450POSTER ABSTRACTSThe kidney is a vital organ required for waste excretion as well as water and solute reabsorption. Its functionality relies on a range of highly specialized cells that are arranged into nephrons, the functional units of the kidney. While we understand many of the properties of the different renal cell types, we lack effective methods to generate large quantities of terminally differentiated human-derived kidney epithelial cells from human pluripotent stem cells. To address this critical knowledge gap, we utilize Quality-by-Design-based methods, in particular the Design-of-Experiment (DoE) theory, to provide a systematic, data driven approach towards renal cell differentiation. In a single experiment we simultaneously test the effect of up to 12 morphogen inputs on more than 50 target genes. Experimental design and statistical methods provide an in-depth understanding of the input parameters. Moreover, compilation of a series of these experiments lead to a systems-developmental biology representation of the signaling logics underlying renal epithelial cell differentiation. Here we now report our progress for the generation of podocytes, a specialized renal epithelial cell type found in the glomerulus of the kidney. Starting from established protocols we differentiate human embryonic stem cells into nephric mesenchyme precursors. Using DoE, we then identify a complex combination of signaling agonists and/or antagonists that differentiate these cells along the podocyte lineage in a stepwise protocol. These cells not only express transcription factors (e. g. WT1 or MAFB) as well as structural proteins (e. g. NPHS1 or PODXL) characteristic for podocytes, but also exhibit structural and physiological properties of these highly specialized renal epithelial cells. Importantly, the conditions are highly robust and lead to the formation of a homogenous culture of human podocytes. The cells will not only provide a valuable tool to understand the signaling inputs required for podocyte development/homeostasis but will also provide a critic tool towards developing treatments for human kidney diseases characterized by podocyte dysfunction.Funding Source: (Re)Building the Kidney Consortium (NIH/NIDDK, 4UH3DK107357). Lisa Dean Moseley Foundation (227G18a).F-2067DEFINING PROGENITOR TYPES AND PROGENITOR PROGRAMS IN STRATIFICATION OF THE MOUSE AND HUMAN RENAL COLLECTING SYSTEMParvez, Riana K - Department of Stem Cell Biology and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USA Rutledge, Elisabeth - Department of Stem Cell Biology and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USA Ransick, Andrew - Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA McMahon, Andrew - Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAIn the adult kidney, the collecting duct is responsible for maintenance of water and pH homeostasis. These two important functions are carried out by distinct cell populations, the principal cells and the intercalated cells. Recently generated single RNA-seq (scRNA-seq) data in the laboratory illuminates diversity in these cell types in relation to their position along the cortical-medullary axis of the kidney. During development, a progenitor pool within each branch tip of the arborizing ureteric epithelium lays down the cellular template for the epithelial network of the collecting system. The mechanisms responsible for the tight temporal and spatial regulation of mature cell fates originating from this progenitor pool are not well understood. We are combining lineage tracing with scRNA-seq and in vivo expression studies to better understand how molecular and cellular changes within the progenitor pool relate to diverse developmental outcomes in assembly of the mature collecting system. Further, we are exploring signaling networks and transcriptional mediators to identify relevant regulatory mechanisms. We expect these approaches will give new insights into basic mechanisms of progenitor programming with potential relevance beyond the kidney model. An understanding of these programs is essential for improving current kidney organoid models which lack a functional collecting duct epithelium.F-2069FOXA TRANSCRIPTION FACTORS SPECIFY HUMAN ENDODERM-DERIVED ORGAN LINEAGES BY PRIMING SIGNAL-RESPONSIVE ENHANCERSGeusz, Ryan - Pediatrics, University of California, San Diego, CA, USA Wang, Allen - School of Medicine, Center for Epigenomics, University of California, San Diego, La Jolla, CA, USA Lam, Dieter - Pediatrics, University of California, San Diego, La Jolla, CA, USA Kefalopoulou, Samy - Pediatrics, University of California, San Diego, La Jolla, CA, USA Vinckier, Nicholas - Pediatrics, University of California, San Diego, La Jolla, CA, USA Qiu, Yunjiang - Bioinformatics and Systems Biology, University of California, San Diego, La Jolla, CA, USA Chiou, Joshua - Biomedical Sciences, University of California, San Diego, La Jolla, CA, USA Ren, Bing - San Diego, Ludwig Institute for Cancer Research, La Jolla, CA, USA Gaulton, Kyle - Pediatrics, University of California, San Diego, La Jolla, CA, USA Sander, Maike - Pediatrics, University of California, San Diego, La Jolla, CA, USADuring development, cell fate decisions are driven by transcription factors (TFs) that initiate cell type-specific gene expression programs. Earlier studies of differentiation of human embryonic stem cells (hESCs) into pancreatic cells have suggested epigenetic priming of pancreatic enhancers as a mechanism by which pancreatic genes are primed in early endodermal intermediates prior to pancreas induction.
451POSTER ABSTRACTSHowever, the mechanism that underlies priming of lineage-specific enhancers remains unclear. We tested the hypothesis that enhancer priming is initiated by FOXA family TFs which have the ability to engage with condensed chromatin. We show that pancreatic enhancers are broadly occupied by FOXA TFs and that association of FOXA TFs with pancreatic enhancers correlates tightly with a gain of the H3K4me1 histone modification characteristic of primed enhancers, as well as a gain in ATAC-seq signal, indicative of accessible chromatin. Supporting a role of FOXA TFs in pancreatic lineage specification, FOXA1-/-/FOXA2-/- lines fail to initiate expression of pancreatic master regulator genes. Analysis of pancreatic enhancers in FOXA1-/-/FOXA2-/- hESC-pancreatic progenitors further revealed a lack of H3K27ac and H3K4me1 deposition and ATAC-seq signal. To explore the mechanism by which FOXA TFs activate pancreatic enhancers, we examined FOXA occupancy throughout the differentiation time course. This analysis revealed FOXA recruitment to a subset of pancreatic enhancers prior to enhancer activation and pancreatic gene expression. Compared to the remainder of pancreatic enhancers, this enhancer subset was enriched for retinoic acid receptor (RAR) motifs and binding of the RAR subunit RXR. These findings suggest that priming by FOXA TFs is limited to a subset of pancreatic enhancers that are signal responsive. Using a hESC-based model of liver development, we determined that the association of early FOXA recruitment and signal-dependency also exists at hepatic enhancers. Consistent with liver induction being BMP-dependent, “FOXA-primed” liver enhancers were enriched for motifs of BMP-dependent SMAD TFs. Combined, our analysis suggests a role for pioneer TFs in selectively priming signal-dependent enhancers with broader activation of the entire enhancer repertoire occurring through other mechanisms.Funding Source: This work was supported by R01DK078803 and U01DK105541.F-2071STEM CELL DERIVED PANCREATIC ISLETS FOR TRANSPLANTATION INTO ANTERIOR CHAMBER OF RABBIT EYESWu, Siqin - Clinical Sciences, Intervention and Technology (CLINTEC), Division of Obstetrics and Gynecology, Karolinska Institutet, Stockholm, Sweden Efstathopoulos, Paschalis - Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden Åstrand, Carolina - Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden André, Helder - Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden Hedhammar, My - Protein Technology, KTH Royal Institute of Technology, Stockholm, Sweden Kvanta, Anders - Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden Lanner, Fredrik - Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden Ortega Melin, Yesenia - Clinical Neuroscience, Karolinska Institute, Stockholm, SwedenType 1 Diabetes is a rapidly expanding endocrine disease affecting millions of people worldwide. Patient with type 1 Diabetes can be successfully treated with transplantation of cadaveric pancreatic islets, but this approach is limited by shortage of donor pancreatic tissue and low viability of islets post transplantation due to inflammation after blood contact. Transplantation of human pluripotent stem cell (hPSC) derived islets into the anterior chamber of eye (ACE) can potentially solve these problems by utilizing a renewable cell source and the possibility of non-invasive longitudinal evaluation post transplantation. In this study, we differentiated hPSCs into pancreatic islet-like cell aggregates. The islet-like aggregates were then incorporated into 3D porous scaffolds made of recombinant spider silk protein. The cell aggregate containing silk matrices were injected into ACE of a large-eyed pre-clinical animal model. Engraftment and maturation into functional islets in vivo were examined one month after transplantation. Our results show that the injected aggregates were integrated onto the iris tissue and became vascularized. The aggregates closely resemble human islets in both size and morphology, and contain mainly mono-hormonal endocrine cells expressing either insulin, glucagon or somatostatin. No major adverse effects on eye morphology and function were found during the transplantation period. Glucose stimulated insulin secretion (GSIS), transplant rejection and tolerance, and biosafety are currently under investigation. Together these results indicate that hPSC-derived islets could be utilized instead of primary human pancreatic islets for transplantation therapy for diabetes in the future.Funding Source: This work is supported by Sweden’s innovation agency Vinnova.F-2073GENERATION OF HUMAN IPSC-DERIVED PANCREATIC DUCTAL CELLS FOR IN VITRO DISEASE MODELLING OF PANCREATIC DUCTAL ADENOCARCINOMAShaharuddin, Syairah Hanan Binti - Regenerative Medicine, Cedars-Sinai Health System, West Hollywood, CA, USA Santos, Roberta - Regenerative Medicine Institute, Cedars Sinai, West Hollywood, CA, USA Ramos, Michael - Regenerative Medicine Institute, Cedars Sinai, West Hollywood, CA, USA Gross, Andrew - Regenerative Medicine Institute, Cedars Sinai, West Hollywood, CA, USA Pandol, Stephen - Department of Medicine, Cedars Sinai, West Hollywood, CA, USA Sareen, Dhruv - Regenerative Medicine Institute, Cedars Sinai, West Hollywood, CA, USA
452POSTER ABSTRACTSPancreatic Ductal Adenocarcinoma (PDAC), which makes up the majority of pancreatic cancer cases, begins as a tumor in the pancreatic duct. Each year, more than 350, 000 people are diagnosed with PDAC and about 96% of them die. The 5-year survival rate of PDAC is very low, and even with surgical removal of the tumor, the 5-year survival rate only increases to about 15%. Even so, due to rapid metastasis and resistance, surgery and standard treatments like chemotherapy are not effective. While there has been advancement in discerning the molecular background of the disease, there is limited understanding of early drivers – genetic and environmental – PDAC transformation, mainly due to limited models of human PDAC. Here, we aim to develop an in vitro model of PDAC by first establishing a robust protocol for generating human induced pluripotent stem cell (hiPSC)-derived pancreatic ductal cells. Our lab has previously developed a consistent and effective protocol to generate pancreatic progenitors across multiple iPSC cell lines. Using this protocol, we have collected cells from pluripotency stage throughout the sequential induction of definitive endoderm, posterior foregut, and pancreatic progenitor, to find a pivotal time-point to direct cells toward ductal lineage. Our results indicate a temporal window with the highest gene expression of ductal progenitor markers, such as SOX9, KRT19, HNF1B, and CA2, which is the best period to drive the PDX1+NKX6.1+PTF1A- pancreatic bipotent trunk progenitors toward the pancreatic ductal lineage. Notch, BMP, EGF, FGF, Shh signaling pathway modulation was tested to determine the optimal generation of pancreatic bipotent trunk progenitors, in favor of generating ductal epithelium from iPSC-derived pancreatic progenitors, and differentiated ductal cells were functionally assayed to assess enzymatic activity by measuring carbonic anhydrase (CA) levels. Our next goal will be to derive PDAC patient iPSCs from a panel of familial PDAC patients, differentiate to ductal cells and expose them to different microenvironments, such as hypoxia, and/or assess their interaction with the immune system, which are all known to be implicated in PDAC, to develop a more comprehensive and reliable disease model.F-2075SOX9+/PTF1A+ CELLS DEFINE THE TIP PROGENITOR DOMAIN WITHIN THE HUMAN DEVELOPING PANCREASVillani, Valentina - Division of Urology, Children’s Hospital Los Angeles, CA, USA Thornton, Matthew - Maternal-Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Grubbs, Brendan - Maternal-Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Orlando, Giuseppe - Department of Surgery, Wake Forest School of Medicine, Winston-Salem, NC, USA De Filippo, Roger - Division of Urology, Children’s Hospital Los Angeles, CA, USA Ku, Teresa - Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA, USA Perin, Laura - Division of Urology, Children’s Hospital Los Angeles, CA, USAThe developing mammalian pancreas is a highly organized structure, in which subpopulations of progenitor and committed cells can be identified based on their molecular profile and location, after branching morphogenesis has initiated. The pancreatic multipotent progenitor cells (MPCs) in early mouse embryos are known to express several key transcription factors, including Pdx1, Sox9, and Ptf1a, which are indispensable for their establishment, maintenance and proliferation. Recent advances have been made in understanding human pancreatic development, however, little is known regarding the presence of pancreatic progenitors, particularly in the second trimester, and their characteristics. We have identified pancreatic human multipotent progenitor cells (hMPC) between 13.5 and 17.5 weeks of gestation, which are characterized by the expression of SOX9 and PTF1A. These cells reside within the tips of the branching epithelium and were isolated by combining an RNA-based probe technology with fluorescence activated cell sorting. These cells are in a proliferative state, in the process of branching morphogenesis, and their pool decreases over time. RNA-sequencing profiling revealed that SOX9+/PTF1A+ cells are enriched for genes characteristic of MPCs rather than committed cells and show activation of NOTCH and WTN/ -βcatenin signaling pathways, known regulators of the MPC niche and pancreatic specification. The SOX9+/PTF1A+ cells thus display the full spectrum of characteristics that define the MPC niche, including maintenance of key transcription factors expression, proliferative and branching capabilities. Based on our results, we propose that SOX9+/PTF1A+ cells in the second trimester human pancreas are uncommitted MPC-like cells that reside at the tips of the expanding pancreatic epithelium, directing self-renewal and inducing pancreatic differentiation. This novel insight into the genetic signature of human fetal MPCs will allow for further understanding of the molecular programs and interactions that sustain the human pancreatic MPC niche. It will also help improve pluripotent stem cell differentiation protocols and allow for better manipulation of the stem cell-derived ß-like cell for regenerative medicine and cell replacement therapy.Funding Source: This work was supported in part by grants from the National Institutes of Health (NIH) R21HD090545 to L. P. and R01DK099734 to H.T.K.EPITHELIAL TISSUESF-2079DORSOVENTRAL DIFFERENCE IN TRACHEAL BASAL STEM CELLSTadokoro, Tomomi - Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan
453POSTER ABSTRACTSTanaka, Keisuke - NODAI Genome Research Center, Tokyo University of Agriculture, Setagaya, Japan Hogan, Brigid - Department of Cell Biology, Duke University School of Medicine, Durham, NC, USA Kobayashi, Hisato - Department of Embryology, Nara Medical University, Kashihara, Japan Taniguchi, Hideki - Department of Regenerative Medicine, Yokohama City University School of Medicine, Yokohama, JapanAirway epithelium is composed of mucociliary epithelium and basal stem cells underneath. Mucociliary epithelium secretes mucous to capture viruses, noxious substances, and allergens, and ciliated cells move them out from airway to protect lung from infection or inflammation. When mucociliary epithelium is damaged, basal stem cells proliferate and give rise to both secretory cells and ciliated cells to repair mucociliary epithelium. In the previous study, we showed that proliferation and differentiation of basal stem cells are regulated by mesenchymal cells during repair of airway epithelium. Airway is surrounded by cartilages and smooth muscles, which are mainly located at ventral side and dorsal side of trachea, respectively. Thus, we hypothesized that behavior of basal stem cells could be different in ventral side and dorsal side of trachea. To observe the difference in proliferation of basal stem cells in ventral and dorsal trachea, we utilized the 3D clonal organoid culture system. This assay revealed that basal stem cells from dorsal trachea showed higher colony forming efficacy superior to those from ventral trachea. In vivo clonal analysis using lineage tracing model for basal stem cells (Keratin5-CreER; Rosa-Confetti) showed that there are more clones and clones contain more cells in dorsal trachea compared to ventral trachea. Since the distribution of blood vessels is not different between ventral and dorsal trachea, characteristics of basal stem cells itself is thought to be cause the difference in proliferation. Gene ontology analysis from RNA-seq data revealed that genes expressed in basal stem cells of dorsal trachea are enriched in wound healing and response to wounding while those in ventral trachea are enriched in response to external stimulus and immune response. Interestingly, basal stem cells in ventral trachea expressed Myostatin, which inhibits the growth of smooth muscle cells, and HGF, which facilitates cartilage repair. This is consistent with a model in which basal stem cells in dorsal trachea are a main source for repair of airway epithelium and basal stem cells in ventral trachea protect airway from infection and also maintain cartilages. Taken together, these results revealed different roles in basal stem cells for the maintenance of airway and airway epithelium.F-2081BMAL1-DRIVEN PERIPHERAL CLOCKS RESPOND INDEPENDENTLY TO LIGHTZinna, Valentina M - Aging and Metabolism Department, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Welz, Patrick-Simon - Aging and Metabolism, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Koronowski, Kevin - Biological Chemistry, Center for Epigenetics and Metabolism, University of California, Irvine, CA, USA Kinouchi, Kenichiro - Biological Chemistry, Center for Epigenetics and Metabolism, University of California, Irvine, CA, USA Smith, Jacob - Biological Chemistry, Center for Epigenetics and Metabolism, University of California, Irvine, CA, USA Symeonidi, Aikaterini - Aging and Metabolism, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Castellanos, Andrés - Aging and Metabolism, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Marín Guillén, Inés - Aging and Metabolism, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Prats, Neus - Histopathology Facility, Institute for Research in Biomedicine IRB Barcelona, Barcelona, Spain Martin Caballero, Juan - Animal Facility, Barcelona Biomedical Research Park PRBB, Barcelona, Spain Sassone-Corsi, Paolo - Biological Chemistry, Center for Epigenetics and Metabolism, University of California, Irvine, CA, USA Aznar Benitah, Salvador - Aging and Metabolism, Institute for Research in Biomedicine IRB Barcelona, Barcelona, SpainOrganisms adjust their daily physiology to anticipate the environmental changes resulting from the Earth rotating around its own axis. At the cellular level, circadian regulation is established by a Bmal1-dependent molecular network that oscillates in a self-sustained manner and imposes daily oscillations on the expression of thousands of genes. In turn, circadian synchronization of cells within tissue depends on environmental entrainment cues, such as changes in light levels. However, it is still unclear whether the circadian clocks of different tissues respond independently to environmental signals, or rather require interactions with each other to achieve a synchronized rhythmicity. This is a relevant issue, since clock alterations can have drastic physiological consequences, such as shortened mammalian lifespan and changes in the predisposition to tumorigenesis. Here, we show that, unexpectedly, light can synchronize the Bmal1-dependent core circadian machinery in the epidermis, in the absence of Bmal1-driven clocks in other tissues. Strikingly, this circadian rhythmicity is lost in constant darkness, indicating that Bmal1 provides a “memory” of time to synchronize peripheral clocks when external entraining signals are absent. Importantly, we find that tissue autonomous Bmal1 suffices for sustaining tissue homeostasis in otherwise arrhythmic and prematurely-aging animals. Our results, therefore, support a two-branched model for the daily synchronization of peripheral tissues: an immediate response branch, whereby light can entrain circadian clocks without any commitment of other Bmal1-dependent clocks; and a memory branch that uses other Bmal1-dependent clocks to “remember” circadian rhythmicity in the absence of external cues.Funding Source: The project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 713673, and from “la Caixa” Banking Foundation (LCF/BQ/IN17/11620018).
454POSTER ABSTRACTSF-2083OPTIMIZING CULTURE CONDITIONS FOR EXPANSION OF HUMAN EPIDERMAL STEM CELLS FOR SKIN REGENERATIONEshwara Swamy, Vinutha – NITTE (Deemed to be University), K S Hegde Medical Academy, Alape ,Padil, Mangalor, India Rao, Shama – NITTE (Deemed to be University), University Centre for Stem Cell Research and Regenerative Medicine (NUCSReM), NITTE (Deemed to be University), Mangaluru, India Shetty, Nikhil - Department of Plastic Surgery, NITTE (Deemed to be University), Mangaluru, India Shetty, Veena – NITTE (Deemed to be University), University Centre for Stem Cell Research and Regenerative Medicine (NUCSReM), NITTE (Deemed to be University), Mangaluru, India Noronha, Tonita - Department of Dermatology, NITTE (Deemed to be University), Mangaluru, India Shetty, Jayaprakashak - NITTE(Deemed to be University), University Centre for Stem Cell Research and Regenerative Medicine (NUCSReM), NITTE (Deemed to be University), Mangaluru, India Shetty, Neha - Faculty of Health and Wellbeing, Canterbury Christ Church University, Kent, UK Mohana Kumar, Basavarajappa - NITTE(Deemed to be University), University Centre for Stem Cell Research and Regenerative Medicine (NUCSReM), NITTE (Deemed to be University), Mangaluru, IndiaThe human epidermal stem cells (EpiSCs) are particularly suited for cellular therapy due to their accessibility and low immunogenicity. The conventional means of expanding EpiSCs uses the support of feeder layer cells. There are still superior techniques needed to culture these cells, ideally without the feeder layer. Therefore, the present study attempted to optimize in vitro culture methods for establishing a pure population of EpiSCs. Human skin samples were obtained with informed written consent taken priorly. The isolation and expansion of EpiSCs was performed by suspension and explant methods using different culture media (alpha-MEM, DMEM/F12 and EpiLife) with supplementation of fetal bovine serum (FBS at 0, 10 and 20%) and growth factors [2 ng/ml keratinocyte growth factor, (KGF) and 2 ng/ml epidermal growth factor (EGF), 1% human keratinocyte growth supplement (HKGS)]. Among the culture methods, explant culture showed the attachment of cells in alpha-MEM at 20% FBS supplementation by exhibiting a typical “cobblestone” epithelial pattern of growth at passage 0, but did not retain the similar morphology at passage 1. In contrast, no cells were firmly attached in suspension culture either in alpha-MEM or DMEM/F12 in all concentrations of FBS. Interestingly, a higher number of proper EpiSCs from suspension culture was observed when alpha-MEM was supplemented with FBS and growth factors, such as KGF, EGF and HKGS. In EpiLife media with HKGS, primary culture did not support the attachment of cells. Whereas, the EpiLife media supplemented with KGF, EGF and HKGS resulted in proper attachment of EpiSCs with typical morphological features. Following this, EpiSCs established from alpha-MEM and EpiLife supplemented with various growth factors were analysed for the proliferation rate and the expression of stem/progenitor and keratinocyte specific markers, such as p63, K5, K14, K15, integrin 1, K1, βK10 and filaggrin. The analyses showed that all EpiSCs had high clonogenic potential with varied levels of expression of selected markers. In summary, our findings illustrated that established EpiSCs in alpha-MEM and EpiLife media with growth factors possess plasticity with high proliferation ability. Thus EpiSCs may represent an ideal source for skin regenerative medicine applications.Funding Source: This work was supported by NITTE (Deemed to be University), Mangaluru, India.F-2085CELL EXTRACT FROM HUMAN LABIAL GLAND STEM CELLS RESCUES THE HYPOFUNCTION OF IRRADIATED SALIVARY GLANDS IN A MOUSE MODEL.Su, Xinyun - Dentistry, McGill University, Montreal, QE, Canada Liu, Younan - Dentistry, McGill University, Montreal, QE, Canada Bakkar, Mohammed - Dentistry, McGill University, Montreal, QE, Canada Tran, Simon - Dentistry, McGill University, Montreal, QE, CanadaNowadays, adult stem cells play an important role in tissue repair and regeneration. However, cell-based therapies still possess potential risks, such as tumorigenesis and immunogenicity. The main objective in this study was to test the therapeutic effect of a cell-free/ cell extract therapy to restore the hypofunction of irradiation-induced salivary glands (SGs). Human labial gland derived-stromal (mesenchymal) stem cells (LSC) were harvested by explant culture method. Stem cell properties of LSC were characterized and confirmed by flow cytometry. Then LSCs were lysed into labial glands stem cell extract (LSCE) by three freeze-thaw cycles. Either LSCE or normal saline (as a vehicle control) was injected intravenously into mice post- local irradiation to SGs (13Gy). Using explant culture method of small pieces of human labial glands, we successfully harvested a large number of LSCs with self-renewal and multipotent differentiation capacities. Our results demonstrated that several angiogenesis-related factors, such as FGF-1/ -2, KGF and VEGF, were detected in LSCE. For the in vivo study, LSCE restored 50%-60% of saliva secretion, protected acinar cells, blood vessels and parasympathetic nerves, promoted the cell proliferation and up-regulated the expression of tissue repair/regeneration proteins and genes. These findings improved our understanding of human LSCs and indicated the potential therapeutic application of labial gland stem cell extract (LSCE) to treat irradiation-induced salivary hypofunction for head and neck cancer patients.
455POSTER ABSTRACTSFunding Source: Supported by: Canadian Institutes of Health Research, MOP-119585.F-2087TRANSCRIPTOME PROFILING IN AMELOBLAST-LIKE CELLS DERIVED FROM ADULT GINGIVAL EPITHELIAL CELLS : IDENTIFICATION OF AMELOBLAST-SPECIFIC CELL SURFACE MARKERSJang, Young-Joo - Department Nanobiomedical Science, Dankook University, Cheonan, Korea Hyun, Sun-Yi - Nanobiomedical Science, Dankook University, Cheonan, Korea Kang, Kyung-Jung - Nanobiomedical Science, Dankook University, Cheonan, Korea Mun, Seyoung - DKU-Theragen Institute for NGS analysis (DTiNa), Dankook University, Cheonan, KoreaDental enamel is the highly mineralized tissue covering the tooth surface and is formed by ameloblasts. Ameloblasts have been known to be impossible to detect in adult tooth because they are shed by apoptosis during enamel maturation and tooth eruption. Owing to these, little was known about appropriate cell surface markers to isolate ameloblast-like cells in tissues. To overcome these problems, epithelial cells were selectively cultivated from the gingival tissues and used as a stem cell source for ameloblastic differentiation. When gingival epithelial cells were treated with a specified concentration of BMP2, BMP4, and TGFb-1, the expression of ameloblast-specific markers was increased, and both the MAPK and Smad signaling pathways were activated. Gingival epithelial cells differentiated into ameloblast-like cells through epithelial-mesenchymal transition. By RNA-Seq analysis, we reported 20 ameloblast-specific genes associated with cell surface, cell adhesion, and extracellular matrix function. These cell surface markers might be useful for the detection and isolation of ameloblast-like cells from dental tissues.Funding Source: The Bio and Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9C6029130).F-2089A MOUSE MODEL OF PRECISION RADIATION INDUCED CHRONIC HYPOSALIVATION FOR HUMAN SALIVARY STEM CELL TRANSPLANTSEmperumal, ChitraPriya - Otorhinolaryngology, Mayo Clinic, Rochester, NY, USA Aalam, Musheer - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USA Xu, Xuewen - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USA Walb, Mathew - Radiation Oncology, Mayo Clinic, Rochester, NY, USA Tryggestad, Erik - Radiation Oncology, Mayo Clinic, Rochester, NY, USA Shi, Geng - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USA Garcia, Joaquin - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USA Sarkaria, Jann - Radiation Oncology, Mayo Clinic, Rochester, NY, USA Janus, Jeffrey - Otorhinolaryngology, Mayo Clinic, Rochester, NY, USA Kannan, Nagarajan - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USARadiation leads to salivary dysfunction and xerostomia (also known as hyposalivation or dry mouth), a major unmet medical need among head and neck cancer survivors. A cure for this chronic debilitating condition is “non-existent”. Since this condition is ‘irreversible’, treatment methods for regeneration of salivary glands and physiological secretion of saliva from reconstituted glands are urgently needed. Stem cells have been identified in salivary gland, which are capable of differentiating into functionally competent salivary units. A preclinical model to test such cells is currently lacking. We have developed the first precision-X-radi ation induced mouse model of radiation induced chronic hyposalivation. We have used highly immunodeficient transgenic NSG-3GSM mice expressing three human cytokines including human stem cell factor. NSG-3GSM mice owing to Prkdcscid mutation are radiosensitive, thus we exploited the stereotactic targeting and dose delivery capabilities of X-Rad SmART system to radio-ablate salivary glands and induce xerostomia. Further, we have established an orthotropic survival surgery procedure to enable transplant of salivary stem cells into radio ablated sub-mandibular glands of these mice to study regeneration and restoration of salivary functions. This protocol includes neck incision and injection of cells in buffered-trypan blue dye into right and left sub-mandibular glands under anesthesia. Mice recovered from surgery remained healthy. Moreover, we have established a robust method for reflex saliva flow measurement using Schirmer strips in irradiated and sham mice. Our data from the se measurements, demonstrates significant X-ray dose-dependent reduction in saliva production, where 10 Gy irradiated mice showed ~50% reduction in saliva production and this remained consistent for over a period of six months. In summary, we have developed the first humanized mouse model that mimics a challenging, chronic lifestyle problem in cancer survivors and plan to use this robust platform to measure regenerative outcome following patient-derived salivary cell transplants.EYE AND RETINAF-2091IPSC BASED CELLULAR MODEL USED TO CHARACTERIZE THE MOLECULAR PATHOLOGY OF CORNEAL ENDOTHELIAL DYSTROPHYDobrovolny, Robert - First Faculty of Medicine, Charles University, Prague, Czech Republic
456POSTER ABSTRACTSBrejchova, Kristyna - First Faculty of Medicine, Charles University, Prague, Czech Republic Dudakova, Lubica - First Faculty of Medicine, Charles University, Prague, Czech Republic Skalicka, Pavlina - First Faculty of Medicine, Charles University, Prague, Czech Republic Liskova, Petra - First Faculty of Medicine, Charles University, Prague, Czech RepublicCongenital hereditary endothelial dystrophy (CHED) is a rare disease caused by biallelic mutations in the SLC4A11 gene. The disease is clinically characterized by cloudy cornea that may be present from birth or may be infantile in onset. Because of the endothelial dystrophy the patient’s vision degrade in time. We have used direct and genome sequencing to screen six probands with CHED, collectively we identified four novel and seven previously reported disease-associated SLC4A11 variants. To analyze the effect of c.2240+5G>A on pre-mRNA splicing, and thus to prove pathogenicity of the variant, we have developed a corneal endothelial-like (CE-like) cell model differentiated from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB and DKK-2. CE-like cells were demonstrated to express several endothelial cell-specific markers including SLC4A11. Total RNA was extracted, and reverse transcriptase PCR was performed. The c.2240+5G>A variant was demonstrated to introduce a cryptic splice donor site leading to an insertion of six bp and the subsequent introduction of a premature stop codon (p.Thr747*). This proof-of-concept study highlights the potential of using iPSC derived CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.Funding Source: This work was supported by GACR 17-12355S, institutional support was provided by UNCE 204064 and PROGRES Q26 programs of the Charles University. PS was supported by grants GAUK 250361/2017 and SVV 260367/2017.F-2093GENERATION OF RETINAL PIGMENT EPITHELIAL CELLS FROM MOUSE CHEMICAL INDUCED PLURIPOTENT STEM CELLS BY SMALL MOLECULESPan, Shaohui - Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medic, WenZhou, China Jin, Zi-bing - Laboratory for Stem Cell and Retinal Regeneration Laboratory for Stem Cell and Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University, Wenzhou, ChinaSomatic cells can be reprogrammed into pluripotent stem cells by pure chemicals, without genetic manipulation. Therefore, chemical induced pluripotent stem cells (CiPSCs) provide a promising approach for generating clinically desirable cell types for regenerative medicine. Retinal pigment epithelium (RPE) cells derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been clinically translated. However, ESC-derived RPE cells face ethical and possible immune rejection problems. The safety of exogenous gene also limits the clinical application of iPSC-derived RPE. In this study, we report highly efficient generation of CiPSCs from mouse fibroblasts and differentiation into RPE cells using chemically defined conditions. We utilize a cocktail of small molecules to generate pluripotent stem cells can be generated from mouse somatic cells without genetic manipulation. The CiPSCs resemble ESCs in terms of their morphology, gene expression and differentiation potential. Then, the CiPSCs were differentiated into functional RPE cells by a cocktail of small molecules and a three-dimensional (3D) culture system. . The results showed that differentiation toward RPE fate within 4 weeks. The RPE cells displayed characteristics of apical basal polarity, cellular structure and gene expression tight junctions, phagocytose photoreceptor outer segments similar with native RPE. We report here development of a high-efficient, small molecule-based methodology for generation of RPE from mouse fibroblast, which we suppose more suitable for clinical application. Alternatively, CiPSCs may be generated from patient-derived somatic cells and provide a readily accessible source of autologous RPE cell grafts.Funding Source: This work was supported by the National Natural Science Foundation of China (No. 81600749), the National Key R&D Program of China (2017YFA0105300), and the Zhejiang Provincial Natural Science Foundation of China (LD18H120001LD).F-2095MODULATION OF OSMOTIC STRESS-INDUCED TRPV1 EXPRESSION RESCUES HUMAN IPS-DERIVED RETINAL GANGLION CELLS THROUGH PKAChiou, Shih-Hwa - Pharmacology, National Yang-Ming University, Taipei, Taiwan Chen, Shih-Jen - Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan Hsu, Chih-Chien - Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, TaiwanTransient receptor potential vanilloid 1 (TRPV1), recognized as a hyperosmolarity sensor, is a crucial ion channel involved in the pathogenesis of neural and glial signaling. Recently, TRPV1 was determined to play a role in retinal physiology and visual transmission. In this study, we sought to clarify the role of TRPV1 and the downstream pathway in the osmotic stress-related retina ganglion cell (RGC) damage. First, we modified the RGC differentiation protocol to obtain a homogeneous RGC population from human induced pluripotent stem cells
457POSTER ABSTRACTS(hiPSCs). Subsequently, we induced high osmotic pressure in the hiPSC-derived RGCs by administering NaCl solution and observed the behavior of the TRPV1 channel and its downstream cascade. We obtained a purified RGC population from the heterogeneous retina cell population using our modified method. Our findings revealed that TRPV1 was activated after 24 hours of NaCl treatment. Upregulation of TRPV1 was noted with autophagy and apoptosis induction. Downstream protein expression analysis indicated increased phosphorylation of CREB and downregulated brain-derived neurotropic factor (BDNF). However, hyperosmolarity-mediated defective electrophysiological activity, morphological change and apoptosis of RGCs, CREB phosphorylation, and BDNF downregulation were abrogated after concomitant treatment with the PKA inhibitor H89. Collectively, our study results indicated that the TRPV1–PKA pathway contributed to cellular response under high levels of osmolarity stress; furthermore, the PKA inhibitor had a protective effect on RGCs exposed to this stress. Therefore, our findings may assist in the treatment of eye diseases involving RGC damage.Funding Source: Division of Experimental Surgery of the Department of Surgery and the Animal Center of Taipei Veterans General Hospital Ministry of Science and Technology Academia Sinica Tri-Service General Hospital National Defense Medical CenterF-2097PURIFICATION OF HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CORNEAL EPITHELIAL CELLS USING CELL TYPE-SPECIFIC ADHESIVENESS ON LAMININ ISOFORMSShibata, Shun - R&D Division, ROHTO Pharmaceutical Co., Ltd.,/Osaka University Graduate School of Medicine, Suita, Japan Hayashi, Ryuhei - Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Japan Kudo, Yuji - R&D Division, ROHTO Pharmaceutical Co., Ltd./Osaka University Graduate School of Medicine, Suita, Japan Okubo, Toru - R&D Division, ROHTO Pharmaceutical Co., Ltd./Osaka University Graduate School of Medicine, Suita, Japan Katayama, Tomohiko - Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan Ishikawa, Yuki - Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan Kobayashi, Yuki - Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan Toga, Junko - Institute for Protein Research, Osaka University, Suita, Japan Taniguchi, Yukimasa - Institute for Protein Research, Osaka University, Suita, Japan Honma, Yoichi - R&D Division, ROHTO Pharmaceutical Co., Ltd./Osaka University Graduate School of Medicine, Osaka, Japan Sekiguchi, Kiyotoshi - Institute for Protein Research, Osaka University, Suita, Japan Nishida, Kohji - Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, JapanThe products differentiated from human induced pluripotent stem cells (hiPSCs) which replace damaged tissues, are expected as a novel treatment for intractable diseases. Previously, we established a multilayered structure that reproduces the whole eye development, generated from hiPSCs on laminin-511 fragment (LN511E8). Our recent work showed that LN isoforms largely affects ocular cell differentiation from hiPSCs. For realization of hiPSC-based therapy, purification of the target cells is an important step. In this study, we examined use of LN isoforms for purification of hiPSC-derived corneal epithelial cells (iCECs) and manufacturing iCEC sheets. We found that differentiated ocular lineage cells, derived from hiPSCs have unique adhesion specificities and growth characteristics on distinct LN isoforms. iCECs and the other type cells showed specific cell adhesion to LN332, 411, 511 and LN211E8, respectively. Furthermore, LN332E8 promoted proliferation for epithelial cells including iCECs, but not for non-epithelial cells. By using the combination of specific adhesion of LN isoforms and magnetic separation, we successfully fabricated iCEC sheet with high purity without using fluorescence activated cell sorting (FACS). Thus, use of specific LN isoforms contributes to establishment of a simple and efficient method for manufacturing of iCEC sheets.F-2099TRANSPLANTATION OF HUMAN RETINAL PIGMENT EPITHELIAL SHEETS IN THE MACAQUE MONKEY EYEBlenkinsop, Timothy - Cell Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Liu, Zengping - Ophthalmology, National University of Singapore, Singapore Lingam, Gopal - Ophthalmology, National University Hospital, Singapore, Singapore Parikh, Bhav - Ophthalmology, National University of Singapore, Singapore Tan, Shu - Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore Stanzel, Boris - Ophthalmology, Augenklinik, Sulzbach, Germany Hunziker, Walter - Institute of Molecular and Cell Biology, Institute of Molecular and Cell Biology, Singapore, Singapore Su, Xinyi - Ophthalmology, National University Health System, Singapore, SingaporeAge-related macular degeneration (AMD) the most common cause of vision loss in the industrialized country and the third cause of blindness globally. Retinal pigment epithelial (RPE) cell death occurs before the majority of vision loss occurs implying that if these lost RPE are replaced, AMD progression can be mitigated. Adult human donor derived RPE (ahRPE) as a source for cell therapy for AMD is promising as this would allow HLA matching and has already been shown rescue vision in a rat model RPE dysfunction. Here we explored a surgical approach
458POSTER ABSTRACTSfor transplanting a sheet of ahRPE on a porous polyester scaffold under the macula of the macaque monkey. We followed the transplant for three months and evaluated retinal physiology and function. We found transplantation of ahRPE underneath the macula is safe and preserves vision and normal retina physiology.Funding Source: This research was funded by start-up lab funds at the Icahn School of Medicine at Mount Sinai and a grant from Singapore’s National Research Foundation.F-2101ISOLATION, CHARACTERIZATION AND COMPARISON OF HUMAN EXTRAOCULAR MUSCLE-DERIVED STROMAL CELLS IN NORMAL INDIVIDUAL AND PATIENTS WITH THYROID ASSOCIATED OPHTHALMOPATHYLew, Helen - Ophthalmology, CHA University, Seongnam, Korea Nepali, Sarmila - Ophthalmology, CHA University, Seongnam, Korea Park, Mira - Ophthalmology, CHA University, Seongnam, Korea Lew, Barklin - Dermatology, Kyunghee University, Seoul, KoreaMesenchymal stem cells (MSCs) are promising cell for various purposes like tissue engineering, regeneration and gene therapy. MSCs isolated from human extraocular muscles can be easily expanded in vitro, and can undergo multilineage differentiations, such as adipogenesis, chondrogenesis, osteogenesis, and even neuronal and myogenic differentiation. This study aimed to isolate, characterize and compare the extraocular muscle derived stromal cells (MDSCs) from normal individual and patients with thyroid-associated ophthalmopathy (TAO) patients. Extraocular muscles were obtained during strabismus surgery. Flow cytometric analysis was done to determine CD surface antigens, such as CD31, CD34 CD45, CD73, CD90, CD44 and CD59. We quantified various cytokines secreted from MDSCs such as, IL-1 , IL-1 , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17A, αβTNF- , IFN- and GM-CSF by using multi-analyze ELISA array αγkit. We performed Oil Red O staining for adipogenesis, Alzarin Red for osteogenesis and Alcian blue for chondrogenesis and real time PCR to measure the mRNA levels for myogenesis. Our results showed MDSCs from normal and TAO patient have similar potential tendency of characterization in terms of surface antigens and secretions of cytokines. Also, there was no significant difference between the tendency of MDSCs from normal and TAO patients in terms of multilineage differentiation, such as adipocytes, osteocytes and chondrocytes. However, myogenic differentiation of MDSCs from normal human showed higher than TAO patients. In conclusion, MDSCs from human extraocular muscles in normal individuals showed similar properties with TAO patients, and are good candidate for stem cell based therapies for treating various disordersFunding Source: This research was supported by the Ministry of Health and Welfare, Republic of Korea (Grant/Award Number: HI16C1559).F-2103CHARACTERIZATION OF RETINAL ORGANOIDS BY 2-PHOTON MICROSCOPY PRIOR TO TRANSPLANTATION INTO RETINAL DEGENERATE RAT MODELSXue, Yuntian - Biomedical Engineering, University of California, Irvine, CA, USA Kalakuntla, Tej - Stem Cell Research Center, University of California, Irvine, CA, USA McLelland, Bryce - AIVITA Biomedical Inc., Irvine, CA, USA Nistor, Gabriel - AIVITA Biomedical Inc., Irvine, CA, USA Keirstead, Hans - AIVITA Biomedical Inc., Irvine, CA, USA Browne, Andrew - Ophthalmology, University of California, Irvine, CA, USA Tang, William - Biomedical Engineering, University of California, Irvine, CA, USA Seiler, Magdalene - Stem Cell Research Center, University of California, Irvine, CA, USAStem cell-derived retinal organoids develop similar cell types and structures as in vivo tissue. Previous studies have shown that retinal organoid sheet transplantation improves visual function in retinal degenerate rats. Evaluating organoid quality and characteristics prior to transplantation is of substantial importance as it may directly determine the quality of its differentiation, integration in the host, and restoration of function. Traditional methods to monitor organoid development includes empirical observation through brightfield microscopy and histological immunostaining. Brightfield microscopy is highly user-dependent and histology requires organoid destruction. Therefore, a quantitative, non-invasive real-time imaging technique is needed for discerning tissue objectively. In this study, we applied 2-photon microscopy approaches to evaluate organoids: fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSpec), both using 740nm pulsed excitation. Organoids were produced and provided by AIVITA Biomedical Inc. FLIM phasor analysis of endogenous NADH provided indicated free and bound NADH distribution, which identifies the cells as glycolytic or oxidative and reflects the metabolism of the tissue. HSpec fluorescence emission light in the range of 420 nm to 690 nm analyzed via the phasor approach highlighted the distribution of retinol. We found that distinguishing between high- and low-quality organoids were very subjective by brightfield microscopy alone. However, FLIM and HSpec showed different structural and developmental stages of organoids while highlighting metabolic features and retinol distribution. Retinal photoreceptors are more glycolytic with higher retinol concentration, thus, the increased distribution of free NADH and retinol is a marker for photoreceptor cells. FLIM and HSpec are two powerful label-free imaging techniques to monitor retinal organoid development. Our future goal is to develop a standard criterion to evaluate organoid quality comprehensively and ultimately enhance the survival rate and efficacy of retinal sheet transplantation.Funding Source: California Institute for Regenerative Medicine: TRAN-1 Grant 10995
459POSTER ABSTRACTSSTEM CELL NICHESF-2105FROM SKIN TO NERVOUS SYSTEM: KERATINOCYTE DERIVED NEURAL CREST STEM CELLS, AN AUTOLOGOUS MULTIPOTENT CELL SOURCE FOR NEURODEGENERATIVE DISEASE TREATMENTAndreadis, Stelios T - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Tseropoulos, Georgios - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Moghadasi Boroujeni, Samaneh - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Mehrotra, Pihu - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Koontz, Alison - Biology and Biological Engineering, Caltech, Pasadena, CA, USA Polanco, Jessie - Pharmacology and Toxicology, University at Buffalo, SUNY, Buffalo, NY, USA Papili Gao, Nan - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Bajpai, Vivek - Stem Cell Biology, Stanford University, School of Medicine, Stanford, CA, USA Gunawan, Rudiyanto - Chemical and Biological Engineering, University at Buffalo, SUNY, Amherst, NY, USA Sim, Fraser - Pharmacology and Toxicology, University at Buffalo, SUNY, Buffalo, NY, USA Bronner, Marianne - Biology and Biological Engineering, Caltech, Pasadena, CA, USANeural crest (NC) cells play a central role in development of the peripheral nervous system, craniofacial skeleton, and skin pigmentation due to their broad multilineage differentiation potential into neurons (Neu), Schwann cells (SC), melanocytes (MC), and smooth muscle cells (SMC). Recently, we identified an easily accessible source of multipotent NC stem cells from human inter-follicular keratinocyte (KC) cultures (termed KC-NC) isolated from glabrous neonatal foreskin. Using small molecules FGF2 and IGF1 as well as inhibition of TGF- 1 we are able βto maintain SOX10+/FOXD3+ KC-NC in vitro. Genome wide transcriptomic analysis, as well as single cell RNA-seq showed upregulation of NC-specific genes and cell differentiation trajectories indicative of the epidermal origin of these cells. Clonal analysis verified the clonal multipotency of KC-NC towards all NC specific lineages (Neu, SC, MC, SMC) with varying efficiencies. Each NC derivative was also examined using functional assays, including electrophysiological tests for neurons, L-Dopa assays for MC and contractile force generation for SMC. Most notably, upon transplantation into chick embryos, KC-NC migrated along stereotypical pathways contributing to all NC-derivatives including Neu, SC, SMC and putative MC. Surprisingly, KC-NC could also be derived from aged adult donors (67-93 years old), maintained their multipotency in vitro and contributed to all NC derivatives in vitro and in ovo. In addition, CpG methylation analysis showed that the epigenetic age of adult KC-NC was significantly lower than that of KC and the chronological age of the donors. We are examining the regenerative potential of KC-NC by transplantation into the corpus callosum of the shiverer mouse model (shi/shi), an established model of demyelinating disease. Our data so far show that KC-NC migrated extensively and continued to proliferate a few weeks post-transplantation. Current efforts focus on long-term transplantation experiments to determine the potential of KC-NC for axonal myelination. Given the accessibility of human skin and the high proliferation capacity of KC and KC-NC, these cells represent a potentially useful source of autologous stem cells for studying or treatment of neurodegenerative diseases.Funding Source: NIBIB R01 EB023114 NYSTEM C30290GG NYSTEM C32601GG-3450000F-2107FEEDER-FREE CULTURE OF NAIVE HUMAN PLURIPOTENT STEM CELLS IN NORMOXIC CONDITIONSPijuan-Galito, Sara - School of Pharmacy, The University of Nottingham, UK Thompson, Jamie - School of Medicine, University of Nottingham, UK Lewis, Lara - School of Medicine, University of Nottingham, UK Tamm, Christoffer - IMBIM, Uppsala University, Uppsala, Sweden Annerén, Cecilia - IMBIM, Uppsala University, Uppsala, Sweden Merry, Cathy - School of Medicine, University of Nottingham, UKThe ‘naïve’ or ground state in pluripotent stem cells (PSCs) is distinctively different from traditional human PSC culture, relating to an earlier embryonic stage. The key characteristics of naïve pluripotency are: hypomethylation, double X chromosome activation, cloning capability, contribution to chimeras, and LIF-dependency. Human PSCs have shown resistance towards reversion to the ground state, with the different protocols described requiring feeder cells and hypoxic conditions, and reportedly showing DNA instability over long-term culture. The complex culture conditions also complicate their handling, with a selection step typically required before any analysis technique. We have previously shown that a human serum-derived protein, Inter-alpha inhibitor (IaI), supports human PSCs in coating-free conditions for long-term culture without loss of pluripotency or DNA instability. Here we present successful generation and culture of human and mouse naïve PSCs in coating-free conditions using the addition of IaI to the medium. In mouse PSCs, the cells show increased genomic stability, naïve marker expression and differentiation potential. In human PSCs, reversion to the naïve state is achieved without mouse feeder cells, and the method is compatible with normoxic conditions. Two different strategies are used: RSeT Medium (StemCell Technologies) and 2iGöY. For the adequate validation of this human naïve PSC culture method, Super-Resolution confocal microscopy has been used to show overall decreased DNA
460POSTER ABSTRACTSmethylation and distinct naïve marker expression in human PSCs reverted using IaI. Integrin engagement and expression is also assessed, showing lower focal adhesion formation and subsequent signalling in IaI culturing conditions, with naïve cells presenting overall decreased integrin expression. Phospho-proteome studies are also performed to investigate early signalling events linked to IaI-mediated attachment of both traditional and naïve hPSCs. After thorough characterisation, this new culture method has the potential to stream-line naïve human PSC culture for high-throughput and pharmaceutical applications, as IaI greatly simplifies the culturing method and is readily available at clinical grade.Funding Source: This research is supported by the Wellcome Trust and The Swedish Research Council.F-2109CHRONIC SENESCENCE AFTER SULFUR MUSTARD EXPOSURE RESULTED IN A SENESCENCE-ASSOCIATED SECRETORY PHENOTYPERothmiller, Simone - Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Jäger, Niklas - Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Bürkle, Alexander - Department of Biology, University of Konstanz, Germany Steinritz, Dirk - Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Thiermann, Horst - Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Schmidt, Annette - Bundeswehr Institute of Pharmacology and Toxicology, Munich, GermanyChronic wound healing disorder after sulfur mustard (SM) exposure is still not completely understood. As mesenchymal stem cells (MSC) are essential for wound healing, it is our hypothesis that SM induces chronic senescence in MSC. Such cells would be able to persist over long time periods and secrete proinflammatory cytokines known as the senescence-associated secretory phenotype (SASP). Therefore, in this initial study it was assessed whether SM is able to induce chronic senescence in MSC and whether changes in their secretome correspond to SASP which may be an underlying cause of the wound healing disorder. Human MSC were obtained from the bone marrow of donor femoral heads. Their quality was determined using differentiation techniques and specific cell surface markers by flow cytometry (CD14-/CD34-/CD45-/CD105+/CD106+). The cells were exposed to single doses of SM (1 - 40 μM) or H2O2 (200 μM) and the senescence-associated -galactosidase (SA- -gal) ββwas stained with X-gal substrate up to 31 days afterwards. Cell culture supernatants were collected at different time points and analyzed for over 70 chemokines, cytokines and growth factors using Bioplex assay. Senescence induction was increasing with time and concentration. Single dose exposure resulted in a stable senescence about 21 days later verified by SA- -gal βstaining. Secreted factors of senescent cells were observed to be early (i.e. MCP-1), only intermediate (i.e. IL-12 (p70)), late (i.e. CXCL9) or constantly elevated (i.e. IL-6), compared to correspondent controls. Moreover, the two analytes sTNF-R1 and CXCL16 showed differences between the SM- and the H2O2-induced senescent cells. In conclusion, SM single dose exposure is sufficient to induce chronic senescence in human MSC. These SM-induced senescent MSC may be unable to fulfil their regenerative role and contribute to the wound healing disorder. This is in line with our results, that these cells secreted a variety of proinflammatory cytokines. The time-dependence as well as the SM-induced specific upregulation of some analytes provide deeper insight into SM-induced senescence. Further research is necessary to prevent the secretion of these cytokines or selectively remove senescent cells which would be an innovative treatment strategy after SM exposure.F-2111HUMAN PERINATAL STEM CELL-DERIVED MATRIX SUPPORTS EXPANSION AND MAINTENANCE OF PLURIPOTENCY IN HUMAN STEM CELLSBlock, Travis J - R&D, StemBioSys, Inc., San Antonio, TX, USA Navarro, Mary - R&D, StemBioSys, Inc., San Antonio, TX, USA Sheldrake, Anne - R&D, StemBioSys, Inc., San Antonio, TX, USA Leeth, Rachel - R&D, StemBioSys, Inc., San Antonio, TX, USA Zeb, Adam - Biology, UTSA, San Antonio, TX, USA Navara, Christopher - Biology, UTSA, San Antonio, TX, USA Griffey, Sy - Operations, StemBioSys, Inc., San Antonio, TX, USAHuman induced pluripotent stem cells (iPSCs) hold promise for the study, diagnosis, and treatment of a variety of diseases based on their theoretical ability to proliferate indefinitely and differentiate into any cell type found in the body. However, in practice it is difficult to achieve mature phenotypes in differentiated cells, and iPSC cultures are notoriously inconsistent and difficult to maintain. Recently, a variety of substrates have been proposed to improve the consistency, ease-of-use, and cost of culturing pluripotent stem cells. It is now widely accepted that the microenvironment is critical for determining cell fate. Previously, our group has reported the development of scalable methods for production of bone marrow mesenchymal stem cell-derived matrices (BM-ECM) for isolation and expansion of mesenchymal stem cells without loss of stemness. Recently, we have reported tissue-specific differences in matrices that are critical for determining cell fate and function in vitro. We hypothesized that highly potent perinatal stem cells may create a matrix that better supports the maintenance of pluripotent stem cells. To test our hypothesis, we produced a cell-derived matrix from human amniotic fluid stem cells (AF-ECM). While possessing many of the same major components, the AF-ECM is distinct in structure and composition from BM-ECM. Relative to BM-ECM, the AF-ECM is an order of magnitude less stiff (p<.00001), twice as adhesive (p<.001), and much less rough (p<.0001). Importantly, these differences result in distinct function. While the BM-ECM does not support iPSC culture,
461POSTER ABSTRACTSAF-ECM supports proliferation of iPSCs comparable to that of Matrigel. Furthermore, AF-ECM also supports expansion and/or maintenance of other more mature cell types, including mesenchymal stem cells, chondrocytes, cardiomyocytes, and endothelial progenitor cells. Together, these data suggest that AF-ECM may possess extracellular cues that are ideal for the maintenance of iPSCs, as well as their differentiated progeny.F-2113DECODING THE MOUSE TOOTH DEVELOPMENT IN VIVO BY LARGE-SCALE SINGLE CELL RNA-SEQWang, Yaofeng - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Zhao, Yifan - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Chen, Xiaoming - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences Guangzhou, China Chen, Shubin - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Zheng, Hui - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Feng, Bo - School of Biomedical Sciences, The Chinese University of Hong Kong, China Cai, Jinglei - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Pei, Duanqing - Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, ChinaTooth develops from a serial of epithelial and neural crest-derived mesenchymal interactions throughout the initiation, bud, cap and bell stages with several signaling pathway families. The odontogenic potential is believed to shift from the dental epithelium to the dental mesenchyme at bud stage. However, the key signals for pulse-on the odontogenic potential in the regenerative organ germs are still unclear. Here we performed single-cell RNA-seq on the mouse dental development in vivo. We analysed the transcriptome profiles of ~96,000 cells of the dental regions in mouse embryo from day 10 to day 16. Cell developmental trajectories were clearly divided into the dental epithelial and mesenchymal paths. Besides, we revealed the key dental signaling pathways between the dental epithelium and mesenchyme, which activate the odontogenic potentials. Our study demonstrated the detailed molecular mechanisms in mouse tooth development. In addition, we discovered a small population of “seed” cells for dental development by the large-scale single-cell RNA-seq technique. Collectively, our study suggests the possible signaling pathway candidates for the tooth regeneration.Funding Source: This study was supported by the grants from National Key Research and Development Program of China (2017YFA0104800), the “Strategic Priority Research Program” of CAS (XDA16010401), and NFSC (81570944, 31300901).F-2115REDUCING EDGE EFFECT IN 96-WELL PLATE MSC SCREENING ASSAYS BY CONTROLLING THERMAL CONDITIONS DURING CELL PLATINGHenn, Alicia - BioSpherix, BioSpherix Medical, Parish, NY, USA He, Yan - Scientific, BioSpherix, Parish, NY, USA Darou, Shannon - Scientific, Bio, Paris, NY, USA Alm, Kersti - Laboratory, Phase Holographic Imaging, Lund, Sweden Yerden, Randy - Scientific, BioSpherix, Parish, NY, USAThe standard 96-well plate is often used for stem cell screening or tox assays. Researchers avoid using edge wells for cells, eliminating over one third of assay space, because of high variability. Others have reported that increased humidity or extended periods of time at room temp during cell settling can reduce edge effect. We previously reported that plating cells at constant 37°C, which is less stressful for cells, also reduces edge effect. Here we extend those studies to test the ability of thermal control of plating conditions to reduce variability in human bone marrow Mesenchymal Stromal/Stem Cells (MSC) toxicity assays. Our hypothesis was that when chamber temperatures were controlled to a constant 37°C during cell plating and settling we would see no difference between the variances in cell density in groups of wells including and excluding edge wells. We used the Xvivo System to provide fully controllable gas and temperature levels for both cell incubation and handling. We performed 24-hour MSC toxicity assays with low concentrations of disinfectants including isopropyl alcohol and Spor-Klenz (peracetic acid, acetic acid, and hydrogen peroxide). Using the HoloMonitor M4 microscope, we showed that cell settling in edge wells was random when MSC were plated at constant 37°C, unlike the directional rolling seen in edge wells when cells were plated at room temp and transferred to the incubator. Using standard MTT-based metabolic and crystal violet cell density assays, we showed that MSC cells were affected in a dose-dependent manner by exposure to commonly used disinfectants. Using a one-tailed F-test for this analysis, we saw no significant difference in variance when edge wells were used along with non-edge wells. We concluded that constant 37°C for cell plating and settling may improve reproducibility of 96-well based cell assays and save money, materials and time.CANCERSF-2117THE WNT CANONICAL PATHWAY CONTROLS CELL NUTRITION BY ACTIVATING MACROPINOCYTOSIS AND LYSOSOMAL DEGRADATION OF EXTRACELLULAR PROTEINSTejeda Munoz, Nydia - Biological Chemistry, University of California, Los Angeles (UCLA), CA, USA Albrecht, Lauren - Biological Chemistry, University of California,
462POSTER ABSTRACTSLos Angeles (UCLA), CA, USA De Robertis, Edward - Biological Chemistry, University of California, Los Angeles (UCLA), CA, USAActivation of the Wnt pathway is at the core of many human cancers. During canonical Wnt signaling, the Lrp6 and Frizzled bind Wnt and the complex is endocytosed. GSK3, Dishevelled and Axin are sequestered inside the intraluminal vesicles of late endosomes, also known as multivesicular bodies (MVBs). Formation of MVBs is required for Wnt signaling, by reducing levels of GSK3 in the cytosol. Recently, we have found that Wnt causes a great increase in levels of non-receptor mediated endocytosis of BSA-De-Quenched (BSA-DQ), a marker that fluoresces upon degradation in lysosome and that the activity of protein arginine methyltransferase 1 (PRMT1) is required for Wnt activation. From these and other experiments involving siRNAs Wnt emerges as a global regulator of endocytosis. We now report our discovery that the increased protein uptake was caused by macropinocytosis, in which membrane ruffles accompanied by F-actin reorganization folded over large amounts of extracellular fluid within minutes of Wnt treatment. PRMT1 activity and the ESCRT machinery were necessary for Wnt signaling. The endocytic uptake of high molecular weight tetramethylrhodamine dextran (TMR dextran 70 kDa, which has a hydrated diameter of 200 μM) was used as a macropinocytosis marker and was inhibited by ethyl-isopropyl amiloride (EIPA) and amiloride (a diuretic commonly used in the clinic that inhibits of plasma membrane Na+/H+ exchanger). The emerging connection between Wnt signaling, arginine methylation, lysosomal trafficking and endocytosis will lead to novel therapies for human cancers and stem cells in which Wnt signaling is activated.F-2119AUTOLOGOUS IMMUNOTHERAPY USING CANCER STEM CELLS AS ANTIGENIC SOURCEDillman, Robert O - Executive Department, AIVITA Biomedical, Irvine, CA, USA Hsieh, Candace - Clinical and Regulatory Affairs, AIVITA Biomedical, Irvine, CA, USA Poole, Aleksandra - Research and Development, AIVITA Biomedical, Irvine, CA, USA Nistor, Gabriel - Executive Department, AIVITA Biomedical, Irvine, CA, USA Keirstead, Hans - Executive Department, AIVITA Biomedical, Irvine, CA, USAIn certain conditions, populations of progenitor cells can restart the growth-proliferation cycle and produce cells with regenerative scope. This stem cell niche is dormant until proper signals trigger re-entry into the proliferation cycle. The re-entry signals can originate from events such as trauma, cell damage, or microorganism aggression. Genetic variants or acquired mutations can perturb the cell cycle, resulting occasionally in survival and proliferative advantages. Cells in the tumor stem cell niche, or tumor initiating cells (TIC) are in low frequency, usually representing < 0.5% of the cells in the tumor mass and may only temporarily express neoantigens, thus eluding an immune response or becoming subject to immune-editing. In previous trials in patients with metastatic melanoma, vaccines designed to express antigens from TICs were associated with minimal toxicity, and encouraging survival. The vaccine consisted of autologous dendritic cells loaded with antigens from autologous TICs (DC-TC). In a single-arm trial 54 patients had a projected 5-year survival rate of 54%,. In a randomized phase II clinical trial patients treated with DC-TC had superior survival compared to those treated with a tumor cell vaccine (median 43 vs 20 months, 3-year OS 65% vs 25%, and a 70%reduction in the risk of death. In contrast, similar therapies using whole tumor preparations or single peptide antigens failed to show significant survival benefit in other trials. We concluded that for the success of the immune therapy using ex-vivo loaded antigen-presenting cells, the isolation and purification of the autologous TIC was a critical step in manufacturing. Our manufacturing methods to isolate and amplify TICs consist of dissociating the tumor, exposing the cells to a serum-free media, and promoting TIC spheroids formation. The growth media lacks differentiation signals usually found in serum and is enriched with mTOR pathway stimulating factors. In addition, spheroid formation encourages paracrine and juxtacrine signaling that is essential for stem cell niche maintenance. Here we present the characterization of the generated TIC population by commonly recognized phenotype markers for stem cells, by DNA exome and methylation analysis, as well by a tumorigenesis assay in NOD/SCID mice.F-2121CALCIUM CHANNELS AS NOVEL THERAPEUTIC TARGET OF OVARIAN CANCER STEM CELLSMin, Sang Hyun – New Drug Development Center (NDDC), Daegu Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu, KoreaOvarian cancer has been known as the most lethal gynecologic malignancy and the overall 5-year survival rate for epithelial ovarian cancer is only almost 30%. Epithelial ovarian cancer shows drug resistance, frequent recurrence and poor prognosis. Although chemotherapy removes most cancer cells, a few cancer stem cells (CSCs) still remain, and could be a major contributor of drug resistance. To isolate compounds with an therapeutic effect on cancer stem cells in ovarian tumor, we used ovarian cancer stem-like cells (CSLCs) via sphere culture of A2780 epithelial ovarian cancer cells for screening FDA-approved library based on a high-throughput screening (HTS) system. Four compounds (known as calcium channel blockers, CCB) which block voltage-gated calcium channels were selected to inhibit the proliferation of ovarian CSLCs. Interestingly, voltage-gated calcium channels are overexpressed in ovarian CSLCs, whereas down-regulation of calcium channel genes reduced the properties of ovarian CSLCs. The treatment of CCBs decreased sphere formation and viability of ovarian CSLCs as well as induced apoptosis. In addition, CCBs destroyed stemness and inhibited AKT and ERK signaling pathway in ovarian CSLCs. Furthermore, their effects on combination therapy with cisplatin showed synergistic effect
463POSTER ABSTRACTSon the inhibition of ovarian CSLCs viability and proliferation. Taken together, the four calcium channel blockers can be potential therapeutic drugs for prevention of ovarian cancer recurrence.Funding Source: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (2015M3A9C7030181).F-2123MOLECULAR NETWORK PATHWAY MECHANISM IN DRUG RESISTANCE, CANCER AND STEM CELLSTanabe, Shihori - Division of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Japan Aoyagi, Kazuhiko - Department of Clinical Genomics, National Cancer Center Research Institute, Tokyo, Japan Hirose, Akihiko - Division of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Japan Ono, Ryuichi - Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Japan Quader, Sabina - Kataoka-Kinoh Lab, Innovation Centre of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, Kawasaki, Japan Sasaki, Hiroki - Department of Translational Oncology, National Cancer Center Research Institute, Tokyo, Japan Yokozaki, Hiroshi - Department of Pathology, Kobe University of Graduate School of Medicine, Kobe, JapanMolecular network pathways interact with each other and regulate cellular phenotypes. Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance. To reveal the molecular signaling network pathway mechanism, gene expression in mesenchymal stem cells (MSCs) and diffuse-type gastric cancer (GC) have been analyzed and compared. Microarray analysis was performed using total RNA purified from MSCs and diffuse-type GC in GeneChip U133Plus 2.0 platform. The gene expression profiling demonstrated that gene expression of cadherin 1 (CDH1), erb-b2 receptor tyrosine kinases (ERBBs), and patched 1 (PTCH1) were up-regulated in diffuse-type GC compared to MSCs, whereas CDH2 and fibronectin 1 (FN1) were down-regulated. Wnt/beta-catenin signaling, as well as ERBB and PTCH1 signaling networks, involved in EMT, CSCs and drug resistance, have been investigated and profiled in bioinformatics. In conclusion, the EMT-related molecular network pathways have been revealed in MSCs and diffuse-type GC, which may contribute into the elucidation of mechanism in the drug resistance of CSC population.F-2125THE bHLH FACTOR E47 DOWNREGULATES ONCOGENIC C-MYC IN PANCREATIC CANCER THROUGH EPIGENETIC MODIFICATIONSCheng, Xiuyuan - Biology, San Diego State University, San Diego, CA, USA Scully, Kathleen - Development, Aging and Regeneration, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Signaevskaia, Lia - Development, Aging and Regeneration, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA Itkin-Ansari, Pamela - Development, Aging and Regeneration, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USADysregulation of c-MYC is a key factor in the malignant transformation of pancreatic ductal adenocarcinoma (PDA). Recent studies have shown: high levels of MYC drive down expression of bHLH transcription factor, E47 in the pancreas. Moreover, the most common mutations in noncoding regions are binding sites of E47 and its homologs suggesting this protein may serve a tumor-suppressor role in pancreatic cancer. Consistent with this, our data indicate that restoring the transcriptional activity of E47 can induce growth arrest in PDA cells by downregulating the number of MYC RNA transcripts. To better understand the molecular mechanism of this reciprocal relationship between MYC and E47, we performed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing). One finding is that the chromatin accessibility around E47 enhancer regions is diminished during pancreas pathogenesis. In contrast, chromatin accessibility of a discrete regulatory element near the 3’ end of MYC locus is elevated in cancerous cells compared to pre-cancerous cells, and this corresponds with the increased amount of MYC transcripts in cancer. Remarkably restoration of E47 activity in pancreatic cancer reduced the chromatin accessibility of the regulatory element in the MYC locus. Thus, we hypothesize that E47 is acting as a tumor suppressor by downregulating MYC transcription through epigenetic modifications. Further efforts we want to further investigate the functional interplay between E47 and MYC during the progression of pancreatic cancer.Funding Source: California Institute for Regenerative MedicineF-2127ANALYSIS OF CHEMOTHERAPY SENSITIVITY IN HUMAN PLURIPOTENT STEM CELLSNissenbaum, Yonatan (Jonathan) - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Hebrew University of Jerusalem, Israel Segal, Emanuel - Genetics, The Hebrew University of Jerusalem, Israel Peretz, Mordecai - Genetics, The Hebrew University of Jerusalem, Israel
464POSTER ABSTRACTSAvior, Yishai - Genetics, The Hebrew University of Jerusalem, Israel Benvenisty, Nissim - Genetics, The Hebrew University of Jerusalem, IsraelThe study of cancer has benefited dramatically from the availability of tumor tissues and the development of cancer cell lines. Nevertheless, the cancer genome complexity of and differences between species frequently limit clinical translation. In contrast, human pluripotent stem cells (hPSCs) offer a reliable and efficient platform with the advantage of a normal and uniform genetic background. Resistance to anti-cancer drugs represents a major barrier for an efficient treatment and subjects a significant number of patients to ineffective treatment and to excessive chemotherapy side-effects. To assess the potential of hPSCs as a tool to predict anti-cancer drugs sensitivity, we exposed the cells to 107 different chemotherapies from the Approved Oncology Drugs Set, obtained from the NCI. These drugs target over 20 different pathways and mechanisms of actions affecting malignant processes. As expected, hPSC sensitivity (monitored by cell viability assays) varied between the different groups ranging from low to extreme sensitivity. Significant sensitivity has been observed to the Topoisomerase-inhibitors group and the epigenetic inhibitors (DNA methyltransferase and histone deacetylase inhibitors) group. We then evaluated the predictive power of hPSCs compared to human cancer cell lines (hCCls), utilizing Sanger’s Genomics of Drug Sensitivity in Cancer and the NCI’s Growth Inhibition Data. This comparison consisted of 88 shared drugs among the independent studies. This analysis produced a significant positive correlation between hPSCs and hCCls (averaged between all cancer cell lines). This high correlation highlights the usefulness of hPSCs in drug response prediction. In contrast, differences have been identified between hPSCs and hCCls in response to epigenetic and topoisomerase inhibitors, highlighting the unique sensitivity of undifferentiated cells to these substances. Together, our results demonstrate the potential of hPSCs in anti-cancer drug research as well as offer new avenues to the study of hPSC and hCCLs cellular differences.F-2129TRIB2 IS AN IMPORTANT REGULATOR OF CANCER STEM CELL-PROPERTIES IN OVARIAN CANCER CELLSKim, Yuna - Department of Physiology, Pusan National University, Yangsan, Korea Kim, Daekyoung - Physiology, Pusan National University, Yangsansi, Korea Park, Insoo - Physiology, Pusan National University, Yangsansi, Korea Lee, Seoyul - Physiology, Pusan National University, Yangsansi, Korea Shin, Minjoo - Physiology, Pusan National University, Yangsansi, Korea Kim, Jaeho - Physiology, Pusan National University, Yangsansi, KoreaIt is important to find the target gene of recurring cancer cells to reduce ovarian cancer. The tribbles homolog 2 (TRIB2) interacts and modulates signaling pathway in significant cellular processes. The relevance of TIRB2 in relation to the tumorigenic potential of ovarian cancer is currently unknown. In the current study, we find that TRIB2 promote cancer stem cell property in ovarian cancer cells through WNT signaling. TRIB2 knockdown in A2780 ovarian cancer cells decreased sphere forming ability, expression of stemness and EMT related genes, cell migration, drug resistance, whereas TIRB2 overexpression of stemness and EMT related genes, cell migration, drug resistance.TRIB2 knockdown considerably decreased tumor size. Furthermore, TRIB2 overexpression increase promoter activity of TOP-Flash, Beta-catenin stability and nuclear beta-catenin accumulation. These results suggest that TRIB2 function as a transcriptional factor for beta-catenin and as and oncogene by enhancing cancer stem cell property in ovarian cancer cells. Together, these data identify that TRIB2 can be important target for developing a therapeutic agent for ovarian cancer patients.NEURAL DEVELOPMENT AND REGENERATIONF-3001THE KYNURENINE PATHWAY OF TRYPTOPHAN METABOLISM MODULATES MOUSE NEURAL STEM CELL PROLIFERATIONLovelace, Michael D - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Sardesai, Varda - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Ayeni, Femi - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Walters, Edward - Department of Science, Notre Dame University, Sydney, Australia Suzuki, Kazuo - HIV Laboratory, St Vincent’s Centre for Applied Medical Research, Sydney, Australia Walker, David - Department of Physiology, Hudson Institute, Monash University, Melbourne, Australia Jones, Simon - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Taylor, Rosanne - Faculty of Veterinary Science, University of Sydney, Sydney, Australia Croitoru-Lamoury, Juliana - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Lamoury, Francois - Peter Duncan Neurosciences Research Unit, St. Vincent’s Centre for Applied Medical Research, Sydney, Australia Brew, Bruce - Peter Duncan Neurosciences Research Unit,
465POSTER ABSTRACTSSt. Vincent’s Centre for Applied Medical Research, Sydney, AustraliaThe search for molecules which critically regulate neural stem cell (NSC) proliferation is ongoing, underpinning future production of cell lineages for therapy, while helping understand why innate repair in neurodegenerative and neuroinflammatory diseases fails. Our ongoing research has investigated a role of the kynurenine pathway (KP) in healthy metabolism and neurological diseases. The KP critically regulates bioavailability of the essential amino acid tryptophan, and is induced by interferon treatment. In MS the KP is dysregulated, producing high levels of metabolites including the potent neurotoxin Quinolinic acid. We investigated the hypothesis that modulating the KP by interferon treatment drove changes in NSC proliferation. E14 mouse neurospheres were generated and cultured. Agonists, antagonists or siRNAs to KP enzymes were used to dissect the pathways. Interferon-gamma (IFN-g) significantly upregulated (4.1 fold) indoleamine-2,3-dioxygenase (IDO-1; the initial rate-limiting enzyme that metabolises Tryptophan) mRNA in NSCs cultured in proliferative conditions, and to a lesser extent with differentiation. NSCs express all KP enzymes and notably, IFN-g upregulates other KP enzymes and has the most prominent effect on mRNA of Kynureninase (KYNU), then partial IDO-1, full IDO-2 and Kynurenine Monooxygenase (KMO). 10 IU/mL IFN-g leads to impaired NSC proliferation (*p=0.0323), and alteration of the metabolic state of NSCs including their NAD+/NADH ratio (representing cellular energy levels) via Trp depletion (needed for protein biosynthesis). KP metabolites themselves can modulate NSC proliferation, as co-treatment with the neuroprotective Kynurenic Acid partially reversed the reduced proliferation in IFN-g-treated NSCs. IFN-beta negligibly affected IDO-1 levels, but induced IDO-2, and significantly decreased proliferation and downstream enzyme KMO. This study is the first characterization of KP enzyme expression in NSCs. We show that KP enzymes play a specific role in the biology of NSCs and tryptophan metabolism, including the dominant regulation of the KP by interferons. Selective KP inhibition could minimize cell death and impaired regeneration during inflammatory episodes and optimize NSC proliferation and differentiation with direct therapeutic applications.Funding Source: This study was supported by the National and Health Medical Research Council (NHMRC), St. Vincent’s Clinic Foundation Sydney, and the University of New South Wales (Sydney, Australia).F-3003RAT HIPPOCAMPAL NEURAL STEM CELL MODULATION USING PDGF, VEGF, PDGF/VEGF AND BDNFGomila Pelegri, Neus - School of Life Sciences, University of Technology Sydney, Australia Gorrie, Catherine - School of Life Sciences, University of Technology Sydney, Ultimo, Australia Santos, Jerran - School of Life Sciences, University of Technology Sydney, Ultimo, AustraliaNeural stem cells have become the focus of many studies as they have the potential to differentiate into all three neuronal lineages. This may be utilised to develop new and novel ways to treat neurological conditions such as spinal cord and brain injury, especially if the stem cells can be modulated in vivo without additional invasive surgical procedures. This research aimed to investigate the effects of the growth factors Vascular Endothelial Growth Factor, Platelet Derived Growth Factor, Brain Derived Neurotrophic Factor and Vascular Endothelial Growth Factor/Platelet Derived Growth Factor on hippocampal derived neural stem cells. Cell growth and differentiation were assessed using immunohistochemistry and glutaminase enzyme assay. Cells were cultured for 14 days and treated with different growth factors at two different concentrations 20ng/mL and 100ng/mL. At 2 weeks, cells were fixed, and immunohistochemistry was conducted to determine cellular differentiation using antibodies against GFAP, Nestin, OSP and NF200. Cell media supernatant was also collected during treatment to determine glutaminase levels secreted by the cells as an indicator of neural differentiation. VEGF/PDGF at 100ng/mL had the greatest influence on cellular proliferation of HNSC, which also stained positively for Nestin, OSP and NF200. In comparison, HNSCs in other treatments had poorer cell health and adhesion. HNSC in all treatment groups displayed some differentiation markers and morphology but this is most significant in 100ng/ml VEGF/PDGF treatment. VEGF/PDGF growth factor combination produced the optimal effect on the HNSCs inducing the differentiation pathway exhibiting oligodendrocytic and neuronal markers. This is a promising finding that should be further investigated in brain and spinal cord injuryFunding Source: University of Technology Sydney internal funding Australian Government Research Training Program Stipend scholarshipF-3005CENPJ REGULATES CILIA DISASSEMBLY AND NEUROGENESIS IN THE DEVELOPING MOUSE CORTEXWu, Qian - Beijing Normal University, China Ding, Wenyu - Chinese Academy of Sciences, ChinaPrimary cilia are microtubule-based protuberances that project from the eukaryotic cell body to sense the extracellular environment. Ciliogenesis is closely correlated to the cell cycle and defects of cilia are related to human systemic diseases such as primary ciliary dyskinesia. However, the role of ciliogenesis in cortical development remains unclear. Here, we demonstrate that Cenpj, a protein that is required for centriole biogenesis, plays a role in regulating cilium disassembly in vivo. Depletion of Cenpj in neural progenitor cells results in long cilia and abnormal cilia disassembly. Radial glial cell (RG cells) with Cenpj depletion exhibit uncompleted cell division, reduced cell proliferation, and increased cell apoptosis in the developing mouse cerebrum cortex, leading to microcephaly. In addition, Cenpj depletion causes long and thin primary cilia and motile cilia in adult neural stem cells and reduced cell proliferation
466POSTER ABSTRACTSin the subventricular zone. Furthermore, we show that Cenpj regulates cilia disassembly and neurogenesis through Kif2a, a plus-end-directed motor protein. These data collected from mice of both sexes provide insights into how ciliogenesis plays roles in cortical development and primary microcephaly induced by Cenpj mutations in humans.Funding Source: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDA16020601, XDB32010100) and the National Basic Research Program of China (Grants 2017YFA0103303 and 2017YFA0102601).F-3007LINEAGE ANALYSIS OF EMBRYONIC NEURAL PRECURSORS FROM THE SUBPALLIAL GERMINAL ZONE AT SINGLE CELL RESOLUTIONYammine, Samantha - Molecular Genetics, University of Toronto, ON, Canada Burns, Ian - Molecular Genetics, University of Toronto, ON, Canada Gosio, Jessica - Molecular Genetics, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada Merritt, Daniel - Institute of Medical Science, University of Toronto, ON, Canada Innes, Brendan - 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, CanadaWe define two distinct types of neural stem cells (NSCs) and their downstream neural progenitor cells (NPCs) from the embryonic ventral germinal zone (GZ) using clonal lineage tracing and single cell transcriptomics. Primitive (p)NSCs express Oct4 but do not express GFAP, and mouse embryo-derived pNSCs form clonogenic neurospheres proliferate rapidly when grown in LIF. pNSCs arise earlier in development than GFAP-expressing definitive (d)NSCs that form clonogenic neurospheres in FGF2/EGF. To assess the differences in functional outputs of both NSC types, we performed clonal lineage tracing within clonal neurospheres grown in either LIF or EGF/FGF2, to enrich for neural progenitor cells (NPCs) directly downstream pNSCs or dNSCs, respectively. PNSCs from the E17.5 ventral forebrain GZ gave rise to more unipotent neuronal progenitor cells than dNSCs, and dNSCs gave rise to more unipotent astrocyte progenitor cells. Both NSCs gave rise to bipotent NPCs that produce neurons and astrocytes, which consistently proliferated more than unipotent NPCs. Surprisingly, pNSCs give rise to these more unipotent neuronal progenitor cells that expressed GFAP+ before they became post-mitotic neurons. These clonal progenitor lineage tracing data allowed us to construct a hierarchy of progenitor subtypes downstream of pNSCs and dNSCs. To validate this hierarchy, identify markers for distinctly specified NPCs, and assess single cell RNA expression differences between these NPCs, we performed Drop-seq on E17.5 neural stem and progenitors cells from clonal neurospheres grown in either LIF or FGF2/EGF. UMAP-based Monocle 3alpha and p-Creode served as additional methods to map out the transition states of NPCs. These bioinformatic lineage analyses revealed a unique pathway for making OB interneurons downstream of pNSCs compared to other neuronal cell types. Combined, these data provide single cell resolution of NPCs present in the pre-natal brain, including NPCs downstream of rare pNSCs that would likely be missed from population level analyses in vivo.F-3009DIRECT CONVERSION OF HUMAN EMBRYONIC STEM CELL-DERIVED GLIAL PROGENITORS INTO MIDBRAIN DOPAMINERGIC NEURONSNolbrant, Sara - Department of Experimental Medical Science, Lund University, Lund, Sweden Hoban, Deirdre - Department of Experimental Medical Science, Lund University, Lund, Sweden Giacomoni, Jessica - Department of Experimental Medical Science, Lund University, Lund, Sweden Rylander Ottosson, Daniella - Department of Experimental Medical Science, Lund University, Lund, Sweden Goldman, Steven - Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA Parmar, Malin - Department of Experimental Medical Science, Lund University, Lund, SwedenDirect in vivo conversion is emerging as a novel route for generating new neurons for brain repair. Oligodendrocyte progenitor cells (OPCs) present an interesting target for direct neuronal conversion since these cells are replenishable and since they are found in relative abundance throughout the adult brain. To date, all in vivo conversion studies have been conducted using resident rodent glia and the outstanding question whether human glia can be converted into neurons within the adult brain remains to be resolved. To address this question we have differentiated human embryonic stem cells (hESCs) into glial progenitor cells (GPCs), to obtain a human glial cell source that is highly expandable and possible to cryopreserve. Using these hESC-derived GPCs we have identified a conversion factor combination that efficiently convert human GPCs into midbrain dopaminergic (mDA) neurons, the cell type that selectively degenerates in Parkinson’s disease (PD). The resulting neurons express markers characteristic of DA neurons and are functionally mature. Building on our in vitro data, we are now transplanting our hESC-derived GPCs into a rat model of PD to convert them into mDA neurons in vivo using a doxycycline inducible system. This study will provide us with novel insight of the possibility to directly convert human glial cells into subtype specific neurons in vivo, and to assess the therapeutic potentials of this approach.Funding Source: The New York Stem Cell Foundation, the Swedish Research Council, Swedish Parkinson Foundation (Parkinsonfonden), and Knut and Alice Wallenberg Stiftelse. M.P is a New York Stem Cell Foundation Robertson Investigator.
467POSTER ABSTRACTSF-3011SPINAL CORD INJURY: EFFECT OF BIOMATERIAL AND IMMUNE RESPONSE ON HUMAN NEURAL STEM CELLS FATENekanti, Usha - Anatomy and Neurobiology, University of California, Irvine, CA, USA Guardamondo, Glenn - Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USA Lawmaster, Lindsey - Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USA Ngotran, Anthony - Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USA Seidlits, Stephanie - Brain Research Institute, University of California, Los Angeles, CA, USA Dumont, Courtney - Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA Cummings, Brian - Physical Medicine and Rehabilitation/Neurological Surgery, University of California Irvine, CA, USA Shea, Lonnie - Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA Anderson, Aileen - Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USASpinal Cord Injury (SCI) is a devastating condition, which results in loss of sensory, motor, and reflex function below the level of injury. Following the initial injury, disruption of the blood spinal cord barrier allows infiltration of peripheral immune cells from the blood into the spinal cord tissue. We have recently shown that immune cells and their components can influence the outcome of reparative strategies, including human neural stem cell (hNSC) transplantation, resulting in changes in fate, migration and efficacy. Additionally, we have demonstrated that implantation of a poly (lactide-co-glycolide) (PLG) biomaterial bridge can lead to regeneration of the sensory and motor axons including the corticospinal tract (CST) in association with recovery of function. My overall goal is to test the combinatorial effect of two regenerative approaches, implantation of a poly (lactide-co-glycolide) (PLG) biomaterial bridge and transplantation of hNSC. However, PLG bridges become extensively cellularized after implantation, including recruitment of macrophages and other innate immune cells, suggesting the potential for the innate immune response to modulate hNSC within the PLG-bridge. Additionally, macrophages are the dominant cell population within the implanted PLG-bridge during axonal regeneration. These macrophages contribute to the biodegradation of PLG-bridge and remain in proximity to the regenerating fibers, suggesting that PLG-bridges may support regeneration by modulating the immune environment. Accordingly, we have investigated the combined effect of PLG and immune cues on hNSC fate in-vitro to understand the synergetic outcome of this combinatorial approach. Thus, as a secondary objective we studied the immunomodulatory effect of the PLG scaffold on innate immune cell intrinsic properties that can alter stem cell fate. These studies provide insight into the interaction between PLG, hNSC, and innate inflammatory cells in vitro in order to enhance in vivo testing of combinatorial administration of PLG bridges to promote axonal regeneration and hNSC to support myelination of new axons after SCI.Funding Source: National Institutes of Health (NIH) 5R01EB005678-12 (L.D.S., A.J.A., and B.J.C.)F-3013SOXC TRANSCRIPTION FACTORS ARE CRUCIAL REGULATORS OF SENSORY PROGENITOR DIFFERENTIATION IN THE MOUSE ORGAN OF CORTIWang, Xizi - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USA Gnedeva, Ksenia - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Tao, Litao - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Llamas, Juan - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Yu, Haoze - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Trecek, Talon - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Makmura, Welly - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Segil, Neil - Department of Development, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAAll vertebrates use sensory organs containing hair cells to perceive sound and motion. These sensory receptors are rare in number and after damage do not regenerate, leading to permanent hearing loss and balance disorders. The cells that give rise to the organ of Corti in the mammalian inner ear first exit the cell cycle within a well-defined prosensory domain and then initiate a process of differentiation that gives rise to a stereotyped mosaic of hair cells and supporting cells. The mechanisms governing prosensory fate commitment are not known, but are important for a full understanding of the failure of regeneration in this system. Sox genes are well-established regulators of cell fate determination. We show that during sensory epithelia development in the organ of Corti, two members in the SoxC family, Sox4 and Sox11, are highly expressed in the sensory progenitors at the onset of hair cell differentiation. Heterozygous knockout of SoxC genes causes partial hearing loss and vestibular dysfunction, while homozygous loss blocks progenitor cell differentiation towards the hair cell fate. Using transcriptomic and epigenomic analysis, we characterized the
468POSTER ABSTRACTStransition from proliferating progenitors to differentiated hair cells, and assayed the effects of the loss on SoxC genes on this process. By assessing chromatin accessibility (ATAC-seq), we observe that the potential regulatory elements associated with hair cell fate-commitment emerge in the postmitotic progenitor cells, and are enriched for Sox transcription factor binding motifs. Using conditional inactivation of Sox4 and Sox11 genes in the inner ear, combined with Chip-seq analysis, we demonstrate that accessibility of these newly emerged regulatory elements is directly dependent on SoxC gene expression. Single cell sequencing confirms that conditional loss of SoxC genes leads to downregulation of the predicted prosensory gene targets in the organ of Corti progenitor cells. Consistent with this observation, overexpression of SoxC prior to cell fate determination enhances sensory differentiation. Our results reveal that through chromatin remodeling, and consequent hair cell-specific gene expression, SoxC transcription factors are crucial for sensory cell fate determination in the progenitor population of the organ of Corti.F-3015ANATOMICAL AND SINGLE CELL TRANSCRIPTOMIC PROFILING OF THE DEVELOPING ENTORHINAL CORTEX NEURONAL CIRCUIT IN THE PIGLiu, Yong - Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark Bergmann, Tobias - Department of Veterinary and Animal Science, University of Copenhagen, Denmark Lee, Julie - Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark Pfisterer, Ulrich - Biotech Research and Innovation Centre, University of Copenhagen, Denmark Handfield, Louis-Francois - Wellcome Sanger Institute, Hinxton, UK Mori, Yuki - Center for Translational Neuromedicine, University of Copenhagen, Denmark Martinez, Andrea - Biotech Research and Innovation Centre, University of Copenhagen, Denmark Seemann, Stefan - Center for non-coding RNA in Technology and Health, University of Copenhagen, Denmark Vargas, Irene - Biotech Research and Innovation Centre, University of Copenhagen, Denmark Vidal, Juan - Department of Veterinary and Animal Science, University of Copenhagen, Denmark Pihl, Maria - Department of Veterinary and Animal Science, University of Copenhagen, Denmark Kornum, Birgitte - Department of Neuroscience, University of Copenhagen, Denmark Thomsen, Preben - Department of Veterinary and Animal Science, University of Copenhagen, Denmark Mollgard, Kjeld - Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark Hyttel, Poul - Department of Veterinary and Animal Science, University of Copenhagen, Denmark Khodosevich, Konstantin - Biotech Research and Innovation Centre, University of Copenhagen, Denmark Witter, Menno - Kavli Institute for Systems Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway Gorodkin, Jan - Center for non-coding RNA in Technology and Health, University of Copenhagen, Denmark Hemberg, Martin - Wellcome Sanger Institute, Hinxton, UK Pers, Tune - Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark Hall, Vanessa - Department of Veterinary and Animal Science, University of Copenhagen, DenmarkThe entorhinal cortex (EC) acts as a gateway for information traveling in and out of the hippocampal formation and is important for spatial memory. The stellate cell (SC) resides in layer II (LII) of the medial EC, projects to the dentate gyrus and contributes to both the grid and border cell phenotypes. However, very little is known about the molecular identity of SCs. SCs express reelin (RELN+) and are negative for calbindin (CALB1-) and this differentiates them from the other main principle neurons. We, therefore, decided to probe the developing EC to identify key growth factors and cytokines that are critical for the formation of SCs in the pig. We selected the pig as a model since its advantageous compared to the rodent in reflecting human EC development due to the pig brains larger size, gyrencephalic anatomy and longer gestation period (gestation length 114 days). Nissl staining and postmortem structural MRI indicated that entorhinal-like cells present at the superficial border of LII at E50 and a more developed EC architecture from E60 onwards. We characterized cortical lamination by performing immunohistochemistry using antibodies against SATB2, CTIP2, GFAP, BLBP, SOX2, PAX6, TBR1 and TBR2 during EC development. We identified the formation of the EC between E26 and E33 and a distinct LII forming from E33 to E39. Interestingly, LV/LVI formed after LII, and prior to LIII/IV at E39 to E50. LIII/IV was first observed at E60 indicating the cortex forms disparately to normal cortical lamination in a sandwich-like manner. Furthermore, we identified a population of RELN+/CALB1-/MAP2+/CTIP2+ neurons at the superficial border of LII at E60, which we presume to be the SCs. The percentage of these cells in LII increased from 1.87% of the population in the cortical plate at E60 to 51.77% within LII by E100. Single-cell RNA sequencing data was performed on EC, which has led to the identification of unique gene signatures for 4 progenitors, 10 excitatory neuron and 7 inhibitory neuron populations. This study has led to the characterization of the developing EC in a new species and documents when SCs arise in the EC. We are now using this data to develop in-vitro protocols to produce SCs from iPSCs.
469POSTER ABSTRACTSNEURAL DISEASE AND DEGENERATIONF-3017NEURAL STEM CELLS FOR DISEASE MODELING AND EVALUATION OF THERAPEUTICS FOR TAY-SACHS DISEASELi, Rong - National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA Vu, Mylinh - National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA Beers, Jeanette - Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Zou, Jizhong - Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Zheng, Wei - National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USATay-Sachs disease (TSD) is a rare neurodegenerative disorder caused by autosomal recessive mutations in the HEXA gene that encodes -hexosaminidase. Deficiency of -hexosaminidase ββresults in accumulation of GM2 ganglioside, a glycosphingolipid, in lysosomes. Currently, there is no effective treatment for TSD. In this study, we generated induced pluripotent stem cells (iPSCs) from two TSD patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs), which exhibited the disease phenotype of accumulations of GM2 ganglioside and other lipids in lysosomes. Treatment with recombinant human Hex A enzyme abolished the lipid accumulation in these cells. We also found that hydroxypropyl- cyclodextrin (HP CD) and ββδ-tocopherol significantly ameliorated the lipid accumulations in patient cells. A combination of HP CD and -tocopherol βδsignificantly decreased the lipid accumulation to levels comparable to wild-type cells. Furthermore, a drug combination screening of a small compound library was performed to identify compounds that enhance the effect of HP CD in the patient βcells. These results provide insights to better understand disease pathophysiology and suggest a potential drug development path for treatment of Tay-Sachs diseases.F-3019SYNERGISTIC EFFECTS OF EXERCISE AND CELL THERAPY FOR PARKINSON’S DISEASE MODEL RATSTorikoshi, Sadaharu - Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Morizane, Asuka - Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Takahashi, Jun - Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, JapanCell transplantation is expected to be a promising treatment for Parkinson’s disease (PD), in which re-innervation of the host striatum by the grafted dopamine (DA) neurons is essential. Especially, the dorsolateral part of the striatum is important because it is the target of midbrain A9 DA neurons, which are degenerated in PD pathology. The effect of exercise on survival and maturation of the grafted neurons has been reported in several neurological diseases models, but never in PD models. In this study, we transplanted the ventral mesencephalic neurons of embryonic (E12.5) rats into the striatum of adult chronic PD model rats, and combined treadmill training as exercise after transplantation. Then the survival and neurite extension of the grafted DA neurons were evaluated. Exercise significantly increased the number of survived DA neurons. Moreover, it promoted the neurite extension from the graft toward the dorsolateral part of the striatum. These effects are supposed to be mediated through the modulation of neurotrophic factors and inflammatory cytokines in the host brain environment. This study indicates a beneficial effect of exercise after cell transplantation in PD.F-3021BREAST CARCINOMA AMPLIFIED SEQUENCE 2 (BCAS2) REGULATES ADULT NEUROGENESIS THROUGH -CATENINΒChen, Show-Li - Microbiology, National Taiwan University, Taipei, TaiwanBreast carcinoma amplified sequence 2 (BCAS2) regulates β-catenin gene splicing; the loss of BCAS2 expression in forebrain (BCAS2 cKO) show the impaired learning and memory with -catenin declination. Due to -catenin regulates adult ββneurogenesis, here we extensively found that BCAS2 cKO mice showed low Sox2+-neuron stem cells (NSC) proliferation and self-renew. Similarly, the stereotaxic intracranial injection of lentivirus-shBCAS2 to knockdown BCAS2 in hippocampus; and confirmed BCAS2-regulating adult neurogenesis via -catenin. βMoreover, AAV-BCAS2 gene therapy to cKO could rescue the proliferation of Sox2+-NSCs with the increasing -catenin βexpression. Collectively, BCAS2 regulating adult neurogenesis through a -catenin are autocrine and paracrine effects. βFurthermore, the lithium treatment to BCAS2 cKO mice reveals the improvement the spatial learning and memory capabilities coupled with increasing -catenin expression and restoring βthe declined number of NSC number and proliferation, thereof, BCAS2 cKO mice can act as a neuron degeneration model for future drug screening.F-3023EFFECTS OF TDP-43 LOCALIZATION ON STRESS RECOVERY IN STEM CELL-DERIVED IN VITRO HUMAN MOTOR NEURONSGill, Stanley P - Department of Stem Cell and Regenerative Biology, Harvard University, Boston, MA, USA
470POSTER ABSTRACTSEggan, Kevin - Department of Stem Cell and Regenerative Biology, Harvard University, Boston, MA, USATar DNA-Binding Protein 43 (TDP-43) cytoplasmic aggregates are known to be associated with several neurodegenerative conditions, including Amyotrophic Lateral Sclerosis (ALS). Although TDP-43 mutations are found in roughly 4% of familial ALS patients, TDP-43 positive inclusions are found in 97% of sporadic and familial ALS cases, suggesting that TDP-43 mutation is not the only contributor to the proteinopathy. Previous work has additionally shown that cytoplasmic TDP-43 levels will transiently increase in response to physical, proteotoxic, and oxidative stress. However, the effect of TDP-43 subcellular localization on human motor neurons’ ability to adapt to these stressors has yet to be tested. We hypothesize that TDP-43 cytoplasmic-nuclear shuttling is required to mount the appropriate compensatory response that ensures neuronal survival and proper recovery in the face of stress. In this study, we examine the capacity for axonal outgrowth after axotomization in the context of expressing different structural variants of TDP-43 from the AAVS1 safe harbor locus in an in vitro system of human motor neurons derived from HUES3 human embryonic stem cells. We believe that these findings represent basic characteristics of TDP-43 regulation that will be informative to multiple neurodegenerative contexts.F-3025INVESTIGATING THE POTENTIAL OF HUMAN UMBILICAL CORD MESENCHYMAL STROMAL CELLS TO TARGET NEUROVASCULAR DYSFUNCTION AFTER TRAUMATIC BRAIN INJURYBarretto, Tanya - St. Michael’s Hospital, University of Toronto, ON, Canada Telliya, Tamar - Trauma Research, St. Michael’s Hospital, Toronto, ON, Canada Park, Eugene - Trauma Research, St. Michael’s Hospital, Toronto, ON, Canada Liu, Elaine - Trauma Research, St. Michael’s Hospital, Toronto, ON, Canada Gallagher, Denis - Research Department, Create Fertility Centre, Toronto, ON, Canada Librach, Clifford - Research, Create Fertility Centre, Toronto, ON, Canada Baker, Andrew - Anesthesia, Critical Care, St. Michael’s Hospital, Toronto, ON, CanadaTraumatic Brain Injury (TBI) is the leading cause of morbidity and life-years lost in North America thus advancement of clinical therapeutics is an urgent issue. Primary injury occurs when the brain is impacted with sufficient force to cause trauma; a secondary injury phase that is composed of cellular and molecular mechanisms follows and contributes to neurovascular dysfunction and axonal breakdown among other events. Human umbilical cord perivascular cells (HUCPVCs) are a source of mesenchymal stromal cells (MSCs); they express the pericyte markers NG2 and CD146 and PDGFR-b. The use of HUCPVCs in the treatment of TBI has the potential to contribute to through paracrine signaling, and through the structural stabilization of damaged vasculature. Collectively, the pleiotropic properties of HUCPVCs make them a potential cell-based therapeutic approach to target multiple pathophysiological pathways associated with secondary injury after TBI. Rats subjected to a fluid percussion injury (FPI) and systemically infused with 1.5 x 106 cells at 1.5h post-injury were sacrificed at acute time points. Vascular leakage was assessed using an Evan’s blue assay and expressed in microgram of dye per gram of tissue. At 24h and 48h vascular leakage was 6.4 microgram and 15.5 microgram vs. 1.7 microgram in sham rats. HUCPVC treated rats had 5.5 microgram and 3.3 microgram at 24 and 48 hours, respectively. Vascular density assessed by RECA-1 immunohistochemistry at 24h and 48h demonstrated a reduction by 40% in injured animals relative to sham and cell-treated animals. Cortical tissue at the injury site was extracted at 24h and 48h for western blot analysis to examine the expression of tight junction complexes as an indicator of vascular dysfunction. Expression of occludin was increased 400% and 200% in FPI and cell treated animals respectively at 24h and was comparable to sham by 48 hours. However, evaluation of the Occludin-ZO1 complex formation by immunoprecipitation and western blot analysis indicated only a 100% increase in complex formation at 24h in FPI animals but a 650% increase in cell treated animals. The infusion of HUCPVCs following modelled TBI injury was associated with reduced vascular leakage suggesting a potential therapeutic strategy to address vascular disruption after TBI.Funding Source: PSI FoundationF-3027THE STRENGTH OF CLINICAL DATA IN INFORMING IPSC-DERIVED MODELS OF AMYOTROPHIC LATERAL SCLEROSISTracey, Timothy - Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia Ovchinnikov, Dmitry - Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia Wolvetang, Ernst - Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia Ngo, Shyuan - Australian Institute for Bioengineering and Nanotechnology, Brisbane, AustraliaIn amyotrophic lateral sclerosis (ALS), disease heterogeneity is often described as one of the largest problems impacting the effective study and treatment of disease. A deeper understanding of the source of disease heterogeneity may aid in elucidating underlying disease mechanisms. By matching clinical data to patient-specific pathophysiological outcomes, we can begin to understand the drivers of disease processes. With this in mind, fibroblasts from ALS patients with familial mutations in SOD1 and C9orf72 genes, as well as a patient with no known mutation (termed a sporadic ALS patient), were reprogrammed into iPSCs. Patients met the revised El-Escorial criteria for ALS, and clinical demographics were collected at the time of enrolment. ALS patient iPSCs, along with iPSCs generated from age- and sex-matched controls, were differentiated into
471POSTER ABSTRACTSlower motor neurons. Neurons displayed all characteristic markers of this cell type, and morphologically resembled lower motor neurons. Evaluation of cellular electrophysiological (3Brain - Biocam X), as well as metabolic (Agilent - Seahorse) phenotypes was performed to align with disease-associated pathology. Fibroblasts were also incorporated into the analysis pipeline to determine if disease-specific phenotypes were uniform between fibroblasts and neurons. In conclusion, we have established a valuable resource of iPSC lines that display hallmark pathophysiological features of ALS. The strength of this resource is exemplified through the matching of in vitro cellular phenotypes with extensive clinical data. These models are now being used to investigate the role of neurometabolic defects in ALS, with specific interest in biochemical pathways that drive pathogenic change. Such pathways are also being specifically targeted with lead compounds in an attempt to ameliorate disease-associated phenotypes.F-3029POOLED KINOME WIDE CRISPR/CAS9 SCREEN IN HUMAN STEM CELL DERIVED NEURONS TO IDENTIFY PHARMACOLOGICAL TARGETS FOR ALS/FTDGuo, Wenting - Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven Stem Cell Institute, KU, Leuven, Belgium Balusu, Sriram - KU Leuven, Laboratory for the Research of Neurodegenerative Diseases, Leuven, Belgium Fan, Yannan - Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven Stem Cell Institute, KU, Leuven, Belgium Gajjar, Madhavsai - Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven Stem Cell Institute, KU, Leuven, Belgium Moisse, Matthieu - Laboratory of Neurobiology, VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Belgium Van Den Bosch, Ludo - Laboratory of Neurobiology, VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Belgium Verfaillie, Catherine - Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven Stem Cell Institute, KU Leuven, BelgiumAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that overlap in their clinical presentation, pathology and genetics. So far, no effective treatment is available. Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of ALS/FTD. The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which contribute to neurodegeneration. In addition, neuroinflammation is widely regarded as chronic cell stress which promote the pathogenesis of ALS/FTD. Cell reprogramming technology enabled the generation of human induced pluripotent stem cells (hiPSCs) to model disease in a dish. The recent developed CRISPR/Cas9 genome editing has expanded the scope and reliability of genetic deletion screens of the human genome. As kinase activation is crucial for neuronal survival and are good targets for novel drug development, we here perform a kinome-wide CRSIPR/Cas screen to identify novel candidate drug targets for ALS/FTD. We created iPSC lines of different genetic backgrounds with a doxycycline inducible CRISPR/Cas9 expressing cassette in the AAVS1 locus. We have transduced the sgRNA kinome wide library in the cells. Following activation of Cas9 with doxycycline and following incubation in the presence and absence of neuroinflamatory cytokines and DPR. To identify the kinases, that when eliminated, enable neurons to survive, we have collected surviving cells and currently performing next generation sequencing (NGS) to identify the sgRNAs that have been depleted or are enriched. This screen combined with ALS/FTD related validation will enable us to discover kinases involved in neuron degeneration and lay the foundation for discovering novel pharmacological targets for ALS/FTD.F-3031A GERMLINE HOMOZYGOUS MUTATION IN HUMAN OXIDATION RESISTANCE 1 GENE CAUSE DEVELOPMENTAL DELAY, EPILEPSY AND CEREBELLAR ATROPHYLin, Xiaolin - Department of Biochemistry, Department of Microbiology, Oslo University Hospital, University of Oslo, Norway Wang, Wei - Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway Yang, Mingyi - Department of Biochemistry, Department of Microbiology, Oslo University Hospital, University of Oslo, Norway Siller, Richard - Department of Molecular Medicine, Norwegian Centre for Stem Cell Research, Oslo University Hospital, University of Oslo, Norway Edvardson, Simon - Hadassah-Hebrew University Medical Center, Jerusalem, Israel Eide, Lars - Department of Biochemistry, Oslo University Hospital, University of Oslo, Norway Sullivan, Gareth - Department of Immunology, Department of Molecular Medicine, Norwegian Centre for Stem Cell Research, Oslo University Hospital and University of Oslo, Oslo, Norway Elpeleg, Orly - Hadassah-Hebrew University Medical Center, Jerusalem, Israel Bjoras, Magnar - Department of Cancer Research and Molecular Medicine, Department of Biochemistry, Department of Microbiology, Norwegian Centre for Stem Cell Research, NTNU, Oslo University Hospital, University of Oslo, Trondheim, Oslo, NorwayOxidation Resistance 1 (OXR1) is a highly conserved gene that plays an essential role in antioxidation in eukaryotes, from yeast to human. Here using whole exome sequencing, we identified three patients with a homozygous mutation in OXR1 that leads to clinical developmental delay, epilepsy and marked
472POSTER ABSTRACTScerebellar atrophy with onset in early childhood. Patient derived lymphoblasts showed impaired cell proliferation, increased apoptosis, and abnormally high sensitivity to oxidative stress with elevated oxidative DNA damage. The patient iPSC-derived neuroepithelial cells and brain organoids display defects in neural aggregate formation, neurite outgrowth and neuronal differentiation, as well as increased apoptosis. RNA sequencing analysis reveals that OXR1 regulates the transcriptional networks important for ROS protection, regulation of apoptosis and neuronal development. The core pathways involved in neuronal differentiation, proliferation and axonal targeting as well as axon guidance are dys-regulated in OXR1 deficient neurons. The landscape of histone modification has been altered in OXR1 deficient neurons. These findings provide the first description of a human disease associated with OXR1 deficiency, indicating an essential role for OXR1 in neuronal protection and brain development.F-3033GENETICALLY CORRECTED IPSC-DERIVED NEURAL STEM CELL GRAFTS DELIVER NAGLU-IGFII FUSION PROTEIN TO AFFECT CNS DISEASE IN SANFILIPPO B MICEPearse, Yewande - Pediatrics, Division of Medical Genetics, Los Angeles Biomedical Research Institute, Torrance, CA, USA Clarke, Don - Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Kan, Shih-Hsin - Children’s Hospital of Orange County, Children’s Hospital of Orange County, CA, USA Le, Steven - Department of Pediatrics, Washington University School of Medicine in St. Louis, MO, USA Sanghez, Valentina - Pediatrics, Division of Medical Genetics, Los Angeles Biomedical Research Institute, Torrance, CA, USA Cooper, Jonathan - Pediatrics, Division of Medical Genetics, Washington University School of Medicine in St. Louis, MO, USA Dickson, Patricia - Department of Pediatrics, Washington University School of Medicine in St. Louis, MO, USA Iacovino, Michelina - Pediatrics, Division of Medical Genetics, Los Angeles Biomedical Research Institute, Torrance, CA, USASanfilipposyndrometypeB(mucopolysaccharidosistype IIIB [MPS IIIB]) is a recessive genetic disorder that severely affects the brain and is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. We carried out ex vivo, lentiviral correction of Naglu−/− neural stem cells derived from Naglu−/− mice (iNSCs) using a modified NAGLU, consisting of this enzyme fused to a receptor binding peptide of IGFII. This enables receptor-mediated uptake and delivery to lysosomes, a process that is very inefficient for natve NAGLU. Corrected iNSCs secreted a functional NAGLU-IGFII enzyme that could be taken up by deficient cells. Following long-term transplantation into Naglu−/− mice, we detected NAGLU-IGFII activity in all engrafted animals. Transplanted Naglu−/− mice showed a significant restoration of Naglu activity, and a decrease in storage material, astrocytosis, and microglial activation, with beneficial effects extending along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu−/− mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver modified enzymes and treat MPS IIIB.Funding Source: This work was supported by grants from the NINDS (R21NS096044) .F-3035DISEASE MODELLING OF ZIKA WITH HUMAN NEURAL STEM CELLSFan, Yiping - Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore Lum, Fok Moon - Singapore Immunology Network, Agency for Science, Technology and Research, Singapore Yee, Wearn Xin - Singapore Immunology Network, Agency for Science, Technology and Research, Singapore Choolani, Mahesh - Obstetrics and Gynaecology, National University Health System, Singapore Ng, Lisa - Singapore Immunology Network, Agency for Science, Technology and Research, Singapore Chan, Jerry Kok Yen - Reproductive Medicine, KK Women’s and Children’s Hospital, SingaporeZika virus caused worldwide concern by its association with severe fetal brain injury including, but not limited to, microcephaly in the fetus of infected pregnant females, with an epidemic in Brazil in 2015. Using human fetal neural stem cells (hfNSC) from several anatomical locations of a developing central nervous system in the second trimester, we explored their role in disease modelling by investigating their infection with Zika. Regionally derived hfNSC (15-21 wks) were derived as neurospheres and cultured on laminin coated plates for 7-14 days before infection with the virus at MOI of 10 for 2 hours. Fresh media is added after removal of virus and collected after 96 hours and analysed, alongside with the infected cells. Cells were also replated on coated dishes for 7 days before harvested for analysis. Viral infectivity was assessed by detection of virus antigen with FACS and effects of infectivity ascertained by qPCR and immunocytochemistry. An average of 10.2±6.7, 7.9±3.5 and 4.6±1.9% of the forebrain derived hfNSC were found infected with the French Polynesia (FP), Brazilian (PE) and Singapore strains of Zika virus respectively. An average of 15.9±4.5, 7.6±2.2 and 10.4±3.8% of the midbrain derived hfNSC were found infected with the same Zika strains respectively. This illustrates that hfNSC were infected at varying degrees with the different strains of ZIKV (Range: 0.63-29.8%) with the higher infection rates observed from infection with the French Polynesia strains. In the forebrain derived hfNSC, Zika infection resulted in a significantly lowered neuronal differentiation by means of neurofilament-M expression (34.4±4.6 (mock-infected) vs 6.0±3.5 (PF, p-value=0.004) vs 0.8±0.8% (PE, p-value=0.002)). In the midbrain derived hfNSC, an increased
473POSTER ABSTRACTSdifferentiation into oligodendrocytes by means of PDGFR αexpression was observed (42.9±2.0 (mock-infected) vs 97.6±1.0 ( PF, p-value<0.001) vs 78.8±3.3% (PE, p-value<0.001)). These studies demonstrated the use of hfNSC in parsing out the effects of congenital ZIKA infection on human neural cells at a clinically relevant gestation and could be useful for in vitro studies on related work, such as vaccine testing or determining the effects of viruses before in vivo work.F-3037ESTABLISHMENT OF A PARKINSON’S DISEASE NOG MOUSE MODEL FOR EVALUATING IN VIVO FUNCTIONS OF HUMAN IPS CELL-DERIVED DOPAMINERGIC NEURONSHiguchi, Yuichiro - Laboratory Animal Research Department, Central Institute for Experimental Animals, Kanagawa, Japan Kawai, Kenji - Pathological Analysis Center, Central Institute for Experimental Animals, Kawasaki, Japan Nishinaka, Eiko - Testing Department, Central Institute for Experimental Animals, Kawasaki, Japan Haga, Hideyuki - Testing Department, Central Institute for Experimental Animals, Kawasaki, Japan Ahmad, Muzammil - Elixirgen Scientific, LLC, Baltimore, MD, USA Suemizu, Hirhoshi - Laboratory Animal Research Department, Central Institute for Experimental Animals, Kawasaki, Japan Ko, Minoru S.H. - Department of Systems Medicine, Keio University School of Medicine, Tokyo, Japan Hata, Jun-ichi - Central Institute for Experimental Animals, Kawasaki, JapanParkinson’s disease (PD) is a neurodegenerative condition characterized by symptoms, such as shaking at rest, muscular rigidity, akinesia, and postural instability. As PD is caused by degeneration and loss of dopaminergic neurons in the substantia nigra, human ES/iPS-derived dopaminergic neurons are a potential source for cell transplantation therapy. Many research groups have reported successful differentiation of functional dopaminergic neurons from human ES/iPS cells and, in 2018, the first clinical trial of human iPS-derived dopaminergic neurons for PD patients was announced in Japan. In the present study, we aimed to establish a novel model for evaluating in vivo functions of human iPS-derived dopaminergic neurons using an immunodeficient mouse. The NOG mouse, which is severely immunodeficient, was established by introducing the IL2r null γgene of IL2r knockout mice into immunodeficient NOD/ShiJic-γscid mice and backcrossing 10 times. As various human cells can be engrafted in NOG mice, a PD model NOG mouse could be used to evaluate in vivo functions of human iPS-derived dopaminergic neurons as well as tumorigenicity. Moreover, the NOG mouse makes it possible to evaluate in vivo functions using a small number of cells. Based on previous work, we injected 6-hydroxydomaine (6-OHDA) into the left striatum of NOG mice. Four weeks later, we administrated apomorphine and scored the number of contralateral rotations. As a result, 6-OHDA-treated NOG mouse demonstrated clockwise rotations (>7 rpm/min). Immunohistochemical analysis revealed clear degeneration of tyrosine hydroxylase-expressing dopaminergic neurons in the striatum of 6-OHDA-treated NOG mice. We are currently examining the transplantation of human iPS-derived dopaminergic neurons into the striatum of 6-OHDA-treated NOG mice. We expect that PD model NOG mouse will be a simple, low-cost model for evaluating in vivo functions of human iPS-derived dopaminergic neurons.ORGANOIDSF-3041DIFFERENTIATION AND CULTURE OF EXOCRINE HUMAN PANCREATIC ORGANOIDS FROM STEM CELL-DERIVED PANCREATIC PROGENITORSQuiskamp, Nina - Research and Development, STEMCELL Technologies Inc, Vancouver, BC, Canada Segeritz-Walko, Charis - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Lam, Stephanie - Research and Development, STEMCELL Technologies, Vancouver, Canada Wu, Cheryl - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Stingl, John - Research and Development, STEMCELL technologies, Vancouver, BC, Canada Riedel, Michael - Quality Control, STEMCELL Technologies, Vancouver, BC, Canada Thomas, Terry - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Eaves, Allen - Executive Committee, STEMCELL Technologies, Vancouver, BC, Canada Louis, Sharon - Research and Development, STEMCELL Technologies, Vancouver, BC, CanadaThe exocrine compartment makes up approximately 98% of the human pancreas and is composed of enzyme producing acinar cells connected by a network of bicarbonate-secreting ductal cells. Pancreatic cancer, chronic pancreatitis and cystic fibrosis are severe pathologic conditions which arise in the exocrine tissue but in vitro models for their study and the development of efficient treatments are lacking. Recent publications describe the in vitro differentiation of human pluripotent stem cells into pancreatic exocrine organoids and demonstrate their value as a physiological 3D model for the investigation of pancreatic pathologies and development. To standardize the generation of these organoids across multiple labs using different hPSC lines, we are developing the STEMdiff™ Pancreatic Exocrine Organoid Kit. Embryonic (H9, H1) and induced pluripotent (M001) stem cell lines previously maintained in mTeSR™1 were seeded as monolayers at a density of 2.6–3.7x105 / cm3 and directed through 12–14 days of differentiation to sequentially generate definitive endoderm, primitive gut tube, foregut endoderm and PDX1+/NKX6.1+ pancreatic progenitor cells. Pancreatic progenitors were harvested and seeded into 50 μL Corning® Matrigel® domes and subjected to a 10–14 day differentiation protocol to promote exocrine pancreatic cell
474POSTER ABSTRACTSdifferentiation. The cells formed E-cadherin+/EpCAM+/ZO-1+ polarized organoids with a central lumen, basolateral cell-contacts, and a collagen IV+/laminin+ basement membrane. The organoids also express progenitor (PDX1, NKX6.1), ductal (CFTR, CA2, SOX9, KRT19), and acinar (amylase) cell markers and contain aldehyde dehydrogenase-expressing cells, as detected by the ALDEFLUOR™ Kit. Organoid cultures can also passage every 8–14 days for a minimum of 3 passages. Our results demonstrate that the STEMdiff™ Pancreatic Exocrine Organoid Kit is an efficient tool to generate exocrine pancreatic tissue.F-3043EFFICIENT, REPRODUCIBLE AND HIGH-THROUGHPUT-COMPATIBLE PROTOCOLS FOR DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELL LINES INTO KIDNEY ORGANOIDSKramer, Philipp M - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Umali, Colleen - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Conder, Ryan - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Stingl, John - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Thomas, Terry - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Eaves, Allen - STEMCELL Technologies Inc., Vancouver, BC, Canada Louis, Sharon - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, CanadaThe ability to differentiate human pluripotent stem cells (hPSC) into kidney organoids provides a next generation cell-based assay and platforms for basic research studies, patient-specific disease modeling and nephrotoxic drug screening. However, current protocols for the derivation of kidney organoids from hPSCs are highly variable and are not standardized. We developed the STEMdiff™ Kidney Organoid Kit, containing specialized serum-free media to enable efficient and reproducible differentiation across multiple human embryonic and induced pluripotent stem cell lines (H1, H9, WLS-1C and STiPS-M001), previously maintained in mTeSR™1. Cells were seeded into Corning® Matrigel® coated 96-well plates. After 24 hours, adherent cells were overlaid with an additional layer of Corning® Matrigel®, which resulted in the formation of cavitated hPSC spheroids within the next 48 hours. On the following day, differentiation of cavitated hPSC spheroids was initiated by switching media from mTeSR™1 to the STEMdiff™ Kidney Organoid Kit. During the next 18 days of differentiation, cells were directed through stages of late primitive streak, posterior intermediate mesoderm, and metanephric mesoderm to give rise to kidney organoids that are composed of podocytes, proximal and distal tubules. Samples were collected prior to and post differentiation and analyzed by immunocytochemistry, RT-qPCR and/or flow cytometry to verify lineage-specific marker expression. Cavitated hPSC spheroids display a uniform expression of pluripotency markers OCT4 and SOX2 prior to the start of differentiation. All tested hPSC lines are highly efficient in generating self-organizing kidney organoids that form convoluted tubular structures with typical nephron-like segmentation. Kidney organoids reliably establish with efficiencies of 98.7 ± 4.1 (mean ± SD; n = 1 - 3 per cell line) organoids per cm2. Organoids, analyzed on day 18, express markers of podocytes (PODXL, NPHS1), proximal (LTL, CUBN) and distal tubules (CDH1, GATA3) (n = 8). In summary, STEMdiff™ Kidney Organoid Kit supports highly efficient and robust differentiation of hPSCs into kidney organoids in high-throughput-compatible microwell plates, which can be used for nephrotoxic compound screening.F-3045UTILIZING PATIENT-DERIVED BRAIN ORGANOIDS TO MODEL NEURAL NETWORK PATHOLOGY IN EPILEPSYSamarasinghe, Ranmal - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Miranda, Osvaldo - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Kurdian, Arinnae - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Mitchell, Simon - Computational Biology, University of California, Los Angeles (UCLA), CA, USA Fernando, Isabella - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Mody, istvan - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Golshani, Peyman - Neurology, University of California, Los Angeles (UCLA), CA, USA Lowry, William - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), CA, USA Watanabe, Momoko - Neurobiology, University of California, Los Angeles (UCLA), CA, USA Novitch, Bennett - Neurobiology, University of California, Los Angeles (UCLA), CA, USAHuman pluripotent stem cell-derived brain organoids recapitulate many aspects of human brain development and cytoarchitecture, and could provide invaluable insights into neurological diseases such as epilepsy. To realize this potential, we generated interneuron-rich ganglionic eminence (G) and excitatory neuron predominant cortical (C) organoids and fused these structures to create complex brain organoids with excitatory-inhibitory neuron interconnections. We then utilized live two-photon calcium imaging methodologies and extracellular electrode recording of local field potentials to determine the electrophysiological signatures of fusion organoids. We showed that human embryonic stem cell derived C+G fusion organoids display unique inhibitory interneuron-modulated spontaneous neural network activities including complex rhythmic oscillations. Moreover, human induced pluripotent stem cell-derived fusion organoids from a patient with Rett syndrome, a genetic disorder highly associated with epilepsy, had epileptiform-like activity
475POSTER ABSTRACTSand altered network oscillations compared to isogenic control organoids derived from the same patient. We next leveraged the fusion technique to mix isogenic control (C) with Rett (G) and vice versa, and subsequently showed a predominant role of interneurons in the Rett epileptiform electrophysiologic signatures. Finally, treatment of Rett fusions with the anti-seizure medication sodium valproate reduced spike frequency without rescuing oscillatory activity, while treatment with the p53 inhibitor pifithrin- reduced spike frequency and rescued some αoscillatory activities. Together, these findings show that we have developed methodologies for reproducibly measuring complex neurophysiologic activities in human brain organoids. Moreover, we generated data that suggest the presence of ictogenic networks in Rett organoids that recapitulate electrographic features of epilepsy. This opens the door to deeper exploration of both Rett-associated and other seizure disorders and for novel therapeutic interventions.F-3047EFFICIENT ESTABLISHMENT AND GROWTH OF HUMAN INTESTINAL ORGANOID-DERIVED MONOLAYERSConder, Ryan K - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada Stahl, Martin - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Simmini, Salvatore - Research and Development, STEMCELL Technologies, Waterbeach, Cambridge, UK Brown, Tyler - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Kramer, Philipp - Scientist, STEMCELL Technologies, Vancouver, BC, Canada Chang, Wing - Research and Development, STEMCELL Technologies, Waterbeach, Cambridge, UK Stingl, John - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Thomas, Terry - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Eaves, Allen - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Louis, Sharon - Research and Development, STEMCELL Technologies, Vancouver, BC, CanadaOrganoids are a cutting-edge tool for regenerative medicine, disease modelling and drug screening. The STEMdiff™ Intestinal Organoid Kit and IntestiCult™ Organoid Growth Medium (OGM; Human) are used for the derivation and expansion of human intestinal organoids from human pluripotent stem cells (hPSC) and primary tissues, respectively. The focus of the current study is to develop protocols for growing and differentiating organoid-derived intestinal cells in a monolayer culture, and to compare the electrophysiological properties of these cells to the commonly used Caco-2 cancer cell line. To do this, primary human intestinal organoids were seeded in IntestiCult™ OGM onto Corning® Matrigel® -coated tissue culture and transwell plates. After 7 days, the cultures were analyzed by immunocytochemistry, transepithelial electrical resistance (TEER) measurements to measure barrier integrity, and by Ussing Chamber analysis to measure ion transport. Organoid-derived monolayers are composed of VIL1-, EpCAM-, E-cadherin- and claudin-expressing enterocytes that have a polarized morphology complete with tight junctions and a brush border, as well as MUC2 expressing goblet cells. Intestinal organoid-derived monolayers have measured TEER values averaging 358.5 ± 22.6 *cm2 (SEM; n=12), compared to an Ωaverage of 301.1 ± 22.3 *cm2 (SEM; n=10) for comparable ΩCaco-2 monolayers, demonstrating that these cells have equivalent or greater barrier function. Treatment with the cAMP elevating agents IBMX and forskolin increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) by a change in short-circuit current ( ISC) of 40.3 ± 7.62 μA/Δcm2 (SEM; n=6) in human colonic monolayers, significantly more than 6.7 ± 0.61 μA/cm2 (SEM; n=4; p=0.04) for Caco-2 monolayers. Additionally, treatment with the CFTR inhibitor-172 reduced CFTR activity by a ISC of 80.6 ± 5.96 μA/cm2 (SEM; Δn=6) compared to 15.5 ± 1.98 μA/cm2 (SEM; n=4; p<0.0001) in Caco-2 cells, demonstrating increased sensitivity in organoid-monolayer compared to Caco-2 cultures. The new IntestiCult™ OGM monolayer protocol provides a novel experimental platform that better mimics the in vivo intestinal epithelium, demonstrated by performance of functional assays that matches or exceeds those of the cell lines currently in widespread use.F-3049THE EFFECTS OF MICROGRAVITY ON MICROGLIA 3-DIMENSIONAL MODELS OF PARKINSON’S DISEASE AND MULTIPLE SCLEROSISNoggle, Scott - New York Stem Cell Foundation Research Institute, New York Stem Cell Foundation, New York, NY, USA Clements, Twyman - CEO, Space Tango, Lexington, KY, USA Nijsure, Madhura - Research Institute, New York Stem Cell Foundation, New York, NY, USA Barbar, Lili - Research Institute, New York Stem Cell Foundation, New York, NY, USA Stein, Jason - Aspen Neuroscience, La Jolla, CA, USA Stoudemire, Jana - Commercial Innovation, Space Tango, Lexington, CA, USA McClelland, Randall - SciKon Innovation, Durham, NC, USA Monsma, Frederick - Research Institute, New York Stem Cell Foundation, New York, NY, USA Loring, Jeanne - Department of Molecular Medicine, The Scripps Research Institute and Aspen Neuroscience, La Jolla, CA, USA Fossati, Valentina - Research Institute, New York Stem Cell Foundation, New York, NY, USA Bratt-Leal, Andres - Aspen Neuroscience, La Jolla, CA, USAExposure to microgravity and radiation, as occurs in the International Space Station (ISS), causes significant mechanical unloading of mammalian tissues, resulting in rapid physiological alterations. While many studies have focused on the impact of microgravity on the cardiac and musculoskeletal systems,
476POSTER ABSTRACTSmicrogravity is also known to have a significant impact on the central nervous system. Research using a variety of tissue types has demonstrated that microgravity increases proliferation and delays differentiation of stem cells. Cell-cell interactions are critical for neuronal communication, but to date no studies have yet evaluated these effects in human cells in microgravity. Taking advantage of the potential of the induced pluripotent stem cells (iPSC) to differentiate into any cell type of the human body, we propose to study 3D neuroglial cell cultures derived from iPSCs of patients with Parkinson’s disease and multiple sclerosis, to analyze the migratory capability of iPSC-derived microglial cells in microgravity. Based on this, we will evaluate retrospectively cell–cell interactions and migratory capabilities of iPSCs-derive microglial cells from patients with Parkinson’s disease (dopaminergic neurons) and multiple sclerosis (cortical neurons), versus co-cultures of the same cell types from healthy control subjects. The cells will be maintained on the International Space Station (ISS) for one month. This groundbreaking study will be the first long-term cell culture experiment conducted in microgravity using human patient-derived iPSCs to study Parkinson’s disease and multiple sclerosis. This research will provide valuable insights into the mechanisms by which neuronal cells mature in 3D cultures, factors influencing migration of glial cells in organoids, and changes in gene expression that may play a role in these disease processes, and may well have an impact on discovery and development of biomarkers and therapeutics.F-3051TRANSPLANTATION OF HUMAN EMBRYONIC STEM CELL-DERIVED CEREBRAL ORGANOID AFTER LONG-TERM CULTURE INTO MOUSE MOTOR CORTEXKitahara, Takahiro - Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Sakaguchi, Hideya - Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto, Japan Takahashi, Jun - Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto, JapanCell replacement therapy is an expected future treatment for reconstructing the neural circuits of cerebral cortices injured by stroke or trauma. The transplantation of cerebral organoids derived from pluripotent stem cells is attracting attention because cerebral organoids can generate three-dimensional structures of the cerebral cortex. Recent studies reported that the transplantation of human embryonic stem cell (hESC)-derived cerebral organoids into mouse cerebral cortex promoted graft survival with vascularization and axonal growth to the host brain. However, it remains unclear which developmental stage of the organoid is optimal for the transplantation. Additionally, it is unclear whether cerebral organoids can reconstruct motor pathways of a damaged motor cortex following transplantation. To clarify these problems, we induced cerebral organoids from hESCs using SFEBq (Serum-free Floating culture of Embryoid Body-like aggregates with quick reaggregation) method. We transplanted the cerebral organoids at early (day 42–43) and late (day 71–84) developmental-stage into cerebral cortices of early postnatal mice. Cerebral organoids at both developmental stages survived and promoted axonal growth to the host brain. Late-stage cerebral organoids did not cause tumor-like graft growth and were considered more suitable for the transplantation. Finally, we transplanted late-stage cerebral organoids into the motor cortex of adult mice. The engrafted cerebral organoids provided fiber extensions along the corticospinal tract when the graft was transplanted one week after lesioning the motor cortex. We confirmed that hESC-derived cerebral organoids at the late developmental stage promote axonal growth along the corticospinal tract without tumor-like graft growth after transplantation into the lesioned motor cortex of adult mice. Our approach is an important step for the reconstruction of motor pathways after cerebral cortex injury.F-3053DECIPHERING THE ROLE OF TSC1 IN A CORTICAL ORGANOID MODEL OF TUBEROUS SCLEROSISIefremova, Vira - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, Germany Loehlein, Simone - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, Germany Braun, Nils - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn,Germany Doll, Roman - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, Germany Till, Andreas - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, Germany Stappert, Laura - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, Germany Bruestle, Oliver - Institute of Reconstructive Neurobiology, University of Bonn School of Medicine and University Hospital Bonn, GermanyTuberous sclerosis complex (TSC) is a developmental disorder caused by a mutation of one of the two tumor suppressor genes, TSC1 or TSC2. Protein products of those genes form a TSC1/TSC2 complex, which plays a pivotal role as a negative regulator of the mTOR pathway. One of the hallmarks of TSC pathology is the formation of cortical tubers (i.e., benign lesions with abnormal glial and neuronal cells) that disrupt the classical six-layered cytoarchitecture of the cortex and can cause epileptic seizures. First signs of the disease can be seen already during the early stage of human fetal development. So far, experimental studies on TSC were largely conducted in mice. However, human and mouse cortical development differs significantly, creating a need for appropriate human model systems. Here we address this need by generating TSC1-deficient human iPSC lines using CRISPR/Cas9 technology and subjecting these lines to cortical
477POSTER ABSTRACTSdifferentiation in 2D and 3D organoid cultures. In agreement with the role of TSC1 in the regulation of mTOR signaling, TSC1-deficient cortical cultures show evidence of increased mTOR signaling, including elevated phospho-S6 levels and an increase in cell size. When compared to isogenic controls, TSC-1-deficient cortical organoids display unaltered overall growth rates as well as stratification into a ventricular zone-like area and neuronal cell layers. However, a more in-depth analysis of division patterns revealed a significant increase in the number of cells with vertical cleavage planes at the expense of horizontal cleavage planes in TSC1-deficient organoids. These observations may serve as an entry point for dissecting potential alterations in the dynamics of neural stem cell proliferation and differentiation associated with TSC1-deficiency and impaired mTOR signaling. Our data underline the notion that cerebral organoids provide a valuable tool to decipher pathomechanisms underlying the emergence of human cortical malformations.F-3055HUMAN SLICED NEOCORTICAL ORGANOIDS ESTABLISH SPECIFIED UPPER AND DEEP CORTICAL LAYERSQian, Xuyu - Neuroscience, University of Pennsylvania, Philadelphia, PA, USA Christian, Kimberly - Neuroscience, University of Pennsylvania, Philadelphia, PA, USA Song, Hongjun - Neuroscience, University of Pennsylvania, Philadelphia, PA, USA Ming, Guo-li - Neuroscience, University of Pennsylvania, Philadelphia, PA, USABrain organoids, human pluripotent stem cell(hPSC)-derived self-organizing three-dimensional (3D) tissues with cell types and cytoarchitecture resembling the embryonic human brain, have emerged as valuable model systems to investigate human brain development and disorders. Previously, we have developed methodologies to generate forebrain organoids that resemble human cerebral cortex development around mid-gestation period with remarkable fidelity. Engineering mature cortical organoids representative of late gestation stages could fill the current gaps in our knowledge of human corticogenesis due to the inaccessibility of fetal tissues in the third trimester. However, due to a lack of functional vascular circulation system, growth and maturation of cortical organoids are limited by an insufficient supply of oxygen and nutrients via diffusion. Here we report a slicing method to bypass the diffusion limit by exposing the organoid interior while maintaining its structural integrity, which leads to the generation of organoids representative of human late-stage neocortical development. Substantially reduced cell death and sustained cell proliferation allow the progenitor and neuronal layers to expand larger beyond previous size limits and produce well-separated upper and deep cortical neuronal layers. Within the cortical layers, spontaneously active neuronal networks fire coordinated bursts across long distance. We further identify the critical role of WNT/ -Catenin βsignaling in regulating cortical neuron fate specification and layer separation, which is disrupted by a mutated risk gene for psychiatric disorder in patient iPSC-derived organoids. Overall, our sliced neocortical organoids introduce a new approach to overcome diffusion limit in 3D cultures, and offer an instrumental platform for investigating previously-intangible human-specific features of late-stage brain development.Funding Source: NIH grant U19AI131130F-3057SCALABLE AND HIGH-FIDELITY DRUG INDUCED LIVER INJURY SCREEN USING HUMAN IPSC-LIVER ORGANOIDSCai, Yuqi - Development Biology and Gastroenterology, Cincinnati Children’s Hospital and Medical Center, Cincinnati, OH, USA Dunn, Andrew - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA Kimura, Masaki - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA Shinozawa, Tadahiro - Pharmacology, Takeda Pharmaceutical Company, Tokyo, Japan Takebe, Takanori - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA Thompson, Wendy - Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USABillions of dollars are lost annually from drug development in the pharmaceutical industry due to the failures of drug candidates in initial screens, and nearly a third of drugs are later withdrawn from the market. Preclinical prediction of which compounds have drug induced liver injury (DILI) risk in humans is still a significant challenge in drug development. Here, we developed an method to generate unique human pluripotent stem cell derived liver organoids (HLOs), that contain cells from multi-lineages, secrete albumin, and have CYP enzyme activity. Polarized hepatocytes with bile canaliculi-like architecture support unidirectional bile acid transportation. Furthermore, coupled with a high-speed imaging microscope and high through-put image processing, we established an innovative Liver organoid-based Toxicity screen (LoT) system, wherein we tested 4 doses of 238 drugs from an Enzo hepatotoxicity library in 384-microwell plates with multiplexed readouts: cholestasis, mito-tox, and cell viability data simultaneously. The results demonstrated that HLOs can not only respond in a dose dependent manner to known DILI risk drugs but can also positively predict potential DILI risk drugs. Further, selected 10 DILI risk drugs were successfully validated the results in 6 different donors. More intriguingly, we were also able to predicted genomic predisposition (CYP2C9*2) for Bosentan-induced cholestasis. Thus, LoT is a scalable, high-fidelity PSC based model for drug safety with a cost-effective platform, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.Funding Source: Cincinnati Children’s Research Foundation grant PRESTO grant from Japan Science and Technology Agency
478POSTER ABSTRACTSTISSUE ENGINEERINGF-3059DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS INTO TESTICULAR ORGANOIDS FOR STUDYING DE NOVO MUTATIONS IN INFERTIILE MENAlbert, Silvia - Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands Oud, Manon - Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands van der Heijden, Godfried - Gynecology, Radboud University Medical Center, Nijmegen, Netherlands Ramos, Liliana - Gynecology, Radboud University Medical Center, Nijmegen, Netherlands Veltman, Joris - Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UKHuman reproduction is vital to our species but worldwide infertility affects 1 in 7 couples, of which one third of the cases are explained by a male factor. Although thousands of genes are known to be involved in spermatogenesis, the genetic causes behind severe spermatogenic failure remain largely unknown. Exome sequencing studies performed in our department will help the investigation of the aetiology of male infertility. With this research we found a prominent role for de novo mutations (DNMs) and identified many potential genes essential for spermatogenesis. Human germ cell induction research is a valuable platform for modelling infertility and congenital anomalies that have been difficult to study in animals. iPS cells were differentiated for one month into germ cells and testicular organoids, in a 2D and 3D culture, respectively. Germ cell type and content were assessed by immunofluorescence staining and quantitative PCR. The analysis performed after one month of culture showed in both methods the increased expression of CXCR4, IFITM3 and VASA, indicating the successful differentiation of iPSCs into spermatogonia. However, a longer culture is needed to obtain further differentiated germ cells. Genetic screening of the testicular organoids will help to link with more confidence the genes affected by a DNM to male infertility. Moreover, the in vitro induction of testicular germ cells and organoids from autologous pluripotent stem cells should lead to a new model for genetic engineering purposes. Putative causative DNM will be mutagenised by Crispr/Cas9 in patient-derived iPSCs, and the effect of the mutation will be assessed in testicular organoids.F-3061CELL-FREE THERAPY USING STEM CELL EXOSOMES FOR TREATMENT OF OSTEOARTHRITISPark, So Young - Biomedical Polymer Research Laboratory, Hanyang University ERICA, Ansan, Korea Kim, Min Kang - Hanyang University, Biomedical Polymer Research Laboratory, Ansan, Korea Cho, Yong Woo - Hanyang University ERICA, Exostemtech Inc., Ansan, KoreaOsteoarthritis (OA) is a common degenerative joint disease characterized by cartilage destruction. Despite the steady increase in the incidence of OA, the recent treatments focused on pain management. Driven by the demand of an alternative to overcoming these drawbacks, recent studies focused on the development of effective therapeutic strategies capable of regenerating the damaged cartilage. Stem cells secrete extracellular vesicles composed of microvesicles and exosomes. Particularly, exosomes derived from stem cells contain various proteins, lipids and genetic materials (mRNA, ncRNA and miRNA etc.). Stem cell exosomes are expected to provide biochemical cues for tissue regeneration through cell-to-cell communication. In this study, we hypothesized that exosomes derived from human adipose stem cells (hASCs) could induce the therapeutic effect of degenerative arthritis. Exosomes were isolated by pre-filtration in 0.2 μm, followed by tangential flow filtration (TFF) system (300 kDa MWCO). The isolated exosomes were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), flow cytometry, cytokine arrays, etc. Exosomal marker proteins such as CD9, CD63 and CD81, were confirmed by western blot (WB). The mRNA expressions of MMP-1, MMP-3, MMP-13, and ADAMTS-5 which activate cartilage degradation were analyzed by real-time polymerase chain reaction (qPCR). A mixture of exosomes (1 ´ 108 particles) and hyaluronic acid hydrogel (1%) were intra-articularly injected 3 times for 3 weeks in MIA-induced subacute OA mouse models. After four weeks, knee joints were harvested and analyzed histologically by safranin O-fast green and hematoxylin and eosin (H&E). Various factors related to anti-inflammatory and cartilage regeneration were found in exosomes. The in vivo studies demonstrated that exosomes prevented proteoglycan degradation and attenuated the cartilage destruction in the damaged articular cartilage. Overall results suggest that exosomes derived from hASCs have great potential for cartilage repair and alleviate the inflammatory reaction caused by OA.F-3063EXPANSION OF HAIR FOLLICLE STEM CELLS IN MICROFABRICATED OXYGEN-PERMEABLE CULTURE VESSEL FOR HAIR REGENERATIVE MEDICINEHirano, Sugi - Graduate School of Engineering Science, Yokohama National University, Yokohama, Japan Kageyama, Tatsuto - Graduate School of Engineering Science,Fukuda Group, Yokohama National University, Kanagawa Institute of Industrial Science and Technology (KISTEC), Yokohama, Japan Fukuda, Junji - Graduate School of Engineering Science,Fukuda Group, Yokohama National University,Kanagawa Institute of Industrial Science and Technology (KISTEC), Yokohama, Japan
479POSTER ABSTRACTSHair regenerative medicine is a promising treatment strategy for hair loss. Considering that the embryonic development of hair follicles is triggered by the formation of a hair follicle germ (HFG), we have proposed an in vitro preparation approach of HFGs and demonstrated efficient hair follicle generation upon transplantation into mice (T. Kageyama, et al, Biomaterials 2018). A challenge is however to expand the number of follicular stem cells (i.e., hair follicle stem cells (HFSCs)) while maintaining stemness prior to the HFG preparation. In this study, we fabricated a microwell array culture vessel where HFSCs formed three-dimensional aggregates and were then encapsulated in Matrigel. The culture vessel was made with oxygen-permeable silicon rubber to improve oxygen supply from the bottom through the culture vessel. The diameter of mouse HFSC aggregates in the culture vessel were uniform and increased from 100 μm to 350 μm during 2 weeks of culture. A quantitative analysis revealed that the number of CD34+ HFSCs increased up to 15 times in 2 weeks of culture. Interestingly, gene expression of trichogenic stem cell markers (e.g., Nfatc1, Tcf3, CD34) was significantly higher than that in a conventional suspension culture in Matrigel. Further, HFSCs grown in this approach were mixed with freshly isolated embryonic mesenchymal cells and formed HFGs. After 18 days of transplantation into the back skin of nude mice, newly generated hair shafts were observed at transplanted site. The hair regeneration efficiency was significantly improved compared to those prepared with a conventional suspension culture in Matrigel. This approach may be useful for large-scale preparation of HFSCs for hair regenerative medicine.Funding Source: This work was supported in part by the ministry of education, culture, sports, science and technology (MEXT) of Japan (Kakenhi) and Kanagawa Institute of Industrial Science and Technology.F-3065THE EFFECT OF MECHANICAL TRAINING ON HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIAC TISSUE SHEET STACKSLopez Davila, Victor - Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Masumoto, Hidetoshi - Center for Biosystems Dynamics Research (BDR), RIKEN, Kobe, Japan Yamashita, Jun - Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, JapanCell sheet technology has been previously applied in our lab, with scaffold-free 3D tissues generated by stacking cardiac tissue sheets (CTSs) showing promising therapeutic effects in vivo, as well as using CTSs to develop an in vitro model of cardiac arrhythmia. While functionally promising, these three-dimensional tissues are still structurally immature and fragile to show sufficient force generation. To overcome this, we applied cyclic stretching forces during cell sheet stack culture in order to drive tissue maturation. CTSs were prepared by differentiating cardiomyocytes and mesenchymal cells from 201B6 iPS cells and co-culturing them on temperature-responsive culture plates. Three layers of CTSs were then stacked and allowed to merge with a biocompatible adaptor prior to loading the stacks on a mechanical training device to undergo cyclic stretching or static stretching. CTS stacks were then analysed for cellular viability, protein expression and force generation. Our results showed that cyclic stretching clearly induced cellular alignment in both mural cells and cardiomyocytes, which is the first step towards cardiomyocyte maturation and improved mechanical properties, while keeping cellular viability above 95% (158/164 (96.4%) stretched; 134/137 (99.8%) suspended control; and 120/120 (100%) attached control). To a lesser extent, the static stress derived from tissue suspension also induced cellular alignment. This indicator of tissue maturation was accompanied by an increase in the expression of cardiac troponin I in cardiomyocytes. Additionally, force generation studies showed a tendency for an increase in Young’s modulus and active force in the stretched samples (8.4kPa and 0.02mN, respectively) compared to the suspended control (1.0kPa and 0.007mN) and the attached control (7.2kPa and 0.006mN). The application of cyclic mechanical training on CTS stacks drives cellular alignment on cardiomyocytes and mural cells, improves the tissue’s mechanical properties and could potentially drive cardiomyocyte maturation, as shown by an increase in cTnI expression. Interestingly, this alignment was perpendicular to the direction of stretching, while the application of static tension resulted in parallel alignment.F-3067EVALUATION OF ELECTROSPUN SCAFFOLDS OBTAINED BY CHITOSAN AND POLYCAPROLACTONE BLEND FOR VESSEL TISSUE ENGINEERINGPranke, Patricia - Hematology And Stem Cells Laboratory, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Iglesias Braghirolli, Daikelly - Hematology and Stem Cells Laboratory, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Klaus, Amanda - Fundação Escola Técnica Liberato Salzano Vieira da Cunha, Novo Hamburgo, Brazil Reis de Paula, Lavínia - Fundação Escola Técnica Liberato Salzano Vieira da Cunha, Novo Hamburgo, BrazilCurrently, there is an absence of vascular grafts suitable for use in replacement surgeries of small diameter vessels. In this context, vascular tissue engineering (VTE) is investigated as a tool for the development of vascular substitutes. In this work, a polymeric blend of PCL and chitosan (CH) was used to produce vascular scaffolds by the electrospinning (ES). Scaffolds with different portions of PCL and CH were evaluated in terms of mechanical properties, cellular adhesion and blood compatibility. PCL and CH solutions with 20 and 2 % (w/v) concentrations were prepared and mixed at different ratios: 4:1, 3:1 and 2:1, and then submitted to ES. A PCL control scaffold was also produced. The morphology of the scaffolds was analyzed by scanning electron
480POSTER ABSTRACTSmicroscopy and analysis of their mechanical properties was made by dynamic mechanical analysis (DMA). For evaluation of cellular adhesion, the mesenchymal stem cells (MSCs) were seeded onto the scaffolds and by the MTT test. The blood compatibility was evaluated by hemolysis assay. All groups of the scaffolds exhibited similar morphology, with well distributed smooth fibers in their structure. The average diameter was 0.73±0.6, 0.81±0.4, 0.80±0.5 and 0.64±0,4 μm for the PCL, PCL: CH 4:1, 3:1 and 2:1 scaffolds, respectively. The DMA tests demonstrated that CH presence did not cause significant changes in the mechanical properties of the scaffolds. The four groups of samples show a similar stress-strain curve. The MTT results showed that the scaffolds produced from blends 4:1 and 3:1 PCL:CH showed higher absorbance than the others. However, their differences were not statistically significant and the average number of adhered cells was 3.0x10-4, 3.7x10-4, 3.2 x10-4 and 2.9 x10-4 for the PCL, PCL: CH 4:1, 3:1 and 2:1 scaffolds, respectively. The hemolysis rates of all groups of the scaffolds were significantly less than the positive control and were similar to the negative control group. PCL is used in VTE due to these desirable mechanical properties. However, PCL shows poor biological properties. Thus, the association of CH with PCL scaffolds could improve their cytocompatibility. Although the cell adhesion results were not conclusive, the addition of CH maintained the mechanical properties and blood compatibility of the scaffolds, which may be interesting for VTE application.Funding Source: MCTI, FINEP, CNPq and Stem Cell Research InstituteF-3069ENGINEERED M13 CADHERIN ASSOCIATED PEPTIDE HAV NANOFIBER ACCELERATES INTERCELLULAR ADHESION STRENGTH AND PROLIFERATION OF FIBROGENIC FIBROBLASTSKim, Yeji - Pusan National University, Pusan National University, Busan, Korea Jo, Soojin - School of NanoenergyEengineering, Pusan National University, Busan, Korea Lee, YeongJu - Department of Nanofusion Technology, Pusan National University, Busan, Korea Oh, Jinwoo - Department of Nanofusion Technology, Busan National University, Pusan National University, Busan, KoreaRecently, there has been considerable effort to develop suitable nanofiber for tissue engineering, and nanotechnology about medicine is being developed with evolution of mammalian cells The M13 phage is nano-sized material and capsulated in 2700 copies of the major coat p8 protein, minor coat p3 proteins. The HAV (histidine alanine valine) sequence in the first extracellular domain (ECM) of E-cadherin is crucial for homophilic interactions between cadherins. The extracellular domain of cadherins contains characteristic repeats that regulate homophilic and heterophilic interactions during adhesion and morphogenesis. The relevance of HAV M13 phage in relation to the fibrogenic potential of fibroblast is currently unknown. We have engineered the p8 position of M13 phage using site-directed mutagenesis PCR and target HAV sequence-displaying M13 phage sequencing and analysis. In the previously study, we find that HAV M13 phage accelerates cell-cell adhesion and morphogenesis through HAV tripeptide motif in the most distal EC (EC1). The number of cell and the fibrogenesis related gene level of HAV-M13 phage treatment of cells were increases in a time-dependent manner compared to wild M13 phage. Additionally, activation of morphogenesis during the HAV-M13 phage with co-culture. These results suggest the HAV-M13 phage nanofiber is beneficial to cellular behavior of normal cells and can be applied as a technique for selectively culturing or removing cells having a specific substanceF-3071ELECTRICAL STIMULATION ENHANCES OSTEOGENESIS OF HUMAN DENTAL PULP-DERIVED STEM CELLS AND ELEVATED THE GENE EXPRESSIONS OF BMP2, BMP3 AND BMP5Cheng, Yu-Che - Proteomics Laboratory, Cathay General Hosp, New Taipei City, TaiwanWe fabricated an electrical stimulation (ES) device which provide direct current electric field (DCEF) treatment to the cells though conductive polypyrrole (PPy) film. We applied human dental pulp-derived stem cells (hDPSCs) on the PPy film and investigated ES effect on osteo-differentiation of (hDPSCs). ES with electrical field of 0.33 V/cm was applied to treat hDPSCs once for 4 h on different days after the chemical induction of osteogenesis. The alizarin red S staining results suggested that ES could accelerate the mineralization rates of hDPSCs. The calcium quantification analysis results revealed a nearly 3-fold enhancement in calcium deposition of hDPSCs by ES at Day 0, 2, and 4, whereas the promotion effect at later stages was in vain. To determine the ES-mediated signalling pathway, osteogenesis of hDPSCs were induced by osteogenic medium and stimulated immediately by ES, and the expression of genes belongs to the bone morphogenesis protein (BMP) family and related receptors were quantified using qRT-PCR. We found that mRNA levels of BMP2, BMP3 and BMP5 were increased significantly in the ES groups, indicating that these genes involved in the specific signalling routes induced by ES. Other BMPs such as BMP1, BMP4, BMP 6, BMP receptor 1B and BMP receptor 2 showed no statistical difference between control and ES group. The expression levels of BMP7 and BMPR1-A were undetectable in hDPSCs. Our results support that ES treatment facilitate bone formation of hDPSCs, and thus may shorten the healing period in clinical application. We showed here a promising applications of ES on hDPSCs in cell-based bone tissue engineering in the near future.Funding Source: Ministry of Science and Technology in Taiwan (MOST 106-2221-E-008-078- and MOST 106-2314-B-281 -001 -MY3)
481POSTER ABSTRACTSF-3073CIRCADIAN OSCILLATORS DURING HEART TISSUE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLSQi, Zhen - School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan Kamoshida, Misato - Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan Tamai, Miho - Faculty of Dental Medicine, Hokkaido University, Sapporo, Japan Tagawa, Yoh-ichi - Life Science and Technology, Tokyo Institute of Technology, Yokohama, JapanCircadian rhythm influences drug effectiveness and toxicity in mammals. It is unknown whether circadian rhythmicity can be established in mouse ES cell-derived heart tissue without a maternal context. It is necessary to synchronize circadian rhythm to all cells in a culture system as well as in animal. Here, we tried to synchronize the circadian rhythm of mES cell-derived heart tissues by the addition of forskolin as a synchronizer for developing a drug test model that exhibit the time dependent cell physiology and drug effectiveness. The expression of the core clock genes, such as Per1 and Bmal1, oscillated in mES cell-derived heart tissues by forskolin, but not in undifferentiated mES cells. This forskolin-synchronized oscillation pattern could be kept at least for three days. Interestingly, the contraction rate had oscillation pattern in this culture system, and also this contraction oscillation could response against -adrenoreceptor βagonist stimulation. In conclusion, these results demonstrate that circadian rhythm develops during cardiomyocyte differentiation process from mES cells. This mES cell-derived heart tissue model having forskolin-synchronized circadian rhythm would be a promising tool for replacing animal experiments for chronotherapy research and circadian rhythms studying in culture.ETHICAL, LEGAL AND SOCIAL ISSUES; EDUCATION AND OUTREACHF-3075INTERNATIONAL COMPARISON OF PUBLIC ATTITUDE TOWARD STEM CELL SCIENCE AND REGENERATIVE MEDICINEShineha, Ryuma - Faculty of Arts and Literatures, Seijo University, Tokyo, Japan Inoue, Yusuke - The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Yashiro, Yoshimi - School of Health Innovation, Kanagawa University of Human Service, Kawasaki, JapanOwing to the rapid progress in stem cell research (SCR) and regenerative medicine (RM), society’s expectation and interest in these fields are increasing. For effective communication on issues concerning SCR and RM, surveys for understanding the interests of stakeholders is essential. For this purpose, we conducted an international comparison between six countries: Japan, South Korea, Germany, France, United Kingdom, and United States of America. We collected 100 valid responses through research company monitors from each country. Results showed that the public is generally interested in the post-realization and responsible governance of SCR and RM. Our data indicate that an increased awareness about RM associated social responsibility and regulatory framework is required among scientists, such as those regarding its benefits, potential accidents, abuse, and other social consequences.Funding Source: JSPS and Secom FoundationF-3077PUBLIC SURVEY IN JAPAN ON HUMAN GENOME EDITING FOR CLINICAL PURPOSESSawai, Tsutomu - Uehiro Research Division for iPS Cell Ethics, Center for iPS Cell Research and Application, Kyoto University, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Akatsuka, Kyoko - Uehiro Research Division for iPS Cell Ethics, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Hatta, Taichi - Uehiro Research Division for iPS Cell Ethics, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Fujita, Misao - Uehiro Research Division for iPS Cell Ethics, Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanIn Japan, guidelines permitting the genome editing of human embryos only for basic research will soon be established. However, we have already seen genome editing of human embryos for clinical purposes, as illustrated by the claims made by He Jiankui in November 2018, who gene-edited two human embryos that came to term. This announcement highlighted a need for further discussion on whether or to what extent human genome editing for clinical purposes is acceptable. Thus far, the International Society for Stem Cell Research (ISSCR) and the National Academy of Sciences and Medicine in the United States brought attention the importance of including diverse stakeholders in the decision-making on human genome editing. In Japan as well, the opinions of the general public on human genome editing such as the human germline and human somatic cells should be reflected in policy decisions. However, there has been only one survey in Japan about the opinions of the general public and patients that focused on the genome editing of human embryos for therapeutic purposes. In short, the Japanese attitude toward genome editing on the human germline such as gametes and embryos as well as on human somatic cells for clinical purposes remains unclear. Hence, we will conduct an Internet-based questionnaire of about 4,000 general public in Japan. Using an explanation about genome editing techniques and human genome editing, we will present multiple therapeutic and non-therapeutic purposes for the genome editing of human germline and human somatic cells and then ask about their attitudes toward human genome editing. In this presentation,
482POSTER ABSTRACTSwe will show the survey results. Based on our survey, we will comprehensively grasp the currently unclear public attitude in Japan toward human genome editing for clinical purposes. We believe our survey framework will provide valuable information for policymaker across the globe.Funding Source: This work was supported by the JSPS KAKENHI Grant Number (17K13843) for T.S and the JSPS KAKENHI Grant Number (18K10000) for M.S. T.S., K.A., T.H., and M.F. was funded by the Uehiro Foundation on Ethics and Education.CLINICAL TRIALS AND REGENERATIVE MEDICINE INTERVENTIONSF-3079CLINICAL EVALUATION OF MINIMAL CONDITIONING FOR ENGRAFTMENT OF AUTOLOGOUS BLOOD STEM/PROGENITOR CELLS GENETICALLY-MODIFIED WITH A LENTIVIRUS VECTOR ENCODING MULTIPLE ANTI-HIV RNASStan, Rodica - Center for Gene Therapy/Beckman Research Institute, City of Hope, Duarte, CA, USA Torres-Coronado, Monica - Center for Gene Therapy, City of Hope, Duarte, CA, USA Gardner, Agnes - Center for Gene Therapy, City of Hope, Duarte, CA, USA Chupka, Jonathan - Center for Gene Therapy, City of Hope, Duarte, CA, USA Li, Xiu-Li - Center for Gene Therapy, City of Hope, Duarte, CA, USA Li, Zhongqi - Center for Gene Therapy, City of Hope, Duarte, CA, USA Gonzalez, Nancy - Center for Gene Therapy, City of Hope, Duarte, CA, USA Ahmed, Amira - Center for Gene Therapy, City of Hope, Duarte, CA, USA Kim, Teresa - Heme/HCT, City of Hope, Duarte, CA, USA Li, Haitang - Mol Cell Biology, City of Hope, Duarte, CA, USA Alluin, Jessica - Mol Cellular Biology, City of Hope, Duarte, CA, USA Duarte, Lupe - Heme/HCT, City of Hope, Duarte, CA, USA Nolan, Cara - Clinical Trials Office, City of Hope, Duarte, CA, USA Palmer, Joycelynne - Information Sciences, City of Hope, Duarte, CA, USA Shovlin, Margaret - Medical Oncology Branch, National Cancer Institute, Bethesda, MD, USA Lucas, Andrea - Medical Oncology Branch, National Cancer Institute, Bethesda, MD, USA Roschewski, Mark - Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Wilson, Wyndham - Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Little, Richard - Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Maldarelli, Frank - Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Rossi, John - Mol Cell Biology, City of Hope, Duarte, CA, USA Cardoso, Angelo - Center for Gene Therapy, City of Hope, Duarte, CA, USA Krishnan, Amrita - Heme/HCT, City of Hope, Duarte, CA, USA Zaia, John - Center for Gene Therapy, City of Hope, Duarte, CA, USAThe lentivirus vector (LV), rHIV7-shI-TAR-CCR5RZ, has been safe when used to transduce hematopoietic stem/progenitor cells (HSPC) that were then transplanted in four patients undergoing complete myeloablative therapy for treatment of AIDS-related lymphoma (ARL) with autologous hematopoietic cell transplantation. To determine if safer and less ablative conditioning regimens could be used as alternative conditioning, we evaluated R-EPOCH [rituximab, etoposide, adriamycin, vincristine, cyclophosphamide, and prednisone] in patients undergoing frontline therapy for ARL (NCT02337985), or low-dose busulfan in HIV-infected subjects in remission from ARL after front-line therapy (NCT01961063). The main goals of these two clinical trials were to demonstrate: a) the safe use of the LV encoding multiple anti-HIV-RNAs in humans, and b) the engraftment of gene-modified progeny cells following minimal conditioning. Long-term follow-up data are still being collected for both clinical trials. In the first study, three subjects with ARL were treated with LV-transduced HSPC, infused two days after they received the last dose of the R-EPOCH regimen. No serious adverse events were attributed to the autologous gene-modified products. With this R-EPOCH regimen, there was some minimal level of marking, tested by ddPCR for WPRE, in the peripheral blood at 1-2 months post infusion, and expression of the shI RNA transgene was detected by RT-loop Q-PCR up to 3 months post infusion. Nevertheless, there was no long-term engraftment of the gene-modified HSPC. Preliminary results showed no apparent effect of treatment on HIV reservoir (cell-associated DNA and RNA and plasma HIV viral load). In the second study, with low-dose busulfan (3.2 mg/kg, intravenous, given once over 4 hours) as conditioning regimen, two research subjects were treated, and as with the other clinical trial subjects, although the therapy was safe, there was negligible vector marking in the peripheral blood. Expression of shI RNA disappeared after Month 2 post infusion. In conclusion, the minimal conditioning regimens chosen here to reduce risk failed to provide adequate preparation for long-term engraftment of LV-modified autologous HSPC patients with ARL.Funding Source: NIH U01 CA183012 and Judith Owens FundF-3081NAVIGATING HUMAN CELL THERAPY TRIALS: A PLATFORM FOR TRANSLATING NOVEL THERAPIES INTO CLINICAL PRACTICEBaer, Meghan - Center for Regenerative Medicine, Mayo Clinic, Saint Johns, FL, USA
483POSTER ABSTRACTSShapiro, Shane - Center for Regenerative Medicine, Mayo Clinic, Jacksonville, FL, USARecent progress in application of human cell and cell-derived therapies around the world demonstrates the rapid pace of discovery and translation of novel approaches to treat unmet patient needs for a number of disease conditions. In order to continue with this ambitious pace, academic, public and private organizations need the requisite tools in place to facilitate further advances in the field. Despite these recent successes, navigating the evolving regulatory and legal environment involves unique challenges in bringing human cell therapy products to market and/or clinical practice. Although common research coordination services exist at many institutions, Mayo Clinic Center for Regenerative Medicine has implemented an integrated system we have titled Translation into Practice Platforms (TIPPs) specifically designed to address the particular challenges within the human cell therapy field. TIPPs is an institution wide program that includes the integral translational components needed to accelerate human cell therapy trials using validated therapies into clinical practice. The objective of the service is to provide partnership with clinicians and research faculty interested in clinical trial research. Components of this partnership include a “Translational Navigator” along with a team consisting of a clinical protocol development specialist, a regulatory support manager, and clinical trial coordinators. We describe how the team connects investigators with resources linking clinical departments and their medical specialty needs with cell therapy manufacturing platforms (cellular, cell-derived, and devices). Collectively, this group provides current and timely education on the scope of the growing human cell therapy field and assists with clinical trial design, IRB approval, regulatory expertise for a given product or device, as well as coordinator support for execution of early clinical trials. As this platform guides clinicians and scientists through education and clinical trial engagement, translational navigation will remain a crucial element ensuring cell therapies progress through the discovery-translation-application continuum via quality clinical trials.F-3083TEG-SEQ: A WORKFLOW FOR IN CELLULO MAPPING OF CRISPR SPECIFICITYTang, Pei-zhong - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA Chesnut, Jonathan - Cell Biology, ThermoFisher, Carlsbad, CA, USA Ding, Bo - GCD, Thermo Fisher, Carlsbad, CA, USA Mozhayskiy, Vadim - GCD, ThermoFisher, Carlsbad, CA, USA Peng, Lansha - Cell Biology, ThermoFisher, Carlsbad, CA, USA Potter, Jason - Cell Biology, ThermoFisher, Carlsbad, CA, USAEngineered nucleases, including the CRISPR/Cas9 system, have been widely used for genome editing, and is now being developed to create gene and cell therapies to treat human disease. However, lack of specificity leading to off-target cleavage is still a concern. To measure this, an in cellulo method, genome-wide unbiased identification of double stranded breaks enabled by sequencing (GUIDE-seq) was developed and has been widely used (Tasi Q et.al). However, this method as originally reported was associated with a significant level of non-specific target amplification which reduced sensitivity and increased the cost to detect low-frequency off-target events. In an attempt to improve robustness and sensitivity, we developed a modified method termed Target-Enriched GUIDE-seq (TEG-seq). The modification improves the sensitivity approximately 10 fold compared to GUIDE-seq. In addition to the increased specificity, we developed high-throughput workflow and data analysis tool that led TEG-seq to become more cost-effective. Using TEG-seq, we evaluated a panel of Cas9 mutants to identify potential high-fidelity Cas9 protein that will be a critical for genome editing, especially for gene and cell therapy. We also used TEG-seq to map on- and off-target cleavage events on 22 gRNAs targeting a set of therapeutically relevant SNPs. Finally, TEG-seq was used to evaluate CRISPR off-target profiling for therapeutic applications in different cells including iPSC and CAR-T cells and an animal model. TEG-seq off-target detection with the use of high-fidelity Cas9 proteins will be one of the crucial steps in genome-editing and gene therapy.F-3085DEVELOPMENT OF A NEEDLE FOR CELL INJECTION INTO A BRAINKikuchi, Tetsuhiro - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Morizane, Asuka - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Doi, Daisuke - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Takahashi, Jun - Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanWe started a clinical trial for a cell transplantation therapy for Parkinson’s disease (PD). Previously, we reported successful transplantation of induced pluripotent stem (iPS) cell-derived dopaminergic neuron progenitors into PD model monkeys, and a few issues remain before the clinical application. One of those problems is a development of a device for cell transplantation. In Japan, some cell injecting devices are approved for clinical use by Ministry of Health, Labor and Welfare. However, these devices are not suitable for our cell transplantation therapy, considering a small amount of transplanted cell suspension (about 5 uL). We decided to develop a brand new needle for our clinical trial. We assessed shape, length and diameter of a needle and manufactured a new needle with a corporation. The newly developed needle is comparable with that we used in the previous preclinical trial in terms of survivability of injected cells and injection efficiency, and cells transplanted with the new needle survived in monkey brains. Accuracy of this stereotactic system is estimated to be within ± 2 mm. We successfully performed the first transplantation of iPS cell-derived dopaminergic neuron progenitors into a PD patient. In conclusion, we developed a brand new needle for cell transplantation for Parkinson’s disease and this system will be applicable for treatment for other brain disorders in a future.
484POSTER ABSTRACTSFunding Source: Japan Agency for Medical Research and Development (AMED)GERMLINE, EARLY EMBRYO AND TOTIPOTENCYF-3087EED REPRESSES SOMATIC GENE EXPRESSION DURING MOUSE PRIMORDIAL GERM CELL DEVELOPMENTLowe, Matthew - Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Yen, Ming-Ren - Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan Hu, Zhongxun - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Hunt, Timothy - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Gorgy, Isaac - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Hosohama, Linzi - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Chen, Pao-Yang - Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan Clark, Amander - Molecular Cell and Developmental Biology, University of California, Los Angeles (UCLA), Los Angeles, CA, USAMammalian Primordial germ cells (PGCs) are specified by induction from the epiblast and serve as the source of all adult germ cells used for reproduction. Following specification, male and female PGCs undergo two-stages of DNA demethylation beginning with global DNA demethylation followed by demethylation of genes responsible for regulating the timing of sex-specific PGC differentiation. As DNA methylation is lost globally from the PGC genome, Histone 3 Lysine 27 trimethylation (H3K27me3) is rapidly enriched. To explore the role of H3K27me3 in PGC development we created a PGC specific conditional knockout of Embryonic Ectoderm Development (EED) in mouse PGCs at the time of induction, which leads to an inability to enrich and maintain H3K27me3 during PGC differentiation. Utilizing immunofluorescence imaging, FACS, and RNA sequencing, we show that H3K27me3 inherited from the epiblast are responsible for regulating somatic cell gene expression. In contrast, H3K27me3 acquired during PGC maturation regulate sex-specific timing of PGC differentiation. Taken together, our data indicate that H3K27me3 plays a complex role in regulating germline cell fate and state in mammals. This work has important consequences for not only elucidating the mechanisms that regulate reproductive health, but also towards developing a fundamental understanding of events that are critical to differentiate germ cells in vitro from stem cells.F-3089MITOCHONDRIAL DYNAMICS CONTROLS GERMLINE STEM CELL MAINTENANCE WITH AGEAmartuvshin, Oyundari - Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Kao, Shih-Han - Institute of Cellular and Organismic Biology, Academia Sinica, Taipei City, Taiwan Hsu, Hwei-Jan - Institute of Cellular and Organismic Biology, Academia Sinica, Taipei City, TaiwanAging is a process which is correlated with inability to maintain tissues’ original function which further increase vulnerability of age-related disease. Asymmetric division of stem cells is crucial for life-long tissue homeostasis where one of the daughter cells receive stemness signaling from niche to maintain its stem cell identity; recent studies have shown that mitochondrial dynamics controls stem cell fate decision, suggesting that stem cell fate is coordinated with mitochondrial dynamics to function properly. However, the link between stemness signaling and mitochondrial dynamics in stem cell during aging is obscure. We have previously reported that decrease of maintenance and division in the germline stem cell (GSC) during aging. Here, by analyzing mitochondria labeled by ATP5ase, we found that mitochondrial content is decreased while the number of fragmented mitochondria is increased in aged GSCs. Consistently, disruption of mitochondrial fusion in the GSCs mimics aging phenotype which exhibits decreased division rate, maintenance and stemness signaling. Interestingly, forcing mitochondrial fusion does not cause decreased GSC division and maintenance; rather, it promotes competitiveness of GSCs for niche occupancy. Taken together, these results suggest that, whereas mitochondrial fusion may be serving as a protective mechanism for GSC persistency, mitochondrial fission is detrimental for GSCs during aging. Although the involved mechanisms by which mitochondrial dynamics controls stemness signaling still need to be further investigated, our study has documented the impact of mitochondrial dynamics change on stem cell maintenance and division with age in Drosophila germline.F-3091MODELING THE DEVELOPMENT OF NEURAL PLATE BORDER AND EPIDERMIS USING HUMAN PLURIPOTENT STEM CELLSXie, Tianfa - Department of Mechanical and Industrial Engineering, University of Massachusetts Amherst, MA, USA Sun, Yubing - Mechanical and Industrial Engineering, University of Massachusetts Amherst, Amherst, MA, USA
485POSTER ABSTRACTSUnderstanding the process of ectoderm development is essential for the prevention and treatment of various birth defects. While the early ectodermal development has been extensively studied using animal models, a model with human genetic background is needed to reveal the human-specific aspect of it. Our previous work has demonstrated that micropatterned culture of human pluripotent stem cells (hPSCs) lead to spatially ordered neuroectoderm and neural plate border differentiation. In this work, we report a fully-defined protocol to induce hPSCs to self-organize into concentric rings of neural plate border cells and epidermis with a clear boundary, mimicking fate patterning in human ectoderm. hPSCs were geometrically confined via micro-contact printing and cultured in E6 medium supplemented with a TGF- inhibitor, a WNT agonist, and BMP4 protein for five βdays. We found that cells at the edge of the micropatterned colonies differentiated to trophectoderm (CDX2+ AP2+ SOX10- TP63- ). Next to those cells was a ring of cells positive for neural crest markers (AP2+ SOX10+ ). Inward was another ring of cells positive for epidermis markers (AP2+ TP63+ ). The cells at the center of the colonies expressed trophectoderm markers. Further studies showed that cell fate patterns depended on the seeding density. Lower seeding density led to patterns without trophectoderm cells at the center while higher seeding density led to patterns without trophectoderm layer at the edge. We further showed that the self-organized ring-shape pattern formation of the neural plate border and epidermis required intermediate WNT and BMP activities. Adding exogenous BMP/WNT Agnoist was required for the formation of complete rings of epidermis and neural plate border, respectively. In addition, a higher dosage of BMP increased the width of trophectoderm and epidermis rings. In summary, by integrating engineering approaches and chemically defined culture conditions, we have developed a highly reproducible, high-throughput in vitro system that can be readily used to quantitatively study the functional role of morphogens and genes in the cell fate patterning of human ectoderm.F-3093DECODING PLURIPOTENCY AND TOTIPOTENCY BY NOVEL LONG INTERGENIC NON-CODING RNASMa, Xun - School of Biomedical Sciences, The Chinese University of Hong Kong (CUHK), Shatin, Hong KongTotipotent cell, which is capable of developing into a complete organism, is expected as an important tool in regenerative medicine and disease modelling. Thus, it is foreseeable that in vitro cultured cell model of mammalian totipotent stem cells is of paramount interests to both biomedical and clinical research. Long intergenic non-coding RNAs (lincRNA), which are critical in cell stemness and cell fate, are expected as a novel important tool to induce cell reprogramming. However, to date, biological roles of lincRNA in totipotency has been rarely uncovered. By integrating extensive public RNA-seq datasets of mES, we have discovered ~5000 novel lincRNAs. In addition, from single cell transcriptome we identified a gene cluster comprising protein coding genes annotated lncRNAs as well as novel lincRNAs, which are highly active in mouse 2-cell embryo. In vivo experiments sugguest those genes express specifically in 2-cell mouse embryo. Besides, selected genes and novel lincRNAs can trigger mouse pluripotent stem cells to totipotent-like cells. Our result suggests a novel path to establish the mammalian totipotent stem cell lines in vitro, and eventually promote the totipotent-related stem cell therapy in biomedical and clinical research.CHROMATIN AND EPIGENETICSF-3095ROLE OF CHROMATIN ORGANIZATION IN DOUBLE STRAND BREAK REPAIR IN MOUSE EMBRYONIC STEM CELLSChechik, Lyuba - Development and Stem Cells, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasbourg, France Furst, Audrey - Development and Stem Cells, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasbourg, France Soutoglou, Evi - Development and Stem Cells, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasbourg, FranceThe ability of pluripotent cells to differentiate into any embryonic or adult cell type raises requirements to maintain the high genome integrity level. Some stem cell-specific mechanisms to prevent accumulation of mutations and chromosomal aberrations have been discovered. However, study of DNA repair is complicated by the influence of local chromatin structure on its kinetics and the balance among repair pathways. Moreover, some chromatin types like facultative heterochromatin and bivalent chromatin have been less studied than the others in this context. Double strand breaks (DSBs) are the most dangerous DNA lesion type as the second strand is not available as a template for repair. Embryonic stem cells (ESCs) have been described to amend this kind of damage in a more error-free way than somatic cells. However, the underlying mechanisms are not clear yet. We aimed to assess them by following the repair outcome in ESCs in comparison with differentiated cells. To this end, we developed a mouse ESC line stably expressing Cas9 and designed guide RNAs to induce DSBs in different chromatin types: euchromatin, facultative heterochromatin and bivalent regions. We used 3T3 fibroblasts as a differentiated cell model for comparison. We assessed repair fidelity by TIDE, a sequencing-based method that render the readout of particular insertions and deletions as well as total proportion of mutated sequences. To evaluate the efficiency of homologous recombination (HR), we co-transfected guide RNAs and homologous templates with a small insert at the place of expected DNA break. Predictably we observed that ESCs perform higher rate of HR than the differentiated cells. However, to our surprise, this was the case in both active and inactive genes, implying that facultative heterochromatin is HR permissive. Additionally, we can also conclude that bivalent regions are repaired the same way as euchromatin. Interestingly, preliminary results from blocking cell cycle suggest
486POSTER ABSTRACTSthat shortened G1 phase in ESCs compare to differentiated cells might be one of the main reasons of the low rate of erroneous repair. To conclude, our work improves the understanding of the interplay between chromatin and DNA repair in ESCs and provides insights to potential mechanisms of genome integrity maintenance.F-3097THE EFFECT OF MATERNAL DIET AND NUTRITIONAL STATUS ON MAMMALIAN SEX DETERMINATIONShingo, Miyawaki - Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Tachibana, Makoto - Graduate School of Frontier Biosciences, Osaka University, Suita, JapanMammalian sex is determined by the expression of Sry in the fetal gonadal somatic cells (pre-Sertoli cells). We previously found that epigenetic regulation plays an important role on Sry regulation. The H3K9 demethylase Jmjd1a directly targets Sry and removes H3K9 methylation mark from this locus to activate Sry. In XY Jmjd1a-deficient mice, male-to-female sex reversal was observed at certain frequency. The discovery of the fundamental role of epigenetic regulation of mammalian sex-determining gene Sry led to a new research area, namely, on the mechanistic link between environmental cues and sex determination. Embryonic development may be influenced by the maternal nutritional and metabolic state as fetal environment. Interestingly, we found that the maternal diet affected the frequency of sex-reversal of XY Jmjd1a-deficient mice. Since the maternal diet may directly affect the embryonic metabolism, we speculate that the metabolism and nutritional status of fetal pre-Sertoli cells influence their sex determination process. We first compared the gene expression profile between pre-Sertoli cells and the other gonadal somatic cells at the sex-determining period. Gene ontology analysis showed that metabolism-related process is clearly enriched in pre-Sertoli cells. To investigate the role of metabolic genes on sex determination that were highly expressed in pre-Sertoli cells, we generated knockout mice of these genes using CRISPR-Cas9 system. At this conference, we will report on the progress of phenotype analysis of several knockout lines. Some metabolites play as the substrates or co-factors for certain epigenetic enzymes. Therefore, it is considered that metabolites can regulate gene expression epigenetically. Our study lead to identification of novel pathway that links metabolites and mammalian sex determination.Funding Source: This work was supported by JSPS KAKENHI grant numbers 17H06423, 17H06424, and 17K17924.F-3099FOLATE SUPPLEMENTS NEURAL TUBE DEFECTS BY TRANSCRIPTIONAL ACTIVATION OF NEURAL LINEAGE GENES VIA SUPPRESSING MDM2-MEDIATED H2A UBIQUITYLATION (MOUSE EMBRYO AND HUMAN EMBRYO)Pei, Pei - Capital Institute of Pediatrics, Capital Institute of Pediatrics, Beijing, China Wang, Shan - Department of Biochemistry and Immunology, Capital Institute of Pediatrics, Beijing, China Zhang, Ting - Department of Biochemistry and Immunology, Capital Institute of Pediatrics, Beijing, ChinaFolate has been widely used in clinics to prevent neural tube defects (NTDs) during the pregnancy. However, the mechanisms by which folate regulates the neural crest lineage cell differentiation is unknown. In this study, we studied methotrexate (MTX)-induced differential gene expression and histone modifications in mouse embryonic stem cells (mESCs), brain/spine tissues from mouse and human NTD fetuses by advanced biochemical and molecular approaches. We found that MTX suppressed embryonic cell differentiation and caused NTDs by repressing neural lineage gene expression in mESCs. Treatment of mESCs with MTX caused an increase in MDM2 expression and binding to lineage genes, leading to H2AK119 monoubiquitylation (H2AK119ub1) and transcriptional repression. Knockdown of MDM expression abolished MTX induction of H2AK119 ubiquitylation, and re-activate lineage gene expression. We also demonstrated that MTX treatment induced H2A ubiquitylation at DSB target sites by a mechanism dependent on ATM activation. Treatment of mESCs with Folate suppress MTX-induced MDM2 expression and H2AK119 ubiquitylation in the neural precursor markers genes, restored the transcriptional activation of these genes, and prevented NTDs in mice. In agreement with the data from mouse studies, we observed a dramatic down-regulation of neural precursor genes in fetal NTD brain tissue that was associated with low levels of folate in maternal sera, and enhanced levels of H2AK119ub1 in NTD tissue. Our studies indicate that folate prevents NTD by maintaining normal lineage cell differentiation via repressing MDM2 expression, reducing MDM2-mediated H2AK119ub1 and transactional repression of the neural precursor marker genes.Funding Source: National Science Fund 31571324F-3101TARGETED GENE ACTIVATION DIRECTS TROPHOBLAST TRANS-DIFFERENTIATION USING THE NOVEL DESIGNED EPIGENETIC INHIBITOR, EEDBINDER-DCAS9Levy, Shiri - Biochemistry, University of Washington, Seattle, WA, USA
487POSTER ABSTRACTSBifurcations in cell fates are controlled through epigenetic modifications, however the key loci regulated by PRC2 dependent H3K27me3 repressive marks are not known. To dissect the functional loci regulated by PRC2 in trophoblast to ICM bifurcation we fused a computationally designed protein, EED binder (EB) that tightly binds EED and disrupts PRC2 function, to dCas9 to direct PRC2 inhibition at precise loci using gRNA. Free of DNA manipulations or chemical inducers, EBdCas9 is able to transdifferentiate human induced pluripotent stem cells (iPSC) to human trophoblast fate using gRNA specific to two key transcription factors CDX2 and GATA3. Co-transfection of gRNA targeting GATA3 and CDX2 resulted in 40-80 fold increase of these transcripts, as well as the trophoblast markers. ChIPseq analysis showed H3K27me3 reduction in GATA3 TSS and gene body compared to untransfected samples. RNAseq analysis revealed that our EBdCas9 gRNA transfected, but not control hPSC produced gene expression signature that corresponds to human trophoectoderm and alignment against early cynomolgus monkey single cell transcriptome showed enhanced advancement towards trophectoderm. Epigenetic memory tracing of the newly trans-differentiated trophoectoderm cells confirms a transcript upregulation threshold that is maintained for at least 21 days following initial gRNA transfection. These data reveal for the first time that the first human fate bifurcation between trophoblast and ICM is solely controlled by PRC2 dependent epigenetic H3K27me3 marks in precise loci upstream of Gata3 and CDX2 TSSs. We also tested the other general applicability of EBdCas9, by using tiling to identify the regions where gRNAs induce transcription in the following bivalent genes: TBX18, p16, and Klf4. In total, we have targeted 40 sites upstream of five different genes, and observed significant transcriptional derepression in all genes, all together in 17 loci. EBdCas9 tool is broadly applicable to questions in epigenetic regulation of single locus to pinpoint critical marks for control of gene expression by PRC2.PLURIPOTENCYF-3105CULTURE OF HIGH-QUALITY HUMAN PLURIPOTENT STEM CELLS WITH VERSATILE WORKFLOWS USING MTESR PLUS, A NEW STABILIZED TESR MAINTENANCE MEDIUMWong, Matthew K - Research and Development, STEMCELL Technologies Inc, Vancouver, BC, Canada Warren, Kiera - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Merkulova, Yulia - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Ho, Rowena - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Lee, Tina - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Hirst, Adam - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Watson, Ashley - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Hills, Mark - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Kardel, Melanie - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Hunter, Arwen - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Thomas, Terry - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Eaves, Allen - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Louis, Sharon - Research and Development, STEMCELL Technologies, Vancouver, BC, CanadaSpecialized culture media is required to maintain the self-renewal and pluripotent properties of human pluripotent stem cells (hPSC). To date, the majority of culture systems require daily medium changes in order to replenish levels of critical components and eliminate accumulated metabolic waste. This is time-consuming when maintaining multiple cell lines, especially in hPSC core facilities, and typically requires operators to change medium over the weekend. mTeSR™ Plus, based on the mTeSR™1 formulation, was specifically developed to ensure truly versatile feeding schedules while maintaining high quality hPSC cultures. The stabilization of FGF2 levels over 72 hours at 37°C (83.6% ± 7.3%, n=3) combined with an enhanced buffering capacity that maintains pH 7.0 for up to ≥72 hours without feeding, supports flexibility for every other day or weekend-free schedules. We investigated key cell quality parameters of hPSCs cultured for 10 passages in mTeSR™ ≥Plus with reduced feeding compared with cells in mTeSR™1 with daily feeding. hPSC marker expression was assessed by flow cytometry every 5 passages and hPSCs cultured in mTeSR™ Plus maintained an average of 98.2 ± 2.1% OCT4 and 93.5 ± 3.1% TRA-1-60 for up to 20 passages (n=4 cell lines). Likewise, hPSCs maintained in mTeSR™ Plus with reduced feeding were capable of directed differentiation to all three germ layers using the STEMdiff™ Trilineage Kit (n=4 cell lines). hPSCs maintained in mTeSR™ Plus (>10p) were karyotypically normal by G-banding and no common chromosomal abnormalities were detected by the hPSC Genetic Analysis Kit every 5 passages (n=4 cell lines). Transcriptome analysis of 19,665 genes by RNA sequencing of hPSCs maintained in mTeSR™ Plus resulted in a gene expression profile indistinguishable from cultures maintained in mTeSR™1 (n=2 cell lines). Furthermore, mTeSR™ Plus demonstrated 33.9 ± 1.4% gene knockout efficiency using the ArciTect™ CRISPR/Cas9 system (n=2) and 26.0 ± 4.4% cloning efficiency with CloneR™ supplement (n =3), which is similar to or better than hPSCs cultured in mTeSR™1. In summary, mTeSR™ Plus is an improved medium that promotes a more consistent cell culture environment enabling versatile workflows while maintaining high quality hPSCs that are fully compatible with established genome editing and differentiation protocols.
488POSTER ABSTRACTSF-3107DERIVATION OF PIG EMBRYONIC STEM CELLS USING CHEMICALLY-DEFINED MEDIA.Lee, Mingyun - Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Lee, Chang-Kyu - Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Choi, Kwang-Hwan - Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Kim, Seung-Hun - Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Lee, Dong-Kyung - Department of Agricultural Biotechnology, Seoul National University, Seoul, KoreaPig embryonic stem cells (ESCs) have been considered an important candidate for preclinical research on human therapies. However, the lack of understanding of pig pluripotent networks has hampered the establishment of authentic pig ESCs. Here, we report that FGF2, ACTVIN, and WNT signaling are essential to sustain pig pluripotency in vitro. Pig ESC lines derived by stimulating three signalings formed colonies with a flattened monolayer morphology. Newly derived ESCs were stably maintained over an extended period and were capable of forming teratomas that contained three germ layers. Furthermore, the pig stem cells have the ability of direct differentiation into specific cells including mature neuron, pancreas progenitor and cardiac muscle cells. Immunostaining showed that the stem cells expressed pluripotency markers including OCT4, SOX2, NANOG, SSEA1, and SSEA4. Interestingly, the pig ESCs had distinct features such as coexpression of SSEA1 and SSEA4, two active X chromosomes, and a unique transcriptional pattern. In conclusion, we derived authentic pig ESCs using novel cell culture conditions. Our findings will facilitate both the development of large animal models for human stem cell therapy and the generation of pluripotent stem cells from other domestic animals for agricultural use.Funding Source: Korea Institute of Planning and Evaluation for Technology (IPET), through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-1-HD020).F-3109A NOVEL GPCR FAMILY GENE HECAT5 MODULATES CELL CYCLE PROGRESSIONRoh, Seung Ryul - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea Kim, Min Woong - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwan, Korea Shin, Jeong A - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwan, Korea Kim, Dong Chul - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwan, Korea Kim, Jiin - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwan, Korea Lee, Myung Ae - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwan, KoreaEmbryonic stem (ES) cells are originated from the pluripotent inner cell mass of the blastocyst stage. They have two important properties: self-renewal and pluripotency. For these reasons, ES cells can be used in potential therapeutic applications in regenerative medicine. But, there are ethical problems and adverse effects regarding the use of ES cells. To investigate the mechanism regulating stemness, especially cell cycle, of ES cells, we identified HECAT5, the gene co-expressed in both ES and tumors, but not normal tissues, using Digital Differential Display (DDD). Overexpression of HECAT5 dramatically increased S phase population and cell proliferation in vitro in hNSC and HEK293, but not NIH3T3 cells. In addition, it enhanced tumorigenicity and growth in soft agar assay and in xenograft tumor assay. HECAT5 had effects on those through interaction with M3R because M3R-specific antagonists specifically inhibit cell proliferation induced by HECAT5 overexpression. All our results demosntrated that novel HECAT5 gene may play a role in the regulation of stemness in hES cells.Funding Source: This work was supported by the National Research Foundation of Korea, a grant funded by the Korean Government [2015M3A9C6028956].F-3111EIF4A1-MEDIATED SPECIFIC PROTEIN TRANSLATION IN HUMAN EMBRYONIC STEM CELLZhou, Xiaoxiao - Faculty of Health Sciences, University of Macau, ChinaProtein translation is critical for numerous cellular functions such as transcription, metabolism, stress response and cellular proliferation. However, it is unclear how translation is regulated in human embryonic stem cells(hESC), and how it affects specific cellular functions. In this report, we show that eIF4A1-dependent translation initiation plays critical roles in hESC maintenance. EIF4A1 is essential for Cap-dependent translation, and its inhibition by small chemical silverstrol leads to severe cell type specific cell death in hESC, but not in other somatic cells. Genome-wide ribosome profiling analysis reveals that silvestrol treatment specifically suppresses pathways involved in TGF signaling, MAPK signaling, apoptosis and so βon. Further study shows that silvestrol specifically suppressed NANOG and CDH1; but not OCT4 and SOX2. These data show eIF4A1-mediated translation differentially control pluripotency related proteins, and influence stem cell survival and cell fate determination.
489POSTER ABSTRACTSFundingSource:MYRG2018-00135-FHS,CellFateDetermination by Pyruvate in Human Pluripotent Stem CellsF-3113HLA DR GENOME EDITING WITH TALENS IN HUMAN IPSCS PRODUCED IMMUNE-TOLERANT DENDRITIC CELLSKwon, Yoo-wook – Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea Ahn, Hyo-Suk - Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea Kim, Hyo-Soo - Internal Medicine, Seoul National University Hospital, Seoul, Korea Cho, Hyun-Jai - Internal Medicine, Seoul National University Hospital, Seoul, KoreaAlthough human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by high costs and long time required for generation. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune non-responsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN- . Teratomas formed by HLA γDR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.F-3115THE IMPACT OF DOUBLE X-DOSAGE ON SIGNALING PATHWAYS IMPLICATED IN PLURIPOTENCYSultana, Zeba - Regulatory Networks in Stem Cells, Max Planck Institute for Molecular Genetics, Berlin, Germany Dorel, Mathurin - Computational Modelling in Medicine, Charite - Universitätsmedizin, Berlin, Germany Klinger, Bertram - Computational Modelling in Medicine, Charite - Universitätsmedizin, Berlin, Germany Sieber, Anja - Computational Modelling in Medicine, Charite - Universitätsmedizin, Berlin, Germany Bluethgen, Nils - Computational Modelling in Medicine, Charite - Universitätsmedizin, Berlin, Germany Schulz, Edda - Regulatory Networks in Stem Cells, Max Planck Institute for Molecular Genetics, Berlin, GermanyFor a short time window during early development of mammalian embryos, both X chromosomes in females are active, before dosage-compensation is ensured through X-chromosome inactivation (XCI) during the exit from the pluripotent ground state. The double dose of X-linked genes compared to male cells, in female mouse embryonic stem cells (mESC) that still have both X chromosome active, has been found to increase expression of pluripotency factors, attenuate differentiation and decrease global DNA methylation. To identify X-dosage dependent effects on the signaling network in we combine systematic perturbation experiments with mathematical modeling. mESCs with either one (XO) or two active X chromosomes (XX) were treated with exogenous stimulants or inhibitors targeting 5 different pathways (Fgf/Mapk, Lif/Stat3, Pi3k/Akt, Bmp4/Smad1, Activin/Smad2), either individually or in combinations (53 treatments per cell line). Signaling intermediates were measured using a bead-based multiplexed phosphorylation assay and western blotting. We then apply a semi-quantitative modeling approach based on Modular Response Analysis to this perturbation data set to reconstruct the signaling networks in XX and XO mESCs. Specifically, a literature-derived starting network comprised of only the canonical linear cascades is optimized, by adding or removing links, to best fit the perturbation data. In this way several previously reported interactions were identified, such as a negative feedback loop in the Mapk pathway and cross talk from Lif pathway towards Mapk and Akt activation. Additionally, not yet described cross talk between the Bmp4, Activin and Mapk pathways was identified, which is currently being validated in experiments. Through comparison of the networks reconstructed for XX and XO cells we can now identify X-dosage dependent differences in their signaling networks. We find that cells having a single active X are more sensitive to the differentiation promoting Activin stimulation, than cells with two active X chromosomes. In summary, a data-driven systems biology approach allows us to identify novel cross-talk between different signaling pathways and to narrow down the putative point(s) in the network via which a double dose of active X-chromosomes might mediate its global effect on the pluripotent state.PLURIPOTENT STEM CELL DIFFERENTIATIONF-31193D MICRO-CULTURE PLATFORM ENABLES ADVANCED, HIGH-THROUGHPUT SCREENING FOR DIFFERENTIATION OF HUMAN PSC-DERIVED CELL THERAPIESMuckom, Riya J - Chemical Engineering, University of California, Berkeley, CA, USA Bao, Xiaoping - Chemical Engineering, Purdue University, West Lafayette, IN, USA Tran, Eric - Chemical Engineering, University of California, Berkeley, CA, USA Chen, Evelyn - Molecular and Cellular Biology, University of
490POSTER ABSTRACTSCalifornia, Berkeley, CA, USA Murugappan, Abirami - Chemical Engineering, University of California, Berkeley, CA, USA Clark, Douglas - Chemical Engineering, University of California, Berkeley, CA, USA Schaffer, David - Chemical Engineering, University of California, Berkeley, CA, USAThe promising outlook for human Pluripotent Stem Cell (hPSC) derived cell therapies motivates the development of manufacturing processes to meet the patient demand for such therapeutics. Toward this aim, 3D culture systems for hPSC differentiation are emerging because of their potential for higher expansion and yield of target cell types compared to 2D culture systems. Therefore, the ability to screen through a multifactorial parameter space of exogenous biochemical cues for 3D hPSC cultures would greatly accelerate the pace of discovery and development of efficient in vitro differentiation protocols for target cell types of interest. Here, we demonstrate the advanced capabilities of a 3D micro-culture platform to screen dosage, duration, dynamics, and combinations of 12 culture parameters, totaling more than 1000 unique 3D culture environments, to derive Olig2+Nkx2.2+ oligodendrocyte progenitor cells (OPCs) from hPSCs with 0.2% of the reagent volumes used in 96-well plates. Additionally, we leverage novel fluorescent hPSC reporter cell lines, engineered using a Cas9 knock-in strategy, to monitor proliferation and differentiation in situ for over 80 days in the 3D micro-culture system. We identified several early culture parameters that could be tuned to increase the Olig2 expression 10-fold. In addition, we observed different sensitivities to signaling pathways across time. Finally, a holistic analysis using statistical models uncovered and ranked parameters and combinations thereof according to their positive effect on OPC differentiation to prioritize them in future optimizations. To show the generalizability of the platform, we then applied it to simultaneously assay 90 unique differentiation protocols to derive TH+ midbrain dopaminergic neurons from hPSCs. Overall, we demonstrate a strong methodology for upstream microscale screening/optimization to inform downstream scale-up processes to improve 3D production strategies of hPSC-derived cell replacement therapies.Funding Source: This work was funded by California Institute of Regenerative Medicine grant number DISC2-08982.F-3121DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS TOWARDS MESENCHYMAL STROMAL CELLS IS TRIGGERED BY FLAT SUBSTRATESGoetzke, Roman - Stem Cell Biology and Cellular Engineering, RWTH Aachen School of Medicine, Aachen, Germany Keijdener, Hans - Applied Medical Engineering, RWTH Aachen University Medical School, Aachen, Germany Franzen, Julia - Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany Ostrowska, Alina - Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany Nüchtern, Selina - Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany Mela, Petra - Applied Medical Engineering, RWTH Aachen University Medical School, Aachen, Germany Wagner, Wolfgang - Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, GermanyDifferentiation of induced pluripotent stem cells (iPSCs) towards mesenchymal stromal cells (MSCs) remains a major challenge in regenerative medicine. On conventional tissue culture plastic, this differentiation process is incomplete, at least on epigenetic level. However, there is evidence, that differentiation of iPSCs can be guided by mechanical cues and therefore, we investigated if this process can be triggered by differentiation in a 3D matrix. iPSCs were embedded into fibrin hydrogels to enable a one-step differentiation procedure towards MSCs within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like morphology and immunophenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, and faint expression of CD73 and CD105. Global gene expression profiles of differentiated iPSCs demonstrated that MSC-specific genes were only up-regulated on flat substrates, whereas genes of neural development were up-regulated in 3D culture. Furthermore, global DNA methylation profiles were very similar if iPSCs were differentiated towards MSCs either on tissue culture plastic or on top of hydrogels. In contrast, there were marked epigenetic differences if iPSCs were differentiated within fibrin gels, pointing towards neural differentiation. Taken together, the 3D culture conditions within fibrin hydrogel supported growth and differentiation of iPSCs, but they hampered differentiation towards MSCs. These results indicate that the typical fibroblastoid and adhesive growth pattern on flat substrates might not only be a cellular characteristic of MSCs, but it might also trigger differentiation towards this cell type.F-3123TRANSPLANTATION OF IPSC-DERIVED RETINAL PIGMENT EPITHELIAL (RPE) CELLS RESCUE OF PHOTORECEPTOR CELL DEATH IN RETINAL DEGENERATIVE RCS RATSShrestha, Rupendra - Institute of Medical Sciences, Tzu Chi university, Hualien, Taiwan Wen, Yao-Tseng - Medical Research, Institute of Eye Research, Hualien Tzu Chi General Hospital, Hualien, Taiwan Tsai, Rong-Kung - Medical Research, Institute of Medical Sciences, Tzu Chi University and Institute of Eye Research, Hualien Tzu Chi General Hospital, Hualien, TaiwanThe physiological basis of vision is associated with the interaction between retinal pigment epithelium (RPE) and photoreceptor cells. Thus, RPE transplantation is extensively used as cell-based therapy to restore the subretinal anatomy in age-
491POSTER ABSTRACTSrelated macular degeneration (AMD). Here, we aim to use the suspension of hiPSC-derived RPE cells to inject into subretinal space of RCS rats to study the biological behavior in the host retina. For this experiment, RPE cells were generated from human epidermal keratinocytes using non-integrating CytoTune 2.0 Sendai reprogramming kit. The primary hiPSC colonies were observed in 12 days that showed the expression of pluripotency markers like Oct4, Sox2, Nanog, Tra1-60 and SSEA4 analyzed by immunohistochemistry and reverse transcription-PCR. Then, the neuroectodermal differentiation of RPE was performed by monolayer culture embryoid bodies in vitronectin coated plates containing retinal differentiation medium supplemented with B27. RPE cells were characterized using immunostaining, Western blot, and PCR. The differentiated RPE showed characteristic flat hexagonal morphology that expressed markers for tight junctions (ZO1), and RPE cells (MITF, CRALBP, Bestrophin, and RPE65). After eight weeks of differentiation, the pigmentation was observed in RPE cells analyzed using a transmission electron microscope (TEM). In a long-term culture, the formation of RPE fluid-filled sac was observed and a layer of sac expressed marker, such as Bestrophin. The phagocytic activity of RPE was evident as phagocytose of latex beads. Furthermore, suspension of GFP labeled hiPSC-RPE cells were injected in the subretinal spaces of 7 RCS rats. In vivo study of transplantation showed the improvement of visual function apparent by electroretinogram (ERG) analysis. This indicates the rescue of photoreceptor loss in retinal degenerative rats, but validation with histological assessments are required to confirm the improvement. To conclude, hiPSC was generated from keratinocytes were differentiated into functional RPE cells. Also, the implantation of hiPSC-RPE cells showed transient improvement of visual function in RCS rats.Funding Source: This research was funded by the Buddhist Tzu Chi Foundation under the research grant number, “TCMMP104-05-01” (Hualien, Taiwan).F-3125IMPROVED HEPATIC MATURATION OF CGMP-COMPLIANT HUMAN PLURIPOTENT STEM CELLS USING BIOMIMETIC PEG SUBSTRATES OF PHYSIOLOGICAL STIFFNESSBlackford, Samuel J - Centre for Stem Cells and Regenerative Medicine, King’s College London, UK Yu, Tracy - Centre for Craniofacial and Regenerative Biology, King’s College London, UK Norman, Michael - Centre for Craniofacial and Regenerative Biology, King’s College London, UK Gentleman, Eileen - Centre for Craniofacial and Regenerative Biology, King’s College London, UK Rashid, Tamir - Centre for Stem Cells and Regenerative Medicine, King’s College London, UKcGMP-compliant human pluripotent stem cells (hPSCs), which are suitable for cell therapy, can be successfully differentiated into hepatocytes (hPSC-Heps); however, hPSC-Heps often fail to reproduce the transcriptomic and functional profile of freshly isolated hepatocytes. During development, hepatic cell fate is impacted by the local microenvironment. However, the hepatic 3D milieu is complex and the biological and physical factors that drive hepatic maturation remain unidentified. Using 2D and 3D biomaterial culture platforms, extracellular cues, such as stiffness and adhesive ligand concentration can be dissected and their impact on hepatic maturation studied. hPSC-Heps were differentiated using standard chemical cocktails on a modifiable 4-armed polyethylene glycol (PEG) hydrogel system conjugated with RGD sequence-containing peptides. Hydrogels with two different solid content concentrations (2.5% and 10%) with Young’s moduli mimicking healthy adult (1kPa) and fibrotic liver (7kPa) were examined. Hepatic phenotype was measured by ELISA (albumin secretion), luciferin-IPA luminescence (CYP3A4 activity) and qPCR (gene expression). When comparing hPSC-Heps cultured for 3 weeks on soft 1kPa PEG substrates to those on 7kPa substrates, significantly greater cytochrome p450 CYP3A4 enzyme activity, albumin secretion, and expression of key hepatic genes were measured. Whereas, for those on 7kPa PEG, expression of genes associated with liver fibrosis (TGFB, IHH/SHH, VEGF, PDGFB) were elevated, along with the expression of Yes-associated protein (YAP) and WWTR1 (TAZ) target genes. Notably, the mRNA levels for RGD binding integrin subunits ITGB1 and ITGB3 were significantly greater in hPSC-Heps matured on 7kPa rather than 1kPa substrates, despite ligand concentration remaining constant. Moreover, when encapsulated within 1kPa PEG, further phenotypic improvement to hPSC-Heps (increased albumin secretion) was measured compared to 2D cultures. This data implies that stem cell culture platforms can be optimised through tuning of their biomechanical properties. Understanding the interplay between mechanical and biochemical factors in cell fate determination will help the development of patient-specific research tools, as well as provide hPSC-Heps better suited for clinical therapies.Funding Source: This research was funded/supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London.F-3127RELIABILITY OF HUMAN CORTICAL ORGANOID GENERATIONYoon, Se-Jin - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA Elahi, Lubayna - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA Pasca, Anca - Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Marton, Rebecca - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA Gordon, Aaron - Neurology, University of California, Los Angeles, CA, USA Revah, Omer - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA Miura, Yuki - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA
492POSTER ABSTRACTSWalczak, Elisabeth - BD Biosciences, Menlo Park, CA, USA Holdgate, Gwendolyn - BD Biosciences, Menlo Park, CA, USA Fan, Christina - BD Biosciences, Menlo Park, CA, USA Huguenard, John - Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA Geschwind, Daniel - Neurology, University of California, Los Angeles, CA, USA Pasca, Sergiu - Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USAThe differentiation of pluripotent stem cells in three-dimensional (3D) neural cultures can recapitulate key aspects of brain development in vitro, but protocols are prone to variable results. Here, we differentiated multiple human pluripotent stem cell (hPSC) lines for over 100 days to generate brain-region specific organoids called human cortical spheroids (hCS), which are 3D floating cultures that resemble the cerebral cortex. Human induced pluripotent stem cells (hiPSC) were cultured in feeder-free and xeno-free conditions on human recombinant vitronectin, aggregated in AggreWell-800 plates to obtain uniform 3D spheroids, and moved to ultra-low attachments plates for neural differentiation. We differentiated multiple hiPSC lines for 4 to 11 times each in 100 day-long experiments. As we have previously shown, hCS showed an internal cytoarchitecture that included proliferative zones and contained neurons resembling deep and superficial cortical layers as well as non-reactive astrocytes. Neurons were electrophysiologically mature and displayed synaptic activity. Single-cell RNA-seq at day 105 showed a similar distribution of the cell types across multiple lines, and bulk transcriptional profiling showed high consistency across hiPSC lines and experiments. We anticipate that this reliable and scalable brain-region specific organoid can be used for large scale differentiation experiments and disease modeling.F-3129BIOCHEMICAL AND PHYSICAL CUES COMBINE TO AUGMENT IPSC-DERIVED SKELETAL MUSCLE DIFFERENTIATION AND MATURATION FOR IMPROVED DISEASE MODELINGLuttrell, Shawn - Department of Rehabilitation Medicine, Institute for Stem Cell and Regenerative Medicine at the University of Washington, Anacortes, WA, USA Dupont, Jean-Baptiste - I-Stem, Université Evry Val d’Essonne, Evry, France Smith, Alec - Department of Bioengineering, Institute for Stem Cell and Regenerative Medicine at the University of Washington, Seattle, WA, USA Kim, Deok-Ho - Department of Bioengineering, Institute for Stem Cell and Regenerative Medicine at the University of Washington, Seattle, WA, USA Mack, David - Department of Rehabilitation Medicine, Institute for Stem Cell and Regenerative Medicine at the University of Washington, Seattle, WA, USAInherited myopathies encompass a wide range of debilitating diseases and affect millions of people worldwide. These diseases can impact mobility, strength, speech, and respiration and are ultimately fatal in many cases. Human induced pluripotent stem cells (hiPSCs) present a feasible and exciting model to study inherited myopathies and their potential remedies in vitro. This model would allow for comprehensive drug testing on diseased cells derived from affected individuals, thereby eliminating the need for invasive, time-consuming, and often costly therapeutic trials in vivo. Attempts to recapitulate myogenesis in vitro using hiPSCs, however, have been challenging and often result in heterogeneous cell cultures, immature neonatal myofibers, and disorganized fiber arrangements with asynchronous myotube contractions. This could limit the usefulness of these cells for modeling mature or late onset disease phenotypes. Here, we report biochemical and physical cues that augment myogenic differentiation and maturation. Surface patterning and nanoscale topographical cues have been shown to control cell alignment. hiPSC-derived myoblasts cultured on nanopatterned plates with ridges and grooves align and fuse to form myotubes that are structurally ordered, creating anisotropic muscular tissue in both normal and diseased cell types. Furthermore, addition of Dexamethasone, a synthetic glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, promotes enhanced sarcomeric assembly and increased myotube width compared with untreated controls. The cytokine interleukin-4, which acts as a myoblast recruitment factor, promotes myoblast fusion resulting in longer, multinucleated myotubes. Our combinatorial differentiation strategy produces robust skeletal muscle myofibers which may display more clinically relevant phenotypes as muscle matures in vitro. Single cell RNA-sequencing shows that normal iPS cells and cells derived from a patient with Duchenne muscular dystrophy diverge in their developmental trajectories soon after dystrophin is expected to be expressed during myogenesis. This validates that our differentiation protocol yields myogenic cells that manifest disease phenotypes which can be used for more accurate disease modeling and drug discovery.F-3131MICROENVIRONMENT OPTIMISATION FOR THE DIFFERENTIATION OF INNER EAR ORGANOIDS FROM MOUSE EMBRYONIC STEM CELLSZingaro, Simona - Ear Institute, University College London (UCL), London, UK Goddard, Nicola - Department of Biochemical Engineering, University College London, UK Wall, Ivan - School of Life and Health Sciences, Aston University, Birmingham, UK Gale, Jonathan - Ear Institute, University College London, UKHearing loss is one of the most common forms of sensory impairment in humans, affecting > 5% of the world’s population. To date, our understanding of the mechanisms of hearing loss and the generation of therapies have been hampered by the lack of a human in vitro model. Previous work showed that it is
493POSTER ABSTRACTSpossible to generate inner ear organoids containing functional hair cells from mouse (mPSCs) or human pluripotent stem cells (hPSCs) through a three-dimensional (3D) culture system; however, their translation into an in vitro model for drug screening or developmental modelling is limited by low differentiation yield, lack of reproducibility and standardisation of the differentiation protocol. This study aims to generate an engineered stem cell niche in which pluripotent stem cells could be cultured and differentiated into inner ear organoids and ultimately combined with microfluidic systems for translational research. We obtained inner ear organoids containing hair cells, supporting cells and neurons with a mouse embryonic stem cell (mESC) Atoh1/nGFP line (a gift from Stefan Heller). We then examined how micro-environment and size control may impact mESC otic differentiation, varying culture chamber size (96-well plate v AggreWellTM) and extracellular matrix (ECM). Results show that microwells allow the control of organoid size and also increase expression of Atoh1/nGFP. However, we did not observe the morphological and phenotypical changes expected during otic differentiation. Preliminary data on organoids embedded in ECM revealed that they are able to grow and undergo the morphological changes observed during the first seven days of the standard differentiation protocol. Our work provides a step forward in the development of a stem cell niche to sustain PSCs in vitro and regulate differentiation into inner ear organoids. The translation of this niche in a microfluidic device would, for example, allow the generation of human Inner-Ear-On-A-Chip for personalised therapies for hearing loss and also for drug screening. The model will be important for understanding the mechanisms underlying inner ear development and hearing loss.F-3133TRANSCRIPTOME ANALYSIS OF HUMAN SOMATIC CELL NUCLEAR TRANSFER-EMBRYONIC STEM CELL DERIVED RETINAL PIGMENT EPITHELIAL CELLSJung, Sookyung - CHA Stem Cell Institute / CHA Advanced Research Institute, CHA University, Gyeonggi, Korea Lee, Jeoung Eun - CHA University, CHA Stem Cell Institute / CHA Advanced Research Institute, Gyeonggi-do, Korea Shim, Sung Han - CHA University, CHA Stem Cell Institute, Gyeonggi-do, Korea Lee, Dong Ryul - CHA University, CHA Stem Cell Institute / CHA Advanced Research Institute, Gyeonggi-do, KoreaDysfunction of retinal pigment epithelial cells (RPE) in retina causes serious visual impairments and retinal degenerative disease such as age-related macular degeneration (AMD) and retinitis pigmentosa. The defects in RPE function can affect the integrity and viability of photoreceptors, so RPE become the main target for treating many retinal degenerative diseases. One of the most promising treatments for RPE related disorders is cell replacement of the dysfunctional RPE. Because of proliferation and differentiation abilities of human embryonic stem cells (hES), hES derived RPE is proposed as a potentially suitable resource of cell replacement therapy for AMD. Recently, we produced RPE lines using somatic cell nuclear transfer(SCNT)-hES derived from a normal healthy donor (CHA-hES NT4) and AMD patients (CHA-hES NT5 and NT8). Then, we have analyzed the characteristics and functional efficacy of the RPE lines compared to hES derived RPE (MA09-RPE), which have been used in clinical trials, for their future applications. In the present study, we conducted the microarray-based analysis comparing SCNT-hES derived RPE lines with MA09-RPE. All of the SCNT-hES derived RPE lines showed high similarity to MA09-RPE in typical RPE characteristics; pigmented polygonal shape, expression of RPE-related markers, epithelial polarization and phagocytosis activity. The pattern of gene expression related in cell proliferation, migration, and immune response was very similar among all RPE lines. Regarding the expression of genes related to the functions of retinal cells, most of them were similar among all RPE lines, but there was a difference in the expression of several genes between the patient group (CHA-hES NT5-RPE and NT8-RPE) and the normal group (CHA-hES NT4-RPE and MA09-RPE). Forward, it should be studied whether the difference in some gene expression affects the actual RPE function. Since there is no difference between healthy donor derived SCNT-hES-RPE and MA09-RPE, HLA-matched allogenic SCNT-hES derived RPE might be one of the effective solutions for the treatment of retinal disease.Funding Source: This research was supported by grants (No.2017M3A9C6061284,2017M3A9C8029318and2017M3A9F8072235) from the Bio and Medical Technology Development Program of the National Research Foundation funded by the Korean government (MSIP).F-3135OFF THE SHELF BLOOD PRODUCTS FROM HUMAN INDUCED PLURIPOTENT STEM CELLSOh, Steve K - Bioprocessing Technology Institute, Bioprocessing Technology Institute, Singapore Loh, Jonathan - Stem Cells, Institute of Molecular and Cell Biology, Singapore Sivalingam, Jaichandran - Stem Cell Group, Bioprocessing Technology Institute, SingaporeManufacture of red blood cells (RBC) as an “off the shelf” cell therapy is an extremely challenging goal, as a packed unit of blood has 10e12 cells in 500mls. A renewable source of cells such as human pluripotent stem cells must be identified and the process optimized over 4 stages to generate this dose of RBC. We began this journey by optimizing the first stage of hemangioblast differentiation by creating a cell microcarrier aggregate differentiation process that increased the yields 60 fold over an embryoid body method. Next optimizing the Wnt activation pathway with CHR99021 and BMP4 resulted in a further 5 fold improvement in yields of haemopoietic precursors at the second stage of differentiation. Thereafter at the third step, erythroblasts (CD 235a+) were expanded another 1000 fold from these precursors. Six human pluripotent stem cell lines successfully differentiated to erythroblasts following this protocol. Cumulatively, one of the lines X13 achieved 12,605
494POSTER ABSTRACTSfold expansion reaching a cell density of 15x10e6 cells/ml. Enucleation efficiency was effected with OP9 co-cultures to 79% and oxygen carrying capacity was validated for all 6 cell lines. Finally, this 4 stage bioprocess was reproduced in 125ml bioreactors in a fully integrated suspension mode as proof of concept for small scale manufacturing. Each step of improvement has led to a 4 stage process which can be intensified in a bioreactor that can concentrate erythroblast cell production by a factor of 5 times to 10e8 cells/ml which will bring the cost of manufacturing to the $1000s/unit of blood. Further optimization through media replacement of growth factors with alternatives, combined with process engineering will bring the RBC production costs to even lower levels.Funding Source: Agency for Science Technology and Research (A*STAR)F-3137INDUCING HALLMARKS OF INTERNEURON MATURATIONAllison, Tom - Biological Chemistry, University of California, Los Angeles (UCLA), Los Angeles, CA, USA De La Torres, Luis – Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, CA, USA Geschwind, Daniel – Institute for Precision Health, University of California, Los Angeles, CA, USA Langerman, Justin - Biological Chemistry, University of California, Los Angeles, CA, USA Lowry, William - M Molecular Cellular and Developmental Biology, University of California, Los Angeles, CA, USA Lund, Andrew – Molecular Cellular and Developmental Biology, University of California, Los Angeles, CA, USA Plath, Kathrin - Biological Chemistry, University of California, Los Angeles, CA, USA Sabri, Shan - Biological Chemistry, University of California, Los Angeles, CA, USAHuman pluripotent stem cells (hPSCs) provide the unique opportunity to derive all somatic cell types from an unlimited source in a patient specific manner. Since the derivation of hPSCs, many somatic cell types have been successfully generated, including neurons, which have been shown to represent cells of the central nervous system (CNS). A huge step forward in better understanding how the CNS develops, and consequently, how diseases arise in the brain, the neurons that are generated from hPSCs in vitro nevertheless do not fully resemble the mature neurons that are present in the human brain. This therefore limits the use of these hPSC-derived neurons for applications such as drug discovery and disease modelling for adult diseases, including Alzheimer’s, Autism, Epilepsy, amongst others. By using high-throughput single cell techniques, we have interrogated the gene expression patterns of hPSC-derived, fetal and adult interneurons and cataloged key differences between their development. We have identified key genes that are absent in hPSC-derived interneurons and by over-expressing these genes we have been able to push these cells to a more adult, mature like state. We hope this work will offer a platform for more effective treatments of CNS diseases.F-3139DISTINCT REGULATORY MECHANISMS OF TUMOR SUPRESSOR PROTEIN P16/INK4A IN SELF-RENEWING HUMAN PLURIPOTENT AND NEURAL STEM CELLSFedorova, Veronika - Department of Histology and Embryology, Masaryk University, Brno, Czech Republic Barak, Martin - Department of Histology and Embryology, Masaryk University, Brno, Czech Republic Bohaciakova, Dasa - Department of Histology and Embryology, Masaryk University, Brno, Czech Republic Elrefae, Lina - Department of Histology and Embryology, Masaryk University, Brno, Czech Republic Petrasova, Martina - Department of Histology and Embryology, Masaryk University, Brno, Czech RepublicHuman pluripotent stem cells (hPSCs) have the ability to differentiate and to unlimitedly self-renew. This is partially ensured by rapid cell division and specific cell cycle regulatory mechanisms. Importantly, length of G1 phase, and activity of specific cell cycle regulators determine the cell fate decision and differentiation. Here we aimed to study the cell cycle inhibitor p16/INK4A (p16), an important regulator of G1 phase transition that has been associated with cellular senescence and is frequently deregulated in human glioblastoma. Initially, we noticed that expression of p16 in hPSCs is low in early passages (<40) and increases with high number of passages (>60). Surprisingly, the elevated level of p16 in cells of high passage does not affect their proliferation. We analysed the cellular localisation of p16 in hPSCs expressing p16 and found that, in addition to nucleus, p16 also localizes to cytoplasm, a phenotype previously found in numerous tumours.. We further studied possible regulations of p16 expression in hPSCs and in hPSC-derived neural stem cells (NSCs) and our functional experiments suggest that regulations of p16 change upon differentiation of hPSCs into NSCs. While p16 protein level increased in hPSCs after inhibition of miRNA biogenesis, suggesting regulation by miRNA, in NSCs p16 level is modulated by proteasomal degradation. Finally, we also investigated the relationship between p16 and its known regulators Ets1, p53, and Bmi1. In undifferentiated hPSCs, our results point to an interesting regulatory loop between p16 and p53. Ets1, although elevated in high passages of hPSCs does not seem to directly regulate p16. Upon differentiation, in NSCs, the expression of p16 is partly inhibited by polycomb protein Bmi1, oncogene necessary for self-renewal. Altogether, our results reveal novel expression patterns of protein p16 during early human development in vitro and point to several molecular pathways by which its expression is being regulated.
495POSTER ABSTRACTSFunding Source: This study was supported by Masaryk University, Faculty of Medicine (ROZV/24/LF/2016), (ROZV/25/LF/2017), and Czech Science Foundation (GJ15-18316Y and GJ18-25429Y).F-3141THE LONG NON-CODING RNA SNHG16 REGULATES NEURAL DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLSKumar, Vivek - Institute of Biosciences, University of São Paulo, Brazil Kaid, Carolini - Human Genome and Stem-cell Center (HUG-CELL) Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Brazil Kuriki, Patricia - Human Genome and Stem-cell Center (HUG-CELL) Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Brazil Okamoto, Oswaldo - Human Genome and Stem-cell Center (HUG-CELL) Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, BrazilThe human genome encodes many thousands of non-coding RNAs, but little is known about lncRNAs regulating neurogenesis. Here we identified SNHG16 as a lncRNA whose stable expression in human embryonic stem cells and derived neural stem cells (NSCs) decreases along with neural differentiation. Silencing of SNHG16 potentiates neuronal differentiation and depletes the NSCs population, whereas SNHG16 overexpression induces NSCs self-renewal. We further explored the underlying mechanism of SNHG16 in neurogenesis and found that SNHG16 directly binds miR-124 and inhibits its action in neural differentiation. In addition, SNHG16 reversed the effect of miR-124 by targeting the transcription factor SOX8. Our data highlight the pivotal role of SNHG16 as a neurogenic lncRNA that acts as a microRNA sponge and transcription factor regulator in human NSCs.Funding Source: Financial support by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Grants FAPESP-CEPID (2013/08028-1), FAPESP (2014/23043-0).F-3143THE EFFECTS OF METFORMIN ON HUMAN CORTICAL DEVELOPMENTAlsanie, Walaa - Deanship of Scientific Research, Taif University, Taif, Saudi Arabia Alswat, Khaled - Deanship of Scientific Research, Taif University, Taif, Saudi Arabia Gaber, Ahmed - Department of Biology, Taif University, Taif, Saudi Arabia Habeeballah, Hamza - Deanship of Scientific Research, Taif University, Taif, Saudi ArabiaMetformin is widely used as an antidiabetic drug to treat patients who have been diagnosed with type 2 diabetes mellitus. Although it is known to cross the placenta, it is one of only two oral medications that can be prescribed for pregnant diabetic women. To date, the effects of metformin on the development of human neurons, particularly cortical neurons in the fetus, have not been evaluated in any study. Human pluripotent stem cells (hPSCs) are considered to be invaluable in the modulation of normal development and drug testing in vitro. Thus, the effects of metformin on cortical neurons derived from hPSCs were evaluated in the current study. Metformin has been added to cortical differentiation cultures established from hPSCs to examine whether metformin has an effect on this early developmental process. The expression of several genes and transcription factors were evaluated at different times points throughout the differentiation. Metformin was shown to delay the differentiation of PAX6-positive early cortical progenitors into TBR2-positive intermediate progenitors (p = < 0.050). During maturation, a reduction in the number of CTIP2-positive neurons, which normally reside in the deep layers of the cerebral cortex, was evident in the metformin-treated differentiation culture compared to the control differentiation culture (p = < 0.050). By contrast, a difference was not observed in the number of TBR1-positive neurons identified in the two differentiation cultures. The delay in differentiation in the metformin-treated culture was found to be due to the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway on further analysis. By inhibiting the AMPK signaling pathway in the metformin-treated cultures, differentiation of the human cortical neurons therein was similar to that in the control group. To the best of our knowledge, this is the first study to have demonstrated the effect of metformin on human cortical development in vitro. Further studies are warranted to examine the effects of metformin on the morphogenesis and synaptogenesis of cortical neurons.F-3145THE ROLE OF SOX1 IN DETERMINING REGIONAL IDENTITY OF HUMAN EMBRYONIC STEM CELL-DERIVED NEURAL PROGENITOR CELLSLiu, Xinyuan - Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Jin, Ying - Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, ChinaDuring human embryogenesis, primitive neural cells start to be generated at the time of gastrulation and gradually acquire regional identities, which is a process called neural patterning and is precisely controlled. However, how intrinsic factors of the primitive neural cells respond to exogenous patterning signals remains poorly understood. Human embryonic stem cells (hESCs) provide a useful model to recapitulate this process. Through exogenous manipulation of canonical Wnt signaling during neural differentiation of hESCs, dose-dependent specification
496POSTER ABSTRACTSof regionally defined neural progenitor cells (NPCs) ranging from the telencephalon to spinal cord could be rapidly and efficiently induced. Unexpectedly, we find that SOX1, generally referred as a pan-neural gene, displays a regional specific distribution in the neural patterning process. To investigate the expression and function of SOX1 efficiently, we generate the SOX1-EGFP reporter and SOX1-knockout (KO) hESCs lines using the CRISPR/Cas9 system. We find that SOX1 expression peaks in the anterior hindbrain NPCs at the early stage. Its depletion leads to the up-regulation of midbrain markers and down-regulation of anterior hindbrain markers. Our in-depth analysis of SOX1 ChIP-sequencing and transcriptomic data indicate that SOX1 may bind to the distal enhancer of GBX2 and activate its expression. Taken together, this study identifies SOX1 as one of the intrinsic factors key for the patterning establishment in NPCs, particularly for defining the midbrain and hindbrain identity.F-3147DIFFERENTIATION AND FUNCTIONAL COMPARISON OF MONOCYTES AND MACROPHAGES FROM HIPSCS WITH PERIPHERAL BLOOD DERIVATIVESCao, Xu - Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands Yakala, Gopala - Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands Van Den Hil, Francijna - Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands Cochrane, Amy - Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands Mummery, Christine - Anatomy and Embryology, Leiden University Medical Centre, Leiden, Netherlands Orlova, Valeria - Anatomy and Embryology, Leiden University Medical Centre, Leiden, NetherlandsA renewable source of human monocytes and macrophages would be a valuable alternative to primary cells from peripheral blood (PB) in many biomedical applications. Here we developed a fully defined, efficient protocol to derive monocytes and macrophages subtypes from human induced pluripotent stem cells (hiPSCs). They were phenotypically and functionally similar to PB-derived cells but also showed important differences. First, hiPSC-derived monocytes were functional after cryopreservation and exhibited comparable gene expression profiles as PB-derived monocytes. Notably, hiPSC-derived monocytes were more activated and showed greater adhesion to endothelial cells under physiological flow. Second, hiPSC-derived monocytes could be polarized to pro- or anti-inflammatory macrophage subtypes. hiPSC-derived macrophages showed similar pan- and subtype-specific gene and surface protein expression and cytokine secretion to PB-derived macrophages. Transcriptomic analysis of hiPSC-derived macrophages indicated a clear subtype specific signature and functional pathways expressed in these cells. Third, functionally, hiPSC-derived macrophages exhibited similar bacterial phagocytosis but higher AcLDL endocytosis and efferocytosis (phagocytic clearance of dead cells) compared to PB-derived macrophages. Last, hiPSC-derived macrophages showed similar ability to phagocytose tumour cells as PB-derived macrophages, indicating their potential value in cancer immunotherapy. In summary, we developed a robust protocol to generate hiPSC-monocytes and macrophages from independent hiPSC lines that showed aspects of functional maturity comparable with those from PB.F-3149EFFECTS OF COMPOUND A IN THE DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO MATURE CARDIOMYOCYTESKim, Yeseul - Physiology, Pusan National University, Yangsan, Korea Kim, Jae Ho - Physiology, Pusan National University, Yangsan, Korea Yoon, Jungwon - Physiology, Pusan National University, Yangsan, KoreaHuman embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be used for a wide range of applications such as cardiac drug toxicity screening, modeling and therapy of cardiac diseases. However, increasing evidence demonstrates that currently available hESC-CMs represent immature embryonic or fetal stage and structurally and functionally different from mature human cardiomyocytes. Therefore, the application of hESC-CMs is largely limited by their immature phenotypes. In order to overcome the immature problem of hESC-CMs, it is needed to stimulate maturation of hESC-CMs. In this study, for the first time, we identified compound A as a novel factor stimulating maturation of hESC-CMs. Treatment of the hESC with compound A during cardiomyocyte differentiation stimulated expression of several cardiomyocyte-specific markers, including cTnT, -sarcomeric actinin, and myosin light chain isoforms. αMoreover, Compound A treatment increased the density of T-tubule and the expression of T-tubule-associated proteins, BIN1 and JPH2. In addition, the contents of mitochondria in hESC-CMs was also increased after treatment with compound A. Consistently, Compound A-treated hESC-CMs exhibited increased mitochondrial membrane potential compared with the immature hESC-CMs. Compound A not only improved the activity of mitochondrial activity, but also made the whole structure of mitochondrial and cristae elongate and clear. This is also functionally increased in mitochondrial oxygen consumption measurements, such as maximum respiratory capacity and ATP production. However, by using a patch clamp analysis, we observed that the activity and expression of cardiomyocyte-specific ion channels were not change in compound A-treated hESC-CMs compared with the control cells. These results suggest that compound A promotes maturation of hESC-CMs through mitochondrial maturation and healthy condition, which can be used for cardiac toxicity screening and cell therapy.Funding Source: This research was supported by the MRC Program (NRF-2015R1A5A2009656)
497POSTER ABSTRACTSF-3151NON-DISRUPTIVE EVALUATION OF DIFFERENTIATION EFFICIENCY FROM HUMAN PLURIPOTENT STEM CELLS TO DIFFERENTIATED CELLS BY MONITORING THE AMOUNT OF MIRNAS IN CULTURE SUPERNATANTSAihara, Yuki - Central Research Laboratories, Sysmex Corporation, Kobe, Japan Tohyama, Shugo - Department of Cardiology, Keio University School of Medicine, Tokyo, Japan Masumoto, Kanako - Central Research Laboratories, Sysmex Corporation, Kobe, Japan Miyagawa, Mao - Central Research Laboratories, Sysmex Corporation, Kobe, Japan Fujita, Jun - Department of Cardiology, Keio University School of Medicine, Tokyo, Japan Fukuda, Keiichi - Department of Cardiology, Keio University School of Medicine, Tokyo, JapanIn recent years, several types of therapeutic products derived from human iPS cells (hiPSCs) are being developed for use in regenerative medicine. One of the important problems is that differentiation efficiency is unstable in order to use these products clinically. Therefore, to realize regenerative medicine using hiPSCs in clinical practice, quality control of differentiated cells and differentiation processes are necessary. Although various methods for evaluating differentiated cells and differentiation processes have been used, most of them are disruptive methods that involve irreversibly lysing or dying a portion of the hiPSCs-derived products. In this study, we report, in order to monitor differentiation processes and evaluate hiPSCs-derived products non-disruptively and high-sensitively, identification of specific miRNAs for hiPSCs or differentiated cells in the culture supernatants and evaluation of variations in the amount of their miRNAs during differentiation. First, the culture supernatants were collected for each cell 1 day after medium change to obtain culture supernatants of hiPSCs and cardiomyocytes as differentiated cells, and cell debris were removed by centrifuging the culture supernatant at 1,500 g for 10 minutes. Thereafter, RNAs were purified from 50 μl of the culture supernatant, and miRNAs copy numbers were mesured by RT-PCR method. As a result, we were able to find specific miRNAs for hiPSCs or cardiomyocytes in the culture supernatants and verify that copy numbers of specific miRNAs for hiPSCs decreased, while copy numbers of specific miRNAs for cardiomyocytes increased, as the differentiation progresses. In addition, this method could estimate differentiation efficiency non-disruptively at an early stage of differentiation. Our method has high potential to contribute the clinical applications of hiPSCs, especially in terms of monitoring the differentiation processes and quality assessment of hiPSCs-derived products.F-3153A NEW CULTURE MEDIUM THAT REDUCES PHOTOTOXICITY AND AUTOFLUORESCENCE WHILE SUPPORTING ACTIVITY IN LONG-TERM PRIMARY TISSUE- AND HPSC-DERIVED NEURONSLee, Vivian M - Research and Development, STEMCELL Technologies Inc., Vancouver, BC, Canada McCormack, Kasandra - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Mak, Carmen - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Thomas, Terry - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Eaves, Allen - Administration, STEMCELL Technologies, Vancouver, Canada Louis, Sharon - Research and Development, STEMCELL Technologies, Vancouver, BC, Canada Lee, Vivian - Research and Development, STEMCELL Technologies, Vancouver, BC, CanadaThe development of imaging technologies coupled with fluorescent sensors has dramatically increased the number of live-cell imaging applications. However, autofluorescence of culture media reduces signal-to-noise and repeated light exposure can result in accumulation of toxic byproducts. We have developed a new medium that overcomes these issues, while maintaining functional neuronal activity, called BrainPhys™ Imaging Optimized Medium (BrainPhys™ IO) based on the original BrainPhys™ formulation (Bardy et al. 2015). For experiments using primary tissues, E18 rat cortices were dissociated into single cell suspensions and plated in NeuroCult™ Neuronal Plating Medium supplemented with NeuroCult™ SM1 Neuronal Supplement (SM1). After 5 days, cultures were transitioned to BrainPhys™ (control) or BrainPhys™ IO both supplemented with SM1 for up to 21 days. Neurons ( III tubulin positive) were quantified in 25 random βfields per well, in triplicate. BrainPhys™ IO supported equivalent survival relative to BrainPhys™ (8293±642 vs. 7582±655 neurons per well; mean ± SE; n = 11) at 21 days in culture. To assess phototoxicity, neurons at 14 days were exposed to blue LED light for 12 hours. Neurons in BrainPhys™ showed disintegrated neurites and cell bodies post-light exposure while BrainPhys™ IO-cultured neurons remained healthy. To assess autofluorescence of the culture media, BrainPhys™ or BrainPhys™ IO was exposed to 450-490 nm light and mean emission at 525 nm across three fields of view was recorded. Autofluorescence in BrainPhys™ IO was reduced by 41.1% ± 3.6% (mean ± SE; n = 3) relative to BrainPhys™. We also tested BrainPhys™ IO for the culture of hPSC-derived neurons. Neural progenitor cells derived from the iPS cell line XCL-1 were differentiated in BrainPhys™ plus growth factors and cultured for 8 weeks in multielectrode array plates. Subsequently, for half the wells, the media was exchanged with BrainPhys™ IO rather than BrainPhys™ (control) for 3 weeks and activity was recorded twice weekly. In BrainPhys™ IO, neurons had an average mean firing rate of 0.63±0.10 Hz compared to control neurons with
498POSTER ABSTRACTS0.51±0.06 Hz (n=1; mean±SE 6 recordings). Together, these data demonstrate that BrainPhys™ IO is an effective medium for fluorescent imaging applications while supporting survival and functional neuronal activity.F-3155INSULIN SUPPLEMENT IS ESSENTIAL IN DIFFERENTIATING HUMAN INDUCED PLURIPOTENT STEM CELLS INTO PANCREATIC BETA CELLSSim, Zixuan Erinn - Department of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan Shiraki, Nobuaki - Department of Science and Technology, Tokyo Institute of Technology, Yokohama, Japan Kume, Shoen - Department of Science and Technology, Tokyo Institute of Technology, Yokohama, JapanInsulin (INS) and insulin-like growth factor 1 (IGF1) are ubiquitous hormones that play important roles in regulating important metabolic and developmental processes, including pancreatic islets. Instead of IGF1 receptors, INS receptors are critical in mediating pancreatic beta cell growth response, which was reported by Ueki K. et. al., 2006, using beta cell specific INS or IGF1 receptor knockout mice. However, there is still less known about the specificity of INS versus IGF1 in human pancreatic development. In this research, we aim to examine this specificity in human cells by focusing on the INS or IGF1 supplements used in cell culture media. Using a five-step rotating culture system established in our lab, we attempted to generate pancreatic beta cells from human induced pluripotent stem cells (hiPSCs) in vitro under cell culture media supplemented with INS or IGF1 conditions. The differentiated cells in each step were collected and assayed for protein and gene expression levels to examine cell maturation. Here, we first report that both INS and IGF1 are capable of maintaining cell culture of pluripotent hiPSCs. Next, our results demonstrated that either INS or IGF1 can be used as medium supplements in differentiating definitive endoderm cells derived from hiPSCs. However, during pancreatic progenitor cell differentiation, instead of IGF1, INS signaling is necessary for the induction of a larger population of pancreatic progenitor, PDX1-expressing cells. We also found that endocrine cells that were cultured under INS supplemented medium condition generated a higher ratio of insulin and NKX6.1-double positive cells. While cells cultured in IGF1 supplemented medium did not turned into insulin-expressing cells. In pancreatic beta cells, both insulin and NKX6.1 are expressed. Therefore, our results were consistent with those reported in mice, proposing INS as an essential component in pancreatic development in comparison to IGF1. This in vitro cell culture model we used precisely demonstrated the effect of INS or IGF1 supplemented cell culture media in pancreatic development. Using this differentiation model, we can find key pancreatic development stages and signalling molecules that might lead to beta cell destruction and dysfunction in both type 1 and type 2 diabetes.PLURIPOTENT STEM CELL: DISEASE MODELINGF-3159HUMAN IPS CELL-DERIVED ARTIFICIAL SKELETAL MUSCLES FOR MODELING MUSCULAR DYSTROPHY AND DEVELOPING THERAPIESKhedr, Moustafa - Cell and Developmental Biology, University College London (UCL), UK Pinton, Luca - Cell and Developmental Biology, University College London, UK Sarcar, Shilpita - Cell and Developmental Biology, University College London, UK Moyle, Louise - Cell and Developmental Biology, University College London, UK Steele-Stallard, Heather - Cell and Developmental Biology, University College London, UK Maffioletti, sara - Cell and Developmental Biology, University College London, UK Hoshiya, Hidetoshi - Cell and Developmental Biology, University College London, UK Henderson, Alexander - Cell and Developmental Biology, University College London, UK Mannhardt, Ingra - Experimental Pharmacology and Toxicology, University Medical Centre Hamburg-Eppendorf, DZHK (German Center for Cardiovascular Research), Hamburg, Germany Newmann, Katrin - Stem Cell Engineering, BIOTEC, Universitaet Dresden, Dresden, Germany Muntoni, Francesco - Dubowitz Neuromuscular Centre, University College London, UK Eschenhagen, Thomas - Experimental Pharmacology and Toxicology, University Medical Centre Hamburg-Eppendorf, DZHK (German Center for Cardiovascular Research), Hamburg, Germany Anastassiadis, Konstantinos - Stem Cell Engineering, BIOTEC, Universitaet Dresden, Germany Zammit, Peter - Randall Centre for Cell and Molecular Biophysics, Kings College London, UK Tedesco, Francesco - Cell and Developmental Biology, University College London, UKSkeletal muscle morphology and function is impaired in severe genetic disorders named muscular dystrophies. Most muscular dystrophies are rare, which makes relying on patient biopsies for research a major hurdle towards the development of therapies; moreover, various animal models poorly recapitulate the phenotype. Recent advances in experimental therapies for muscular dystrophies, such as exon-skipping, gene-editing and read-through technologies promise to ameliorate disease phenotype. However, the development of these therapies is limited by the lack of a temporally, economically and ethically viable platform. Therefore, generating human skeletal muscle models able to faithfully recapitulate disease-specific features is instrumental for investigating pathology and developing therapies. However, several muscle engineering platforms
499POSTER ABSTRACTSare challenged by the limited proliferation and differentiation of primary myogenic cells. To this aim, we developed three-dimensional (3D) artificial skeletal muscles from human induced pluripotent stem cells (iPSCs) derived from patients with Duchenne (DMD), limb-girdle and congenital muscular dystrophies. Artificial muscles recapitulated key characteristics of skeletal muscle tissue, contained up to four isogenic lineages and could be implanted into immunodeficient mice. Notably, we used this novel platform to model features of muscular dystrophy in vitro in order to develop possible treatments. In the case of limb-girdle and congenital muscular dystrophies caused by mutations in the LMNA gene, we showed that pathological cellular hallmarks can be modeled with higher fidelity using this 3D platform than standard 2D cultures. Notably, we have identified nuclear length as a robust and objective mutation-specific outcome to measure response to treatments. In parallel, to facilitate the development of therapies for DMD we exploited CRISPR/Cas9 gene editing to develop dystrophin-detectable iPSCs and artificial muscles by inserting a novel reporter cassette able to track dystrophin expression levels temporally and spatially, in real time and in fixed cells. These results lay the foundation for next-generation complex disease modeling of muscle diseases, accelerating development of personalized therapies for incurable neuromuscular disorders.F-3161FUNCTIONAL STUDY OF NON-CODING VARIANTS IN HIRSCHSPRUNG DISEASE USING HUMAN PLURIPOTENT STEM CELL-BASED MODELLui, Nga Chu - Department of Surgery, The University of Hong Kong, Hong Kong Fu, Alexander Xi - Department of Computer Science and Engineering, The Chinese University of Hong Kong, Hong Kong Lai, Frank Pui-Ling - Department of Surgery, The University of Hong Kong, Hong Kong Lau, Cynthia Sin-Ting - Surgery, The University of Hong Kong, Hong Kong LI, Peng - Department of Surgery, The University of Hong Kong, Hong Kong YIP, Kevin Yuk-Lap - Department of Computer Science and Engineering, The Chinese University of Hong Kong, Hong Kong Ngan, Elly Sau-Wai - Surgery, The University of Hong Kong, Hong KongHirschsprung disease (HSCR) is a complex multigenic disorder which is caused by incomplete colonization of enteric neural crest cells (ENCCs) in the gut. The aganglionic gut of HSCR patients is functionally obstructed due to uncoordinated muscular peristalsis. Previously, our whole genome sequencing study has identified thousands of HSCR-associated genetic variants residing in the non-coding regions of the human genome. Intriguingly, many of these variants are spanning throughout the non-coding regions of the RET gene and they are highly associated with HSCR susceptibility. rs2435357 (C>T), in particular, has been suggested to increase the risks of HSCR by >4-fold, especially in Chinese patients. In this study, we established an experimental paradigm to interconnect the HSCR associated non-coding variants with the disease phenotypes and severity using human pluripotent stem cell (hiPSC)-based model. By CRISPR-Cas9 genome editing technology, C allele of rs2435357 was changed to T allele in the control hiPSCs or vice versa in the patient-derived hiPSCs. The hiPSCs were directed to ENCC lineage through inhibition of BMP and TGF pathways, βfollowed by activation of the WNT pathway and caudalized with retinoic acid. The ENCCs would then be differentiated into neurons by addition of various neurotrophic factors. We examined the RET expression in different developmental stages of cells and found that rs2435357 (C>T) alone is not sufficient to alter RET expression level in either ENCCs or their neuronal derivatives. Based on in silico analysis, we have identified more HSCR susceptible variants in the putative enhancers of RET gene. In further study, multiplex approach will be used to examine the accumulative effect of multiple variants in the development of enteric neurons.Funding Source: The work was supported by TRS T12C-714/14 from RGC, HMRF 06173306 from the Health Department of HKSAR and LDS Seed Funding for Stem Cell and Regenerative Medicine Research (LDS-IS-2016/17) to E Ngan and Hong Kong PhD Fellowship to NC Lui.F-3163DRUG SCREENING PLATFORM DEVELOPMENT USING A HIGHLY RESPONSE NADH/NAD+ SENSOR IN MELAS CARDIOMYOCYTESKargaran, Parisa - Regenerative Medicine, Mayo Clinic, Rochester, NY, USA Secreto, Frank - Regenerative Medicine, Mayo Clinic, Rochester, MN, USA Nelson, Timothy - Regenerative Medicine, Mayo Clinic, Rochester, MN, USANicotinamide adenine dinucleotide (NAD+) is a central metabolic cofactor in eukaryotic cells that plays a critical role in regulating cellular metabolism and energy homeostasis. The reduced form, NADH, serves as the primary electron donor in the mitochondrial respiratory chain, essential for generating adenosine triphosphate (ATP) by oxidative phosphorylation. Here, we present an assay capable of monitoring in real time the level of NADH/ NAD+ redox state in MELAS cardiomyocytes, using the highly responsive, genetically encoded fluorescence sensor SoNar (sensor of NAD (H) redox). We initially determined the dynamic range of SoNar by measuring the level of NADH/NAD+ in the presence of different concentration of lactate and pyruvate in our system. Next, we used SoNar sensor to determine whether there is a difference in the level of NADH/ NAD+ ratio in both MELAS cardiomyocytes with high (disease) and low (healthy) heteroplasmy. Our data demonstrated a positive correlation between high levels of heteroplasmy (94%) and an increased
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