ISSCR 2019 Poster Abstracts_clone

350POSTER ABSTRACTSRehovot, Israel Yavriyants, Karina - Diabetes Cell Therapy Unit, Kadimastem ltd, Rehovot, Israel Zimerman, Michal - Diabetes Cell Therapy Unit, Kadimastem ltd, Rehovot, Israel Hasson, Arik - Cell Therapy, Kadimastem ltd, Rehovot, Israel Itskovitz-Eldor, Joseph - Cell Therapy, Kadimastem ltd, Rehovot, Israel Chebath, Judith - Diabetes Cell Therapy Unit, Kadimastem ltd, Rehovot, Israel Revel, Michel - Cell Therapy, Kadimastem ltd, Rehovot, IsraelThe limiting factor in islet transplantation treatment remains the low numbers of available islets. This has shed light on the usage of hPSCs as a potential renewable source for insulin-producing cells. By following embryonic developmental pathways, hPSCs were differentiated successfully into the pancreatic lineage and specifically to Islet-Like-Clusters (ILCs) containing insulin and other hormones needed to control glucose homeostasis. Large scale production of hPSC-derived islets are composed from heterogeneous populations that include targeted therapeutic population (~40%) along with functionally irrelevant populations that include pancreatic progenitors and poly-hormonal cells (~50%). Using cell-capture antibody array, novel antibodies to distinguish these populations were identified. Utilizing MACS technology, large scale cultures were enriched using a novel antibody combination and re-aggregated. The enriched islet-like clusters (ILCs) preparations were tested both in vitro and in vivo. In vitro, sorted ILCs exhibited an improved mature pancreatic gene expression profile, manifested by higher expression of MAFA, Insulin and NKX6.1. In addition, significantly lower expression levels of immature pancreatic genes like of AFP and CK19 was found, along with a marginal decrease in intestinal gene expression markers like CDX2. In vivo, sorted ILCs (~1,000) were macro-encapsulated using hydrogel polymer disks and transplanted under the skin of SCID-Beige mice. Already at day 7, human insulin secretion was detected at higher physiological levels (~2 fold higher) in the blood of mice implanted with sorted ILCs as compared to the non-sorted ILCs control. The secretion was highly dependent on glucose, being low in fasted animals and increased ~10 fold after IP glucose administration. Such secretion was observed weekly for over 6 weeks, implying that indeed a functional enrichment was achieved. Hence, by using the novel antibodies combination, the purity of hPSCs-derived islets can be increased by 2 fold, generating improved and more defined therapeutic cell therapy product. It is suggested that our novel cell surface markers combination strategy could be used to reduce the required cell mass loaded into an encapsulation device and potentially to decrease the device’s physical dimensions.T-2072INSULIN NEGATIVE PANCREATIC PROGENITORS ARE DERIVED FROM THE HUMAN AND MOUSE PANCREATIC ENDODERM STAGE OF DEVELOPMENTJacques-Smith, Krystal - Institute of Medical Science, University of Toronto, ON, Canada Khalili, Saeed - Department of Molecular Genetics, University of Toronto, ON, Canada Takabe, Brenda - Department of Molecular Genetics, University of Toronto, ON, Canada Nostro, Cristina - Physiology, University of Toronto, ON, Canada van der Kooy, Derek - Department of Molecular Genetics, Institute of Medical Science, Medical Biophysics, University of Toronto, ON, CanadaAdult insulin positive (INS+) pancreatic multipotent progenitors (PMPs) are found in the adult human and mouse pancreas but their origin during embryonic development has not been fully characterized. PDX1 is expressed at mouse embryonic day 8.5 (E8.5) and marks the pancreatic endoderm stage of pancreatic development. NKX6.1 expression begins at E10.5 marking the PDX1+/NKX6.1+ pancreatic progenitor stage. The first four stages of in-vitro cytokine-induced differentiation of human embryonic stem cells (hESCs) to beta-like cells follow similar transitions of hESCs to the definitive endoderm, posterior foregut, pancreatic endoderm, and pancreatic progenitor cells, modelling early, mouse pancreatic development. Using this human in-vitro model, we report the clonal isolation of clonal sphere-forming cells at each of these early stages. Using an ins-GFP hESC line, 80% of clonal spheres generated at the pancreatic endoderm stage contain cells that are ins-GFP+, while only 10% of spheres derived from the later pancreatic progenitor stage are ins-GFP+. This percentage drop is due either to a loss of pancreatic sphere-forming cells or to an expansion of non-pancreatic sphere forming cells, at the last pancreatic progenitor stage. Flow cytometry immediately after hESCs have transitioned to each of the four stages of cytokine-induced differentiation reveals that insulin is not expressed at any of these early differentiation stages. However, the last two stages produce sphere forming cells that then give rise to ins-GFP clonal spheres. This suggests that pancreatic endoderm-derived INS+ spheres emerged from an INS- precursor cell. Using PDX1 Cre x Rosa YFP transgenic mice, clonal YFP+ spheres were formed from E10.5 pancreatic buds - a timepoint characterized as the PDX1+/NKX6.1+ pancreatic progenitor stage when insulin is not yet expressed. Pan-differentiation (non-cytokine induced-differentiation) of E10.5 pancreatic bud-derived spheres reveals that some of the progeny of PDX1+/ NKX6.1+/ INS- sphere-forming precursor cells differentiated into INS+ beta-cells. These data suggest that INS- PMPs give rise to INS+ beta cells. Isolation of PMPs from embryonic development implies a novel timepoint in which to enrich for expandable lineage-restricted PMPs for cell replacement therapy in diabetes.Funding Source: CIHR, JDRF, MbD

351POSTER ABSTRACTST-2074DECODING SINGLE-CELL GENE REGULATORY NETWORKS IN THE HUMAN PANCREAS AND BETA CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELLSVanheer, Lotte - Department of Development and Regeneration, KU Leuven, Belgium Pasque, Vincent - Department of Development and Regeneration, KU Leuven, BelgiumHuman induced pluripotent stem cells (iPSCs) hold tremendous potential for providing replacement insulin-producing beta ( ) βcells in unlimited quantities to treat diabetics worldwide. Despite considerable progress, the -like cells derived by differentiating βhuman iPSCs in vitro are not fully mature and fail to recapitulate the full functional properties of primary cells isolated from βcadaveric donor organs. In order to further improve the maturity of human iPSC-derived (iPSC- ) cells, additional insight is ββneeded into how cell identity is established and maintained βin vivo. Here, we combine single cell RNA sequencing data with machine learning algorithms to reveal gene regulatory networks active in human pancreatic cells as well as in human iPSCs- cells. Specifically, we used single-cell regulatory βnetwork inference and clustering to identify transcription factors, regulatory states, and cell lineages based on cis-regulatory information. This approach provides critical biological insights into the mechanisms of human pancreas development and the maintenance of cell identity. In addition, this allows for βa comprehensive exploration of transcriptional and regulatory states of pancreatic cell types. Comparing in vivo cells to βthose derived from human iPSCs will provide a basis to generate human iPSC- cells functionally more equivalent to in vivo ββcells. Understanding and defining the cellular mechanisms surrounding the establishment and maintenance of functionally mature cells will have major implications for regenerative βmedicine in the context of diabetes treatment.Funding Source: FWO SB PhD Fellowship 1S29419N to L.V. ; FWO Odysseus Return Grant G0F7716N to V.P. ; KU Leuven Research Fund (BOFZAP starting grant StG/15/021BF and C1 grant C14/16/077) to V.P.T-2076XENOTRANSPLANTATION OF HUMAN STEM CELL-DERIVED ENCAPSULATED LIVER TISSUE EFFECTIVELY AND SAFELY TREATS ACUTE LIVER FAILURE IN IMMUNOCOMPETENT MICE WITHOUT THE NEED OF IMMUNOSUPPRESSIONPaganelli, Massimiliano - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QC, Canada Raggi, Claudia - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QE, Canada M’Callum, Marie-Agnes - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QE, Canada Pham, Toan - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QE, Canada Selleri, Silvia - Immunology, Sainte-Justine UHC, University of Montreal, QE, Canada Benabdallah, Basma - Pharmacology, Sainte-Justine UHC, University of Montreal, QE, Canada Beausejour, Christian - Pharmacology, Sainte-Justine UHC, University of Montreal, QE, Canada Haddad, Elie - Immunology, Sainte-Justine UHC, University of Montreal, QE, CanadaAcute liver failure (ALF) consists in a dramatic loss of liver functions resulting in a survival rate <50%. There is an urgent need for new therapies capable of replacing liver functions in patients with ALF. We developed a human induced pluripotent stem cell (iPSC)-derived Encapsulated Liver Tissue (ELT) capable of consistently performing mature liver functions in vitro and in vivo. Thanks to the combination of complex iPSC-derived liver organoids and tailored biomaterials, the ELT performs liver-specific synthetic and metabolic functions as effectively as human hepatocytes. Here we assessed the efficacy and safety of the ELT in treating ALF in immunocompetent mice without immunosuppression. The human ELT effectively replaced liver functions and prevented death (68% survival at 5 weeks vs. 28% in controls) in immunocompetent mice with CCl4-induced ALF (xenotransplant into the peritoneal cavity without immunosuppression), with no rejection or tumor formation. Mice receiving the ELT showed lower plasma ammonia levels and less severe hepatic encephalopathy. The ELT also accelerated the host liver regeneration, which allowed, once the ELT explanted, long term survival of mice with normal liver function. The immune-isolating capacity of the biomaterial was tested by mixed lymphocyte reaction: the biomaterial isolated embedded organoids from allogeneic T cells completely. Upon implantation into immunocompetent healthy mice, the ELT did not trigger any inflammatory reaction, with no adhesions or foreign body reaction. At explant, after 4 weeks, the organoids within the ELT were alive and functional, without signs of rejection. No tumor or teratoma was observed when encapsulated highly tumorigenic cells or undifferentiated iPSCs were transplanted subcutaneously into immunodeficient NSG mice, confirming the protective capacity of the biomaterial towards tumor formation. Overall, we illustrate here the first stem cell-derived liver tissue capable of immediate, effective allogeneic/xenogeneic replacement of liver functions in immunocompetent subjects, without the risk of rejection or tumor formation. The ELT has the potential of being developed into a good-for-all, off-the-shelf, regenerative medicine product to replace liver functions in patients with ALF without the need of immunosuppression.Funding Source: Funded by the Stem Cell Network (Disease Team grants), CIHR (New Investigator grant) and FRQS (Junior 1)

352POSTER ABSTRACTST-2078AN IN VITRO THREE DIMENSIONAL MODEL OF LIVER DEVELOPMENT FROM HUMAN PLURIPOTENT STEM CELLSOgoke, Ogechi - Chemical and Biological Engineering, The State University of New York at Buffalo, NY, USA Mon, Tala - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Hasan, Osama - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Hoang Anh, Tram - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Ott, Cortney - Chemical and Biological Engineering, State University at New York, Buffalo, NY, USA Kalinousky, Allison - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Wayne, Lin - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Shamul, Claire - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Ross, Shatoni - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USA Parashurama, Natesh - Chemical and Biological Engineering, State University of New York at Buffalo, NY, USAThe liver arises during embryogenesis from the liver diverticulum (LD), an out-pocketing of hepatic specified endoderm (HE) at 26 d.p.c (human). We aimed to design an in vitro model of the LD to mimic pivotal stages of liver formation. The LD is composed of a thin pseudostratified layer of HE and endothelial cells surrounded by a collagen type II-rich septum transversum mesenchyme (STM). Liver formation initiates from the coordinated interactions of the liver progenitor cells that invade the STM as finger-like projections. In our in vitro approach, human pluripotent stem cells (hPSCs) were differentiated into hepatic endoderm cells using a robust protocol involving activators of Wnt and TGF βsignaling pathways at low oxygen (5% O2) (endoderm), KGF (gut tube endoderm) and FGF2 and BMP4 (hepatic endoderm). To model the LD, in vitro derived hepatic endodermal cells, were then replated into ultralow attachment (ULA) round bottom 384 well plates (Corning) and co-cultured with endothelial cells (HUVEC) to form compact, miniature microtissues (MCTs) over a 24 hour period. These MCTs were then embedded in Matrigel to model interactions between the hepatic endoderm and the STM. These embedded MCT demonstrated radial, uniform, cord-like projections of into the matrix, similar morphologically to hepatic cords that form during liver development between E9 to E10 (mouse). These MCT grew 23% in overall diameter over a 72 hour period, and displayed enhanced gene expression consistent with migration, including epithelial-to mesenchymal transition (EMT) markers and early liver differentiation genes that was not as pronounced in controls. Immunolocalization of MCT demonstrated liver and biliary markers such as albumin, CK18, Sox9 and HNF6. Live cell permeant dyes were utilized to track individual hepatic and endothelial cell movements during the morphogenic cord development. The LD model presented here represents a novel approach to modeling hepatic cord formation, a rate limiting step during liver development. Further studies will investigate the genomic and biophysical cues that regulate such multicellular events. These studies will improve models to create 3D liver tissue in vitro for various liver regenerative medicine applications, including modeling human development, liver disease, and tissue regeneration.Funding Source: Stem Cells in Regenerative Medicine (SCiRM) Training Program. Western New York Prosperity FellowshipEPITHELIAL TISSUEST-2080CHARACTERIZATION OF THE RABBIT ORAL MUCOSA EPITHELIAL STEM CELLS FOR CAOMECS ENGINEERINGOliva, Joan - Medicine, Emmaus Lifes Sciences, Inc., Torrance, CA, USA Sanghez, Valentina - Medical Genetics, LA BIoMed, Torrance, CA, USA Iacovino, Michelina - Medical Genetics, LA BioMed, Torrance, CA, USA Bardag-Gorce, Fawzia - Medicine, LA BioMed, Torrance, CA, USA Niihara, Yutaka - Medicine, Emmaus Medical, Inc., Torrance, CA, USAOral mucosa epithelial stem cells (OMECS) are commonly used to engineer epithelial cells sheets, to repair epithelium damaged tissues. Esophagus, skin and cornea are the major’s targets of these cell sheets called Cultured Autologous Oral Mucosa Epithelial Cell Sheet (CAOMECS). After isolating rabbit OMECS (Animal Study 21379-01), cells of different size and shape were identified: small, medium and large. The specific identity of these cells is unknown but this heterogeneric population of cells is usually seeded together to engineer CAOMECS. We propose to seed the OMECS sorted based on their size or epithelial stem cell surface markers p75 (limbus and mucosa epithelium) to determine which cells are important for CAOMECS engineering. First, cells were sorted-based on their size (Side-scattered light). Small and medium size cells cultured together, or large cells cultured by themselves, in co-cultured with 3T3 NIH Fibroblasts mitomycin-C treated. Large cells died very quickly, when the group of small/medium cells size formed few small colonies of dozens of cells, after 9 days in culture, but they were not able to form a CAOMECS. Second, we sorted isolated OMECS using a progenitor stem cells marker p75 in 2 populations (FACS (fluorescence-activated cell sorter)): p75+ representing progenitor stem cells (10.1% of the total cell population) and p75- (89.9% of the total cell population). After sorting the cells, p75– and + cells didn’t attach well on the transwell, died, and CAOMECS were not engineered. At this point, it is difficult to know if the OMECS are sensitive to the FACS/SSC sorting, or

353POSTER ABSTRACTSif OMECS need to be a heterogenous population to survive and engineer CAOMECS. Further studies should be conducted to characterize the OMECS phenotype and the raw material necessary for the CAOMECS engineering.Funding Source: Emmaus Medical, Inc.T-2082NEUROENDOCRINE CELL DIFFERENTIATION IS A CONSERVED TISSUE RESPONSE TO HYPOXIARajagopal, Jayaraj - Center for Regenerative Medicine, MGH/Harvard Medical School, Boston, MA, USA Shivaraju, Manjunatha - Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USANeuroendocrine cells (NE cells) are specialized secretory cells found in diverse metazoan tissues. They have myriad proposed functions based in part on the peptides and amines they secrete. Aberrant NE cell differentiation and increased NE cell numbers are associated with a spectrum of lung disorders including small cell cancers. In the case of endocrine disorders like diabetes, cell βnumbers are known to be regulated by physiologic parameters, such as blood glucose level. But whether NE cell numbers are generally responsive to physiologic cues is unknown. Here, we report that hypoxia stimulates neuroendocrine differentiation in multiple non-neuroendocrine cell lineages of the adult mouse trachea including basal stem cells. We show that hypoxia-dependent neuroendocrine differentiation is mediated through hypoxia-inducible factor 1 (Hif1 ). Strikingly, hypoxia-induced ααneuroendocrine differentiation is conserved, not only across multiple murine epithelial tissues, but also in human. These findings point to a conserved tissue response in which hypoxia leads to the generation of a cell type that can in turn secrete neuropeptides. Since most NE cells are not associated with a known function, hypoxia-induced NE cells may be useful to dissect their mechanism of action. Moreover, our findings suggest the broad conservation of a tissue level response that involves the differentiation of a specific cell type. Many molecular mechanisms have been identified within single cells to respond to various stresses: heat, nutrient limitation, and oxidative stress to name a few. Here we identify a tissue level response to a stress that actually involves the production of a unique type of cell that is poised to secrete peptides and amines in response to a hypoxic assault.Funding Source: HHMIT-2084MESENCHYMALSTEM CELLS DECREASE OXIDATIVE STRESS IN THE BOWEL OF IL-10 KNOCKOUT MICEJung, Kyong-Jin - Anatomy, Yeungnam University College of Medicine, Daegu, Korea Song, In-hwan - Anatomy, Yeungnam University College of Medicine, Daegu, KoreaInflammatory bowel disease (IBD) is an autoimmune disease characterized chronic inflammation mainly in large intestine. The interleukin-10 knockout (IL-10 KO) mouse is a well-known animal model of IBD which develops spontaneous intestinal inflammation that resembles Crohn’s disease. Oxidative stress is considered a leading cause of cell and tissue damage. Reactive oxygen species (ROS) itself can cause direct cell injury and/or cause indirect cell injury by secretion of cytokines from damaged cell. In this study, human bone marrow-derived mesenchymal stem cells (MSCs) was injected to IL-10 KO mice (MSC) and oxidative stress and inflammation levels were evaluated in the large intestine and compared with those of control IL-10 KO mice (CON) and wild control mice (Wild). The levels of ROS (superoxide and hydrogen peroxidase) and secondary end product of lipid peroxidation (malondialdehyde) were significantly higher in CON but superoxide dismutase (SOD) and catalase levels were lower in MSC. Inflammation related markers (INF- , TNF- , IL-4, and CD8) expression and γαinflammatory changes in histological analysis were much milder in MSC compare than CON. We conclude that MSCs have effects for redox balance this lead to suppression of IBD.Funding Source: This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (Ministry of Education; NRF-217C000377, NRF-218C000401).T-2086IGF1 PROMOTES PROLIFERATION OF MURINE SKIN-DERIVED PRECURSOR CELLS AND REDUCES OXIDATIVE STRESS OF THE CELLS VIA ACTIVATION OF ANTIOXIDANT REGULATORY FACTORSRoh, Sangho - School of Dentistry, Seoul National University, Seoul, Korea Park, Sangkyu - Biomedical Research Institute, NeoRegen Biotech, Seoul, Korea Kim, Kichul - School of Dentistry, Seoul National University, Seoul, Korea Kim, Hyewon - School of Dentistry, Seoul National University, Seoul, KoreaIGF1 has an important role in cell growth, differentiation and transformation of various stem cells and organogenesis during mammalian development. However, the effect of IGF1 on skin-derived precursors (SKPs) is not yet clear. The present study demonstrated the effects of IGF1 on the proliferation of murine SKPs (mSKPs). The antioxidant effect was also investigated. The proliferation was analyzed by WST-1 assay during the culture of mSKPs in IGF1-supplemented medium. To test its antioxidant effect, IGF1 was treated to the mSKPs after the induction of oxidative stress by H2O2. Western blotting, qPCR, and immunofluorescence staining were used to analyze the expression of genes related to stemness, epithelial–mesenchymal transition (EMT), antioxidant response, ageing. In results, the number, size of spheres and proliferation rate were higher in the IGF1-treatment group than those in non-treated

354POSTER ABSTRACTScontrol group. In addition, reactive oxidative species production and oxidative stress were reduced by IGF1 treatment from 24 to 72 h of culture. The mRNA and protein level of EMT markers (Cdh2, Fn1, S100a4, Snai2, Vim and Tgfb1) and antioxidant-related markers (GPX1, HO-1 and Nrf2) were increased by IGF1 treatment. These findings show that IGF1 enhances sphere formation and cellular proliferation by stimulation of EMT genes and also reduces oxidative damage by upregulation of antioxidant enzymes. This study suggests that IGF1 can be used as the supplement for skin regeneration by its proliferation and antioxidant effects on skin cells.Funding Source: This study was supported by a grant from the National Research Foundation of Korea (NRF-2016R1D1A1B03931864).T-2088TRANSCRIPTOME AND EPIGENOME ANALYSES FOR REGENERATED SKIN IN A RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA MOUSE MODELShimbo, Takashi - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Yamazaki, Sho - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Kitayama, Tomomi - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Ouchi, Yuya - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Yamamoto, Ryoma - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Takaki, Eiichi - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Kikuchi, Yasushi - Department of Stem Cell Therapy Science, Osaka University, Suita, Japan Bruckner-Tuderman, Leena - Department of Dermatology, University of Freiburg, Freiburg, Germany Uitto, Jouni - Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, USA Kaneda, Yasufumi - Department of Gene Therapy Science, Osaka University, Suita, Japan Tamai, Katsuto - Department of Stem Cell Therapy Science, Osaka University, Suita, JapanRecessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin disease caused by mutations in COL7A1 (coding type VII collagen) and known as a one of the most severe form of EB. Loss of functional type VII collagen compromises the dermal-epidermal junction. As a result, RDEB patients suffer from the repetitive blistering and have high risk of early-onset aggressive squamous cell carcinoma. Recently, we reported that a domain of HMGB1 stimulates bone marrow stem cells, and the activated stem cells mobilized into the damaged tissue to support efficient tissue regeneration. This HMGB1 treatment model in the RDEB mouse serves as a unique opportunity to describe how the damaged skin can be reconstructed. Here, we performed single cell RNA-seq and single cell ATAC-seq to comprehensively characterized the transcriptome and epigenome of the HMGB1 treated RDEB model mouse skin. Both single cell RNA-seq and ATAC-seq analyses showed a reduction of inflammatory cells after the HMGB1 treatment. In addition, single cell RNA-seq analysis identified that the treatment increased keratinocytes marked by keratin 1/keratin 10, suggesting a functional reconstruction of the RDEB skin. Furthermore, single cell ATAC-seq analysis delineated a massive change in epigenome. These singe cell level analyses suggested that the HMGB1 treatment induced the reconstruction of the damaged skin by introducing massive changes in transcriptome and epigenome, and also serve as a foundation to design more effective RDEB treatments.EYE AND RETINAT-2090OPTIMIZING A CONE RICH POPULATION FROM HUMAN PLURIPOTENT STEM CELLSMartin, Heather M - Biology, California State University, San Marcos, Escondido, CA, USA Diaz, Emily - Ophthalmology, University of California, San Diego, La Jolla, CA, USA Ogata, Anna - Ophthalmology, University of California, San Diego, La Jolla, CA, USA Wahlin, Karl - Ophthalmology, University of California, San Diego, La Jolla, CA, USAMillions of Americas suffer from some form of retinal degeneration. While these can be the result of inherited mutations, the vast majority, such as in age-related macular degeneration (AMD) have a cause that is less well understood. As our population ages and we are seeing an increase in individuals with symptoms of AMD, there is a need for better cell based models to study retinal disease and potential therapies. The discovery of pluripotent stem cells and new methods to differentiate them into a variety of cell types and tissue makes them well suited to address these needs. Using CRISPR-Cas9 gene editing, we were able to create cell type specific fluorescent reporters that allow tracking of retinal development in both 2D and 3D cultures. Our current protocol produces 3D laminar retinal structures (organoids) containing all the respective neuronal cell types typical of the human retina. Like human retinas, these organoids are generally rod rich, and although they do contain cones, they lack a dense cone rich structure resembling the macula. Previous studies have suggested that human recombinant COCO pushes iPSCs grown in 2D towards a blue-cone specific fate through Wnt, BMP, and TGF inhibition. A cell was engineered to incorporate βa fluorescent reporter into the SIX6 gene—an early eyefield marker—and CRX—a gene associated with the production of photoreceptors Using this dual fluorescent reporter system, we are able to track target gene expression in real-time. These cells were differentiated in both 2D and 3D cultures either in a standard medium, or a medium that had been enhanced with COCO, FGF-2, and IGF. Movement from hypoxic to normal conditions were also studied for the 2D cultures. Morphological and gene expression differences between control and treatment

355POSTER ABSTRACTSgroups were observed, as well as differences in how the treated groups expressed SIX6 in 2D versus 3D. Immunohistochemistry further detailed the photoreceptor makeup of the samples. The successful creation of a cone rich retinal organoid would allow for the study of an in vitro macula-like structure, which could aid in the search for treatments. This structure could also, with the use of patient derived iPSCs, provide a cone-rich section for transplant into individuals who have already suffered photoreceptor loss due to AMD.T-2092RETINAL ORGANOIDS REVEAL MECHANISM OF VISION LOSS IN ATF6 ACHROMATOPSIA PATIENTSKroeger, Heike - Pathology, UC San Diego, La Jolla, CA, USA Chiang, Wei-Chieh - Pathology, UC San Diego, La Jolla, CA, USA Nguyen, Amanda - Pathology, UC San Diego, La Jolla, CA, USA Lin, Jonathan - Pathology, UC San Diego, La Jolla, CA, USALoss of function of the ATF6 transcription factor causes heritable αphotoreceptor diseases including achromatopsia and cone-rod dystrophy. These patients have severely impaired vision, loss of cone photoreceptor function, and foveal hypoplasia from infancy. The mechanism by which ATF6 defects lead to vision loss in these patients is unknown. Here, we generated 3D retinal organoids from ATF6 mutant patient iPSC cells and isogenic ATF6 knock-out human embryonic stem cells (hESCs) . We used cellular tomography, combined with OCT cryosectioned and immunohistological analysis to visualize developing rod and cone photoreceptors. We performed RNAseq analysis of retinal organoids in the presence and absence of functional ATF6, and revealed that the lack of functional ATF6 relates to a significant disruption of the ATF6 transcriptional program. We found that the loss of ATF6 signaling affects early differentiation events by suppressing loss of pluripotency and increasing the expression of early eye development markers, such as PAX6, SIX3 and RX. Microscopic analysis at day 120 and 290 confirmed the presence of rod and cone photoreceptor cells in ATF6 wild-type retinal organoids. Interestingly, retinal organoids derived from ATF6 knockout hESCs and ATF6 mutant patient iPSCs showed no development of cone photoreceptors cells at the same ages. By contrast, the development of rod photoreceptor cells appeared to be normal in all 3D retinal cups. Retinal organoid surface scanning analysis showed a similar morphology seen in the retina of ATF6 patient achromats. ATF6 mutant alleles show a selective failure to form cone photoreceptors during retinal development while rods appear to be spared. We propose that the vision defect found in ATF6 patients arise as a consequence of the development of a rod-dominant retina.T-2094IN VIVO RETINAL STEM CELL PROLIFERATION, EXPANSION AND RETINAL MIGRATION IS INDUCED VIA INHIBITION OF BMP AND SFRP2 IN THE ADULT MOUSE EYEGrise, Kenneth N - Molecular Genetics, University of Toronto, ON, Canada Coles, Brenda - Molecular Genetics, University of Toronto, ON, Canada Bautista, Nelson - Molecular Genetics, University of Toronto, ON, Canada van der Kooy, Derek - Molecular Genetics, University of Toronto, ON, CanadaAdult retinal stem cells (RSCs) are rare cells that reside in the pigmented ciliary epithelium (CE) of the mammalian eye. In vivo, RSCs do not proliferate or generate new retinal cells in adult mammals. Previously, we identified BMP and sFRP2 proteins as mediators of adult RSC quiescence with in vitro experiments. Here, we investigated whether BMP and sFRP2 inhibition could induce RSC proliferation in vivo in adult mice. Intravitreal injections of the BMP antagonist Noggin or an anti-sFRP2 antibody were administered once a day for 3 days. At 1 day after the injection period, each inhibitor alone induced a ~5-fold increase in proliferating (EdU+) CE cells (Pax6+) compared to PBS injection. Noggin and anti-sFRP2 combined with the growth factors FGF2 and Insulin (FINS) induced an additive increase in EdU+/Pax6+ cells (~12-fold), while the growth factors alone showed a ~5-fold increase in EdU+/Pax6+ cells. At both 7 and 31 days after BMP or sFRP2 inhibition, clonal sphere assays revealed an over 2-fold increase in the number of sphere-forming RSCs present in the eye. Next, we employed inducible CE lineage tracing (Msx1-CreERT2;Rosa26-TdTomato mice) to investigate whether adult CE cells migrate into the retina after FINS treatment, and whether MNU-induced retinal injury could impact their migration. In uninjured eyes, we observed a ~3-fold increase in TdTomato+ cells in the retinas of FINS-treated eyes compared to PBS injection. MNU injury in PBS-treated eyes slightly increased retinal migration from the CE, whereas MNU injury did not further enhance retinal migration in FINS-treated eyes. Notably, a decreased total eye diameter was observed in 50% of the PBS+MNU eyes that was not observed in any other group, suggesting the FINS+MNU group may have been protected from this pathology. However, in eyes with normal diameter, there was a similar reduction in outer nuclear layer thickness in PBS+MNU and FINS+MNU eyes. Together, these results demonstrate that blocking BMP and sFRP2 in the adult mouse eye can bring RSCs out of quiescence, induce them to proliferate and expand, and induce CE cells to migrate into the retina. Currently, we are determining if CE cells that migrate into the retina differentiate into retinal cell types, which would establish this method as a new paradigm to stimulate adult retinal neurogenesis.Funding Source: Canadian Institutes of Health Research, Medicine by Design, National Sciences and Engineering Research Council, Foundation Fighting Blindness

356POSTER ABSTRACTST-2096EFFECT OF CHROMOSOME 20 GENETIC ABNORMALITIES ON HUMAN EMBRYONIC STEM CELL DIFFERENTIATION INTO RETINAL PIGMENT EPITHELIUM CELLSVitillo, Loriana - Institute of Ophthalmology, University College London, UK Anjum, Fabiha - Institute of Ophthalmology, University College London, UK Weightman, Richard - Department of Biomedical Sciences, University of Sheffield, UK Gregory, Sian - Department of Biomedical Sciences, University of Sheffield, UK Shaw, Allan - Department of Biomedical Sciences, University of Sheffield, UK Hewitt, Zoe - Department of Biomedical Sciences, University of Sheffield, UK Andrews, Peter - Department of Biomedical Sciences, University of Sheffield, UK Coffey, Peter - Institute of Ophthalmology, University College London, UKThe ability to maintain self-renewing human pluripotent stem cells (hPSCs) indefinitely in culture is cornerstone for research and clinical studies of pluripotent cells. However, the regenerative medicine field has come to the realisation that karyotype and sub-karyotype aberrations arise in hPSCs as a result of continuous expansion. Understanding the biological consequences of carrying common and subtle variances into a clinically relevant differentiation protocol is necessary to provide much-needed evidence of the mechanisms at play and support an educated risks/benefit assessment during regulatory evaluation. In this study, we aimed to assess the effect of frequent and commonly detected chromosome 20 (Chr20) genetic aberrations during the differentiation of hPSCs towards retinal pigment epithelium (RPE) cells, currently in Phase 1 clinical trial. Experiments were conducted on clonal lines derived from human embryonic stem cells as follows: normal karyotype; 20q11.21 copy number variation (CNV) and trisomic for the long arm and monosomic for the short arm of Chr20 (called normal, CNV and i20q, respectively). Our data show that hPSCs carrying the 20q11.21 CNV amplification undergo RPE differentiation faster and with a higher yield compared to normal cells. Moreover, both types of mutated hPSCs have a proliferative advantage over normal cells, which is also reflected in the speed of differentiation. Interestingly, the more severe aberration of the i20q clones led the population to completely abort the differentiative pathway and instead undergo apoptosis. This study illustrates that hPSCs carrying the CNV Chr20 variants can efficiently progress into RPE differentiation and outperform the normal counterparts. At the same time, variants that favour the balance towards self-renewal (i20q) fail quickly in our protocol, suggesting that the RPE differentiation culture conditions are not permissive to the survival of undifferentiated cells. Comprehensive RNA-seq examination of early stem cell fate decision regulatory networks modified by gains of Chr20 regions could explain the clonal differences in differentiation propensities and lead to the discovery of new therapeutic targets applicable to retina-associated disorders.T-2098DISCOVERY OF TWO STEM CELL POPULATIONS WITH DISTINCT MARKER EXPRESSION PROFILES DURING DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS TOWARDS LIMBAL EPITHELIAL STEM CELLSVattulainen, Meri - Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Ilmarinen, Tanja - Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Skottman, Heli - Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Viiri, Keijo - Faculty of Medicine and Health Technology, Tampere University, Tampere, FinlandDifferentiation of corneal limbal epithelial stem cells (LESCs) from human pluripotent stem cells (hPSCs) represents an attractive future therapeutic option for patients suffering from bilateral limbal stem cell deficiency (LSCD). In addition, it provides new tools for studying the biological properties of LESCs in humans. Here, two genetically distinct hPSC lines were subjected to corneal differentiation in previously published conditions, with specific aim to study the hierarchy of putative limbal stem cell markers in more detail. Characterization of undifferentiated hPSC and derivative cells in several time points between 7 and 24 days was carried out using immunofluorescence labeling against a panel of selected limbal/corneal epithelial and pluripotency markers. Along the differentiation process, we observed a decrease of pluripotency markers OCT3/4 and SSEA4, accompanied by ascending expression of several widely proposed LESC-related markers PAX6, Np63 , CK15 and CK14. Interestingly, ABCG2, ∆αa marker for slow-cycling limbal cells in vivo and a universal marker of stemness, was only transiently expressed at a very early phase of the differentiation. Hence, we performed further comparison of the early and latter hPSC-LESC cell populations by quantifying the protein expressions with cell counting and flow cytometry, and gene expressions with qRT-PCR. With this, two noticeably different cell populations were identified during hPSC-LESC differentiation, the earlier population expressing robustly the stemness marker ABCG2 and the latter expressing the other proposed LESC markers Np63 , CK15 and CK14. ∆αBased on our results acquired with hPSC-derived LESCs, we propose a marker protein hierarchy that potentially reflects the limbal stem cell differentiation in vivo. Furthermore, novel culture conditions stabilizing the expression of ABCG2 in hPSC-LESC cultures were optimized in order to identify the exact functional role and as a continuum, the therapeutic relevance of these two identified cell populations with distinct LESC-associated marker profiles.

357POSTER ABSTRACTSFunding Source: This study has been supported by the Academy of Finland, Business Finland, Sigrid Juselius Foundation, Finnish Cultural Foundation as well as Finnish Eye and Tissue Bank Foundation.T-2100HUMAN PLURIPOTENT STEM CELL DERIVED RETINAL ORGANOIDS FOR MODELING LEBER CONGENITAL AMAUROSISKambli, Netra K - Shiley Eye Institute, University of California, San Diego (UCSD), La Jolla, CA, USA Ray, Sunayan - Ophthalmology, Shiley Eye Institute UCSD, La Jolla, CA, USA Jones, Melissa - Ophthalmology, Shiley Eye Institute UCSD, La Jolla, CA, USA Wahlin, Karl - Ophthalmology, Shiley Eye Institute UCSD, La Jolla, CA, USAModeling human retinal disease in vitro using pluripotent stem cells has opened new avenues of research in regenerative medicine. Importantly, these stem cells have the potential to differentiate into any cell type in the body, including the neural retina, and thus hold great promise for modeling human retinal disease. Leber Congenital Amaurosis (LCA) is an inherited retinal degeneration which affects vision at birth or within the first few years of life. It causes dysfunction and death of photoreceptor cells resulting in severe loss of vision and eventually leads to blindness. Though relatively rare, mutations in close to 20 genes, including the CRX (cone-rod homeobox) gene, are known to cause LCA. Using CRISPR-Cas gene -editing, a patient-specific mutation was created in the transactivating domain of the CRX gene. This mutation was introduced into iPSC bearing a Six6-Ruby3 reporter which allows for the selective enrichment of 3D retinal structures which will improve the reliability and reproducibility of differentiation experiments. We have demonstrated that these reporter organoids develop discrete laminar structures with appropriate reporter expression in progenitors and photoreceptor gene expression in photoreceptors. The similarity to human retinal development will facilitate the analysis of any retinal disease phenotype. To evaluate disease progression, organoids were collected at different time points and evaluated by immunohistochemistry and RT-qPCR to analyze gene expression. Organoids expressed ruby3 signal indicating endogenous Six6 expression and immunohistochemistry of older organoids indicated the presence of photoreceptor genes. RT-qPCR was performed to further validate the presence of genes involved in retinal development and to determine the changes involved in the mutant and control organoids. Studying human retinal degeneration in a dish allows us to understand the mechanism of disease progression and offers a reliable platform to develop assays for future drug screening to treat retinal degenerative diseases.Funding Source: A CIRM Bridges II Grant (EDUC2-08381)T-2102POST-TRANSPLANTATION MOUSE IPSC-DERIVED RGCS ESTABLISH NEURONAL POLARITY WITHIN NMDA-DAMAGED HOST RETINAS AND SHOW ENHANCED SURVIVAL FOLLOWING GROWTH FACTOR CO-TREATMENTBaranov, Petr - Ophthalmology, Harvard Medical School, Schepens Eye Research Institute, Boston, MA, USA Oswald, Julia - Ophthalmology, The Schepens Eye Research Institute, Boston, MA, USATransplantation of iPSC-derived neurons as a strategy to replace host cells, lost in progressive neurodegenerative diseases has gained traction over the past years across the field of neuroscience. The advantage of the eye as a target organ for cell replacement therapy has been demonstrated in pre-clinical and clinical studies of retinal pigment epithelium and photoreceptor transplantation. To address visual decline associated with Glaucoma our laboratory has been working on the replacement of retinal ganglion cells (RGCs), the neuronal link between the retina and the brain. We have demonstrated that within mouse models, iPSC-derived RGCs are able to integrate within pups (83%, n=12) and adult retinas, including both healthy (57%, n=14) and diseased (67%, n=12) hosts. While the rate of transplantation success was high compared to previously published primary cell transplants, the number of surviving donor RGCs per host retina remained below 1%. To address this challenge, we have developed a co-treatment regime using known neuroprotective factors BDNF, GDNF and CNTF, delivered in a slow-release formulation. For transplantation Thy1-GFP+ cells were isolated by magnetic micro-beads at day 21 of retinal organoid culture and injected into the vitreous of the eye either alone or with co-treatment in healthy mice and mice subjected to NMDA one week prior. Electroretinography performed at 6 weeks post-transplant, showed that growth factor cotreatment partially preserved inner retina function as measured by pSTR response (32uV in co-treatment group vs 17uV in control). Furthermore, growth factor co-treatment enhanced both overall grafting success from 50% to 73% and donor cell survival. Axonal and dendritic outgrowth could be detected as early as two weeks post-transplant in healthy and NMDA treated retinas underlining the potential of iPSC-derived RGCs to establish neuronal polarity even within diseased microenvironments.Funding Source: BrightFocus Foundation, Department of Ophthalmology, Massachusetts Lions Fund

358POSTER ABSTRACTSSTEM CELL NICHEST-2104DESIGNING A BIOLOGICAL INTERFACE COMBINING A COCHLEAR IMPLANT WITH HUMAN EMBRYONIC STEM CELLSChang, Hsiang-Tsun - Otolaryngology, Northwestern University, Chicago, IL, USA Ameer, Guillermo - Biomedical Engineering, Northwestern University, Evanston, IL, USA Heuer, Rachel - Otolaryngology, Northwestern University, Chicago, IL, USA Matsuoka, Akihiro - Otolaryngology, Northwestern University, Chicago, IL, USA Nella, Kevin - Otolaryngology, University of Miami, Miami, FL, USA Oleksijew, Andrew - Otolaryngology, Northwestern University, Chicago, IL, USASensorineural hearing loss is the one of the most common sensory deficits worldwide. Currently, cochlear implant (CI) is the only clinically available surgical intervention to restore the sensory deficit. There are, however, limitations to current CI designs. First, a patient’s pathology typically entails different degree of loss in the spiral ganglion neurons (SGNs). The loss can create an “electrode-neuron gap” that can reduce spatial selectivity. Second, current CI design in the electrode array contain 22 or less electrode channels; far less than the number of hair cells. These limitations can decrease available information that is sent to the brain. We, therefore, propose a biological interface that combines a CI technology with human embryonic stem cell (hESC) replacement therapy. By introducing human embryonic stem cell-derived otic neuronal progenitors (hESC-derived ONPs), which are neuronal precursors to human SGNs, into the scala tympani, we expect to pave electrode-neuron gaps to improve the spatial selectivity, which can further improve CI’s performance. The benefits of neurotrophins such as brain-derived neurotrophic factor (BDNF) in neuronal differentiation has been shown to play a pivotal role in axonal growth and synaptic plasticity. Successful, long-term delivery of BDNF in the inner ear has yet to be established; partially because one-time administration of BDNF to the scala tympani is not biologically effective due to its short half-life. Alternatively, encapsulating BDNF in a thermoresponsive polymer-poly (polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN), which is suitable for delivery of therapeutics, could be our solution. It possesses a lower critical solution temperature (LCST) of 26 ; liquid status ℃efficiently entraps chemokine when temperature is below 26 and gelation appears to slowly release chemokine as ℃temperature reach above 26 . The ELISA result demonstrates ℃a steady long-term release when BDNF entrapped in PPCN, and our current goal is to assess the bioactivity of BDNF on ONP’s neurite outgrowth. Furthermore, PPCN has the potential to develop in vivo cell entrapment near the CI electrode arrays, which could increase cell survival in post-transplantation to the inner ear.FundingSource:TheDepartmentofDefence(W81XWH-18-1-0752), NIH (NIDCD) K08 ((K08DC01382910), and the Triological Society/American College of Surgeons Clinician Scientist AwardT-2106INFUSED BONE MARROW DRIVED CELLS ARE REPAIRED FIBROSIS AND ENGULFED DAMAGED CELLSYamamoto, Naoki - Gastroenterology and Hepatology /Health Administration Center, Yamaguchi University, Ube Yamaguchi, Japan TAakami, Taro - Gastroenterology and Hepatology, Yamaguchi University, Yamaguchi, Japan Fujisawa, Koichi - Gastroenterology and Hepatology, Yamaguchi University, Yamaguchi, Japan Matsumoto, Toshihiko - Gastroenterology and Hepatology, Yamaguchi University, Yamaguchi, Japan Uchida, Koichi - Human Nutrition Faculty of Nursing and Human Nutrition, Yamaguchi Prefectural University, Yamaguchi, Japan Terai, Shuji - Gastroenterology and Hepatology, Niigata University, Niigata, Japan Tani, Kenji - Veterinary Surgery, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan Taura, Yasuho - Veterinary Surgery, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan Nishina, Hiroshi - Development and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Sakaida, Isao - Gastroenterology and Hepatology, Yamaguchi University, Yamaguchi, JapanThe autologous bone marrow cells were useful for the repair therapy in liver cirrhosis and many kind of diseases. We developed the GFP/CCl4 model which monitor the GFP-positive bone marrow cell (BMC) repopulated under liver cirrhosis mice (Hepatology). In this study, we estimated characterization and function of infused BMC in liver cirrhosis using Electron Microscopy (EM) in recipient liver. C57BL/6 mice were injected with CCl4 twice a week for 4 weeks to make the liver cirrhosis. GFP-positive BMC were infused from tail vein and sacrificed at 4 weeks after BMC infusion. The liver sample was fixed using both paraformaldehyde+glutaraldehyde and made epon section. We analyzed the characterization of the infused GFP-postive BMC using both EM and Immune EM (IEM). We analyzed the image of IEM, comparing with the character of positive cells by immunohistochemistry and double fluorescent staining(Antibody:GFP, EpCAM, A-6, Liv8-CD44,hepatoblastmarker-Liv2,MMP9,MMP13,AK4,CXCR4,p62,CD68,TGF-beta,alfa-SMA,transcription regulator-maternal of inhibitor of differentiation -Maid). We analyzed some kind of gene by Real-TimePCR(Gene: p16,p21,p62,EpCAM,AFP,A-6,HNF4,Sirt1,2,3,6,AK-4,Hmox,Ncam,ATF,XBP1 etc). We had two kinds of GFP positive BMCs in recipient cirrhosis liver using IEM method. One group of GFP positive BMCs was similar to

359POSTER ABSTRACTShepatocyte in size(15-30um) and located around fiber. MMP9 positive cells, Liv8 positive cells, Maid positive cells, CXCR4 positive cells were same. These cells were round forms and different from stellate cell or Kupffer cell in feature and had the increase of lysosome structure in cytoplasm. These cells were located on fiber in hepatic cord and repaired fibrosis. The other group cells were small size (2-5um) and located in destructive area and A6 positive cells, Liv2 positive cells, EpCAM positive cells were same. These cells had high N/C ratio and smaller than hepatocyte. These cells migrated into damaged cell area and had the phagocytic capacity. These cells were few F4/80 positive cells and smaller than Kupffer cell in size. In conclusion, we detected two kind of infused BMCs. The round BMCs repaird liver fibrosis and the small BMCs worked the phagocytized damaged hepatocyte and maintenance of liver.T-2108ELECTROMAGNETIZED GOLD NANOPARTICLES MEDIATE DIRECT LINEAGE REPROGRAMMING INTO INDUCED DOPAMIN NEURONS INVIVO FOR PARKINSON’’S DISEASE THERAPYYoo, Junsang - Chemistry, Dongguk University, Seoul, Korea Kim, Jongpil - Chemistry, Dongguk University, Seoul, KoreaElectromagnetic fields (EMF) are physical energy field generated by electrically charged objects and specific ranges of EMF can influence numerous biological processes, including control of cell fate and plasticity. In this study, we show that electromagnetized goldnanoparticles (AuNPs) in the presence of specific EMF condition facilitate efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to specific activation of the histone acetyltransferase Brd2, resulting in histone H3K27 acetylation and robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and noninvasively alleviated symptoms in the mouse Parkinson’s disease models. This study provides proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for treatment of neurodegenerative disorders.T-2110A TIE2-NOTCH1 SIGNALING AXIS DRIVES ENDOTHELIAL REGENERATION AND RECOVERY OF THE BONE MARROW HEMATOPOIETIC NICHEPajcini, Kostandin V - Center for Stem Cell and Regenerative Medicine/Pharmacology, University of Illinois at Chicago, IL, USA Lucas, Daniel - Division of Experimental Hematology and Cancer Biology, University of Cincinnati, OH, USA Malik, Asrar - Pharmacology, University of Illinois at Chicago, IL, USA Rehman, Jalees - Pharmacology, University of Illinois at Chicago, IL, USA Shao, Lijian - Pharmacology, Center for Stem Cell and Regenerative Medicine, Chicago, IL, USA Sottoriva, Kilian - Pharmacology, University of Illinois at Chicago, IL, USARegeneration of the hematopoietic niche is of critical importance to the recovery of blood after chemotherapeutic and irradiation treatment. Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain (TAD) we generated a viable, post-natal, Notch signaling hypomorph. Heterozygous Notch1-deltaTAD (Notch1+/deltaTAD) mice appear normal and have no endothelial or hematopoietic phenotype, aside for an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, Notch1+/deltaTAD mice exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our hypomorphic model system, Notch1-deltaTAD mutant protein accumulated in endothelial cells and interfered with optimal activity of Notch1 the transcriptional complex. Failure of the Notch1-deltaTAD mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc caused prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required as the penultimate signaling event for regeneration of bone marrow endothelial niche and hematopoietic recovery.Funding Source: This study was funded by NIH 1R011HL134971.T-2112TARGETING THE LEUKEMIA PROPAGATING CELLS WITHIN THERAPY-INDUCED NICHES BY CELL-BIOMIMETIC NANOPARTICLESMu, Lili - Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School Of Medicine, Shanghai, China Dong, Xiao - Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao-Tong University School of Medicine, Shanghai, China Hong, Dengli - Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, China Fang, Chao - Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao-Tong University School of Medicine, Shanghai, ChinaAcute lymphoblastic leukemia (ALL) is one of the most common pediatric cancers. Although the treatment has achieved great success, relapse remains the major challenge. Recently we reported a therapy-induced niche (TI-niche) shielding residual leukemia cells. Precisely targeting the residual cells as well as interfering its interaction with the niche can be a promising strategy. Here, we develop a leukemia cell-biomimetic nanoparticles (NPs) and co-deliver the anti-TGF RII to blockade β

360POSTER ABSTRACTSthe drug-resistance signaling pathway being activated by the TI-niche. We coat NPs with leukemia cell membrane which can anchor on the leukemic niches during blood circulation. The anti-TGF RII embedded in cell membrane will release under hypoxia-βenvironment in the bone marrow, neutralize the TGF RII in ALL βcell membrane and sensitize the cells to chemotherapy which the NPs released. Therefor, we have developed a sequential drug delivery system which can target the therapy-resistant leukemia cells, improve the drug efficiency.T-2114CELL CYCLE ACTIVATION OF HUMAN IPSC-DERIVED CARDIOMYOCYTES BY EXPOSURE TO CONDITIONED MEDIUM FROM MESENCHYMAL STEM CELLSSantin Velazque, Natalia L - Research Laboratory Applied to Neurosciences, Fleni, Tigre, Argentina Amin, Guadalupe - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Biani, Maria - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Colli, Carolina - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina La Greca, Alejandro - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Lombardi, Antonella - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Luzzani, Carlos - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Miriuka, Santiago - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Mobbs, Alan - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Moro, Lucia - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Neiman, Gabriel - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Scarafia, Agustina - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Sevlever, Gustavo - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, Argentina Waisman, Ariel - LIAN, Fundacion para la Lucha contra las Enfermedades Neurologicas de la Infancia, Buenos Aires, ArgentinaHuman heart has little regenerative capacity after myocardial damage due to the low proliferation potential of cardiomyocytes (CM). Hence, there is a growing interest in the production of human induced pluripotent stem cell-derived CM (hiPSC-CM) as a potentially promising strategy for regenerative therapies, and in identifying factors playing relevant roles in the regulation of the hiPSC-CMs cell cycle. Moreover, recent findings highlights the regenerative capacity of mesenchymal stem cells (MSC) and the many properties of MSC-conditioned medium and extracellular vesicles (EV) in cell culture. The aim of this study is to investigate the ability of the hiPSC-CM to re-enter cell cycle after exposure to conditioned medium from WJ-MSC. Human iPSC-CMs were obtained with a monolayer protocol. We established FUCCI-hiPSCs, a line constitutively expressing Fluorescent Ubiquitination-based Cell Cycle Indicator, which consists of system that employs red fluorescence for G1 state and green for S/G2/M. FUCCI-hiPSC-CMS were incubated with WJ-MSC-conditioned medium for 24, 48 and 72 hours. At the first 24hs, hiPSC-CM in S/G2/M increased from 14.7% to 28.7% with conditioned medium. Interestingly, while replication activity decrease over time with control medium (near 5% at 48 and 72hs), with MSC-conditioned medium maintain up to 20%. Enhanced DNA synthesis was confirmed by EdU assay and subsequent immunofluorescence. Finally, we used LC-MS/MS proteomic approach to identify proteins presents in the secretome from WJ-MSC that could explain the effect in the hiPSC-CM. Through Gene Ontology analysis we found 147 proteins involved in cell cycle. Particularly, some of them were related to the G2/M checkpoint and mTOR pathway. In conclusion, this study reveals that the exposure to MSC-contidionated medium induce proliferation of hiPSC-CMs. Our next goal is to study the differential expressed genes in the proliferative hiPSC-CM.Funding Source: CONICET. Fundación para la Lucha contra Enfermedades Neurológicas de la Infancia.CANCERST-2116NOVEL INSIGHTS IN CANCER STEM CELL BIOLOGY: WILMS’ TUMOR AND HUMAN FETAL NEPHRON PROGENITORS AS AN EXAMPLEPetrosyan, Astgik - GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics/Urology/Saban Research Institute, Children’s Hospital Los Angeles, CA, USA Villani, Valentina - Urology, GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, The Saban Research Institute, Children’s Hospital Los Angeles, CA, USA Aguiari, Paola - Urology, GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, The Saban Research Institute, Children’s Hospital Los Angeles, CA, USA Thornton, Mattew - Maternal-Fetal Medicine Division, USC Keck School of Medicine, Los Angeles, CA, USA Grubbs, Brendan - Maternal-Fetal Medicine Division, USC Keck School of Medicine, Los Angeles, CA, USA

361POSTER ABSTRACTSDe Filippo, Roger - Urology, GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, The Saban Research Institute, Children’s Hospital Los Angeles, USC Keck School of Medicine, Los Angeles, CA, USA Da Sacco, Stefano - Urology, GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, The Saban Research Institute, Children’s Hospital Los Angeles, USC Keck School of Medicine, Los Angeles, CA, USA Perin, Laura - Urology, GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, The Saban Research Institute, Children’s Hospital Los Angeles, USC Keck School of Medicine, Los Angeles, CA, USAWilms’ tumor (WT) accounts for 95% of renal malignancies in children and is characterized by postnatal uncontrolled proliferation of nephron progenitors (NP) without generation of functional nephrons. Little is known about WT involvement of NP in tumor progression and their relation to the microenvironment. Using our validated Smartflares technique we isolated, for the first time, NP expressing SIX2 and CITED1 (the master genes regulating nephrogenesis) from WT samples and human fetal kidneys (hFK) and compared them by RNA-seq. We established conditions for long-term culture of NP cells and studied in vitro mechanism of self-renewal vs. differentiation of NP from both WT and hFK. We also transplanted WT-NP in vivo to study tumorigenesis and targeted tumor growth and progression using neutralizing antibodies against integrins. We verified the presence of NP (SIX2+CITED1+ cells) in WT samples; RNA-Seq data confirmed their nephrogenic signature but also highlighted differences in expression of pluripotency and self-renewal related genes like OCT4, FOXO1, NANOG along with a lower expression of -catenin compared to hFK-NP. We confirmed that βboth WT-NP and hFK-NP can be cultured for multiple passages in vitro and are tumorigenic in vivo. We identified for the first time a disruption in integrin expression patterns within the WT-NP that, when specifically targeted, regulates tumor stem cell self-renewal or differentiation via modulation of the -catenin/βLEF1 pathway, opening new avenues for the treatment of WT. In conclusion, this work represents the first characterization of SIX2+CITED1+ cells from WT and suggests the importance of matrix-cell interaction in development and during WT formation. These studies have the potential to increase our knowledge of human nephrogenesis and facilitate the development of new strategies aimed at halting tumor progression.Funding Source: GOFARR Laboratory for Organ Regenerative Research and Cell TherapeuticsT-2118MULTIPLE TREATMENT CYCLES OF NEURAL STEM CELL DELIVERED ONCOLYTIC ADENOVIRUS FOR THE TREATMENT OF GLIOBLASTOMABatalla-Covello, Jennifer - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Adileh, Lana - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Gonzaga, Joanna - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Flores, Linda - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Gnai, Hoi Wa - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Hyde, Caitlyn - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Mooney, Rachael - Developmental and Stem Cell Biology, City of Hop, Duarte, CA, USA Aboody, Karen - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USATumor tropic neural stem cells (NSCs) can improve the anti-tumor efficacy of oncovirotherapy agents by protecting them from rapid clearance by the immune system and delivering them to multiple distant tumor sites. We recently completed a first-in-human trial assessing the safety of a single round of NSC-delivered CRAd-Survivin-pk7 (NSC.CRAd-S-pk7) in combination with radiation and chemotherapy within newly diagnosed GBM patients. While safety of NSC.CRAd-S-pk7 to tumor sites has been demonstrated, we expect the therapeutic efficacy of a single NSC.CRAd-S-pk7 treatment cycle to be suboptimal. Inspired by multiple treatment cycles of oncovirotherapy to treat peripheral tumors, here we investigate the potential therapeutic enhancements of multiple treatment cycles within the brain tumor setting. We believe that continuing the therapy after our initial dose will be a requisite for providing a regular immune system boost and update of the tumor antigen profile. Multiple treatment rounds involving adenovirus has often led to both humoral and cellular anti-AD immunity within animal models, with many studies demonstrating that repeat administration of Ad Vectors has limited efficacy over the single round treatment. In contrast, here we report an improvement in treatment efficacy when 3 rounds are administered to B6 mice with pre-established GL261 tumors. In fact, in 25% of mice, the tumor burden was reduced to occult levels after 3 rounds of treatment. Ongoing studies are investigating if differences in the vector dose, strength of the pre-existing immune response, or intratumoral administration route within tumor tropic NSCs is responsible for these favorable results. We are also monitoring the development of humoral and cellular immunity after each treatment round to establish a rationally-based treatment schedule for Pre-IND enabling studies.Funding Source: Ben and Catherine Ivy Foundation, Alvarez Family Charitable Foundation, Rosalinde and Arthur Gilbert Foundation, Jeanne and Bruce Nordstrom.T-2120CELLULOCYTOSIS BETWEEN KUPFFER CELLS AND TUMOR-INITIATING STEM-LIKE CELLS PROMOTES METASTATIC COLONIZATION VIA CD47 AND FGFR2 SIGNALINGHernandez, Juan Carlos - USC, California Institute for Regenerative Medicine (CIRM), Irvine, CA, USA

362POSTER ABSTRACTSHighly self renewing, CD 133+ liver tumor-initiating stem-like cells (TICs), are immensely invasive/metastatic cells that perpetuate secondary tumor even after eradication of primary Hepatocellular carcinoma (HCC); a process independent of propensity for lineage commitment. Here we probed into the cause for plasticity in TIC’s that empowers TIC’s to derail lineage commitment. We identified cellulocytosis, and define cellulocytosis as a process in which phagocytic cell (F4/80+ Kupffer cells; KC) transports an entire non-phagocytic cell (CD133+ TIC) onto itself thereby leading to a hybrid cell formation (F4/80+ CD 133+ TIC). The hybrid TIC acquire the Macrophage’s tropism such that TICs gravitate to macrophage-rich metastasis sites. We performed genetic screen based on transplantation of tumor cells infected with a viral cDNA library into Mouse Livers. This identified metastatic promoting genes such as CD47, MGAT-5 (GNT-V) and Lin28. We performed Metaphase chromosome spreading analysis on co cultured cells and then FISH analysis on TIC’s and Kupffer cells to identify an interaction between CD47 and SIRP. Immunohistochemistry of paraffin embedded normal, tumor and metastatic Brain tissue, revealed double positive expression of GNT-V and CD133. Flow cytometry analysis (FACS) also showed a double positive population in LPS stimulated Macrophage(F4/F80) and TICs (CD133). This proposed link between cell fusion and metastasis, not only advances our understanding of molecular mechanisms responsible for metastatic TIC generation in HCC but also represents a new target in the treatment development for metastatic HCC.Funding Source: California Institute for Regenerative MedicineT-2122ACTIVATION OF ANTITUMOR IMMUNITY BY HUMAN NEURAL STEM CELL MEDIATED CPG-STAT3 ANTISENSE OLIGONUCLEOTIDES AND OX40 ANTIBODIES IN A SYNGENEIC BREAST CANCER MOUSE MODELAdileh, Lana - Developmental and Stem Cell Biology, City of Hope, Newbury Park, CA, USA Aboody, Karen - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Adamus, Tomasz - Immuno-oncology, City of Hope, Duarte, CA, USA Flores, Linda - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Gonzaga, Joanna - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Hammad, Mohamed - Developmental and Stem Cell Biology, City of Hope, Duarte, CA, USA Kortylewski, Marcin - Immuno-oncology, City of Hope, Duarte, CA, USABreast cancer is the most common cancer among women in the US. Patients diagnosed at advanced stages have limited treatment options, due in part to tumor cell immune tolerance caused by Signal Transducer and Activator of Transcription 3 (STAT3). Treatment with CpG-STAT3 antisense oligonucleotides (ASOs) may inhibit STAT3 and trigger immunostimulation. The CpG moiety tethered to STAT3-ASO enables cellular uptake of the conjugate by immune cell expression of Toll-like receptor 9 (TLR9). The current study investigates the novel approach of delivering CpG-STAT3-ASOs in a clinically relevant neural stem cell (NSC) line (HB1.F3.CD21) to increase their tumor-targeted delivery and local retention at breast cancer sites. These NSCs have demonstrated tumor tropism to invasive tumors including brain, ovarian, and breast cancers. In vitro studies showed rapid uptake of CpG-STAT3 ASOs by NSCs. Following uptake, NSCs continuously released extracellular vesicles (EVs) containing the CpG-STAT3 ASOs (CpG-STAT3 ASO-NSCs) for 2–3 days. In an in vivo immunocompetent mouse model of breast cancer, in which tumors were established in left and right mammary fat pads. When NSC-delivered versus free CpG-STAT3 ASO was administered intratumorally, we observed an enhanced distribution of CpG-STAT3-ASOs in the tumor microenvironment, attracting more tumor infiltrating lymphocytes and generating a systemic anti-tumor effect. Moreover, because OX40 expression is induced on T-cells upon TLR9 activation, the addition of OX40 agonist antibodies (Abs) led to greater anti-tumor activity in CpG-STAT3 ASO-NSC treated mice. This resulted in reduced volume of both the injected and non-injected tumor, and extended long term survival. These data support further translational development of the NSC delivered CpG-STAT3-ASO and OX40 agonist Ab combination treatment, which has the potential to overcome immune tolerance and induce a systemic anti-tumor immune response in patients with advanced breast cancer.Funding Source: Rosalinde and Arthur Gilbert Foundation, CIRM, Alvarez Family Charitable Foundation, and Anthony and Susan Markel Foundation, Jeanne and Bruce Nordstrom.T-2124MULTIPLE MYELOMA BM-MSCS INCREASE THE TUMORIGENICITY OF MM CELLS VIA TRANSFER OF VLA4 ENRICHED MICROVESICLESDabbah, Mahmoud - Tel Aviv University, Kfar Saba, Israel Attar, Oshrat - Oncogenetic Laboratory, Meir Medical Center, Kfar Saba, Israel Drucker, Liat - Oncogenetic Laboratory, Meir Medical Center, Kfar Saba, Israel Jarchowsky, Osnat - Hematology, Meir Medical Center, Kfar Saba, Israel Lishner, Michael - Hematology Unit and Meir Research Center, Meir Medical Center, Kfar Saba, IsraelMultiple myeloma (MM) cells accumulate in the bone marrow (BM) where their interactions impede disease therapy. We have shown that microvesicles (MVs) derived from BM mesenchymal stem cells (MSCs) of MM patients promote the malignant traits via modulation of translation initiation (TI), whereas MVs from normal donors (ND) do not. Here, we observed that this phenomenon is contingent on a MVs’ protein constituent, and determined correlations between the MVs from the tumor microenvironment, e.g. MM BM-MSCs, and patients’ clinical characteristics. BM-MSCs MVs (ND/MM) proteomes were

363POSTER ABSTRACTSassayed (mass spectrometry) and compared. Elevated integrin CD49d (X80) and CD29 (X2) was determined in MM-MSCs’ MVs and correlated with patients’ staging and treatment response (Free light chain, BM plasma cells count, stage, response to treatment). BM-MSCs’ MVs uptake into MM cell lines was assayed (flow cytometry) with/without integrin inhibitors (RGD, Natalizumab and anti-CD29 monoclonal antibody) and recipient cells were analyzed for cell count, migration, MAPKs, TI and drug response (doxorubicin, velcade). Their inhibition, particularly together, attenuated the uptake of MM-MSCs MVs (but not ND-MSCs MVs) into MM cells and reduced MM cells’ signaling, phenotype, and increased drug response. This study exposed a critical novel role for CD49d/CD29 on MM-MSCs MVs and presented a discriminate method to inhibit cancer promoting action of MM-MSCs MVs while retaining the anti-cancer function of ND-MSCs-MVs. Moreover, these findings demonstrate yet again the intricacy of the microenvironment involvement in the malignant process and highlight new therapeutic avenues to be explored.Funding Source: Cancer Biology Research Center (CBRC) #0601242482 Dream Idea Grant, Dotan Hemato-Oncology seed award 2017, Tel Aviv University, The study presentation costs were supported by the Israel Ministry of Science, Technology and Space.T-2126KIT D816V INDUCED PLURIPOTENT STEM CELLS FOR MODELLING LEUKEMIA AND COMPOUND SCREENINGZenke, Martin - Cell Biology, Institute for Biomedical Engineering, Aachen, Germany Szymanski de Toledo, Marcelo - Institute for Biomedical Engineering - Cell Biology, RWTH Aachen University, Aachen, Germany Gatz, Malrun - Institute for Biomedical Engineering - Cell Biology, RWTH Aachen University, Aachen, Germany Sontag, Stephanie - Institute for Biomedical Engineering - Cell Biology, RWTH Aachen University, Aachen, Germany Gleixner, Karoline - Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria Koschmieder, Steffen - Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University Hospital, Aachen, Germany Valent, Peter - Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria Bruemmendorf, Tim - Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University Hospital, Aachen, Germany Chatain, Nicolas - Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University Hospital, Aachen, GermanyMutations in the stem cell factor (SCF) receptor tyrosine kinase KIT are key to leukemias confined to the mast cell compartment and over 90% of aggressive systemic mastocytosis (ASM) and mast cell leukemias (MCL) are positive for a KIT D816V mutation. The KIT D816V mutation results in ligand-independent tyrosine phosphorylation and constitutive activation of KIT leading to aberrant cell proliferation, migration and function. KIT D816V cells are resistant to the tyrosine kinase inhibitor imatinib (Gleevec/Glivec), which is successfully used for treating chronic myeloid leukemia (CML). Thus, KIT D816V patients in the terminal phase of disease are essentially left without effective targeted therapy, rendering this a fatal disease. We have generated a panel of induced pluripotent stem cells (iPS cells) of KIT D816V ASM and MCL patients, including isogenic controls without mutation. We also introduced the KIT D816V mutation into human ES cells by CRISPR/Cas to generate KIT D816V ES cells. KIT D816V iPS cells and ES cells were induced to differentiate into hematopoietic cells and further into mast cells to recapitulate KIT D816V disease in vitro. As expected KIT D816V progenitors showed characteristics of constitutive KIT signaling in biochemical assays. In addition KIT D816V hematopoietic progenitors were increased in their proliferative capacity and propensity towards mast cell differentiation. Therefore KIT D816V hematopoietic progenitors and mast cells were subjected to compound screening for identification of KIT D816V selective inhibitors and these studies are currently being performed. Our studies should pave the way towards an effective therapy of KIT D816V ASM and MCL.Funding Source: This work was funded in part by IZKF Aachen, RWTH Aachen University, Aachen, Germany and German Federal State North Rhine-Westphalia and European Union, priority area LifeSciences.NRW within the StemCellFactory III project.T-2128METASTATIC POTENTIAL OF NANOG OVEREXPRESSING MELANOMA CELLSSaito, Mikako - Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Japan Kishi, Ryota - Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan Sasai, Tomoko - Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan Hatakenaka, Tomohiro - Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, JapanCancer cell lines with different metastatic potential are important bioresources for the molecular analysis of metastasis and the development of its inhibition methods. Some studies have reported the potential involvement of differentiation-related genes in the regulation of metastatic potency. Here we have selected Nanog as such a gene and aimed at the development of Nanog gene modified melanoma cell lines. B16BL6, B16F10, and B16F1 are melanoma cell lines with different metastatic potential. Nanog overexpression vector was introduced into these 3 cell lines, respectively. Consequently, the increase of proliferation and migration potential was observed most

364POSTER ABSTRACTSmarkedly in Nanog overexpressing B16BL6 (N-BL6). In this cell line, the expression of matrix metalo-proteinase 9 was activated that supported the increase of migration. RNA sequence revealed the increase of TGF- expression and the βdecrease of the expressions of inhibitor of differentiation (ID)1 and ID3. This finding suggested that Nanog overexpression might modify melanoma cells to less differentiated state. Nanog overexpressing melanoma cells as well as the control melanoma cells were administered to 8-week-old C57BL/6 male mice from the tail vein. After breeding for 2 weeks, the number of metastatic colonies and their volume were analyzed. Typically the number of colonies on lung in N-BL6 increased to 1.2 times greater than the control. These indicate that Nanog overexpression was effective for the increase of metastatic potential of melanoma cells.T-2130IMMORTALIZATION OF DIFFERENT BREAST EPITHELIAL CELL TYPES RESULTS IN DISTINCT MITOCHONDRIAL MUTAGENESISAhn, Eun Hyun - Department of Pathology, University of Washington, Seattle, WA, USA Kwon, Sujin - Department of Pathology, University of Washington, Seattle, WA, USA Kim, Susan - Department of Biochemistry, University of Washington, Seattle, WA, USA Nebeck, Howard - Department of Pathology, University of Washington, Seattle, WA, USADifferent phenotypes of normal cells might influence genetic and epigenetic profiles, and tumorigenicities of their transformed derivatives. In this study, we investigated whether the whole mitochondrial genome of immortalized cells can be attributed to different phenotypes (stem versus non-stem) of their originator normal epithelial cells. To accurately determine mutations, we employed Duplex Sequencing, which exhibits the lowest error rates among currently available DNA sequencing methods. Our results indicate that the vast majority of observed mutations of the whole mitochondrial DNA occur at low-frequency. The most prevalent mutation types are C>T/G>A and A>G/T>C transitions. Frequencies and spectra of homoplasmic (high-frequency) point mutations are virtually identical between stem cell-derived immortalized (SV1) cells and non-stem cell-derived immortalized (SV22) cells. However, frequencies of rare point mutations are significantly lower in SV1 cells (5.79E-5) than in SV22 cells (1.16E-4). Additionally, the predicted pathogenicity for rare mutations in the mitochondrial tRNA genes is significantly lower (by 2.5-fold) in SV1 cells than in SV22 cells. Our findings suggest that the immortalization of normal cells with stem cell features leads to decreased mitochondrial mutagenesis, particularly in noncoding RNA regions. The identified mutation spectra and mutations specific to stem (versus non-stem) cell-derived immortalized cells have implications in characterizing heterogeneity of tumors and understanding the role of mitochondrial mutations in immortalization and transformation of human cells.Funding Source: NIEHS P30 ES007033 sponsored-University of Washington (UW) EDGE grant (to EH Ahn), UW RRF (to EH Ahn), and NCI P30 CA015704-39 FHCRC-UW CCSG (to EH Ahn), NCI R21 CA220111 (to EH Ahn), and NCI P01 AG001751 and R33 CA181771 (to LA Loeb).NEURAL DEVELOPMENT AND REGENERATIONT-3002ACCUMULATION OF 7-DEHYDROCHOLESTEROLS-DERIVED OXYSTEROLS CAUSES NEURODEVELOPMENTAL DEFECTS IN SMITH-LEMLI-OPITZ SYNDROMETomita, Hideaki - Medicinal Chemistry, The University of Washington, Seattle, WA, USA Hines, Kelly - Medicinal Chemistry, University of Washington, Seattle, WA, USA Xu, Libin - Medicinal Chemistry, University of Washington, Seattle, WA, USASmith-Lemli-Opitz Syndrome (SLOS) is a developmental and metabolic disorder characterized by distinct facial features, microcephaly, intellectual disability and Autism-like behaviors. SLOS is caused by a defect in cholesterol synthesis with mutations in the gene encoding 7-dehydrocholesterol reductase (DHCR7). DHCR7 catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol in the final step of cholesterol biosynthesis. Defective DHCR7 resulting from the mutations leads to a greatly decreased level of cholesterol and the accumulation of 7DHC in affected individuals. 7DHC was found to be extremely reactive toward free radical oxidation, leading to the formation of several oxidative metabolites (i.e., oxysterols), many of which have been found in-vivo. Although the combination of low cholesterol level and accumulation of 7DHC-derived oxysterols likely contributes to the disease pathophysiology, it still remains unknown how these biochemical changes lead to specific neurodevelopmental defects in SLOS. Here we asked whether DHCR7 mutations cause abnormal neural development by disrupting neurogenesis in the murine cortex and SLOS patient-derived human induced pluripotent stem cells (hiPSCs). We found that 7DHC-derived oxysterols begin to accumulate at embryonic day 12.5 and continue to increase during cortical neural development by using liquid chromatography-tandem mass spectrometry. The endogenous levels of these oxysterols, such as as 3 ,5 -dihydroxycholest-β α7-en-6-one (DHCEO), 4a-hydroxy-7-DHC, and 4b-hydroxy-7-DHC, range from sub-mM to over 10 mM. We showed that loss of DHCR7 caused decreased proliferation and self-renewal of cortical neural precursors and aberrantly increased neurogenesis in both mouse and human neural precursor cells. Importantly, treatment of mouse neural precursor cells with individual oxysterols at physiological concentrations led to a similar phenotype. Furthermore, we found that inhibition of 7DHC-derived oxysterol production by antioxidants rescued the increased neurogenesis caused by knockdown

365POSTER ABSTRACTSand homozygous loss of DHCR7. These results suggest the accumulation of 7DHC-derived oxysterols is a causal factor in SLOS neural pathophysiology and cholesterol biosynthesis plays a critical role in the normal neural development.Funding Source: National Institutes of Health (R01HD092659)T-3004IDENTIFYING THE OPTIMAL AGE OF HUMAN STEM CELL-DERIVED DOPAMINERGIC PROGENITORS FOR TRANSPLANTATION IN PARKINSON’S DISEASEde Luzy, Isabelle R - Stem Cells and Neural Development, The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia Hunt, Cameron - Stem Cells and Neural Development, Florey Institute of Neuroscience and Mental Health, Melbourne, Australia Niclis, Jonathan - Stem Cells and Neural Development, Florey Institute of Neuroscience and Mental Health, Melbourne, Australia Parish, Clare - Stem Cells and Neural Development, Florey Institute of Neuroscience and Mental Health, Melbourne, Australia Thompson, Lachlan - Stem Cells and Neural Development, Florey Institute of Neuroscience and Mental Health, Melbourne, AustraliaDonor age can have a significant impact on transplantation outcomes. Surprisingly, despite the rapid advancement of human pluripotent stem cell derived dopaminergic progenitors for transplantation into Parkinson’s Disease patients, little documentation exists regarding the influence/s progenitor state has on transplantation survival, composition and integration – as well as reproducibility across cell lines. To address this, we transplanted ventral midbrain progenitors at varying stages of differentiation (13, 19, 22, 25 and 30 days in vitro, DIV) into 6OHDA mice. Employment of two GFP reporter lines under the promoter PITX3 (iPSC and ESC), enabled specific tracking of graft derived dopamine neurons. Post-mortem histological analysis at 6months revealed surviving transplants at all time points for both cell lines. Graft of 13 and 29 DIV progenitors were small, whilst grafts derived from 19, 22 and 25 DIV cells were larger yet contained a higher number and proportion of non-dopaminergic cells. Cells implanted at 13DIV generated discrete grafts, containing the highest proportion of dopaminergic neurons with the least incorrectly specified cells and maintained re-innervation of developmentally relevant dopaminergic targets. IPSC grafts showed greater yield of dopaminergic neurons at 22DIV, unlike ESC grafts which generated the highest at 13DIV, and had a 3-fold increase in total DA neurons and density compared to ESC grafts, highlighting previously recognised differences in PSC lines. These findings indicate that the timing of implantation (or donor age) is directly related to the survival and outgrowth of dopaminergic neurons, and that careful assessment, on a line to line basis is required prior to translation. Ongoing analysis will determine if donor age impacts the sub-type identity (A8, A9 or A10) of transplanted dopaminergic neurons.T-3006UNCOVERING THE MOLECULAR IDENTITY OF CONE PHOTORECEPTOR RESTRICTED PROGENITORS IN THE DEVELOPING MOUSE RETINABelair-Hickey, Justin J - Molecular Genetics, University of Toronto, ON, Canada Khalili, Saeed - Molecular Genetics, University of Toronto, ON, Canada Ballios, Brian - Department of Ophthalmology and Visions Sciences, University of Toronto, ON, Canada Coles-Takabe, Brenda - Molecular Genetics, University of Toronto, ON, Canada Grisé, Kenneth - Molecular Genetics, University of Toronto, ON, Canada Liu, Jeff - Molecular Genetics, University of Toronto, ON, Canada Bader, Gary - Molecular Genetics, University of Toronto, ON, Canada van der Kooy, Derek - Molecular Genetics, University of Toronto, ON, CanadaThe developing retina represents a robust and tractable model to understand how precursor cells are fate specified to produce a diversity of cell types in the central nervous system. Over the course of retinal development, seven major cell types are produced in a temporally stereotyped manner from retinal progenitor cells (RPCs). Much remains to be determined as to the extent cell intrinsic vs. extrinsic environmental factors dictate cell fate specification, and to what degree RPCs are multipotential or more lineage restricted. The retinal photoreceptor lineage illustrates both a therapeutically useful and developmentally interesting fate specification, as photoreceptors are lost in retinal degenerative diseases and produced at distinct non-overlapping timepoints in development. Here, using both adult retinal stem cells (RSCs) and RPCs isolated from the embryonic retina, we report the existence of cone photoreceptor lineage restricted progenitors that are induced by a secreted inhibitory retinal factor. Single adult RSCs proliferate in culture to produce a colony containing RPCs and pigmented epithelial progenitors. When single RSC-derived RPCs were exposed to COCO (a retinal secreted inhibitor of TGFb, BMP, and WNT signalling), they produced clones of nearly pure cone photoreceptors, indicating that COCO induces RPCs to assume a cone photoreceptor restricted lineage. Moreover, when RPCs were isolated directly from the murine retina across multiple early and late embryonic timepoints and exposed to COCO, they produced nearly pure populations of cone photoreceptor cells, indicating that the RSC cultures accurately model this developmental lineage. The ability to molecularly identify this cone restricted progenitor type would allow for in vivo analysis of lineage tracing

366POSTER ABSTRACTSand ablation/expansion. Accordingly, we have performed an RNA-sequencing analysis on COCO exposed RPCs over their differentiation timecourse. This analysis suggests that Sox15 may be a novel marker of cone progenitors. This previously uncharacterized retinal gene seems to be expressed in RPCs at early E12-14 timepoints when cones are first starting to differentiate. Ongoing experiments exploring mouse Sox15 K/O and in vivo overexpression will further uncover the potential of this gene to dictate cone fate in the retina.Funding Source: Medicine by Design. NSERC. CIHR. Vision Science Research Program. Foundation Fighting Blindness.T-3008SINGLE MOLECULE IMAGING OF ASCL1 DURING DIRECT REPROGRAMMING OF MOUSE EMBRYONIC FIBROBLASTS TO INDUCED NEURONAL (IN) CELLSSchaukowitch, Katie M - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA Meng, Lingjun - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA Wernig, Marius - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USAThe ability to directly convert differentiated somatic cell types into neuronal cells provides critical information about what defines a neuron. Elucidation of the mechanisms underlying this direct conversion has the potential to generate more comprehensive models of neurological disorders with which to study disease biology and perform drug screening, as well as possibilities for more efficient regenerative medicine methods. Ascl1, a proneural basic helix loop helix (bHLH) transcription factor, directly converts mouse embryonic fibroblasts into functional induced neuronal (iN) cells and has a unique ability among other reprogramming factors to independently locate its cognate binding sites not only in its endogenous neural context, but also in the different chromatin context of fibroblasts. However, it is unclear how precisely Ascl1 locates its targets in closed chromatin. Recent advances in single molecule imaging significantly increase the spatial and temporal resolution of tracking individual factors within the nucleus, and it is now possible to analyze their dynamic interactions with DNA. This project aims to utilize highly inclined and laminated optical sheet (HILO) microscopy to understand how Ascl1 independently activates its targets in the different epigenetic landscapes of neurons and fibroblasts. The search kinetics of Ascl1 in its physiological context will be compared to Ascl1 overexpression in reprogramming fibroblasts, as well as to the context-dependent reprogramming factor Brn2. These characteristics will also be measured in heterochromatin to determine whether Ascl1’s ability to orchestrate reprogramming is due to a more efficient search pattern in closed chromatin.Funding Source: This work was funded by an NINDS F32 NRSA Postdoctoral Fellowship.T-3010EVALUATION OF SUBTYPE SPECIFIC ION CHANNEL FUNCTIONS ON AXONAL CONDUCTION OF CULTURED NEURONS WITH MICROFABRICATED RECORDING DEVICEShimba, Kenta - School of Engineering, The University of Tokyo, Japan Sakai, Koji - School of Engineering, The University of Tokyo, Japan Kotani, Kiyoshi - Research Center for Advanced Science and Technology, The University of Tokyo, Japan Jimbo, Yasuhiko - School of Engineering, The University of Tokyo, JapanFunctional evaluation of neurons derived from human pluripotent stem cells is an important task for regenerative medicine and disease modeling. Normally, gene or protein expression analysis, and electrophysiological recording by patch clamp method are performed. However, neither method is difficult to evaluate functions from a single sample during a long term enough for functional maturation. In addition, although it is possible to evaluate the function more than one year by using a measuring device called microelectrode array, there remains a problem that it is impossible to obtain information correspond to intracellular protein expression. In this study, we developed a culture device and analysis method to evaluate propagations of action potential along individual axons with the aim of establishing a method for continuously and indirectly monitoring intracellular protein expression for a long time. As a proof of concept study, we performed three experiments: 1) continuous monitoring of developmental change, 2) pharmacological blockade of sodium ion channels, 3) subtype specific blockade. First, conduction velocity of mouse primary cultured neurons was calculated at various time points. The conduction velocity significantly increased with days, suggesting that developing neurons change their conduction properties. Second, axons were treated with various sodium ion channel blockers, TTX and lidocaine, at various concentration. The conduction velocity gradually decreased as increasing concentrations of reagents, suggesting that partial blockade of sodium ion channels decreased conduction velocity. Finally, axons were treated with subtype specific inhibitors to sodium ion channels. Addition of a subtype specific inhibitor showed that Nav 1.2 is dominant for conduction in axons of cerebral cortical neurons. Taken together, our method is suggested to be a useful tool for long term functional evaluation of cultured neurons.Funding Source: This work was supported by the Japan Society for the Promotion of Science through Grants-in-Aid for Scientific Research (16H03162, 18H03512, 18K19902 and 18K18362), and the Casio Science Promotion Foundation.

367POSTER ABSTRACTST-3012GENERATION OF TWO DIFFERENT HUMAN MICROGLIA CELLS SEPARATELY FROM IPSC-DERIVED PRIMITIVE OR DEFINITIVE HEMATOPOIETIC PROGENITORSChoi, Kyung-Dal - BrainXell, Madison, WI, USA Du, Zhong-wei - BrainXell, Madison, WI, USAHuman microglia are vital residents of the brain, where they play important roles in development, injury and disease, such as Alzheimer’s (AD) disease. Arising from yolk sac primitive progenitors that colonize in the brain during embryogenesis, microglia are unique among tissue macrophages in that they are thought to remain primitive progenitors derived throughout life, without contribution from the definitive hematopoiesis. After insults such as stroke and neurodegenerative disease; however, microglia dramatically change their phenotype and are joined by infiltrating macrophages from blood. These definitive hematopoietic progenitors-derived occupants can resemble microglia in morphology and surface marker expression but appear to participate differently in disease pathogenesis, making it essential to further clarify their functions. Currently, there is no method reported to generate these two microglia cells from the same iPSCs. To understand how naive microglia and infiltrating microglia cells affect the brain in disease condition, we developed a novel protocol to efficiently produce two different microglia cells separately from primitive or definitive hematopoietic progenitors. By manipulating WNT signaling and Activin-Nodal signaling, human iPSCs were first induced to separate hematopoietic fates, primitive hematopoietic progenitors (CD43+, CD235a+) and definitive hematopoietic progenitors (CD34+, CD43-, CD235a-). Next, these two hematopoietic progenitors were grown in serum-free differentiation medium containing CSF-1 and IL-34 to differentiate into macrophages, then cocultured with our BrainXell cortical neuron and astrocyte mixed culture to induce microglia identity. We identified the different gene expression between these two microglia cells by RNA-seq profiling, and validated by qPCR, immunostaining and FACS analysis. In summary, two different microglia cells, derived separately from iPSC derived primitive or definitive hematopoietic progenitors, will provide useful models to understanding microglia function in neurological diseases like AD diseaseT-30143D BIOPRINTING INTO TISSUE SPECIFIC HYDROGELS FOR THE STUDY OF MICROENVIRONMENTAL CONTROL OF STEM CELL FATEZamponi, Martina - Biomedical Engineering, Old Dominion University, Virginia Beach, VA, USA Mollica, Peter - School of Medical Diagnostic and Translational Sciences, Old Dominion University, Norfolk, VA, USA Reid, John - Biomedical Engineering, Old Dominion University, Norfolk, VA, USA Bruno, Robert - School of Medical Diagnostic and Translational Sciences, Old Dominion University, Norfolk, VA, USA Sachs, Patrick - School of Medical Diagnostic and Translational Sciences, Old Dominion University, Norfolk, VA, USAThe cellular microenvironment has been shown to play a fundamental role in the regulation of cell function, stem cell fate determination, maintenance of cell potency and tissue homeostasis. Our laboratory focuses on the study of the effects of cellular microenvironment in the context of cancer and neurological models, based on the observation that a healthy environment can induce the suppression of tumorigenesis in mouse models. Insights concerning the molecular mechanisms that drive these processes are very limited, partly due to the inability of the current traditional methods of investigation, such as two-dimensional cell cultures and animal models, to accurately represent the human in vivo cellular microenvironment. Three-dimensional cell cultures allow us to overcome the structural limitations posed by monolayer cultures, and maintain the ease of experiment design, monitoring and data analysis associated with in vitro procedures. Our laboratory has established systems to overcome some of these limitations and rely on the strengths of three-dimensional culture methods to elucidate mechanisms that govern stem cell differentiation. A customized 3D extrusion-based bioprinter was developed starting from a commercially available model, allowing for precise and controlled injection of cells within three-dimensional substrates. This tool allows for design of highly controlled experiments, in which the effects of cellular microenvironment on stem cell differentiation can be studied at a single-cell resolution. For increased levels of biomimicry, tissue specific substrates are generated from extracted tissue. Collected tissue is subjected to a chemical decellularization process, followed by lyophilization, enzymatic digestion and neutralization, to generate a self-gelling product upon incubation at 37°C. Mammary and brain extracellular matrix-derived substrates have been shown to support the growth of cells of the epithelial and neuronal lineages, respectively. Here, we apply these established systems to study the effects of the environment constituted by the three-dimensional substrates on the differentiation of injected stem cells.Funding Source: Jeffress Trust Awards Program in Interdisciplinary Research; NIH R15 GM131330T-3016RAPID GENERATION AND MATURATION OF CORTICAL LAYER II-III GLUTAMATERGIC NEURONS FROM HUMAN IPSCSHendrickson, Michael - BrainXell, Inc., Madison, WI, USA Hjelmhaug, Julie - BrainXell, Madison, WI, USA Du, Zhong-Wei - BrainXell, Madison, WI, USA Xu, Kaiping - BrainXell, Madison, WI, USANumerous neurological and psychiatric disorders involve glutamatergic (Glut) neurons specific to one or more of the six cortical layers. Death or malfunction of layer II-III specific Glut neurons underlies disease pathophysiology and disrupts higher cognitive function in Alzheimer, Schizophrenia and Autism

368POSTER ABSTRACTSdiseases. Study of these neurons may reveal the molecular mechanisms behind their vulnerability and enable development of more relevant disease models. Toward this goal, we developed a novel protocol to rapidly and efficiently produce layer II-III cortical Glut neurons. Human iPSCs were first induced to a neural epithelia fate, patterned to Glut neuronal progenitors (>95% SOX1+/PAX6+), and then rapidly switched to layer II-III Glut progenitors (>85% CUX1+). Using novel combinations of small molecules, the layer II-III progenitors can be expanded up to 500-fold, which allows for production of large and consistent batches of neurons. Finally, the progenitors were plated in medium containing a specialized maturation supplement that rapidly promotes morphological and functional maturation. After treating with this maturation supplement, layer II-III Glut neurons displayed extensive neurite outgrowth within 3 days, expressed pre- and post-synaptic mature markers within 7 days, and exhibited electrophysiological activity within 2 weeks. Seven days post-plating, the cultures were >90% neurons (MAP2+) with the following breakdown by layer identity: ~85% layer II-III (CUX1+/ CTIP2-/ FOXP2-), ~10% layer V (CTIP2+), and ~2% layer VI (FOXP2+). Thus, our novel differentiation protocol generates a pure neuronal culture that is highly enriched for cortical Glut neurons with a layer II-III identity. To validate whether this production protocol can be applied to other iPSC lines, we generated cortical layer II-III Glut neurons from three healthy control and three Schizophrenia iPSC lines, and we identified disease-specific phenotypes by RNA-seq profiling and MEA analysis. In conclusion, coupled with the ability to generate very large batches (>1 billion neurons) and bring about rapid maturation, layer II-III cortical Glut neurons present a highly relevant model system for disease study and drug discovery.NEURAL DISEASE AND DEGENERATIONT-3018HYPERPHOSPHORYLATED TAU IN HUMAN IPSC-DERIVED ASTROCYTES SHIFTS THE FOCUS FROM NEURONAL TAU PATHOLOGY TO ASTROCYTIC TAU PATHOLOGYSantiago, Isaac E - Neuroscience, UCSD, San Marcos, CA, USA Yuan, Shauna - Neuroscience, UCSD, La Jolla, CA, USA Aulston, Brent - Neuroscience, UCSD, La Jolla, CA, USA Mishra, Priyanka - Neuroscience, UCSD, La Jolla, CA, USA Liu, Qing - Neuroscience, UCSD, La Jolla, CA, USAPathological accumulation of the microtubule associated protein tau is hypothesized to underlie cell death in neurodegenerative diseases such as Alzheimer’s disease (AD), frontotemporal lobe dementia (FTD), Pick’s disease and others. Despite numerous studies, the mechanisms that underlie aberrant tau aggregation in CNS tauopathies remain unknown. We and others have recently found that both neuronal and microglial-derived tau is sufficient to initiate tau propagation in the rodent brain. However, despite evidence that astrocytes accumulate and release pathogenic tau species, the role of astrocytic-derived tau in human tauopathies remains unknown. Therefore, with this work, we examined the expression of tau and tau phosphorylation status in human astrocytes derived from induced pluripotent stem cells (iPSCs). Our data show that overall expression of tau in cultured iPSC-astrocytes is lower than tau expression in iPSC derived neurons, however, iPSC-astrocytes display higher levels of tau phosphorylation compared to neuronal cultures. Remarkably, we found that the total level of phosphorylated tau was greater in astrocytes compared to neurons and that even in the absence of a stress-inducing stimuli, astrocytic tau was hyperphosphorylated. These results suggest that astrocytic derived tau may be more susceptible to phosphorylation than neuronal tau and that hyperphosphorylation of astrocytic tau may be an early feature of neurodegenerative disease. Future studies will examine the pathogenic potential of astrocytic-derived tau and determine the role of astrocytes in disease-associated tau propagation. In total, the work presented here provides preliminary evidence that astrocytic tau dynamics may play a role in AD-related pathologies.Funding Source: The California Institute for Regenerative MedicineT-3020MODELING CALCIUM KINETICS OF NEURAL COGNITIVE DISORDERS WITH HIPSC DERIVED NEURONAL CULTURESVillalba, Isaura A - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Research Institute, San Diego, CA, USA Sampson, Joshua - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Snyder, Evan - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Winquist, Alicia - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Donnell, Ryan - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Pernia, Cameron - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Tobe, Brian - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Tolcher, Heather - Sanford Consortium for Regenerative Medicine, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USAHuman induced pluripotent stem cells (hiPSCs), have been beneficial for modeling poorly understood diseases, specifically psychiatric disorders. By utilizing hiPSCs, animal modeling, and primary human brain material, we have generated a strongly supported hypothesis that the psychiatric disease bipolar

369POSTER ABSTRACTSdisease (BPD) is through the protein collapsin response mediator protein (CRMP2), that controls the form and function of dendritic spines (collapsin response mediator protein-2 (CRMP2)) and hence neuronal network activity. It is unknown how this pathway mechanistically changes these neuronal structures. If this were known, better drug targets and drugs might be devised for treating BPD. In addition, there is significant overlap in the etiology of bipolar disease and other neurodegenerative diseases such as Alzheimer’s (AD). Not only is CRMP2 central to BPD, but it is also associated with AD pathophysiology(Nunes et al, 2013 Curr Alzheimer’s Res). Our research focuses on understanding how abnormalities in CRMP2 underlie BPD and drug induced psychosis in conjunction with AD. Through the use of looking at neurons generated from hiPSCs and transgenic mouse models, we will attempt to discern this mechanism. The techniques will involve neurite proteomics, electrophysiological assessments of network formation (via multi-electrode array), and calcium kinetic imaging. By deriving neuronal cultures from human bipolar disorder iPSCs, we can observe how CRMP2 mediates neurological processes relevant to psychiatric disorders in conjunction with testing how stimulants known to induce psychosis impact key psychiatric genes such as CRMP2 and phenocopy psychiatric disorders at the cellular and molecular level.T-3022AGE RELATED AUTOPHAGY IMPAIRMENTS IN DIRECTLY REPROGRAMMED DOPAMINERGIC NEURONS IN PATIENTS WITH IDIOPATHIC PARKINSON’S DISEASEDrouin-Ouellet, Janelle - Faculty of Pharmacy, University of Montreal, Montreal, QC, Canada Birtele, Marcella - Experimental Medicine, Lund University, Lund, Sweden Pircs, Karolina - Experimental Medicine, Lund University, Lund, Sweden Shrigley, Shelby - Experimental Medicine, Lund University, Lund, Sweden Pereira, Maria - Experimental Medicine, Lund University, Lund, Sweden Sharma, Yogita - Experimental Medicine, Lund University, Lund, Sweden Vuono, Romina - Clinical Neuroscience, University of Cambridge, UK Stoker, Thomas - Clinical Neuroscience, University of Cambridge, UK Jakobsson, Johan - Experimental Medicine, Lund University, Lund, Sweden Barker, Roger - Clinical Neuroscience, University of Cambridge, UK Parmar, Malin - Experimental Medicine, Lund University, Lund, SwedenUnderstanding the pathophysiology of Parkinson’s disease (PD) has been hampered by the lack of models that recapitulate all the critical factors underlying its development. Using a novel and highly efficient approach, we generated functional induced dopaminergic neurons (iDANs) that were directly reprogrammed from dermal fibroblasts of patients with idiopathic PD (n=19) as well as sex- and age-matched healthy donor (n=10) to investigate whether such cells have deficits in autophagy. We show that iDANs derived from PD patients exhibit lower basal chaperone-mediated autophagy as compared to iDANs of healthy donors. Furthermore, stress-induced autophagy resulted in an accumulation of macroautophagic structures in induced neurons (iNs) derived from PD patients, independently of the specific neuronal subtype but dependent on the age of the donor. Finally, we showed that these impairments in autophagy in the iNs derived from idiopathic PD patients lead to an increase in phosphorylated alpha-synuclein, a hallmark of PD pathology. Taken together, our results show that direct neural reprogramming provides a patient-specific model to study neuronal features relevant to idiopathic PD.Funding Source: M.P. is a New York Stem Cell Foundation - Robertson Investigator.T-3024A SMALL MOLECULE SIRT3 AGONIST RESCUES ALS PHENOTYPES IN MOTOR NEURONS DERIVED FROM SPORADIC AND FAMILIAL ALS IPSCSHor, Jin Hui - Institute of Molecular and Cell Biology (IMCB), Singapore, Singapore Liou, Yih-Cherng - National University of Singapore (NUS), Singapore, Singapore Ng, Shi-Yan - Institute of Molecular and Cell Biology (IMCB), Singapore, Singapore Soh, Boon-Seng - Institute of Molecular and Cell Biology (IMCB), Singapore, SingaporeAmyotrophic lateral sclerosis (ALS) is a progressive adult-onset motor neuron disease that affect the upper and lower motor neurons, resulting in muscular weakness and ultimately death. Majority of ALS patients (~90%) have sporadic forms of the disease where the disease etiology remains unknown. The other 10% of patients have familial ALS where mutations are commonly found in SOD1, C9ORF72, and TDP43. Despite being intrinsically heterogeneous, the disease manifestation between sporadic and familial ALS are clinically indistinguishable, suggesting a possible converging pathogenic mechanism. Motor neurons rely on mitochondrial respiration to fuel their metabolic needs and any disruption in the mitochondria function will lead to neurological disorders. Recent studies have found a causative relationship between defective energy metabolism and the survival rate of patients with ALS. However, clear mechanistic insights between altered metabolism and ALS disease manifestation is currently unknown. In this study, we investigated if defective mitochondrial respiration could be the common pathway affected in both sporadic and familial ALS. Metabolic flux analyses reveal a deficiency in mitochondrial

370POSTER ABSTRACTSrespiration in both familial and sporadic ALS motor neurons. This defective metabolic profile was attributed by decreased activity of SIRT3, a mitochondrial deacetylase that regulates mitochondrial function and is implicated in aging. Using a small molecule SIRT3 agonist, we were able to reverse the defective metabolic profiles in both familial and sporadic ALS motor neurons, promoting neurite regeneration and enhancing motor neuron health/survival. Comparing SIRT3 agonist with Riluzole and Edaravone, we found that the former was more effective in improving ALS phenotypes in motor neurons derived from sporadic and familial ALS iPSCs. Therefore, our study demonstrated that SIRT3 is involved in the converging pathogenic mechanism between both familial and sporadic ALS, and provides an explanation as of why age is a risk factor for ALS. In addition, we demonstrate that SIRT3 agonists can be exploited as potential therapeutics for ALS.T-3026TUNING THE FUNCTION OF MICROGLIA TO DEVELOP NOVEL TREATMENT OF ADLiu, Jing - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing, China Wang, ZhiMeng - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Zhang, Boya - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Mo, Fan - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Tang, MingMing - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Teng, ZhaoQian - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Hu, Baoyang - State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaMicroglia are the neuroimmune cells that involve surveillance and phagocytosis in the central nervous system. In devastating neurodegenerative disorders such as Alzheimer’s disease (AD), microglia have been shown to play major roles in internalization and degradation of amyloid-beta peptide (A ). Functional βimpairment of microglia eventually causes accumulation of A , βwhich accounts for the development of AD. It is intriguing whether tuning the function of microglia can alleviate such category of disorders. In this study, we named MDR-1, a key regulator for the development of microglia, which is downregulated in brains of aged and AD mice. We demonstrated that overexpression of MDR-1 either in culture mouse primary microglia, or in human and mouse microglial cell lines, can enhance the phagocytic uptake and degradation of A . Elevated MDR-1 in microglia βalso cause decreased secretion of proinflammatory cytokines, such as interleukin-6 (IL-6), TNF , and IL-1 , and increased αβexpression of anti-inflammatory Arg1/IL-10. We thus identify a novel molecule that can potently regulates the accumulation and clearance of A , as well as the inflammatory cytokine profile of βmicroglia, which we expect promising in engineering microglia for cell transplantation and AD therapy.T-3028HUMAN IPSC-DERIVED GABAERGIC INTERNEURON TRANSPLANTS ATTENUATE NEUROPATHIC PAINCaron, Leslie - The Charles Perkins Center, The University of Sydney, Camperdown, Australia Manion, John - The Charles Perkins Centre, The University of Sydney, Camperdown, AustraliaNeuropathic pain causes severe suffering and most patients are resilient to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated hiPSC-derived GABAergic (iGABAergic) neurons provide significant, long-term and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic Neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.T-3030CHARACTERIZATION OF NEUROPSYCHIATRIC DISEASE PHENOTYPES OF IPS CELL DERIVED DOPAMINERGIC NEURONS WITH 16P11.2 COPY NUMBER VARIATIONSSundberg, Maria K - Neurobiology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA Pinson, Hannah - Physics, MIT, Boston, MA, USA Winden, Kellen - Neurology, Boston Children’s Hospital, Boston, MA, USA Wafa, Syed - Neurobiology, Boston Children’s Hospital, Boston, MA, USA Tai, Derek - Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA Gusella, James - Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA Talkowski, Michael - Center for Human Genetics Research, Massachusetts General Hospital, Boston, MA, USA Tegmark, Max - Physics, MIT, Boston, MA, USA Sahin, Mustafa - Neurology, Boston Children’s Hospital, Boston, MA, USA

371POSTER ABSTRACTSPatients with 16p11.2 duplication have an increased risk of psychiatric disorders, including schizophrenia, bipolar disorder, anxiety, and depression. Patients with 16p11.2 deletion have autism spectrum disorder, including impaired social skills as well as delayed development of language and speech. Behavioral problems associated with this copy number variant (CNV) also include attention deficit hyperactivity disorder. At the moment there are no curable treatments for these neuropsychiatric disorders and often the medications cause severe side effects for the patients. In addition, the molecular and cellular deficits predisposing patients to these disorders remain to be resolved. Here, we have established a dopaminergic (DA) neuron cell culturing model to study disease phenotypes of human induced pluripotent stem cell (hiPSC) derived neurons carrying 16p11.2 duplication and 16p11.2 deletion. We have characterized the hiPSC-derived DA neurons with transcriptional gene expression profiling and this analysis showed that genes regulating synaptic development were significantly altered in the DA neurons with 16p11.2 duplication. In addition, genes regulating vesicle formation and neurotransmission were significantly altered in the DA neurons with 16p11.2 deletion compared to control cells. These genetic deficits can affect the formation of the striatal neuron networks during brain development in the patients with 16p11.2 CNVs. To understand the functional development of the DA neurons in vitro we have characterized the network development and DA signaling on the high-density multi-electrode array platform during long term culturing. We detected that the cells carrying 16p11.2 CNVs had altered activity compared to healthy control DA neurons. This data implicates that DA neuron function may be deficient in patients with these copy number variations, which may lead to different physiological and psychological symptoms. In addition, establishment of these cell culturing platforms for disease phenotyping and drug screening assays in vitro will, in the future, facilitate the design of more personalized therapies for the patients with these copy number variations.Funding Source: The Tommy Fuss Center for Neuropsychiatric Disease Research, Translational Neuroscience Center/Boston Children’s Hospital.T-3032REVAMPING THE CRISPR-CAS9 SYSTEM FOR TARGET SPECIFIC METHYLATION OF RISK GENES ASSOCIATED WITH ALZHEIMER’S DISEASECarless, Melanie A - Population Health, Texas Biomedical Research Institute, San Antonio, TX, USA Kumar, Ashish - Population Health, Texas Biomedical Research Institute, San Antonio, TX, USA Kos, Mark - South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley, San Antonio, TX, USAAlzheimer’s disease (AD) is an irreversible, neurodegenerative disease characterized by disruption of neuronal connections and cell death, leading to memory loss and cognitive decline. Pathological hallmarks of AD include inflammation, neuritic plaques, stemming from accumulation of extracellular amyloid β, and neurofibrillary tangles, arising from aggregation of intraneuronal hyperphosphorylated Tau protein. To better understand the progression of these pathological hallmarks, and identify potential therapeutic avenues, we established a model to assess DNA methylation changes in key genes associated with AD. Using induced pluripotent stem cells (iPSCs) from patients harboring a dominant, fully penetrant fAD mutation in the amyloid precursor protein gene, APP (V717I), we successfully developed an epigenetic editing tool by fusing the catalytic domain of the DNA methyltransferase, DNMT3A, to dCas9 for site specific DNA methylation of the neuronal β-secretase gene, BACE1, which plays a critical role in the processing of APP and subsequent generation of amyloid . We βgenerated a stable iPSC line using a third generation lentivirus system, which increased methylation of the BACE1 promoter by ~20-40%, and resulted in ~80% transcriptional repression (compared to a non-targeting gRNA). We are currently deriving additional cell lines to assess PSEN1 and PSEN2 activity, as well as additional patient cell lines. iPSC lines are currently being differentiated into mature neurons to observe changes in amyloid β production, Tau phosphorylation and other pathophysiological changes associated with AD. Our use of AD patient neurons and a clinically relevant third generation lentivirus system allows us to examine previously unrecognized effects of epigenetic modifications on key genes associated with AD, and provides a model system/tool for testing therapeutic strategies.Funding Source: This work was funded by the William and Ella Owens Medical Research Foundation.T-3034GENOME-WIDE CRISPR-CAS9 SCREENS IN ACCELERATED HUMAN STEM CELL-DERIVED NEURAL PROGENITOR CELLS IDENTIFY ZIKA VIRUS HOST FACTORS AND DRIVERS OF CELL PROLIFERATIONWells, Michael F - Stanley Center for Psychiatric research, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Salick, Max - NIBR, Novartis, Cambridge, MA, USA Piccioni, Federica - GPP, Broad Institute, Cambridge, MA, USA Hill, Ellen - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Mitchell, Jana - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Worringer, Kathleen - NIBR, Novartis, Cambridge, MA, USA Chan, Karrie - NIBR, Novartis, Cambridge, MA, USA Kommineni, Sravya - NIBR, Novartis, Cambridge, MA, USA Raymond, Joseph - NIBR, Novartis, Cambridge, MA, USA Ho, Daniel - NIBR, Novartis, Cambridge, MA, USA Peterson, Brant - NIBR, Novartis, Cambridge, MA, USA Siekmann, Marco - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Nehme, Ralda - Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA Pietilainen, Olli - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

372POSTER ABSTRACTSKaykas, Ajamete - NIBR, Novartis, Cambridge, MA, USA Eggan, Kevin - Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USANeural progenitor cells (NPCs) are essential for brain development and their dysfunction is linked to several disorders, including autism, Zika Virus (ZIKV) Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements in stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, and are proliferative and multipotent. Like other human NPCs, SNaPs are susceptible to ZIKV infection and viral-mediated cell death, while also being able to support active replication of this virus. Importantly, the SNaP method his highly reproducible, having worked in 48 of 50 human stem cell lines on the first attempt. We demonstrate that SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our screens identified hundreds of new and previously characterized ZIKV host factors in a microcephaly-relevant cell type, while also detecting dozens of genetic drivers of NPC proliferation, including PTEN, NF2, and PTCH1, through a built-in fitness assay. Finally, leveraging the reproducibility of the SNaP system, we performed “population-in-a-dish” experiments in which human stem cell lines from dozens of different donors were maintained as pooled cultures of SNaPs in single flasks prior to genomic and/or transcriptomic analyses. This mixed-culture approach revealed inherent growth differences among SNaP cell lines, and located several expression quantitative trait loci (eQTLs) that are relevant to autism spectrum disorders. As a whole, this work demonstrates the utility of novel stem cell-based technologies and their application towards high-throughput investigations of human neurodevelopment.Funding Source: Harvard University Faculty of Arts and Sciences Dean’s Competitive Fund for Promising Scholarship, NIH/NIMH grants U01MH105669 and U01MH115727. M.F.W. is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Program.T-3036INVESTIGATING THE EFFECTS OF PATHOGENIC AND PROTECTIVE GENE MUTATIONS ASSOCIATED WITH ALZHEIMER’S DISEASE (AD) OVER INJURY-INDUCED AMYLOID BETATran, My - Sanford Consortium of Regenerative Medicine, Monterey Park, CA, USA Gutierrez, Edgar - Department of Physics, University of California, San Diego, San Diego, CA, USA Goldstein, Lawrence - Sanford Consortium for Regenerative Medicine, San Diego, CA, USA Almenar, Angels - Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA Shah, Sameer - Departments of Orthopedic Surgery and Bioengineering, University of California, San Diego, La Jolla, CA, USA Chaves, Rodrigo - Sanford Consortium for Regenerative Medicine, La Jolla, CA, USAAlzheimer’s disease (AD) is a progressive, neurodegenerative disorder characterized by the formation of protein aggregates containing amyloid beta (A ) and hyperphosphorylated tau. AD βis a multifactorial disease that involves a complex interaction between genetic susceptibility and environmental exposures. Mutations in PSEN1 and APP genes, such as PSEN1DE9 and APPSwedish, can lead to early onset of AD, whereas APPIcelandic mutation is reported to confer a protective effect against AD. Moreover, while traumatic brain injury (TBI) also increases the risk of developing AD, the effects of TBI in AD-associated outcomes in individuals carrying these mutations are still unknown. To start addressing these questions, here we used neurons derived from TALEN/CRISPR-engineered human induced pluripotent stem cell (hiPSC) lines harboring either APPSwedish, PSEN1DE9, or APPIcelandic mutations. Neurons were rapidly stretched using a novel microfluidic device to simulate mild injury. As expected, among non-injured neurons expressing PSEN1DE9 and APPSwedish, but not APPIcelandic, presented higher levels of extra- and intracellular A² than control neurons. Twenty-four hours after injury, control neurons exhibited increased extracellular levels of A² when compared to non-stretched control group. Intriguingly, A² levels in neurons carrying AD-pathogenic or -protective mutation were not affected following mechanical trauma. These results suggest that, consistent with its protective potentials against AD pathologies, APPIcelandic mutation also demonstrates neuroprotective effects against mechanical injury. Strikingly, despite the increased levels of A² at steady state, mechanical stretch did not enhance amyloidogenesis in neurons expressing familial AD (FAD) pathogenic mutants, implying they reached saturated amounts of A² prior to the injury, thus, they are impervious to the mechanical damage. In future experiments, we will investigate whether A² generation in FAD mutants can be further upregulated using pharmacological compounds expected to enhance A².Funding Source: We acknowledge grant support from CDMRP/DOD (AZ140064) and California Institute of Regenerative Medicine (CIRM).T-3040EFFECTS OF LIPOPOLYSACCHARIDE ON THE SELF-RENEWAL AND NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS AND NEURAL STEM CELLSHsu, Yi-chao - Institute of Biomedical Sciences, Mackay Medical College, New Taipei City

373POSTER ABSTRACTSPrenatal infections in early brain development can engender adverse neurological outcomes in children. We hypothesized that prenatal infections may affect the neural differentiation of embryonic stem cells (ESCs) and further interfere with neural development and differentiation. To mimic the most common scenario of prenatal infection, mouse ESCs and ESC-derived neural stem cells (NSCs) were treated with lipopolysaccharide (LPS). In the ESC stage, 65% ± 1.3% of toll-like receptor 2 (TLR2)-positive cells and 1.6% ± 0.5% of TLR4-positive cells in ESCs were detected by flow cytometry. Notably, the percentage of TLR2(+) cells significantly decreased to 1.6% ± 0.4% and that of TLR4(+) cells significantly increased to 7.0% ± 0.9% after the ESC-to-NSC transition at days in vitro (DIV) 7. Furthermore, LPS did not affect the viability of mouse ESCs but significantly increased the number of ESC-derived neurospheres during the ESC-to-NSC transition. When LPS was administered during the ESC-to-NSC transition, we further observed that LPS significantly upregulated the mRNA expression levels of neuronal markers (Tuj1, Map2), astrocytic marker (Gfap), and oligodendrocyte marker (O4 and Oliog2) in the differentiated neural cells at DIV14, by using quantitative reverse transcription polymerase chain reaction analyses. Whole transcriptome analysis further revealed that significant upregulation of membrane dynamics of plasma, lysosomal and vacuole membranes and intracellular protein activities are involved. Notably, administration of LPS during the NSC-to-neural differentiation resulted in the downregulation of the above neural markers through significant upregulation of cell death pathways, such as p53 signalling pathway, and ferroptosis. In conclusion, this study revealed the dynamic expression profiles of TLRs during early neural development and that the timing of prenatal infection could be crucial for determining the types and severity of neural disorders developed in adulthood. Our findings may facilitate the development of preventative and therapeutic strategies for the adverse neurological side effects of prenatal infections.ORGANOIDST-3042DEVELOPMENT OF COLLAGEN BASED 3D MATRIX FOR GASTROINTESTINAL TRACT-DERIVED ORGANOID CULTURESJee, JooHyun - Organoid Research Center, CHA University, Seongnam-si, Korea Kim, Han Kyung - School of Medicine, CHA University, Seongnam, Korea Yoo, Jongman - School of Medicine, CHA University, Seongnam, KoreaAn organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin tissue. This organ-like structure requires a supporting matrix to maintain its characteristics and functions. Matrigel is mainly used as a matrix for 3D culture systems; however, it may not be used for clinical applications, as it is created from mouse sarcoma and contains unidentified components. This study attempts to develop a new, collagen-based matrix that may serve as a substitute for Matrigel in the organoid culture method. A collagen-based matrix was made, using type 1 collagen, Ham’s F12 nutrient mixture, and bicarbonate. Characteristics of mouse colonoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colonoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine–derived enteroids, stomach-derived gastroids, and human colon–derived colonoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties to those cultured in Matrigel. This suggests that the collagen-based matrix could be used for gastrointestinal tract–derived organoid culture as an extracellular matrix instead of Matrigel. This technology will render organoid cultivation inexpensive and clinically applicable, which may facilitate various therapeutic clinical trials of gastrointestinal tract–derived organoids.Funding Source: Supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Republic of Korea (HR16C0002, HI16C1634, HI17C2094, HI18C2458).T-3044NEURAL TISSUE ENGINEERING WITH IPSC-DERIVED NEURAL STEM CELLS USING A HYPER-CROSSLINKED CARBOHYDRATE POLYMERJudd, Justin - Cell Biology and Human Anatomy, University of California, Davis, CA, USA Pham, Kristen - Cell Biology and Human Anatomy, University of California, Davis, CA, USA Koleva, Plamena - Cell Biology and Human Anatomy, University of California, Davis, CA, USA Hirasawa, Naoki - Cell Biology and Human Anatomy, University of California, Davis, CA, USA Ebinu, Julius - Department of Neurological Surgery, University of California, Davis, CA, USA Lee, Charles - Cell Biology and Human Anatomy, University of California, Davis, CA, USASpinal cord regeneration is at the fingertips of developing tissue engineering efforts, but complete functional recovery of spinal cord injury is still out of reach. Scaffolds provide a physical substrate for cells to adhere in three dimensions, thus enabling de novo reconstruction of gross tissue architecture by loaded neural stem and progenitor cells or through immigration of native cells. Some scaffolds have been shown to facilitate partial functional recovery in spinal cord injury animal models, but the gold standard is not yet developed. Scaffolds can also be used to grow neural organoids in vitro, providing a scalable model for neural tissue in high-throughput neurotoxicity screens or developmental experiments. Another major advantage in this approach is the ability to use iPSC-derived stem and progenitor cells, allowing the study and use of autologous cells. Scaffold

374POSTER ABSTRACTSdesign is important to the successful growth of native-like tissue for several reasons. We present a novel hyper-crosslinked carbohydrate polymer (HCCP) for neural tissue engineering for the first time. HCCP exhibits key requirements of an ideal tissue engineering scaffold, including absence of host immune response, high porosity, bioresorbability, biocompatibility, and biomechanical properties similar to native tissues. HCCP has been approved by the U.S. FDA for clinical utilization for bone repair and regeneration and also shown to promote the generation of cancer, cardiac, and renal organoids. In this study, HCCP is seeded with iPSC-derived neural stem cells and used to grow large scale neural organoids in vitro and screen for drug neurotoxicity. HCCP is also used to regenerate the spinal cord in a hemi-section spinal cord injury model in vivo. The polymer is seeded with neural stem cells prior to implantation and gait analysis is used to evaluate functional recovery during regeneration. The favorable characteristics of HCCP provide an attractive scaffold for tissue engineering and stem cell biology in developing organoid models. The unique properties of HCCP also provide an effective tool for screening drugs, which targets the central nervous system, and investigating their effects on human brain during development.T-3046THE DEVELOPMENT STUDY APPLICATION OF BRAIN ORGANOIDS BY HUMAN PLURIPOTENT CELLSSo, Kyoung-Ha - Institute for Stem Cell and Regenerative Medicine (ISCRM) and Veterinary Medicine, Chungbuk National University, Cheongju, Korea Park, YoungSeok - Institute for Stem Cell and Regenerative Medicine (ISCRM). Department of Neurosurgery, Chungbuk National University Hospital, Chungbuk National University, Cheongju, Korea Hyun, Sang-Hwan - Institute for Stem Cell and Regenerative Medicine (ISCRM) and Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Chungbuk National University, Cheongju, KoreaAround 2010 year, several researchers had report floating 3D culture method as like serum-free culture of embryoid body-like aggregates (SFEB) that is source for brain organoid generation. First, we were isolated human primary somatic cell from human teeth and human brain tumor patient’s tissue. Second, obtained somatic cells were reprogramming using mRNA tools and characterized. Third, reprogrammed induced pluripotent stem cells (iPSCs) were generated cerebral organoid for development study as follows. Human primary somatic cells were isolated using enzymatic method and cell banking. And then these cells were reprogrammed using mRNAs (OSKMNL) mixed with evasion mRNAs (EKB) for 4 days. Human iPSCs like colonies were pick up around 2 weeks after transfection. Human iPSCs were stained with Alkaline Phosphatase as a stemness marker and characterized by immunocytochemistry to detection of pluripotency-associated genes as like TRA-1-60 and TRA-1-81 on live. Reprogrammed colonies were characterized and aggregated to generate cerebral organoid which organoid were cultured 3D floating method. Human iPSCs were collected and aggregated for embryonic body (EB) formation during germ layer differentiation, 9,000 cell/well for 6 days. And then EBs were induced of neural differentiation step to use neural induction media, contained with inducer of neural morphogen that differentiated neural ectoderm. And then neuroepithelial like tissues were transfer to matrigel droplets for the expansion of neuroepithelial buds and expansion culture until one month and two months. During organoid generation, each step was measured on bright filed and organoid like 3D cell were immunostained with neural marker as follow, Tuj1, PAX6, and Sox2. Reprogramming of human somatic cell is good source to regeneration study and Organoids may be valuable in the investigating developmental biology study. In the further study, generated brain organoids will be required to investigate their availability of the interaction of neural cell-to-cell signaling.Funding Source: This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2015H1D3A1066175, 2016R1D1A1B03933191, 2017R1A2B4002546, 2017K1A4A3014959), Republic of Korea.T-3048DEVELOPING HUMAN VASCULARIZED BRAIN ORGANOIDSLai, Jesse D - Stem Cell Biology and Regenerative Medicine, University of Southern California/Amgen, Los Angeles, CA, USA Griffin, Casey - Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA Bajpai, Ruchi - Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA, USA Yu, Violeta - Neuroscience, Amgen, Cambridge, MA, USA Ichida, Justin - Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAIncreasing evidence suggests that the neurovasculature is compromised throughout the initiation and development of neurodegenerative diseases. While clinical observations and rodent models have provided invaluable insight into these processes, generating an experimental human model facilitates a more mutable system with scalable potential for therapeutic screening. Human brain organoids derived from induced pluripotent stem cells (iPSCs) provide significant advantages with regards to cellular complexity and organization compared to traditional 2-dimensional cell culture methods. However, current organoid models are deficient in vascular tissues that likely play a significant role in brain development. Using iPSCs, we simultaneously generated brain microvascular endothelial cells, pericytes derived from the neural crest, and cortical brain organoids by directed differentiation. In conjunction with the neurogenesis phase, we embedded organoids in a droplet of extracellular matrix containing a single-cell suspension of endothelial cells and pericytes, generating a self-organizing vascularized cortical organoid (vCO). Over 30 days of culture,

375POSTER ABSTRACTSvCOs develop endothelial cell networks exhibiting extensive interactions specifically with a subset of S100 + radial glia. βMoreover, we also observed regions of PDGFR + pericyte-βendothelial cell interactions. One of the hallmarks of the blood-brain barrier is the presence of contiguous tight junctions. In vCOs, endothelial cell networks exhibited cell borders delineated by the claudin-5, suggesting that the local microenvironment promotes the development of barrier function. Vascular networks within the organoid can be maintained over 100 days, at which point endothelial cell networks are encapsulated by S100 + and βGFAP+ astrocytes. Morphological features were comparable across vCOs and were replicated in 2 iPSC lines. Collectively, these data represent the first in vitro model of a human brain organoid containing elements of a functional blood-brain barrier, and may provide insights into neurovascular development, as well as neurodegenerative and neurovascular disease. In particular, this model enables an unprecedented view into the purely cellular aspects of these pathologies in the absence of the circulatory system.Funding Source: Amgen, New York Stem Cell Foundation, Tau Consortium, NINDS, John Douglas French Alzheimer’s FoundationT-3050GENERATION OF 3D RETINAL ORGANOIDS FROM HUMAN NAÏVE PLURIPOTENT STEM CELLS ELIMINATES INTERLINE VARIABILITY OF RETINAL DIFFERENTIATIONPark, Tea Soon - Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Kanherkar, Riya - Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Zimmerlin, Ludovic - Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Evans-Moses, Rebecca - Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Liu, Ying - Ophthalmology, Johns Hopkins School of Medicine, Baltimore, MD, USA Singh, Mandeep - Ophthalmology, Johns Hopkins School of Medicine, Baltimore, MD, USA Lutty, Gerard - Ophthalmology, Johns Hopkins School of Medicine, Baltimore, MD, USA Zambidis, Elias - Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USARetinal Organoid (RO) development from human pluripotent stem cells (hPSCs) has thus far been optimized only in selected ‘permissive’ conventional hPSC lines with efficient neural differentiation capacities. Reproducible generation of ROs with photoreceptor progenitors from any hPSC line without interline variability would facilitate therapeutic regeneration in ophthalmologic disorders in a universal, defined manner. We have established that conventional, lineage-primed hPSC can be stably-reverted to naïve hPSC (N-hPSC) with enhanced multi-lineage differentiation potential following chemical inhibition with LIF-2i and the tankyrase inhibitor XAV939 (LIF-3i). To test whether LIF-3i reversion improves RO development amongst ‘non-permissive’ hPSC lines with poor neural potential, isogenic primed vs N-hPSC were differentiated with an established 3D retinal protocol. Differentiated neural ‘horse-shoe’ (HS) shaped domains were cultured in suspension and matured into retinal cups (RC) with laminated neural layers. 3 hESC and 3 hiPSC lines were differentiated into ROs +/- LIF-3i reversion. 2 of 6 non-permissive conventional hPSC lines incapable of HS development differentiated into RC organoids following LIF-3i reversion with efficiencies comparable to “permissive” hPSC lines (e.g. H9, IMR90-4). Eye field-specific gene expression at 2 weeks and photoreceptor lineage markers at 8-20 weeks in N-hPSC were robustly detected in RCs generated via qRT-PCR and immunofluorescent confocal microscopy, including Rhodopsin+ CRX+ Recoverin+ photoreceptor progenitors, HuC/D+ ganglion/amacrine cells, PROX1+ horizontal cells, and MITF+ pigmented epithelium. N-hPSC-derived RO displayed improved long-term maturation of in vitro Rhodopsin+ photoreceptors with proper histo-architecture. 13 week-old RCs were dissected into neural retinal sheets and transplanted into the subretinal space of ischemia reperfusion-injured NOG-SCID mice eyes, and human cell specification and engraftment was evaluated at 3-6 months post-transplant. We propose that improved RO development from N-hPSC will facilitate the clinical development of autologous or universal donor-based ocular cell therapies and enhance the engraftment and long-term survival of retinal cell lineages following cellular transplantation.Funding Source: TEDCO 2014-MSCRFE-118153 (TSP), NIH/NEI R01EY023962), TEDCO 2013-MSCRFII-0032-00, and Novo Nordisk Diabetes and Obesity Science Forum Award, RPB Stein Innovation Award (all ETZ), EY001765 (Wilmer Core Grant for Vision Research)T-3052VASCULARIZATION AND MATURATION OF HUMAN IPSC-DERIVED KIDNEY ORGANOIDS THROUGH TRANSPLANTATION IN THE COELOMIC CAVITY OF CHICKEN EMBRYOS INCREASES GLOMERULAR MODELLINGKoning, Marije - Internal Medicine - Nephrology, Leiden University Medical Center, Leiden, Netherlands Avramut, Cristina - Cell and Chemical Biology, Leiden University Medical Center, Netherlands Howden, Sara - Department of Paediatrics, Murdoch Children’s Research Institute, Melbourne, Australia Jaffredo, Thierry - Developmental Biology Laboratory, Sorbonne Université, Paris, France Lievers, Ellen - Internal Medicine - Nephrology, Leiden University Medical Center, Netherlands Little, Melissa - Department of Paediatrics, Murdoch Children’s Research Institute, Melbourne, Australia Rabelink, Ton - Internal Medicine - Nephrology, Leiden University Medical Center, Netherlands van den Berg, Bernard - Internal Medicine - Nephrology, Leiden University Medical Center, Netherlands

376POSTER ABSTRACTSvan den Berg, Cathelijne - Internal Medicine - Nephrology, Leiden University Medical Center, Netherlands Wiersma, Loes - Internal Medicine - Nephrology, Leiden University Medical Center, NetherlandsHuman induced pluripotent stem cell (hiPSC) derived kidney organoids closely resemble in vivo kidney tissue, offering unprecedented possibilities for modelling disease and development. Despite recent advances in kidney organoid generation, currently available protocols still yield organoids that lack a functional vasculature. As kidneys are highly vascularized organs that depend on the presence of vascular endothelial cells and blood flow for their correct development and functionality, it is not surprising that the organoids remain immature. Unfortunately, this limits their applicability. Particularly problematic is the modelling of glomerular diseases, since all three components of the glomerular filtration barrier, namely endothelial cells, glomerular basement membrane (GBM) and podocytes, are either absent or immature in in vitro kidney organoids. Recently, we have shown that transplantation of hiPSC-derived kidney organoids under the renal capsule of mice leads to functional vascularization and maturation of the organoids. However, this model is labor-intensive and time consuming, requiring 28 days for optimal maturation. Here, we describe a new model where we transplanted kidney organoids inside the coelomic cavity of early chicken embryos. We observed rapid vascularization by host vasculature as well as reorganization of organoid-derived human endothelial cells. Through intravascular injection of fluorescently-labeled lectins, we show functionality of and anastomosis between the chicken- and human- derived blood vessels. Upon 8 days of transplantation we did not observe misdirected differentiation to stromal tissue. Furthermore, unbiased wide field transmission electron microscopy analysis of these organoids demonstrated glomerular maturation with the formation of podocyte foot processes, slit diaphragms and a GBM. Tubular structures also showed signs of maturation, with the appearance of cell junctions and microvilli. These data demonstrate that intracoelomic transplantation inside chicken embryos is an efficient method to vascularize and mature kidney organoids. This highly accessible model could be employed to rapidly mature organoid glomerular structures in order to improve the modelling of glomerular kidney disease.T-3054A SERUM-FREE DEFINED CULTURE SYSTEM FOR STUDYING THE DIFFERENTIATION AND FUNCTION OF STROMAL PROGENITOR CELLS IN KIDNEY ORGANOIDSNishikawa, Masaki - Chemical System Engineering, University of Tokyo, Bunkyo, Japan Chang, Hsiao-Min - David Geffen School of Medicine, University of California, Los Angeles, North Hills, CA, USA Kimura, Hiroshi - Mechanical Engineering, Tokai University, Hiratsuka, Japan Yanagawa, Naomi - Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA, USA Hamon, Morgan - David Geffen School of Medicine, University of California, Los Angeles, North Hills, CA, USA Sakai, Yasuyuki - Chemical System Engineering, University of Tokyo, Hongo, Japan Yanagawa, Norimoto - Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA, USAThe adult kidney is a complex organ consists of more than 20 different types of cells, but it develops from a rather simple structure, i.e., metanephros, consists of three types of progenitor cells: ureteric bud (UB), cap mesenchymal (CM), and stromal (SM) cells. Recent studies have reported that aggregates made of dispersed cells from mouse metanephroi, or hiPSC-derived CM and UB cells, can self-organize and differentiate to create nephron-like structures. These kidney organoids can thus serve as a model system for disease modeling and drug screening. However, most of these studies have focused mainly on CM and UB cells without detailed insights on how SM progenitor cells differentiate and function in kidney organoids.Here we present a serum free defined culture system suitable for studying the differentiation and function of SM progenitor cells in kidney organoids. For this purpose, cells derived from E13.5 Foxd1-EGFP mouse metanephroi were used to prepare either the conventional aggregates that contain SM progenitor cells (AG+SM) or aggregates devoid of SM progenitor cells (AG-SM), where Foxd1-EGFP+ SM progenitor cells were excluded by FACS sorting. Aggregates were cultured on membranes placed at air-liquid interface for up to 7 days. When aggregates were cultured in 10% FBS containing medium, self-organization and differentiation of UB and CM cells were observed in both AG+SM and AG-SM organoids, but no differentiated SM structures emerged in either organoids. In contrast, when AG+SM were cultured in a serum free defined medium, containing a cocktail of inhibitors and growth factors, structures stained positive for differentiated SM markers, such as Renin, Pdgfrb, and alpha smooth muscle Actin, were detected in AG+SM but not in AG-SM. We also noticed prominent growth of CM cells in AG-SM as compared to AG+SM, consistent with the function of SM progenitor cells to induce the differentiation of CM cells. In conclusion, we report here a novel culture system which is suitable for studying the differentiation and function of SM progenitor cells in kidney organoids. This culture system may serve as a useful platform to study the role of SM cells in kidney development, pathology and regeneration.T-3056EFFICIENT DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELL TO BRANCHING LUNG BUD ORGANOIDS and MATURATION INTO ALVEOLAR LUNG ORGANOIDSLu, Min - Cellular Assays, Biological Reagents and Kits, MilliporeSigma, Temecula, CA, USA Asbrock, Nick - Cellular Assays, Biological Reagents and Kits, MilliporeSigma, Temecula, CA, USA

377POSTER ABSTRACTSChu, Vi - Cellular Assays, Biological Reagents and Kits, MilliporeSigma, Temecula, CA, USAWe describe a stepwise protocol to efficiently differentiate human iPSCs to branching lung bud organoids and then further matured into alveolar lung organoids. Branching lung bud organoids (LBOs) are formed by day 20 and exhibit airway-like structures with morphological and functional characteristics similar to developing lungs in vivo. LBOs express SFTPB, a pulmonary-associated surfactant protein B that is secreted by alveolar lung cells, pulmonary endoderm markers SOX9, NKX2.1 and EPCAM, the mesenchymal marker VIMENTIN, and MUC5AC, a marker for airway goblet cells. By days 30-40, large branching structures with rounded expansion at the distal tips resembling alveolospheres could be observed. To enable high-throughput (HTP) screens, large numbers of lung organoids would be required. We show that branching lung organoids can be maintained for an extended period and moreover can be cryopreserved. Upon thawing, cryopreserved lung organoids can recover their branching morphologies upon encapsulation in Matrigel and can be further differentiated into spatially organized alveolar lung organoids. The ability to culture and cryopreserve lung bud organoids that retained the ability to differentiate into alveolar lung organoids is a significant step towards widespread adoption of LBOs for disease modeling and HTP screens.T-3058EFFICIENT DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS TO COLONIC ORGANOIDS AND THEIR APPLICATIONS IN TOXICOLOGY SCREENSSu, Kevin - Cellular Assay, MilliporeSigma, Temecula, CA, USA Hoffmann, Stefanie - Early Investigative Toxicology, Chemical and Preclinical Safety, Merck KGaA, Darmstadt, Germany Hewitt, Philip - Early Investigative Toxicology, Chemical and Preclinical Safety, Merck KGaA, Darmstadt, Germany Chu, Vi - Cellular Assay, MilliporeSigma, Temecula, CA, USAIn recent years, 3D organoids have emerged as a new tool for disease modeling and for pre-clinical toxicity screening applications. Organoids are self-organizing three-dimensional structures derived from either pluripotent stem cells or from primary tissues with the ability to recapitulate some of the spatial architecture and in vivo function of organs. Here, we describe a stepwise protocol in serum-free medium to efficiently differentiate human pluripotent stem cells to colonic organoids. Two human iPS cell lines were used; one derived from PBMCs and the other from human foreskin fibroblasts. Colonic organoids generated from both hiPS lines could be propagated and expanded in long-term culture and expressed the appropriate colonic markers. In comparison to previous 2D in vitro cell culture models, organoids resemble very closely the intestinal epithelial layer in vivo. They can be used in applications including high throughput screenings for agents that target the intestinal epithelium and for the identification of potential toxicity of new compounds. Human iPS derived colon organoids could be cryopreserved and upon thawing were highly viable and could be further expanded and re-developed into 3D colon organoids. Efforts are underway to further characterize the functional attributes of the colon organoids and validate their use in a low to mid-throughput toxicology screen.TISSUE ENGINEERINGT-3060POSITIVE EFFECTS OF PRE-COATING DECELLULARIZED LIVERS WITH HEPATIC CELLS CONDITIONED MEDIUMde Caires Junior, Luiz Carlos - Department of Biology, University of São Paulo, Brazil Goulart, Ernesto - Department of Biology, University of São Paulo, Brazil Telles-Silva, Kayque - Department of Biology, University of São Paulo, Brazil Musso, Camila - Department of Biology, University of São Paulo, Brazil Kobayashi, Gerson - Department of Biology, University of São Paulo, Brazil Assoni, Amanda - Department of Biology, University of São Paulo, Brazil Ribeiro-Junior, Antonio - Department of Biology, University of São Paulo, Brazil Caldini, Elia - Department of Biology, University of São Paulo, Brazil Rangel, Thadeu - Department of Biology, University of São Paulo, Brazil Passos-Bueno, Maria Rita - Department of Biology, University of São Paulo, Brazil Raia, Silvano - Department of Biology, University of São Paulo, Brazil Lelkes, Peter - Department of Bioengineering, Temple University, Philadelphia, PA, USA Zatz, Mayana - Department of Biology, University of São Paulo, BrazilCurrently, liver transplantation from compatible donors main alternative therapy for patients with irreversible hepatic injuries. Hepatic tissue engineering is an important approach for the development of new organs aiming future human transplantation. Bioengineering technologies have been developed to produce decellularized liver scaffolds which could be recellularized with human cells. However, high quality liver recellularization methods are still under investigation. In order to enhance the recellularization process, the aim of this work was to improve the hepatic matrices recellularization process by pre-coating the extra cellular matrix (ECM) with human hepatic cells-conditioned medium (CM). Furthermore, we are investigating the potential of human liver cells to adhere on several ECM proteins as well as CM components. Livers from male Wistar rats were collected and decellularized by perfusion of 1% Triton-X solution with 0.05% NaOH. The samples were analyzed by histology, immunohistochemistry, scanning electronic microscopy and

378POSTER ABSTRACTSDNA-content analysis. Decellularized livers were coated with HepG2-CM, recellularized up to 5 weeks with hepatoblasts, mesenchymal stem cells (MSC), both differentiated from hiPSCs and human aortic endothelial cells (HAEC). iPSCs and differentiated cells were characterized by RT-qPCR, flow citometry and immunofluorescence. The organization and integrity of the hepatic ECM was maintained after decellularization. The recellularization steps were successfully improved on pre-coated liver ECM as compared with non-coated liver ECM. Absence of nuclei and cellular residues were confirmed by optical and electronic microscopy. Residual DNA-content was below the immunogenic limits (10ng/mg; p< 0.001). Cell adhesion assay showed high capability of hepatic adhesion on collagen I + CM, fibronectin + CM, and laminin + CM but not only on CM. Thus, recellularization of pre-coated livers-ECM was significantly improved showing the positive effects of the association of liver ECM with CM components.Funding Source: FAPESP, CNPq, CAPES, Ministry of HealthT-3062BMAL1 FUNCTIONALIZED 3D RECONSTRUCTED EPIDERMIS: HUMAN MODELS RECAPITULATING CHARACTERISTICS OF DEREGULATED CIRCADIAN RHYTHM SKIN PRECURSORSParis, Maryline - L’Oreal Advanced Research, L’Oreal R&I, Aulnay-sous-Bois, France Denat, Laurence - L’Oreal Advanced Research, L’Oreal R&I, Aulnay, France Genty, Gaïanne - L’Oreal Advanced Research, L’Oreal R&I, Aulnay, France Deshayes, Nathalie - L’Oreal Advanced Research, L’Oreal R&I, Aulnay, France Boissout, Florian - L’Oreal Advanced Research, L’Oreal R&I, Aulnay, France Dimitrov, Ariane - L’Oreal Advanced Research, L’Oreal R&I, Aulnay, FranceIn mammals, desynchronized circadian rhythm leads to various biological symptoms. In skin and hair follicle, human epidermal stem cells function in vitro is regulated by circadian oscillations, thus contributing to tissue aging when deregulated. The impact of circadian oscillations, through a feedback loop involving Clock pathway on hair and skin stem cells function in vitro is well described in mice. We recently demonstrated that the in vivo deregulation of the Clock pathway affects regenerative properties of human skin and hair precursor cells. The present study aimed at developing functionalized 3D reconstructed skin models mimicking the premature aging features of regenerative defect observed in human Epidermal precursor cells (hEpi) after a long term deregulated circadian rhythm in vivo. Using primary culture of human epidermal keratinocytes two modified primary lines of hEpi were generated: hEpi overexpressing BMAL1 (constitutive overexpression of BMAL1) and hEpi shBMAL1 (knockdown for BMAL1). Cell cultures, measurement of colony area, count of clones and 3D reconstructed skin were carried out using these two lines. Our results demonstrate that BMAL1 overexpression and BMAL1 knockdown affect hEpi keratinocytes clone-forming efficiency as well as the reconstructed epidermis process. In this study, two BMAL1 functionalized 3D reconstructed skin models were developed that recapitulate the observations previously described in human precursor cells that were isolated from donors with a long term in vivo deregulated circadian rhythm. These models will allow i) the Clock pathway deregulation mechanisms leading to an alteration of the skin precursors properties to being better understood and ii) compounds apt at rescuing the regenerative potential of keratinocyte precursors to being screened.T-3064B2M AND CIITA DEFICIENT HUMAN EMBRYONIC STEM CELLS LACK HLA CLASS I AND II SURFACE PRESENTATIONWinblad, Nerges - Clinical Science, Intervention and Technology, Karolinska Institutet, Huddinge, Sweden Petrus-Reurer, Sandra - Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Kvanta, Anders - Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Lanner, Fredrik - Clinical Science, Intervention and Technology, and Ming Wai Lau Centre for Reparative Medicine-Stockholm Branch, Karolinska Institutet, Stockholm, SwedenFor clinical translation of an allogenic stem cell-derived treatment, matching of HLA molecules or extensive immunosuppression will be essential to avoid graft rejection. In order to minimize this burden, we generated human embryonic stem cells (hESC) lacking human leukocyte antigen (HLA)-I and -II molecules . Since HLA genes are highly polymorphic, we edited B2-microglobulin (B2M) to prevent HLA-I proteins anchoring in the cell membrane, and class II major histocompatibility complex transactivator (CIITA) to hinder activation of HLA-II genes. Using CRISPR/Cas9 technology, we first created an hESC-B2M-knockout (hESC-B2M-KO) line and evaluated pluripotency and HLA-I gene and protein expression in several single-cell clones. Next, to assess the differentiation competence of the edited clones we differentiated them into retinal pigment epithelium (RPE) cells and further characterized them. Results demonstrated loss of B2M on transcriptional and protein levels. Intracellular HLA-I was still detected but the cells failed to present HLA-I on the cell surface. Mature hESC-RPE-B2M-KO also exhibited proper cobblestone morphology as well as upregulation of RPE-related genes, comparable to wildtype cells. We selected one of the hESC-B2M-KO clones to edit CIITA aiming to generate a double-knockout hESC-B2M/CIITA-KO line. HLA-II transcript levels were undetected in hESC-B2M/CIITA-KO , while CIITA expression was comparable to wildtype. Following differentiation of the double-knockout line into RPE cells, we demonstrated that these cells retained cobblestone morphology, pigmentation and expression of RPE-related genes but lacked surface expression of HLA-II proteins. In conclusion, we demonstrate that hESC-B2M/CIITA-KO can be generated

379POSTER ABSTRACTSusing CRISPR/Cas and that they can be differentiated into clinically relevant cells such as RPE cells. These edited hESCs may provide a valuable cell source for regenerative medicine and cell replacement therapies.T-3066MODELLING THE CONGENITAL HEART CONDITION OF TETRALOGY OF FALLOT WITH HUMAN INDUCED PLURIPOTENT STEM CELLS AND ENGINEERED CARDIAC TISSUESChan, Chun Ho - Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, Keung, Wendy - Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong Lam, Yin Yu - Department of Pediatric and Adolescent Medicine, The University of Hong Kong, Kowloon, Hong Kong Lin, Geng - Dr. Li Dak-Sum Research Centre, The University of Hong Kong Wong, Nicodemus - Department of Pediatric and Adolescent Medicine, The University of Hong Kong, Kowloon, Hong Kong Li, Ronald Adolphus - Karolinska Institute Ming Wai Lau Centre for Reparative Medicine, Hong Kong Cheung, Yiu Fai - Department of Pediatric and Adolescent Medicine, The University of Hong Kong, Hong KongCongenital heart disease (CHD) is the most common congenital malformation with a prevalence of 8 to 10 per 1000 live births. Tetralogy of Fallot (TOF), as characterized by the presence of ventricular septal defect, pulmonary stenosis, hypertrophic right ventricle and an overriding aorta, is the most common cyanotic CHD. Its pathogenesis is, however, unclear. Indeed, primary defects of TOF are difficult to be discerned from other defects secondary to anatomical changes. Our study aim to employ human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) differentiated from normal individuals and TOF patients together with state-of-the-art cardiac tissue engineering to examine the intrinsic abnormalities of TOF, while single-cell-RNA-sequencing further provides their molecular bases. As a first step, single-cell transcriptomes from TOF and control constructs were compared across different engineered tissue configurations: hiPSCs, hiPSC-vCMs at day 30 after initiation of cardiac differentiation (hiPSC-vCMs), human ventricular cardiac anisotropic sheets (hvCAS) and tissue strips (hvCTS) fabricated from hiPSC-vCMs. Cardiac genes including TNNI3 and TAGLN were downregulated in TOF-hiPSC-vCMs. Comparisons between normal and TOF hvCAS further revealed the pathway involved in myocardial contraction as the top under-represented pathway in the TOF group, with MYH7, MYL7, MYL2, TNNI3 and TPM2 being significantly downregulated. By contrast, the pathway involved in glycolytic process was found to be the top over-represented pathway in the same tissue construct with ALDOA, ENO1, ENO2, GPI, GAPDH, LDHA, PFKP and PGK1 being significantly upregulated. Taken together, our findings provide evidence of possible intrinsic impairment of contractility of cardiomyocytes in TOF independent of the secondary effects of the structural cardiac malformation. Further studies are underway to determine the force generation in hvCTS generated from control- and TOF-hiPSC-vCMs.T-3068HARNESSING THE POTENTIAL OF 3D TOPOGRAPHY AND GRAPHENE FOR EFFICIENT DIFFERENTIATION OF MESENCHYMAL STEM CELLS INTO NEURONS AND CARDIOMYOCYTESMohanty, Sujata - Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India Aggarwal, Ashwini - Smita Lab, Department of Textile Technology, Indian Institute of Technology, IIT, New Delhi, India Debnath, Debika - Department of Biomedical Engineering, University of Bridgeport, Bridgeport, CT, USA Gupta, Deepika - SMITA LAB, IIT, New Delhi, India Gupta, Suchi - Stem Cell Facility, AIIMS, New Delhi, India Jain, Krishna - Stem Cell Facility, AIIMS, New Delhi, India Jassal, Manjeet - Smita Lab, Department of Textile Technology, Indian Institute of Technology, Delhi, India Kalluri, Ankarao - Department of Biomedical Engineering, University of Bridgeport, Bridgeport, CT, USA Patra, Prabir - Department of Biomedical Engineering, University of Bridgeport, Bridgeport, CT, USA Rawat, Sonali - Stem Cell Facility, AIIMS, New Delhi, India Vig, Sanjana - Stem Cell Facility, AIIMS, New Delhi, IndiaNeurons and cardiomyocytes are terminally differentiated post-mitotic cells with very limited regenerative potential. Hence, injuries or degeneration to these cells might incapacitate normal body functions. The present study aims at developing PCL scaffolds with nano-graphene fillers to differentiate human Mesenchymal Stem Cells (MSCs) into functional neurons and cardiomyocytes. Graphene may provide appropriate microenvironment with topographical and electrical cues for MSCs differentiation owing to its excellent electrical and mechanical properties. Scaffolds with 15% of PCL and 0.01, 0.05 and 0.1 wt% of graphene as filler in the polymer matrices were prepared by electrospinning. For biological studies, sterile scaffolds were seeded with human Mesenchymal Stem Cells. For neuronal differentiation, induction medium was supplemented with EGF, FGF2 and Oxysterol while for cardiac differentiation; Oxytocin was used as an inducer. Physical characterization of the scaffolds showed highly porous mimicking the extracellular matrix of native tissue. Live and dead staining and proliferation studies showed that all scaffolds were biocompatible and support cell attachment and proliferation respectively. Cell differentiation over PCL 15% G0.05 scaffolds revealed better neuronal and cardiac differentiation as evaluated by morphological analysis , alignment and expression of proteins specific to neurons (MAP2 and TH) and cardiomyocytes(MLC and CTnI). This study indicates the role of graphene nanocomposite scaffold in enhanced differentiation of MSCs into DA neurons and cardiomyocytes. Presence of graphene in the scaffolds not only improved MSC adhesion, morphology but also upregulated

380POSTER ABSTRACTSthe cell membrane calcium ion efflux, thereby indicating better functionality of differentiated cells. Thus, we envisage that low amount of graphene nanofiber based scaffold can serve as a potential graft for developing future therapies for regeneration of degenerated neuronal and cardiac cells. These in vitro studies will further be validated in in vivo animal models.Funding Source: The work done in this study was generously supported by Department of Biotechnology, India.T-3070GENERATION OF LUNG ORGANS FROM MOUSE EMBRYONIC STEM CELLS VIA BLASTOCYST COMPLEMENTATION IN MICEZhou, Qiliang - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Ran, Qingsong - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Kitahara, Akihiko - Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Ye, Xulu - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Sasaki, Kenta - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Matsumoto, Yoshifumi - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Moriyama, Masato - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Oda, Kanako - Brain Research Institute Center for Bioresource-based Researches/Bioresource Science Branch Department of Comparative and Experimental Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Sasaoka, Toshikuni - Brain Research Institute Center for Bioresource-based Researches/Bioresource Science Branch Department of Comparative and Experimental Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Ajioka, Yoichi - Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Yasue, Akihiro - Orthodontics and Dentofacial Orthopedics, Tokushima University Graduate School of Oral Sciences, Tokushima, Japan Saijo, Yasuo - Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, JapanGeneration of the pancreas and kidney from stem cells via blastocyst complementation has been reported, but to our knowledge, there has been no report regarding lung generation. Fibroblast growth factor 10 (Fgf10) is a gene involved in the development of limbs and lungs in mice. Fgf10-/- mice present phenotypes indicating limb and lung deficiencies. We attempted to generate lung organs from embryonic stem cells via blastocyst complementation in this study. Previously, we developed Fgf10-/- mice by the CRISPR/Cas system. Because Fgf10-/- mice die immediately after birth, Fgf10+/- genotype mice are maintained in Fgf10 Exon1-/+ and Fgf10 Exon3-/+. Four hundred and seven compound heterozygous mutant embryos (Exon1-/+ × Exon3 -/+) in total were prepared and then enhanced green fluorescent protein (EGFP)-positive murine ES cells were microinjected into 8-cell-stage embryos. Three hundred sixty microinjected embryos were transferred into pseudopregnant mice and 91 littermates (25.3%) were obtained. Fifty-two littermates (57.1%) were EGFP positive (chimeric) and 19 littermates of these died at birth, while 11 littermates of these were able to survive until they have been weaning. Nine out of 91 littermates (9.9%) showed limb defects and died at the time of birth. No EGFP expression were detected in these littermates and genomic analysis using surveyor mutation test and DNA sequence analysis indicated a compound heterozygous genotype (Fgf10 Ex1 -/+ and Fgf10 Ex3 -/+). Other 20 out of 91 littermates (22%) showed ventral hernias and/or opened eyes with EGFP expression and a wild genotype of Fgf10. Two out of 11 weaned chimeric mice were indicated deriving from compound heterozygous embryo (Fgf10 Ex1 -/+ and Fgf10 Ex3 -/+). Histological analysis exposed that the lung tissues of the two weaned compound heterozygous mice were well developed and showing positive expression of the EGFP. This study suggests the possibility of lung organ generation from ES cells via blastocyst complementation in a lung aplastic mouse model.Funding Source: This work was supported by JSPS KAKENHI Grant Number 18K15921 and 18H02817GT-3072MULTIPARAMETRIC STUDY OF HUMAN HEPATOTOXICITY ON A NOVEL MICROLIVER DEVICESharma, Ruchi -, Stemnovate Limited, Cambridge, UK Lawrence, Nathan - ANB Sensors, Cambridge, UK Akande, Femi - Stemnovate Limited, Cambridge, UK Cumberland, Max - Stemnovate Limited, Cambridge, UK Fisher, Adrian - Stemnovate Limited, Cambridge, UK Hay, David - Stemnovate Limited, Cambridge, UK Legg, Ben - Stemnovate Limited, Cambridge, UK Sur, Sumon - Stemnovate Limited, Cambridge, UKHepatotoxicity remains one of the biggest reasons for the attrition of candidate drugs during the later stages of drug development. The cell lines and animal models due to species differences provide limited physiological relevant information and have resulted in ‘silent’ hepatotoxic drugs being introduced into clinical trials, garnering huge financial

381POSTER ABSTRACTSlosses for drug companies through withdrawals and late-stage clinical failures. Therefore, interest in new systems to identify human drug toxicity are high, particularly at preclinical phase. We have developed a novel ‘MicroLiver’ device that provides bioinspired microfluidic environments to allow spatiotemporal control of various chemical and physical culture conditions that are unavailable with other methods, which in turn allows for manufacturing of more physiologically relevant drug screening platforms. Our microengineering approach using microfluidic technology is designed to simulate liver structure and function. The ‘MicroLiver’ system is integrated with Hepatocyte-like cells (HLCs) derived from patient-specific, and genomically diverse human induced pluripotent stem cells (iPSCs) and are a promising alternative to animal studies to enable greater precision, safer and personalised targeting for the drug development process. Human somatic cells reprogrammed into iPSCs utilising the viral-free delivery system to introduce (Yamanaka) reprogramming factors (OCT3/4, KLF4, SOX2, MYC). The iPSCs were characterised for pluripotency and then differentiation into HLCs for 18 -30 days. The HLCs evaluated through assessment of cytochrome (CYP) P450 (CYP1A2, CYP3A4 and CYP2D6) activity and the expression of liver-specific genes were integrated onto the microfluidic devices, and the toxicity of selected chemicals evaluated. ATP assays assessed the cell viability and proliferation after drug exposure. The ‘MicroLiver’ system to study hepatotoxicity allows spatiotemporal control of various chemical and physical culture conditions that are unavailable with other methods. Our approach through the use of functional and stable patient-specific iPSC-derived HLCs counteracts the disadvantages associated with utilising primary hepatocytes, cell lines and animal models while reducing animal research and improving drug safety.Funding Source: The study is co-funded by Innovate UKETHICAL, LEGAL AND SOCIAL ISSUES; EDUCATION AND OUTREACHT-3076INFORMED CONSENT FOR IMPLANTATION OF HUMAN GENOME-EDITED EMBRYOS INTENDED TO RESULT IN LIVE BIRTHSJonlin, Erica C - Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Mathieu, Julie - Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USAWe now know that the consent form and actual consent process that were employed in Dr. He Jiankui’s infamous and secret “CRISPR baby” clinical trial were misleading at best and deceptive at worst. The language was over-technical, benefits were exaggerated, stated risks were underplayed, the most serious risks were omitted, and responsibility for untoward effects was shifted to the vulnerable population of research subjects recruited to the clinical trial. In that off-target editing did occur, the effects on the children are unknown, and monitoring into the future is unclear. While it is arguable whether implantation of genome-edited human embryos should not be done at all, because it has already been attempted, it is realistic to expect that it will happen again. Thus, in this poster we present consent form language that would meet the regulatory requirements for informed consent, while sensitively and accurately explaining the benefits and all of the risks of human embryo genome-editing to would-be recipients of these embryos, and to the offspring themselves. Additionally, we discuss potential appropriate parent populations to enroll in these studies, and factors to consider to avoid coercion in recruitment. Because informed consent is a process, we present points to consider for who should and who should not administer informed consent, how the consent process might be conducted, and how long-term participant oversight and protection should be ensured before, during, and following the birth of any offspring for this very sensitive research.T-3078AN INTERACTIVE LEARNING APPROACH TO ADVANCE STEM CELL SCIENCE EDUCATION IN THE MIDDLE AND HIGH SCHOOL CLASSROOM: A NOVEL STUDENT-CENTERED TEACHING MODELLu, Karol - Natural Sciences, Pasadena City College, Pasadena, CA, USA Eversole-Cire, Pamela - Natural Sciences, Biological Technology Program at Pasadena City College, Pasadena, CA, USAThe California Institute for Regenerative Medicine (CIRM)-funded program: Bridges to Stem Cell Research at Pasadena City College was designed to include an outreach component to advance stem cell education and awareness amongst diverse student populations at local participating high schools. While initial outreach efforts included high school visits by CIRM Bridges interns to discuss stem cell-related projects and a follow-up Stem Cell Training Workshop at Pasadena City College (PCC), the workshop limited the number of students that could participate. To extend outreach activities to the classrooms of participating teachers and allow more students to learn about stem cells in an interactive way, a portable Stem Cell Demonstration Laboratory for high school students and accompanying teaching module were developed. The Stem Cell Demonstration Laboratory includes a mini laminar flow hood, a small CO2 incubator, and a microscope with imaging capabilities which allows the students to perform many of the techniques offered in the workshop in their classrooms. The Stem Cell Demonstration Laboratory and module have been offered since 2012 and has reached out to over 734 high school sophomores, juniors, and seniors. Within the last two years, the Stem Cell Demonstration Laboratory for high school students was extended to the middle school classroom, reaching over 278 seventh grade students. The teaching module has recently been developed into a short course to include lectures and facilitated group discussions on stem cell science and regenerative medicine, as well as complementary laboratory activities.

382POSTER ABSTRACTSThe preliminary descriptive data suggests that the Stem Cell Demonstration Laboratory and its complementary teaching module may be an appropriate student-centered teaching model for the middle school science classrooms as well as high school biology or biotechnology classes. In addition, providing hands-on stem cell training activities directly in the classroom may motivate students to eventually pursue stem cell research, biological sciences or related disciplines at the post-secondary level to build the STEM pipeline and increase diversity in STEM-fields to mirror that of California.CLINICAL TRIALS AND REGENERATIVE MEDICINE INTERVENTIONST-3082CLINICAL-GRADE HLA HOMOZYGOUS IPS CELL STOCK AT CIRADohi, Hiromi - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Hanatani, Tadaaki - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Takasu, Naoko - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Yamanaka, Shinya - Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanHuman induced pluripotent stem cell (iPSC) repositories from human leukocyte antigen (HLA) haplotype homozygous donors have been called iPSC haplobank. The haplobank is aimed for minimizing the influence of immune rejection and considered important to a future clinical strategy for the allogeneic cell transplantation. Since 2013, we have built an iPSC stock which is a clinical-grade HLA homozygous iPSC haplobank optimized for Japanese population and provided the stock to research institutions and companies for developing iPSC-based regenerative medicine. Our iPSC stock is generated from peripheral blood mononuclear cells and umbilical cord blood cells. For the healthy HLA homozygous donor recruiting, we are collaborating with Japanese Red Cross Society, the Japan Marrow Donor Program and several cord blood banks which have already performed HLA typing for huge numbers of people. As of January 2019, we have already established five donor-derived 21 iPSC lines with three types of HLA haplotypes, and provided to 13 institutions and 12 projects. The three haplotypes cover approximately 32% of the Japanese population in terms of HLA matching. In the manufacturing of our iPSC stock, we have developed a feeder-free and xeno-free culture system for complying with the regulatory requirements. In order to assuring the quality, a comprehensive quality testing is performed, including microbiological sterility, morphology, vector clearance, viability, marker expression and genomic analysis. Here, we share our efforts and experience of establishing clinical-grade iPSC stock.Funding Source: This work was supported in part by the Core Center for iPS Cell Research grant from Japan Agency for Medical Research and Development (AMED).T-3084IMMUNOMODULATORY EXOSOMES DERIVED FROM HUMAN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF ACUTE RESPIRATORY DISTRESS SYNDROMETieu, Alvin - Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Lansdell, Casey - Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Stewart, Duncan - Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Lalu, Manoj - Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, CanadaAcute respiratory distress syndrome (ARDS) is an inflammatory disease that leads to impaired gas exchange and respiratory failure. Despite decades of research, there is no specific therapy for this condition and mesenchymal stem cells (MSCs) are currently in clinical trials for ARDS. The protective effects of MSCs appear to be largely mediated by paracrine mechanisms, which includes release of small extracellular vesicles, such as exosomes. This pilot study aims to provide an estimate of therapeutic efficacy for MSC-derived exosomes (MEX) in a lipopolysaccharide (LPS) induced mouse model of acute lung injury (ALI) to be used in the design of a definitive preclinical study. Human bone marrow-derived MSCs were cultured with serum-free media and MEX were isolated by ultracentrifugation. MEX displayed a mean size of 72nm using nanoparticle tracking analysis, cup-shaped morphology by electron microscopy, and enrichment of surface markers CD63 and CD81 by Western Blot analysis. In an in vitro wound healing assay, MEX markedly increased the rate of endothelial cell migration compared to fibroblast exosomes as a control, at 12h and 24h (P<0.05), which was dose-dependent. An ALI time-course study was conducted to determine temporal changes in local inflammatory response. Total cell count and IL-1beta in BALF peaked at 24h post-LPS, whereas IL-6 was highest 10h post-LPS. In a separate study, ALI mice (N=4-9 per group) were randomized to treatment with saline, MSCs, MSC conditioned media, or MEX given 30 mins post-LPS. Bronchoalveolar lavage fluid (BALF) and plasma were collected 72h later for analysis of inflammatory cytokines and total cell count. In ALI mice, MSC conditioned media significantly attenuated the increase in IL-1beta levels in BALF, whereas MSCs and MSC-exosomes resulted in a trend towards decreased levels of IL-1b and total cell count. This preliminary study suggests that MSC-derived exosomes can improve endothelial cell migration in vitro and potentially reduce the inflammatory response to ALI in vivo. Power calculations based on this data will determine the sample size necessary

383POSTER ABSTRACTSfor a definitive preclinical study. Based on findings from the time-course study, delivery of MSC-exosomes at a time of elevated inflammation (10h post-LPS) may augment lung tissue accumulation of MEX in future experiments.T-3086SAFETY AND EFFICACY OF HUMAN EMBRYONIC STEM CELLS DERIVED ASTROCYTES FOLLOWING INTRATHECAL TRANSPLANTATION IN SOD1G93A AND NSG ANIMAL MODELSIzrael, Michal - Neurodegenerative Diseases Dept, Kadimastem Ltd, Rehovot, Israel Chebath, Judith - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Hasson, Arik - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Itskovitz-Eldor, Joseph - General, Kadimastem Ltd, Nes-Zionna, Israel Krush Paker, Lena - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Kuperstein, Graciela - QA, Kadimastem Ltd, Nes-Ziona, Israel Lavon, Neta - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Slutsky, Shalom Guy - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Volman, Ella - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Yehezkel Ionescu, Shiran - Neurodegenerative Diseases, Kadimastem Ltd, Nes-Zionna, Israel Revel, Michel - CSO, Kadimastem Ltd, Nes-Zionna, IsraelALS is a Motor Neuron (MN) disease characterized by the loss of MNs in the central nervous systems. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocyte play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes. we developed a GMP-grade protocol for generation of astrocytes from human embryonic stem cells (hESC). The first stage of our protocol is derivation of astrocyte progenitor cells (APC) from hESCs. These APC can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APC yields an enriched population of astrocytes with more than 90% GFAP expression (AstroRx). In vitro, these cells possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that AstroRx cells secrete also several inhibitors of metalloproteases as well as variety of neuroprotective factors (e.g. TIMP-1&2, OPN, MIF and Midkine). In ALS animal models, hSOD1G93A high-copy number transgenic mice and rats, intrathecal injections of the AstroRx significantly delayed disease onset and improved motor performance compared to sham-injected animals. A nine-month safety study conducted in immunedeficient NSG animal model under GLP conditions showed that intrathecal transplantation of AstroRx to the cerebrospinal fluid is safe. Transplanted AstroRx attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight as the sham injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control was observed in hematological parameters or blood chemistry. These findings demonstrate the feasibility, safety and potential efficacy of intrathecal injections of AstroRx for the treatment of ALS and allowed the initiation of the first in human phase I/IIa clinical trial in ALS patients (clinicaltrials.gov identifier: NCT03482050).Funding Source: This work was supported by the Israel Innovation Authority National Natural (grant No. 59652).GERMLINE, EARLY EMBRYO AND TOTIPOTENCYT-3088ACCURATE ANNOTATION OF ACCESSIBLE CHROMATIN IN MOUSE AND HUMAN PRIMORDIAL GERM CELLSChen, Jiayu - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Li, Jingyi - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Shen, Shijun - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Wang, Mingzhu - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Liu, Wenqiang - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Li, Xiaocui - Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Shanghai, China Wang, Beiying - Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Shanghai, China Jiang, Cizhong - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China Gao, Shaorong - Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, ChinaExtensive and accurate chromatin remodeling is essential during primordial germ cell (PGC) development for the perpetuation of genetic information across generations. Here, we report that distal cis-regulatory elements (CREs) marked by DNase I-hypersensitive sites (DHSs) show temporally restricted activities during mouse and human PGC development. Using

384POSTER ABSTRACTSDHS maps as proxy, we accurately locate the genome-wide binding sites of pluripotency transcription factors in mouse PGCs. Unexpectedly, we found that mouse female meiotic recombination hotspots can be captured by DHSs, and for the first time, we identified 12,211 recombination hotspots in mouse female PGCs. In contrast to that of meiotic female PGCs, the chromatin of mitotic-arrested male PGCs is permissive through nuclear transcription factor Y (NFY) binding in the distal regulatory regions. Furthermore, we examined the evolutionary pressure on PGC CREs, and comparative genomic analysis revealed that mouse and human PGC CREs are evolutionarily conserved and show strong conservation across the vertebrate tree outside the mammals. Therefore, our results reveal unique, temporally accessible chromatin configurations during mouse and human PGC development.Funding Source: National Key R&D Program of China (2016YFA0100400) and the National Natural Science Foundation of China (31721003 and 31871446).T-3090HYPERBARIC OXYGEN EXPOSURE INCREASED CDX2 AND DECREASED OCT4 EXPRESSION IN MOUSE BLASTOCYSTS VIA UPREGULATING NRF2-NOTCH1 AND DOWNREGULATING NF2-HIPPO SIGNALINGChen, Yi-Hui - Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan Wu, Yung-Fu - Department of Medical Research, National Defense Medical Center, Taipei, Taiwan Yang, Yung-Yu - Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Liang, Chang-Min - Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Kang, Bor-Hwang - Department of Otorhinolaryngology-Head and Neck Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan Huang, Kun-Lun - Division of Pulmonary and Critical Care Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, TaiwanIt has been reported that exposure of pregnant female rats or hamsters in the early-to-mid stages of gestation to 100% oxygen under 3−4 atm for over 90 min/day led to reduced rat fetal weight and increased placental weight as well as malformed hamster fetuses. To further elucidate the etiology and molecular mechanisms underlying the hyperbaric oxygen (HBO)-induced maldevelopment of the placenta and embryo, we conducted HBO exposure to female mice in the preimplantation stages of pregnancy. C57BL/6 female mice post coitum were exposed to 100% O2 under 3.0 atm for 90 min/day for totally four days, followed by removal of the uterus and collection of E4.0 blastocysts. In comparison with the blastocysts derived from normoxia-exposed female mice, immunofluorescence analyses in the blastocysts derived from HBO-exposed mothers revealed significantly increased CellROX® oxidative stress signals and nuclear Nrf2 staining whereas significantly decreased membranous Nf2 staining throughout the whole embryo, while significantly decreased Oct4 expression, increased nuclear NF- B and active caspase-3 signals as well as ectopic nuclear κstaining of Cdx2 and Yap and the Notch1 intracellular domain (N1ICD) were detected in the inner cell mass (ICM) of the HBO-exposed blastocysts. Interestingly, we found that microinjection of Nf2 overexpressor cDNA into the zygotes followed by oviduct transfer before HBO exposure significantly decreased the nuclear levels of Yap, NF- B and active caspase-3 without affecting κectopic Cdx2 and decreased Oct4 expression in the ICM of the HBO-exposed blastocysts. On the other hand, microinjection of Nrf2 siRNA into the zygotes significantly decreased ectopic Cdx2 and N1ICD expression without increasing Oct4 expression or decreasing Yap, NF- B and active caspase-3 signals in the κICM of the HBO-exposed blastocysts. Only co-microinjection of both Nf2 cDNA and Nrf2 siRNA into the zygotes was capable of preventing aberrant expression of Cdx2, Yap, N1ICD, NF- B κand Oct4 in the blastocysts following HBO exposure. Therefore, our study demonstrates for the first time that the Nrf2-Notch1 and Nf2-Hippo signaling pathways synergistically regulate normal expression of Cdx2 in the trophectoderm and Oct4 in the ICM at the blastocyst stage.Funding Source: This work was supported by grants MAB105-056 and MAB106-026 to YHC from the Medical Affairs Bureau–Ministry of National Defense, R.O.C.T-3092SELF-ORGANIZATION OF THE IN VITRO ATTACHED NON-HUMAN PRIMATE EMBRYOKishimoto, Keiko - Department of Marmoset Research, Central Institute for Experimental Animals, Kawasaki, Japan Hu, Huaiyu - Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY, USA Sasaki, Erika - Department of Marmoset Research, Central Institute for Experimental Animals, Kanagawa, JapanImplantation of the blastocyst is a developmental milestone in mammalian embryonic development. Before implantation of the embryos into the uterus, the mammalian embryo establishes three founding populations of cells, epiblast, hypoblast and trophoblast. The epiblast gives rise to the fetus, while hypoblast and trophoblast form vital extraembryonic tissues, such as the placenta. Furthermore, embryonic stem cells (ESCs), extraembryonic endoderm (XEN) and trophoblast stem cells (TSCs) have been successfully derived from rodent epiblast, hypoblast and trophoblast and these stem cells have been good models to understand embryonic development in mouse. However, human and non-human primate early development radically diverges from the rodent paradigm. Although, gene expression analysis revealed that primates use a variety of different factors to regulate early embryonic development, ESCs, XEN and TSCs that captured biologic features completely of epiblast, hypoblast and trophoblast have not been established in primate. Therefore molecular mechanisms of early primate development are poorly understood. Recently established

385POSTER ABSTRACTSin vitro implantation platforms in the human have expanded the knowledge of post-implantation development however, human embryos cannot extensively use because of ethical reasons. Common marmoset (Callithrix jacchus, marmoset) is an excellent model to study primate embryogenesis, because they allow access to naturally conceived embryos. In this study, we established in vitro post implantation culture in marmoset embryos. As the result, all embryos successfully attached to in vitro implantation platform. In these embryos, 4 out of 10 (40%) embryos successfully developed and their morphologies were similar to human Carnegie stage 5 embryos which is consist of inner cell mass, bilaminar embryo, trophoblast and hypoblast. These embryos expressed Oct4 in the epiblast and Gata6 in the hypoblast. This in vitro post implantation embryo culture system in marmoset would provide unprecedented insights into human development, with far-reaching implications for stem cell research, placental research and treatments for implantation failure.T-3094CHEMICAL REVERSION OF HUMAN PLURIPOTENT STEM CELLS TO A NAÏVE EPIGENOMIC STATE WITH HIGH FUNCTIONAL PLURIPOTENCY REDUCES THE DEVELOPMENTAL BARRIERS TO HUMAN-MURINE CHIMERISMZimmerlin, Ludovic - Division of Pediatric Oncology / Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD, USA Evans-Moses, Rebecca - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Park, Tea Soon - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Thomas, Justin - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Huo, Jeffrey - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA He, Alice - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Kanherkar, Riya - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Kulpa, LeighAnn - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA He, Yunlong - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA Considine, Michael - Division of Cancer Biology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA Cope, Leslie - Division of Cancer Biology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA Zambidis, Elias - Division of Pediatric Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USANaïve human pluripotent stem cells (N-hPSC) may allow development of human tissues in interspecific chimeras for human disease modeling. However, human chimerism following injection of N-hPSC into animal embryos has thus far proven inefficient, ephemeral, and restricted to early fetal stages. Efficient human chimerism faces barriers that minimally includes: discordant interspecific developmental kinetics, N-hPSC methods producing unstable epigenomes, and inefficient interspecific cell-cell adhesion. Herein, we evaluated these barrier variables utilizing N-hPSC derived via supplementing LIF-2i with the tankyrase inhibitor XAV939. N-hPSC upregulated protein expressions (CD77, NANOG, KLF2, DNMT3L), and signalling pathways (pSTAT3) of the human preimplantation epiblast. N-hPSC transcriptional signatures clustered with E6 human epiblast, and global CpG methylation decreased by ~50%. N-hPSC maintained normal karyotypes, and PD0325901 inhibition of pERK1/2 did not destabilize DNMT1 or CpG methylation at imprinted loci. Improved multilineage differentiation correlated with reduced lineage-primed gene expression, CpG demethylation of PRC2 targets, JARID2 downregulation, diminished H3K27me3 bivalency, and global H3K9 methylation loss. To test interspecific chimerism potency, we injected single N-hPSC cells expressing GFP/puromycin transgenes into murine blastocysts. Robust human cell contribution was confirmed in >10% of E9.5-E11.5 fetuses using fluorescent reporters (GFP), human-specific antigens (HNA, STEM121), PCR amplification of human mitochondrial/GAPDH sequences, and expansion of puromycin-resistant human cell lineages. Human chimerism was ~25% at E7.5, 1% at E9.5, and lost by E12.5. Chemical blastocyst complementation dramatically improved human cell contribution at E9.5, but disrupted normal morphogenesis. N-hPSC expressing murine-E-cadherin and injected into morulae robustly contributed human cells to CDX2+ trophectoderm of hatching blastocysts and E11.5 murine embryos and placenta, but not thereafter. These data demonstrate that XAV939-regulated N-hPSC with naïve epigenomes and high functional pluripotency efficiently contribute to early fetal murine organogenesis; unknown barriers may impair further human chimerism at later murine fetal stages.Funding Source: NIH/NEI R01HD082098 (ETZ) NIH/NICHD R01HD082098 (ETZ) RPB Stein Innovation Award (ETZ) Lisa Dean Mosely Foundation (ETZ)CHROMATIN AND EPIGENETICST-3096XIST MEDIATES BOTH X-CHROMOSOME DAMPENING AND X- CHROMOSOME INACTIVATION IN HUMAN DEVELOPMENTDror, Iris - Department of Biological Chemistry, University of California, Los Angeles, CA, USA Chitiashvili, Tsotne - Department of Biological Chemistry, University of California, Los Angeles, CA, USA Sahakyan, Anna - Department of Biological Chemistry, University of California, Los Angeles, CA, USA

386POSTER ABSTRACTSQuinodoz, Sofi - Division of Biology and Biological Engineering, California Institute of Technology, Los Angeles, CA, USA Guttman, Mitchell - Division of Biology and Biological Engineering, California Institute of Technology, Los Angeles, CA, USA Plath, Kathrin - Department of Biological Chemistry, University of California, Los Angeles, CA, USAFemale placental mammals silence one of the two X-chromosomes through X-chromosome inactivation (XCI). This essential process is mediated by the lncRNA XIST that spreads along the X to mediate gene silencing. During female human pre-implantation development, a unique mechanism for dosage compensation takes place, where instead of silencing of one of the two X-chromosomes as in XCI, gene expression is reduced from both X-chromosomes in a process called X-chromosome dampening (XCD). Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI. Moreover, XIST accumulates on both dampened X chromosomes at this stage, but unable to induce complete gene silencing. Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI. To understand the unique mechanism of dosage compensation that takes place during human pre-implantation development, we are addressing the fundamental questions of how XCD is achieved mechanistically and why XIST is not inducing silencing during XCD. To study these questions, we are taking advantage of naïve human embryonic stem cells (hESCs) and somatic cells, which capture the XCD and XCI state, respectively. To this end, we have explored XIST localization in female naïve hESCs and somatic cells and discovered that XIST spreads over the entire X, but displays striking differences in enrichment along the X between XCD and XCI, which are explained by differences in the 3D folding of the X. We also made the remarkable observation that XIST spreads to autosomal regions in naïve hESCs but not in somatic cells, consistent with XIST dispersal observed by imaging. Importantly, in naïve hESCs and blastocysts, X-linked and autosomal genes enriched for XIST are downregulated, demonstrating that XIST mediates XCD as well as autosomal repression. Additional data demonstrate that XIST fulfills its function in XCD and autosomal repression through SPEN, similar to its mechanism of action in XCI. We propose that XIST concentration is lower within the dampened X-chromosome compared to the inactive X, which prevents complete gene silencing in naïve hESCs. Taken together, our data show that XIST mediates both XCD and XCI, uncover an unprecedented function of XIST in autosomes, and define new principles that govern the regulation of gene expression by lncRNAs.T-3098CRISPR-CAS9 BASED SCREENINGS TO INTERROGATE THE MOLECULAR BASIS OF DEVELOPMENTAL COMPETENCE IN ESC LINEAGE SPECIFICATIONPulecio, Julian - Sloan Kettering Institute, MSKCC, New York, NY, USA Huangfu, Danwei - Sloan Kettering Institute, New York, NY, USAA central question in developmental biology is to understand how cell-lineage intermediates interpret and execute inductive cues from their environment during embryo differentiation. It is believed cell-intermediates acquire two types of competences at defined developmental windows, a signaling competence to decode inductive signals from their environment and an epigenetic competence to undertake the chromatin modifications required to promote gene expression. However, the molecular mechanisms that sustain epigenetic competence are poorly understood. Previous approaches to discover chromatin regulatory regions (CRR) are mostly based on descriptive techniques that led to inferred correlations but are not well suited to decipher causal relationships between chromatin modifications and gene expression. In contrast, the CRISPR system has made possible to unbiasedly discover and functionally interrogate CRR. In this study, we used the specification axis from human ESC to the pancreatic lineage as a proof of principle to study how CRR are established through development. We combined the analysis of chromatin conformation, histone modification and transcription factor (TF) binding dynamics with CRISPR genomic and epigenomic screenings to identify and characterize the CRR with the potential to regulate PDX1 expression. Using a PDX1 reporter line and a tiled gRNA library targeting 100kb around PDX1, we interrogated the presence of CRR using three CRISPR-based screenings. Cas9 and dCas9-KRAB screenings identified a set of CRR necessary to activate PDX1 expression along pancreatic differentiation, discovering some previously unannotated regions both upstream and downstream the PDX1 gene body. In parallel, we performed a dCas9-VP64-based screening, as well as functional interrogation assays to uncover the CRR that are sufficient to promote PDX1 expression at different developmental stages. The combined analysis of TF binding and chromatin features revealed the molecular determinants that characterize the potential of each CRR to promote PDX1 expression along differentiation. Collectively, we will establish an unbiased approach to dissect the dynamics of CRR along development and determine the basis to discover the molecular mechanisms that support epigenetic competence.T-3100H3 K-TO-M MUTATIONS REVEAL CRUCIAL ROLES FOR H3K36 AND H3K9 METHYLATION IN DIFFERENTIATION, BOTH IN VITRO AND IN VIVOBrumbaugh, Justin - Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, CO, USA Kim, Ik Soo - Department of Pathology and Center for Cancer Research, Broad Institute, Boston, MA, USA Ji, Fei - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Di Stefano, Bruno - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Huebner, Aaron - Molecular Biology, Massachusetts General

387POSTER ABSTRACTSHospital, Boston, MA, USA Choi, Jiho - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Charlton, Jocelyn - Molecular Genetics, Max Planck Institute, Berlin, Germany Coffey, Amy - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Schwarz, Benjamin - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Anselmo, Anthony - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Walsh, Ryan - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Schindler, Jeffery - Cancer Center, Massachusetts General Hospital, Boston, MA, USA Meissner, Alexander - Molecular Genetics, Max-Planck Institute, Berlin, Germany Sadreyev, Ruslan - Molecular Biology, Massachusetts General Hospital, Boston, MA, USA Bernstein, Bradley - Department of Pathology and Center for Cancer Research, Broad Institute, Boston, MA, USA Hock, Hanno - Cancer Center, Massachusetts General Hospital, Boston, MA, USA Hochedlinger, Konrad - Molecular Biology, Massachusetts General Hospital, Boston, MA, USADevelopment and differentiation are associated with profound changes to histone modifications. However, the direct biological consequences of individual modifications remain incompletely understood due to the paucity of selective tools. Here, we describe a transgenic strategy to globally but specifically suppress the methylation of target histone residues in pluripotent stem cells and mice. Our system takes advantage of lysine (K) to methionine (M) mutants of histone H3, which function as dominant negative inhibitors of methylation at their respective sites. When expressed in embryonic stem cells, these mutants induced widespread changes to the chromatin landscape and a corresponding block in differentiation. Upon induction of H3K9M or H3K36M in adult mice, we observed potent differentiation defects in a variety of stem/progenitor cells and regenerative tissues, including intestine, testes, and blood-related organs. Focusing on hematopoiesis, H3K36M expression led to severe anemia and rapid lethality due to a block in erythropoiesis. H3K36M mice also exhibited aberrant lymphoid, megakaryocyte, and hematopoietic stem cell defects. By contrast, mice expressing H3K9M survived long term and exhibited distinct hematopoietic phenotypes including an expansion of multipotent progenitors. H3K36M and H3K9M induction led to genome-wide reduction of H3K36 and H3K9 trimethylation patterns, respectively. We observed corresponding changes in chromatin accessibility and gene expression landscapes, providing a mechanistic explanation for the observed phenotypes. Strikingly, we find that discontinuation of mutant histone expression largely restores differentiation programs, suggesting that the effects of methylation loss are reversible. Collectively, our work provides direct evidence that individual chromatin modifications are required at multiple stages of differentiation and offers powerful tools to interrogate the physiological consequences of these modifications in vivo.T-3102REGULATION OF 3D GENOME REORGANIZATION DURING CELL FATE TRANSITIONShevade, Kaivalya - Center for Craniofacial Molecular Biology, USC, Los Angeles, CA, USA Bajpai, Ruchi - Center for Craniofacial Molecular Biology, USC, Los Angeles, CA, USA3D genome reorganizes when normal cells switch fate, respond to stimuli, undergo senescence etc. Yet, real time visualization of genome reorganization has remained a challenge. We used Neural Crest Cell (NCC) to Ectomesenchymal Cell (EMC) transition as a model to study the genome organization changes that happen during differentiation. When induced to differentiate rapidly proliferating NCCs transiently exit the cell cycle and homogenously differentiate to ectomesenchymal cells over a period of 96hr. After 96hr of differentiation the genome of NCCs gets organized into DAPI dense and less dense regions and H3K9Me3 foci begin to appear. Nuclei of NCCs undergo a 3-5 times volume expansion within the first 20hr of differentiation during which massive chromatin redistribution occurs, followed by formation of multi lobed nuclei which are rapidly resolved in the final stages of the transition to reform the spherical nucleus. This massive chromatin redistribution occurs without an increase in transposon accessible DNA sites. Insitu HiC analysis has revealed the progressive nature of reorganization. We have also identified CHD7, a chromatin remodeling protein to be necessary for this large scale genomic restructuring. Knockdown experiments with shRNA where we can get 75% knockdown of CHD7 show that the knockdown completely inhibits the nuclear expansion and the subsequent ectomesenchymal transition. NCCs derived from CHARGE patient cells with a haploinsufficieny for CHD7 show a similar defect in the nuclear expansion stage followed by inability to upregulate key mesenchymal genes as shown by RNAseq analysis of the patient cells compared to the mutation corrected cells. We for the first time, have identified a cellular transition during normal development where 3D genome reorganization occurs and have also discovered that a chromatin remodeler is necessary for this 3D genome reorganization.

388POSTER ABSTRACTSPLURIPOTENCYT-3104HIGH-DENSITY DYNAMIC SUSPENSION CULTURE SYSTEM OF HUMAN INDUCED PLURIPOTENT STEM CELLS IN MINIATURIZED MULTICOMPARTMENT DIALYSIS DEVICERizal, Gandhi - Department of Bioengineering, The University of Tokyo, Bunkyo-ku, Japan Horiguchi, Ikki - Department of Biotechnology, Osaka University, Osaka, Japan Sakai, Yasuyuki - Department of Chemical Systems Engineering, The University of Tokyo, JapanHuman induced pluripotent stem cells (hiPSCs) is a cell type which has decent self-renewal and exhibit capability to regenerate a broad type of organ. In order to realize their clinical and industrial application, a large number of cells were required. The three-dimensional dynamic suspension culture of the hiPSCs spheroid is a promising method for improving their expansion. However, some problem such as the requirement of expensive growth factor usage and difficulties to preserve the pluripotency has still remained difficult. In this study, we would like to improve and evaluate the advantage of high density (HD) hiPSCs culture using our proposed miniaturized dialysis device. To maintain the small molecule nutrient and waste product exchange, the HD hiPSCs expansion was performed in miniaturized multicompartment culture system separated by 12 kDa MWCO dialysis membrane. The gellan gum was also administrated in culture medium to reduce the shear-induced effect caused by rotational culture. The results showed that the increase of hiPSCs expansion efficiency was affected by the accumulation of their native secreted macromolecular substance in well-maintained culture environment. Several important growth factors such as bFGF, TGF -1, and Nodal βwere successfully recycled and accumulated in culture compartment during the expansion which achieved in minimum growth factor supplementation. As a result, the hiPSCs can be expanded at a very high density up to 32 millons cells/ml with 8 times efficiency in well-preserved pluripotency indicated by the higher gene expression level of OCT4, SOX2, and Nanog in comparison with conventional suspension culture system. Resulted hiPSCs also showing better differentiation potential into organoid-like spheroid consisted cell type from three germ layer confirmed by higher expression of trilineage differentiation marker. This novel miniaturized medium refinement system represent the potential utilization of the native cellular interaction in high-density suspension culture to enhance the cost-effective production of hiPSCs for various application.T-3106IDENTIFICATION AND CHARACTERIZATION OF SURFACE ANTIGENS RECOGNIZED BY NAÏVE HPSC-SPECIFIC MONOCLONAL ANTIBODIESChoi, Hong Seo - Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea Lee, Hyun Min - Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea Kim, Min Kyu - Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea Ryu, Chun Jeih - Integrative Bioscience and Biotechnology, Sejong University, Seoul, KoreaPluripotent stem cells exist in two distinct states in mammals: (1) naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and (2) primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. The different pluripotent states in hPSCs can be interconvertible by growth conditions with 2iL (PD0325901, CHIR99021 and LIF) and various chemicals for inhibitors of signaling pathways. However, the conversion conditions for naïve hPSCs are still controversial. Therefore, the generation of human naive pluripotency in vitro requires specific culture conditions and signalling pathways that differ from naive mESCs. To identify and characterize the molecules that are critical for the maintenance of naïve pluripotency of hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve H9 hPSCs cultured in (1) 2iL-F/A (2iL, Forskolin and Ascorbic acid), (2) 2iL-X/F/P (2iL, XAV939, Purmorphamine) or (3) LCDM (hLIF, CHIR99021, (S)-(+)-Dimethundene maleate, Minocycline hydrochloride, IWR-1-endo, Y-27632) medium by decoy immunization. We finally selected 56 surface or 107 intracelluar MAbs which bind to the naïve H9 cells, but only weakly or not at all to primed H9 and MEFs by flow cytometry analysis. To identify the cell surface antigens of the MAbs, biotinylated naïve H9 or various cancer cells were subjected to immunoprecipitation. Of these, MAb N1-A4, N15-F8, N16-F2(c1/c2), N25-G12, N32-B6, N38-B2 and N49-A7 immunoprecipitated approximately 60, 30, 45, 63, 50, 80 or 80KDa, respectively. LC-MS/MS identified that N1-A4 recognized heat shock protein 60 (HSP60). HSP60 is identified in the natural environment of undifferentiated hPSCs, namely, the blastocoel fluid, which is in contact with all the cells in the blastocyst. HSPs containing HSP60 are also essential for successful preimplantation development. N16-F2(c1/c2) recognized membrane associated carbonic anhydrase 14 (CA14) which is a zinc-binding metalloproteinase that catalyzes reversible hydration of carbon dioxide. The molecular function of HSP60 or CA14 in naïve hPSCs and the identification and characterization of the target antigens of other naïve hPSC-specific MAbs is under investigation.

389POSTER ABSTRACTST-3108PRECISE LEVELS OF SNF5 ARE REQUIRED FOR HUMAN PLURIPOTENT STEM CELLS DEVELOPMENTAL FATE REGULATIONCarmel Gross, Ilana - The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Giv’at Shmuel, Israel Urbach, Achia - The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, IsraelSNF5 is one of the core subunits of the SWI/SNF chromatin-remodeling complex. Therefore, loss of function (LOF) or gain of function (GOF) of this protein might have significant effects on the epigenetic state of the cells and on their phenotype. Indeed, SNF5 LOF is the solely genetic lesion in Rhabdoid tumor of the kidney and in AT/RT tumor in the brain. SNF5 is also required for early embryonic development as it was shown that SNF5-/- mouse blastocysts lose their hatching capacity and that SNF5 regulates the expression of Nanog during the differentiation of mouse embryonic stem cells. Here we aimed to study the effect of SNF5 LOF and SNF5 GOF on human pluripotent stem cells (hPSCs). For this purpose, we introduced a conditional SNF5 over-expressing cassette into the AAVS1 locus and targeted the endogenous SNF5 gene. Using this system, we show that both SNF5 GOF and SNF5 LOF lead to rapid changes in cell fate, however in a different manner. SNF5 GOF leads to down-regulation of pluripotency markers such as Oct4 and to spontaneous differentiation of the cells mainly into neuronal lineage. By contrast, SNF5 LOF significantly affects the morphology of the cell colonies, which start to grow as 3D structures, but does not appear to directly affect the expression of pluripotency markers. These morphological changes suggest that SNF5 LOF affects the interactions between the cells and the extra cellular matrix. Indeed, global gene expression analysis reveals significant down-regulation in pathways related to the extra cellular matrix upon SNF5 LOF. To further study the effect of SNF5 LOF on the pluripotency of the cells, we evaluated their in-vitro differentiation capacity toward embryonic bodies (EBs). Notably, the LOF cells show compromised capability to differentiate into EBs compared to normal cells. Taken together, our results reveal the crucial role of SNF5 in human pluripotent stem cell biology and show that SNF5 has to be strictly regulated to maintain the self-renewal and differentiation capacity of the cells.T-3110TRANSPOSABLE ELEMENTS ARE REGULATED BY CONTEXT- SPECIFIC PATTERNS OF CHROMATIN MARKS IN MOUSE EMBRYONIC STEM CELLSHe, Jiangping - Guangzhou Institutes of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, ChinaThe majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that sig- nificantly deregulated at least one type of TE. The loss of Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.T-3112ALIGNING HUMAN NAIVE AND PRIMED STATES OF PLURIPOTENCY WITH HUMAN PRE- AND POST-IMPLANTATION USING SINGLE-CELL TRANSCRIPTIONAL ANALYSISSchell, John P - CLINTEC, Karolinska Institutet, Huddinge, Sweden Panula, Sarita - CLINTEC, Karolinska Institutet, Stockholm, Sweden Wong, Frances - Department of Physiology, University of Toronto, ON, Canada Ortega, Nicolás - CLINTEC, Karolinska Institutet, Stockholm, Sweden Kumar, Pankaj - CLINTEC, Karolinska Institutet, Stockholm, Sweden Reyes, Alvaro - CLINTEC, Karolinska Institutet, Stockholm, Sweden Cox, Brian - Department of Physiology, University of Toronto, ON, Canada Sandberg, Rickard - Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Petropoulos, Sophie - Department of Medicine, University of Montreal, QE, Canada Lanner, Fredrik - CLINTEC, Karolinska Institutet, Stockholm, SwedenPluripotent stem cells are categorized into two distinct culture condition states that represent progressing developmental physiologies: naive and primed. While pluripotency itself describes an intrinsic potential for self-renewal and a definitive capacity for differentiation into all germ lineages, the molecular signaling required for maintenance of this property significantly changes during endogenous developmental progression. As pluripotency arises in the developing epiblast cells during pre-implantation blastocyst expansion, it is then maintained exclusively in the post-implantation epiblast lineage. Based on functional in-vivo mouse studies, naïve stem cells represent pre-implantation pluripotency, while primed stem cells represent a

390POSTER ABSTRACTSpost-implantation stage. These states are characterized by their differences in cell morphology, metabolism, epigenetic signature, transposable element signature, surface protein composition and transcriptional profile. Pluripotent states have been classified based on their resemblance to endogenous cells, although because mouse has been the canonical model for mammalian pre-implantation and post-implantation studies, conserved terminology has yielded classifications in human self-renewing states. Advancements in single-cell transcriptomics have allowed us to compare and contrast transcriptional circuitry between in-vitro pluripotent states and cells picked from human preimplantation stages (day 3-7) and in-post-implantation model (day 10). Our analysis resolves that human naïve hPSCs transcriptionally are more representative of an early peri/post-implantation epiblast cell, than a pre-implantation epiblast cell. E10 epiblast maintains expression of naïve specific transcripts, while also co-expressing several primed specific genes.Funding Source: Vetenskapsrådet SSF- Swedish Foundation of Strategic Research KID Funding, Karolinska InstitutetT-3114GENETIC VARIATION INFLUENCES PLURIPOTENT GROUND STATE STABILITY IN MOUSE EMBRYONIC STEM CELLS THROUGH A HIERARCHY OF MOLECULAR PHENOTYPESReinholdt, Laura - The Jackson Laboratory, Bar Harbor, ME, USA Skelly, Dan - The Jackson Laboratory, Bar Harbor, ME, USA Czechanski, Anne - The Jackson Laboratory, Bar Harbor, ME, USA Byers, Candice - The Jackson Laboratory, Bar Harbor, ME, USA Spruce, Catrina - The Jackson Laboratory, Bar Harbor, ME, USA Aydin, Selcan - The Jackson Laboratory, Bar Harbor, ME, USA Stanton, Alexander - The Jackson Laboratory, Bar Harbor, ME, USA Choi, Ted - Predictive Biology, Carlsbad, CA, USA Churchill, Gary - The Jackson Laboratory, Bar Harbor, ME, USA Munger, Steven - The Jackson Laboratory, Bar Harbor, ME, USA Baker, Christopher - The Jackson Laboratory, Bar Harbor, ME, USAMouse embryonic stem cells (mESCs) occupy a ground state where pluripotency-associated transcriptional and epigenetic circuitry are highly active. However, we observed variability in expression of core pluripotency genes in genetically diverse mESCs grown in ground state conditions. To dissect the genetic basis of this variability, we profiled gene expression and chromatin accessibility in 185 mESC lines derived from genetically heterogeneous Diversity Outbred mice, and grown in the absence of ERK1/2 inhibition. We mapped thousands of loci that affect chromatin accessibility (caQTL) and/or transcript abundance (eQTL). We found eleven instances where distant QTL co-localize in clusters or hotspots, suggesting a common regulator. These hotspots include one on Chr. 10 that influences the expression of the rare 2C-like state and another on Chr. 15 that influences the expression of 254 genes including many known pluripotency-related genes. We applied causal mediation analysis and identified Lifr (leukemia inhibitory factor receptor) transcript abundance as the causal intermediate. Moreover, a joint mediation analysis of gene expression with chromatin accessibility yielded a single peak upstream of Lifr containing one SNP. Orthogonal functional assays and CRISPR allele swap experiments verified that this SNP strongly influences Lifr expression and pluripotency. Thus, we detected a causal chain of molecular events: a single SNP modulates regulatory element accessibility, which affects Lifr expression, leading to large-scale transcriptional shifts including known and novel pluripotency-associated genes. These results reveal that genetic variation controls mESC ground state through interacting gene expression networks, and these differences in ground state stability are likely to influence differentiation propensity. It is now widely recognized that genetic variation influences differentiation propensity of human pluripotent stem cell lines. Our study highlights the power of mouse genetic reference populations for dissecting these molecular regulatory networks using relatively small panels of cells.T-3116VARIABILITY AND MULTIPOTENCY EVALUATION OF MESENCHYMAL STEM CELLS DERIVED FROM EQUINE MUSCLES BEFORE AND AFTER DRUG LOADING: THE EXAMPLE OF CURCUMINColin, Margaux - Department of Pharmacotherapy and Pharmaceutics, Université Libre de Bruxelles, Belgium Dechene, Lola - CORD, Universite de Liege, Belgium Calvo Esposito, Rafaele - Department of Pharmacotherapy and Pharmaceutics, Universite Libre de Bruxelles, Belgium Ceusters, Justine - CORD, Universite de Liege, Belgium Lagneaux, Laurence - Laboratory of Clinical Cell Therapy, Universite Libre de Bruxelles, Belgium Van Antwerpen, Pierre - Pharmacognosy, Bioanalysis and Drug Discovery Unit and Analytical Platform, Universite Libre de Bruxelles, Belgium Goormaghtigh, Erik - Center for Structural Biology and Bioinformatics, Universite Libre de Bruxelles, Belgium Renard, Patricia - URBC, UNamur, Namur, Belgium Serteyn, Didier - CORD, Universite de Liege, Belgium Mathieu, Veronique - Department of Pharmacotherapy and Pharmaceutics, Universite Libre de Bruxelles, Brussels, BelgiumA new therapeutic perspective of stem cells (MSCs) concerns their potential use for drug delivery purposes. However, when considering MSCs as delivery agents, it is important to consider that incorporation of the therapeutic agent may possibly affect their biology and particularly their mesenchymal stem cell properties. A minimally-invasive process to obtain MSCs from muscles of different species has been previously

391POSTER ABSTRACTSdeveloped (WO2015091210). These muscle’s derived MSCs are easy to sample but their characterization remains more limited than bone-marrow derived MSCs. In our study, we aim to evaluate i) variability of MSCs sampled from different horses and overtime in culture and ii) whether drug loading affects their mesenchymal stem cell properties. For this purpose, we started first with a well-tolerated and widely used medicinal polyphenol, i.e. curcumin whose pharmacokinetic properties are not favourable for use as a drug. The preliminary analyses of the biochemical signature of MSCs in culture by means of Fourier transformed infrared spectroscopic microscopy (FTIR) suggest high degree of similarity among MSCs from 5 different donors, a feature that seems to remain till passage 8. Those cells efficiently uptake a hydrosoluble curcumin salt complexed with cyclodextrin (NDS27; WO2009144220A1). Importantly, we have found that this drug loading does not alter their viability, cellular proliferation or their multipotency characteristics, at least in terms of expression of CD29, CD44, CD73, CD90, CD105 and OCT4 markers as evaluated by means of qRT-PCR. Finally, the immunomodulation potential of the MSCs on T lymphocytes is also not modified according to their proliferation in co-cultures. Those preliminary results encourage further investigations of muscle-derived MSCs as therapeutic delivery agents for various purposes including inflammatory diseases.Funding Source: Région Wallonne, WalInnov 1610151PLURIPOTENT STEM CELL DIFFERENTIATIONT-3120WHEN DOES EMBRYONIC STEM CELLS BEGIN TO SHOW THYROID HORMONE MARKERS DURING DEVELOPMENT?Calza, Laura - CIRI-SDV, University of Bologna, Ozzano Emilia, Italy Baldassarro, Vito Antonio - CIRI-SDV, University of Bologna, Ozzano Emilia, Italy Fernandez, Mercedes - DIMEVET, University of Bologna, Ozzano Emilia, Italy Giardino, Luciana - CIRI-SDV, University of Bologna, Ozzano Emilia, Italy Pannella, Micaela - CIRI-SDV, University of Bologna, Ozzano Emilia, ItalyThe key role of thyroid hormone (TH) during development is well recognized for its effect on cell lineage determination, tissue and organ maturation, morphogenesis and adult phenotype establishment. On the contrary, very little is know on the embryo sensitivity to thyroid hormone during early differentiation, at blastocyte stage and until implantation. In this study, we investigated the expression level of molecular machinery involved in the TH cellular signalling and determining the intracellular content of active TH (triiodothyronine, T3) in the rat embryonic stem cells at different very early stages, cultured as single cells, clusters and embryonic bodies (EBs), which are formed in 7 days. The different differentiation stages were characterized by the expression of pluripotency and early differentiation markers Oct4, Stella, AFP. While AFP and Stella increases in EBs compared to single cells, Oct4 expression level and immunoreactivity decreases. We then analysed the expression level of the membrane transporters MTC8 and MTC 10, the TH activating enzymes deiodinase 1 and 2, the TH inactivating enzymes deiodinase 3, the nuclear receptors TR αand TR by RT-PCR and immunocytochemistry. In the EBs, βwe observed a strong up-regulation (up-to 9 times) of the TH activating enzyme D1 and D2 compared to single cells, such as the up regulation of the membrane transporters (up-to 4 times) and all the forms of thyroid hormone nuclear receptors (up-to 4 times). This preliminary results indicate that in rat embryonic stem cells the TH molecular machinery appears very early during development, in experimental conditions mimicking the pre-implant phase of blastocytes. The functional significance of this data remain to be establish, but, notably, in conventional culture protocols embryonic stem cells are grown in the presence of serum, that contains thyroid hormones.T-3122DEVELOPING A VISUAL MODEL AND GENOMIC MAP FOR CARDIOMYOCYTE DIFFERENTIATIONGunawardane, Ruwanthi - Stem Cells and Gene Editing, Allen Institute, Seattle, WA, USAThe Allen Institute for Cell Science is creating a visual model of cell organization and behavior from pluripotency through cardiomyocyte differentiation using human induced pluripotent stem cells (hiPSCs). We have used gene edited iPSC-derived cardiomyocytes and live imaging to study the organization of the major cellular structures in cardiomyocytes and are coupling live imaging with single cell transcriptomics to identify and follow the cell states and transitions that accompany the differentiation of hiPSCs into cardiomyocytes. Using the WTC hiPSC line and CRISPR/Cas9, we have fluorescently tagged ~40 loci representing key cellular organelles, sub-structures, and signaling molecules. We have also developed a scarless gene editing strategy to endogenously tag transcriptionally silent loci and used this methodology to tag five sarcomeric proteins (troponin I1, alpha actinin 2, myosin light chain 2a, myosin light chain 2v, and titin). Following genetic, cell biological, and stem cell QC, these clonal hiPS cell lines are differentiated and replated onto glass using optimized protocols for live cell imaging, allowing us to study the organization of the endogenously tagged structure within the cardiomyocytes. We are additionally using single cell RNA-seq and RNA FISH to profile the transcriptional heterogeneity at various stages of differentiation. Using SPLiT-seq, we have generated transcriptomic data for 4 timepoints from pluripotency through day 90 of cardiomyocyte differentiation and identified sub-populations of cells based on gene expression profiles. We observed changes in expression of genes encoding for structural proteins, transcription factors, metabolic switches and calcium handling and confirmed these findings with RNA FISH in cardiomyocyte populations. We are also conjoining high-resolution live cell imaging and parallel transcriptomic profiling

392POSTER ABSTRACTSusing RNA FISH. Here, we present our optimized methods for silent gene editing and cardiomyocyte differentiation, live cell images of various organelles and sarcomeric structure in the edited cardiomyocytes, and transcriptomic analysis and validation using RNA FISH of various cardiomyocyte populations to explore cell states and transitions.T-3124NFIA AND NFIB ARE JOINTLY REQUIRED FOR MOUSE NEURAL STEM CELL SELF-RENEWAL AND DIFFERENTIATIONWebber, Karstin - Genetics, Genomics and Bioinformatics, University of Buffalo, NY, USA Campbell, Christine - Biochemistry, University of Buffalo, NY, USA Osinski, Jason - Biochemistry, University of Buffalo, NY, USA Gronostajski, Richard - Biochemistry, University of Buffalo, NY, USAUnderstanding postnatal neural stem/progenitor cells (pNSPC) self-renewal and lineage specification is key to future neural stem cell therapies. Here we assess the effects of loss of single or multiple Nfi genes on murine pNSPC self-renewal and differentiation in vitro. We showed previously that germline loss of either Nfia or Nfib reduces astrogenesis in cortex and spinal cord and results in prenatal dysgenesis of the corpus callosum. Conversely, germline loss of Nfix has minor effects on astrogenesis but promotes oligodendrogenesis. To assess their role in pNSPC self-renewal and differentiation in vitro we generated floxed alleles of Nfia, Nfib and Nfix. Mice homozygous for these alleles and carrying R26CreERT2 are viable and pNSPCs were cultured from the subventricular zone (SVZ). pNSPCs were cultured in the presence of EGF and FGF (proliferation (prolif.) conditions) then placed into medium lacking these growth factors (differentiation (diff.) conditions). Transcript levels of markers of self-renewal and differentiation were assessed by qPCR from RNA of cells cultured without (WT) or with 4OHT (NFI-deleted) during both prolif. and diff. conditions. Treatment with 4OHT during prolif. efficiently deleted all floxed alleles with >99% loss of transcripts within 3 days. Single deletion of Nfia or Nfib resulted in no obvious changes in self-renewal or neuronal differentiation but reduced the expression of astrocyte markers upon differentiation, consistent with our previous studies on loss of Nfia or Nfib in vivo. Surprisingly, simultaneous deletion of Nfia and Nfib resulted in a major reduction in self-renewal as seen by reduced PCNA and Nestin expression and the loss of colony forming ability. In addition, increases in the neuroblast marker DCX and oligodendrocyte marker MBP were seen under prolif. conditions. Upon differentiation, there were increases in DCX but relatively reduced expression of astrocyte and oligodendrocyte markers. This loss of self-renewal appears specific for the combined loss of Nfia and Nfib as combined loss of Nfia and Nfix or Nfib and Nfix does not. We are currently assessing the molecular mechanisms that influence these changes in self-renewal and lineage-specification by RNA-seq analysis of prolif. WT and NFI-deleted pNSPCs and quantification of the cell types formed upon differentiation.Funding Source: NYSTEMT-3126ELASTIN-SECRETING VASCULAR SMOOTH MUSCLE CELLS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLSTsang, Kit Man - NHLBI, National Institutes of Health (NIH), Bethesda, MD, USA Kozel, Beth – NHLBI, National Institutes of Health (NIH), Bethesda, MD, USA Liu, Delong – NHLBI, National Institutes of Health (NIH), Bethesda, MD, USAElastin (ELN) plays an essential role in providing recoil capacity to elastic tissues, including the skin and blood vessels. Abnormalities in elastin expression and deposition cause vasculopathy throughout the body that contributes to significant morbidity and mortality in infancy and childhood. Even though prescribed blood pressure medications could reduce vascular stiffness in patients with elastin deficiency, such treatment had no effect on vessel remodeling in elastin deficient mice, instead led to functionally decreased vessel diameter and reduced end organ blood flow. Therefore, it would be of benefit to identify drugs that could increase elastin deposition and improve the biomechanical properties of vessels. We have developed induced pluripotent stem cell (iPS) lines from elastin-deficiency patients and control fibroblasts. Currently, we are working to generate iPS-derived vascular smooth muscle cells (iVSMC) from these lines using chemically defined and serum free media. The differentiated cells remain relatively immature and express SMA, SM22a, SMEMb, and calponin, but do not express additional maturity markers like MYH11. As in human and mouse tissues, elastin is expressed by our cell lines for only a brief period, with initial elastin mRNA expression on day 2 and peaking on day 3 of differentiation and disappears by day 5. Single cell RNA-seq study further confirms the transition of TBXT+ mesoderm cells to SM22a+ iVSMC from day 2 to day 3 of differentiation. More than 90% of elastin-expressing cells co-express SM22a at day 3 (ELN+/SM22a+) revealing they are of smooth muscle origin. Gene enrichment analysis demonstrates an increased level of genes involved in signal-recognition particle (SRP)-dependent protein-membrane targeting as well as extracellular matrix generation in ELN+/SM22a+ cells. Elastin protein is detectable in cells by day 5 and in the matrix in robust quantities by day 9. Additionally the protein appears to remain within the cell for a prolonged time, not entering the matrix until other elastic fiber assembly genes such as FBLN4, FBLN5 and LOX are up-regulated. Our study first demonstrated that iPS-derived vascular smooth muscle cells expressed ELN in early phase of differentiation, while ELN deposition machinery was initiated by the presence of other elastic fiber assembly genes.

393POSTER ABSTRACTST-3128IDENTIFYING SMALL MOLECULE MODULATORS OF OTIC PROGENITOR CELL PROLIFERATION AND DIFFERENTIATION FOR THE TREATMENT OF HEARING LOSSMeitz, Lance E - Department of Chemistry, The Scripps Research Institute, Poway, CA, USA Lairson, Luke - Department of Chemistry, The Scripps Research Institute, San Diego, CA, USA Spagenberg, Stephan - Department of Chemistry, The Scripps Research Institute, San Diego, CA, USAHearing loss is statistically suggested to affect 5% of the global population. This is most often caused by damage to the hair cells of the cochlea. While these cells, re-enter the cell cycle to replace lost hair cells in birds and other animals, in adult mammals, this process is inactive. But gene therapeutic approaches have suggested that this process could be possible in adult mammals. Previous studies established and characterized a LGR5+ progenitor cell population in the cochlea, that have the capacity to undergo proliferation and asymmetric division in a manner that leads to the generation of new support cells and hair cells. This preliminary research has generated a technique to culture otic organoids and primary otospheres that are positive for LGR5, ATOH1, and MYOVII markers. To date, high throughput screening (HTS), for hearing loss therapies are limited, in part, by the difficulties of isolating and purifying these cell types of interest. We have observed that specific otic progenitor markers can be expressed from hiPSC cultures in a dish. We aim to establish a high-throughput phenotypic-based screening in small molecules, that modulate LGR5+ progenitor cells for hair cell regeneration. First, we used design-of-experiment based optimization to establish a system for generating hiPSC-derived LGR5+ progenitor cells into hair cells in vitro. We then designed an assay to identify compounds which selectively proliferate and differentiate LGR5+ progenitor cell populations. The objective of this research is to identify small molecule drug candidates that will mitigate damaged-induced hearing loss in humans.Funding Source: California Institute for Regenerative Medicine (CIRM), The Scripps Research InstituteT-3130EARLY PREDICTION OF THE DIFFERENTIATION TENDENCY CONTRIBUTES TO GENERATION OF FUNCTIONAL MELANOCYTES DERIVED FROM INDUCED PLURIPOTENT STEM CELLSLiu, Liping - Institute of Regenerative Medicine, Jiangsu University, Zhenjiang, China Guo, Ningning - Institute of Regenerative Medicine, Jiangsu University, Zhenjiang, China Zhang, Yixuan - Institute of Regenerative Medicine, Jiangsu University, Zhenjiang, China Li, Yumei - Institute of Regenerative Medicine, Jiangsu University, Zhenjiang, China Zheng, Yunwen - Faculty of Medicine, University of Tsukuba, Tsukuba, JapanInduced pluripotent stem cells (iPSCs) provide a promising source for cellular therapy; however, distinct iPSC lines present different differentiation capacities even in the same condition, which is always a relatively complicated and time-consuming process. To predict the differentiation tendency of iPSCs in the early stage, we compared four iPSC lines in the capacity of embryonic bodies (EBs) formation and maintenance, the expression of specific germ layer markers as well as in the melanocyte differentiation capability. Furthermore, to confirm whether the prediction was correct or not, in vivo experiments were performed and induced melanocytes (iMels) were transplanted into immunodeficient mice using a modified hair follicle reconstitution assay. As a result, we found that iPSCs which could form EBs with regular and smooth morphology and express higher levels of ectoderm marker genes (SALL3, EDNRB and SOX1) and lower levels of mesoderm and endoderm marker genes (MEIS2, GATA4, MEF2C, FOXA2 and HOXA1), had a better differentiation capability into melanocytes. After transplantation, these human-origin iMels were detected in the mouse hair bulb and epidermis up to 7 weeks, showing DOPA positive staining and expressing melanocytic markers, MITF, PAX3 and TYRP1. They also produced melanin which were localized in the hair bulb, epidermis and hair shaft. In addition, PAX3+MITF+TYRP1- melanocyte stem/progenitor cells were co-localized with hair follicle stem cells marked with KRT15 and ITGA6 in the bulge region, not only in the anagen hair follicles but also in the catagen/telogen hair. In conclusion, our data demonstrate that the differentiation tendency of iPSCs might be predicted using parameters in EBs stage, specifically, the formation and maintenance of optimal EBs and the expression level of germ layer-specific markers. In addition, it is the first time to show the long-term survival and function maintenance of iPSC-derived melanocytes in vivo and to demonstrate their hair follicle and epidermis reconstitution capacity. The bulge region with existing hair follicle stem cells functions as a niche for melanocytic stem cells and should support and provide the long-term maintaining of functional iMels.Funding Source: National Natural Science Foundation of China ( 81573053 and 81770621); MEXT of Japan, Kakenhi (16K15604 and 18H02866 ) ; Natural Science Foundation of Jiangsu Province (BK20180281).T-3132MOLECULAR MECHANISMS UNDERLYING THE BLASTULA STAGE SPECIFICATION OF NEURAL CREST FROM A PLURIPOTENT STEM CELL STATEPrasad, Maneeshi S - Biomedical Sciences, University of California, Riverside, CA, USA Charney, Rebekah - Biomedical Sciences, University of California Riverside, CA, USA Garcia-Castro, Martin - Biomedical Sciences, University of California Riverside, CA, USA

394POSTER ABSTRACTSNeural crest (NC) is a multipotent stem cell population that gives rise to multiple derivatives in vertebrate embryos. The specification of NC has been a topic of great discourse over the past two decades. Studies in chick, rabbit, and Xenopus embryos have eluded to NC specification during early gastrulation. However, molecular mechanisms involved in the earliest specification of NC from a pluripotent stem cell state remain unknown. Here we present for the first time the earliest NC cell fate specification in amniotes using avian and human NC (hNC) models. In avian embryos, specification assays and fate map analysis identified a population of specified NC in the intermediate epiblast region of the blastula embryo, and this specification is independent of mesodermal and neural contributions. The specification of human NC (hNC) was assessed using a robust 5-day model of hNC development based on Wnt-signaling activation in human embryonic stem cells (hESC). Functional analysis of prospective NC (pNC) using mesendoderm differentiation assays suggest a restricted stem cell potential of pNC unlike hESC. Further molecular characterization of pNC at high temporal resolution identified stoichiometric changes in the expression of pluripotency genes, accompanied by a distinct expression profile of pNC and NC genes within 6 hours of NC induction from hESCs. Perturbation of pluripotency genes during the first 48 hours of NC induction drastically affects hNC formation, suggesting a role for these pluripotency factors during hNC specification. This study demonstrates for the first time the restriction in NC cell fate in avian and human NC that diverges from the epiblast/ESC fate at unprecedented early facets of development, and provides novel insight into the role of pluripotency genes during cell fate specification from a pluripotent stem cell state.Funding Source: NIH/NIDCR R01DE017914 to Martin I Garcia-CastroT-3134PARTIAL REPROGRAMMING: A HARD-TO-FIND CAUSE OF HUMAN INDUCED PLURIPOTENT STEM CELLS’ REDUCED PROPENSITY FOR ENDODERM DIFFERENTIATIONM’Callum, Marie-Agnes - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QC, Canada Raggi, Claudia - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QC, Canada Pham, Toan - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QC, Canada Legault, Lisa-Marie - Developmental Epigenetics, Sainte-Justine UHC, University of Montreal, QC, Canada Gaub, Perrine - Neuronal Energy Metabolism, Sainte-Justine UHC, University of Montreal, QC, Canada Joyal, Jean-Sebastien - Neuronal Energy Metabolism, Sainte-Justine UHC, University of Montreal, QC, Canada McGraw, Serge - Developmental Epigenetics, Sainte-Justine UHC, University of Montreal, QC, Canada Paganelli, Massimiliano - Hepatology and Cell Therapy, Sainte-Justine UHC, University of Montreal, QC, CanadaThe propensity for hepatic differentiation varies significantly among induced pluripotent stem cell (iPSC) clones. What determines the susceptibility of a population to differentiate into definitive endoderm (DE) and its derivatives remains to be determined. Here we investigate the causes of such a variability and assess the role of the state of pluripotency and reprogramming stage. We compared three TRA-1-60-positive, well characterized human iPSC populations with good (P01 and P02), and suboptimal (P03) propensity for hepatic differentiation in terms of pluripotency markers, self-renewal capability, methylation and gene expression profiles, metabolism. We demonstrate that P03 population proliferates more actively, shows lower expression of genes characterizing “primed” embryonic stem cells (ESC), and responds with delayed dynamics upon differentiation into DE. While P01 and P02 use anaerobic glycolysis, P03 preferentially uses oxidative phosphorylation. This has a direct effect on the overall state of DNA methylation, with P03 characterized by global DNA hypomethylation compared to other iPSCs. Such data, together with MDB2-dependent repression of Nanog and a characteristic microRNAs expression profile, suggest that P03 is partially reprogrammed. We identified a peptide hormone capable of promoting full reprogramming of iPSC clones by enhancing expression of key pluripotency markers (TET1, NANOG, LIN28, TRIM71, CDH1) and consequently switching the cells from a state more evocative of “naïve” ESCs to a “primed” state. A short treatment with such a growth factor can, at least partially, restore the propensity of P03 for DE differentiation. Overall, our findings suggest that iPSC populations that might be mistakenly considered well reprogrammed according to standard characterization parameters are indeed partial reprogrammed. Such a status is responsible for their reduced propensity to differentiate into endoderm derivatives, propensity that can be restored with a targeted intervention.T-3136SINGLE CELL ANALYSIS ELUCIDATES THE MECHANISM OF CARDIAC AND HEMATOPOIETIC DIFFERENTIATION FROM HUMAN PLURIPOTENT STEM CELLSUmeda, Masayuki - Center For iPS Cell Research And Application (CiRA), Kyoto University, Kyoto, Japan Okada, Chihiro - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Watanabe, Akira - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Takaori, Akifumi - Department of Hematology and Oncology, Kyoto University, Kyoto, Japan Yoshida, Yoshinori - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, JapanClinical application of human induced pluripotent stem cells (hiPSCs) such as disease modeling requires efficient differentiation of the desired populations. Signals that specify the fates of mesodermal cells to the Primitive and Definitive hematopoiesis, and the Ventricular and Atrium cardiomyocytes

395POSTER ABSTRACTShave been extensively investigated. However, transcriptional control within each cell and the mechanisms of cell fate decisions toward different lineages including cardiac and hematopoietic cells have not been elucidated sufficiently. Here we conducted a comprehensive study of mesodermal differentiation mechanism at a single cell level, using two methods of single-cell analysis, single cell qPCR and single cell RNA seq. We performed single cell qPCR analysis of 93 genes from 1,725 cells in total from each hematopoietic and cardiac differentiation from hiPSCs, which revealed heterogeneity of differentiating mesodermal cell at day2-4. Knockdown of transcription factors such as EOMES and FOXH1, which were expressed at the bifurcation of each lineage significantly increased the hematopoietic differentiation efficiency while they decreased the cardiac differentiation efficiency, affecting the downstream genes like GATA6 and CDX2. These results suggest that these transcriptional factors specify the fate of each cell promoting one fate while suppressing others. To examine a novel mechanism involved in the early stage specification of the mesoderm, we conducted single-cell RNA sequencing from 71 cells at day 0-3 of differentiation. Pseudo-time analysis that evaluates temporal changes of expression revealed that specific pathways are temporarily activated in addition to known WNT signaling and TGF signaling. To investigate the transcriptional regulation, βwe performed the single-cell regulatory network inference and clustering (SCENIC) analysis. SCENIC revealed that upstream genes such as SALL3 regulated the downstream genes involved in the epithelial-mesenchymal transition, suggesting that these genes were involved in the differentiation of mesoderm. Taken together, these findings provide new understandings of cardiac and hematopoietic differentiation, which applies to yield the desired population efficiently for clinical application.Funding Source: Grants from Research Center Network for Realization of Regenerative Medicine of Japan Agency for Medical Research and DevelopmentT-3138METHOD FOR 3D HEPATIC DIFFERENTIATION IN STIRRED BIOREACTORS OF HUMAN PLURIPOTENT STEM CELLS IN CHEMICALLY DEFINED, ANIMAL-ORIGIN-FREE CULTURE MEDIA, STEMFITChiba, Mayumi - Institute For Innovation, Ajinomoto Co., Inc., Kawasaki, Japan Ito, Kenichiro - Institute For Innovation, Ajinomoto.Co.,Inc., Kanagawa, Japan Konishi, Atsushi - Institute For Innovation, Ajinomoto.Co.,Inc, Kanagawa, Japan Ogawa, Shimpei - Institute For Innovation, Ajinomono. Co.,Inc., Kanagawa, Japan Wagatsuma, Hirotaka - Institute For Innovation, Ajinomoto.Co.,Inc, Kanagawa, Japan Chang, Jessica - Institute For Innovation, Ajinomoto.Co.,Inc, Kanagawa, JapanHuman pluripotent stem cells (hPSCs) are promising cell sources for regenerative therapy, drug discovery, because of their potential to expand long-term in vitro and ability to differentiate into various types of somatic cells. Currently, differentiation protocols of hPSCs are often based on 2D manner and a limited number of 3D differentiation methods is available. 3D cell culture platforms are reported to better mimic in vivo conditions and have advantages such as scalability and automatability. For applications such as cell therapy, a large number of cells are required, on scales that will require 3D differentiation systems for large scale manufacturing. In this study, we demonstrate an efficient 3D differentiation system of hPSCs using clinical-grade (Japan), defined media. First, hPSC spheroids were formed from single cell suspensions for uniform and controllable spheroid size using “StemFit Basic03”, an animal component-free and chemically defined hPSC maintenance medium. Next, hPSC spheroids in the spinner flask were directly differentiated in dynamic suspension into definitive endoderm (~90% CXCR4+), immature hepatocytes, and then mature hepatocytes using “StemFit AS400”, which is animal component-free and chemically defined nutrient supplement for hPSC differentiation. Obtained hepatic spheroids highly expressed markers such as albumin, AFP, and OTC. Our results indicate that the combination of hPSC spheroid formation with “StemFit Basic03” and differentiation with “StemFit AS400” nutrient supplement will enable high density, scalable differentiation of hPSCs under chemically defined, animal-origin-free conditions.T-3140SINGLE-CELL TRANSCRIPTOMICS REVEALS A CD31+CD105- SUBPOPULATION OF EARLY ENDOTHELIAL CELLS WITH ELEVATED HEMATOPOIETIC POTENTIAL FROM HUMAN PLURIPOTENT STEM CELLSWang, Mengge - State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Wang, Hongtao - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Xu, Changlu - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Wen, Yuqi - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Chen, Xiaoyuan - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College,

396POSTER ABSTRACTSState Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Liu, Xin - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Gao, Jie - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Su, Pei - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Shi, Lihong - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China Zhou, Jiaxi - Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, ChinaAccumulating evidence shows that endothelial cells are the origin of blood cells both in vitro and in vivo. However, the heterogeneous nature of endothelial cells and the mechanism regulating their derivation remain largely elusive. In this study, we found by using single-cell transcriptomics analysis that CD31+ endothelial cells are highly heterogeneous even at its emergence from human pluripotent stem cells (hPSCs). Among them, a CD31+CD105- subpopulation of cells are highly potent for hematopoietic differentiation. Interestingly, genetic deletion of CD105 promotes the hematopoietic differentiation of hPSCs. While inhibition of TGF signaling facilitates the generation of βCD31+CD105- subpopulation of cells, overexpression ETS1 suppresses its derivation. Our findings indicate that CD105 can be used as a reliable surface marker to enrich hematopoietic potent endothelial cells. The discovery of the underlying mechanism should also benefit the derivation of functional blood cells from hPSCs for translational medicine.FundingSource:NationalKeyResearchandDevelopmentProgramofChinaStemCellandTranslationalResearch(2016YFA0102300,2017YFA0103100,2017YFA0103102) CAMS Initiative for Innovative Medicine (2016-I2M-1-018, 2016-I2M-3-002)T-3142ANALYSIS OF EXTRACELLULAR RNAS DURING PANCREATIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLSFoster, Mikelle - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Touboul, Thomas - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Bennett, Shania - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA DeHoff, Peter - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Srinivasan, Srimeenakshmi - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA To, Cuong - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Morey, Robert - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USA Laurent, Louise - Reproductive Medicine, University of California, San Diego (UCSD), La Jolla, CA, USAEver since human embryonic stem cells (hESCs) were first successfully cultured, it was recognized that hESCs had the potential to be used for cell replacement therapies to treat traumatic injury, developmental disorders, and degenerative diseases. hESC-derived pancreatic ß cells are of particular interest for the treatment of Type 1 Diabetes Mellitus. Multiple methods involving sequential addition of growth factors and small molecules to direct in vitro pancreatic differentiation of hESCs have been developed, but existing protocols have failed to produce a pure population of mature and functional ß cells. Extracellular RNAs (exRNAs) have been shown to mediate cell-cell communication in several biological contexts, but their potential roles as autocrine or paracrine effectors in hESC differentiation and maturation are not well understood. In this study, we aim to use small RNA sequencing to generate expression profiles of exRNAs isolated from conditioned hESC culture media at each stage of in vitro pancreatic differentiation up to the pancreatic progenitor stage. To discern differences in exRNA cargo among carrier subclasses (CSs), including extracellular vesicles and ribonucleoprotein complexes, immunoaffinity separation is performed with magnetic beads conjugated to antibodies raised against CS markers, such as CD63, CD81, CD9 and AGO2. ExRNAs purified from the purified CSs are sequenced and the data are compared to those from the source cells and unfractionated total supernatant. Identification of exRNAs released at each stage of differentiation will provide information necessary for characterizing their roles in intercellular communication and regulation of differentiation in vitro. This will lay the foundation for further investigations into the roles of exRNAs in pancreatic differentiation.

397POSTER ABSTRACTST-3144MOUSE ZSCAN5B DEFICIENCY IMPAIRS DNA DAMAGE RESPONSE AND CAUSES CHROMOSOME ABERRATIONS DURING MITOSISYamada, Mitsutoshi - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Ooka, Reina - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Nakamura, Akihiro - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Sugawara, Toru - Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan Ogawa, Seiji - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Miyado, Kenji - Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan Akutsu, Hidenori - Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan Hamatani, Toshio - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Tanaka, Mamoru - Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan Umezawa, Akihiro - Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, JapanZygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of -H2AX despite elevated expression of the γDNA repair genes Rad51l3 and Bard. To determine whether DNA damage repair is dependent on Zscan5b, a piggyBac vector carrying wild-type Zscan5b-GFP driven by a CAG promoter for long-term stable expression was transfected into Zscan5b-deficient ESCs. We found a significant increase in DNA damage after irradiation in Zscan5b deficient irradiated ESCs. However, a lower frequency of H2AX foci was present in γtransfected Zscan5b deficient cells, suggesting that restoration of Zscan5b expression in these cells was sufficient to restore the genome stability. To test whether Zscan5b binds either directly or indirectly to chromosomes, and helps to form nucleosome structures, we performed a co-immunoprecipitation analysis using protein extracted from ESCs transfected with 3XFLAG-Zscan5b-EGFP. The components of bands from 3XFLAG-Zscan5b-EGFP combined proteins were analyzed and the histone H1 family, H1.1, H1.2 and H1.4, was specifically detected in bands. We cross- validated these results by transfection and by an immunoprecipitation assay using the cells transfected with a pcDNA3.1+C-eGFP plasmid carrying mouse Zscan5b cDNA and three pcDNA3.1-MYC-HIS A plasmids carrying mouse Histone 1,1, 1.2, and 1.4 cDNAs. The sizes of these bands correspondeds to the molecular sizes of histones H1 family, H1.1, H1.2 and H1.4 of in the antibody-added protein sample. These results supports that ZSCAN5B binds to the linker histone H1, and may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis.T-3146HUMAN PLURIPOTENT STEM CELL-DERIVED HEPATOCYTES SHOW STRONGER CORRELATION AND HIGHER SIMILARITY TO HUMAN ADULT LIVER THAN TO HUMAN FETAL LIVERGhosheh, Nidal – School of Bioscience, The University of Skovde, Sweden Sartipy, Peter - School of Bioscience, University of Skovde, Sweden Synnergren, Jane - School of Bioscience, University of Skövde, Sweden X. Andersson, Christian - R&D, Takara Bio Europe AB, Gothenburg, Sweden Asplund, Annika - R&D, Takara Bio Europe AB, Gothenburg, Sweden Kuppers-Munther, Barbara - R&D, Takara Bio Europe AB, Gothenburg, SwedenHuman pluripotent stem cell derived hepatocytes (hPSC-HEP) display many properties of mature hepatocytes, including expression of many important genes of the drug metabolizing machinery, glycogen storage, and production of multiple serum proteins. However, hPSC-HEP have not yet been shown to fully recapitulate the complete functionality of in vivo mature hepatocytes, and they typically express some fetal hepatocyte markers. In this study, we applied the COMBAT algorithm to two transcriptomics data sets generated applying different microarray platforms, to obtain one data set containing samples from different developmental stages during hPSC-HEP differentiation, human fetal liver tissues (FL) from different gestational weeks and human adult liver tissues (AL). We performed Similarity and correlation analysis on this data set. In addition, we performed functional analysis including cytochrome P450 activities, albumin secretion and urea production on hPSC-HEP from cells generated by our group. Our results showed that the transcriptional correlation of hPSC-HEP to adult liver tissues was higher than to fetal liver tissues (0.83 and 0.70, respectively). Moreover, hPSC-HEP showed higher similarity to AL than to FL (0.75 and 0.56 respectively). Functional analysis showed cytochrome P450 activities of hPSC-HEP and albumin secretion that are comparable to cryoplateable human primary hepatocytes. The results showed also the ability of hPSC-HEP to produce urea. To conclude, our results show for the first-time

398POSTER ABSTRACTShigher similarity of hPSC-HEP to AL both on the transcriptional and the functional levels. These results demonstrate substantial improvement from previous studies categorizing hPSC-HEP as immature and resembling fetal hepatocytes.T-3148TIME-COURSE AND DOSE-DEPENDENT TRANSCRIPTOME PROFILING REVEAL KEY REGULATORS FOR NEURAL CONVERSION OF HUMAN IPSCS UNDER CHEMICALLY DEFINED CONDITIONSChu, Pei-Hsuan - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Malley, Claire - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Braisted, John - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Tristan, Carlos - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Ormanoglu, Pinar - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Simeonov, Anton - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Singec, Ilyas - National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USAThe systematic study of cell differentiation provides deeper insights into developmental pathways and how they control complex genetic programs. Neural induction of human pluripotent cells can be used as a model system to investigate the interplay between pathway manipulation and gene activation/silencing in chemically defined E6 medium. Small molecule based inhibition of bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF- ) pathways β(dual SMAD inhibition) is a widely used approach to convert pluripotent cells into neuroectoderm and neural crest. While previous studies characterized human neural induction in bulk cultures, high-resolution analysis should capture the dynamic molecular changes more comprehensively. Here, we performed seven day time-course single-cell sequencing (scRNA-Seq) to reconstruct differentiation trajectories induced by blocking BMP and TGF- pathways separately and in combination. Using dual βSMAD inhibition and single concentrations of small molecules (0.1 uM LDN 193189, 2 uM A83-01), we identified distinct transition stages characterized by transiently expressed genes (e.g. SIX3, HESX1, and LMO1), which led to expression of PAX6, DLK1, TPBG, TMSB15A, HES4, IGFBP5, FOXG1, SOX11. Next, to systematically investigate gene expression dynamics, we used RASL-Seq for gene expression profiling upon BMP inhibition and TGF- inhibition alone or in combination across βseven different small molecule concentrations. During BMP inhibition, FOXG1, SOX1, FZD5, ZIC4, HEXS1, SIX3, PTN, HES4 were strongly upregulated while PAX3, FOXD3, SOX10, SNAI2, S100B were antagonized in a dose-dependent manner. Conversely, TGF- inhibition alone positively regulated PAX3 βand SOX10, suggesting that this strategy favors the induction of neural crest. In summary, modulation of cell signaling pathways via high-throughput gene expression profiling and small molecule titration can control expression of transcription factors determining cell specification. This strategy should help to optimize cell differentiation protocols using precisely calibrated small molecule combinations to produce functional phenotypes for clinical therapies.T-3150POTENTIAL ROLE OF TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 CHANNELS IN MOUSE EMBRYONIC STEM CELL-DERIVED CARDIOMYOCYTESDing, Qianqian - School of Life Sciences, The Chinese University of Hong Kong (CUHK), Hong Kong SAR, ChinaTransient receptor potential (TRP) channels are broadly expressed in a variety of tissues and cell types. They are able to respond to a wide range of stimuli in the cellular environment, which makes them act as cellular vanguard sensors involved in nociception, taste perception, temperature and osmolarity sensation. Among TRP channels, TRP ankyrin 1 (TPRA1) channel was initially described as a cold-sensitive non-selective cation channel expressed in neuron. Recently, emerging evidence indicates that TRPA1 is expressed in various cell types including cardiomyocytes (CMs) and plays an important role in CM contractile function. Mouse embryonic stem cells (mESCs) are able to self-renew and maintain pluripotency to differentiate into all cell lineages including CMs. In CMs, mitochondria can not only supply energy to cells but also has a key role in the regulation of calcium homeostasis and cell contraction. The function of mitochondrion is tightly related to its morphology, which is determined by continuous fission and fusion, called mitochondrial dynamics. Up till now, there is limited knowledge on how TRPA1 regulates intracellular calcium ([Ca2+]i) and action potential in CMs and whether TRPA1 exerts an effect on mitochondria function. Our preliminary results indicated that mESC-derived CMs (mESC-CMs) expressed TRPA1. In addition, we found that the activities of TRPA1 are positively associated with the Ca2+ transients (CaTs) in mESC-CMs. Moreover, TRPA1 activator increased mitochondrial fusion while TRPA1 blocker increased mitochondrial fission. However, the exact mechanism is still under investigation. In the future, our research will focus on elucidating how the TRPA1 activity influences mitochondrial dynamics and their effects on the function of mESC-CMs.Funding Source: General Research Fund (14148116) from the University Grants Committee (UGC) of the Hong Kong SAR

399POSTER ABSTRACTST-3152ISOLATION AND ENRICHMENT OF HUMAN STEM CELL DERIVED PROXIMAL LUNG SUBPOPULATIONSMcvicar, Rachael N - Sanford Burnham Prebys Medical Discovery Institute, Sanford Burnham Prebys, San Diego, CA, USA Leibel, Sandra - University of California San Diego, University of California San Diego, CA, USA Snyder, Evan - SBP, Stanford Burnham Prebys, San Diego, CA, USA Winquist, Alicia - SBP, Stanford Burnham Prebys, San Diego, CA, USAThe human lung is a complex organ composed of over 40 cell types, including epithelial, mesenchymal, immune and endothelial populations. Directed differentiation from human embryonic (hESC) and induced pluripotent stem cells (iPSC) into respiratory epithelial cells using small signaling molecules and growth factors can be used as a robust platform to study human lung development and disease in vitro. However, even with an optimized proximal lung differentiation protocol, the resulting cell culture is a mix of various types of lung cells, which makes studying cell specific molecular pathways difficult. By identifying cell surface markers of specific proximal lung cells, desired subtypes can be isolated and enriched via FACS to enhance lung development and disease modeling research. We aim to screen hESC derived proximal lung cells with a library of CD antigens using a SOX2-GFP reporter cell line by sorting cells positive for both GFP and the target CD antigen. Proximal cell types of the sorted cells will be verified using gene expression profiles and immunocytochemistry. We have performed an investigative bioinformatic screen of 350 CD antigens using Ingenuity Pathway Analysis (IPA) to predict likely surface markers of proximal lung subpopulations including basal, secretory, goblet, and ciliated cells. We then generated proximal lung cells in a 2D culture system from a H9 hESC line with a SOX2-GFP reporter and confirmed the presence of proximal markers such as P63, SCGB3A2, MUC5A and FOXJ1 via immunostaining. Lastly, we performed FACS on the hESC derived proximal lung cells by selecting for cells with both endogenous GFP expression as well as the CD antigen of interest. In summary, we have narrowed down the 350 CD antigens to our top 10 hits using IPA as well as FACS analysis of SOX2+ proximal lung cells. We aim to determine the cell types that these CD antigens correlate with through gene expression profiles and immunostaining of proximal markers.Funding Source: UCSD Senate grantT-3154DEFINING THE TRANSCRIPTIONAL NETWORK THAT GOVERNS PERIPHERAL GLIA SPECIFICATION BY USING HUMAN PLURIPOTENT STEM CELLSRamos Calcada, Raquel M - Institute Of Anatomy, Department of Stem Cell Biology, University of Zurich, Switzerland Varum Tavares, Sandra - Institute of Anatomy, Department of Stem Cell Biology, University of Zurich, Switzerland Marzorati, Elisa - Institute of Anatomy, Department of Stem Cell Biology, University of Zurich, Switzerland Sommer, Lukas - Institute of Anatomy, Department of Stem Cell Biology, University of Zurich, SwitzerlandThe neural crest (NC) is a transient and multipotent embryonic stem cell population that migrates throughout the embryo to populate numerous derivatives. It generates peripheral neurons and glia, smooth muscle cells of the outflow track of the heart, craniofacial bone and cartilage, and skin melanocytes. Thus, human neural crest stem cells (hNCSCs) offer the possibility to study cell-fate decisions during embryonic development. The peripheral glia or Schwann cell lineage not only plays an essential role in nerve repair and regeneration, but is also responsible for several demyelination disorders, such as the Charcot-Marie-Tooth disease. Despite its importance, the transcriptional program that regulates hNCSC specification into human Schwann cells (hSCs) remains unknown. Moreover, recent findings in different stem cell types suggest that fundamental metabolic processes are actively implicated in stem cell maintenance and lineage specification, although in NC this remains to be elucidated. Therefore, the overall goal of this project is to define gene regulatory networks, translational landscapes and metabolic profiles that distinguish hNCSCs from hSCs. To accomplish this aim, we take advantage of the human embryonic stem cell (hESC) system. We efficiently differentiated hESCs into hNCSCs using a combined TGF- and GSK-3 ββinhibition. Afterwards, hNCSCs were treated with neuregulin-1 and forskolin to induce the specification into the glial lineage. To characterize the specification process, we analyzed the expression of various hNCSC and hSC markers, such as Sox10 and p75, during the course of differentiation. We observed that Sox10+ p75+ cells acquire a bipolar morphology at the end of the treatment, resembling Schwann cell precursors. With this strategy we plan to recapitulate the neural crest specification into peripheral glia by using hESCs, and to unravel which are the main drivers for this fate decision. These findings will allow a better understanding of hSC development, and consequently of its role in nerve repair and neuropathies.