Fu, Yunpeng - Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA Hidalgo San Jose, Lorena - Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA, USAThe inability to maintain and expand somatic stem cells in culture represents a major barrier to their use in cell-based therapies. As such, there is significant need to uncover why somatic stem cell self-renewal is diminished ex vivo, and to discover conditions that support stem cell growth in vitro. We recently discovered that hematopoietic stem cells (HSCs) exhibit low protein synthesis in vivo, regardless of whether they are quiescent or undergoing self-renewing divisions. Low protein synthesis is necessary for HSCs, as modest (~30%) increases in protein synthesis impair HSC self-renewal. In the present study, we determined that cultured HSCs rapidly

446POSTER ABSTRACTSupregulated genes that promote translation and exhibited a ~2000% increase in protein synthesis. Increased protein synthesis overwhelmed protein quality control systems within HSCs, caused an imbalance in protein homeostasis, and was associated with nuclear translocation of Hsf1. Hsf1 is the master regulator of the heat shock pathway, and induces transcription of heat shock proteins that coordinate protein folding, trafficking and degradation to sustain protein homeostasis in response to proteotoxic stress. Inactive Hsf1 is typically sequestered in the cytoplasm through binding to Hsp90 and TRiC, and was rarely seen in the nucleus within HSCs in vivo. Genetic deletion of Hsf1 exacerbated HSC depletion in vitro, but had little effect on HSC function in vivo. These data indicated that Hsf1 promotes ex vivo HSC maintenance, and raised the possibility that increasing Hsf1 activation could enhance HSC self-renewal. To test this, we cultured purified HSCs in the presence of a Hsp90 or TRiC inhibitor that each promoted Hsf1 nuclear translocation, and assessed HSC function in serial transplantation assays. Over the 10-day culture period, HSCs proliferated extensively and fully retained serial long-term multilineage reconstituting activity. The positive effect of both Hsp90 and TRiC inhibitors on HSC growth was completely ablated in the absence of Hsf1. Furthermore, Hsf1 activation reduced the unfolded protein load and partially rebalanced protein homeostasis. These findings indicate that maintaining protein homeostasis is a key factor in promoting ex vivo stem cell self-renewal, and reveal new strategies that enable sustained HSC growth in culture.F-2051FORGOTTEN GEMS: HUMAN CD34- HEMATOPOIETIC STEM CELLSPopova, Semiramis - Haematopoietc Stem Cell Lab, The Francis Crick Institute, London, UK Anjos-Afonso, Fernando - European Cancer Stem Cell Research Institute, University of Cardiff, Cardiff, UK Bonnet, Dominique - Haematopoietic Stem Cell Lab, The Francis Crick Institute, London, UK Gacia Albornoz, Manuel - Haematopoietic Stem Cell Lab, The Francis Crick Institute, London, UKHuman HSC research effort has been largely restricted to CD34+ cells both in prenatal and adult life. However, recent evidence has highlighted that both adult bone marrow and umbilical cord blood contain CD34- SCID-repopulating cells (SRCs). CD34- SRCs exhibit distinct in vivo repopulation kinetics and capacity to produce functional CD34+ HSCs, and accordingly have been speculated to reside at the apex of the human haematopoietic hierarchy. However, only one out of 5000 cells within the CD34-/CD38-/CD93+ population is estimated to have in vivo repopulation capacity, which has significantly hindered efforts to elucidate the function and molecular mechanisms regulating this HSC compartment in comparison to CD34+ HSCs. We thus aim to enhance our ability to purify bona-fide CD34- HSCs and further expand the knowledge of this immature stem pool. We have successfully identified an additional positive selection marker, enriching for CD34- SRCs by 20-fold, thus facilitating our efforts to elucidate the functionality and molecular pathways maintaining this population. Through serial transplantations of enriched CD34- SRCs in NSG and enhanced NSG mouse models (NSG-S, NSBGW, and NSG-hmSCF), we observed more potent and efficient repopulation and lineage commitment kinetics. Resulting data and single cell-transcriptomic profile of this population represent a further step toward a better understanding of its role in human hematopoiesis. With these new data we have asserted the importance of human CD34- HSCs. In view of their highly quiescent nature, yet robust population capacity, it is easy to speculate that the CD34- HSCs hold enormous therapeutic potential that cannot be ignored.F-2053IDENTIFICATION OF A MACROCYCLIC DITERPENE COMPOUND WITH COMBINATORIAL DIFFERENTIATING EFFECT VIA PKC ACTIVATION AND BET INHIBITION IN PRIMARY HUMAN MONOCYTIC LEUKEMIAHultmark, Simon - Department of Laboratory Medicine, Molecular Medicine and Gene Therapy Lund Stem Cell Center, Lund, Sweden Baudet, Aurelie - Laboratory Medicine, Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund, Sweden Schmiderer, Ludwig - Laboratory Medicine, Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund, Sweden Larsson, Christer - Laboratory Medicine, Translational Cancer Research, Lund, Sweden Lehmann, Soren - Department of Medical Sciences, Uppsalaa University Hospital, Department of Medicine, Karolinska Institutet, Stockholm, Sweden Juliusson, Gunnar - Laboratory Medicine, Department of Hematology Skane University Hospital, Lund Stem Cell Center, Lund, Sweden Ek, Fredrik - Department of Experimental Medical Science, Chemical Biology and Therapeutics, Lund, Sweden Magnusson, Mattias - Laboratory Medicine, Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund, SwedenSafer and more efficient therapy for patients with acute myeloid leukemia (AML) is needed and differentiation therapy is an appealing option to eradicate leukemic stem cells blocked in differentiation. With the aim of identifying new AML differentiating molecules, we screened 528 natural compounds on primary AML samples in our co-culture system mimicking the niche. Compounds that upregulated the expression of the myeloid differentiation markers CD11b/CD15 were considered as positive hits. Among those, a macrocyclic diterpene called H4 upregulated expression of CD11b (DMSO 22±5%, H4 66±8% SEM), which was accompanied with a clear decrease in nuclear-cytoplasmic ratio, on patient cells with AML subtype M5. No toxicity was observed to healthy control cells. In order to unravel the signaling pathways contributing to the differentiation response, we performed a combinatorial treatment with H4 and 176 molecules with defined drug targets. Inhibition of PKC, RTKs,

447POSTER ABSTRACTSJNK and Raf/MEK/ERK impeded the differentiation-promoting effect of H4 with complete block upon combined treatment with the PKC inhibitor GFX109203X. Furthermore, PKC-GFP-isoforms ( , , , ) were translocated to the plasma membrane α β ε δwithin 60-120 seconds upon H4 treatment. Demonstrating that H4 induced myeloid differentiation is mediated by PKC activation. Gene expression profiling on primary M5 AML cells treated for 16h with H4 revealed a significant enrichment for MYC target genes. The increase in MYC transcription was confirmed by qPCR and was accompanied by an increased protein translation rate (OP-Puro incorporation assay). To evaluate the role of MYC activation upon H4 treatment, MYC expression was inhibited using the BET inhibitor CPI-203 in combination with H4. The combination increased the H4 induced differentiation capacity synergistically from a 3-fold (H4 or CPI alone) to 11-fold increase in CD11b MFI in comparison to DMSO. A similar trend was seen in two additional patient samples of M5 AML. In summary, the compound H4 promotes differentiation by activation of PKC. Combinatorial treatment with BET inhibition results in stronger promotion of differentiation with reduced expression of MYC. Thus, the combination of PKC agonists and BET inhibitors has therapeutic potential that warrants further studies as a differentiation option for AML.Funding Source: This work was supported by the Swedish Cancer Foundation, the Swedish Research Council, the Swedish Society for Medical Research, and Kamprad foundation.F-2055MOLECULAR CHARACTERIZATION OF T CELL RECEPTOR AND HLA IN T-CELL DERIVED IPSCSSwitalski, Stephanie - Cell Biology Division, Thermo Fisher Scientific, Carlsbad, CA, USA Pradhan, Suman - Cell Biology Division, Thermo Fisher Scientific, Carlsbad, CA, USA Lakshmipathy, Uma - Cell Biology Division, Thermo Fisher Scientific, Carlsbad, CA, USAAs the immunotherapy market continues to grow, significant progress has been made in treating diseases and cancer by leveraging the potential of immune cells, particularly T cells. Autologous CAR T cell therapy has shown high levels of complete remission in treatment of blood-based cancers, but the personalized medicine is hard to scale. Off-the-self, allogenic CAR T treatments derived from donor T cells are being explored as a potential to address issues with the autologous treatments. CAR T cell therapies could be advanced through the use of induced pluripotent stem cells (iPSC). iPSCs derived from T cells offer advantages over traditional somatic cell iPSCs by conservation of T cell receptor (TCR) through reprogramming. Additionally, the ability to engineer chimeric antigen receptors (CAR) into iPSCs, which can then be expanded and differentiated back to T cells, provides a cost-effective method to generate large number of allogeneic CAR-T cells. The therapeutic success of such allogeneic T cell therapies can be further increased with high-resolution HLA typing to reduce the risk of any post-transplant complications. In this study, we utilize qPCR and next generation sequencing tools for the comparison and thorough characterization of TCR and HLA in iPSCs derived from Fibroblast, PBMC and T cells. Since reprogramming is carried out from a heterogeneous population of T cells, detailed phenotyping of the starting donor cells was performed. The parent and resulting iPSC clones that were subjected to comprehensive characterization to confirm pluripotency were used for downstream molecular characterization. To determine the T cell sub types that were reprogrammed, a qPCR assay was developed to assess the germline configuration of iPSCs. Next Generation Sequencing-based Immune Repertoire was used to generate high-throughput sequencing data of TCR populations. These assays revealed the diversity, or the number of unique TCR sequences, present in the starting T cells and the resulting iPSC clones. Further, HLA typing was carried out on the parental cells and iPSC clones to qualify the cells for use in immune therapy applications. The development a robust workflow to characterize the starting immune cell population and the iPSC lines will benefit the development of translational therapies.F-2057GENERATION OF HYPOIMMUNOGENIC HUMAN PLURIPOTENT STEM CELLSMeissner, Torsten B - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Han, Xiao - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Wang, Mengning - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Duan, Songwei - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Franco, Paul - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Kenty, Jennifer - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Hedrick, Preston - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Xia, Yulei - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Allen, Alana - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Ferreira, Leonardo - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Strominger, Jack - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Melton, Doug - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Cowan, Chad - Cardiology, Beth Israel Deaconess Medical Center, Boston, MA, USAPolymorphic human leukocyte antigens (HLA) form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA class Ia and class II in human pluripotent stem cells (hPSCs). Furthermore, to prevent

448POSTER ABSTRACTSinnate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage ‘don’t-eat me’ signal CD47, from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells was minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.Funding Source: This work was supported by awards from the Harvard Stem Cell Institute (HSCI) and the Blavatnik Biomedical Accelerator Program, as well as the Juvenile Diabetes Research Foundation (JDRF).F-2059EPIDERMAL GROWTH FACTOR REJUVENATES AGING MOUSE HEMATOPOIETIC STEM CELLSChang, Vivian - Pediatrics, University of California, Los Angeles, CA, USA Chute, John - Division of Hematology/Oncology, Department of Medicine, University of California, Los Angeles, CA, USA Fang, Tiancheng - Division of Hematology/Oncology, Department of Medicine, University of California, Los Angeles, CA, USA Himburg, Heather - Division of Hematology/Oncology, Department of Medicine, University of California, Los Angeles, CA, USA Pang, Amara - Division of Hematology/Oncology, Department of Medicine, University of California, Los Angeles, CA, USA Pohl, Katherine - Division of Hematology/Oncology, Department of Medicine, University of California, Los Angeles, CA, USA Tran, Evelyn - Division of Hematology/Oncology, Department of Medicine, University of California,Los Angeles, CA, USAAging hematopoietic stem cells (HSCs) display distinct abnormalities such as myeloid skewing, decreased repopulating capacity, and leukemia predisposition. Radiation exposure potentiates aging of the hematopoietic system. Our lab demonstrated that EGF promotes HSC regeneration after radiation. Therefore, we hypothesized that EGFR signaling may rejuvenate aging HSCs. We discovered that aged >18-24 month old C57BL/6 mice have decreased levels of EGF in blood compared to young 2-4 month old mice and decreased expression of EGFR on bone marrow (BM) ckit+sca-1+lin- (KSL) stem/progenitor cells. Aged BM KSL displayed increased DNA damage in culture with thrombopoietin, SCF and Flt-3 ligand (TSF) compared to young KSL while EGF treatment decreased DNA damage in aged BM KSL compared to TSF. Treatment with EGF also increased colony forming capacity of aged BM KSL and increased primary donor cell engraftment compared to TSF. We next tested whether systemic administration of EGF for 4 weeks could alter the hematopoietic characteristics of aged mice. EGF treatment decreased BM myeloid skewing and increased CD3 T cell content in aged mice compared to saline-injected controls. Furthermore, EGF treatment of aged mice increased functional HSCs capable of competitive multilineage engraftment of recipient congenic mice in primary and secondary transplants. We next sought to determine if deficiency in EGFR signaling could accelerate hematopoietic aging in mice by using a doxycycline-inducible, hematopoietic cell specific EGFR dominant negative mutant model (SCL-tTA;EGFR-DN mice). Aged mutant mice displayed increased myeloid skewing and generated significantly decreased colony forming cells, compared to age-matched, EGFR-expressing mice. Mechanistically, BM KSL cells from aged mutant mice demonstrated increased senescence and increased expression of p16 compared to young mice. Additionally, in vivo treatment of aged C57BL/6 with EGF decreased reactive oxygen species level compared to saline, and EGF stimulation of aged BM KSL cells in vitro resulted in decreased phosphorylation of p38. These studies suggest that EGF/EGFR signaling declines with age and that reactivation of EGFR signaling via EGF treatment can ameliorate clinically relevant features of hematopoietic aging, including HSC self-renewal capacity.Funding Source: NHLBI 1K08HL138305 (VYC) UCLA CDI Seed (VYC) NHLBI 2RO1 HL 086998-05 (JPC) CIRM Leadership Award (JPC) NIAID U01AI-107333 (JPC)F-2061THE ROLE OF BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR SWITCHING IN DETERMINING CHROMATIN LANDSCAPE AND T CELL FATE COMMITMENT IN MOUSE EARLY T CELL DEVELOPMENTWang, Xun - Biology and Biological Engineering, Caltech, Pasadena, CA, USA He, Peng - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Williams, Brian - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Ungerback, Jonas - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Mitra, Anik - Biotechnology, Indian Institute of Technology Kharagpur, India Romero-Wolf, Maile - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Wold, Barbara - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Rothenberg, Ellen - Biology and Biological Engineering, Caltech, Pasadena, CA, USAFor hematopoietic precursors establishing their T cell identity in mouse thymus, a key question is how the precise coordination of key transcription factors activates the T lineage program and shuts off the precursor program. To identify the key regulatory changes in a non-biased way, we profiled the global chromatin accessibility changes during the T lineage commitment process using ATAC-seq. Many regulatory elements that became accessible during commitment were highly enriched for E protein heterodimer E2A-HEB binding motifs. Using E protein

449POSTER ABSTRACTSinhibitor ID2 to block the E2A-HEB activity in a committed pro-T cell line, we observed that E2A-HEB is indispensable to maintain the chromatin accessibility of T cell specific elements and to activate their associated T cell genes. Paradoxically, E2A-HEB expression levels are nearly constant throughout the developmental process, and ID2 level is nearly absent. We therefore have tried to identify the mechanism that must be counteracting E2A-HEB activity before commitment, specifically testing the precursor-specific form of E2A heterodimer, E2A-Lyl1, and its cofactor Lmo2. Forced expression of Lmo2 and Lyl1 in primary early committed T cells induces closing of commitment specific sites and loss of T lineage commitment, evident by the downregulation of T cell gene expression, including Bcl11b, Rag1 and Cd3e. More strikingly, it shows dramatic backward chromatin and transcriptome reprogramming to a precursor-like state, with the reopening of precursor-specific sites and the reactivation of many precursor genes including PU. 1, Bcl11a, c-Kit. These results indicate that in precursors, E2A-Lyl1-Lmo2 can not only establish the precursor specific chromatin landscape and activate precursor genes, but can also strongly compete against existing HEB proteins and suppress E2A-HEB activity on initiating T lineage program. Our work suggests that the E2A heterodimer partner switching during early T cell development is crucial in determining T lineage progression through regulating the chromatin landscape both before and after commitment. Our work also suggests new strategies of hematopoietic reprogramming.F-2063INHIBITION OF PROTEIN TYROSINE PHOSPHATASE-SIGMA PROMOTES HEMATOPOIETIC REGENERATION VIA ACTIVATION OF RAC1 SIGNALING PATHWAYZhang, Yurun - Molecular Biology Institute, University of California, Los Angeles, CA, USA Roos, Martina - Division of Hematology/Oncology, University of California Los Angeles, CA, USA Himburg, Heather - Division of Hematology/Oncology, University of California, Los Angeles, CA, USA Li, Michelle - Division of Hematology/Oncology, University of California, Los Angeles, CA, USA Quarmyne, Mamle - Division of Hematology/Oncology, University of California, Los Angeles, CA, USA Fang, Tiancheng - Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA Jung, Michael - Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA McBride, William - Department of Radiation Oncology, University of California, Los Angeles, CA, USA Chute, John - Division of Hematology/Oncology, University of California, Los Angeles, CA, USAReceptor tyrosine kinases, such as c-kit, Flt-3 and Tie2, regulate hematopoietic stem cell (HSC) proliferation, differentiation and maintenance. Substantially less is known regarding the function of protein tyrosine phosphatases (PTPs) in regulating HSC fate. We previously discovered that receptor protein tyrosine phosphatase-sigma (PTP ) was highly expressed by murine σand human HSCs and constitutive deletion of PTP caused a σmarked increase in HSC repopulating capacity in vivo (Quarmyne et al. J Clin Invest 2015). We hypothesized that pharmacologic inhibition could increase HSC repopulating capacity or regenerative capacity following injury. Utilizing a small molecule screen, we identified small molecule, 5483071 (Chembridge), with predicted PTP inhibitory activity. We synthesized a σchemical homologue, DJ001, and demonstrated that DJ001 had strong PTP inhibitory activity in vitro (IC50 = 1.54μM). Systemic σadministration of the PTP inhibitor, DJ001, to irradiated σmice promoted HSC regeneration, accelerated hematologic recovery and improved survival compared to control, irradiated mice. Similarly, following chemotherapy, DJ001 administration accelerated hematologic recovery in mice. DJ001 displayed high specificity for PTP and antagonized PTP via unique σσnon-competitive, allosteric binding. Mechanistically, DJ001 suppressed radiation – induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. DJ001 concordantly induced HSC proliferation after irradiation via RAC1 – dependent induction of CDK2. Treatment of irradiated human HSCs with DJ001 also promoted the regeneration of human HSCs capable of multilineage in vivo repopulation in NOD/SCID IL2 receptor-gamma null (NSG) mice. These studies demonstrate the therapeutic potential of a selective, small molecule PTP inhibitor for human hematopoietic regeneration.σFunding Source: NIH/NIAID: AI-067769 (JPC) CIRM Leadership Award (JPC) CIRM Quest - Discovery Stage Research Award (JPC) UCLA Eli and Edythe Broad Stem Cell Research Center Pre-doctoral Fellowship (YZ)PANCREAS, LIVER, KIDNEYF-2065A DESIGN-OF-EXPERIMENT APPROACH TOWARDS DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS INTO DEFINITIVE KIDNEY CELL LINEAGESWessely, Oliver - Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Tran, Uyen - Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA Bukys, Michael - Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA Curry, Caleb - Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA Nallappan, Akila - Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA Jensen, Jan - Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA