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EXHIBITOR/SPONSORS TRENDBIO - STAND 130 WILEY - STAND 144 Phone: 1300 720 574 1 Fusionopolis Walk Email: [email protected] #07-01 Solaris South Tower www.trendbio.com.au 138628 Singapore +65 6643-8000 TrendBio is a leading supplier of specialised instrumen- tation for the Life Sciences. We are proudly supporting www.wiley.com ISSCR 2018 and will showcase the following products; Wiley, a global company, helps people and organizations Holographic Live Cell Imaging, Maestro MEA Platform, develop the skills and knowledge they need to succeed. Wes, the Fully automated Western Blotting System, ELI- Our online scientific, technical, medical, and scholarly SA, hands-free, single or multi-analyte with no cross-re- journals, combined with our digital learning, assessment activity, Animal Imaging – Optical, CT, Optoacoustic, and certification solutions help universities, societies, PET/SPECT businesses, governments, and individuals increase the academic and professional impact of their work. UNION BIOMETRICA, INC. - STAND 48 84 October Hill Rd. ZHEJIANG HOPSTEM BIOENGINEERING Holliston, MA 01746 COMPANY LIMITED - STAND 133 United States +1 508-893-3115 3F,Build#16,Chitic Sci&Tech Park,No.260, 6th Street, Xia- www.unionbio.com sha, Hangzhou, Zhejiang, China Hangzhou 310018 Union Biometrica Large Particle Flow Cytometers auto- China mate the analysis and sorting of objects that are too big +86 571-88197776 / fragile for traditional cytometers. Examples include www.hopstem.com large cells / cell clusters, cells in/on beads and small model organisms. COPAS and BioSorter models cover Zhejiang Huode (Hopstem) Bioengineering Ltd Co is the full 10-1500um range of particle sizes. A special ro- founded by neuroscience and stem cell scientists from tating horizontal sample chamber is available for intro- Johns Hopkins University on January 2017 in Hangzhou. ducing fragile samples. Hopstem has world leading advantages in human iP- SCs/ESCs neural differentiation, gene editing, cell bank- ing and cell engineering. Hopstem aims to use the most WICELL - STAND 81 cutting edge technologies to promote the diagnoses, 504 S Rosa Rd research and therapies of neurological and other disor- Suite 101 der Madison, WI 53719, U.S.A. +1 (888) 204-1782 www.wicell.org Celebrating 20 years of human pluripotent stem cell re- search. Stop by our booth #81 to join in the celebration and for your chance to win WiCell gear. About WiCell: As a recognized world leader in pluripotent stem cell banking, distribution, and characterization services, Wi- Cell provides the stem cell community with high quality cell lines as well as accurate and reliable characterization testing. Products and Services offered: - Stem Cells (1200+ ES, iPS, disease model and con- trols, modified and GMP compliant cell banks) - Cell Banking and Distribution Services for research- ers and companies generating cell lines - Characterization Testing (G-banded karyotyping, SKY, FISH, SNP microarray, and Identity testing via STR) - Reagent Testing assays customized to meet your needs 99

INNOVATION SHOWCASES including machine learning for smart drug classifi- THURSDAY, 21 JUNE cation, markedly enhancing Novoheart’s capabilities for next-generation drug discovery. APPLIED STEMCELL THERMO FISHER SCIENTIFIC Room 105, Level 1 8:00 – 8:30 Room 212/213, Level 2 HUMAN IPSC-BASED DISEASE MODELING & 8:00 – 8:30 DRUG SCREENING GMP RAW MATERIALS FOR CELL AND GENE THERAPY MANUFACTURING Xianmin Zeng, Scientific Advisory Board, Applied Stem Cell, Inc. United States Kasey Kime, Regulatory Affairs, Clinical and Com- Human induced pluripotent stem cell (iPSC) tech- pliance Manager, Thermo Fisher Scientific, United nology offers the benefits of a cell line coupled with States the advantage of using human primary cells. We Eric Roos, Strategic Alliance Manager, Cell Therapy, have developed a large panel of iPSC lines including Thermo Fisher Scientific, United States isogenic and reporter lines for disease modeling and Have you ever wondered what to consider when you drug discovery. I will discuss the utility of these cells are selecting raw materials for your cell therapy clin- for modeling neurodegenerative disorders including ical research? This presentation will cover regulatory Parkinson’s disease and Alzheimer’s disease, as well requirements for cGMP raw materials and the impor- as neurotoxicity and neuroprotective assays with iP- tance of regulatory documentation and support to SC-derived neurons, glia and their co-culture. enable successful clinical translation. As you move NOVOHEART through clinical trials toward commercialization you’ll need solutions that can scale with you to meet Room 106, Level 1 the clinical need. The second part of this session, 8:00 – 8:30 will address aspects you should consider now such as scalability, consistency of supply and commer- MYHEART™ PLATFORM: UNVEILING cial use rights to help clear your path to commercial NOVOHEART’S NEXT-GENERATION manufacturing in the future. DRUG DISCOVERY TOOLS INCLUDING 3D BIOENGINEERED HUMAN HEART-IN-A-JAR UNION BIOMETRICA Kevin D. Costa, CSO, Novoheart, United States Room 219/220, Level 2 Drug development is a notoriously lengthy, ex- 8:00 – 8:30 pensive and inefficient process, with cardiotoxici- AUTOMATION FOR ANALYSIS, IMAGING, ty being a frequent cause for failure. Conventional AND HANDLING OF CELLS AND CELL non-human cell and animal models are poorly pre- CLUSTERS IN STEM CELL RESEARCH dictive of human responses, leading to false nega- tive and positive results that compromise overall Rock Pulak, Director of Life Science Technologies, success rates. Novoheart’s MyHeart™ Platform aims Union Biometrica, Inc., Holliston to revolutionize this process, offering a hierarchy Cells growing in clusters communicate with each of bioartificial human pluripotent stem cell-derived other and behave differently than cells grown as heart tissue constructs designed as screening tools monolayers or in suspension. These interactions are for predicting cardiotoxicity as well as drug efficacy. likely to be important for proper function. Union Bio- This includes our human ventricular Cardiac Aniso- metrica Large Particle Flow Cytometers automate tropic Sheet (hvCAS) assay for electrophysiology, the analysis, sorting, and dispensing of objects too our human ventricular Cardiac Tissue Strip (hvCTS) big or too fragile for traditional cytometers, some of assay for contractility, and our unique human ven- which are studied by stem cell researchers. Sample tricular Cardiac Organoid Chamber (hvCOC, or types include embryoid bodies, neurospheres and “human heart-in-a-jar”) assay, the only macroscop- other spheroids, and organoids. Flow cytometry ic human cardiac model on the market capable of data, Profiles and brightfield images of the sample pumping fluid, with unprecedented biofidelity mim- constituents are collected for analysis and used to icking the native human heart. We will also introduce make sorting decisions. This technology provides the new MyHeart™ Platform 2.0, which delivers ad- automation for the analysis and handling of these ditional value in terms of throughput, sensitivity and sample types in multiwell plate format and increases accuracy, with hardware and software innovations 100 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

INNOVATION SHOWCASES reproducibility by removing some of the day-to-day ALLEN INSTITUTE FOR CELL SCIENCE variability that can be introduced between research- ers and by the same researcher from one day to the Melbourne Room 1, Level 2 next. 11:30 – 12:30 MAXWELL BIOSYSTEMS PROVIDING STEM CELL & GENE EDITING RESOURCES TO EMPOWER YOUR RESEARCH Room 203/204, Level 2 Ruwanthi Gunawardane, Allen Institute for Cell Sci- 8:00 – 8:30 ence, Seattle, U.S. NOVEL FUNCTIONAL ANALYSIS Allen Institute for Cell Science; providing stem TECHNIQUES FOR ACCURATE PHENOTYPE cell & gene editing resources to empower your CHARACTERIZATION OF HUMAN IPSC- research. DERIVED NEURONS We will share details about our legacy collec- Michele Fiscella, PHD – MaxWell Biosystems / ETH tion of endogenous, fluorescently-tagged hiPS Zurich cell lines highlighting key structures within the iPSC technology enabled in vitro studies with hu- cell. You will hear how these lines were generat- man neurons for investigating disease mechanisms ed, including gene editing strategies, the plas- and for finding treatments. mids used to generate them and the substantial We will present techniques to dissect the functional quality controls performed before making these phenotype of A53T α-synuclein dopaminergic neu- cells and plasmids publicly available. We will rons, modeling Parkinson’s disease. We will discuss share details about our efforts to differentiate how the electrical activity of whole cell networks can these lines to cardiomyocytes, featuring images be monitored label-free and long-term, at high spa- from some of our newer ‘cardio-specific’ lines. tio-temporal resolution. By observing cell networks across multiple days, the capability of iPSC-derived We will touch briefly on the other resources neurons to form synaptic connections can be ana- available on our website (www. allencell.org), lyzed. In addition, we will show how the activity of including our large, high replicate 3D image single neurons can be isolated and studied, together data sets showing the subcellular localization of with subcellular details, such as the propagation of each of our tagged structures, our microscopy action potentials along single axons. pipeline workflow and 3D segmentations. We Our methods are powered by a high-throughput will also discuss machine learning and label-free electrophysiology platform using high-resolution approaches to build integrated and predictive microelectrode array (MEA) technology. The plat- models of cell organization, and will wrap up form allows access to individual cells simultaneously the presentation with a guided tour through through 26’400 electrodes. With this platform, we our website to familiarize you with where all found differences in physiological activity between these resources can be found. the A53T α-synuclein cell line and the isogenic con- trol cell line. Phenotype differences were detected at different scales, ranging from network connectiv- BIOLAMINA ity to subcellular structures as axons. Room 105, Level 1 11:30 – 12:30 CTG BIOLAMININ™ 521 – A BIOLOGICALLY RELEVANT CULTURE MATRIX, ENABLING PRE-CLINICAL RESEARCH PROTOCOLS TO BE TRANSLATED FOR CLINICAL TRIALS Kristian Tryggvason, CEO, BioLamina, Sweden Malin Parmar, Professor, Lund University, Sweden Fredrik Lanner, Karolinska Institute and Karolinska University Hospital, Sweden As a complement to our portfolio of defined and xe- no-free laminin stem cell substrates, we now offer a 101

INNOVATION SHOWCASES cell therapy grade (CTG) Biolaminin 521 cell culture cal analysis and single-cell RNAseq. The integration substrate For Research Use or Non-commercial Man- of single-cell electrophysiology, morphology and ufacturing of Cell, Gene, or Tissue-Based Products. transcriptomics analysis of live human neural cells Biolaminin 521 CTG (CT521) has been developed and enables a bridging of human neurophysiology with manufactured to allow customers to qualify the ma- gene expression. This novel approach allows the terial for use in the manufacturing of cells for clini- identification of neurons in specific functional states cal research. USP Chapter 1043: Ancillary materials and can compensate for variations among cell lines. for cell, gene and tissue-engineered products has been considered in the design of the product. The STEMCELL TECHNOLOGIES product is animal origin component free and has supporting documentation, such as Certificate of Room 203/204, Level 2 Analysis, Animal Origin Free Statement and Bill of 11:30 – 12:30 Material provided with every lot to support regulato- GI TRACT ORGANOIDS: USING ADVANCED ry filings. CT521 is a full-length, human, recombinant TISSUE MODELS TO INTERROGATE laminin 521 substrate, the only one of its kind on the ABSORPTION AND REGULATION market, providing an optimal environment for feed- er-free culture of human PSCs, MSCs and most an- Heather A. McCauley and Ryan K. Conder, STEM- chorage-dependent progenitor cell types. With this CELL Technologies Inc., Vancouver, Canada new clinical grade product, scientists are supported Organoid cultures have redefined the type of bio- throughout their cell therapy development process logical data that can be obtained using in vitro mod- – from concept to commercialized therapy. It recre- els. These techniques enable researchers to main- ates a biologically relevant milieu in vitro, promot- tain and manipulate cells that recapitulate many of ing high survival and robust expansion of hPSCs, the intra- and intercellular characteristics of their and subsequent cell lineage specification. The cells specific tissue-of-interest, including disease phe- grow in a homogeneous monolayer, easy to monitor. notypes. We have developed specialized, robust The substrate is flexible and compliant with any cell organoid culture kits that reduce the variability in culture medium. It allows an operator-independent these systems and make organoids a more accessi- culture maintenance and reliable, standardized pro- ble research tool. Enteroendocrine cells (EECs) are tocols which can easily be adapted to automation gastrointestinal nutrient-sensing cells that secrete platforms. hormones in response to nutrient ingestion. While CDI EECs only comprise 1-2% of the intestinal epitheli- um, they are essential regulators of nutrient absorp- Room 106, Level 1 tion. The mechanisms underlying how EECs control 11:30 – 12:30 this process, however, are poorly understood. Using CRISPR/Cas9, we introduced a specific mutation in NOVEL APPLICATIONS OF HUMAN human pluripotent stem cells to generate human in- IPSC-DERIVED NEURONS: FROM HIGH- testinal organoids lacking EECs. This organoid mod- THROUGHPUT SCREENING TO PATCH-SEQ el system allowed us to investigate how EECs couple ANALYSIS an epithelial-neurohormonal signal with nutrient and ion transport to regulate nutrient absorption. Gener- Anne Bang, Director, Cell Biology, Sanford Burn- ation of EEC-free intestinal organoids also provided ham Prebys Medical Discovery Institute insight on the potential role for ECCs in regulating Cedric Bardy, Assistant Professor - South Austra- intestinal cell differentiation lian Health and Medical Research Institute (SAHMRI Mind & Brain) Dr. Anne Bang will discuss the use of hiPSC-derived neurons, including iCell® GABANeurons and iCell DopaNeurons, in high-throughput assays for pheno- typic analyses and drug screening. She will describe the development of a suite of foundational assays in higher throughput formats to monitor neuronal morphology, mitochondrial function, and electro- physiology, and address the challenges of balancing higher throughput with relevance. Dr. Cedric Bardy will discuss patch-seq characterization of hiPSC-de- rived neurons with electrophysiology, morphologi- 102 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

INNOVATION SHOWCASES THERMO FISHER SCIENTIFIC characterize disease-relevant neuronal subtypes, in- cluding motor neurons, dopaminergic neurons, and Room 212/213, Level 2 cortical neurons made from pluripotent cells engi- 11:30 – 12:30 neered to express cell-type specific fluorescent re- FROM BENCH TO BEDSIDE: GENERATION porters. These studies utilize live-cell imaging anal- ysis to determine the cellular changes that underlie OF IPSCS FOR CELL THERAPY AND DISEASE neurodegenerative diseases and to design and per- MODELING APPLICATIONS form new types of drug screens. Measurements of Kapil Bharti, Earl Stadtman Tenure-Track Investiga- neuronal properties, such as soma size and neurite tor, National Eye Institute; Adjunct Group Leader, growth, can be paired with single cell tracking for National Center for Advancing Translational Scienc- prolonged time courses (up to weeks). These stud- es, National Institutes of Health ies have revealed some of the earliest morphological changes that foretell impending death. Further, we David Piper, Director, Research and Development, have identified properties of degenerating neurons Thermo Fisher Scientific that determine whether or not they can be rescued. Today, iPSC are most prominently used in disease These types of analyses provide the basis for a phys- modeling and the most promising iPSC application iologically relevant human neuron-based approach of tomorrow is the emerging area of iPSC-derived to finding new treatments for neurodegenerative cell therapies. In this session, Dr. Kapil Bharti will diseases. discuss his journey to the clinic and Dr. David Piper will review new methods to facilitate disease mod- BIO-TECHNE eling. Dr. Kapil Bharti will discuss the preparation of a phase I clinical trial using iPS cell derived oc- Room: Melbourne Room 2, Level 2 ular tissue to treat age-related macular degenera- 11:30 – 12:30 tion (AMD). Combining efforts in developmental bi- UNFOLDING ORGANOIDS: A NEW PLATFORM ology and tissue engineering, Dr. Bharti will review FOR GENERATING ACCESSIBLE 3-D the development of a clinical-grade iPS cell derived EPITHELIAL ORGAN TISSUE RPE-patch on a biodegradable scaffold for poten- tial transplantation of an autologous iPS cell therapy. Scott Schachtele and Fabrizio Rinaldi, Bio-Techne, Dr. David Piper will review the use of iPSCs to cre- Minneapolis, U.S. ate diseased and disease-corrected lines for poten- Three dimensional (3-D) cell culture models are tial use in screening and for clinical research. In his quickly being adopted for toxicology, drug discov- talk he will delve into methods developed to over- ery, and disease modeling. These models, including come current bottlenecks in delivery, identification, organoids, experience difficulties with variability, selection and subsequent clonal outgrowth. These tissue viability, and experimental accessibility. Over- optimizations facilitate consistent and reliable gene coming these obstacles is paramount for the logis- knock-out and knock-in to create engineered iPSC tical incorporation of 3-D tissues into high through- lines resulting in isogenic disease model cell lines put toxicity and disease modeling workflows. In that can be differentiated into the cell type of inter- this showcase we introduce MimEX™ Tissue Model est for downstream applications. Systems, a new technology for the generation of NIKON sustainable and accessible 3-D human organ tissue. Using MimEX GI, a gastrointestinal model system, Room 219/220, Level 2 we demonstrate the principle and benefits of the 11:30 – 12:30 technology, including methods for the efficient iso- lation, expansion, and differentiation of adult human QUANTIFYING NEURODEGENERATION “ground-state” stem cells from the gastrointestinal USING LIVE CELL IMAGING epithelium. The second part of the showcase focus- es on the flexibility of MimEX Tissue Model Systems, Lee L. Rubin, Professor and Director of Transla- tional Medicine Harvard University and the Harvard including demonstrating how the technology can be used to generate 3-D models of other epithelial-de- Stem Cell Institute, Boston, U.S. rived organ tissues. In addition, we show that adult Live cell imaging of specialized cell types derived epithelial stem cells can be isolated from pathogenic from stem cells holds much promise as a new ap- tissue and will differentiate back into the diseased proach to drug discovery and personalized medi- tissue in vitro, yielding a new method for modeling cine. In a long-term collaboration with Nikon Cor- human disease. poration, we have been using the BioStation CT to 103

INNOVATION SHOWCASES LONZA AND GUEST FROM SEMMA FRIDAY, 22 JUNE THERAPEUTICS STEMBIOSYS Room 106, Level 1 Room 219/220, Level 2 11:30 – 12:30 8:00 – 8:30 INDUSTRIALIZATION OF CELL & GENE THERE IS NO PLACE LIKE HOME! CELL THERAPY MANUFACTURING- FROM DERIVED MATRICES THE NEXT EVOLUTION CONCEPT TO PATIENTS IN CELL CULTURE Thomas Fellner, Head of Cell & Gene Therapy, Sy Griffey, StemBioSys Inc. Lonza Pharma & Biotech Julie Carson, Principal Scientist, Semma Therapeu- In standard cell culture procedures, cells are first ex- tracted from their native tissue and then placed in a tics foreign environment (polystyrene, collagen coated The cell and gene therapy market has experienced an dish, etc). Cells react to this by altering their gene immense growth driven by the recent landmark ap- expression to construct a provisional matrix or dif- provals of therapies such as Novartis’ Kymriah, Kite/ ferentiate based on the signals (or lack thereof) Gilead’s Yescarta and Spark’s Luxturna. A pipeline from this new environment. filled with promising pre-clinical and clinical can- didates provides confidence that the cell and gene StemBioSys has developed technologies and meth- therapy field is becoming a key player in the phar- ods to produce a natural cell culture substrate that will revolutionize cell culture methods. The CELLvo™ maceutical world. While the products are showing remarkable therapeutic efficacy, there is an emerg- Matrix is a cell-constructed extracellular matrix that provides cells with a natural microenvironment and ing need for flexible and robust manufacturing plat- forms capable of both scaling up and scaling out, substrate for attachment and growth. Cells grown on an intact cell derived ECM proliferate more quick- while maintaining control over the cell processing parameters to achieve consistent product of highest ly, maintain a phenotype more consistent with that expressed while in their native environment and are quality and safety. Further production of these ther- apies at a large scale will be key to bringing these more responsive than cells grown on other sub- strates. We will discuss our matrix technology as therapies to patients globally. Production costs of cell and gene therapies remain another obstacle the well as several important potential applications in tissue engineering and regenerative medicine. field has been facing; this is mostly driven by manu- al processing, manufacturing footprint requirements THERMO FISHER SCIENTIFIC and expensive raw materials and testing. Automa- tion represents itself as a potential solution for at Room 105, Level 1 least some of these challenges and therefore driv- 11:30 – 12:30 ing commercial viability of these therapies. During VALIDATING ANTIBODIES TO DISTINGUISH this session we will outline the specific challenges development and manufacturing of these ground BETWEEN NAIVE AND PRIMED HPSCS breaking therapies are facing and how innovative Andrew Laslett, CSIRO Manufacturing, Clayton, platforms may help overcome some of the issues Victoria, Australia faced by the industry. This session will discuss the validation and charac- STEMCELL TECHNOLOGIES terisation of a panel of new monoclonal antibodies to defined cell surface proteins found on human plu- Room 203/204, Level 2 ripotent stem cells. This panel of antibodies is use- 11:30 – 12:30 ful for the detection and enrichment of human plu- HUMAN PLURIPOTENT STEM CELL QUALITY: ripotent stem cells and can be used to distinguish ESSENTIAL CONSIDERATIONS FOR GENE between naive and primed human pluripotent stem cells. Additionally, the session will cover the Thermo EDITING, CLONING, MAINTENANCE AND Fisher Scientific Antibody Validation Initiative and DISEASE MODELING using data show the value of using well-validated Adam Hirst and Vivian Lee, STEMCELL Technolo- monoclonal antibodies. gies Inc., Vancouver, Canada Human pluripotent stem cells (hPSCs) hold tremen- dous promise for a wide range of applications, in- 104 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

INNOVATION SHOWCASES cluding regenerative medicine, disease modeling, BIOLOGICAL INDUSTRIES drug discovery and toxicology. Recently, there has been a dramatic increase in the generation of ge- Room 212/213, Level 2 nome-edited hPSC lines due to widespread use of 11:30 – 12:30 the CRISPR-Cas9 technology. As a result, research- THE PROMISE OF INDUCED PLURIPOTENT ers are becoming more aware of the critical impor- tance of cell quality for generating robust and rele- STEM CELLS: BASIC RESEARCH AND vant data. This seminar will focus on how to maintain CLINICAL GRADE MANUFACTURING and assess high-quality hPSC cultures throughout Micha Drukker, Leader of the junior research group the various stages of your research with an empha- Human Pluripotent Stem Cell Lineage-Choice Re- sis on five critical quality control aspects: genomic search and the Human Induced Pluripotent Stem integrity, pluripotency, gene and marker expression, Cell Unit at the Institute of Stem Cell Research, the epigenetic landscape and culture morphology. Helmholtz Zentrum München Speakers will also discuss the ArciTect™ family of products for ribonucleoprotein-based CRISPR-Cas9 Achia Urbach, Institute of Nanotechnology and Ad- genome editing, providing you with a rapid, flexible vanced Materials, Bar-Ilan University and precise protocol to modify the hPSC genome David Fiorentini, VP of Scientific Affairs, Biological for various applications. Finally, methods to model Industries neurological disease with cerebral organoids de- Pluripotent Stem cells research has generated tre- rived from high-quality hPSCs will be discussed. mendous excitement, and holds promise for the MINERVA BIOTECHNOLOGIES treatment of many uncured diseases. Early stage clinical trials using differentiated induced plurip- Melbourne Room 1, Level 2 otent stem cells (iPS) show great potential in cell- 11:30 – 12:30 based therapeutics. Bridging the gap between re- search models and clinical applications is the utmost THE IMPACT OF VARIOUS STEM goal to ensure clinically relevant stem cell products. CELL GROWTH MEDIA ON LINEAGE The first part of this innovation showcase will pres- DETERMINATION ent a research work on SNF5 , which is one of the core subunits of the SWI/SNF chromatin-remodel- Cynthia Bamdad, CEO of Minerva Biotechnologies ing complex. Mutations in this protein might have Minerva and end-users will present studies using Al- significant effects on the epigenetic state of human phaSTEM® System, which uses a serum-free single embryonic stem cells and on their phenotype and growth factor (NME7AB) media, an antibody adhe- precise levels of SNF5 are required in order to pre- sion surface and a synthetic peptide that breaks the serve the pluripotent state of the cells. The second pluripotency interaction and induces differentiation, part will focus on the manufacturing of clinical grade thus eliminating risk of teratoma. human iPS under cGMP conditions as a critical part Side-by-side comparisons of AlphaSTEM® to mTeSR of the translation from research to clinical therapeu- and E8 for differentiation of iPSCs to hepatocytes, tics. The innovation showcase will address the chal- neural progenitors, MSCs and cardiomyocytes will lenges of the generation, expansion and storage of be presented. Comparisons will include ease of use, iPS cells, including tissue biopsies, xeno-free culture yield and quality of the differentiated cells. A dis- procedures and raw materials, testing and cryopres- cussion of the effect of stem cell media on manu- ervation. facturability, scale-up, and cost of stem cell derived therapeutics will follow. Lunch and discount coupons provided. 105

INNOVATION SHOWCASES MILTENYI IRVINE SCIENTIFIC Room 219/220, Level 2 Room: Melbourne Room 2, Level 2 11:30 – 12:30 11:30 – 12:30 TOWARDS A THERAPY FOR PARKINSON’S IMPORTANCE OF USING SERUM-FREE MEDIA DISEASE: LATEST RESEARCH HIGHLIGHTS IN THE CELL THERAPY FIELD AND CONCEPTS FOR MANUFACTURING OF Vanda S. Lopes, Senior Scientist, Irvine Scientific ATMPS Cell therapy is a growing area that focuses on the Malin Parmar, Senior Project Manager R&D Stem application of cells as the therapeutic product, and Cells, Miltenyi where cell culture is the manufacturing process. Sebastian Knöbel, Biotec GmbH, Bergisch Glad- Currently the main challenges in this approach re- bach side in the generation of enough cell numbers, with consistent clinical quality. While this may seem rath- Cell therapy for Parkinson’s disease (PD) based on pluripotent stem cell (PSC) derived products is ap- er straightforward, cell culture is a process that in- volves a large number of interlinked variables, such proaching clinical trials. Malin Parmar will give an up- date on latest developments on her road to the clin- as cell quality and culture media. In an attempt to move towards a consistent manufacturing process, ic aiming at the first in man study for PD in Europe using ES derived dopaminergic progenitors. This will the removal of undefined components is critical. In here we will address the importance of using se- include GMP manufacturing and cryopreservation of the cells, as well as a discussion of the key pre-clin- rum-free media in the development of cell therapy. We will also introduce some alternative products for ical in vivo assays for safety and efficacy. Sebastian Knöbel will highlight recent developments towards the culture of several cell types (HSC, T Cell and NK cells) that can help in addressing some of the chal- an automated manufacturing platform for ATMPs, the CliniMACS Prodigy. A closed system Tubing Set lenges in translational cell applications. and software package adapted for adherent cells together with high quality QC and cell culture re- agents constitute major advances for development and scaling of cell manufacturing processes for a variety of applications. Besides PSCs expansion and DA differentiation, MSC isolation from bone marrow plus subsequent cultivation will be presented. Final- ly, cells sorting using the GMP compliant cell sorter MACSQuant Tyto will be discussed in the context of ATMPs. 106 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 107

STEM CELL REPORTS EDITOR-IN-CHIEF Christine Mummery, PhD, Leiden University Medical Center OPEN ACCESS Stem Cell Reports offers rapid consideration of your research At Stem Cell Reports our editorial team provides you with fast and fair consideration of your research. Read some of the most recently published research: • Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs • Directed Differentiation of Human Pluripotent Stem Cells to Microglia • Fetal Therapy Model of Myelomeningocele with Three-Dimensional Skin Using Amniotic Fluid Cell- Derived Induced Pluripotent Stem Cells • Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells • RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis • Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing • Responsible Translation of Stem Cell Research: An Assessment of Clinical Trial Registration and Publications For more information visit cell.com/stem-cell-reports INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH 108 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 14:20 – 14:45 WEDNESDAY, 20 JUNE, 13:00 – 15:15 EPITHELIAL STEM CELLS AND REGENERATIVE MEDICINE PLENARY I: PRESIDENTIAL SYMPOSIUM 1 3 2 Plenary Room, Ground Level Pellegrini, Graziella , Rama, Paolo , Hirsch, Tobias , Rothoeft, Tobias , Teig, Nortbert , Bauer, Johann and 4 5 4 Sponsored by Fate Therapeutics De Luca, Michele 6 1 University of Modena and Reggio Emilia, Modena, 13:30 – 13:55 Italy, Cornea and Ocular Surface Unit, San Raffaele 2 INTEGRATED CONTROL OF STEM CELL Scientific Institute, Milan, Italy, Department of 3 ACTIVITY IN PLANTS Plastic Surgery, Burn Centre, BG University Hospital Bergmannsheil, Ruhr University Bochum, Germany, Scheres, Ben, Chakrabortty, Bandan, Mulder, Bela, ten 4 Department of Neonatology and Pediatric Tusscher, Kirsten and Mahonen, Ari-Pekka Intensive Care, University Children’s Hospital, Ruhr Wageningen University and Research, Wageningen, University Bochum, Germany, EB House Austria and 5 Netherlands Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, Plants grow continuously throughout their life cycle and 6 Centre for Regenerative Medicine, University of ‘behave’ by adaptation of developmental programs to the environment. Growth is fuelled by somatic stem cells that Modena and Reggio Emilia, Modena, Italy reside in stem cell niches. The specification of stem cell Regenerative medicine has generated many efforts to niches is dynamic and depends on signals, of which the explore new therapeutic potentials of both somatic and indolic small molecule auxin is the most important, that pluripotent stem cells with many possibilities envisaged are positioned by continuous interactions among many for therapeutic applications. Hematopoietic and epithe- cells. The same signals regulate rapid responses of growth lial cells are extensively adopted for tissue regeneration, to the environment. We have investigated how signal ac- due to their high proliferative capacity and their acces- cumulation leads to the activation of a small suite of tran- sibility. 30 years ago, the method for producing epider- scription factors that together maintain the multipotent mis was discovered by cultivation from a small skin biop- state, and how a gradient of these transcription factors sy, allowing life-saving treatment of thousands severely regulates the transition from stem cell to differentiation burned patients, in the following years. The importance states. We show how this gradient separates develop- of stem cell content was proven for tissues or organs in mental and environmental responses in growth regions to different pathologies. For instance, recent developments the fluctuating auxin signal that is upstream of both. Final- in cell-based therapy for ocular burns provided support ly, we provide evidence how control of the direction of cell for improvement and standardization of the cure for this division, which is of key importance for the formation of disabling disease, causing depletion of limbal stem cells. plant tissues, is connected to the auxin signalling process. Indeed, biopsies taken from the healthy eye, or other au- tologous source as oral mucosa in bilateral blindness, can 13:55 – 14:20 be used for their content of stem cells. The combined use COORDINATING TISSUES DURING AXOLOTL of cell and gene therapy represents a further scientific LIMB REGENERATION approach for the treatment of congenital diseases. This approach has recently been established using genetical- Tanaka, Elly ly modified epidermal cells for life-saving treatment on Institute of Molecular Pathology, Vienna, Austria severe genetic diseases, as epidermolysis bullosa. Gene therapy, cell therapy, and tissue engineering have the po- Regrowth of a severed limb in the axolotl is a spectac- tential to revolutionize the treatment of disease and inju- ular example of regeneration—the remaining portion of ry.  Attaining marketing authorization for such Advanced the limb is able to activate stem cells so that the replace Therapy Medicinal Products (ATMPs) requires a rigorous the appropriate part of the limb, and rebuild the complex scientific evaluation by the European Medicines Agen- architecture of the different, interacting cells types. This cy - authorization is only granted if the product can fulfil process occurs in three morphologically and molecular- stringent requirements for quality, safety and efficacy.  Ex ly definable stages, wound-healing, blastemal formation, vivo expanded autologous human corneal epithelium, a and patterning. I will talk about our work that has iden- novel treatment for eye burns, is one of the few ATMPs, tified wound signals that initiate regeneration, and how to have been granted marketing authorization and is the the connective tissue cells orchestrate the building of the first claiming a specific amount of stem cells.  This pre- blastema and the patterning of the regenerate. A stable sentation highlights the peculiarities of EU rule compliant positional code is maintained in the adult limb that is cen- medicinal products, and specifically discusses how the tral for controlling the amputation specificity and con- manufacture had to be updated to achieve authorization.  trolling that the correct parts of the limb are produced. The result is that patients will have access to a therapy that is manufactured to high commercial standards, and is supported by robust clinical safety and efficacy data. 109

WEDNESDAY 20 JUNE 2018 14:50 – 15:10 bust capacity both for spermatogenesis and oogenesis. THE ISSCR TOBIAS AWARD LECTURE: We have also shown that human iPSCs (hiPSCs) with a primed pluripotency robustly generates human PGCLCs A PROSPECTIVE ANALYSIS OF HUMAN (hPGCLCs) with a property of human early PGCs. More- LEUKEMOGENESIS over, by investigating the development of cynomolgus Eaves, Connie J. monkeys, we have defined a developmental coordinate of Terry Fox Laboratory, BC Cancer Agency, Vancouver, the spectrum of pluripotency among mice, monkeys, and humans, and have made an unexpected finding that the BC, Canada germ cell lineage in primates is specified in the amnion. I would here discuss our efforts towards understanding Targeting early events in the leukemogenic process has the mechanism of and reconstituting in vitro of germ cell been a long sought strategy to more effectively treat and development in mice, monkeys, and humans. perhaps eventually prevent human leukemias. To this end, the increasing resolution and decreasing cost of genomic sequencing has enabled a large spectrum of acquired mu- 16:30 – 16:55 tations associated with this process to be identified from PANCREAS ORGANOIDS TO DECONSTRUCT analyses of patients’ samples. The sequence of acquisition NORMAL AND PERTURBED DEVELOPMENTAL of mutations in these cells and their subclonal evolution MECHANISMS IN MOUSE AND HUMAN can then be retrospectively inferred from analyses of the representation of each mutation in the sample. Draw- Grapin-Botton, Anne, Gonçalves, Carla, Figueiredo- backs of this approach are the limitations in the size and Larsen, Manuel, Yennek, Siham, Nakamura, Akino, site of the samples obtainable, their genomic diversity Azad, Ajuna, Larsen, Michael, Beydag-Tasöz, Belin both between and within patients, and the contributions Selcen, Borup Kjær Petersen, Maja, Ramond, Cyrille, of non-genomic mechanisms to the altered properties Kim, Yung Hae, Honoré, Christian and Scharfmann, of the cells. An alternative approach is to use protocols Raphael that allow the process of leukemogenesis to be tracked DanStem, University of Copenhagen, Denmark prospectively. However, until recently, this was difficult to achieve de novo starting from primary sources of human Organoids representing a diversity of tissues have re- hematopoietic cells. As a result, most studies have relied cently flourished, bridging the gap between cell lines or on the use of mouse models or immortalized cell lines to primary cells grown on the bottom of culture plates and generate transformed hematopoietic cells, despite the experiments performed in vivo. Being small and amena- recognition that these poorly recapitulate the leukemias ble to continuous monitoring they offer the opportunity that appear in patients. This presentation will summarize a to scrutinize the dynamics of organ development, which new description of the diversity of primitive human hema- includes the exciting prospect of observing aspects of hu- topoietic cell states that are potential targets for leukemic man embryo development live. Though not recapitulating transformation, their ability to undergo this process, and all facets of physiology, these miniature organs generated factors that affect it. in a dish are simpler than the whole organ and offer an opportunity to manipulate culture conditions in isolation from the rest of the embryo and from the mother. Their WEDNESDAY, 20 JUNE, 16:00 – 18:00 ability to self-organize, that is to differentiate and orga- nize cells in space, calls for the identification of the simple rules that underlie this capacity. We initially established PLENARY II: RECAPITULATING 3D culture conditions that enable the efficient expansion DEVELOPMENT FROM STEM CELLS of dissociated mouse embryonic pancreatic progenitors. Plenary Room, Ground Level With this system, we revealed that pancreas progenitors behave differently in 2D and 3D and that they are sen- sitive to the stiffness and nature of the 3D environment. 16:05 – 16:30 Two media compositions were established that unfold dif- MECHANISM AND RECONSTITUTION IN VITRO ferent responses in progenitors. A first medium balances OF GERM CELL DEVELOPMENT IN MICE, long lasting progenitor expansion and endocrine cell pro- MONKEYS, AND HUMANS duction in spheres. A second medium balances progeni- tor expansion and acinar cell production and enables the Saitou, Mitinori formation of a branched network of ducts, in a manner Kyoto University, Kyoto, Japan and timing similar to the developmental process. Focus- ing on the initial conditions leading to these organoids, The germ cell lineage ensures the creation of new individ- we observed that the organoids formed if enough cells uals, perpetuating/diversifying the genetic and epigenetic were clustered and identified a cooperative community information across the generations. We have been inves- effect. Assembling defined numbers of Notch active and tigating the mechanism for germ cell development, and inactive cells shows that their interaction is needed to ini- have shown that mouse embryonic stem cells (mESCs)/ tiate organoid formation and fuel growth. We also used induced pluripotent stem cells (miPSCs) are induced this model to investigate how the branched structure of into primordial germ cell-like cells (mPGCLCs) with a ro- the pancreatic ducts emerges from the initial small cell 110 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS aggregates. Developing the model to study human devel- 17:20 – 17:30 opment and model disease, we will present recent data showing the robust expansion, differentiation and mor- POSTER TEASERS phogenesis of human pancreatic spheres and organoids derived from embryonic stem cells. Benchmarking these W-2064 systems to human embryos enables us to assess their rel- evance and their use to model neonatal diabetes. HETEROCHROMATIN CONDENSATION IS MEDIATED BY JMJD1A AND JMJD2C DURING 16:55 – 17:20 PHYSILOGICAL STEM CELL AGING VASCULAR NICHE ANGIOCRINE SIGNALS Jiang, Xiaohua (Cynthia) DICTATE ORGANOTYPIC STEM CELL The Chinese University of Hong Kong, Hong Kong REGENERATION W-3069 Rafii, Shahin INTEGRATIVE MOLECULAR ANALYSES Cornell Medical College and Angiocrine Bioscience, REVEAL DISTINCT REPROGRAMMING New York, NY, U.S. TRAJECTORIES INTO STATES OF NAIVE AND Stem cell self-renewal and fate determination require PRIMED HUMAN INDUCED PLURIPOTENCY? niche-derived signals. However, the source of the niche Liu, Xiaodong cells and signals that regulate regeneration are unknown. Monash University, Australia Tissue-specific endothelial cells (ECs) by oscillating the ultradian production of stimulatory and inhibitory angio- W-1081 crine factors establish an instructive vascular niche that choreographs organ regeneration and stem cell self-re- THE ROLE OF TRANSLATION IN HUMAN newal, such as hematopoietic stem cells (HSCs). To un- KERATINOCYTE CELL FATE DETERMINATION cover the mechanism by which ECs regulate stem cell Lewicka, Aleksandra homeostasis, we have devised a tissue-specific vascular University of Cambridge, U.K. niche platform for expansion of HSCs and for generating vascularized tissue-specific 3D organoids. Co-culture of W-1020 adult marrow-derived mouse or human HSCs with ECs re- sults in > 50 fold clonal HSC self-renewal with long-term MOLECULAR MECHANISMS REGULATING multi-lineage engraftment potential. Vascular niche cells MUSCLE STEM CELLS QUIESCENCE AND also specify pluripotent-independent conversion of readily EARLY ACTIVATION accessible adult ECs into engraftable HSCs. To prove this, we transduced human or mouse adult mature ECs with Relaix, Frederic Runx1 /Spi1/Gfi1/FosB transcription factors along with INSERM U955-E10 IMRB, Faculté de médecine UPEC, vascular niche-induction enabling step-wise conversion of France ECs into immunocompetent HSCs. Clonal populations of converted HSCs expanded on vascular niche and recon- W-1100 stituted hematopoiesis in rodents. Co-infusion of the ECs A METHOD TO ISOLATE AND TRANSPLANT along with HSCs augmented hematopoietic recovery. To MOUSE HEMATOPOIETIC STEM CELLS ALONG translate the potential of vascular niche to clinic, we have WITH THEIR NICHE ALLOWING FUNCTIONAL engineered ECs capable of vascularizing epithelial, hepat- ic, pancreatic, neural and cardiac 3D organoid cultures. HEMATOPOIETIC STEM CELL ENGRAFTMENT Cross talk of ECs with tissue-specific stem cells promotes WITHOUT MYELOABLATION proper patterning of organoids into functional tissues. We Borrelli, Mimi R. show that transplantation of ECs stimulates organ repair Department of Plastic and Reconstructive Surgery, without provoking maladapted fibrosis. We have acquired Stanford University, U.S. an IND approval from FDA to perform the First-In-Human co-transplantation of HSCs with vascular niche cells to ac- celerate hematopoietic recovery. These clinical trials will set the stage for reconstructing and remodeling vascular niche in vivo for treatment of acquired, inherited, and ma- lignant stem cell disorders. Tissue-specific vascular-stem cell organoid cultures facilitate screening by gene-editing and small molecule libraries to identify unknown vascular niche signals that coordinate stem cell self-renewal and differentiation for functional organ repair. 111

WEDNESDAY 20 JUNE 2018 17:30 – 17:55 THE ANNE MCLAREN MEMORIAL LECTURE: DEVELOPMENTAL TRAJECTORY OF THE MOUSE EPIBLAST DEFINES THE DEVELOPMENTAL CORRELATE OF EMBRYO-DERIVED PLURIPOTENT STEM CELLS Tam, Patrick P.L Children’s Medical Research Institute, Westmead, NSW, Australia The basic body plan of the embryo is visualised by the regionalization of cell fates in the primary germ layers during gastrulation. The collation of stage-wise fate maps enables the reconstruction of the developmental trajec- tory of lineage differentiation of cells in the germ layers. Transcriptome analysis of the mouse embryo across the developmental stages from pre-gastrulation to late gas- trulation has defined the developmental progression and provides the molecular annotation of the genealogy and the transcriptional and signalling activity of spatially reg- istered sub-populations of cells in the germ layer. The developmental-spatial transcriptome presents a refined perspective of the developmental trajectory and lineage relationship of the multipotent tissue progenitors during germ layer differentiation. In relevance to the biology of stem cells, the transcriptome knowledge provides the developmental correlates for tracking the state of plurip- otency, the embryological counterpart and the lineage trajectory of the embryo-derived pluripotent stem cells and the molecular activity underpinning the acquisition of lineage propensity, and for the rationalization of the methodology of directed differentiation. 112 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 09:40 – 10:05 THURSDAY, 21 JUNE, 09:00 – 11:10 SINGLE CELL ANALYSIS OF HUMAN PREIMPLANTATION EMBRYOS AND PLENARY III: SYSTEMS BIOLOGY OF PLURIPOTENT CELLS HETEROGENEITY Plenary Room, Ground Level Lanner, Fredrik Karolinska Institute, Stockholm, Sweden 09:15 – 09:40 Our research focuses on understanding how pluripotency STEM CELL HETEROGENEITY IN THE ADULT is established and controlled in the human embryo. We have HIPPOCAMPUS recently undertaken two approaches to explore how the pluripotent cells emerge in the human embryo using sin- Guillemot, Francois, Blomfield, Isabelle, Harris, gle cell RNA sequencing and developed new tools to track Lachlan and Urban, Noelia naïve and primed pluripotent cells. These data show that The Francis Crick Institute, London, UK pluripotent epiblast cells emerge coincidently with blasto- cyst formation. The data further suggest that female cells Stem cells in the hippocampus of the adult brain produce undergo X-chromosome dosage compensation prior to neurones that have important functions in memory and implantation. In contrast to the mouse, XIST is transcribed mood control. Most adult hippocampal stem cells are from both alleles throughout the progression of this ex- quiescent while a small fraction proliferate and produce pression dampening, and X-chromosome genes maintain neurones in response to various physiological stimuli or biallelic expression while dosage compensation proceeds. to injury. How stem cells compute the diverse stimuli and In vitro, pluripotent cells are thought to exist in naïve and downstream niche signals they receive to produce ap- primed states, and provide important models to investi- propriate numbers of adult neurones remains an open gate the earliest stages of human development. Naïve cells question. We found that the transcription factor Ascl1 is can be obtained through primed-to-naïve resetting, how- essential for activation of hippocampal stem cell. We also ever, there are no reliable methods to prospectively isolate obtained evidence that Ascl1 expression is controlled by unmodified naïve cells during this process. Comprehen- different post-translational mechanisms at different stages sive profiling of cell-surface proteins by flow cytometry in the hippocampal stem cell lineage. Ascl1 is transcribed in naïve and primed pluripotent cells. Several naïve-spe- in most quiescent stem cells but Ascl1 protein accumu- cific, but not primed-specific, proteins were expressed by lation in these cells is suppressed by the transcriptional pluripotent cells in the human preimplantation embryo. repressor Id4, via sequestration of Ascl1 dimerisation part- Naïve-specific cell-surface proteins were induced during ner and degradation of monomeric Ascl1. Ascl1 protein is primed-to-naïve resetting and enabled the isolation and also actively degraded in proliferating hippocampal stem characterization of live naïve cells and intermediate cell cells, by a different mechanisms involving the E3 ubiquitin populations. This analysis revealed distinct transcriptional ligase Huwe1. We are characterising the niche signals that and X-chromosome inactivation changes associated with controlling Ascl1 protein levels via regulation of Id4 and early and late stages of naïve cell formation. Thus, identi- Huwe1. Further investigation of Huwe1 function in prolifer- fication of state-specific proteins provides a robust set of ating hippocampal stem cells has shown that active elim- molecular markers to unambiguously define human plu- ination of the pro-activation factor Ascl1 is essential for a ripotant state, and allows new insights into the molecular fraction of these cells to return to quiescence. Moreover, events leading to naïve cell resetting. examination of Huwe1 function in mice of different ages has revealed that stem cells that have previously prolif- 10:05 – 10:30 erated and have returned to quiescence (which we call ‘resting stem cells’) have a unique role in maintaining ho- THE HEMATOPOIETIC AND ERYTHROID meostatic hippocampal neurogenesis. In contrast, stem HIERARCHIES THROUGH THE LENS OF SINGLE cells that have not previously proliferated (‘dormant stem CELL TRANSCRIPTOMICS cells’) have a limited role in homeostatic neurogenesis, Socolovsky, Merav, Tusi, Betsabeh, Wolock, Sam, suggesting they may serve as a reserve stem cell popu- lation. We are currently investigating whether resting and Weinreb, Caleb, Hwang, Yung and Hidalgo, Daniel dormant stem cell populations are differentially activated Department of Molecular, Cell and Cancer Biology, by niche signals and by physiological neurogenic and in- University of Massachusetts Medical School, jury stimuli. Worcester, MA, U.S. The formation of red blood cells begins with the differ- entiation of multipotent haematopoietic progenitors. Re- constructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snap- shots, to demonstrate that mouse hematopoietic pro- 113

THURSDAY 21 JUNE 2018 genitors differentiate through a continuous, hierarchical T-2003 structure into seven blood lineages. We uncovered cou- CLONAL LINEAGE TRACING AND SINGLE pling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to eryth- CELL ANALYSES OF NEURAL STEM AND roid stress and novel growth factor receptors that regu- PROGENITOR CELLS IN THE MOUSE late erythropoiesis. We defined a flow cytometry sorting EMBRYONIC VENTRAL FOREBRAIN GERMINAL strategy to purify early stages of erythroid differentiation, ZONE completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell Yammine, Samantha cycle is progressively remodelled during erythroid devel- Department of Molecular Genetics, University of opment and during a sharp transcriptional switch that Toronto, Canada ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and 10:40 – 11:05 provides insights into lineage development in vivo. THE ERNEST MCCULLOCH MEMORIAL LECTURE: MAKING ISLET CELLS FOR 10:30 – 10:40 DIABETICS POSTER TEASERS Melton, Douglas A. Harvard University and Harvard Stem Cell Institute, T-2074 Cambridge, MA, U.S. GENERATION OF INNER EAR ORGANOIDS Beta cell insufficiency in Type 2 diabetes and beta cell loss ENRICHED WITH MECHANOSENSITIVE in Type 1 diabetes lead directly to a dependence on insu- VESTIBULAR HAIR CELLS DERIVED FROM lin injections. An alternative approach is produce human HUMAN PLURIPOTENT STEM CELLS beta cells, and other islet endocrine cells, for transplanta- Mattei, Cristiana tion which would, in principle, relieve patients of regular Melbourne School of Engineering, The University of finger pricks and insulin injections and provide superior Melbourne, VIC, Australia metabolic control. Advances in controlling the directed differentiation of stem cells into functional human islets cells, and protecting them from immune attack following T-2162 transplantation, will be described. UTILIZING RNA SEQUENCING TO IDENTIFY CANCER-RELATED MUTATIONS IN HUMAN PLURIPOTENT STEM CELLS THURSDAY, 21 JUNE, 13:15 – 15:15 Avior, Yishai The Hebrew University, Israel CONCURRENT IA: MECHANISMS OF REPROGRAMMING 1: TO PLURIPOTENCY T-1030 SINGLE-CELL TRANSCRIPTOME ANALYSIS Melbourne Room 1, Level 2 OF EARLY MOUSE CARDIOGENESIS AND 13:20 – 13:45 PERTURBATION UPON HAND2 LOSS ACQUISITION OF NAÏVE PLURIPOTENCY IN De Soysa, Yvanka MICE AND MEN BY MODULATING TGF-BETA University of California, San Francisco and Gladstone Institutes, U.S. SIGNALING PATHWAY: TWO SIDES OF THE SAME COIN? T-1051 Baharvand, Hossein SINGLE CELL RNA-SEQ REVEALS DACH1 Royan Institute, Teheran, Iran EXPRESSION SEGREGATES LYMPHOID AND MYELOID FATE IN EARLY HAEMATOPOIESISA Mouse embryonic stem cells (ESCs) cultured in the pres- ence of inhibitors of TGFβ and FGF signaling pathways Tian, Luyi (dubbed R2i) exhibit typical features of ground state plu- Walter and Eliza Hall Institute of Medical Research, ripotency in which stem-cell pluripotency is established Melbourne, Australia and maintained by efficient blockade of endogenous dif- ferentiation pathways. R2i allows for efficient pluripotent stem cell generation from mouse and rat preimplantation blastocysts as well as from primordial germ cells (PGCs). R2i as same as the well-known 2i condition which include the FGF and GSK3 inhibitors could support the criteria of 114 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS ground state pluripotency. However, R2i exhibits more ef- sorted in each clone and their differentiation potency ficiency in controlling pluripotency, genomic integrity and was compared. From this screen, a clone showing distinct ESC generation from single blastomeres obtained from characteristics between Venus-positive and -negative mouse pre-implantation embryos and embryonic germ cells was identified. Expression of Venus was reversible: cell (EGCs) derivation from mouse PGCs. High through- Venus-positive cells could be derived from Venus-nega- put transcriptome analysis indicated that in spite of the tive cells, and vice versa. In this clone, a noncoding gene high similarity between 2i and R2i grown ESCs, BMP4 sig- with no known function was inserted with Venus. In vitro, naling pathway is highlighted in R2i cells. Moreover, the Venus-positive cells formed dome-shaped colonies with proteome and miRNome analysis indicated the differenc- sharp edges on culture compared with Venus-negative es in traditional serum/LIF cultured- and the ground state cells. Distinct differentiation potency was noted between (2i and R2i)-cultured ESCs. We found that the focal adhe- Venus-positive and -negative cells by in vitro differentia- sion signaling pathway significantly downregulated and tion assays, indicating the heterogeneity of differentiation glycolysis signaling pathway upregulated in ground state potency of undifferentiated ESCs. This hypothesis was conditions. Moreover, the ground-state ESCs express a further supported by RNA-seq analysis of undifferentiat- distinct set of miRNAs compared with serum/LIF grown ed ESCs, in which distinct gene ontology was enriched ESCs in that way most ‘‘ground-state miRNAs’’ are encod- between Venus-positive and -negative cells. Unexpect- ed by an imprinted region on chromosome 12 within the edly, Venus-negative cells showed high expression of Dlk1-Dio3 locus. Our time course transcriptome analysis two-cell stage embryo-specific markers (e.g. Zscan4 and during ESC generation from inner cell mass (ICM) in R2i MERVL), suggesting that Venus-negative cells have ear- regimen indicated that DNA methyltransferases and ep- ly stage embryonic characteristics. Finally, these results ithelial to mesenchymal transition blockage play pivotal demonstrate the functional heterogeneity of ground state roles in launching the ESC self-renewal program. In con- pluripotency and that this condition is not in a static state trast to the mouse context, however, it seems that TGF-β but is dynamically fluctuating. signaling plays a stimulatory role in generation and main- tenance of human naïve pluripotency, which is obtained 14:00 – 14:15 through the activation of nuclear receptors. We found that the brief treatment with two chemical agonists of nu- TFAP2C REGULATES TRANSCRIPTION IN clear receptors or TGFβ as the alternative is sufficient to HUMAN NAIVE PLURIPOTENCY BY OPENING induce naivety. Indeed, activation of TGFβ pathway pro- ENHANCERS motes key features of naivety in human pluripotent stem Clark, Amander , Pastor, William , Liu, Wanlu , Chen, 3 1 2 cells. TGFβ signaling might be the key feature that distin- Di , Ho, Jamie , Kim, Rachel , Hunt, Timothy and 3 3 3 3 guishes mouse from human naivety. Jacobsen, Steven 4 1 Molecular Cell and Developmental Biology, University 13:45 – 14:00 of California, Los Angeles, CA, U.S., McGill University, 2 IDENTIFYING HETEROGENEITY OF GROUND Montreal, Canada, University of California, Los 3 STATE PLURIPOTENCY IN MOUSE EMBRYONIC Angeles, CA, U.S., Howard Hughes Medical Institute, 4 STEM CELLS Los Angeles, CA, U.S. 1 Horie, Kyoji and Yoshida, Junko 2 Naïve human embryonic stem cells (hESCs) largely reca- 1 Department of Physiology II, Nara Medical University, pitulate the transcriptional state of pre-implantation epi- Kashihara, Japan, Nara Medical University, Kashihara, blast. In contrast, primed hESCs more closely resemble 2 Japan post-implantation epiblast. Therefore, naïve and primed hESCs constitute a developmental model for understand- Mouse embryonic stem cells (ESCs) are maintained in plu- ing the earliest pluripotent stages in human embryo devel- ripotent states in serum free medium in the presence of opment. To identify new transcription factors that differen- Mek and Gsk3 inhibitors and LIF (2i/LIF), which is called tially regulate the unique pluripotent stages, we mapped a ground state culture condition. Core pluripotency tran- open chromatin using ATAC-Seq and found enrichment of scription factors known to fluctuate under serum/LIF the AP2 transcription factor binding motif at naïve-spe- such as Nanog are homogenously expressed under 2i/LIF, cific open chromatin. We determined that the AP2 family implicating that ground state pluripotency is static in na- member TFAP2C is upregulated during primed to naïve ture. However, recent reports of single cell transcriptome reversion and becomes widespread at naïve-specific en- analyses revealed heterogeneously expressed gene mod- hancers. Using CRISPR/Cas9 we show that TFAP2C func- ules in ground state, with the significance of this hetero- tions to maintain pluripotency and repress neuroectoder- geneity remains elusive. Here we report the heterogeneity mal differentiation during the transition from primed to of ground state ESCs was associated with their differenti- naïve by facilitating the opening of enhancers proximal to ation potency. A gene trap vector using Venus as a report- pluripotency factors. Additionally, we identify a previously er was randomly inserted genome-wide for the trapped undiscovered naïve-specific OCT4 enhancer enriched for gene expression. Thousands of single cell-derived colo- TFAP2C binding. Taken together, TFAP2C establishes and nies were microscopically observed and clones showing maintains naïve human pluripotency and regulates OCT4 heterogeneous Venus expression at the single cell level expression by mechanisms that are distinct from mouse. were identified. Venus-positive and -negative cells were 115

THURSDAY 21 JUNE 2018 14:15 – 14:30 Understanding the molecular programs that guide cellu- EPIGENETIC AND GENETIC EFFECTS OF lar differentiation during development is a major goal of modern biology. Here, we developed an approach, WAD- GENDER ON REPROGRAMMING TO IPS CELLS DINGTON-OT, for inferring developmental landscapes, AND PLURIPOTENCY probabilistic cellular fates and dynamic trajectories from Pasque, Vincent large-scale single-cell RNA-seq (scRNA-seq) data col- Department of Development and Regeneration, KU lected along a time course. We demonstrated the pow- er of WADDINGTON-OT by applying it to study around Leuven - University of Leuven, Belgium 300,000 scRNA-seq profiles collected during reprogram- ming of fibroblasts to iPSCs by the Yamanaka factors. We Pluripotency can be established from somatic cells by re- applied this strategy to an additional 200,000 scRNA-seq programming approaches and also captured from early profiles of other reprogramming cocktails, with the goal embryos. However, how gender affects reprogramming processes and pluripotency remains unclear. We have re- of discovering the inherent mechanisms of iPSC repro- gramming. We construct a high-resolution map of repro- cently isolated isogenic male and female mouse induced pluripotent stem cells (iPSCs). Here, I will present new gramming that rediscovers known features; uncovers new alternative cell fates; predicts the origin and fate of any studies combining DNA methylation profiling, transcrip- tional profiling, pluripotency exit measurements, chroma- cell class; highlights senescent-like cells that may support tin profiling, growth and genetic analyses, and functional reprogramming through paracrine signaling; and impli- experiments to investigate the transcriptional, epigenetic cates regulatory models in particular trajectories. Our ap- and genetic effects of gender on the induction, mainte- proach provides the first high resolution roadmap of dif- nance and exit from pluripotency. I will show that the tran- ferent reprogramming cocktails and a general framework scriptional state, DNA methylation, exit from pluripotency for cell fate conversions in natural and induced settings. and cellular growth of female iPSCs differs from that of male iPSCs, partly mimicking early mammalian embryo 14:45 – 15:10 development. I will present evidence that X chromosome NOVEL FUNCTIONS OF RNA-BINDING loss in female iPSCs resolves gender-specific differences PROTEINS IN TRANSCRIPTION REGULATION but does not restore imprint methylation. I will show that DNA hypomethylation and delayed pluripotency exit can AND STEM CELL PLURIPOTENCY be molecularly uncoupled in female embryonic stem (ES) Shen, Xiaohua and Bi, Xianju cells through manipulation of the X-linked MAPK inhibitor Tsinghua University, Beijing, China Dusp9. I will also present evidence that the open chroma- tin landscape of ES cells is modulated by gender at thou- Much of the developmental complexity of higher eu- sands of chromatin regions and reveal the transcriptional karyotes is thought to arise from gene regulation rather regulatory logic by which gender influences pluripotency. than from an increase in the number of protein-coding Defining the mechanisms regulating the establishment, genes. RNA may represent a hidden layer of regulatory maintenance and exit from pluripotency in vitro and in information in complex organisms. Noncoding RNAs have vivo and understanding how these mechanisms are influ- been increasingly recognized as important regulators enced by gender will have important implications for de- of transcription and chromatin structure. The fact that velopment and regenerative medicine.  RNA-binding proteins (RBPs) must be enlisted to medi- Funding Source: The Research Foundation - Flanders ate RNA functions raises the possibility that RBPs might (FWO) (Odysseus Return Grant G0F7716N to V.P.), the participate in transcription control. I will discuss recent KU Leuven Research Fund (BOFZAP starting grant St- progresses we have made in RBP-mediated regulations of G/15/021BF to V.P., C1 grant C14/16/077 to V.P. and Proj- gene expression and stem cell pluripotency. ect financing). 14:30 – 14:45 RECONSTRUCTION OF CELLULAR REPROGRAMMING LANDSCAPES AND TRAJECTORIES BY ANALYSIS OF LARGE- SCALE SINGLE-CELL GENE EXPRESSION Shu, Jian , Tabaka, Marcin , Schiebinger, Geoff , Cleary, 1 1,2 1,2 Brian , Subramanian, Vidya , Solomon, Aryeh , Berube, 1 1 1 Peter , Lee, Lia , Brumbaugh, Justin , Hochedlinger, 3 1 1,2 Konrad , Regev, Aviv , Jaenisch, Rudolf and Lander, 2 1 4 Eric 1 1 Broad Institute, Cambridge, MA, U.S., Whitehead 2 Institute, Cambridge, MA, U.S., Massachusetts 3 General Hospital, Boston, MA, U.S., Harvard 4 University, Cambridge, MA, U.S. 116 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS variants are often incompletely penetrant or of weak-ef- THURSDAY, 21 JUNE, 13:15 – 15:15 fect, it would be enormously valuable if cellular studies could learn from vast numbers of variants simultaneous- CONCURRENT IB: DISEASE MODELING ly. We have been working to develop a novel strategy for Melbourne Room 2, Level 2 population-scale cellular studies: pooling cell lines from increasing numbers of individuals into reaction chambers, then using sequencing-based strategies for deconvolut- 13:20 – 13:45 ing cells’ identities as part of a phenotypic read out. This MOUSE MODELS OF GASTRIC CANCERS allows us to address two key challenges; 1) Scale - we can investigate phenotypes from hundreds of cell lines at once; Yang, Xiao and 2) Variance, control, and reproducibility -pooling cell State Key Laboratory of Proteomics, Beijing Institute lines reduces technical noise arising from well-to-well vari- of Lifeomics, Beijing, China ation and downstream molecular processing. Using this experimental paradigm, the polygenicity of these disor- Gastric cancer is the second leading cause of cancer-re- ders - which historically has been viewed as a weakness lated death worldwide, the molecular mechanisms under- - can be transformed into a scientific strength. Our ap- lying the pathogenesis of gastric carcinomas remain to proach combines single-cell RNA sequencing with mosaic be fully defined. To better understand the genetic mecha- culture systems to allow us to link RNA expression profiles nisms controlling the function of gastric epithelial cells in with an individual’s genotype. We found that co-culturing the homeostasis maintenance of the gastric epithelium, cell lines in this way greatly reduces variance; the coeffi- we established 4 transgenic mouse lines in which the Cre cient of variation between cell lines cultured individually recombinase were expressed in various gastric epithelial with those co-cultured simultaneously dropped marked- cells and generated different kinds of mouse models of ly from 0.20 to 0.08. We directly tested the association gastric cancers. We have previously shown that inactiva- between gene expression and cis-regulatory variation to tion of PTEN in mouse gastric epithelium initiates spon- identify expression quantitative trait loci (eQTL). These taneous carcinogenesis with complete penetrance by 2 “population-in-a-dish” analyses allow us to map changes months of age, providing the in vivo causal link between in direction and magnitude of gene expression directly the dysregulation of PTEN/Akt signaling and gastric tu- back to regulatory variants implicated in disease. We have morigenesis. Using the inducible Cre-LoxP system to de- identified hundreds of eQTLs in pools of pluripotent stem lete Smad4 and PTEN genes in murine gastric Lgr5+ stem cells, and in pools of cells differentiated in-vitro towards cells as well as marked mutant Lgr5+ stem cells and their neural precursors and more mature, upper-layer cortical progeny with Cre-reporter Rosa26tdTomato, rapid onset excitatory neurons. These experiments lay the framework and progression from microadenoma and macroscopic for future investigations aimed towards investigating the adenoma to invasive intestinal-type gastric cancer (IGC) effects of disease-associated variation on neuronal phys- were found in the gastric antrum of double mutant mice. iology, and represent a crucial step towards elucidating In contrast, Smad4 and PTEN deletions in differentiated the molecular pathways onto which these genetic signals cells, including antral parietal cells, pit cells and corpus converge. Lgr5+ chief cells, failed to initiate tumor growth. All these data demonstrated that gastric Lgr5+ stem cells were 14:00 – 14:15 cancer-initiating cells and might act as cancer-propagat- ing cells that contribute to malignant progression. The NUTRACEUTICAL RESCUE OF PATHOLOGICAL function of E-cadherin in gastric antral Lgr5+ cells in the CHANGES IN IPSC-DERIVED NEURAL maintenance of gastric epithelial homeostasis will also be CELL TYPES FROM A CHILDHOOD discussed. LEUKODYSTROPHY CAUSED BY MUTATIONS IN ASPARTATE TRNA SYNTHETASE (DARS) 13:45 – 14:00 Wolvetang, Ernst J. , He, Ruojie , Endes, Carola , 1 3 2 GENETIC NEUROSCIENCE: HOW HUMAN Ovchinnikov, Dmitry , Sun, Jane , Mar, Jessica , Powell, 3 3 3 4 3 4 GENES AND ALLELES SHAPE NEURONAL Joseph , Froehlich, Dominique , Klugmann, Matthias , 5 5 PHENOTYPES Salomons, Gaia Salomons , Wolf, Nicole , van der Knaap, Marjo and Vanderver, Adeline 6 5 Mitchell, Jana M. , Nemesh, James , Mello, Curtis , 1 2 2 Ghosh, Sulagna , Eggan, Kevin and McCarroll, Steven 2 1 Stem Cell Engineering Group, The Australian 3 3 1 Harvard Stem Cell and Regenerative Biology, Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Harvard University, Cambridge, MA, U.S., Harvard Australia, Sun Yat-Sen University, Guangzhou, China, 2 2 Medical School, Boston, MA, U.S., Harvard University, 3 The University of Queensland, Brisbane, Australia, 3 Cambridge, MA, U.S. 4 The University of New South Wales, Sydney, 5 The high polygenicity of neuropsychiatric disorders - Australia, VU University Medical Center, Amsterdam, 6 shaped by thousands of common and rare variants, and Netherlands, Children’s Hospital of Philadelphia, PA, by different combinations of risk alleles in each individu- U.S. al - presents formidable challenges for biology. As these 117

THURSDAY 21 JUNE 2018 The DARS gene codes for Aspartate-tRNA synthetase for ototoxin-induced cell death. Time-lapse data showed (AspRS) and is mutated in patients with Hypomyelin- a clear dose-dependent loss of iHCs in response to the ation with Brain Stem and Spinal Cord Involvement and ototoxins. A preliminary otoprotectant screen was per- Leg Spasticity (HBSL). How AspRS mutations lead to fo- formed on iHCs. Our screen examined 640 compounds cal hypomyelination and cognitive impairment, and why for their ability to rescue the previously identified oto- there is inter-patient variability in severity and onset has toxic effect of either gentamicin or cisplatin. The screen remained unclear, and no treatment is available. We re- identified 8 potentially protective compounds that sig- programmed HBSL patient fibroblasts into foot-print free nificantly extended the survival of iHCs by greater than IPSC, and show that HBSL-IPSC derived cortical neu- 3 standard deviations from the mean survival of the oto- rons exhibit increased ER-stress, increased apoptosis, toxin treated control. Some compounds identified are in- and  gene expression changes consistent with defective volved in known pathways of hair cell degeneration, and neuronal function and protein translation. We further find some identified novel targets. We are pursuing these hits HBSL-iPSC derived astrocytes show defects in activation, by testing them on human iHCs, whole organ of Corti ex- whereas single cell RNAseq reveals specific mitochondri- plants and in vivo in mouse models. So far one compound al gene expression differences in HBSL oligodendrocytes, has demonstrated a significant rescue in the whole organ indicating AspRS deficiency affects each of the three cell explant. Taken together, in vitro iHCs will help us pursue types that control CNS myelination differently. Important- protective and regenerative initiatives for the vulnerable ly, we are able to show that supplementation of cortical hair cells of the cochlea. neuronal cultures with the nutraceutical L-Ornithine-L-As- partate rescues the neuronal phenotypes, indicating this 14:30 – 14:45 may be a therapeutic option for HBSL patients. ENUCLEATION IN INDUCED RED BLOOD Funding Source: Supported by: The Mission Massimo CELLS: A PLATFORM FOR AUTOLOGOUS Foundation and the ARC SRI “Stem Cells Australia”. CELL THERAPY AND IN VITRO MODELING OF SICKLE CELL ANEMIA 14:15 – 14:30 Rosanwo, Tolulope O. , Clark, Martha , Vo, Linda , 1 2 3 USING INDUCED SENSORY HAIR CELLS FOR Kinney, Melissa , North, Trista and Daley, George 1 1 1 HIGH THROUGHPUT SCREENING TO IDENTIFY 1 OTOPROTECTANTS Boston Children’s Hospital, Boston, MA, U.S., 2 Harvard TH Chan School of Public Health, Boston, Menendez, Louise , Gopalakrishnan, Suhasni , Trecek, U.S., University of California San Francisco, U.S. 1 2 3 Talon , Yu, Haoze , Llamas, Juan , Makmura, Welly , 2 2 2 2 Segil, Neil and Ichida, Justin 2 Human induced pluripotent stem cells (hiPSCs) hold tre- 2 1 Department of Neuroscience, University of Southern mendous promise for disease modeling and the devel- 2 California, Los Angeles, CA, U.S., University of opment of novel therapeutic treatments for sickle cell anemia (SCA). hiPSCs can theoretically produce all cell Southern California, Los Angeles U.S. types including induced red blood cells (iRBCs). Sickle Hearing loss affects 360 million people worldwide and cell patients could benefit from autologous, engineered the leading cause is loss of sensory hair cells in the co- red blood cells as these patients have rare blood types, chlea. Hair cells are scarce and very fragile, making stud- are frequently allo-sensitized to blood products, and at ies difficult. Here we used direct reprogramming to gen- risk of iron overload from recurrent transfusions. However, erate induced sensory hair cells (iHCs) in vitro. Our results in vitro modeling of SCA as well as iRBC production from demonstrate that a specific set of hair cell transcription hiPSCs has been hampered by their inability to differen- factors is sufficient for reprogramming mouse fibroblasts tiate into terminally-mature, enucleated, beta globin-ex- towards a hair cell fate. The iHCs resemble primary mouse pressing red blood cells. Here, we describe strategies to hair cells at a transcriptional and functional level. The improve in vitro production of iRBCs. We generated hiP- transcriptional profile of iHCs successfully recapitulates SCs from sickle cell patients with hemoglobin SS disease 72% of primary hair cell genes and shuts down 79% of seen at our hematology clinic at Boston Children’s Hospi- the starting fibroblast genes. The functionality of the iHCs tal. Using a cocktail of transcription factors that promote has been assayed by their ability to take up styryl dyes in self-renewal and multipotency expressed under the con- a similar manner as primary hair cells, indicative of a ru- trol of a doxycycline-regulated promoter (ERG, HOXA9, dimentary mechano-transduction apparatus. Additionally, RORA, SOX4, MYB), we generated conditionally immor- whole cell patch clamping of the iHCs repeatedly demon- talized hematopoietic progenitors that serve as a renew- strated positive outward currents that have activation able source of robust erythroid cells in vitro. Erythroid kinetics characteristic of primary hair cell currents. One progenitors differentiated from these lines underwent goal of this in vitro model is to provide a high-through- globin-switching once transfused into immunodeficient put system to study the selective vulnerability of senso- mice, with a 27% induction of beta globin expression. An ry hair cells to environmental insults, such as well-known in vitro protocol incorporating human plasma can be used ototoxins like gentamicin, an aminoglycoside antibiotic, to produce 30-40% beta-globin-expressing cells. 10-50% and cisplatin, a chemotherapy drug. We performed a lon- of generated iRBCs are also enucleated. Preliminary iRBC gitudinal survival tracking experiment with iHCs to assay analysis reveals nearly 36% of the enucleated population to be RNA negative erythrocytes and 64% RNA positive 118 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS reticulocytes. With an expandable source of erythroid versican, brevican and tenascin C, a downregulation of progenitors capable of producing mature red cells, we basement membrane constituents (e.g., laminins, colla- hope to assess the feasibility of this platform for autolo- gens and fibrillins) and higher expression of synaptic and gous cell therapies. In future studies, we anticipate the im- ion transport machinery. pR-neurospheroids were gener- provement of the sickling model via a robust induction of ated using hiPSC-NSC derived from Mucopolysaccharido- beta-globin expression. The generation of hiPSC-derived sis type VII (MPS VII) patient. MPS VII is a neuronopathic SCA models will be critical in broadening the current un- lysosomal storage disease caused by deficient β-glucu- derstanding of the molecular mechanisms of this disease, ronidase (β-gluc) activity, leading to glycosaminoglycan and the development of improved pharmacological treat- (GAGs) accumulation in the brain. The main MPS VII mo- ments for the treatment of SCA. lecular hallmarks were recapitulated, e.g., accumulation of GAGs. MPS VII pR-neurospheroids showed reduced neu- Funding Source: Howard Hughes Medical Institute, ronal activity and disturbance in network functionality, Doris Duke Charitable Foundation, National Institute of Diabetes and Digestive and Kidney Disease, Na- with alterations in connectivity and synchronization, not observed in 2D cultures. These data provide insight into tional Heart Lung and Blood Institute: Progenitor Cell Translational Consortium. the interplay between reduced β-gluc activity, GAG accu- mulation, alterations in the neural network, and its impact on MPS VII-associated cognitive defects. 14:45 – 15:00 Funding Source: SFRH/BD: /78308/2011; /52202/2013; IPSC-DERIVED NEUROSPHEROIDS /52473/2014, FCT, Portugal and iNOVA4Health-UID/ RECAPITULATE DEVELOPMENT AND Multi/04462/2013, FCT/ MEC, Portugal. PATHOLOGICAL SIGNATURES OF HUMAN BRAIN MICROENVIRONMENT 1,2 1, 2 Terrasso, Ana P. , Simão, Daniel , Bayó-Puxan, THURSDAY, 21 JUNE, 13:15 – 15:15 1,2 1,2 Neus 3,4,5 , Arez, Francisca , Silva, Marta M. , Sousa, 6 1,2 1,2 Marcos F. , Gomes-Alves, Patricia , Raimundo, Nuno , CONCURRENT IC: CARDIAC Kremer, Eric J. and Brito, Catarina 1,2 DEVELOPMENT AND DISEASE 3,4 1 Instituto de Biologia Experimental e Tecnológica (iBET), Portugal Instituto de Tecnologia Química Room 219/220, Level 2 2 e Biológica António Xavier, Universidade Nova de 3 Lisboa, Portugal, Institut de Génétique Moléculaire 13:20 – 13:45 de Montpellier, CNRS UMR 5535, Montpellier, CARDIAC MICROTISSUES FROM HPSC IN France, Université de Montpellier, France, Neural MODELLING CARDIOVASCULAR DISEASE 4 5 Commitment Differentiation Department, The Institute of Biomedicine of the University of Mummery, Christine, Bellin, Milena, Giacomelli, Elisa, Barcelona (IBUB), Spain, Universitätsmedizin van Meer, Berend, Orlova, Valeria, Sala, Luca and 6 Göttingen, Institut für Zellbiochemie, Göttingen, Tertoolen, Leon Germany Leiden University Medical Center, Leiden, Netherlands Brain microenvironment plays important roles in neuro- Derivation of cardiovascular cell types from human plu- development and pathology. Neural cell culture typical- ripotent stem cells derived from patients or introducing ly relies on the use of heterologous matrices that poorly targeted mutations is an area of growing interest as a resemble brain ECM or reflect its pathological features. platform for drug discovery and toxicity. Our lab has been We have shown that perfusion bioreactor-based 3D dif- investigating organs on chip and microtissue solutions in ferentiation of iPSC-derived human neural stem cells which cardiomyocytes and cardiac vascular and stromal (hiPSC-NSC) sustains the concomitant differentiation of cells are present. This promotes cardiomyocyte matura- the three neural cell lineages (pR-neurospheroid). Here, tion and in combination with new methods for function- we hypothesized that if the pR-neurospheroid strategy al phenotyping, we have been able to quantify the out- would also allow deposition of native neural ECM, it would comes of drug and disease mutation responses in situ. be possible to (i) mimic cellular and microenvironment re- The use of isogenic pairs has proven very important since modeling occurring during neural differentiation, without variability between “healthy control” hiPSC lines is often the confounding effects of exogenous matrices and (ii) greater than the difference between a diseased cells and recapitulate pathological phenotypic features of diseas- its isogenic control. hiPSC derived cardiomyocytes with es in which alterations in homotypic/ heterotypic cell-cell mutations in ion channels and other genes can accurately interactions and ECM are relevant. Quantitative transcrip- predict changes in cardiac electrical properties and reveal tome (NGS) and proteome (SWATH-MS) analysis showed drug sensitivities also observed in patients. that neurogenic developmental pathways were recapitu- lated in our system, with significant changes in cell mem- brane and ECM composition, diverging from 2D differen- tiation. We observed a significant enrichment in structural proteoglycans typical of brain ECM, such as neurocan, 119

THURSDAY 21 JUNE 2018 13:45 – 14:00 advantage of protective mechanisms such as the one PATIENT DERIVED-IPS CELLS IDENTIFY A established in this study is of paramount importance to combat the ongoing CVD global epidemic. We believe NOVEL PROTECTIVE FACTOR AGAINST this avenue of research into AADAC has tremendous fu- ATHEROSCLEROSIS ture therapeutic potential. Toyohara, Takafumi , Roudnicky, Filip , Florido, Mary , 2 1 1 Nakano, Toshiaki , Ptaszek, Leon , Yu, Haojie , Lee, 14:00 – 14:15 1 3 4 Minjin , Friesen, Max , Davidow, Lance , Rubin, Lee , PROFILING PROLIFERATIVE CELLS AND THEIR 5 5 1 5 Pereira, Alexandre , Aikawa, Masanori and Cowan, PROGENY IN DAMAGED MURINE HEARTS 6 3 Chad 1 1 1 1 Beth Israel Deaconess Medical Center, Boston, MA, Kretzschmar, Kai , Post, Yorick , Bannier-Hélaouët, 1 3 2 U.S., Roche pRED (Pharmaceutical Research and Marie , Mattiotti, Andrea , Drost, Jarno , Basak, 2 2 4 1 Early Development), Basel, Switzerland, Center Onur , van den Born, Maike , Gunst, Quinn , Versteeg, 3 1 1 1 for Interdisciplinary Cardiovascular Sciences, Danielle , Kooijman, Lieneke , van der Elst, Stefan , Li, 5 1 1 Cardiovascular Division, Brigham and Women’s Vivian , van Es, Johan , van Rooij, Eva , van den Hoff, 1 2 Hospital, Harvard Medical School, Boston, MA, U.S., Maurice and Clevers, Hans 2 4 Cardiac Arrhythmia Service, MGH Heart Center, 1 Hubrecht Institute, Utrecht, Netherlands, Academic 3 Massachusetts General Hospital, Boston, MA, U.S., Medical Centre Amsterdam, Netherlands, Princess 5 Department of Stem Cell and Regenerative Biology, Maxima Centre for Paediatric Oncology, Utrecht, 4 Harvard University, Cambridge, MA, U.S., Laboratory Netherlands, University Medical Centre Utrecht, 6 5 of Genetics and Molecular Cardiology, Heart Institute, Netherlands, The Francis Crick Institute, London, University of São Paulo Medical School, Brazil Netherlands Cardiovascular disease (CVD) is the most common cause The capacity of the adult mammalian heart to functional- of death in patients with type II diabetes mellitus (T2DM). ly regenerate upon injury remains controversial. Different Susceptibility to CVD varies, and some T2DM patients cardiac stem cell (CSC) populations have been reported appear protected from CVD. The mechanism for this to contribute to tissue renewal after insult, yet their func- protective effect has not been clarified. We propose to tional significance for myocardial regeneration remains study this protective phenotype with patient-derived in- disputed. Furthermore, the proliferative capacity of res- duced pluripotent stem cells (iPSCs), which can be used ident non-cardiomyocyte cell lineages has been largely to model CVD in a dish and shed light on the underlying neglected. Here we perform single-cell messenger RNA mechanisms. To this end we have generated iPSCs from sequencing and genetic lineage tracing using two Ki67 T2DM patients with or without CVD, and subsequently knock-in mouse models that allow sorting and unbiased differentiated these into human endothelial cells (ECs) mapping of proliferating cells and their progeny in ho- as well as vascular smooth muscle cells (VSMCs). In com- meostatic and regenerating murine hearts. We find that paring the gene expression profiles in the ECs and the cardiomyocyte proliferation is largely restricted to the VSMCs, we found an esterase, arylacetamide deacetylase early postnatal growth phase, while non-cardiomyocyte (AADAC) increased significantly in VSMCs derived from cardiac cell lineages actively cycle also in the homeostatic T2DM patients without CVD. To investigate the function and damaged adult myocardium. Proliferative post-dam- of AADAC, we overexpressed AADAC in human primary age fibroblasts display a gene-expression pattern similar and human iPSC-derived VSMCs. AADAC overexpression to that of neonatal cardiac fibroblasts, while no signifi- reduced the number of lipid droplets per cell significantly. cant numbers of cardiomyocytes re-enter the cell cycle Cellular migration, proliferation and apoptosis were also upon damage. We find follistatin-like 1 (Fstl1), previously significantly decreased with higher levels of AADAC. We described as a cardiomyogenic factor of epicardial origin, attempted to validate these findings in a murine model by to be specific to cardiac fibroblasts and to be strongly in- generating VSMC-specific AADAC overexpressing mice duced upon cardiac injury. Genetic lineage tracing from on an apolipoprotein E (ApoE) knockout background. the Fstl1 locus reveals that these fibroblasts generate the Mice with increased AADAC showed dramatically amelio- fibrotic scar tissue, yet do not transdifferentiate into car- rated atherosclerotic lesions in their aorta compared to diomyocytes. Genetic deletion of Fstl1 in cardiac fibro- non-overexpressing ApoE mice. Murine VSMCs isolated blasts results in a severe phenotype with high mortality from mice overexpressing AADAC displayed a phenotype due to cardiac rupture upon cardiac injury. In sum, we find of decreased lipid droplets, cell migration, proliferation no evidence for the existence of a quiescent CSC popu- and apoptosis, all of which are consistent with our human lation, for transdifferentiation towards cardiomyocytes, or data. Our findings suggest that AADAC protects T2DM for significant numbers of cardiomyocytes that can re-en- patients from CVD by upregulating lipid metabolism, re- ter the cell cycle in response to cardiac injury and contrib- ducing migration, proliferation and apoptosis thereby ute to the myocardial lineage. However, resident cardiac protecting VSMCs from atherogenic phenotypes. Our fibroblasts that proliferate upon myocardial infarction are data indicate that iPSCs are a robust tool to interrogate a critical to maintain post-damage tissue integrity, as they patient’s clinical status, and that we can identify CVD-pro- generate a fibrotic scar to prevent cardiac rupture. tective mechanisms by leveraging this technology. Taking 120 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 14:15 – 14:30 scription factor regulatory networks linking the trajectory NEW STRATEGIES FOR ACCELERATING of subpopulations in vitro with cell types derived during cardiac development in vivo. We leveraged this data to CARDIAC REGENERATION study gene networks governing cardiomyocyte differ- Rosenthal, Nadia A. entiation in vivo to advance translational applications of The Jackson Laboratory, Bar Harbor, ME, U.S. stem cells in disease modelling and therapies. Among a network of known genetic drivers of differentiation, we Regeneration in vertebrates is an evolutionary variable identified dysregulation of the non-DNA binding home- that hinges on the rapid transition from wound healing odomain protein, HOPX as a candidate cause for the im- to restoration of organ form and function after tissue mature state of in vitro derived cardiomyocytes. Utilizing damage. Analysis of regenerative capacity in an unbiased genetic approaches coupled with engineered heart tis- screen of genetically defined recombinant inbred mouse sues, we determined that HOPX is functionally required panels has yielded dramatic intra-specifies variation, with for molecular and physiological cardiac maturation in vi- distinct gene networks underlying the repair of heart, tro. While HOPX is expressed in cardiac progenitor cells muscle and skin. Using combination of genetic manipula- (CPC) in vivo, we show during in vitro differentiation that tion and pharmacological blockades we have shown how HOPX has a repressive chromatin state in day 5 CPCs with modifying the profile of immune cell infiltration can facil- delayed onset of expression in only 16% of day 30 defini- itate or prevent cardiac repair, revealing that diversity in tive hPSC-derived cardiomyocytes. To recapitulate in vivo regenerative capacity is dependent on immune composi- development, we screened for mechanisms promoting tion and response, and uncovering immune tolerance as a HOPX expression in CPCs compared to small molecule dif- critical component of the regeneration process, suggest- ferentiation alone. Our results identified mechanisms driv- ing new cell targets for clinical intervention. The diverse ing hypertrophy in CPCs as essential for HOPX expression regenerative capacity of adult mammalian organs is also and gene networks associated with cardiomocyte mat- reflected in the tissue-specific composition and transcrip- uration.Taken together, we utilized single cell analysis of tomic profiles of resident immune cell subsets and the cardiac in vitro differentiation to identify mechanisms for stromal cells with which they interact, highlighting the po- activating gene networks in CPCs as they occur during in tential for more genetically precise, organ-specific treat- vivo heart development. These findings will facilitate the ments of degenerative diseases. utility of hPSCs for cardiac translational applications.  Funding Source: This work was supported by the 14:30 – 14:45 Australian Research Council (SR1101002). THE TRANSCRIPTIONAL LANDSCAPE OF CARDIAC DIFFERENTIATION AT SINGLE CELL 14:45 – 15:10 RESOLUTION CARDIAC DEVELOPMENT: BASIS FOR DISEASE AND REGENERATION 2 2 Palpant, Nathan , Friedman, Clayton , Nguyen, Quan , 1 2 Lukowski, Samuel , Helfer, Abbigail , Chiu, Han , Srivastava, Deepak 2 2 2 2 Voges, Holly , Baillie, Greg , Senabouth, Anne , Christ, Gladstone Institutes, San Francisco, CA, U.S. 2 Angelika , Bruxner, Timothy , Murry, Charles , Wong, 2 3 2 3 Emily , Ding, Jun , Wang, Yuliang , Hudson, James , Heart disease is a leading cause of death in adults and 4 2 2 5 4 Bar-Joseph, Ziv , Tam, Patrick and Powell, Joseph 2 children. We, and others, have described complex signal- 1 Institute for Molecular Bioscience, The University ing, transcriptional and translational networks that guide 2 of Queensland, Brisbane, Australia, The University early differentiation of cardiac progenitors and later mor- 3 of Queensland, Brisbane, Australia, The University phogenetic events during cardiogenesis. By leveraging these networks, we have reprogrammed disease-specific of Washington, Seattle, U.S., Carnegie Mellon human cells in order to model genetically defined human 4 5 University, Pittsburgh, U.S., Children’s Medical heart disease in patients carrying mutations in cardiac de- Research Institute, Sydney, Australia velopmental genes. These studies revealed mechanisms of haploinsufficiency and we now demonstrate the contri- Differentiation into diverse cell lineages requires the or- chestration of gene regulatory networks guiding diverse bution of genetic variants inherited in an oligogenic fash- ion in congenital heart disease. We also utilized a combi- cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from nation of major cardiac developmental regulatory factors to induce direct reprogramming of resident cardiac fibro- 43,168 cells across five time points over a thirty day time- course of in vitro cardiac-directed differentiation. We blasts into cardiomyocyte-like cells with global gene ex- pression and electrical activity similar to cardiomyocytes, used unsupervised clustering to identify transcriptional networks underlying lineage derivation of 15 subpopula- and now have revealed the epigenetic mechanisms under- lying the cell fate switch. Most recently, we identified an tions including mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cardiac approach to unlock the cell cycle in adult cardiomyocytes by introducing fetal cyclins and cyclin dependent kinases, fates including contractile cardiomyocytes and non-con- tractile derivatives. Utilizing customized machine learning and have been able to induce resident, post-mitotic car- diomyocytes to undergo cell division efficiently enough to algorithms, we analyzed scRNA-seq data to identify tran- regenerate damaged myocardium. Knowledge regarding 121

THURSDAY 21 JUNE 2018 the early steps of cardiac differentiation in vivo has led 13:45 – 14:00 to effective strategies to generate necessary cardiac cell BEYOND GOOSEBUMPS: INTERACTIONS types for disease-modeling and regenerative approaches, and may lead to new strategies for human heart disease. BETWEEN THE HAIR FOLLICLE, THE ARRECTOR PILI MUSCLE, AND THE SYMPATHETIC NERVE DURING DEVELOPMENT AND HAIR FOLLICLE REGENERATION THURSDAY, 21 JUNE, 13:15 – 15:15 Hsu, Ya-Chieh , Schwartz, Yulia , Gonzalez Celeiro, 2 1 3 2 CONCURRENT ID: EPITHELIAL STEM Meryem , Chen, Jyhlong and Lin, Sung-Jan 3 Department of Stem Cell and Regenerative Biology, CELLS 1 Harvard University Cambridge, MA, U.S., Harvard 2 Room 212/213, Level 2 University, Cambridge, MA, U.S., National Taiwan 3 University, Taipei, Taiwan 13:20 – 13:45 Piloerection, commonly known as goosebumps, involves LET’S TALK ABOUT STROMA: ADIPOCYTES three interconnected cell types: the hair follicle, the ar- AND FIBROBLASTS IN TISSUE REGENERATION rector pili muscle (APM), and the sympathetic nerve. The AND REMODELING interactions between these three cell types during de- Horsley, Valerie, Shook, Brett and Zwick, Rachel velopment and adult tissue maintenance remains poorly understood. Here, we identify a central role of the devel- Yale University, New Haven, CT, U.S. oping hair follicle in regulating the formation of APMs, which then attract sympathetic innervation to the hair Multiple stromal cell types support the homeostasis and regeneration of epithelial tissues. However, the function follicle stem cells. Although dispensable for hair follicle development, impulses from the sympathetic nerves are and heterogeneity of mesenchymal cells in epithelial tis- sues is not well understood. Here, we examined the con- crucial for regulating hair follicle stem cell activity during hair follicle regeneration. Formation of the APMs requires tribution of mesenchymal cells and mature adipocytes to the regeneration of epithelial tissues using mouse skin Sonic Hedgehog secreted from the developing hair fol- licles. Once developed, APMs do not undergo turnover, wound healing and mammary gland lactation as model systems. activated mesenchymal cells generate myofibro- providing a stable anchor that maintains sympathetic innervations to the hair follicle stem cells. APM ablation blasts that produce extracellular matrix (ECM) molecules for tissue resilience and strength and can produce exces- leads to concurrent loss of sympathetic nerve innervation to the hair follicles. Our results uncover a novel function sive ECM during fibrotic disorders. Using extensive analy- sis of pro-fibrotic cells during mouse skin wound healing, of APM in bridging the body’s sympathetic modulations to influence hair follicle stem cell activity, and illustrate fibrosis and aging; we identify three major functionally distinct subpopulations of myofibroblasts. Our findings an example for how a developing tissue regulates the es- tablishment of the niche to modulate its regeneration in identify multiple populations of fibrotic cells and suggest the pro-fibrotic environment dictates functional myofi- adulthood. Our results may also explain why hair loss is a common side effect of beta-blockers, which suppress the broblast heterogeneity; which is driven by fibroblast-im- mune cell interactions after wounding. In the mammary sympathetic tones, and why loss of APMs is commonly associated with permanent hair loss conditions such as in gland, stromal adipocytes rapidly repopulate the stroma as the epithelial cells regress during involution of epitheli- androgenic alopecia. al cells following lactation. Using genetic lineage tracing, lipid tracking, and lipidomics, we show that during involu- 14:00 – 14:15 tion, mature adipocytes undergo hypertrophy to regen- AN EVOLUTIONARILY CONSERVED erate the stromal MG adipose depot by filling with milk RIBOSOME-RESCUE PATHWAY MAINTAINS lipid trafficked from epithelial lumen. We find that milk MOUSE EPIDERMAL STEM CELL HOMEOSTASIS lipid are recycled into adipocytes after lactation, facilitat- 1 2 ing the epithelial regression and stromal remodeling that Liakath-Ali, Kif , Mills, Eric , Sequeira, Inês , 1 1 occurs during MG involution. Furthermore, we developed Lichtenberger, Beate , Pisco, Angela Oliveira , Sipilä, 1 a unique tissue and cell type-specific method to ablate Kalle , Mishra, Ajay , Yoshikawa, Harunori , Wu, Colin 1 1 3 MG adipocytes after lactation and demonstrate that in the Chih-Chien , Ly, Tony , Lamond, Angus , Adham, 3 3 2 MG, mature adipocytes are necessary for lipid uptake into Ibrahim , Green, Rachel and Watt, Fiona 1 2 4 epithelial cells and proper epithelial alveolar regression 1 King’s College London, UK, Howard Hughes 2 during involution. Our work has profound implications for our understanding of tissue regeneration and remodeling Medical Institute, Johns Hopkins School of Medicine, 3 and the contribution of the stroma to these processes. Baltimore, MD, U.S., University of Dundee, UK, 4 University of Goettingen, Germany Ribosome-associated mRNA quality control mechanisms ensure fidelity of protein translation. Although extensively studied in yeast, little is known about their role in mam- 122 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS malian tissues, despite emerging evidence that stem cell levels of stem cell markers. The potential role of Nrg1/ErbB fate is controlled by translational mechanisms. One evolu- signalling during tissue regeneration was investigated us- tionarily conserved component of the quality control ma- ing two mouse models of injury/regeneration (irradiation chinery, Dom34/Pelota (Pelo), rescues stalled ribosomes. and 5-FU treatment). Interestingly, the expression of Nrg1 Here we show that Pelo is required for mammalian epi- in both models was elevated 5-10 fold during the intestinal dermal homeostasis. Conditional deletion of Pelo in those regenerative phase. This was reinforced by using in vitro murine epidermal stem cells that express Lrig1 results in organoids where Nrg1 significantly promoted organoid hyperproliferation and abnormal differentiation. In con- growth and the formation of colonies from single ISCs. trast, deletion in Lgr5+ stem cells has no effect and dele- Importantly, the intestinal regenerative response follow- tion in Lgr6+ stem cells has only a mild phenotype. Loss of ing damage was significantly improved when mice were Pelo results in accumulation of short ribosome footprints treated with Nrg1. Taken together, our results demonstrate and global upregulation of translation rather than affect- that Nrg1 is a crucial niche signal that regulates ISC pro- ing expression of specific genes. Translational inhibition liferation. Manipulating the Nrg1/ErbB signalling pathway by rapamycin-mediated down regulation of mTOR res- during intestinal tissue regeneration has potential thera- cues the epidermal phenotype. Our study reveals a nov- peutic applications. el role for the ribosome-rescue machinery in mammalian Funding Source: This work is supported by an Nation- tissue homeostasis and an unanticipated specificity in its al Health and Medical Research Council project grant impact on different stem cell populations. and a Monash University Strategic grant. 14:15 – 14:30 14:30 – 14:45 ACTIVATION OF NEUREGULIN1/ERBB ELUCIDATING THE RADIATION RESISTANCE SIGNALLING PROMOTES INTESTINAL STEM MECHANISMS IN THE REGENERATIVE CELL PROLIFERATION AND IMPROVES TISSUE MOUSE INTESTINE USING SINGLE CELL RNA REGENERATION FOLLOWING DAMAGE SEQUENCING 1 2 Jarde, Thierry , Rossello, Fernando , Kurian Arackal, Ayyaz, Arshad , Kumar, Sandeep , Ghoshal, Bibaswan , 1 1 1 Teni , Flores, Tracey , Giraud, Megane , Prasko, Trcka, Daniel , Gregorieff, Alex and L. Wrana, Jeffrey 1 2 2 2 1 2 Mirsada , Nefzger, Christian , Abe, Shin-ichi , Polo, 2 2 3 2 Jose and Abud, Helen 2 1 Lunenfeld-Tanenbaum Research Institute, Toronto, 2 1 Department of Anatomy and Developmental Biology, ON, Canada, McGill University, Montreal, PQ, Canada Monash Biomedicine Discovery Institute, Monash The intestinal epithelium completely turns over every University, Clayton, VIC, Australia, Monash University, week in mice and every 2-3 weeks in humans. The mul- 2 Clayton, Australia, Kumamoto University, Kumamoto, tipotent Lgr5+ crypt base columnar (CBC) cells are re- 3 Japan sponsible for constantly replenishing the epithelium with fresh cells under homeostatic conditions throughout the Defining signals that regulate intestinal stem cells (ISCs) animals’ life span. However, in response to injury, such as may enable stem cell pools to be manipulated in degen- ionizing radiation (IR), the majority of Lgr5+ cells are lost, erative diseases and intestinal pathologies. The Neureg- but then re-emerge post-IR and are indispensable for a ulin1/ErbB signalling pathway plays a pivotal role in regu- successful recovery. It remains unclear how these resur- lating aspects of tissue homeostasis and regeneration in gent Lgr5+ stem cells are generated. To investigate their the nervous system. However, the function of this path- population dynamics at the single cell level, we performed way in the intestinal epithelium is currently unknown. We single cell RNA sequencing on 4500 cells isolated from examined the expression of Nrg1 and its receptors in the the mouse small intestine without or after 3 days whole small intestine using immunofluorescence and qRT-PCR. body IR exposure. Unsupervised clustering classified dif- We observed that supporting niche cells express Nrg1, ferentiated cell types into separate groups that could be while ISCs express ErbB receptors, suggesting that Nrg1/ identified by the expression of their corresponding lineage ErbB signalling may directly regulate ISCs. To investigate markers. In response to IR we found significant changes the functional activity of Nrg1 in the intestine, 12 week-old in the cellular composition of the intestine that included mice were injected with 15ug Nrg1 for 5 days. Activation depletion of about 75% of Paneth cells, 50% of Tuft cells of Nrg1/ErbB signalling increased cell proliferation in the and 30% of Goblet cells, with a concomitant increase in intestinal crypts by 43% and caused alterations in cellular the proportion of enterocytes (ECs) and enteroendocrine differentiation. The requirement for Nrg1 was assessed by (EE) cells. We also observed a loss of 90% of Lgr5+ cells inducing loss of Nrg1 in transgenic mice. Loss of Nrg1 re- and in particular Lgr5high cells were undetectable after sulted in a significant decrease in cell proliferation within 3 days post-IR. To enrich for stem cells, we next repeat- crypts in both ISCs and progenitor cells. To characterise ed this strategy on isolated crypts, which led to a 2.1-fold the molecular changes induced by Nrg1 signalling in ISCs, enrichment of Lgr5+ cells under homeostatic conditions, RNA sequencing was performed on ISC and progenitor whereas the number of Lgr5high cells in IR-treated sam- cell populations isolated from Nrg1-treated and control ples remained negligible (2 of 4500 cells). Intriguingly, animals. Nrg1 treatment produced a proliferative molecu- unsupervised clustering of crypt cells revealed a cell pop- lar signature in both cell types. Importantly, the ISC popu- ulation that displayed heterogeneous expression of stem lation became more homogeneous and expressed higher 123

THURSDAY 21 JUNE 2018 cell markers and could be divided by low or high expres- to injury. A key control point is the decision between qui- sion of proliferative markers. We called these two class- escence and proliferation. Drosophila neural stem cells es the ‘revival’ and ‘proliferative’ groups. The revival stem (NSCs) enter quiescence in late embryogenesis and are cell compartment was composed of slowly cycling cells reactivated post-embryonically in response to a nutri- and was marked by YAP target gene expression, while the tion-dependent signal from the fat body. The fat body proliferative compartment expressed high levels of CBC performs many of the storage and endocrine functions of markers. We propose that damage to the intestine leads the vertebrate liver and adipose tissue and acts as a sen- to the emergence of a distinct regenerative stem cell that sor, coupling nutritional state to organismal growth. We reconstitutes the homeostatic Lgr5 compartment. showed that the nutritional stimulus transduced by the fat body induces the expression of insulin/IGF-like pep- Funding Source: Canadian Institutes of Health Re- search (CIHR). Terry Fox Research Institute. University tides (dILPs) in the blood brain barrier glia, which overlie the quiescent stem cells. We found that insulin signalling of Toronto’s Medicine by Design which receives fund- ing from the Canada First Research Excellence Fund is essential for NSCs to exit quiescence. Insulin signalling can also promote proliferation in vertebrate neural stem (CFREF). cells, suggesting that the mechanisms controlling stem cell reactivation may be conserved. We are investigating 14:45 – 15:10 the systemic and local signals that regulate neural stem REGULATION OF EPIDERMAL STEM CELL FATE cell quiescence and reactivation. BY NICHE-DERIVED SIGNALS AND FORCES Wickström, Sara 13:45 – 14:00 Max Planck Institute for Biology of Ageing, Cologne, EPIGENETIC REGULATION OF LIPID Germany METABOLISM IN NEURAL STEM CELL FATE DECISION Our research aims to uncover how complex but stereo- Syal, Charvi , Sarma, Sailendra Nath , Seegobin, 2 1 typed tissues are formed, maintained and regenerated 2 3 2 through local growth, differentiation and remodeling. To Matthew , Thomas, Jacob and Wang, Jing decipher this fundamental question we need to under- 1 Regenerative Medicine Program, Ottawa Hospital 2 stand how single cell behaviors are coordinated on the Research Institute, Ottawa, ON, Canada, Ottawa population level and how population-level dynamics is Hospital Research Institute, Ottawa, ON, Canada, coupled to tissue architecture. Uncovering these regula- 3 University of Western Ontario, London, ON, Canada tory principles will further facilitate development of stem cell (SC) therapies and effective treatments against can- Lipids, often considered little more than structural com- cers. As a self-renewing organ maintained by distinct stem ponents of a cell, have recently been identified as promi- cell populations, the epidermis represents an outstanding, nent regulators of neural stem and progenitor cell (NPC) clinically highly relevant research paradigm to address function, under both physiological and pathological con- these questions. We apply mouse genetics and molecu- ditions. However, our knowledge of molecular aspects lar cell biology, combined with state-of-the art biological of regulation of lipid metabolism and its specific role in imaging, biophysics, biochemistry and theoretical ap- determination of NPC function is lacking. Previously, we proaches to study stem regulation and tissue homeosta- identified that the atypical protein kinase C (aPKC)-me- sis/aging in this system. In my presentation I will discuss diated phosphorylation of CBP (CREB binding protein) our recent research on stem cell-niche interactions in cell at Ser436, activated by age-related intrinsic signals and fate decisions and plasticity, and the role of mechanical metformin stimulation, was able to promote neuronal dif- forces in these processes. ferentiation of adult NPCs. We have now identified mono- glyceride lipase (Mgll) as the direct target of this path- way, wherein an active aPKC-CBP pathway represses Mgll expression in adult NPCs to promote their differentiation. THURSDAY, 21 JUNE, 13:15 – 15:15 Mgll, an enzyme that hydrolyses the endocannabinoid, 2-arachidonoyl glycerol (2-AG) to produce arachidonic CONCURRENT IE: HOMEOSTASIS, acid (ARA), is thus, a key regulator of two critical lipid METABOLISM AND AGING components in the brain as well as a potential modulator of NPC function. We observed elevated Mgll levels, con- Room 203/204, Level 2 comitant with neuronal differentiation deficits in NPCs isolated from sub-ventricular zone of transgenic CBP- 13:20 – 13:45 S436A mice, that lack a functional aPKC-CBP pathway. Genetic knockdown of Mgll in these CBPS436A NPCs res- AWAKENING STEM CELLS IN THE BRAIN cued their differentiation defects. In addition, we found Brand, Andrea and Otsuki, Leo that CBPS436A NPCs exhibit enhanced proliferation at University of Cambridge, U.K. the expense of differentiation as an outcome of increased Mgll levels, implicating Mgll as a dynamic switch that reg- The systemic regulation of stem cells ensures they meet ulates NPC function by altering lipid composition (2-AG the needs of the organism during growth and in response versus ARA). Interestingly, we also found that NPCs from 124 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS + an Alzheimer’s disease (AD) model, the 3xTg mice, closely D -dependent class III histone deacetylase, is abundantly mimic CBPS436A NPC behaviour in vitro. 3xTg NPCs ex- expressed in hESCs and has been reported to play a role hibit attenuation of the aPKC-CBP pathway, which is as- in regulating early differentiation and telomere elongation. sociated with elevated Mgll expression and increased NPC We found that blocking the function of SIRT1 in hESCs in- proliferation at the expense of differentiation in culture, duced massive cell death, thus leading us to hypothesize similar to CBPS436A NPCs. Reactivation of the aPKC- that SIRT1 is required for hESC survival. Either blocking CBP-Mgll pathway in these AD NPCs mitigates their dif- the function or decreasing the level of SIRT1 dramatically ferentiation deficits. These findings thus suggest that at- promoted cell death in hESCs, but not in differentiated tenuation of aPKC-CBP mediated Mgll repression results cells such as fibroblasts. SIRT1 inhibition-mediated cell in neuronal differentiation deficits in NPCs, contributing to death was preceded by increased DNA damage. Our de- AD predisposition. tailed mechanistic study showed that the increased DNA damage caused by SIRT1 down-regulation was at least Funding Source: Natural Sciences and Engineering Research Council of Canada (NSERC); Scottish Rite partially due to decreased levels of DNA repair enzymes such as MSH2, MSH6, and APEX1. Furthermore, we ob- Charitable Foundation of Canada. served p53 activation followed by the overexpression of PUMA and BAX, two pro-apoptotic p53 target genes, af- 14:00 – 14:15 ter SIRT1 inhibition. Owing to these events, apoptotic cell CONTROL OF PLURIPOTENT STEM CELL FATE death was induced in hESCs in the absence of SIRT1, thus DECISIONS BY METABOLIC FLUX suggesting that SIRT1 acts as a guardian of pluripotent stem cells. Together, our results demonstrated that SIRT1 2 1 Dalton, Stephen , Cliff, Tim and Wu, Tianming 2 is required to maintain a high level of the DNA repair pro- 1 Center for Molecular Medicine, University of Georgia, teins MSH2, MSH6, and APEX1 and to prevent massive Athens, GA, U.S., University of Georgia, Athens, GA, hESC death. This study provides valuable insights into the 2 U.S. mechanism of SIRT1-mediated hESC survival and should contribute to the development of safe and effective cell As human pluripotent stem cells (hPSCs) exit pluripoten- replacement therapies. cy they are thought to switch from a glycolytic mode of Funding Source: This research was supported by energy generation to one more dependent on oxidative the Bio and Medical Technology Development Pro- phosphorylation. In this presentation, we show that al- gram of the NRF funded by the Korean government, though metabolic switching occurs during early meso- MSIT(2017M3A9B4042580). derm and endoderm differentiation, elevated glycolytic flux is maintained and essential during early ectoderm specification. Metabolic switching is therefore not an 14:30 – 14:45 obligatory event required for exit from pluripotency. Ele- FUNCTIONAL REJUVENATION OF vated glycolysis is required for self-renewal of hPSCs and AGED INTESTINAL STEM CELLS BY requires elevated MYC/MYCN activity. In endoderm and METABOLIC INTERVENTION AND DIRECT mesoderm, decreased MYC/MYCN transcriptional activity coincides with metabolic switching, but this is reversed by REPROGRAMMING ectopically restoring MYC activity. MYC activity is there- Nefzger, Christian M. , Jarde, Thierry , Horvay, Katja , 1 1 1 fore necessary and sufficient for metabolic switching. In Rossello, Fernando , Srivastava, Akanksha , Prasko, 1 2 nascent ectoderm, sustained MYCN activity maintains the Mirsada , Paynter, Jacob , Sun, Yu , Liu, Xiaodong , 1 1 1 1 transcription of ‘switch’ genes that are rate-limiting for Chan, Eva , Li, Jinhua , Pflueger, Jahnvi , Knaupp, Anja , 1 1 1 2 metabolic activity and lineage commitment. This study Nilsson, Susan , Rackham, Owen , Lister, Ryan , Abud, 3 2 4 identifies MYC and MYCN as developmental regulators Helen and Polo, Jose 1 1 that couple metabolism to pluripotency and cell fate de- 1 2 termination. This general mechanism is likely to have di- Monash University, Clayton, Australia, The University rect implications for MYCs role in reprogramming to the of Western Australia, Perth, Western Australia, 3 pluripotent state. Australia, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC, 4 14:15 – 14:30 Australia, Duke-National University of Singapore (NUS) Medical School, Singapore SIRT1 ENHANCES THE SURVIVAL OF HUMAN EMBRYONIC STEM CELLS BY PROMOTING Intestinal stem cells (ISCs) drive epithelial homeostasis DNA REPAIR and regeneration following damage and these processes are impaired with age, however the exact reason of this Kim, Dong-Wook decline is unknown. In order to uncover the underlying Department of Physiology, Yonsei University College mechanism for functional differences in aged stem cells of Medicine, Seoul, Korea we determined the molecular and functional changes occurring in the ISC niche during the aging process. We Human embryonic stem cells (hESCs) hold great prom- found widespread transcriptional changes associated with ise for the treatment of many currently incurable diseases reduced Wnt signalling and decreased metabolic activity. through cell replacement therapy. Sirtuin1 (SIRT1), an NA- Importantly, we show that elevation of Wnt signalling only 125

THURSDAY 21 JUNE 2018 partially rescued ISC defects. However, metabolic inter- vention restored Wnt signaling state, transcriptional sig- THURSDAY, 21 JUNE, 16:00 – 18:00 nature and organoid formation frequency to levels found in young animals. Furthermore, we identified key drivers CONCURRENT IIA: GENE EDITING of an “aged” transcriptional network and demonstrat- ed that forced expression of these factors in organoids Melbourne Room 1, Level 2 derived from aged stem cells rescued their regenerative Sponsored by The Allen Institute for Cell defects and re-established metabolic potential. Our data Science demonstrate that different molecular changes associated with aging stem cells can be efficiently reversed, which 16:05 – 16:30 has implications for aging intervention strategies. Collec- tively, we anticipate that our findings will open the door GENE EDITING: OPTIMIZATION AND for future clinical applications in improving regeneration APPLICATION IN PRIMARY CELLS of the gastrointestinal tract. Wang, Haoyi, Mu, Wei, Xiang, Guanghai and An, Chenrui 14:45 – 15:10 Institute of Zoology, Chinese Academy of Sciences, METABOLIC DETERMINATION OF CELL FATE Beijing, China THROUGH SELECTIVE INHERITANCE OF MITOCHONDRIA CRISPR-Cas9 system has become the tool of choice for gene editing. To facilitate its application in primary cells, Katajisto, Pekka, Dohla, Julia, Englund, Johanna, we optimized the sgRNA to improve its stability and re- Amaral, Ana, Gebert, Nadja, Salminen, Ella, duce its immunogenicity. We enhanced the sgRNA stabili- Gopalakrishnana, Swetha, Kakela, Reijo and Ori, ty by adding 5' cap and 3' polyA tail to in vitro transcribed Alessandro (IVT) sgRNA (indicated as CTsgRNA). Using CTsgRNAs, University of Helsinki, Finland, and Karolinska we achieved significantly higher gene editing and acti- Institutet, Helsinki, Sweden vation efficiency in human cells compared to unmodified sgRNAs. In addition, we found that IVT sgRNAs induced In order to maintain homeostasis, tissue renewing adult type I interferon (IFN) release, which led to poor surviv- stem cells are controlled by multiple mechanisms balanc- al of human CD34+ HSPCs, CD3+ T cells and chimeric ing self-renewal and differentiation. As fate changes in antigen receptor (CAR)-T cells. By treating the IVT sgR- stem cells are paralleled by profound metabolic rewiring, NA with calf intestine phosphatase (CIP), we were able cellular metabolism provides one possible level for such to eliminate the IFN induction effect of IVT sgRNAs, and control. We have studied the role of cellular metabolism achieve significantly better cell survival without affecting in the context of asymmetric cell divisions to address how gene editing efficiency. To improve the gene editing effi- metabolism is determined immediately after cell division, ciency in human pluripotent stem cells, we compared the and whether asymmetric fate determination is controlled efficiency of introducing indels, precise point mutation, by metabolism. We have discovered that the chronological and transgene integration in primed human embryonic age of mitochondria inherited from the mother cell, deter- stem cell culture and three naïve culture systems. Naïve mines daughter cell metabolism and fate upon asymmet- culture conditions in general had higher efficiency. 5i naïve ric division in stem-like human mammary epithelial cells system in particular had a more consistent improvement, (HMECs). Old mitochondria are apportioned selectively to with targeted transgene insertion efficiency reaching 20% the differentiating daughter cell, while progeny omitting without any selection. old mitochondria retains stem-like properties. Age-spe- cific isolation and profiling of mitochondria revealed that 16:30 – 16:45 protein and lipid composition, as well as organelle func- tion, changes as mitochondria mature, and results in more ENDOGENOUS GENE TAGGING WITH CRISPR/ active oxidative metabolism in old mitochondria. Upon CAS9 TO ILLUMINATE CELL ORGANIZATION asymmetric segregation, inherited mitochondrial metab- AND DYNAMICS olism prompts metabolic bias in daughter cells, and has Gunawardane, Ruwanthi the capacity to preserve stem-like properties or induce differentiation. Pharmacologic modulation immediate- Allen Institute for Cell Science, Seattle, WA, U.S. ly after cell division can mitigate the inherited metabolic bias and alter fate of daughter cells. Our results demon- The Allen Institute for Cell Science is creating a dynam- strate that cell fate programs are susceptible to modula- ic visual model of hiPSC organization to understand and tion by metabolism immediately after division of stem-like predict normal and pathological cell states. Towards this cells, and that the asymmetric apportioning of old mito- goal, we have created the “Allen Cell Collection”; an open chondria may be one of the first fate determinants in adult source collection of fluorescently tagged human induced stem cell divisions. pluripotent stem cell (hiPSC) lines representing the major organelles of the cell. Our approach utilizes CRISPR/Cas9 to introduce fluorescent tags via homology driven repair (HDR) into the genomic locus of interest. Editing yields 126 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS isogenic hiPSC lines expressing fusion proteins unique to that flanking gRNAs with self-cleaving ribozymes permits each cell line under endogenous regulation. Live cell im- proper processing of active gRNA from pol2 transcripts. -/- aging, image analysis, computational modeling, and open In Fah mice, loss of Hpd< or Hgd, genes upstream of FAH distribution of tools and data to the scientific commu- in tyrosine degradation, provides a selective advantage nity define our endeavor. Here we present the CRISPR/ for hepatocytes. We observed strong in vivo selection of Cas9- based gene editing strategy and workflow used to albuminF9 generide AAV vectors harboring self-cleaving generate ~25 GFP-tagged clonal hiPSC lines represent- gRNA (scgRNA) against Hpd and Hgd. Superphysiologic ing the major organelles of the cell including the nucleus, levels of human F9 were seen in Fah mice treated with -/- mitochondria, adhesions, cytoskeleton, golgi and ER. We these vectors and then taken off NTBC. In order to broad- have also developed related editing methods for intro- en this paradigm, we engineered a system that renders ducing red fluorescent proteins, tagging dual structures, hepatocytes resistant to the hepatotoxic drug acetamin- safe harbor edits, and differentiation specific gene tag- ophen. A scgRNA designed to knock out NADPH-cyto- ging. We will describe our genotyping strategy to identify chrome P450 reductase (Cypor) was incorporated into clones harboring precisely incorporated FP tags includ- the albumin-hF9 gene ride vector. Loss of Cypor prevents ing off-target and NGS analysis and present trends ob- the conversion of acetaminophen into its toxic metabo- served during precise editing. Clonal lines also undergo lite. Acetaminophen treatment resulted in the expansion various quality control assays for ensuring genomic stabil- of Cypor null hepatocytes to ~45% of the liver mass and ity, pluripotency, and confirmation of subcellular localiza- therapeutic levels of human F9 expression. tion prior to use in live cell imaging and distribution. We will highlight the utility of these cell lines for generating 17:00 – 17:15 image-based integrated models of cell organization and dynamics and discuss the potential applications of these IN VIVO SIMULTANEOUS TRANSCRIPTIONAL endogenously tagged lines for basic science and disease ACTIVATION OF MULTIPLE GENES IN THE modeling.  BRAIN USING CRISPR-DCAS9-ACTIVATOR TRANSGENIC MICE 16:45 – 17:00 Zhou, Haibo , Liu, Junlai , Zhou, Changyang , Gao, Ni , 1 1 2 1 SELF-CLEAVING GUIDE RNAS FOR SELECTIVE Rao, Zhiping , Li, He , Hu, Xinde , Li, Changlin , Yao, 1 1 1 1 1 1 1 1 3 EXPANSION OF PRECISELY GENE EDITED Xuan , Shen, Xiaowen , Sun, Yidi , Wei, Yu , Liu, Fei , 1 1 1 HEPATOCYTES IN VIVO Ying, Wenqin , Zhang, Junming , Tang, Cheng , Zhang, 1 1 1 Xu , Xu, Huatai , Lin, Linyu , Cheng, Leping , Huang, 1 1 Grompe, Markus , Tiyaboonchai, Amita , Nygaard, Pengyu and Yang, Hui 1 2 2 2 Sean and Vonada, Anne 2 1 Institute of Neuroscience, Shanghai, China, 1 Papé Family Pediatric Research Institute, Oregon 2 ShanghaiTech University, Shanghai, China, Chinese 3 Health and Science University, Portland, OR, U.S., Academy of Sciences Max Planck Society (MPG) 2 Oregon Health and Science University, Portland, OR, Partner Institute for Computational Biology, U.S. Shanghai, China Transplantation of cells that have been manipulated by Despite rapid progresses in the genome-editing field, in precise gene editing is an attractive approach for cell- vivo simultaneous overexpression of multiple genes re- based therapies, especially for genetic disorders. Howev- mains challenging. We generated a transgenic mouse us- er, gene editing by homologous recombination is inher- ing an improved dCas9 system that enables simultaneous ently inefficient. One strategy to achieve a higher overall and precise in vivo transcriptional activation of multiple efficiency of gene editing is to selectively expand cells genes and long noncoding RNAs in the nervous system. that have acquired the desired targeting event in vivo As proof of concept, we were able to use targeted activa- after transplantation. This can be achieved by linking the tion of endogenous neurogenic genes in these transgen- wanted genetic modification to a selectable gene disrup- ic mice to directly and efficiently convert astrocytes into tion cassette in cis, such that growth selection can happen functional neurons in vivo. This system provides a flexible only if proper gene targeting has been achieved. Using and rapid screening platform for studying complex gene a factor 9 expressing Generide rAAV vector targeted to networks and gain-of-function phenotypes in the mam- the albumin locus we previously showed that an shRNA malian brain. embedded in a microRNA within an intron can protect hepatocytes from a hepatotoxic drug and result in high Funding Source: National Science and Technology transgene levels. While this approach can work, shRNAs Major Project, CAS Strategic; Priority Research Pro- usually only knock down a gene by ~ 80-90%, thus limiting gram, the MoST863 Program, NSFC grants, China its utility for drug protection regimens. In contrast, com- Youth; Thousand Talents Program, Break through plete gene knockouts can be achieved by CRISPR-cas9 project of Chinese Academy of Sciences. cutting. In order to generate selectable CRISPR-mediated gene knockouts, it is necessary to restrict its endonucle- ase activity to only cells that have proper gene target- ing. This strategy requires that the gRNA expression is driven by a tissue-specific pol2 promoter. Here we show 127

THURSDAY 21 JUNE 2018 17:15 – 17:30 17:30 – 17:55 FIRST-IN-HUMAN STUDY OF FEASIBILITY, HUMAN DEVELOPMENT AND DISEASE SAFETY AND ENGRAFTMENT OF ZINC THROUGH THE LENS OF PLURIPOTENT STEM FINGER NUCLEASE CCR5-MODIFIED CD34+ CELLS HEMATOPOIETIC STEM/PROGENITOR CELLS Huangfu, Danwei IN HIV-1 (R5) INFECTED SUBJECTS Developmental Biology Program, Memorial Sloan Stan, Rodica , Cardoso, Angelo , Krishnan, Amrita , Kettering Cancer Center, New York, NY, U.S. 1 1 1 Kim, Teresa , Torres-Coronado, Monica , Gardner, 1 1 Agnes , Killion, Salena , Holmes, Michael , Lee, Gary , My laboratory is interested in understanding pancreatic 2 2 2 1 Greengard, Judy , Dubois-Stringfellow, Nathalie , development and disease through applying genetic ap- 2 1 Win, Sandra , Pushkin, Richard , Lalezari, Jay , Mills, proaches in human pluripotent stem cells (hPSCs), in- 3 3 3 Anthony , Mitsuyasu, Ronald , Conner, Ed and Zaia, cluding embryonic and induced pluripotent stem cells 5 4 2 John 1 (hESCs and hiPSCs). Combining CRISPR/Cas-mediated 2 1 City of Hope, Duarte, CA, U.S., Sangamo gene editing and stem cell technologies, our reverse ge- netics approach has revealed the roles of key transcrip- Therapeutics, Richmond, CA, U.S., Quest Clinical tion factors pancreatic development and diabetes. Using 3 Research, San Francisco, CA, U.S., Mills Clinical the forward genetics approach, we performed a number 4 Research, Los Angeles, CA, U.S., University of of genome-wide knockout screens for identification of 5 California, Los Angeles CARE Center, Los Angeles, developmental regulators. We also applied both forward CA, U.S. and reverse genetic approaches to our stem-cell based system for study of epigenetic mechanisms underlying We developed a novel therapeutic strategy for HIV-1 in- lineage differentiation focusing on DNA methylation. To- fection by engineering HIV-resistant immune cells via gether our findings establish the use of hPSCs as a genet- gene editing of autologous hematopoietic stem/progen- ic model system for studying human disease and develop- itor cells (HSPC). A Zinc Finger Nuclease (ZFN) mRNA mental mechanisms. construct was designed to selectively disrupt the chemo- kine receptor 5 (CCR5), a co-factor required for HIV-1 in- fection of human cells, in HSPC. ZFN-CCR5-HSPC were manufactured and infused as an autologous “transplant” THURSDAY, 21 JUNE, 16:00 – 18:00 after busulfan conditioning. Preliminary results are now available from the ongoing safety/feasibility clinical trial CONCURRENT IIB: NEURAL (NCT02500849). R5-tropic HIV-1 infected subjects who were aviremic and had suboptimal CD4 counts (≥200 DEVELOPMENT and ZFN-HSPC products were manufactured for 3 sub- Melbourne Room 2, Level 2 jects in each cohort. The products had between 2.15E+06 and 11.5E+06 CD34+ cells/kg body weight. The median 16:05 – 16:30 CCR5 disruption evaluated by MiSeq was 20% indels for the investigational products manufactured for Cohort 1 REGULATION OF NEURAL STEM/PROGENITOR and 25% for Cohort 2. Median exposure to busulfan (AUC) CELL FATE DURING NEOCORTICAL was 9,340 µmol*min in Cohort 1 and 12,773 µmol*min in DEVELOPMENT Cohort 2. All subjects received the anticipated number of Gotoh, Yukiko busulfan doses, except one in Cohort 1, who metabolized busulfan slowly and achieved the target AUC with the test University of Tokyo, Japan dose (3.2 mg/kg). Subjects in Cohort 1 engrafted by Day +15 (ANC ≥ 500/µl for 3 consecutive days), and those in A fundamental question in understanding tissue develop- Cohort 2 engrafted by Day +14. Only one subject had a ment is how resident stem cells or multipotent progenitors platelet count below 20k/µl (Cohort 2) at Day +11. The give rise to the various cell types in appropriate numbers CD4 counts dropped significantly in all subjects post mo- and at the right locations to achieve tissue organization. bilization and collection, and then had a delayed recovery Neural stem/progenitor cells (NPCs) in the mammalian to the levels prior to mobilization. This is the first-in-hu- neocortex initially divide symmetrically to increase their man use of ZFN genome editing of HSPC, and preliminary pool size (expansion phase). They then divide asymmet- data show that conditioning with busulfan and infusion of rically and give rise to neuronal and glial cell types in a autologous CCR5-ZFN-modified HSPC are safe and well region- and developmental stage-dependent manner and tolerated in HIV-infected subjects. with high precision (neurogenic and gliogenic phases, respectively). We have previously shown that Polycomb Funding Source: This study is funded through a stra- group (PcG) complex and high mobility group A (HMGA) tegic partnership between California Institute for Re- proteins play pivotal roles in driving the fate switches of generative Medicine (CIRM), grant SP3A 07536, and NSCs associated with the transition from the neurogenic Sangamo Therapeutics. phase to the gliogenic phase. At this meeting, we would like first to focus on how these and other proteins control the fate of NPCs. Second, we will address the mechanisms 128 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS underlying the transition from the expansion phase to the the SVZ and numbers of transit-amplifying progenitors in neurogenic phase and discuss their potential role in psy- ontogeny and phylogeny. Furthermore, the role of Akna chiatric diseases such as autism spectrum disorder. has broader relevance beyond the developing brain as it is necessary for dissociation of junctional complexes in oth- 16:30 – 16:45 er true epithelial cells undergoing EMT. Akna is required for mesenchymal transformation and thus, highlights the AKNA, A NEW CENTROSOMAL PROTEIN importance of centrosomal microtubule organization in REGULATES EMT-LIKE FEATURES OF other contexts of development and cancer. NEUROGENESIS BY MICROTUBULE ORGANIZATION 16:45 – 17:00 1 1 Camargo Ortega, Germán D. , Falk, Sven , Johansson, MAKE DO AND MAKE NEW: HOW ZEBRAFISH 3 1 Pia , Peyre, Elise , Sahu, Sanjeeb , Broix, Loic , De RAPIDLY REGENERATES SPINAL CORD 2 2 4 1 Juan Romero, Camino , Draganova, Kalina , Vinopal, INJURY Stanislav , Geerlof, Arie , Feederle, Regina , Shao, 5 6 6 2 1 2 Wei , Shi, Song-Hai , Hauck, Stefanie , Bradke, Frank , Kaslin, Jan , Vandestadt, Celia , Castillo, Hozana , 7 6 5 7 2 2 2 4 8 Borrell, Victor , Tiwari, Vijay , Huttner, Wieland , Khabooshan, Mitra , Li, Mei , Hamimi, Mustafa , 9 3 9 2 Wilsch-Braeuninger, Michaela , Nguyen, Laurent and Schulze, Keith and Anko, Minna-liisa 2 Goetz, Magdalena 1 1 Monash University, Australian Regenerative 2 1 Helmholtz Zentrum Muenchen and University of Medicine Institute, Melbourne, Australia, Australian 2 Munich, Germany, University of Liege, Belgium, Regenerative Medicine Institute, Melbourne, VIC, 3 3 Institute of Molecular Biology, Mainz, Germany, Australia, Monash Micro Imaging (MMI), Melbourne, 4 Universidad Miguel Hernandez, Alicante, Spain, Australia 5 Center for Neurodegenerative Diseases, Bonn, Germany, Helmholtz Zentrum Muenchen, Munich, Zebrafish have a remarkable capacity to regenerate fol- 6 lowing spinal cord injury. While many factors controlling 7 Germany, Memorial Sloan Kettering Cancer Center, neurogenesis have been identified, the cellular mecha- 8 New York, U.S., previously at Institute of Molecular nisms regulating global neural regeneration are largely Biology, Mainz, Germany, Max Planck Institute unknown. We used in vivo imaging to pin-point specific 9 of Molecular Cell Biology and Genetics, Dresden, cells and signals that control spinal cord regeneration in Germany zebrafish. Surprisingly, we identified two temporally and mechanistically distinct waves of cellular regeneration in Understanding mechanisms regulating neural stem cell the spinal cord. The initial wave of regeneration relying (NSC) homeostasis and their fate commitment is funda- on cell migration of neural precursors to the lesion site, mental for their efficient manipulation and future use in enabling rapid functional recovery, and the activation of regenerative medicine. Our laboratory interrogates path- quiescent neural stem and progenitor cells (NCSs). This is ways regulating NSC self-renewal or differentiation into then followed by the second wave of regeneration which transit-amplifying progenitor cells by genome-wide ex- largely driven by regenerative neurogenesis. Neurogene- pression analysis of subsets of NSCs in the developing sis compensates for both the loss of tissue at injury site as and adult brain. These studies have proven successful for well as the cells depleted from proximal areas due to ear- the identification of novel regulators of neurogenesis and ly migration. Furthermore, we find that inflammation and brain folding also key in direct neuronal reprogramming. leukocytes play a critical role in differentially regulating Here we choose to examine the role of the putative AT- cell recruitment and activation of NSCs after injury. The hook containing transcription factor Akna as we found it two waves of regeneration demonstrate how the zebraf- to have elevated mRNA expression in differentiating NSCs ish are able to rapidly regain motor function after com- (generating transit-amplifying progenitors) while self-re- plete ablation, but also gradually replenish lost tissue over newing NSCs have lower expression levels. We show that time. Taken together, our data suggest that inflammation Akna is instead a novel bona fide centrosomal protein, driven recruitment of neural precursors play an unantici- mainly localizing at subdistal appendages of the moth- pated role in neural repair. er centriole where it is necessary and sufficient to confer microtubule organizing activity via microtubule anchor- Funding Source: This work was supported by a NHM- ing and to promote microtubule nucleation to the inter- RC project grant. phase centrosome. We show the importance of this pro- cess in the epithelial-mesenchymal transition (EMT)-like delamination of NSC subtypes thereby promoting cells to move towards the subventricular zone (SVZ) where tran- sit-amplifying progenitors reside and remain there until Akna levels decrease again, concomitant with the loss of centrosomal MTOC activity in mature neurons. Together with a similar function in human cerebral organoids, our work demonstrates a novel mechanism regulating NSC maintenance versus differentiation, serving to expand 129

THURSDAY 21 JUNE 2018 17:00 – 17:15 The switch between quiescence and proliferation is cen- NEW INSIGHTS ON HUMAN CORTICAL tral for neurogenesis and its alteration is linked to neu- rodevelopmental disorders such as microcephaly. Howev- DEVELOPMENT AND MICROCEPHALY USING er, intrinsic mechanisms that reactivate Drosophila larval SINGLE ORGANOID AND SINGLE CELL neural stem cells (NSCs) to exit from quiescence are not RNASEQ well established. Here we show that the spindle matrix Elkabetz, Yechiel complex containing Chromator (Chro) functions as a key intrinsic regulator of NSC reactivation downstream of ex- Genome Regulation, Max Planck Institute for trinsic insulin/insulin-like growth factor signalling. Chro Molecular Genetics, Berlin, Germany also prevents NSCs from re-entering quiescence at later stages. NSC-specific in vivo profiling has identified many Methods for deriving cortical progenitors from PSCs are downstream targets of Chro, including a temporal tran- highly variable due to the immense complexity of cortical scription factor Grainy head (Grh) and a neural stem cell cell and identity and the lack of efficient readout for the quiescence-inducing factor Prospero (Pros). We show desired cortical starting population. We generated organ- that spindle matrix proteins promote the expression of oids (3D) and rosettes (monolayers) and systematically Grh and repress that of Pros in NSCs to govern their reac- compared inhibitor-free, dual SMAD inhibition, WNT inhi- tivation. Our data demonstrate that nuclear Chro critically bition and combined dual SMAD and WNT inhibition and regulates gene expression in NSCs at the transition from performed extensive RNA-Seq for multiple, individually quiescence to proliferation. analyzed organoids and single cell RNA-Seq alongside comprehensive cellular and cytoarchitectural analysis for early and late derived cultures.  PCA combined with en- 17:30 – 17:55 richment analysis with human brain transcriptomic data- USING PLURIPOTENT STEM CELLS TO sets reveals that dual SMAD inhibition promotes thalamic DECIPHER HUMAN-SPECIFIC MECHANISMS OF and midbrain/hindbrain fates, while combined dual SMAD BRAIN DEVELOPMENT and WNT inhibition exclusively specifies dorsal pallium progenitors that ultimately develop into the four lobes Vanderhaeghen, Pierre of the neocortex. On the other hand, the medial pallium University of Brussels ULB, Belgium (archicortex) that constitutes the cortical hem and that later develops into the hippocampus can be well induced The cerebral cortex is the most complex structure in our by both treatments and also quite prominent transcrip- brain, and has undergone major and rapid complexifica- tionally in inhibitor-free derived organoids. Cortical deep tion during recent evolution, mostly related to increased and upper layer markers eventually appear in all deriva- neuronal number and connectivity. Pluripotent stem cells tion methods, due to their expression in non-cortical ar- constitute a promising tool for the modelling of human eas in vivo - proving them problematic for demonstrating brain development and diseases. Here we will describe cortical fate. Nonetheless, only under combined inhibi- how corticogenesis from pluripotent stem cells, whether tion, cortical layers including the important CUX2 upper in vitro or using xenotransplantation, can be used to iden- layer marker appears homogeneous. We show that only tify novel molecular and cellular mechanisms linking hu- neocortical progenitors (derived by combined inhibition) man brain development and evolution, from neurogenesis demonstrate early enhanced Notch activation and robust to synaptogenesis. ability to radially organize, and propose that these two features - when overlapping - serve well as an efficient readout for successful transition towards neocortical, but not more posterior brain starting populations. Strikingly, THURSDAY, 21 JUNE, 16:00 – 18:00 organoids harboring microcephaly a centrosomal muta- tion displays specific loss of neocortical cells and massive CONCURRENT IIC: ROAD TO THE CLINIC 1 apoptosis in these Notch active radially organized re- gions only if derived by exclusively under triple inhibition Room 219/220, Level 2 - demonstrating neocortex-specific cell death. Thus, com- bined inhibition is indispensable for standardized model- 16:05 – 16:30 ing of corticogenesis and demonstrating cortex-specific CAR T CELL THERAPY: THE CD19 PARADIGM defects associated with microcephaly. AND BEYOND 17:15 – 17:30 Wang, Xiuyan Memorial Sloan Kettering Cancer Center, U.S. AN INTRINSIC MECHANISM CONTROLS REACTIVATION OF NEURAL STEM CELLS BY Chimeric Antigen Receptor modified T cells targeting SPINDLE MATRIX PROTEINS CD19 have demonstrated remarkable potency in B cell Wang, Hongyan malignancies. Advances in the selection of optimal T cells and genetic engineering are poised to broaden T-cell- Duke-National University of Singapore (NUS) Medical based therapies and foster new applications in infectious School, Singapore diseases and autoimmunity. We recently demonstrated 130 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS that the targeting of CD19-specific CAR to the T-cell re- er management of arrhythmias, human cardiomyocyte ceptor α constant (TRAC) locus results in more uniform grafts can induce similar improvements in human hearts. CAR expression in human peripheral blood T cells, and enhances T-cell potency, with edited cells vastly outper- 16:45 – 17:00 forming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukemia. We are presently THE ADULT HUMAN EPCAM+CD24+CD133+ scaling up the cGMP manufacture of TRAC-CAR T cells CHOLANGIOCYTE AS A BIPOTENTIAL to test their safety and efficacy in a clinical trial where re- HUMAN HEPATIC PROGENITOR AND sponses and toxicities will be compared with those en- TRANSPLANTABLE REGENERATIVE THERAPY countered with current CAR therapies. In addition, CAR T FOR BILIARY DISEASE cells can be generated in vitro from pluripotent stem cells 2 1 2 as an alternative to manipulating mature T cells, offering Hallett, John M. , Lu, Wei-Yu , Ferrira-Gonzalez, Sofia , 2 2 2 potential access to an unlimited source of therapeutic T Man, Tak Yung , Thomson, John , O’Duibhir, Eoghan , 2 2 3 lymphocytes. We have demonstrated that iPSC-derived T Gadd, Victoria , Dwyer, Benjamin , Thirlwell, Kayleigh , 3 2 2 cells expressing a CAR can eradicate tumors in vivo, pro- Fraser, Alasdair , Hay, David , Oniscu, Gabriel and viding a foundation for further exploiting the potential of Forbes, Stuart 2 self-renewing stem cells to engineer therapeutic T cells. 1 Scottish Centre for Regenerative Medicine, University The combination of iPSC technology and immune engi- of Edinburgh, U.K., University of Edinburgh, U.K., 2 neering may thus provide an opportunity to generate T 3 Scottish National Blood Transfusion Service, cells that uniquely combine favorable attributes including Edinburgh, U.K. antigen specificity, lack of alloreactivity, enhanced func- tional properties, and histocompatibility. There is no effective regenerative therapy for advanced biliary diseases. Liver transplantation can be curative 16:30 – 16:45 however demand exceeds organ availability. We have HUMAN ESC-CARDIOMYOCYTES RESTORE analysed EpCAM+CD24+CD133+ cholangiocytes as a transplantable cell therapy. Human livers deemed unsuit- FUNCTION IN INFARCTED NON-HUMAN able for organ transplantation were studied. EpCAM+C- PRIMATE HEARTS D24+CD133+ (triple +ve) cells represented 1% of the total Murry, Charles E. cholangiocyte yield. Following single cell isolation and Institute for Stem Cell and Regenerative Medicine, organoid culture, triple +ve cells had a greater colony forming ability than EpCAM +CD24 +CD133- (dual +) cells University of Washington, Seattle, WA, U.S. (1.67 relative increase p<0.01.). RNA seq analysis showed that triple +ve cells highly expressed Sox 9 and FoxA2. Much of the burden of heart disease arises from the heart’s inability to regenerate new myocardium after in- Fatty livers had increased numbers of K19 +ve cholangio- jury.  Pluripotent stem cell-derived cardiomyocyte grafts cytes (2.2% vs 0.62%) and showed upregulation of gene can remuscularize substantial amounts of infarcted myo- groups associated with positive regulation of cell prolif- cardium and beat in synchrony with the heart but in some eration (p=1.2x10-2) and extracellular matrix organisation settings cause ventricular arrhythmias.  It is unknown (p=2.7x10-3) in keeping with a cholangiocyte driven re- whether human cardiomyocytes can restore cardiac generative response to injury. Triple +ve cells were main- function in a physiologically relevant large animal model.  tained in culture for over 1 year, whilst dual +ve cells could Here we show that transplantation of 750 million cryopre- only be maintained for 15 weeks. Triple +ve cells could served human embryonic stem cell-derived cardiomyo- be differentiated towards a hepatocyte lineage (positive cytes (hESC-CMs) enhances cardiac function in macaque staining for CYP2D6 and albumin and negative for CK19) monkeys with large myocardial infarctions.  Infarction was unlike dual +ve cells which maintained a biliary pheno- induced by inflating a balloon catheter in the mid-LAD ar- type. The Krt19CreERMdm2fl/fl Rag2-/- Il2rg-/- mouse tery for 3 hours followed by reperfusion.  After induction was developed as a novel immunodeficient model of bili- of immunosuppression, hESC-CMs or vehicle control were ary injury and senescence. Induced mice had activation of delivered surgically via trans-epicardial injection.  One p21 and p53 in intra and extrahepatic bile ducts, increased month after hESC-CM transplantation, global left ventric- serum markers of biliary disease, fibrosis (1.44% vs 0.87% ular ejection fraction (LVEF; MRI) significantly improved p=0.005) and histological changes compared to controls. compared to vehicle-injected hearts, and by 3 months When one million triple +ve cells were transplanted into post-engraftment there was >90% recovery of function.  the Krt19CreERMdm2fl/fl Rag2-/- Il2rg-/- model of biliary Grafts averaged 11.6% of infarct size, formed electrome- disease all animals treated survived well to day 42 with chanical junctions with the host heart and by 3 months normalising liver biochemistry and liver histology with hu- contained 99% ventricular myocytes.  A subset of animals man cells engrafting within the biliary tract. Controls had experienced graft-associated ventricular arrhythmias, deranged liver biochemistry, necrotic and inflamed livers shown by electrical mapping to originate from a point- and reached clinical endpoints for euthanization. In con- source functioning as an ectopic pacemaker.  In conclu- clusion, triple +ve cells isolated from discarded human sion, remuscularization of the infarcted macaque heart livers, are clonogenic in vitro, functionally engraft and res- with human myocardium provides durable improvement cue a model of severe biliary disease, indicating a poten- in left ventricular function.  We predict that, with prop- tial future cell therapy. 131

THURSDAY 21 JUNE 2018 17:00 – 17:15 17:15 – 17:30 ISOLATION AND TRANSPLANTATION OF INTRACEREBRAL TRANSPLANTATION HUMAN PLURIPOTENT STEM CELL-DERIVED OF NEURAL STEM CELL LINE, NSI-566, IN MIDBRAIN DOPAMINERGIC PROGENITORS CHRONIC ISCHEMIC STROKE PATIENTS FOR INTO PARKINSONIAN RATS TREATMENT OF PARALYSIS de Luzy, Isabelle R. , Kauhausen, Jessica , Gantner, Johe, Karl K. , Zhang, Guangzhu , Li, Ying , Reuss, 2 2 1 1 2 Carlos , Niclis, Jon , Pouton, Colin , Thompson, James , Ulmer, John , Li, James , Xu, Shuangshuang , 3 4 4 5 3 2 5 Lachlan and Parish, Clare 2 Wang, Feng , Hazel, Thomas , Hand, Holly , Dai, 2 1 1 5 2 2 2 2 1 Stem Cells and Neural Development, Florey Institute Yiwu , Zhang, Hongtian , Hong, Peng , Liu, Nan , He, 2 2 2 2 of Neuroscience and Mental Health, Melbourne, Jianghong , Wu, Cuiying , Zhang, Ping , Feng, Huiru , 2 1 VIC, Australia, Florey Institute of Neuroscience and Lu, Xiangdong , Johe, Karl and Xu, Ruxiang 2 2 Mental Health, Melbourne , Australia, Centre for 1 Neuralstem, Inc., Germantown, MD, U.S., Affiliated 2 3 Translational NeuroMedicine, Copenhagen, Denmark, BaYi Brain Hospital, Army General Hospital of 4 Monash Institute of Pharmaceutical Sciences, People's Liberation Army (PLA), Beijing, China, Melbourne, Australia 3 Prism Clinical Imaging, Inc., Elm Grove, WI, U.S., 4 Medical College of Wisconsin, Milwaukee, WI, U.S., Human pluripotent stem cells are a promising tool for the 5 Suzhou Neuralstem Biopharmaceutical, Suzhou cellular replacement of degenerated ventral dopaminer- Industrial Park, China gic neurons (vmDA) in Parkinson’s disease. Despite the successful generation of bona fide vmDA neurons in vitro, Paralysis due to ischemic stroke is a major cause of pro- the asynchronous and heterogeneous nature of the differ- longed neurological disability world-wide and in partic- entiations results in transplants consisting of proliferative, ular in China and Asian countries. There are currently no immature and terminally differentiated cells. To ensure effective therapies to reverse the paralysis. We have inves- safety and maximal functional benefit of stem cell-de- tigated the feasibility and safety of transplanting human rived vmDA grafts it will be imperative to remove poorly neural stem cells to reverse the paralysis in stably hemipa- specified and potentially tumorigenic cells prior to clinical retic stroke patients. Neural stem cells are the precursor translation. Here, we have utilized two novel hESC knock- cells present in the neuroepithelium along the neuraxis in reporter lines expressing eGFP under the LMX1A and during mammalian fetal development. NSI-566, the inves- PITX3 promoters, enabling selective isolation of early and tigational product used in this study, is a stable cell line late VM progenitors by FACS following in vitro differenti- consisting of neural stem cells derived from a single hu- ation. For both reporter lines, Unsorted, GFP+ and GFP- man fetal spinal cord tissue, expanded only by epigenetic cells were transplanted into Parkinsonian nude rats. Only means with no genetic modification. This cell line is also animals receiving Unsorted or LMX1A-GFP+ cell grafts being tested in clinical trials in the U.S. for treatment of showed progressive improvements in motor function. amyotrophic lateral sclerosis (NCT01348451) and spinal Postmortem histological analysis revealed small grafts cord injury (NCT01772810). In a single-site, Phase I study, from PITX3-GFP+ cells, suggesting isolation of these late 3 cohorts (n=3/cohort) were transplanted with ascending progenitors was not compatible with cell survival and doses of NSI-566, which involved a one-time stereotactic, integration. In contrast, LMX1A-GFP+ grafts were highly intracerebral injection of 1.2×10 , 2.4×10 , or 7.2×10 cells.  7 7 7 enriched for vmDA neurons, and importantly excluded all Immunosuppression therapy with tacrolimus was main- 5-HT serotonergic neurons (known to underpin graft-in- tained for 28 days. All subjects had chronic motor stroke, duced-dyskinesia’s) as well as reduced proliferative pop- verified by MRI, initiated between 5 and 24 months prior ulations (that could lead to neural overgrowths). Grafts to surgery, with Modified Rankin Score of 2, 3, or 4 and derived from LMX1A-GFP+ cells innervate developmen- Fugl-Meyer Motor Score of 55 or less.  Safety was the pri- tally relevant targets whilst LMX1A-GFP- cell grafts largely mary objective. Changes in Fugl-Meyer Motor Scale, Mod- innervated non-dopaminergic nuclei. The identification of ified Rankin Scale, and NIH Stroke Scale were measured a target, such as LMX1A, to isolate vmDA progenitors ca- as secondary outcomes. Changes in FDG-PET, functional pable of surviving and integrating, while simultaneously MRI, and structural MRI were measured as exploratory out- eliminating unwanted cell populations is highly relevant, comes. Twelve-month clinical data of the combined nine given the rapid advancement of PSCs towards the clinic participants were analyzed using the Wilcoxon signed and the likely requirement for more stringent safety and rank test.  At the 12-Month Visit, compared to Baseline, the standardization measures. mean Fugl-Meyer Motor Score (FMMS, total score of 100) showed 15.6 points of improvement (p=0.0078), the mean Modified Ranking Score (MRS) 0.8 points of improvement (p=0.031), and the mean NIH Stroke Scale (NIHSS) 3.2 points of improvement (p=0.016). The stem cell treatment was well tolerated at all doses. Longitudinal MRI studies showed evidence of graft survival and cavity-filling in all 9 patients. There was no death or any serious adverse event related to the treatment. This result warrants further study with larger cohorts with a randomized control arm. 132 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 17:30 – 17:55 the current mismatch in shape, size and lifespan between A PIPELINE OF INNOVATIVE CELLULAR native organs and their in vitro counterparts hinders their even wider applicability. In this talk I will discuss some of MEDICINES FOR CURRENTLY INTRACTABLE, our ongoing efforts in developing programmable organ- ADVANCED-STAGE DISEASES oids that are assembled by guiding cell-intrinsic self-pat- Simmons, Paul J. terning through tissue engineering. Research and New Product Development, Mesoblast Limited, Melbourne, VIC, Australia 16:30 – 16:45 MECHANICS-GUIDED DEVELOPMENTAL Mesoblast’s allogeneic, ‘off-the-shelf’ mesenchymal lin- PATTERNING OF NEUROECTODERM TISSUE eage cell product candidates are being evaluated in a FROM HUMAN PLURIPOTENT STEM CELLS range of advanced-stage diseases with high, unmet med- ical needs, including cardiovascular diseases, musculo- Xue, Xufeng , Sun, Yubing , Resto Irizarry, Agnes , 2 3 1 skeletal disorders, immune-mediated and inflammatory Studer, Lorenz and Fu, Jianping 3 4 conditions and oncologic and hematologic diseases. The 1 Mechanical Engineering, University of Michigan, Ann Company has established what it believes is the industry’s Arbor, MI, U.S., University of Massachusetts, Amherst, 2 most clinically advanced and diverse portfolio of cell- MA, U.S., University of Michigan, Ann Arbor, MI, U.S., 3 based product candidates with three programs in Phase 3 4 Memorial Sloan-Kettering Institute, New York, NY, clinical studies. Consistent and durable clinical outcomes U.S. have been demonstrated across multiple difficult-to-treat diseases. Our lead mesenchymal precursor cell (MPC) Classic embryological studies have successfully applied product candidates under investigation are MPC-150-IM genetics and cell biology principles to understand embry- for advanced heart failure, MPC-06-ID for chronic low onic development. However, it remains unresolved how back pain due to disc degeneration and MPC-300-IV mechanics, as an integral part for shaping development, for biologic refractory rheumatoid arthritis and diabetic is involved in controlling tissue-scale cell fate patterning. nephropathy. The Phase 3 trial of Mesoblast’s allogene- Here we report a micropatterned human pluripotent stem ic mesenchymal stem cell product candidate MSC-100- (hPS) cell-based neuroectoderm developmental model, IV (remestemcel - L) in children with steroid refractory wherein pre-patterned geometrical confinement induces acute Graft versus Host Disease (a GVHD ) has success- emergent patterning of neuroepithelial (NE) and neural fully met the primary endpoint of Day 28 overall response plate border (NPB) cells, mimicking neuroectoderm re- (OR, complete + partial response ) rate. This represents gionalization during early neurulation. Specifically, mi- an important milestone towards delivering a potentially crocontact printing is utilized to generate circular shaped effective treatment for this very serious and life-threaten- hPS cell colonies with defined sizes. Neural induction of ing condition and a further step towards Mesoblast’s goal hPS cell colonies leads to autonomous regionalization to have the first industrially manufactured allogeneic stem of NE and NPB cells, with PAX6+ NE cells preferentially cell product approved in the United States. localized at colony central region and PAX3+, ZIC1+ and MSX1+ NPB cells concentrated at colony periphery, form- ing a concentric ring-shaped tissue sheet consistent with THURSDAY, 21 JUNE, 16:00 – 18:00 neuroectoderm patterning. Importantly, strong correla- tions between spatial regulations of cell shape, cytoskel- etal contractility and BMP activity are observed during CONCURRENT IID: TISSUE emergent neuroectoderm patterning of hPS cell colonies. ENGINEERING Using microcontact printing to obtain patterned single Room 212/213, Level 2 hPS cells with prescribed spreading areas as well as a cus- tom designed microfluidic device for stretching hPS cell Sponsored by eLife Sciences colonies, we further show that cell shape and mechanical force can directly activate BMP-SMAD signaling and thus 16:05 – 16:30 repress NE but enhance NPB differentiation. All together, ORGANOID DEVELOPMENT BY DESIGN we show that autonomous patterning of neuroectoderm tissue with proper NP and NPB regionalization emerges Lutolf, Matthias P. de novo as the tissue physically takes shape and self-as- École Polytechnique Fédérale de Lausanne (EFPL), semble in pre-patterned geometrical confinements. Switzerland Self-organization of morphogenetic cues, including cell shape and cytoskeletal contractility, could provide posi- Organoids form through poorly understood morphoge- tional information and directly feed back to mediate BMP netic processes in which initially homogeneous ensembles activity and thus dictate spatial regulations of NE and of stem cells spontaneously self-organize in suspension or NPB lineage commitments during neuroectoderm pat- within permissive three-dimensional extracellular matri- terning. This study provides a novel hPS cell-based mod- ces. Yet, the absence of virtually any predefined pattern- el to understand the biomechanical principles that guide ing influences such as morphogen gradients or mechan- ical cues results in an extensive heterogeneity. Moreover, 133

THURSDAY 21 JUNE 2018 neuroectoderm patterning, thereby useful for studying cle organoid-like platform for complex disease modelling, neural development and diseases. regenerative medicine and drug development. Funding Source: National Institutes of Health Funding Source: EU FP7 grant 602423 (PluriMes), IMI (R21 EB017078 and R01 EB019436), National Sci- grant 115582 (EBiSC), BBSRC, Fundació La Marató de ence Foundation (CMMI 1129611, CBET 1149401, and TV3, MuscularDystrophyUK and European Research CMMI 1662835), and American Heart Association Council. F.S.T. is funded by an NIHR Academic Clinical (12SDG12180025). Fellowship. 16:45 – 17:00 17:00 – 17:15 THREE-DIMENSIONAL IPSC-DERIVED HUMAN BIO-ENGINEERING TRANSPLANTABLE HUMAN ARTIFICIAL SKELETAL MUSCLES MODEL VASCULARISED LIVER ORGANOIDS MUSCULAR DYSTROPHIES AND ENABLE Yap, Kiryu K. , Gerrand, Yi-Wen , Taylor, Caroline , 2 1,2 1 MULTILINEAGE TISSUE ENGINEERING Poon, Christopher , Kramer, Anne , Pera, Martin , Yeoh, 1 3 4 3 1 Tedesco, Francesco Saverio , Maffioletti, Sara , Sarcar, George , Morrison, Wayne 1,2,5 and Mitchell, Geraldine 1,2,5 2 2 Shilpita , Henderson, Alexander , Mannhardt, Ingra , 1 O’Brien Institute, Department of St Vincent’s 2 3 Pinton, Luca , Moyle, Louise , Steele-Stallard, Heather , Institute, Fitzroy, VIC, Australia, University of 2 2 2 2 4 2 Cappellari, Ornella , Wells, Kim , Ferrari, Giulia , Melbourne Department of Surgery at St Vincent’s 4 2 2 Mitchell, Jamie , Tyzack, Giulia , Kotiadis, Vassilios , Hospital Melbourne, Fitzroy, VIC, Australia, Harry 2 3 2 2 2 Khedr, Moustafa , Ragazzi, Martina , Wang, Weixin , Perkins Institute for Medical Research and Centre for Duchen, Michael , Patani, Rickie , Zammit, Peter , Medical Research, University of Western Australia, 2 2 5 4 Wells, Dominic and Eschenhagen, Thomas 3 Perth, Australia, The Jackson Laboratory, Bar Harbor, 4 5 1 Department of Cell and Developmental Biology, ME, U.S., Australian Catholic University, Fitzroy, VIC, University College London, U.K., University College Australia 2 3 London, U.K., University Medical Center Hamburg Eppendorf (UKE), Hamburg, Germany, Royal Bio-engineered liver organoids offer a regenerative alter- 4 5 Veterinary College, London, U.K., King’s College native to donor-derived organ transplantation and a plat- form for drug testing and disease modelling. Transplant- London, U.K. able liver organoids with intrinsic micro-vasculature were Generating artificial human skeletal muscle is instrumental developed using human liver progenitor cells (LPC) as a for investigating muscle pathology and therapy. Howev- parenchymal cell source, liver sinusoidal endothelial cells er, most bioengineering platforms are challenged by the (LSEC) to generate liver-specific vasculature, and adi- limited expansion potential and differentiation ability of pose-derived mesenchymal stem cells (ASC) as support tissue-derived myogenic cells. Moreover, although there cells. Cells in a 10:10:1 ratio (LPC:LSEC:ASC, total 1 million is an increasing need to develop clinically-relevant, multi- cells) were mixed in a human liver-derived extracellular lineage, patient-specific models, no such isogenic human matrix hydrogel and seeded into a porous polyurethane skeletal muscle model has been derived to date. These scaffold (NovoSorbTM, 3mm diameter, 0.8mm thickness, obstacles negatively impact on the translational potential bio-absorbable and FDA-approved). Between day 1 and 3 of these platforms to develop novel therapies for muscle in culture, organoids upregulated key liver genes (HNF4α, diseases. To overcome these limitations, we generated albumin, HGF, CYP3A4). Functional assays demonstrated three-dimensional (3D) artificial skeletal muscle tissue albumin secretion, urea production, bile acid excretion, from human pluripotent stem cells, including induced plu- and CYP3A4 activity, which all increased over time. Capil- ripotent stem cells (iPSCs) from patients with Duchenne, lary and bile canaliculi-like structures were also observed. limb-girdle and congenital muscular dystrophies. 3D Concurrent organoids were developed by replacing liv- skeletal myogenic differentiation of pluripotent cells was er gel with Matrigel (a commonly used hydrogel derived induced within hydrogels under tension to provide align- from mouse sarcoma extracellular matrix). In all gene and ment. Artificial muscles recapitulated key characteristics functional parameters, liver gel organoids outperformed of human skeletal muscle tissue and could be implanted Matrigel organoids. For in vivo studies, liver gel organoids into immunodeficient mice. Importantly, pathological cel- were transplanted into an \"in vivo bioreactor\" (a protective lular hallmarks of severe (and currently incurable) muscu- chamber surgically placed around an artery/vein to induce lar dystrophies could be modeled with higher fidelity using capillary sprouting and create a highly vascularised space) this 3D platform than standard bi-dimensional cultures. created in the groin of immuno-deficient SCID mice. At 14 Finally, we show generation of fully human iPSC-derived days, functional human blood vessels perfused by mouse complex multilineage models, containing key isogenic blood and clusters of differentiating human hepatocytes cellular constituents of normal skeletal muscle, including were present. Measurable levels of human albumin were vascular endothelial cells, pericytes and motor neurons. secreted into the mouse circulation by transplanted or- These results lay the foundation for a human skeletal mus- ganoids (n=4, human albumin in mouse serum 25.3±9 µg/ mL). In conclusion, functional human vascularised liver or- ganoids can be engineered using a combination of LPC/ LSEC/ASC, enhanced by liver-specific extracellular matrix. 134 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS Notably, all components were human-derived. Proof-of- 17:30 – 17:55 concept transplantation studies indicate organoid surviv- ENGINEERING HUMAN TISSUES FOR al/function in immuno-deficient mice. Longer time-points and transplantation in a humanised mouse model of liver REGENERATIVE MEDICINE AND STUDY OF disease are currently underway to assess the therapeutic DISEASE efficacy of this organoid approach. Vunjak-Novakovic, Gordana, Ronaldson-Bouchard, Kacey, O’Neill, John and Liu, Bohao 17:15 – 17:30 Columbia University, New York, NY, U.S. CO-EMERGENCE OF MULTIPLE RESPIRATORY HINDBRAIN POPULATIONS FROM HUMAN Tissue engineering is becoming increasingly successful PLURIPOTENT STEM CELLS with authentically representing the actual environmen- tal milieu of the development, regeneration and disease. Butts, Jessica, Mihaly, Eszter and McDevitt, Todd The classical paradigm of tissue engineering involves an Gladstone Institutes, San Francisco, CA, U.S. integrated use of human stem cells, biomaterial scaffolds (providing a structural and logistic template for tissue for- The hindbrain is the most conserved central nervous sys- mation) and bioreactors (providing environmental con- tem structure in vertebrates and is critical to the control trol, and dynamic sequences of molecular and physical of autonomic function, including respiration. V2a and V0V regulatory factors). This biomimetic approach is designed interneurons (IN) as well as chemosensitive neurons are to recapitulate the critical aspects of tissue development, critical populations in the phrenic circuit that provide in- regeneration and disease. Living human tissues are being put to respiratory control centers. Damage to these pop- engineered from various types of human stem cells, and ulations by cervical spinal cord injury or disease (i.e. ALS) tailored to the patient and the condition being treated. A dramatically diminishes respiration. There are currently no reverse paradigm is now emerging with the development in vitro sources to study their development and functional of platforms for modeling of integrated human physiolo- interactions, thus, the objective of this work was to co- gy, using micro-tissues derived from human iPS cells and emerge critical hindbrain populations from human plurip- functionally connected by vascular perfusion. In all cases, otent stem cells (PSC). We have previously described a the critical questions relate to our ability to recapitulate protocol to generate V2a IN from PSC using retinoic acid, the cell niches, using bioengineering tools. This talk will sonic hedgehog (Shh), and Notch inhibition. HOX gene discuss some recent advances in regenerative engineer- expression from single cell RNA sequencing on day 17 ing of whole organs (lung, heart, bone) and recapitulation cultures revealed the IN population was primarily hind- of human physiology using microphysiological platforms brain and cervical (HOXA3, HOXB1, HOXB6). K-means with interconnected human tissues derived from the pa- clustering of 15 principle components identified 5 distinct tient’s iPS cells. clusters that described the cell population. Transcription factors expressed in the hindbrain were among the top 10 genes expressed in the 3 largest clusters: PHOX2A/ PHOX2B (chemosensitive neurons), LHX5/PAX2 (V0V THURSDAY, 21 JUNE, 16:00 – 18:00 IN), and CHX10/SOX14 (V2a IN). To modulate relative proportions of co-emerged V0V and V2a IN, the con- CONCURRENT IIE: MUSCLE STEM CELLS centration of purmorphamine (pur), a Shh agonist, was Room 203/204, Level 2 varied analogous to signaling in the developing neural tube, where a ventral-to-dorsal gradient of Shh gives rise to V0v (dorsal, low Shh) and V2a (ventral, high Shh) IN. 16:05 – 16:30 In in vitro cultures, low pur concentration (10nM) result- INTRINSIC AND EXTRINSIC REGULATION OF ed in higher LHX5 (10.48%) and lower CHX10 (3.87%) ex- THE MUSCLE STEM CELL NICHE pression while higher concentration (100nM) resulted in lower LHX5 (2.7%) and higher CHX10 (11.43%) expression, Tajbakhsh, Shahragim consistent with developmental Shh signaling. To inter- Pasteur Institute, Paris, France rogate how these populations organize and mature, the differentiation was performed in suspension. The aggre- The microenvironment is critical for the maintenance gates doubled in size after 100 days in culture to become of stem cell populations, and it can be of cellular and >1mm in diameter. Additionally, NeuN+, Neurofilament+, non-cellular nature, including secreted growth factors and and VGlut2+ neurons were identified throughout the ag- extracellular matrix (ECM) as well as intrinsic regulators. gregate with GFAP+ glia along the edges. Together, this Skeletal muscle satellite (stem) cells are quiescent during study reports a tunable system to co-emerge multiple re- homeostasis and they are mobilised to restore tissue func- spiratory hindbrain populations from human PSC, which tion after muscle injury. Although certain signalling path- can be further evaluated to model disease and develop- ways that regulate quiescence have been identified, the ment. mechanisms by which niche molecules regulate stem cell properties remain largely unknown. We have identified Funding Source: California Institute of Regenerative Medicine (CIRM), The Roddenberry Foundation. Notch signalling as a major regulator of the muscle stem cell niche. Specifically, Notch/RBPJ-bound regulatory ele- 135

THURSDAY 21 JUNE 2018 ments are located adjacent to specific collagen genes in 16:45 – 17:00 adult muscle satellite cells. These molecules are linked to STEM CELL-MACROPHAGE INTERACTIONS the ECM and constitute putative niche components. No- tably, satellite cell-produced collagen V (COLV) is a critical REGULATE VERTEBRATE MUSCLE component of the quiescent niche, as conditional deletion REGENERATION: INSIGHTS FROM ZEBRAFISH of Col5a1 leads to anomalous cell cycle entry and differen- Ratnayake, Dhanushika and Currie, Peter tiation of satellite cells. Strikingly, COLV, but not collagen I and VI, specifically regulated quiescence through Calci- Australian Regenerative Medicine Institute, Clayton, tonin receptor mediated activity, therefore, a Notch/COLV VIC, Australia signalling cascade cell-autonomously maintains the stem cell quiescent state, and raises the possibility of a similar Studying muscle regeneration through in vivo imaging has the potential to reveal phenomena that might not be reciprocal mechanism acting in diverse stem cell popu- lations. In other studies, we have identified a microRNA observed by in vitro studies. Zebrafish are a key verte- brate model system for high-resolution live imaging due pathway that is regulated by Notch, and it required for stabilizing muscle stem cells in their niche. to their optical transparency and the genetic tools avail- able for lineage analysis. Here we use the zebrafish sys- tem to characterize stem cell-macrophage interactions 16:30 – 16:45 during muscle regeneration. Nitroreductase-mediated ERBB3 AND NGFR MARK A DISTINCT genetic ablation of macrophages prior to injury resulted SKELETAL MUSCLE PROGENITOR CELL IN in a muscle regeneration deficit, due at least in part to a HUMAN DEVELOPMENT AND HPSCS failure of stem cell migration into the wound site. Remark- ably, in vivo cell tracking identified distinct macrophage 2 2 Hicks, Michael , Hiserodt, Julia , Paras, Katrina , Xi, populations based on cell behaviour: a “transient” injury 1 2 2 2 Haibin , Young, Courtney , Saleh, Kholoud , Evseenko, responding population that migrate into the wound site 3 2 2 Denis , Spencer, Melissa , Van Handel, Ben and Pyle, and phagocytose cellular debris, making the environment April 2 conducive for repair; and a long-term injury responding 1 Microbiology, Immunology, and Molecular Genetics, macrophage population that “dwells” in the wound site University of California, Los Angeles, CA, U.S., throughout the repair process, continuously extending 2 University of California, Los Angeles, CA, U.S., cytoplasmic protrusions to physically interact with injury 3 University of Southern California, Los Angeles, U.S. responding stem and progenitor cells. We hypothesize that “dwelling” macrophages regulate regeneration by Human pluripotent stem cells (hPSCs) can be direct- providing environmental and positional information to ed to differentiate into skeletal muscle progenitor cells stem cells via these physical interactions, and are current- (SMPCs). However, the myogenicity of hPSC-SMPCs rela- ly focused on characterizing the signalling pathways that tive to human fetal or adult satellite cells remains unclear. mediate these processes. HPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite 17:00 – 17:15 cells. Utilizing RNA-SEQ, we identified cell surface recep- DEFINING HUMAN IN VIVO SKELETAL MUSCLE tors ERBB3 and NGFR that demarcate myogenic popu- DEVELOPMENT AND IN VITRO PLURIPOTENT lations, including PAX7 progenitors in human fetal devel- opment and hPSC-SMPCs. We demonstrated that hPSC STEM CELL MYOGENESIS AT SINGLE CELL skeletal muscle is immature, but inhibition of TGF-β sig- RESOLUTION naling during differentiation improved fusion efficiency, Xi, Haibin , Langerman, Justin , Sabri, Shan , Gonzalez, 2 2 1 ultrastructural organization, and expression of adult my- Karen , Young, Courtney , Fujiwara, Wakana , Mota, 2 2 2 osins. This enrichment and maturation strategy restored Andrea , Marzi, Julia , Liebscher, Simone , Van Handel, 4 3 4 dystrophin in hundreds of dystrophin-deficient myofibers Ben , Evseenko, Denis , Spencer, Melissa , Schenke- 5 2 5 after engraftment of CRISPR/Cas9-corrected Duchenne Layland, Katja , Plath, Kathrin and Pyle, April 2 4 2 muscular dystrophy hiPSC-SMPCs. Post-engraftment, hiPSC-SMPCs not fused with myofibers were re-isolated 1 Microbiology, Immunology and Molecular Genetics, from wild type or diseased muscle and single-cell RNA- University of California Los Angeles, CA, U.S., SEQ used to determine in vivo cell fate and maturation. 2 University of California Los Angeles, CA, U.S., The work provides an in-depth characterization of human 3 California State University Northridge, CA, U.S., developmental myogenesis, and identifies candidates that 4 Eberhard Karls University Tübingen, Germany, improve the in vivo myogenic potential of hPSC-SMPCs to 5 University of Southern California, Los Angeles, CA, levels equal to directly-isolated human fetal muscle cells. U.S. Funding Source: Eli and Edythe Broad Stem Cell Re- Skeletal muscle progenitor cells (SMPCs) derived from hu- search Center Postdoctoral Fellowship. man pluripotent stem cells (hPSCs) are promising sourc- es for regenerative medicine in treating muscle wasting disorders including muscular dystrophies and sarcopenia. Nevertheless, the current hPSC directed myogenic dif- ferentiation protocols result in highly heterogeneous cell 136 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS populations with immature SMPCs that are unsuitable for ing to the switch of muscle fiber type. To test the hypothe- clinical implementation. These drawbacks are reflective sis, TA cross-section staining and single myofiber isolation of insufficient SMPC specification in vitro, due to lack of and immunostaining indicated that BAMBI is detected knowledge of human skeletal myogenesis in vivo. Using in both myofiber and satellite cells. MCK-Cre-mediated single cell RNA-sequencing (scRNA-seq), we profiled hu- deletion of BAMBI in skeletal muscle leads to the spon- man limb myogenic cells from early week 5 embryos to taneous activation of satellite cells with the upregulation adulthood. We observed heterogeneity of in vivo SMPCs of Pax7 and MyoD. Long-term deletion of BAMBI causes at individual stages and more strikingly, across develop- the fiber type switch from TCA-based Type I fiber to gly- ment. We confirmed the dynamic expression pattern of colysis-based Type IIB fiber. Mice with the dysfunction of key myogenic transcriptional factors as well as unique the BAMBI gene in skeletal muscle also fails to regenerate cell surface marker expression in distinct SMPC popula- upon the CTX-induced muscle regeneration compared tions by IHC. Next, to define the in vitro SMPCs derived with the control mice. Further investigation will delve into from hPSCs, we generated a knock-in PAX7-GFP report- the molecular mechanism of BAMBI in fiber type switch er hPSC line using CRISPR/Cas9. We differentiated them, and satellite cell physiology and pathogenesis. sorted the GFP+ (PAX7+) cells at different time points, and performed scRNA-seq. Interestingly, we found that 17:30 – 17:55 the early wave of PAX7+ cells are mainly composed of SOX2+ neural lineage cells, both neurogenic and myo- IN VIVO GENE EDITING IN MUSCLES AND genic cells co-exist during intermediate period, whereas MUSCLE STEM CELLS the myogenic cells dominate at later differentiation time Wagers, Amy points. Similar to the in vivo cells, we observed extensive heterogeneity of the in vitro SMPCs. In mice myofibers are Harvard University, Cambridge, MA, U.S. known to be critical for early SMPC development. Thus, The in vivo delivery of genome modifying enzymes, in- we utilized our reporter cells to enrich PAX7+ SMPCs cluding CRISPR/Cas9 gene editing complexes, holds sig- during myogenic differentiation, cultured them in the ab- nificant promise for both therapeutic applications and sence or presence of myotubes, and re-isolated the PAX7+ functional genetic screening in muscle and other tissues. cells via FACS. Intriguingly, the PAX7+ cells cultured with In this context, delivery to endogenous tissue stem cells, myotubes display gene expression patterns resembling a including muscle satellite cells which provide an enduring transition from early embryonic to later fetal stage, such source of myofiber replacement in homeostasis and in re- as decreased PAX3 and increased COL15A1 expression. sponse to regenerative cues, is of particular interest. We Overall, our study comprehensively profiled the human previously developed an adenoassociated virus- (AAV-) myogenic cells across different stages during both in vivo based system for local and systemic delivery to skeletal development and in vitro hPSC differentiation. This work muscle of experimentally engineered programmable Cas9 can serve as a resource for advancing our knowledge of nuclease. Application of this system to delete specific human myogenesis and guide the generation of optimal gene sequences in a mouse model of Duchenne Muscular cells for translational applications in muscle diseases. Dystrophy (DMD) demonstrated simultaneous targeting in multiple organs of therapeutic interest, with restoration 17:15 – 17:30 of the mutated Dystrophin protein reading frame, recov- BAMBI-MEDIATED SIGNALING PATHWAY IS ery of muscle function, and establishment of a pool of INVOLVED IN SATELLITE CELLS QUIESCENCE modified muscle stem cells capable of participating in AND ACTIVATION subsequent muscle regenerative events. In recent work, we have further adapted this approach to enable templat- 1 1,2 1 Xie, Liwei , Yao, Xiangping , Chen, Shujie , Yu, ed sequence replacement via homology-directed repair, Taiyong , Yang, Gongshe and Xu, Guohuan 1 raising the possibility of accomplishing sequence-direct- 2 2 1 Guangdong Institute of Microbiology, Guangzhou, ed, systemically disseminated DNA replacement in vivo China, Northwest A and F University, Xian, China in postnatal muscle and muscle stem cells. We have also 2 demonstrated the capacity of AAV-delivered genome BAMBI is bone morphogenic protein and activin mem- modifying enzymes to target non-muscle progenitors in brane-bound inhibitor, which regulates cell proliferation distinct anatomical niches, indicating the robustness and and differentiation through the interaction with TGF-beta potential generalizability of this approach as a new exper- and Wnt/beta-catenin signaling pathway. Previous work imental alternative to conventional transgenic/knockout in C2C12 myoblast demonstrated the BAMBI is a key fac- mouse models and ex vivo transduction strategies. Taken tor regulating the differentiation. In present work, our together, these results suggest exciting new opportunities preliminary data suggest that BAMBI protein is detected for manipulating stem cell function experimentally and for both in the cytosolic fraction instead of the membrane of therapeutic intervention to achieve functional recovery of skeletal muscle and in satellite cells. Skeletal muscle-spe- disease-relevant gene products and promote endoge- cific knockout of BAMBI leads to upregulation of Pax7 and nous repair activity across organ systems. MyoD expression, followed with spontaneous activation of satellite cells, which lead us to generate the hypothe- sis that BAMBI-mediated signaling pathway is involved in regulating satellite cells quiescence and activation, lead- 137

FRIDAY 22 JUNE 2018 sion via transcriptional and epigenetic reprogramming. FRIDAY, 22 JUNE, 09:00 – 11:15 We have recently described a novel editing approach that exploits epigenetics and programmable DNA binding do- PLENARY IV: NEW TECHNOLOGIES IN mains to permanently silence gene expression. During my STEM CELL ENGINEERING talk, I will discuss critical aspects affecting efficiency of this novel approach and propose possible therapeutic tar- Plenary Room, Ground Level gets that may benefit from targeted epigenetic editing. Furthermore, I will extend on the power of genome ed- 09:00 – 09:25 iting to discuss a synthetic biology approach aiming at generating immune-stealth human stem cells, which may IN VIVO BRAIN ORGANOID MODEL OF provide an indefinite source of off-the-shelf cells for trans- VASCULARIZED AND FUNCTIONAL PSC- plantation. DERIVED HUMAN BRAIN ORGANOIDS Gage, Fred H., Mansour, Abed AlFattah, Gonçalves, 09:50 – 10:15 Tiago, Johnston, Stephen and Parylak, Sarah 3D EPIGENOME RECONFIGURATION IN BRAIN The Salk Institute for Biological Studies, La Jolla, CA, DEVELOPMENT AND NEURODEGENERATIVE U.S. DISEASE Stem cells have the remarkable ability to self-organize in Phillips-Cremins, Jennifer E. three-dimensional (3D) space into organ-like structures Perelman School of Medicine University of termed Organoids. By harnessing this property, research- Pennsylvania, Philadelphia, PA, U.S. ers have been able to create such organoids for several tissues that better recapitulate the complexity and phys- The Cremins Lab focuses on higher-order folding of the iological properties of tissues and organs. Despite many genome and how epigenetic marks work through long- reports describing the generation of human neural organ- range regulatory mechanisms to govern neural cell fate oids, the generation of vascularized and functional neural in the mammalian brain. Much is already known regard- organoid graft is not described yet. Here we describe the ing how transcription factors and epigenetic marks work generation of vascularized, and electrophysiologically ac- in the context of the linear genome to regulate neuronal tive, human cerebral-organoids by transplantation of or- development and function. Yet, severe limitations still ganoids grown in vitro to an adult mouse brain. Engraft- exist in our ability to apply this knowledge to engineer ed mice were viable, and exhibit long and high survival neuron fate at will or correct brain diseases in vivo. The rates. Moreover, histological and immunostaining analysis overarching goal of the Cremins lab is to obtain detailed revealed intact grafts with mature neurons, and extensive mechanistic understanding of how the genome is folded axonal trajectories from the implant to multiple regions and reconfigured during neural lineage commitment and of the host mouse brain. Importantly, live imaging on of synaptogenesis and how these folding patterns influence the implanted organoids using two-photon microscopy the specificity, maturation and pruning of neuronal con- revealed neuronal activity, and intensive vascular network nections in healthy mammalian brain development. We with active blood flow within the organoid. Moreover, our also study how the genome is misfolded in neurodegen- method creates opportunities for noninvasive recording erative disease and we develop tools to engineer 3D ge- of neuronal activity with high spatial and temporal reso- nome folding on demand. Addressing this knowledge gap lution deep within organoid-brain chimera. This powerful will provide an essential foundation for our long-term goal combination of in vitro 3D human neural structures, and to engineer the 3-D genome to control neural cell fate in an in vivo rich environment in the animal brain provides debilitating neurodevelopmental and neurodegenerative a promising novel approach with broad applications for diseases. degenerative and regenerative medicine. 10:15 – 10:40 09:25 – 09:50 WHOLE-ORGANISM CLONE-TRACING USING EXPLOITING TARGETED (EPI)GENOME SINGLE-CELL SEQUENCING EDITING FOR THERAPEUTIC APPLICATIONS van Oudenaarden, Alexander M. Lombardo, Angelo Hubrecht Institute-KNAW and University Medical San Raffaele Telethon Institute for Gene Therapy, Center, Utrecht, Netherlands Milan, Italy Embryonic development is one of the most crucial peri- The development of targeted technologies able to pre- ods in the life of a multicellular organism. A limited set of cisely edit the genome and its regulatory code is opening embryonic progenitors gives rise to all cells in the adult novel exciting perspectives for the treatment of inherit- body. Determining which fate these progenitors acquire ed and acquired diseases. These technologies hold the in adult tissue is a major challenge and requires the simul- promise of in situ correction of genetic defects, targeted taneous measurement of clonal history and cell-type at integration of exogenous transgene expression cassettes, single-cell resolution. Clonal history has traditionally been and fine-tuning modulation of endogenous gene expres- 138 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS quantified by microscopically tracking cells during devel- F-2088 opment, monitoring the heritable expression of genetical- GUIDED SELF-ASSEMBLY OF SOX2+SOX9+ ly encoded fluorescent proteins and, most recently, by uti- lizing next generation sequencing technology exploiting HUMAN LUNG PROGENITORS INTO SIMPLIFIED somatic mutations, transposon tagging, viral barcoding, DEVELOPMENTALLY INSPIRED TUBULAR and CRISPR/Cas9 genome editing strategies. Single-cell ARCHITECTURE transcriptomics on the other hand, provides a powerful Soleas, John P. technology platform for cell-type classification in an un- Institute of Biomaterials and Biomedical Engineering, biased manner. However, integrating both measurements University of Toronto, Canada for many single cells has been a major hurdle. Here, we present ScarTrace, a single-cell sequencing strategy that allows us to simultaneously quantify information on clon- F-3038 al history and cell type for thousands of single cells ob- PATIENT-DERIVED IPSC-BASED DRUG tained from different organs from adult zebrafish. Using DISCOVERY PLATFORM HIGHLIGHTS this approach we show that all blood cells in the kidney RAPAMYCIN AS A DRUG CANDIDATE FOR marrow arise from a small set of multipotent embryonic FIBRODYSPLASIA OSSIFICANS PROGRESSIVA progenitors that give rise to all blood cell types. In con- trast, we find that cells in the eyes, brain, and caudal tail fin (FOP) arise from many embryonic progenitors, which are more Hino, Kyosuke restricted and produce specific cell types in the adult tis- iPS Cell-Based Drug Discovery, Sumitomo Dainippon sue. Next we use ScarTrace to explore when embryonic Pharma Co., Ltd., Japan cells commit to forming either left or right organs using the eyes and brain as a model system. Lastly we monitor regeneration of the caudal tail fin and identify a subpop- 10:45 – 11:10 ulation of resident macrophages that have a clonal origin that is distinct from other blood cell types. We envision ISSCR DR. SUSAN LIM AWARD FOR that ScarTrace will have major applications in other exper- OUTSTANDING YOUNG INVESTIGATOR imental model systems to match embryonic clonal origin LECTURE: CONTROL HUMAN PLURIPOTENT to adult cell-type to ultimately reconstruct how the adult STEM CELL FATE USING CHEMICAL body was built from a single cell. APPROACHES 10:40 – 10:45 Chen, Shuibing Weill Cornell Medical College, New York, NY, U.S. POSTER TEASERS Human pluripotent stem cells (hPSCs) provide unlimited F-2048 starting material to generate differentiated cells that can be used to build a functional organ. Essential to this pur- BMP PATHWAY AS THE MASTER REGULATOR suit is an efficient way to differentiate hPSCs into specific OF THE SLOW CYCLING, CHEMO-RESISITANT types of mature cells. Cell-permeable small molecules that CANCER STEM CELLL COMPARTMENT IN can modulate the function of specific proteins provide a HUMAN EPITHELIAL CARCINOMA convenient and efficient approach to controlling stem/ progenitor cell fate. Our laboratory has an in house chem- Seyedasli, Naisana ical library containing 6,000 chemicals, including kinase University of Sydney, Australia inhibitors, signaling pathway regulators, nature products and FDA-approved drugs, and protein library containing F-2129 400 growth factors. Using high content and high through- MAPPING CELLULAR REPROGRAMMING VIA put screening approaches, we have identified a series of POOLED OVEREXPRESSION SCREENS WITH small molecules that control stem cell self-renewal, differ- PAIRED FITNESS AND SINGLE CELL RNA- entiation and reprogramming. In addition, we have identi- fied small molecules that direct hPSC differentiation into SEQUENCING READOUT certain cell types, including pancreatic endocrine cells, Parekh, Udit pancreatic ductal epithelial cells, cardiac SA nodal cells, University of California, San Diego, CA, U.S. trophoblast cells, and colonic organoids. Using hPSCs de- rived tissues or organoids, we have established several in vitro and in vivo platforms to study the role of genetic factors and environmental factors in the progression of diabetes, pancreatic cancer, colorectal cancer, and virus infection. Recently, we performed a high content chemi- cal screen using human embryonic stem cell derived cor- tical neuron progenitor cells (hNPCs). Hippeastrine hyd- robromide (HH) was discovered to inhibit ZIKV infection in hNPCs. The hit compound was further confirmed for its 139

FRIDAY 22 JUNE 2018 anti-ZIKV effect using human fetal-like forebrain organ- Most of our current knowledge of mammalian embryolo- oids and adult mouse model. This stem cell-based screen gy is derived from studies of the mouse embryo.  However, followed by validation using human forebrain organoids mammalian development involves substantial divergence and a mouse model identifies drug candidates for the in the mechanism and order of cell-fate allocations among treatment of ZIKV infection and ZIKV-related neurological species, and there has been a critical lack of information complications in fetal and adult patients. regarding human development due to the scarcity of hu- man embryo specimens.  Leveraging the self-organizing properties of human pluripotent stem (hPS) cells, re- cent developments of synthetic human entities with em- FRIDAY, 22 JUNE, 13:15 – 15:15 bryo-like features (SHEEFs) have sparked great interests in using such synthetic models to advance human em- CONCURRENT IIIA: ORGANOIDS bryology, embryo toxicology, and reproductive medicine.  IN MODELING DISEASE AND However, existing SHEEF systems rely on uncontrolled, DEVELOPMENT spontaneous organization and development of hPS cell cultures in ill-defined environments without any spatial Melbourne Room 1, Level 2 constraints.  Such lack of controllability and reproduc- Sponsored by Decibel Therapeutics ibility significantly hinder the full potential of the SHEEFs for their uses in embryo toxicological screening and drug 13:20 – 13:45 testing or mechanistic investigations of human embryo- genesis.  Herein, we report a novel microfluidic strategy INTESTINAL ORGANOIDS FOR CYSTIC to achieve programmable, scalable, robust synthesis of FIBROSIS MODELING SHEEFs using hPS cells.  By asymmetrically applying ag- onists and/or antagonists in a highly controlled manner Beekman, Jeffrey using microfluidics - both spatiotemporally and in terms University Medical Center Utrecht, Netherlands of dosage, we have successfully developed a hPS cell- based, synthetic embryological model of human post-im- We use intestinal organoids to model cystic fibrosis, a plantation development that recapitulates multiple em- life-shortening monogenetic diseases characterised by bryogenic events including amniotic cavity formation, aberrant epithelial ion and fluid transport due to CFTR amnion-epiblast patterning, primordial germ cell (PGC) mutations. Forskolin that raises cAMP and opens the specification, and development of the primitive streak CFTR ion channel induces rapid luminal fluid secretion with controlled anteroposterior polarity.  Enabled by this in rectal organoids, which leads to organoid swelling in novel microfluidic synthesis system, we further discover a fully CFTR-dependent manner. This assay can be used that the amnion, as a unique keystone during early human to quantitate individual CFTR function, and also individual embryo development, functions as a signaling center for response to novel pharmacotherapies (CFTR modulators) PGC specification and controls the proper developmental that restore CFTR function in a CFTR mutation and pa- sequence by triggering the onset of gastrulation from the tient-specific manner. Data will be presented that show posterior epiblast.  We envision that our microfluidic syn- correlations between in vitro forskolin-induced organoid thesis system for SHEEFs, given its controllability, scalabil- swelling and in vivo indicators of disease severity or ther- ity, and compatibility with live-cell imaging, will provide a apeutic response. We also developed a high throughput powerful synthetic embryological platform that open up screening assay to screen organoids from people with previously inaccessible phases of the human life cycle to rare CFTR mutations (n>170) for response to clinically experimental study. available CFTR modulators and >1400 FDA-approved drugs. On a whole, our data demonstrate that organoids Funding Source: National Institutes of Health (R01 recapitulate individual therapeutic response, and support DK089933), Natural Sciences and Engineering Re- that clinically annotated biobanks of individual stem cells search Council of Canada, and University of Michigan could be patient-friendly options to select optimal indi- Mechanical Engineering Startup Fund. vidualised therapies in progressive diseases. 13:45 – 14:00 PROGRAMMABLE MICROFLUIDIC SYNTHESIS OF SYNTHETIC HUMAN EMBRYO-LIKE ENTITIES 2 2 2 1 Fu, Jianping , Zheng, Yi , Xue, Xufeng , Shao, Yue , Nasr Esfahani, Sajedeh , Wang, Sicong and Gumucio, 2 2 Deborah 2 1 Mechanical Engineering, Biomedical Engineering, Cell and Developmental Biology, University of 2 Michigan, Ann Arbor, MI, U.S., University of Michigan, Ann Arbor, MI, U.S. 140 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS 14:00 – 14:15 transcriptomics, and may play a key role in understanding SINGLE CELL TRANSCRIPTOMICS OF GENE- the complex physiology behind tuberous sclerosis. EDITED HUMAN CEREBRAL ORGANOIDS Funding Source: Funding was provided by the Novar- REVEALS NEURON-SPECIFIC PATTERNING tis Institutes for Biomedical Research and the NIBR DEFECTS AND A TREATMENT-RESPONSIVE Postdoctoral Program. GLIAL INFLAMMATORY SIGNATURE IN TUBEROUS SCLEROSIS 14:15 – 14:30 HIGH THROUGHPUT SINGLE CELL RNA-SEQ Salick, Max R. , Goold, Carleton , Ihry, Robert , Biag, 1 2 2 Jonathan , Ho, Daniel , Kommineni, Sravya , Kulkarni, OF DEVELOPING MOUSE KIDNEY AND HUMAN 2 2 2 Tripti , Raymond, Joe , Henry, Beata , Altshuler, Bob , KIDNEY ORGANOIDS REVEALS A ROADMAP 2 2 2 2 Kirkpatrick, Nathaniel , Chen, Julie , Ye, Chaoyang , FOR RECREATING THE KIDNEY 2 2 2 Randhawa, Ranjit , Bilican, Bilada , Fletcher, David , Combes, Alexander , Phipson, Belinda , Zappia, 2 2 2 2 1,2 Worringer, Kathleen , Dolmetsch, Ricardo and Kaykas, Luke , Lawlor, Kynan , Er, Pei , Oshlack, Alicia and 2 2 2 2 2 2 Ajamete 2 Little, Melissa 2 1 Neuroscience, Novartis Institutes for Biomedical 1 University of Melbourne, VIC, Australia, Murdoch 2 Research, Cambridge, MA, U.S., Novartis Institutes Children’s Research Institute, Melbourne, VIC, 2 for Biomedical Research, Cambridge, MA, U.S. Australia Tuberous sclerosis complex (TSC) is a systemic disease Recent advances in our capacity to differentiate human caused by a heterozygous germline mutation in the TSC1 pluripotent stem cells to human kidney tissue are mov- or TSC2 genes, which occurs in 1 out of 6,000 live births. ing the field closer to novel approaches for renal replace- Symptoms arise during development when somatic mu- ment. Such protocols have relied upon our current un- tations in the affected gene lead to loss of heterozygos- derstanding of the molecular basis of mammalian kidney ity, resulting in unchecked mTOR activity. This leads to a morphogenesis. To date this has depended upon popu- range of symptoms in the heart, lung, kidneys, skin, and lation based-profiling of non-homogenous cellular com- other organs; however, the most severe symptoms arise partments. In order to improve our resolution of individual when these somatic mutations occur in the brain. When cell transcriptional profiles during kidney morphogenesis, this happens, hamartomas form in distinct regions of the we have performed 10x Chromium single cell RNA-seq on cortex, along with severe epilepsy, intellectual disability, over 6000 cells from the E18.5 developing mouse kidney, and often autism. There are several unique hallmarks of as well as more than 7000 cells from human iPSC-derived the afflicted region, including dysmorphic neurons, bal- kidney organoids. We identified 16 clusters of cells rep- loon cells, and disrupted cortical architecture. To model resenting all major cell lineages in the E18.5 mouse kid- this disease in vitro we produced TSC2-/- human em- ney. The differentially expressed genes from individual bryonic stem cell and induced pluripotent stem cell lines murine clusters were then used to guide the classification using an inducible CRISPR/Cas9 system. Neurons differ- of 16 cell clusters within human kidney organoids, reveal- entiated from these stem cell lines replicated several of ing the presence of distinguishable stromal, endothelial, the diseases phenotypes, such as S6 phosphorylation nephron, podocyte and nephron progenitor populations. and neuron size, which are then corrected by RAD001, Despite the congruence between developing mouse and an effective mTOR inhibitor. We then produced cerebral human organoid, our analysis suggested limited nephron organoids to mimic human brain development, which we maturation and the presence of ‘off target’ populations treated with DMSO or RAD001, followed by dissociation in human kidney organoids, including unidentified stromal and single cell sequencing using a 10X Chromium system. populations and evidence of neural clusters. This may re- Analysis of the single cell data revealed distinct mutation flect unique human kidney populations, mixed cultures or effects in the neuronal and glial populations, which were aberrant differentiation in vitro. Analysis of clusters with- largely corrected in the RAD001-treated cerebral organ- in the mouse data revealed novel insights into progenitor oids. Network analysis revealed a disruption in telenceph- maintenance and cellular maturation in the major renal alon patterning in the neurons, which was confirmed in lineages and will serve as a roadmap to refine directed follow-up two-dimensional differentiation studies. Sin- differentiation approaches in human iPSC-derived kidney gle-cell analysis revealed a strong inflammatory signal in organoids. the mutant astrocytes, which was particularly rescued in the RAD001-treated samples. Lastly, we conducted an Funding Source: This work was supported by the aptamer-based screen of secreted proteins from WT and Australian Research Council (DE150100652), the Na- TSC2-/- neurons. Considerable differences in the secre- tional Health and Medical Research Council, and the tome of these cultures suggests that the healthy tissue National Institutes of Health Rebuilding a Kidney con- surrounding the hamartomas may also be involved in the sortium (DK107344). disease phenotype. This study marks the first known in- vestigation of isogenically-controlled disease models, along with a treatment response, using droplet single cell 141

FRIDAY 22 JUNE 2018 14:30 – 14:45 14:45 – 15:10 HUMAN INTESTINAL ORGANOIDS AS A BRAIN ORGANOIDS AS A MODEL SYSTEM FOR MODEL FOR INTESTINAL FAILURE AND NEURODEVELOPMENT AND EVOLUTIONARY ABERRANT LIPID METABOLISM IN PATIENTS STUDIES WITH DGAT1 DEFICIENCY Muotri, Alysson van Rijn, Jorik M. , van Haaften Visser, Désirée , van University of California, San Diego, La Jolla, CA, U.S. 1 2 der Doef, Hubert , van Hoesel, Marliek , van Vugt, 3 2 Anke , Kokke, Freddy , Stigter, Edwin , Lichtenbelt, The complexity of the human brain, with thousands of 2 2 2 Klaske , Massink, Maarten , Duran, Karen , Verheij, neuronal types, permits the development of sophisti- 2 2 2 Joke , Lugtenberg, Dorien , Nikkels, Peter , Brouwer, cated behavioral repertoires, such as language, tool use, 3 4 2 Henricus , Verkade, Henkjan , Scheenstra, Rene , self-awareness, symbolic thought, cultural learning and 3 5 3 Spee, Bart , Nieuwenhuis, Edward , Coffer, Paul , van consciousness. Understanding what produces neuro- 2 2 6 Haaften, Gijs , Houwen, Roderick and Middendorp, nal diversification during brain development has been a 2 2 Sabine 2 longstanding challenge for neuroscientists and may bring 1 Pediatric Gastroenterology, UMC Utrecht, insights into the evolution of human cognition. Human pluripotent stem cells have the ability to differentiate in Netherlands, UMC Utrecht, Netherlands, UMC specialized cell types, such as neurons and glia. Moreover, 3 2 Groningen, Netherlands, Radboud University induced pluripotent stem cells can be achieved from living 4 Nijmegen Medical Center, Nijmegen, Netherlands, individuals by reprogramming somatic cells that would 6 5 Elkerliek Hospital, Helmond, Netherlands, Utrecht capture their entire genome in a pluripotent state. From University, Netherlands these pluripotent state, it is possible to generate models of the human brain, such as brain organoids. We have been Patient-derived stem cell organoids provide research- using brain-model technology (BMT) to gain insights on ers with an increasingly well described patient-specific several biological processes, such as human neurodevel- model of disease. Especially human intestinal organoids opment and evolution. We also applied BMT to measure have been shown to reflect the donor’s epithelial char- the impact of genetic variants in autism spectrum disor- acteristics in a highly representative fashion. Here, we ders and for evolutionary studies. The reconstruction of have used small intestinal organoids as a disease model human synchronized network activity in a dish can help for three patients with a novel mutation in the gene en- to understand how neural network oscillations might con- coding diacylglycerol-acyltransferase 1 (DGAT1), a defect tribute to the social brain. Our findings suggest a potential which has previously been described to cause intestinal bridge to the gap between the microscale in vitro neural failure. Symptoms of this chronic condition include se- networks electrophysiology and non-invasive electroen- vere early-onset diarrhoea, vomiting, and protein losing cephalogram. enteropathy on ingestion of dietary lipids. In healthy indi- viduals, DGAT1 catalyses the formation of triacylglycerol from diacylglycerol and acyl-CoA, which is necessary for lipid metabolism in the intestine. The DGAT1 mutation we FRIDAY, 22 JUNE, 13:15 – 15:15 found led to loss of protein due to immediate proteasomal degradation. Using thin-layer chromatography, we show CONCURRENT IIIB: NERVOUS SYSTEM that DGAT1 deficiency specifically altered triacylglycerol metabolism in enterocytes. Furthermore, we found that DISEASE DGAT1 mutant organoids show impaired lipid droplet for- Melbourne Room 2, Level 2 mation upon incubation with oleic acid. In addition, DGAT1 mutant organoids were more sensitive to OA-induced 13:20 – 13:45 caspase-3 mediated cell death compared to healthy con- trol organoids. We confirmed these DGAT1-dependent USING STEM CELLS FOR DRUG DISCOVERY IN functional differences through CRISPR/Cas9-guided de- NEUROSCIENCE letion of DGAT1 in healthy control intestinal organoids. Dolmetsch, Ricardo In conclusion, we show for the first time the importance of DGAT1 in human gut epithelium and link DGAT1 defi- Novartis Institutes for BioMedical Research, Inc., ciency to altered lipid metabolism and fat intolerance. By Cambridge, MA, U.S. developing several functional assays to model DGAT1 de- ficiency in-vitro, we furthered our understanding of the Drug development in neuroscience has been particularly pathophysiology of disease and enabled the screening for challenging over the last two decades. A major limitation potential novel therapeutics. Our data once again empha- has been the lack of predictive preclinical models that can size the strength of organoids in patient-specific disease be used at early stages of drug discovery to select and modelling and their use in personalized drug screening. optimize drug candidates. iPSC-derived neurons have the potential to improve our drug discovery pipeline by Funding Source: This work was supported by the providing human cellular models of neurological and psy- Netherlands Organisation for Scientific Research chiatric disease. We have built a platform for generating (NWO; VIDI 016.146.353) to SM. iPSC derived neurons, engineering them using CRISPR 142 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS Cas9 and using them to conduct both high throughput spinal cord injuries grow deeper, more neurons die and the screens and secondary hit selection and optimization as- normally occurring slight recovery of function is attenuat- says. I will discuss our efforts to optimize this system and ed. Spinal cord ependymal cells are known to show mo- some of the learnings that we have made along the way. I lecular heterogeneity, but it has remained unclear if stem will also discuss our ongoing programs to develop drugs cell properties are shared between all ependymal cells or for neurodevelopmental diseases using human induced held only by some. We examined the functional heteroge- pluripotent stem derived neurons. I will discuss our efforts neity among ependymal cells by fate mapping two small in Spinal Muscular Atrophy, Dravet Syndrome and Phelan subpopulations that can be recombined and heritably McDermid Syndrome and discuss some of the challenges labelled in Glast-CreER and Troy-CreER mice. We found ahead. that Glast-CreER recombined cells self-renew inefficient- ly in vitro, while the Troy-CreER recombined population 13:45 – 14:00 contains almost all efficiently self-renewing cells. Our FINDING YOUR PLACE: CONTROL OF results suggest the Glast-CreER recombined cells act as progenitor cells in vitro, while the Troy-CreER recombined CELL MIGRATION DURING FETAL BRAIN subpopulation harbours nearly the entire in vitro stem cell DEVELOPMENT AND NEURONAL MIGRATION capacity of the adult spinal cord. After spinal cord injury, DISORDER IN HUMANS Glast-CreER recombined ependymal cells do not contrib- ute to glial scar formation. On the contrary, we show that Heng, Julian Troy-CreER recombined ependymal cells divide rapidly to Curtin Health Innovation Research Institute, Curtin generate astrocytes in the glial scar and oligodendrocytes. University, Perth, Western Australia, Australia Using single-cell fate mapping, we explore the expansion and spread of migrating clones generated from individu- The development of neural circuits during fetal brain de- al Troy-CreER recombined ependymal cells. Furthermore, velopment relies on the timely production of neurons, we confirm that Troy-CreER recombined ependymal cells their correct placement as well as their capacity to form typically stay by the central canal when they generate appropriate connections with other brain cells. Abnormal migrating progeny, likely by undergoing asymmetric cell cell migration during fetal brain development can lead division. We conclude that a subpopulation containing to disruptions in the development and functional organ- less than 10% of the ependymal cells harbours nearly the isation of neural circuits which subserve mental func- entire in vitro neural stem cell capacity of the adult spinal tion.  We have identified the heterogeneous ribonuclear cord. Troy-CreER recombined ependymal cells contribute protein U-like 2 (HNRNPUL2) as a mediator of cell migra- to scar formation after injury and maintain their own pop- tion during fetal brain development.  Through a series of ulation while they generate migrating progeny. The small functional studies in mice, we demonstrate that appropri- Troy-CreER recombined neural stem cell population may ate levels of HNRNPUL2 are essential to radial migration pose an interesting target for therapeutic intervention af- by neuroprogenitor/stem cells and postmitotic neurons of ter spinal cord injury. the embryonic cerebral cortex, and that a rare missense variant likely causes periventricular nodular heterotopia, Funding Source: Swedish Research Council, the a neuronal migration disorder in humans.  These findings Swedish Cancer Society, the Swedish Society for Stra- broaden our understanding of the mechanisms for cell tegic Research, Tobias Stiftelsen, AFA Försäkringar, migration and support the accurate genetic diagnosis of StratRegen at Karolinska Institutet and Torsten Söder- neuronal migration disorder in humans. bergs Stiftelse. Funding Source: This research was supported by a 14:15 – 14:30 Career Development Fellowship (ID:1011505) from the National Health and Medical Research Council of Aus- MUTATIONS IN ACTL6B CAUSE AUTISM AND tralia. EPILEPSY AND LEAD TO LOSS OF DENDRITES IN HUMAN NEURONS 14:00 – 14:15 Bell, Scott , Rousseau, Justine , Peng, Huashan , Jefri, 2 1 3 SPINAL CORD EPENDYMAL CELLS ARE Malvin , Wu, Hanrong , Theroux, Jean-Francois , Ernst, 3 3 3 3 FUNCTIONALLY HETEROGENOUS AND Carl and Campeau, Phillipe 2 CONTAIN A SMALL SUBPOPULATION OF 1 Integrated Program of Neuroscience, McGill NEURAL STEM CELLS University, Montreal, Canada, Montreal University, 2 Canada, McGill University, Montreal, Canada 3 Stenudd, Moa, Sabelström, Hanna and Frisén, Jonas Karolinska Institutet, Stockholm, Sweden We identified nineteen families with undiagnosed neu- rodevelopmental disorders that all possessed mutations The neural stem cells in the adult spinal cord are ependy- in ACTL6B, a crucial regulator of dendrite formation that mal cells. Ependymal cells self-renew slowly during physi- had not been previously identified to contribute to hu- ological conditions, but they start dividing rapidly in vitro man disease. Ten families were found to have bi-allelic and after spinal cord injury. After injury, they generate as- mutations in ACTL6B, presenting symptoms of epileptic trocytes in the glial scar and remyelinating oligodendro- encephalopathy and spasticity, and nine families were cytes. Without the injury response by ependymal cells, 143

FRIDAY 22 JUNE 2018 found to have de novo heterozygous missense mutations 6 weeks post-transplant. Post mortem histology revealed and displayed intellectual disability, autism, and Rett-like that transplanted cells were capable of innervating the stereotypies. Generating iPSC-derived neurons from an dopamine depleted striatum in a similar, biologically-rel- affected individual revealed that mutations in ACTL6B evant pattern previously seen in the 6-OHDA model. We result in increased binding of the BAF complex to an en- also used monosynaptic tracing based on modified rabies hancer driving increased expression of SEMA4D, a key virus to assess that the pathology present in this model inhibitor of dendrite outgrowth. Patient cells were also did not inhibit the ability of the graft to integrate into the observed to have abnormal cell differentiation, including host circuitry, meaning that the grafted cells are able to a profound loss of dendrites. Both the increased SEMA4D receive appropriate and sufficient synaptic contact with expression and loss of dendrites was reversed upon CRIS- the host central nervous system. Finally, on closer exam- PR/Cas9-mediated mutation correction of the patient ination, we found preliminary evidence of alpha-synuclein line. To examine the effect of ACTL6B on human neuronal pathology in the grafted region of the striatum, indicating development, a CRISPR/Cas9 mediated ACTL6B knock- possible host-to-graft transfer of alpha-synuclein patholo- out (KO) neuronal line was generated, and was observed gy. Further studies to confirm this observation, and to ex- to also present severe deficits in dendritogenesis and in- amine a longer time-point where we can assess the matu- creased SEMAD4 expression. Moreover, the introduction ration and function of the transplanted cells, and if this is of three different ACTL6B mutations identified in patients affected by the potential pathology transfer, are currently into ACTL6B KO neurons all resulted in further increases underway. This will give us a better understanding of the in SEMA4D expression, whereas re-introduction of wild- performance of these cells in a more clinically relevant, type ACTL6B resulted in downregulation of SEMA4D ex- novel alpha-synuclein model of PD, thus adding to the pression. This study provides the first ever description of body of knowledge required as this cell replacement ther- a novel genetic disease caused by mutations in ACTL6B apy progresses to clinical trials. and identifies aberrantly high inhibition of dendritogene- Funding Source: New York Stem Cell Foundation (NY- sis through SEMA4D signalling as a likely mechanism con- SCF); The Swedish Research Council; The European tributing to the pathology of the disease. Research Council (ERC); NeuroStemCellRepair (EU); Funding Source: Carl Ernst and Phillipe Campeau are Parkinsonfonden; Bagadilc; MultiPark; Malin Parmar is supported by the Canadian Institue for Health Re- a NYSCF-Robertson Investigator. search (CIHR), as well as the Fonds de Recherche du Québec (FRQS). 14:45 – 15:10 GDNF ENHANCES THE FUNCTIONAL 14:30 – 14:45 INTEGRATION OF HUMAN PLURIPOTENT STEM TRANSPLANTATION OF HUMAN EMBRYONIC CELL-DERIVED DOPAMINE GRAFTS IN A RAT STEM CELL DERIVED DOPAMINERGIC MODEL OF PARKINSON’S DISEASE NEURONS IN AN ACCELERATED ALPHA- 1 1 1 SYNUCLEIN RAT MODEL OF PARKINSON’S Parish, Clare , Gantner, Carlos , Kauhausen, Jessica , 1 1 de Luzy, Isabelle R. , Niclis, Jonathan , Penna, Vanessa , 1 DISEASE Bye, Christopher , Pouton, Colin , Kirik, Deniz and 3 2 1 1 1 Hoban, Deirdre B. , Breger, Ludivine , Wahlestedt, Thompson, Lachlan 1 1 1 Jenny , Cardoso, Tiago , Mattsson, Bengt , Luk, Kelvin , 1 The Florey Institute of Neuroscience and Mental 2 1 1 Björklund, Anders and Parmar, Malin 1 Health, Melbourne, Australia, Monash Institute of 2 2 3 1 Lund University, Sweden, University of Pennsylvania, Pharmaceutical Sciences, Melbourne, Australia, Lund Philadelphia, PA, U.S. University, Sweden Preclinical validation studies to assess the therapeutic po- The derivation of neurotransmitter and region specific tential of human embryonic stem cell (hESC) derived do- neuronal populations from human pluripotent stem cells paminergic (DA) neurons have mostly been performed in (hPSC) provides impetus for advancing cell therapies into the 6-hydroxydopamine (6-OHDA) model of Parkinson’s the clinic. At the forefront is our ability to generate mid- disease (PD). However, this model does not reflect the brain dopaminergic (DA) progenitors, suitable for trans- pathological features or progressive nature of PD. Here, plantation in Parkinson’s disease. However, pre-clinical we aim at assessing how the transplanted cells survive, studies have highlighted the relatively low proportions of mature, integrate and innervate the existing circuitry in a DA neurons within these grafts and their inferior plasticity, novel accelerated model of PD, whereby preformed hu- particularly in comparison to human fetal grafts. Here we man alpha-synuclein fibrils and AAV6 human alpha-synu- sought to examine whether modification of the host envi- clein are unilaterally injected into the rat substantia nigra. ronment, through the viral delivery of a developmentally This model gives rise to alpha-synuclein pathology, inflam- critical molecule, glial cell-line derived neurotrophic fac- mation and progressive loss of DA cells from the substan- tor (GDNF), could improve graft integration and function tia nigra and terminals in the striatum. After allowing the in Parkinsonian rodents. Utilising LMX1A- and PITX3-GFP pathology to develop for 8 weeks, we then transplanted hPSC reporter lines, we tracked the response of DA pro- hESC-derived DA neurons into the striatum and assessed genitors implanted into either a GDNF-rich environment their survival, maturation, integration and innervation at or following delayed delivery of the neurotrophin. Early 144 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS exposure of the graft to GDNF promoted survival of DA 13:45 – 14:00 and non-DA cells, leading to enhanced motor recovery USING A PREDICTIVE COMPUTATIONAL in PD rats. Delayed overexpression of intrastriatal GDNF also promoted motor recovery in transplanted rats, not ALGORITHM TO ESTABLISH A UNIVERSAL through neuroprotection, but alternate selective mecha- TRANSCRIPTION FACTOR ENHANCED nisms including enhanced DA graft plasticity, increased DIFFERENTIATION FRAMEWORK activation of striatal neurons and elevated DA metabo- Chen, Joseph , Tiedemann, Mathew , Brewster, Kaitlyn , 1 1 1 lism. These results highlight the potential of targeted neu- Liu, Xiaodong , Manent, Jan , Grubman, Alexandra , 1 1 1 rotrophic gene therapy strategies to improve hPSC graft Gough, Julian , Rackham, Owen , Nefzger, Christian , 3 2 1 outcomes. Polo, Jose 1 2 1 Monash University, Australia Cambridge Biomedical Campus, United Kingdom, Duke-National University 3 FRIDAY, 22 JUNE, 13:15 – 15:15 of Singapore Medical School, Singapore CONCURRENT IIIC: MECHANISMS The ability of pluripotent stem cells (PSCs) to generate OF REPROGRAMMING 2: virtually any cell type in the body holds great promise in regenerative medicine and disease modeling. Many strat- TRANSDIFFERENTIATION BETWEEN egies, adapted from our understanding of developmen- LINEAGES tal biology, have aided us in optimizing specific culture Room 219/220, Level 2 conditions that facilitate the differentiation of target cell types. However, in recent years, a number of studies sug- gested the use of transcription factors (TFs), master reg- 13:20 – 13:45 ulators of cell identity, to improve the yield, speed and/or CELL-TYPE- AND STAGE-SPECIFIC specificity of differentiation. These novel TF-based strate- CONSTELLATIONS OF ENHANCERS CONTROL gies were discovered through empirical testing of a large COMPLEX GENE EXPRESSION PROGRAMS IN number of TF combinations that have been associated THE NERVOUS SYSTEM with the target cell type, a very complex, laborious and inefficient process. Unfortunately, the identification of key Wichterle, Hynek, Closser, Michael, Guo, Xiaoyun, TFs to enhance differentiation of PSCs is currently limit- Kopunova, Rachel, Patel, Tulsi, Guo, Yuchun, Rhee, Ho ed to specific cell lineages which have been extensively Sung, Ruan, Yijun and Gifford, David studied (e.g. neurons, blood and muscle). In spite of this, Columbia University, New York, NY, U.S. we and others have previously developed computational frameworks to accurately predict the TFs required for cell Cellular complexity of the nervous system demands a conversion into target cell types. We adopted our predic- commensurate complexity of the regulatory system con- tive algorithm, Mogrify, to predict TFs which will enhance trolling cell type-specific patterns of gene expression. the differentiation of PSCs into cells of all three germ lay- Using embryonic stem cell-derived motor neurons we ers. We show that co-expression of the predicted key TFs discovered a unique architecture of enhancer elements for PSC to keratinocyte differentiation gave rise to cells associated with neuronal genes. Genome-wide maps of acquiring keratinocyte-like morphologies and expressing enhancer-promoter interactions revealed that most genes keratinocyte markers (K1 and K14) in half the time com- induced in motor neurons are regulated by constellations pared to the standard baseline differentiation method. of distributed enhancers spanning extensive genomic Similarly, enhanced differentiation into cells of other germ territories, rather than by localized super-enhancers. Me- layers using separate sets of predicted TFs yielded the ta-analysis of regulatory regions associated with genes desired cells in less than half the time compared to con- expressed in other neuronal populations suggests that trol cultures. Through this approach, we have established distributed enhancers are not a unique property of spi- new enhanced differentiation protocols for a variety of nal motor neurons, but are broadly employed in the reg- lineages and cell types which were previously unreport- ulation of gene expression within the nervous system. ed. Accordingly, Mogrify enables the identification of key Furthermore, within individual neuronal cell types, these TFs that can enable cell fate conversion as well as devel- constellations of enhancers are highly dynamic, exhibit- op enhanced differentiation protocols for the generation ing temporal specificity. Together, our findings support and enrichment of target cell types from pluripotent stem the view that neuronal genes are associated with large cells for potential therapeutic downstream applications. non-coding genomic territories, accommodating uniquely large numbers of cell-type- and cell-stage-specific regu- latory elements controlling complex patterns of gene ex- pression within the nervous system. 145

FRIDAY 22 JUNE 2018 14:00 – 14:15 When using TF over-expression based methods, a major- DIRECT REPROGRAMMING OF MOUSE ity of the work comes down to identifying the subset of TFs in the genome that can successfully convert cell type FIBROBLASTS INTO FUNCTIONAL SKELETAL ‘A’ to cell type ‘B’ when overexpressed. Historically this MUSCLE PROGENITORS identification process has been rooted in expert knowl- Bar-Nur, Ori , Gerli, Mattia , Di Stefano, Bruno , edge from groups that had extensive expertise in the ge- 2 2 1 Almada, Albert , Galvin, Amy , Coffey, Amy , Huebner, netics of one or more cell type. While this approach has 2 2 2 Aaron , Feige, Peter , Verheul, Cassandra , Ott, Harald , resulted in some significant successes, these methods are 2 2 2 3 Tajbakhsh, Shahragim , Rudnicki, Michael , Wagers, generally low-throughput and many valuable conversions 3 4 Amy and Hochedlinger, Konard 2 are still unknown. Furthermore, recent work in our group 2 has resulted in the definition and production of a ‘TFome’ 1 Center for Regenerative Medicine, Massachusetts of all known TFs in the human genome composed of General Hospital, Harvard University, Boston, MA, ~1768 genes - thus exhaustively screening all possible U.S., Harvard University, Boston, MA, U.S., Ottawa combinations of TFs of even a relatively small size is es- 2 3 Health Research Institute, Canada, Institut Pasteur, sentially impossible with current methods. Some recent 4 Paris, France efforts have instead attempted to approach this problem computationally using transcriptomics data to identify Skeletal muscle harbors quiescent stem cells termed sat- TF combinations that are likely candidates to perform a ellite cells and proliferative progenitors termed myoblasts, specific conversion, although these methods have a few both of which play pivotal roles during muscle regenera- key limitations - they exclusively rely on sometimes sparse tion and hold promise for the treatment of muscle-associ- transcriptomics data, they output one final solution as op- ated disorders. However, current technology does not al- posed to an experimental design, they do not leverage low permanent capture of cell populations with myogenic other types of next-generation sequencing data, and they potential in vitro. We report that ectopic expression of the have no intrinsic feedback loop for evaluating outcomes to myogenic transcription factor MyoD, combined with expo- automatically design the next round of experiments. Here sure to three small molecules, readily reprograms mouse we present a software tool that uses open-chromatin data fibroblasts into induced myogenic progenitors (iMPCs) and transcriptomics data to output an experimental de- that can be propagated extensively while retaining the sign in which a subset of the human TFome is multiplexed ability to produce contractile myotubes. Immature iMPCs to identify cells that seem to be most successful in that express markers of skeletal muscle stem and progenitor screen. The most promising candidates from this screen cells, including Pax7 and Myf5, and can differentiate into are then sequenced for RNA expression and open chro- Dystrophin-expressing myofibers upon transplantation matin and the data is used to inform the next multiplex into a mouse model of Duchenne Muscular Dystrophy. experiment. This tool is first being applied to known TF- Notably, a subset of transplanted iMPCs maintain Pax7 based conversions, but is being developed as generally as expression in vivo and sustain regenerative responses in a possible for use as a tool for any desired novel conversion. serial injury model, consistent with stem cell-like proper- ties. We further provide evidence that functional progen- Funding Source: This work was funded under the itor cells can be established from explanted muscle tissue IARPA Fun GCAT Program PTE Federal Award No: following small molecule exposure alone. These findings W911NF-17-2-0089. reveal a novel and robust approach to derive expandable myogenic stem/progenitor cells with characteristics of 14:30 – 14:45 satellite cells from different somatic tissues. MITOCHONDRIAL DYNAMICS DETERMINES HUMAN EMBRYONIC STEM CELL FATES 14:15 – 14:30 1 1 Lacey, Joanne , Hill, Christopher , Mortiboys, Heather , 1 A SOFTWARE TOOL FOR DESIGNING Rodriguez, Tristan and Barbaric, Ivana 1 2 TRANS-DIFFERENTIATION EXPERIMENTS 1 University of Sheffield, UK, Imperial College London, 2 WITH COMBINATIONS OF TRANSCRIPTION UK FACTORS Appleton, Evan , Ng, Alex and Church, George 2 Human pluripotent stem cells (hPSCs) may provide an 1 2 unlimited source of cells of therapeutically relevant cell 1 Department of Genetics, Harvard Medical School, types, due to their ability to self-renew in vitro for long Boston, MA, U.S., Harvard Medical School, Boston, periods of time, whilst retaining the ability to differentiate 2 MA, U.S. into all of the cell types in the body. Unlocking the ther- apeutic potential of hPSCs relies on our ability to control A current focus in the stem cell field is to develop meth- stem cell fates, i.e. self-renewal, differentiation and death. ods for direct cell-type conversions. This can be accom- However, the detailed molecular mechanisms governing plished in a variety of ways including surface-condition the stem cell fate decisions remain largely unknown. Giv- based methods, media growth factor methods and ge- en the importance of mitochondria for multiple essen- netic methods. Many recent successes rely primarily on tial cellular processes, such as energy metabolism and the use of genetic methods - specifically directed differ- apoptosis, we posited that mitochondrial dynamics can entiation via transcription factor (TF) over-expression. influence hPSC fate decisions. Mitochondria are dynamic 146 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH

SPEAKER ABSTRACTS organelles which undergo cycles of fusion (mediated pre- population size of HHCs in converting cultures correlates dominately by MFN1, MFN2 and OPA1) and fission (medi- with conversion efficiency. By profiling cells early in con- ated by DRP1). In order to elucidate the effects of mito- version, we find that cells driven with hypertranscription chondrial dynamics on hPSC fate determination, we have increase expression of transcriptional machinery and used CRISPR-Cas9 genome editing to generate knock out DNA repair pathways. We specifically identify topoisom- cell lines of the mitochondrial fusion (MFN1, MFN2 and erase expression as a key parameter modulating the cell’s OPA1) and fission (DRP1) factors. We show that deletion ability to maintain the population of HHCs by balancing of MFN1, MFN2, OPA1 and DRP1 generates distinct mito- hypertranscription and hyperproliferation. Finally, hyper- chondrial fragmentation and fusion phenotypes. Our pre- transcription significantly increases functional and molec- liminary data supports our hypothesis that mitochondrial ular properties of engineered cells to mimic their in vivo shape impacts stem cell fate decisions. Lending weight to surrogates. By providing the context to drive and sustain this hypothesis is our finding that the spontaneously aris- hypertranscription, we robustly generate reprogrammed en genetic variants in hPSC cultures display distinct mi- cells with signatures of maturity. tochondrial phenotypes linked to improved survival and reduced differentiation of variant cells. Overall, our find- ings provide important insights into the underlying mech- anisms of stem cell maintenance, which has important im- FRIDAY, 22 JUNE, 13:15 – 15:15 plications for the translational goals of hESC research for regenerative medicine. CONCURRENT IIID: HEMATOPOIESIS Funding Source: This work was supported by a grant Room 212/213, Level 2 from the Medical Research Council (MR/N 009371/1). 13:20 – 13:45 14:45 – 15:00 MODELING NORMAL AND MALIGNANT HYPERTRANSCRIPTION DRIVES CELLULAR HAEMATOPOIESIS USING HUMAN REPROGRAMMING PLURIPOTENT STEM CELLS 1 Babos, Kimberley N. , Galloway, Kate and Ichida, Elefanty, Andrew George, Ng, Elizabeth, Bruveris, 2 Justin 2 Freya, Nafria I Fedi, Monica, Leitoguinho, Ana Rita, 1 Department of Development, Stem Cells, and Motazedian, Ali, Kusur, Jasna, McDonald, Penny, Regenerative Medicine, University of Southern Labonne, Tanya and Stanley, Edouard 2 California, San Gabriel, CA, U.S., University of Murdoch Children’s Research Institute, Parkville, VIC, Southern California, Los Angeles, CA, U.S. Australia Cellular reprogramming requires massive transcriptional It has been an ongoing challenge to dissect the mech- realignment, and only a small fraction of cells successful- anisms underlying the normal development of haema- ly process the demands of transcription factor-mediat- topoiesis. Even with access to developing embryos, the ed reprogramming to convert into an alternative cellular replication of the processes that guide a cell from pluripo- identity. We examined the cellular processes that drive tency to mesoderm to either an extra- or an intra-embry- reprogramming to identify why conversion rarely occurs. onic haemogenic endothelium and then to yolk sac-like Previous work in iPSC reprogramming identified fast-cy- or AGM-like blood cells have remained very difficult. In cling, hyperproliferating cells as a privileged population. this presentation I will discuss what we have learnt about To determine how hyperproliferating cells contribute to the steps involved in these processes from studies using conversion to a post-mitotic cell type, we isolated hyper- gene-targeted and reporter cell lines. Finally, we have be- proliferating cells undergoing conversion to induced mo- gan to model human leukaemias in vitro and I will share tor neurons (iMNs). We observe that hyperproliferating data from our latest insights on the initial steps of the neo- cells reprogram to iMNs with significantly higher frequen- plastic process. cy. Genetic perturbation via a p53 mutant (p53DD) sig- nificantly increases the population of hyperproliferating cells and magnifies conversion 100-fold. We also find that addition of p53DD provides an extensive reprogramming boost across species and protocols. In investigating the mechanisms that promote reprogramming, we identified hypertranscription is a primary driver of conversion. By measuring transcription rate through 5-ethynyl uridine incorporation, we observe that addition of the repro- gramming factors induces a wave of hypertranscription early in reprogramming. Inclusion of p53DD sustains the window of hypertranscription. By driving a global tran- scriptional increase using an hRASV12 mutant, conversion is significantly enhanced. We identified a rare population of hyperproliferating, hypertranscribing cells (HHCs). The 147

FRIDAY 22 JUNE 2018 13:45 – 14:00 vitro human HSC differentiation protocols from pluripo- YAP REGULATES HEMATOPOIETIC STEM tent stem cell sources. CELL FORMATION IN RESPONSE TO THE BIOPHYSICAL FORCES OF BLOOD FLOW 14:00 – 14:15 LIVER-DERIVED SYSTEMIC THROMBOPOIETIN Lundin, Vanessa , Theodore, Lindsay , Wrighton, 1 2 Paul , Sousa, Patricia , Han, Areum , Hwang, Katie , IS REQUIRED FOR BONE MARROW 3 2 3 2 Goessling, Wolfram , Ingber, Donald , Daley, George HEMATOPOIETIC STEM CELL MAINTENANCE 3 4 2 and North, Trista 2 Ding, Lei, Decker, Matthew, Leslie, Juliana and Liu, 1 Stem Cell Program, Division of Hematology/ Qingxue Oncology, Boston Children’s Hospital and Dana- Columbia University Medical Center, New York, NY, Farber Cancer Institute, Boston, MA, U.S., Boston U.S. 2 Children’s Hospital and Dana-Farber Cancer Institute, Boston, MA, U.S., Brigham and Women’s Hospital, Hematopoietic stem cells (HSCs) depend on extrinsic 3 Dana-Farber Cancer Institute and Harvard Medical cues for their maintenance. Currently, only local signals School, Boston, MA, U.S., Harvard University, Boston from the bone marrow niche have been shown to sustain 4 Children’s Hospital, Harvard Medical School and adult HSCs. This has raised the fundamental question of Harvard John A. Paulson School of Engineering and whether systemic factors may play critical roles in HSC Applied Sciences, Boston, MA, U.S. maintenance. We assessed the in vivo functional source of thrombopoietin (TPO), a key cytokine required for Hematopoietic stem cells (HSCs) are specified during em- maintaining bone marrow HSCs. It was previously shown bryonic development from hemogenic endothelial cells that Tpo translation is under stringent control. We engi- (HEC) along the ventral surface of the dorsal aorta (VDA). neered a novel Tpo DsRed-CreER knockin mouse by recombin- Prior studies from our labs demonstrated that HSC emer- ing DeRed and CreER into the endogenous locus of Tpo. gence follows the onset of blood flow, which exposes the This is the first genetic tool for in vivo labeling of cells that vascular endothelium to biomechanical forces. To better translate Tpo transcripts. We found that TPO protein was understand how these forces drive HSC production, we expressed in liver hepatocytes but not cells in the bone engineered a human biomimetic aorta-on-a-chip platform marrow. To functionally identify the physiological source and subjected induced pluripotent stem cell (iPS)-derived of Tpo in vivo, we generated a floxed allele of Tpo and HEC to wall shear stress (WSS) or circumferential stretch conditionally deleted it from a variety of candidate cell (CS). RUNX1, the main transcription factor involved in HSC populations. Deletion of Tpo from hematopoietic cells, os- specification, was upregulated by both WSS and CS com- teoblasts, or bone marrow mesenchymal stromal cells did pared to static control cells. Interestingly, YAP (Yes-as- not affect HSC number or function. Strikingly, when Tpo sociated protein) target genes ANKRD1 and CTGF were was deleted from hepatocytes, HSCs were nearly com- also upregulated after exposure to CS; this induction was pletely depleted from the bone marrow. Inducible dele- abolished in the presence of a Rho inhibitor, indicating a tion of hepatic Tpo in adult mice by a hepatocyte-specific role for Rho-YAP mechano-transduction in mediating the Cre-bearing adenovirus had the same effect as deletion effect of blood flow on HECs. To determine whether these by a transgenic Cre line. This suggests a continuous, rath- findings pertain in vivo, we utilized an inducible zebrafish er than developmental, dependence on hepatic TPO. Thus Yap overexpression (Yap-OE) model, which significantly a long-range regulatory factor - systemic TPO made in the increased runx1+ in the VDA. This effect was sustained liver by hepatocytes - is required for adult bone marrow throughout development, as Yap-OE also significantly in- HSC maintenance. creased the number of Cd41+ HSCs in the caudal hemato- poietic tissue as well as rag1+ lymphoid progenitors in the 14:15 – 14:30 thymus. Similar results were found following knockdown THE SPLICEOSOMAL COMPONENT SF3B1 IS of lats1/2, negative regulators of endogenous Yap. In con- ESSENTIAL FOR ZEBRAFISH HEMATOPOIETIC trast, yap-/- knockout embryos exhibited a significant reduction in runx1 expression. Activation of Yap through STEM CELL FORMATION THROUGH Rho-GTPase stimulation significantly increased runx1 ex- REGULATION OF THE JAK/STAT SIGNALING pression in silent heart morphant embryos that lack circu- PATHWAY lation, but was ineffective in yap mutants, indicating that Potts, Kathryn S. , Cameron, Rosannah , McKinstry, 1 2 Rho-Yap influences HSC formation downstream of blood Mia , Gupta, Varun , Bai, Xiaoying and Bowman, 1 3 1 flow. In vitro, YAP activation in iPS-derived HEC mediat- Teresa 1 ed via Rho-GTPase stimulation or LATS1/2 knockdown increased RUNX1 levels and colony forming potential, 1 Albert Einstein College of Medicine, Bronx, NY, U.S., demonstrating that biophysical forces from blood flow 2 Ferrier Research Institute, Victoria University of 3 can be mimicked by YAP modulation. Together, our find- Wellington, Wellington, New Zealand, University of ings reveal a functional intersection between mechano- Texas, Dallas, TX, U.S. transductive Rho-YAP activation and RUNX1-dependent HSC production, which may be exploited to improve in Hematopoietic stem cells (HSCs) sustain multilineage he- matopoiesis for the lifetime of an organism. During em- 148 INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH