550POSTER ABSTRACTSthroughput screening (HTS) using FCeM, a three-dimensional (3D) cell culture medium with gellan gum, for human ovarian cancer cell line SKOV3 to identify small-molecule compounds that promote cell proliferation under 3D cell culture conditions. As the result of screening, we obtained active compounds named GA series. They promoted the proliferation of various cells including cancer cells, fibroblasts, endothelial cells, mesenchymal stem cells under 3D cell culture conditions. DNA microarray-mediated transcriptional profiling of SKOV3 cells cultured with GA series revealed that GA series led to up-regulation of yes-associated protein (YAP) downstream target genes including CTGF and Cyr61 at higher level than vehicle treated control did. Consistently, cells treated with GA series showed decreased levels of phosphorylated YAP and elevated levels of nuclear YAP. Next, we investigated the effect of GA series in mice with a predamaged liver after a one-third partial hepatectomy. Administration of GA series significantly promoted the recovery of liver weight as compared to that of vehicle. In conclusion, we identified GA series as a consequence of HTS under 3D cell culture conditions. GA series augmented the proliferation of various cells, which were probably caused by activated YAP. In addition, GA series promoted liver regeneration in vivo. GA series will contribute to development of novel and efficient treatment for liver diseases.F-2076GENERATION OF AN INSULIN REPORTER GENE KNOCK-IN HESC LINE USING CRISPR-CAS9 GENE EDITINGNair, Vani Manoharan - Department of Pediatrics, University of California, San Diego, CA, USA Nguyen-Ngoc, Kim-Vy - Pediatrics and Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA Sander, Maike - Pediatrics and Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USALoss of dysfunction of pancreatic insulin-producing beta cells is the hallmark of diabetes mellitus. Human pluripotent stem cells can generate functional pancreatic beta cells, which in turn, can effectively be used in the therapeutics for treating diabetes. However, current protocols differentiate pancreatic beta cells from human embryonic stem cells (hESCs) are unable to generate glucose-responsive, functionally mature beta cells. This is partially due to the heterogenous population of cells that is generated with differentiation. It is suggested that the presence of undifferentiated cells in hESC-derived cultures inhibit glucose stimulated insulin secretion (GSIS). Therefore, a strategy to enrich for insulin-producing mature beta cells would improve beta cell function in these cultures. To accomplish this, we aim to develop a fluorescent-based insulin reporter in hESCs that will enable us to enrich for mature beta cells in our differentiation cultures using fluorescence-activated cell sorting (FACS). Here, we will use CRISPR-Cas9 mediated homologous recombination to knock-in the fluorescent reporter, insulin-T2A-GFP, at the insulin locus of the hESC H1 line. This insulin reporter one would be a powerful tool for diabetes disease modeling as well as for beta cell-specific studies at the molecular level.Funding Source: EDUC2-08381F-2078GENERATION OF HUMAN IPSC-DERIVED QUIESCENT HEPATIC STELLATE CELLS FOR DRUG DISCOVERY AND LIVER DISEASE MODELINGKoui, Yuta - Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Japan Miyajima, Atsushi - Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Kido, Taketomo - Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, JapanHepatic stellate cells (HSCs) are liver-specific mesenchymal cells present in the perisinusoidal space of the liver. HSCs in a healthy liver are quiescent and store vitamin A, whereas they are converted into activated HSCs by virus infection or hepatic toxins. As activated HSCs play a central role for the progression of liver fibrosis by producing collagen, it is necessary to recapitulate the HSC activation process in vitro to develop effective drugs for fibrosis and a liver disease model. However, freshly isolated HSCs from normal liver rapidly undergo activation in culture and the supply of human quiescent HSCs is also limited. Therefore, it is strongly desired to develop a method to prepare quiescent HSCs for drug screening and disease modeling. In this study, we aimed at developing a culture system to generate quiescent HSCs from human induced pluripotent stem cells (hiPSCs). By mimicking the HSC development process, we have established a culture system to generate HSCs highly expressing HSC marker genes, such as NGFR, DES, NES, and LRAT, but not ACTA2, a key activation marker. Moreover, they exhibited a stellate morphology and stored vitamin A. These results indicated that hiPSC-derived HSCs are quiescent state. Upon activation by TGFb, those HSCs started to express activation marker genes, ACTA2, and COL1A1, indicating that they were converted into activated HSCs. The conversion of quiescent to activated HSCs was suppressed by inhibition of TGFb or WNT signaling. Finally, to establish a method for quantitative assessment of HSC activation in vitro, we generated the ACTA2-RFP reporter hiPSC line by inserting the RFP gene at the ACTA2 locus. The quiescent HSCs derived from the reporter hiPSC expressed RFP fluorescence after the conversion into the activated state, indicating that the reporter cell line is a useful tool for monitoring the activation process of HSCs and for drug discovery and liver disease modeling.Funding Source: Japan Society for the Promotion of Sciences (JSPS) and Japan Agency for Medical Research and Development (AMED)
551POSTER ABSTRACTSEPITHELIAL TISSUESF-2080MODELING AND ENHANCING MUCOCILIARY CLEARANCE IN PULMONARY DISEASE USING HUMAN AIRWAY BASAL STEM CELLSAgarwal, Trisha - Department of Pediatrics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Garg, Kartik - Department of Pediatrics, University of California, Los Angeles, CA, USA Shia, David - Department of Pediatrics, University of California, Los Angeles, CA, USA Sandlin, Jenna - Department of Pediatrics, University of California, Los Angeles, CA, USA Gomperts, Brigitte - Department of Pediatrics, University of California, Los Angeles, CA, USA Vijayaraj, Preethi - Department of Pediatrics, University of California, Los Angeles, CA, USAAirway basal stem cells (ABSCs) of the proximal airway can self-renew and differentiate into mucus and ciliated cells for host defense. Effective mucociliary clearance (MCC) is essential for lung health. In conditions with impaired MCC, ABSCs become metaplastic causing the bronchioles to grow too far distally in the lung parenchyma, a finding known as bronchiolization. While bronchiolization is widely acknowledged as a pathological finding, it is unclear why these ABSCs proliferate towards the alveolar region. The pathophysiology of several lung diseases such as Idiopathic Pulmonary Fibrosis (IPF), bronchiectasis, Cystic Fibrosis (CF) and Chronic Obstructive Pulmonary Disease (COPD), involve impaired MCC. To date, no study has directly targeted bronchiolization and MCC for therapeutic intervention. The development of rationally designed treatments for pathologic MCC has been hindered by a lack of understanding of the mechanisms of mucus dysfunction and its clearance. We believe that the bronchiolization observed in the lungs is a futile attempt of the lung to heal itself. It attempts this by the self-renewal and differentiation of ABSCs, but instead becomes hyperplastic and develops a stratified bronchiolar epithelium. In the case of IPF, which is a progressive scarring lung disease of unknown etiology, a gain-of-function promoter variant of the mucus producing MUC5B gene, is a genetic risk factor for the disease. In these patients, we observed desquamation of the ciliated cells of the bronchiolar epithelium, linked to the aberrant activity of the serine proteases KLK10 and KLK7. We observed that the cilia in these desquamated cells continue to beat unidirectionally, causing them to spin and injure the bronchiolar epithelium. We hypothesize that this persistent injury is responsible for the bronchiolization and progression of the disease. To further test our hypothesis, we are currently modeling bronchiolization, using ABSCs from IPF patients with the MUC5B variant, differentiating them into a mucociliary epithelium, and selectively detaching the ciliated cells using recombinant KLK7. Using this model, we aim to characterize the injury response and changes in the rheological properties of the mucus to determine its role in the progression of the disease and provide a target for therapeutic intervention.F-2082HISTONE PHOSPHORYLATION PATTERN IN CORNEAL EPITHELIUM AND IN CAOMECSOliva, Joan - Medicine, Emmaus Life Sciences, Inc., Torrance, CA, USA Bardag-Gorce, Fawzia - Medicine, LA BioMed, Torrance, CA, USA Niihara, Yutaka - Medicine, Emmaus Medical, Inc, Torrance, CA, USAOral mucosa epithelial stem cells (OMECS) are used to engineer cultured autologous oral mucosa epithelium cell sheets (CAOMECS), to repair epithelial damaged tissues. A lot of effort is put in the proteomic characterization of the epithelium and much less is done on the epigenetic modification such as the DNA and the histones (Histone 3: H3). The purpose of the study is to study the pattern of expression of phosphorylated H3S10 and phosphorylated H3S28 in rabbits’ corneal epithelium, oral mucosal epithelium and CAOMECS. Immunostaining were performed on rabbit fixed tissues and CAOMECS. H3S10-p is lightly expressed in the microvilli of the oral mucosa (OM), on the limbal region, on the upper layers of the corneal epithelium and mainly on the basal side of the CAOMECS. H3S28-p is not present on the cornea, but it is present on few cells of the limbal region, on the OM (with a decreasing gradient of expression from the microvilli to the apical side of the OM) and highly present on CAOMECS. The H3S10-p and H3S28-p are new markers that could be used to characterize epithelial progenitor stem cells and confirm the proliferative/apoptotic activity of the cells, on epithelium. H3S10-p is involved in active transcription, chromatin condensation (cell division or apoptosis) and UVB response. The cornea blocks a significant part of the UVB, through the activation of H3S10-p, which could damage the cornea until hazy cornea appears. H3S28-p is involved in cell division and active transcription, and can work in synergy with H3S10-p. In addition, the activation of pathways (Aurora Kinase, A/B, Msk1, JNk1, RSK2, erk1/2) controlling H3S10-p and H3S28-p could ameliorate CAOMECS engineering by shortening the time of growth. The presence of these markers and downstream genes could also be used to show the healthiness of the CAOMECS grafting on cornea, and to show that they are capable to protect the eye from UVB damage, and they play a role in the renewal of the epithelium.Funding Source: Emmaus Medical, Inc.
552POSTER ABSTRACTSF-2084USING SINGLE CELL RNA-SEQ DISSECT DRUG-RESISTANT CANCER STEM CELLS AND THEIR NICHESHe, Xi (CiCi) - Adult Stem Cell/ Linheng Li lab, Stowers Institute, Stowers Institute for Medical Research, Kansas City, MO, USA He, Xi - Adult Stem Cell/ Linheng Li Lab, Stowers Institute, Kansas city, MO, USA Li, Linheng - Adult Stem Cell/ Linheng Li Lab, Stowers Institute, Kansas City, MO, USATreatment or Drug-resistance is a fundamental biological question and clinical challenge. To dissect the cell population that are drug-resistant and also responsible for regrowth of tumor (cancer stem cell, CSC) post chemotherapy will shed light on clinical treatment of tumor or cancer. Here we report our recent work using single cell RNA-seq (scRNA-seq) to dissect CSCs and their microenvironment (TME), particularly myeloid derived cells (MDCs) as well as T cells. We first used the APCMin/+ adenoma model to study the dynamic interaction between MDCs and CSCs in vivo following chemoradiotherapy. Using 3D scanning electronic microscopy, we observed that while proliferating tumor cells were dying following chemoradiotherapy, MDCs or its derivative tumor associated macrophages (TAMs) were recruited to the site where CSCs paired with necroptotic cells. TAMs functioned to promote activation of slow-cycling CSCs. Dissected with single cell RNA-seq, we observed that while proliferating tumor cells were declining following chemotherapy, an increase in MDCs was accompanied with a reduction of T cells as well as an activation and short-term expansion of initially quiescent CSCs. Depletion of TAMs using a drugs or genetic means resulting in reduced CSCs and attenuated tumorigenesis. On the other hands, adding MDCs to the adenoma derived organoid culture increase but with EP (Pge2 receptor) inhibitor reduced organoid growth. Thus, we show that MSCs with secreted PGE2 activated Akt and Wnt signaling to promote activation of CSCs. Hence, targeting TAMs will benefit clinical treatment of colorectal tumor and cancer by reducing drug-resistant CSCs.F-2086ENTEROCOLITIS MOUSE MODEL: PHENOTYPIC AND MECHANISTIC ANALYSIS OF FZD7 DELETION IN MICEChen, Yi-Fan - The Ph.D. Program for Translational Medicine, Taipei Medical University, Taipei, Taiwan Gu, Nai-Xin - The Ph.D. Program for Translational Medicine, Taipei Medical University, Taipei, Taiwan Yen, Yun - Ph.D. Program of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei, TaiwanFrizzled7 (Fzd7) receptor is the important signaling receptor in the Wnt signaling pathway. The purpose of Fzd7 receptor is to receive Wnt signaling molecular and to activate beta-catenin for the downstream pathway processing the transcription of the target genes. Currently, the research had indicated that these target genes involved in regulating the proliferation and differentiation of intestinal stem cells (ISCs). Intestinal epithelial cells are considered to be rapid-renewing cells, and intestinal stem cells keep differentiating to maintain the homeostasis of intestinal epithelial cells. Therefore, it is predicted that the function of intestinal stem cells will be affected when Fzd7 gene undergoes deletion. In this study, we hypothesize the Fzd7 deficiency causes the defects on intestinal epithelium, and the specific aims as follow, (1) to establish and characterize the Fzd7 gene knockout mouse model, especially focus on intestine tract; (2) to analyze the effect of Fzd7 deletion on regeneration/repair in intestine; (3) to verify the mechanisms of Fzd7 in regulating the proliferation and differentiation of intestinal stem cells and enterocolitis formation. It can be seen from the results that the deletion of the gene of Fzd7 affects the expression of goblet cells and causes functional defects, resulting in reduced secretion of mucus and destroyed the intestinal mucosal barrier, finally triggering an inflammatory response. We expect this animal model can facilitate to understand the functions of Fzd7 on intestine development and regeneration, and furthermore explore therapeutic for human patients with enterocolitis.F-2088CDC42 CONTROLLED EPITHELIAL POLARITY REGULATES INTESTINAL STEM CELL TO TRANSIENT AMPLIFYING CELL FATE TRANSITION AND CRYPT EXPANSION VIA HIPPO-MTOR SIGNALINGZhang, Zheng - Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Davies, Ashley - Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Xin, Mei - Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Zheng, Yi - Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USAEpithelial polarity is a fundamental cell function controlled by delicate polarity machinery including the Rho family GTPase Cdc42 and the SCRIBBLE/PAR complex. In Drosophila, polarity regulated hippo signaling is known for epithelial cell proliferation regulation. Previously we have shown that intestinal epithelial deletion of CDC42 by Villin-Cre in CDC42 lox/lox mice causes intestinal hyperplasia and crypt expansion associated with a Microvillus Inclusion Disease-like phenotype. Here, by using intestinal stem cell (ISC)-specific deletion of CDC42 in Olfm4-IRES-eGFPCreERT2;CDC42 lox/lox mice, we found that ISC-
553POSTER ABSTRACTSinitiated CDC42 loss causes similar intestinal hyperplasia with drastic hyper-proliferation of transient amplifying (TA) cells and disrupted epithelial polarity as found in Villin-Cre;CDC42 lox/lox mice, indicating that the epithelium defects are due to ISC-intrinsic effects. Intestinal TA cell population expands in the CDC42-null crypts at the expense of ISCs, accompanied with significantly elevated canonical hippo signaling through YAP/TAZ - Ereg and mTOR activation, without detectable changes in Wnt or Notch signaling. Conditional knockout of three alleles of YAP/TAZ genes is able to restore the balance of ISC/TA cell populations and crypt proliferation but not the altered intestinal morphology in the CDC42 KO small intestine. Administration of mTOR inhibitor rapamycin into CDC42 KO mice also exhibits similar rescuing effects without affecting YAP/TAZ signaling activity, indicating that mTOR acts downstream of YAP/TAZ. Inducible ablation of SCRIBBLE gene in intestinal epithelial cells mimics that of CDC42 KO defects including crypt hyperplasia and TA cell expansion. Our results demonstrate that mammalian epithelial cell polarity is critical for ISC maintenance and progenitor proliferation via a hippo-mTOR signaling cascade.EYE AND RETINAF-2090TRANSCRIPTION FACTOR-MEDIATED REPROGRAMMING OF HUMAN MULLER GLIA INTO PHOTORECEPTOR-LIKE CELLS USING CRISPR ACTIVATIONWong, Raymond C - Cellular Reprogramming Unit, University of Melbourne/Centre for Eye Research Australia, Melbourne, Australia Nguyen, Tu - Cellular Reprogramming Unit, Center for Eye Research Australia, Melbourne, Australia Fang, Lucy Lyujie - Cellular Reprogramming Unit, Centre for Eye Research Australia, Melbourne, Australia Lo, Camden - Private Consultancy, Melbourne, Australia Jabbari, Jafar - Australia Genome Research Facility, Melbourne, Australia Hung, Sandy - Clinical Genetics Unit, Centre for Eye Research Australia, Melbourne, Australia Liu, Rick - Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia Luu, Chi - Macular Research Unit, Centre for Eye Research Australia, Melbourne, Australia Gillies, Mark - Save Sight Institute, University of Sydney, Sydney, Australia Hewitt, Alex - Clinical Genetics Unit, Centre for Eye Research Australia, Melbourne, AustraliaThe loss of photoreceptors is a key hallmark of many incurable blinding diseases and regenerative medicine has great potentials of alleviating blindness in patients. Previous studies have highlighted the feasibility of using transcription factors to reprogram glial cells into different types of neurons both in vitro, and in vivo in the brain, spinal cord and retina. This study aims to use CRISPR activation to develop reprogramming technology to convert human Müller glia into photoreceptors (induced photoreceptors, iPH) as an innovative approach for photoreceptor generation. We have adapted the CRISPR activation (CRISPRa) system to activate expression of endogenous genes, which allow us to activate up to 9 transcription factors simultaneously. Using this CRISPRa platform, we have screened and identified cocktails of transcription factors that allow reprogramming of human Müller glia into iPH in vitro. qPCR and immunocytochemical analysis demonstrated that the reprogrammed iPH expressed a panel of rod markers including RHO, PDE6B and GNAT. To comprehensively analyse the iPH, we performed single cell transcriptome profiling of ~10000 iPH cells using 10X Chromium. Transcriptome analysis demonstrated the transition of glial to neuron through reprogramming, the activation of photoreceptor markers in iPH and the presence of different stages of reprogrammed cells. We also generated a single-cell transcriptome atlas for human retina using ~20000 adult retinal cells as a benchmark. Using this atlas, we showed that iPH reprogramming promoted transcriptome changes and transitions from Müller glia to photoreceptors. Our study demonstrated the feasibility of using CRISPRa to induce cell reprogramming of Müller glia into photoreceptors, providing a potential cell source for tissue engineering and regenerative medicine. Future application for in vivo reprogramming provides an exciting regenerative approach to replace photoreceptor losses in retinal diseases.F-2092IDENTIFICATION OF DIFFERENTIALLY METHYLATED WNT5A AS A DIABETIC MARKER IN HUMAN CORNEOLIMBAL CELLS AND LIMBAL-DERIVED IPSCShah, Ruchi - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Spektor, Tanya - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Punj, Vasu - Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Turjman, Sue - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Ghiam, Sean - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Kramerov, Andrei - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Saghizadeh, Mehrnoosh - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Ljubimov, Alexander - Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USADiabetes mellitus appears to be an epigenetic disease with DNA methylation and histone acetylation changes. Diabetic corneal epithelial alterations include persistent defects and impaired wound healing, which may occur due to the dysfunction of limbal epithelial stem cells (LESC). In this study, DNA methylation analysis was performed using Illumina Infinium Methylation 450k Beadchips to compare the methylation
554POSTER ABSTRACTSpatterns between 3 diabetic and 4 non-diabetic primary cultured human limbal epithelial cells (LEC) enriched in LESC as well as induced pluripotent stem cells (iPSC) derived from these primary cells. A 98-gene cluster showed common significant methylation differences between non-diabetic and diabetic LEC. Conversely, another 90-gene cluster showed similarity between diabetic iPSC and non-diabetic LEC but not with diabetic LEC. In the differentially methylated gene cluster, WNT5A gene was hypermethylated in diabetic LEC. This finding was validated by the decreased WNT5A expression in the limbus and LEC from 5 diabetic vs. 5 non-diabetic donors by Western blot and immunostaining. Scratch wound healing assay in diabetic LEC treated with recombinant WNT5A (200 ng/mL) showed accelerated wound healing as compared to the untreated controls. In contrast, siRNA knockdown of WNT5A expression in non-diabetic LEC showed decreased wound healing as compared to the controls. In summary, this study reported for the first time the similarity in the methylation patterns of certain genes in diabetic iPSC and non-diabetic primary LEC, indicating a possible way of normalizing diabetic LEC through iPSC generation. There is also reproducible differential methylation of diabetic and non-diabetic primary LEC, confirming epigenetic changes in diabetic limbal epithelium. WNT5A was identified as a new diabetic marker in the cornea with increased gene methylation and decreased protein expression, which can be modulated to accelerate diabetic corneal epithelial wound healing.Funding Source: This work is supported by NIH R01 EY13431.F-2094JAGGED 1-MEDIATED NOTCH SIGNALING CONTROLS THE DIFFERENTIATION AND STRATIFICATION OF HUMAN LIMBAL EPITHELIAL STEM/PROGENITOR CELLS IN VITROGonzalez, Sheyla - Ophthalmology/Stein Eye Institute, University of California, Los Angeles, CA, USA Halabi, Maximilian - Ophthalmology, University of California, Los Angeles, CA, USA Deng, Sophie - Ophthalmology, University of California, Los Angeles, CA, USALimbal epithelial stem/progenitor cells (LSCs) maintain the homeostasis and regeneration of the corneal epithelium. Understanding the regulation of the limbal stem cell niche is crucial to efficiently expand the LSC population in vitro and treat patients suffering from limbal stem cell deficiency characterized by the loss or deficiency of these stem cells. Notch signaling plays a pivotal role in regulating stem cell maintenance, differentiation and homeostasis. We investigated the role of Jagged 1-mediated Notch signaling activation in the regulation of human limbal stem/progenitor cells (LSCs) in vitro. To better understand the role of Notch signaling in the LSCs, we analyzed the expression of several Notch family members on the human sclerocorneal tissue at the mRNA and protein level. Primary human LSCs were cultured on Jagged 1-coated (Jag1-Fc) plates to activate Notch signaling. Air-lifting induction was performed in the LSC cultures to study the stratification and differentiation capacity of the limbal epithelium. The expression of Notch receptors (Notch 1, Notch 2), ligands (Jagged 1, Jagged 2, Delta-like 1) and target genes (Hes1, Hey1) was detected in the human corneal epithelium at the mRNA and protein level. Jag1-Fc activated Notch signaling in the LSCs. Upon Notch activation in the cultivated LSCs, cell morphology was less compact, and cell growth and the amount of small cells ( 12 μm) were significantly ≤reduced. We also observed a significant lower expression of the stem cell marker p63 bright. After induction of air-lifting, the αnumber of epithelial layers was reduced (lower stratification), and differentiation, given by the amount of cytokeratin 12+ cells, was increased in the Jagged 1-activated cultures. Our data suggest that Jagged 1-mediated Notch signaling activation in the LSC cultures induces differentiation.Funding Source: This work was supported by the National Eye Institute (R01EY021797 and 5P30EY000331), California Institute for Regenerative Medicine (TR2-01768 and BF1-01768), and an unrestricted grant from Research to Prevent Blindness.F-2096DIRECT PRODUCTION OF CORNEAL ENDOTHELIAL (SUBSTITUTE) CELLS FROM HUMAN IPS CELLS BY OMITTING NEURAL CREST DIFFERENTIATIONHatou, Shin - Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Inagaki, Emi - Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Jin, Honglian - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Niwano, Hiroko - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Suzuki, Saori - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Shimizu, Shota - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Yamashita, Kazuya - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Tsubota, Kazuo - Ophthalmology, Keio University School of Medicine, Tokyo, Japan Shimmura, Shigeto - Ophthalmology, Keio University School of Medicine, Tokyo, JapanIn order to provide regenerative medicine for the unmet millions of patients waiting for corneal transplants globally, we have derived corneal endothelial substitute cells from iPSCs (CECSi cells) for the treatment of corneal endothelial dysfunction (bullous keratopathy). At first we tried to develop CECSi cells step by step via neural crest cells (NCC) differentiated from iPS cells, but the stepwise protocol was a disadvantage for mass production. DNA microarray analysis between NCC from iPS cells and human corneal endothelial cells from donor corneas revealed three important intracellular pathways for corneal endothelial development. We therefore improved the protocol to produce CECSi from iPS cells directly by activating three pathways, omitting neural crest development. Hexagonal
555POSTER ABSTRACTSconfluent monolayer cells with Na,K-ATPase alpha 1 subunit (ATP1A1), ZO-1, N-cadherin, and PITX2 expressions could be produced from two of three different iPS cell clones (Ff-I01s04, Ff-MH09s01, Ff-WJs513). Real time PCR analysis revealed that pump function marker ATP1A1 expression levels of CECSi cells from two different iPS cell clones are equal to human corneal endothelial cells from donor corneas. Since this protocol is simple, it may be more suitable for mass production of corneal endothelial cells compared to previous methods.Funding Source: Fund from Research Project for Practical Applications of Regenerative Medicine, Japan Agency for Medical Research and Development. Fund from Cellusion, Inc.F-2098TRANSPLANTATION OF CONE PHOTORECEPTORS PURIFIED FROM RETINAL ORGANOIDS GENERATED WITH A CONE-SPECIFIC HUMAN REPORTER IPSC LINEGasparini, Sylvia J - Centre for Regenerative Therapies Dresden, Technical University Dresden, Germany Carido Pereira, Madalena - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Tessmer, Karen - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Wieneke, Stephanie - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Kempe, Anka - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Borsch, Oliver - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Voelkner, Manuela - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Busskamp, Volker - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Karl, Mike - Centre For Regenerative Therapies Dresden, Technical University Dresden, Germany Ader, Marius - Centre For Regenerative Therapies Dresden, Technical University Dresden, GermanyHuman vision is particularly reliant on cone photoreceptor dependent high acuity and colour vision. A strategy to restore vision in retinal degenerative diseases is the replacement of lost cells with stem cell-derived photoreceptors, however, efficient human cone-enrichment strategies are lacking. Here, we describe the enrichment and transplantation of purified cones through the use of a human iPSC cone reporter line. Applying piggyBAC technology, hiPSCs were transduced to express GFP under the control of the mouse cone arrestin promoter and subsequently used to generate retinal organoids. Organoids were dissociated at 140 or 200 days of age and FACS sorted for GFP+ cells. Sorted cells were transplanted into the subretinal space of wild-type mice and three mouse lines exhibiting aberrant retinas: CPFL1 (cone degeneration), CPFL1; Rho-/- (cone and rod degeneration) and the Nrl-/- line which displays a cone-like cell enriched retina. Mice were immune-suppressed with triamcinolone and eyes were analysed 3 or 9 weeks later. GFP+ cells comprised 8% of all organoid cells at day 140 and 27% by day 200. Enrichment of cones as well as the cone identity of GFP+ cells were confirmed by immunostaining. This showed 90% of sorted cells co-express GFP, the pan-photoreceptor marker recoverin and the cone marker ARR3 (GFP- fraction: < 4% ARR3+ cells). Grafted cells formed clusters in the subretinal space and expressed human cone markers. While GFP+ cells were almost absent in the photoreceptor layer of wt, CPFL1, and CPFL1; Rho-/- hosts, in Nrl-/- mice some transplanted cones appeared to incorporate into the host retina. The transfer of cytoplasmic material between donor and host photoreceptors, as was recently observed in mouse-to-mouse transplantation, was not detected. In summary, FACS of dissociated retinal organoids derived from a hiPSC cone-GFP line yielded a greatly enriched cone population suitable for transplantation. Human cones survived in the murine subretinal space but did not appear to integrate into the recipient tissue or engage in material transfer, with the exception of Nrl-/- hosts, where a subset of donor cones incorporated into the retina. The viability of organoid-derived cones and the specificity of the reporter line suggests the outlined strategy to be a promising approach for further translational studies.Funding Source: This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) Research Grant 01EK1613AF-2100FORWARD PROGRAMMING OF HUMAN PHOTORECEPTORSBusskamp, Volker - Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany Zuzic, Marta - Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany Kempe, Anka - Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany Karl, Mike - Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, GermanyThe replacement of photoreceptors represents a promising option to counteract retinal degenerative diseases. However, for a viable cell therapeutic intervention, one requires human photoreceptors in high quantity and quality. While it is possible to obtain photoreceptors in low quantities by direct reprogramming from fibroblasts or from human stem-cell-derived 3D retinal organoids, an efficient 2D forward programming protocol to generate photoreceptors in vitro from human induced pluripotent stem cells (hiPSCs) needs to be established. Forward programming relies on transcription factors’ (TF) abilities to activate distinct differentiation pathways in stem cells. Aiming to find TF combinations that drive efficient differentiation of stem cells into photoreceptors, we performed a TF-library-on-library screen. A conditional fluorescent photoreceptor hiPSC reporter line was transduced with lentiviral particles each carrying one of 16 TFs known from in vivo photoreceptor development and with a comprehensive library consisting of 1748 human TFs. We sorted 87 out of 8.4 million cells that
556POSTER ABSTRACTSwere qPCR-tested for photoreceptor markers and sequenced to identify the overexpressed TFs at single cell resolution. 90% of the sorted cells were qPCR-positive for at least one of the tested photoreceptor-specific genes indicating the cell-type-precision of our screen. One validated TF combination (two known TFs and one unbiasedly-screened TF) led to a significant loss of the pluripotency marker TRA-1-60 and upregulation of the neuronal marker NCAM within 5 days of overexpression, indicating that cells are differentiating towards the neuronal lineage. Furthermore, fluorescence microscopy and flow cytometry detected high numbers GFP-positive cells suggesting the presence of photoreceptor-like cells. We are currently characterizing these cells in-depths. Our data suggest that the known TFs were insufficient to drive photoreceptor differentiation, indicating that photoreceptor genesis from hiPSCs requires additional TFs. In-vitro-engineered photoreceptors might serve as donor material for cell transplantation to treat blindness or as biomedical testbeds as sufficient quantities can be generated within few days compared to hundreds of days if dissociated from 3D human retinal organoids.Funding Source: V.B. is supported by a Volkswagen Foundation Freigeist fellowship (A110720), by an ERC starting grant (678071-ProNeurons), by the Paul Ehrlich Foundation and by the Deutsche Forschungsgemeinschaft (DFG).F-2102PEDF PEPTIDE FACILITATES SIMPLE LIMBAL EPITHELIAL TRANSPLANTATION FOR OCULAR SURFACE RECONSTRUCTION IN THE RABBIT MODEL OF TOTAL LIMBAL DEFICIENCYTsao, Yeou-Ping - Department of Ophthalmology, Mackay Memorial Hospital, Taipei, TaiwanTo demonstrate the capacity of a pigment epithelial-derived factor (PEDF) peptide 44-mer to facilitate ocular surface epithelialization by enhancing simple limbal epithelial transplantation (SLET) in a rabbit model of total limbal stem deficiency (LSCD). Total limbal stem cell deficiency (LSCD) was created surgically by total circumferential limbal excision and total corneal epithelial removal. Two months later, ocular surface reconstruction was achieved by removal of the fibrovascular pannus over the cornea and limbus, followed by SLET and PEDF peptide treatment. A 2×2 mm strip of donor limbal tissue was obtained from the contralateral healthy eye and divided into eight to ten small pieces. The structure of the restored ocular surface was analyzed at one- and three-months follow-up. Immunohistochemical analysis was performed with antibodies to DNp63a and BrdU to identify proliferating and slow cycling cells. Cells harvested from the regenerated limbal and corneal epithelium were analyzed for colony forming capacity and expressions of limbal and mesenchymal putative stem cell markers were analyzed by immunostaining assay and quantitative real-time PCR (qPCR). The ocular surface epithelialization occurred in vehicle control eyes with conjunctivalization and vascularization of the cornea and limbus. In PEDF 44-mer treated eyes, a completely epithelialized and stable corneal surface was seen in all recipient’s eyes by three months follow-up. The reconstructed ocular surface showed anatomic and functional recovery of the corneal and limbal epithelia. Immunohistochemical staining demonstrated a wide distribution of BrdU labelled slow-cycling cells throughout the regenerated corneal and limbal epithelia and superficial stromal cells. The PEDF peptide enhances ocular surface reconstruction by SLET and sustains ocular surface integrity. The addition of PEDF peptide facilitates SLET survival and reconstruction of the ocular surface with better recovery of the ocular surface epithelium than SLET only in severe LSCD eyes.STEM CELL NICHESF-2104COMMITMENT OF MOUSE NEPHRON PROGENITOR CELLS TO NEPHROGENESIS IS DRIVEN BY A -CATENIN-DRIVEN SWITCH IN ΒTCF/LEF TRANSCRIPTION FACTOR BINDING TO ENHANCERS OF DIFFERENTIATION PROMOTING TARGET GENESGuo, Qiuyu - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Kim, Albert - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Bugacov, helena - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Chen, Xi - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Lindstrom, Nils - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA McMahon, Jill - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA Brown, Aaron - Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, CA, USA Oxburgh, Leif - Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, CA, USA McMahon, Andrew - Stem Cells Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USAWnt signaling and the Wnt pathway transcriptional co-activator -catenin are required for both the self-renewal βand differentiation of mammalian nephron progenitor cells (NPCs). To investigate the molecular mechanism underlying Wnt/ -catenin-driven NPC self-renewal and differentiation, we βmodeled these processes in nephron progenitor expansion medium (NPEM) culture (Brown et al., 2015) supplemented with low (maintenance/expansion of NPCs) or high (differentiation of NPCs) levels of CHIR99021 (CHIR), a small molecule GSK3 inhibitor. Gene expression profiling detected a downregulation of transcriptional repressor Tcf7l1 and Tcf7l2, and a dramatic
557POSTER ABSTRACTSup-regulation of transcriptional activators Tcf7 and Lef1 in differentiated NPCs. Fluorescent in situ analysis and single-cell RNA-Seq data in the developing kidney suggest Tcf7l1 and Lef1 localized to self-renewing and differentiated NPCs, respectively. As a result of changing -catenin levels, Tcf7/Lef1 factor binding βreplaced Tcf7l1/Tcf7l2 interactions at enhancers regulating expression of key differentiation promoting genes, correlating with the activation of these genes. Notably, Hi-C analysis showed Tcf7/Lef1/ -catenin utilizes both pre-established βand CHIR-induced enhancer-promoter loops to activate gene expression. Gene-editing approaches are being used to mechanistically dissect key interactions in this regulatory framework. Together these data suggest NPCs are in a primed state ready to undergo a rapid commitment to the nephrogenic program in which repressive interactions mediated by Tcf7l1/Tcf7l2 silence differentiation promoting enhancers while a β-catenin driven replacement by activating Lef1/Tcf7/ -catenin βactivation initiates NPC differentiation. We expect the findings here will have broader implications for Wnt pathway regulation of stem/progenitor systems in invertebrate and vertebrate developmental systems and Wnt-driven cancers.Funding Source: NIDDKF-2106MACROPHAGE-HEMATOPOIETIC STEM CELL INTERACTIONS IN THE EMBRYONIC NICHE DETERMINE ADULT HSC CLONE NUMBER IN THE MARROWWattrus, Samuel J - Stem Cell Program, Boston Children’s Hospital/Harvard University, Boston, MA, USA Hagedorn, Elliott - Stem Cell Program, Boston Children’s Hospital/Harvard University, Boston, MA, USA Zon, Leonard - Stem Cell Program, Boston Children’s Hospital/Harvard University, Boston, MA, USAIn development, hematopoietic stem cells (HSCs) emerge and ultimately comprise the fetal and adult hematopoietic system. Using a brainbow color barcoding system, we have previously demonstrated that zebrafish produce 20-30 HSCs in the developing aorta. Following emergence, HSCs traffic to a sinusoidal vascular niche, exit circulation, and divide. The number of HSC clones that support hematopoiesis is thought to be regulated by the niche. Using spinning disk confocal microscopy, we imaged HSPCs in the niche and found surprising HSC ‘grooming’ behavior by primitive macrophages, marked with the specific mpeg1:GFP transgene: macrophages contacted newly lodged HSCs and scanned their cell surface for 30-45 minutes. To evaluate clone number, we made use of a brainbow labeling system in fish in which macrophages were depleted. Unique color barcodes were induced in individual HSCs at 24 hours post fertilization (hpf), just prior to HSC emergence, and clodronate loaded liposomes were injected into circulation at 28 hpf. The resulting embryos showed rapid loss of primitive macrophages and were raised to 3 months for color analysis of the adult marrow. On average, animals injected with clodronate liposomes had only 14 unique HSC color clones, compared to an average of 24.6 HSC color clones in sibling controls (p = 0.0002), with a larger average clone size of 7.8% of total marrow compared to 5% in controls. Similar results were produced when macrophages were reduced through a macrophage differentiation-blocking irf8 morpholino, suggesting that this result is due to a specific loss of macrophages. Based on these data, we undertook further imaging studies. In many cell-cell interactions fluorescent protein can be seen moving from the stem cell (runx1+23:mCherry) into the macrophage. Between 48 – 72 hpf, around 20% of HSPCs interact with a macrophage in this manner. Macrophages harvested from runx1+23:mCherry;mpeg1:GFP embryos exhibit punctate mCherry signal, and in some cases appear to have engulfed entire mCherry+ cells. Our barcoding studies taken together with our imaging establish a role for macrophages in determining the number of HSC clones during development and impacts our understanding of clonal diseases in the adult hematopoietic system.F-2108MICRO-SCALED TOPOGRAPHIES DIRECT DIFFERENTIATION OF HUMAN EPIDERMAL STEM CELLSZijl, Sebastiaan - Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK Vasilevich, Aliaksei - Department of Cell Biology Inspired Tissue Engineering, Maastricht University, Maastricht, Netherlands Viswanathan, Priya - Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK Helling, Ayelen - Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK Beijer, Nick - Department of Cell Biology Inspired Tissue Engineering, Maastricht University, Maastricht, Netherlands Walko, Gernot - Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK Chiappini, Ciro - Centre for Craniofacial and Regenerative Biology, King’s College London, London, UK de Boer, Jan - Department of Cell Biology Inspired Tissue Engineering, Maastricht University, Maastricht, Netherlands Watt, Fiona - Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UKHuman epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. 24h later the proportion of cells expressing the differentiation marker Transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. The topographical features that promoted differentiation of rounded and spread cells were different.
558POSTER ABSTRACTSTopographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Topography coverage was also important: low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant amounts of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions; they did, however, organise the actin cytoskeleton in response to the topographies, suggesting a potential role for actin polymerization and SRF signalling in the topography-induced differentiation of spread cells.Funding Source: UK Regenerative Medicine Platform (UKRMP), Medical Research Council, Biotechnology and Biological Sciences Research Council, Wellcome Trust.F-2110ENDOGENOUS MOUSE NEURAL STEM CELLS ARE IRREPLACEABLEBurns, Terry - Neurosurgery, Mayo Clinic, Rochester, NY, USA Ansari, Kambiz - Neurosurgery, Stanford University, Stanford, CA, USA Palmer, Theo - Neurosurgery, Stanford, Stanford, CA, USAQuiescent tumor-initiating with neural stem cell-like properties are resistant to therapy and enable tumor recurrence. As such, quiescent tumor stem cells are increasingly targeted by novel anti-tumor therapies. Quiescent neural stem cells (NSCs) are extremely similar to tumor stem cells and may be targeted by these therapies. The capacity of the quiescent NSC niche to recover following ablation remains unknown. Nestin-creERT2::diphtheria toxin reporter (DTR) mice were induced to express DTR on NSCs with 3 days via 150mg/kg i.p. tamoxifen. Ablation and control mice were treated with 100ug/kg diphtheria toxin DT or vehicle, respectively. Proliferative cells were labeled with BrdU prior to sacrifice. For cell replacement experiments, embryonic stem cell (ESC)-derived NSCs were stereotactically implanted into the dentate gyrus and lateral ventricle. Results: Ablation caused 50-80% reduction of quiescent NSCs and BrdU+ cells in the subventricular zone and dentate gyrus within 10 days. NSC loss persisted for at least 2 months, and was accompanied by 48% loss of neurogenesis at 2 months (p<0.01), suggesting inability of the partially vacated neurogenic niche to be repopulated over time by surviving NSCs. ESC-derived NSCs increased proliferation of endogenous NSCs, though only modestly impacted the quiescent NSC pool size. Implanted NSCs also differentiated into neurons with appropriate morphology and projections but failed to engraft long term into the partially vacated quiescent NSC niche. We demonstrate for the first time that adult NSCs are unable to repopulate the quiescent NSC niche after partial ablation of quiescent NSCs, even though surviving NSCs remain functional and responsive to proliferative stimuli. Primitive ESC-derived NSC also appear unable to engraft into the partially vacated quiescent NSC niche. These findings suggest that loss of quiescent NSCs irreversibly decreases niche size, and suggest endogenous NSC could comprise an irreplaceable component of the niche itself. Given the importance of neurogenesis for cognition and memory, the potentially irreversible impacts of novel stem cell-targeted antitumor therapies on quiescent NSC pool size should be carefully evaluated.Funding Source: California Institute of Regenerative Medicine Regenerative Medicine MinnesotaF-2112MYOKINE SIGNALING BY MYF6/MRF4 IS REQUIRED TO SUSTAIN ADULT SKELETAL MUSCLE STEM CELL POOLSoleimani, Vahab - Human Genetics, McGill University, Montreal, QC, Canada Blackburn, Darren - Human Genetics, McGill University, Montreal, QE, Canada Corchado, Aldo - Human Genetics, McGill University, Montreal, QE, Canada Jahani-Asl, Arezu - Oncology, McGill University, Montreal, QE, Canada Karam, Nabila - Human Genetics, McGill University, Montreal, QE, Canada Lazure, Felicia - Human Genetics, McGill University, Montreal, QE, Canada Lepper, Christoph - Cell Biology and Physiology, Ohio State University, Columbus, OH, USA Najafabadi, Hamed - Human Genetics, McGill University, Montreal, QE, Canada Nguyen, Duy - Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, QE, Canada Perkins, Theodore - Biochemistry and Microbiology, University of Ottawa, Ottawa, ON, Canada Sahinyan, Korin - Human Genetics, McGill University, Montreal, QE, CanadaIn metazoans, skeletal muscle evolved to contract and produce force. Recent experimental evidence, however, suggests that skeletal muscle has also acquired endocrine functions and produces a vast array of myokines. The mechanisms that regulate myokine production and their effect on the resident stem cell population in skeletal muscle remain unknown. Here, we report that in adult skeletal muscle, Myf6/MRF4 is a major regulator of myokine expression. Genetic deletion of Myf6 in skeletal muscle leads to reduction of the muscle stem cell (MuSCs) pool in adult mice in a myokine-dependent manner but, surprisingly, does not disrupt muscle differentiation. Using ChIP-Seq and gene expression analyses of myogenic factors, we show that Myf6/MRF4 is a direct regulator of many myokines and muscle-secreted proteins, including ligands for canonical signaling pathways such as EGFR and VEGFR. Consequently, in Myf6-deficient animals MuSCs increasingly break quiescence, but can nevertheless undergo differentiation. Lastly, we show
559POSTER ABSTRACTSthat Myf6 and its gene network rapidly respond to aerobic and anaerobic exercise. Thus, Myf6 may play a unique role in regulation of muscle-derived cytokines and growth factor collectively called myokines during exercise.Funding Source: Canadian Institute of Health Research (CIHR), Richard and Edith Strauss Foundation and Natural Resources and Engineering Research Council (NSERC)F-2114IDENTIFYING THE NICHE FOR SPERMATOGENESISPeng, Yi - Department of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Shanghai, China Zhou, Bo - Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, ChinaMammalian spermatogenesis is a complex developmental process based on a robust stem cell system, which is controlled by a special microenvironment in the testis. Although multiple cell types have been proposed as potential niche(s) for spermatogenesis, direct in vivo evidence is lacking among these studies. SCF/c-kit pathway have been implied in the regulation of spermatogenesis and is essential for maintaining normal sperm production. For this purpose, we employed different cell type-specific Scf knockout mice to investigate the physiological source and function of SCF-expressing cells, aiming to identify the niche for spermatogenesis. Using ScfGFP mice, we found that Sertoli cells were the major source of SCF in mouse testis. Conditional deletion of Scf from Sertoli cells by Amh-Cre did not alter the number of spermatogonial stem cells, but depleted most of the differentiating spermatogonial cells, differentiated spermatocytes and spermatids in the testis of six-week-old mice, suggesting that SCF-expressing cells regulate the differentiation but not the self-renewal of spermatogonial stem cells, which was further supported by the result that transplantation of SSC from Scf conditional knockout mice restored spermatogenesis in busulfan-treated wild type recipient mice. Interestingly, overexpression of SCF in DDX4-positive germ cells led to an increase of differentiated germ cells with a tamoxifen-inducible system, suggesting a dose-dependent regulation of spermatogenesis by SCF. In summary, using in vivo systems, our study provided key evidence that SCF-expressing sertoli cells create a niche for spermatogenesis, which regulate the differentiation of spermatogonial stem cells and ensure constant production of sperms.CANCERSF-2116MAYO CLINIC HIGH-RISK MAMMARY GLAND AND FALLOPIAN TUBE LIVING ORGANOID BIOBANKS FOR THE DISCOVERY OF MOLECULARLY GUIDED CANCER PREVENTION APPROACHESKannan, Nagarajan - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Shi, Geng Xian - Lab Medicine and Pathology, Mayo Clinic, Rochester, NY, USA Yang, Wenmei - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Zhao, Hui - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Yu, Yifan - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Aalam, Musheer - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Carter, Jodi - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Couch, Fergus - Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Sherman, Mark - Lab Medicine and Pathology, Mayo Clinic, Jacksonville, FL, USA McLaughlin, Sarah - Surgery, Mayo Clinic, Jacksonville, FL, USA Bakkkum-Gamez, Jamie - Obstetrics and Gynecology, Mayo Clinic, Rochester, MN, USA Boughey, Jodi - Surgery, Mayo Clinic, Rochester, MN, USAA woman’s life-time risk for breast and ovarian cancers are more than 10% and 1. 5% respectively and this risk is further elevated in patients with family history of associated cancers and/or deleterious mutations in genes such as BRCA1/2. A significant proportion of high-risk patients succumb to their disease. The prevention strategy currently available to these patients involve highly invasive and least desirable surgical debulking of breast and/or ovaries/fallopian tubes. Understanding of developmental and mechanistic origins of cancer cells in high-risk patients is urgently needed to develop molecularly guided cancer prevention approaches. The cell-of-origin for breast cancer and high-grade serous ovarian cancer are believed to be from epithelial cells lining the mammary gland and fallopian tubes. Organoids from dissociated tissues or single primitive epithelial cells obtained from high-risk patients are vital resource to study mechanisms regulating tissue homeostasis and track disease origin. Such viable tissue resources are currently unavailable. With a vision to boost women’s cancer prevention research at Mayo, we have established the first and largest clinically and genetically-annotated patient-derived organoid biobank for mammary gland and fallopian tube tissues from average and high-risk patients undergoing surgeries at different sites of Mayo Clinic. We have viably frozen >1000 vials of mammary tissue-organoids from 100 patients including carriers of BRCA1/2 and other breast cancer-associated mutations who underwent prophylactic mastectomy, reduction mammoplasty or autopsy, and >17,000 individual fallopian tube stem cell derived organoids from 27 patients who underwent salpingectomy. Our large-scale and successful effort to create the first, well-annotated ‘living’ organoid biobanks for women’s cancer prevention program, attendant challenges in its establishment will be discussed, and our preliminary characterization of primitive epithelial cells with the intention to identify molecular targets for prevention will be presented.
560POSTER ABSTRACTSFunding Source: This work is partly supported by grants to N. K. from Mayo Clinic’s Breast Cancer SPORE and Ovarian Cancer SPORE.F-2118ACTIVE TARGETING EFFECTS OF THERAPEUTIC NANOCOMPLEX USING NOVEL BIOMARKER OF BREAST CANCER STEM CELL IDENTIFIED BY PROTEOGENOMIC ANALYSISKim, Pyung-Hwan - Department of Biomedical Laboratory Science, Konyang University, Daejeon, Korea Koh, Eun-Young - Department of Biomedical Laboratory Science, Konyang University, Daejeon, KoreaBreast cancer in women is one of the most determined life-threatening malignancy and the leading cause of cancer death. Although many conventional therapies have been applied for its treatment, it still has many handicaps to overcome. Among them, cancer stem cells (CSC) are well known to be responsible for tumor formation, development, and differentiation that offers to cellular heterogeneity as well as the recurrence cause of cancer. Therefore, to fully cure breast cancer, the treatment of both cancer and CSC is required. For CSC targeting, we developed a new surface biomarker (Factor X) of breast-derived CSC (BCSC) via proteogenomic analysis and evaluated the possibility as the specific marker and its biological effects. Next, we generated liposome-based on smart nanocomplex for selective targeting against BCSC. Selective and increased transduction efficiency was observed in BCSC treated with rhodamine included nanocomplex conjugated with anti-Factor X monoclonal antibody depending on the expression level of factor X. Doxorubicin-encapsulated nanocomplex selectively showed increased cell killing effects in BCSC with high Factor X expression level and improved anti-tumor effects in mice. Targeting of our excellent therapeutic agent using novel surface biomarker of cancer stem cell may be a more effective strategy to overcome cancer resistance.Funding Source: This research was supported by grants from the National Research Foundation (NRF) funded by the Ministry of Education (2016R1D1A1B03935498).F-2120QUANTITATIVE EVALUATION AND BIODISTRIBUTION ANALYSIS OF INTRAVENTRICULAR DELIVERY OF THERAPEUTIC NEURAL STEM CELLS TO ORTHOTOPIC GLIOMA IN MOUSE BRAINRockne, Russell - Computational and Quantitative Medicine, Division of Mathematical Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA, USA Gutova, Margarita - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Flores, Linda - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Adhikarla, Vikram - Computational and Quantitative Medicine, Division of Mathematical Oncology, Beckman Research Institute, City of Hope, Duarte, CA, USA Joanna, Gonzaga - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Annala, Alexander - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Metz, Marianne - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Soraya, Aramburo - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Tirughana, Revathiswari - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Tsaturyan, Lusine - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Synold, Timothy - Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA Portnow, Jana - Medical Oncology and Therapeutics, City of Hope, Duarte, CA, USA Karen, Aboody - Stem Cell and Developmental Biology, Beckman Research Institute, City of Hope, Duarte, CA, USANeural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an ‘off-the-shelf’ therapy that can be readily available for patient treatment. The route of NSC delivery and the distribution of NSCs at tumor sites are key determinants in the efficacy of cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes which convert prodrugs to active chemotherapeutic agents; tumor-targeted antibodies; therapeutic nanoparticles; oncolytic viruses; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. Here we quantitatively analyze biodistribution of HB1.F3.CD21 NSCs administered via ventricular injection (IVEN). IVEN delivery enables repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In preclinical studies using multiple glioma cell lines, we quantify NSC migration and biodistribution in mouse brain. We find robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their location of origin. These results establish proof-of-concept and demonstrate the potential of IVEN delivery of therapeutic options using modified NSCs.Funding Source: This work was supported by NIH NCI awards R03CA216142, R01CA198076, P30CA033572, the Arthur and Rosalinde Gilbert Foundation, and the Ben and Catherine Ivy Foundation.
561POSTER ABSTRACTSF-2122AN ASSAY FOR THE DETECTION AND ISOLATION OF SINGLE LIVE CTCS USING ACCUCYTE/RARECYTE PLATFORMSaremi, Shahin - Broad CIRM Center for Stem Cell Research/ University of Southern California, California State University, Channel Islands, Woodland Hills, CA, USA Kamal, Mohammed - Broad CIRM Center for Stem Cell Research, University of Southern California, Los Angeles, CA, USA Yu, Min - Broad CIRM Center for Stem Cell Research, University of Southern California, Los Angeles, CA, USA Klotz, Remy - Broad CIRM Center for Stem Cell Research, University of Southern California, Los Angeles, CA, USA Iriondo, Oihana - Broad CIRM Center for Stem Cell Research, University of Southern California, Los Angeles, CA, USACirculating tumor cells (CTCs) contain critical metastatic precursors with cancer stem cell properties and are at the interface between the primary tumor and the target secondary organs. Identification and analyzing CTCs at single cell level will help understand the properties associated with metastatic precursors. To develop methods for detecting and isolating single CTCs from whole blood with the goal for transcriptomic studies via single cell RNA sequencing and biological characterization via ex-vivo culture of single CTCs. Healthy donor blood samples were spiked with cells from breast cancer patient-derived CTC lines and processed using the AccuCyte system. CTCs are enriched by AccuCyte platform in a layer termed “Buffy Coat”. Buffy coat containing spiked CTCs was stained live with a cocktail of PE-594 antibodies conjugated to immune cell markers including CD14, CD16 and CD45. Buffy coats were simultaneously stained with either a live cell dye or with a cocktail of Alexa Fluor 488 antibodies conjugated to cancer cell surface markers (EpCAM, Her2, and EGFR). Stained cells were then plated on chamber slides in the presence of serum free RPMI media and investigated using RareCyte fluorescence platform for the presence of CTCs. For cells stained with live cell dye and immune markers, CTCs were identified based on the lack of immune cell marker expression and the presence of the live cell dye and hence termed “negative selection”. For cells stained with cancer cell surface markers and immune markers, CTCs were identified based on their surface expression of cancer markers and lack of immune cell markers and hence termed “positive selection”. Single CTCs detected by both approaches were retrieved using an optimized interactive picking protocol. Cells detected by “negative selection” were tested for their ability to proliferate ex-vivo, whereas those isolated by “positive selection” were processed for single cell RNA sequencing analyses. Detection rates of the optimized protocol reached up to 100% (24 out of 24 CTCs detected by negative selection). Furthermore, single CTCs were spiked in whole blood can be detected by positive and negative selection. Moreover, cells picked by “positive selection” are currently validated for RNA sequencing analyses.F-2124AN IN-VITRO ANALYSIS OF THE CROSS TALK BETWEEN DENTAL PULP MESENCHYMAL CELLS(DPSCS) AND CANCER CELLSKheur, Supriya M - Regenerative Medicine, Dr. D.Y.Patil Dental College AND Hospital, Pune, IndiaMesenchymal stem cells (MSCs) have shown to inhibit or proliferate cancer cells depending on the lineage of the cancer cells and the tissue of origin of the MSCs. Compared to other MSCs (bone marrow-derived, placenta-derived, adipose tissue-derived etc) the effect of dental pulp-derived MSCs (DPMSCs) on cancer cells have been relatively less explored. Thus, the present study analysed the effects of DPMSCs on 4 different cancer cell lines including melanoma, oral, breast, and prostate cancer cells. The DPMSCs were isolated from human teeth. The isolated DPMSCs were characterized to confirm their lineage. The cancer cell lines were bought from national center for cell sciences (NCCS), Pune. The DPMSCs were co-cultured with each of the cancer cell lines using both direct and indirect methods (trans-well and conditioned media). Two common phenomena were observed in the direct co-cultures: Homo-fusion between the cancer cells and hetero-fusion (hybrid cells) between the cancer cells and the DPMSCs. In homo-fusion, the fused cancer cells formed polyploid giant cancer cells which were more aggressive than the parent cell line. In the hetero-fusion, the fused hybrid cells exhibited properties different from the parent cell. The properties of the fused cells (from both homo and hetero fusion) were compared with the parent cancer cells using the MTT assay (to estimate metabolic potential), the wound healing assay (to estimate migratory potential), and the colony forming assay (to estimate clonogenic potential). Flow cytometric analysis for cancer stem cell markers like CD44, CD133 and SOX 2 were performed using the hybrid cells and the results were compared to the parent cancer cell line expression. As tumour microenvironment (hypoxic, acidic pH) is favourable for cell fusion, there could be an association between the frequency of cell fusion and cancer progression. An increased proportion of fused cell population in cancer could potentially influence the overall tumour behaviour. Thus, inhibiting cell fusion through manipulation of the tumour microenvironment (oxygen saturation, increasing the pH) could aid in modifying the cancer cells behaviour including its sensitivity to therapeutic modalities.F-2126TOWARDS A MECHANISTIC UNDERSTANDING OF THE TUMOR SUPPRESSOR FUNCTION OF WISKOTT-ALDRICH SYNDROME PROTEINZhou, Xuan - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Jeddah, Saudi Arabia Ramos-Mandujano, Gerardo - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi Arabia
562POSTER ABSTRACTSCortes Medina, Lorena Viridiana - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi Arabia Suzuki, Keiichiro - Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Andijani, Manal - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi Arabia Xu, Jinna - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi Arabia Bi, Chongwei - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi Arabia Izpisua Belmonte, Juan Carlos - Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Li, Mo - Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Jeddah, Saudi ArabiaWiskott-Aldrich syndrome (WAS) is a rare pediatric disorder caused by mutations in the WAS gene. The biological features of this disease include thrombocytopenia, eczema, complex immunodeficiency, and malignancy. WAS protein (WASP), encoded by the WAS gene, is a classical actin nucleation-promoting factor. Yet, the well-known functions of WASP fail to fully explain the high rate (13%~ 22%) of cancer in children with WAS. Recently, WASP was identified as a tumor suppressor by Chiarle’s group; however, the mechanism of its tumor suppressor function is not clear. Mounting evidence has already demonstrated that the ribosomal DNA (rDNA) gene inside the nucleolus is critical for genome stability, chromatin structure, and cancer pathogenesis. In addition, the perinucleolar heterochromatin shows structural alterations in cancer cells. Here, we use induced pluripotent stem cells (iPSCs) from patients with WAS (WAS-iPSC), isogenic gene-corrected cells (cWAS-iPSC), wild type B lymphocytes, and immortalized WASP-deficient cell lines to study the mechanisms of WAS pathogenesis. Our results showed that WASP deficiency results in the abnormal cell proliferation, over-expression of oncogenic genes, and perinucleolar heterochromatin lost. Besides, WASP physically interacts with partners inside the nucleolus. WASP binds to the ribosomal DNA (rDNA). We are examining gene expression, genome stability, and copy number variations. Taken together, our data suggests that WASP is a tumor suppressor in blood cells, and reveals important mechanisms associated with this function.Funding Source: Work in the Li laboratory is supported by grants from the King Abdullah University of Science and Technology under Awards No. BAS/1/1080-01 and URF/1/3007-01F-2128ACTIVATION OF GSK3 BETA SUPPRESSES CANCER STEM CELLS AND OVERCOMES THE CETUXIMAB RESISTANCE IN HUMAN COLORECTAL CANCERCho, Yong-Hee - Department of Biotechnology, Yonsei University, Seoul, Korea Lee, Sang-Kyu - Department of Biotechnology, Yonsei University, Seoul, Korea Kim, Taeil - Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea Choi, Kang-Yell - Department of Biotechnology, Yonsei University, Seoul, KoreaThe limited efficacy of anti-EGFR therapies such as Cetuximab and Erlotinib in advanced cancer patients remains to be resolved to enhance the prognosis of cancer patients. Clonal evolution, which is derived from survived cancer stem cells, is the barrier to effective anti-epidermal growth-factor receptor (EGFR) therapies. In this study, we observed that the protein levels of EGFR and RAS, especially mutant KRAS, are increased in Cetuximab-resistant CRC patient tissues. We also identified that these increases are attributed to the mutations of adenomatous polyposis coli (APC) which occur in 90% of human CRC. In addition, the increment of these proteins by APC loss synergistically promotes activation of cancer stemness of CRC. Therefore, we tested the effects of reducing the levels of EGFR and Ras in overcoming the cetuximab resistance by KYA1797K, a recently identified small molecule that degrades Ras and suppresses transcriptional level of EGFR via GSK3 βactivation. KYA1797K effectively suppresses the anti-EGFR therapy resistances with decreased cancer stem cell markers in the CRC animal models as well as patients derived tumor organoids (PDTOs) having KRAS mutations. Therefore, therapeutic strategy lowering levels of both RAS and EGFR would be an effective strategy that overcomes current limits of the anti-EGFR therapies in CRC and other types of cancer with mutations of EGFR and RAS.Funding Source: This study was supported by National Research of Korea (NRF) grants funded by Korean goverment (MSIP) (Grants: 2018R1D1A1B07050189, 2016R1A5A1004694, 2015R1A2A1A05001873).F-2130HIGHER ACCURACY DETERMINATION OF IMMUNE CELL IDENTITY AND PURITY USING THE PUREQUANT REAL-TIME PCR ASSAYLandon, Mark - Guzman, Jerry - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA DAargitz, Carl - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA Lakshmipathy, Uma - Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, USA Pradhan, Suman - Thermo Fisher Scientific
563POSTER ABSTRACTSIn the past several years significant improvement has been made in treating certain kinds of cancers by harnessing the power of immune system, in particular, T cells. One of the key challenges in developing immune cells as therapeutic agents is the accurate estimation of their identity and purity. Current methods used for characterization of immune cell types rely on flow cytometry. Flow cytometry can accurately estimate CD8+ T lymphocytes and other surface markers. However, this method is challenging to implement in a GMP manufacturing environment posing logistical challenges such as requirement for live cells, variability leading to difficult in standardizing and high throughput. In addition, cytometric methods are not accurate for specific intracellular targets that positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Therefore, there is an emerging need for alternative assay methods. Epigenetic DNA methylation is known to be unique for specific cell types and can thus be used as an identifier in heterogeneous population of cells. Exploiting differences in cell type-specific methylation signatures, we developed assay kits that quantify the percentage of Treg and Th17 by detecting methylation status of FoxP3 and IL17A via qPCR of bisulfite converted genomic DNA. In contrast to flow analysis, sample requirement is minimal and the assay works well with fresh/frozen cells or genomic DNA. This assay has been implemented to accurately identify and estimate different T cell population in Chimeric Antigen Receptor (CAR)-modified T cells. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types specifically critical for therapeutic applications.NEURAL DEVELOPMENT AND REGENERATIONF-3002DISTINCTIVE RESPONSE OF NEUROEPITHELIAL STEM CELLS WITH CORTICAL AND SPINAL CORD IDENTITY TO THE NOTCH SIGNALING IN INJURED SPINAL CORD MICROENVIRONMENTKhazaei, Mohamad - Krembil Research Institute, University Health Network, Toronto, ON, Canada Ahuja, Christopher - Krembil Research Institute, University Health Network, Toronto, ON, Canada Nakashima, Hiroaki - Krembil Research Institute, University Health Network, Toronto, ON, Canada Chan, Priscilla - Krembil Research Institute, University Health Network, Toronto, ON, Canada Varga, Balazs - Mount Sinai Hospital, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada Nagy, Andras - Mount Sinai Hospital, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada Fehlings, Michael - Krembil Research Institute, University Health Network, Toronto, ON, CanadaTransplantation of tripotent neural stem/progenitor cells is a promising therapeutic strategy for traumatic spinal cord injury (SCI), however, the optimal temporal and spatial developmental stage for these cells remains to be determined. In this study, we compared the fate determination of neuroepithelial stem/progenitor cells (NECs) with an anterior cortical identity to NECs patterned to acquire a ventral spinal cord identity in the injured spinal cord microenvironment. Human-induced pluripotent stem cell (hiPSC) derived cortical NECs (cNECs) and spinal NECs (spNECs) were generated and transplanted into the injured spinal cord. cNECs mainly differentiated into neurons, while spNECs mainly differentiated to myelinating oligodendrocytes. The unique differentiation profiles were mainly due to differential Pax6 expression between the two lines and were affected by activation of Notch signaling in the injured spinal cord microenvironment. cNECs excreted their effect in functional recovery in part through differentiation to neurons, migration towards cavity and making a cellular bridge, while spNECs implemented their effect partially through remyelination. Both lines provided trophic support for tissue preservation and regeneration.Funding Source: This study was supported by funding from the Ontario Institute of Regenerative Medicine (OIRM), Wings for Life (WfL), Krembil Fundation and Canadian Institutes of Health Research (CIHR).F-3004MESENCHYMAL STOROMAL CELL DELIVERY THROUGH CARDIOPULMONARY BYPASS FOR NEUROPROTECTION IN A JUVENILE PORCINE MODELIshibashi, Nobuyuki - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Sarkislali, Kamil - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Maeda, Takuya - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Saric, Nemanja - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Somaa, Fahad - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Leonetti, Camille - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Stinnett, Gary - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Sasaki, Toru - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Lewis, Bobbi - Department of Radiology and Imaging Sciences, National Institutes of Health, Bethesda, MD, USA Panchapakesan, Karuna - Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA Ulrey, Robert - Center for Cancer and Immunology Research, Children’s National Health System, Washington, DC, USA Grecco, Krystal - Department of Radiology and Nuclear Medicine, Children’s National Health System, Washington, DC,
564POSTER ABSTRACTSUSA Vyas, Pranav - Department of Radiology and Nuclear Medicine, Children’s National Health System, Washington, DC, USA Imamura Kawasawa, Yuka - Departments of Pharmacology and Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA Hashimoto-Torii, Kazue - Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA Hanley, Patrick - Center for Cancer and Immunology Research, Children’s National Health System, Washington, DC, USA Frank, Joseph - Department of Radiology and Imaging Sciences, National Institutes of Health, Bethesda, MD, USA Jonas, Richard - Cardiac Surgery, Children’s National Health System, Washington, DC, USANeurodevelopmental impairment is emerging as one the most important current challenges for survivors after pediatric cardiac surgery. Cardiopulmonary bypass (CPB) can cause substantial systemic inflammation and trigger prolonged microglial activation. Mesenchymal stromal cells (MSCs) have significant immunomodulatory properties and regulate microglia activation. We hypothesize that intra-arterial MSC delivery through CPB is neuroprotective by modulating systemic and neuro-inflammatory responses. Two-week old piglets were randomly assigned to one of 3 groups: (1) Control, (2) CPB, (3) CPB followed by MSC administration. 18F-FDG or superparamagnetic iron oxide (SPIO)-labeled MSCs (10x106 per kg) were delivered through CPB. PET was performed at 1hr after MSC delivery. Animals were sacrificed 3hrs after CPB. It has been well demonstrated that intra-venous injection of MSCs resulted in high accumulation of cells primarily into lungs. In contrast our PET study showed that intra-arterial delivery through CPB uniformly distributed MSCs to all organs analyzed such as the brain, heart, and kidney except that lungs and intestine showed lower uptake. Brain MRI showed diffuse distribution of hypointense voxels (SPIO particles) throughout the entire brain. We have previously demonstrated an increase in permeability of the blood-brain barrier after DHCA. Consistently we identified MSCs located in the extra-vascular space. MSC delivery through CPB modulated plasma cytokine/chemokine expression following surgery. In the brain MSC treatment reduced microglia expansion/activation and inhibited caspase activation resulting from CPB. Analysis of the RNA sequencing data identified 262 differentially expressed genes. Of these, 53 upregulated genes were significantly enriched for WNT signaling, indicating a potential mechanism through which MSCs mediate their neuroprotective effects. Various biomarkers after MSC delivery did not differ compared with CPB group. No evidence of either embolic events or microstrokes were observed by MRI and histology. MSC delivery during CPB is highly effective and shows translational potential to minimize CPB-induced systemic inflammation and reduce microglial expansion and caspase activation in children with CHD.Funding Source: R01HL128546, R01HL139712, U54HD090257F-3006TFII-I ISOFORMS REGULATE TYROSINE HYDROXYLASE EXPRESSION IN COOPERATION WITH NURR1 AT BOTH THE TRANSCRIPTIONAL AND EPIGENETIC LEVELKausar, Rukhsana - Department of Brain Science, Ajou University School of Medicine, Suwon, Korea Roh, Seung Ryul - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea Seo, Ji Seon - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea Lee, Myung Ae - Department of Brain Science and Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, KoreaOur previous study showed that Nurr1 actively represses human tyrosine hydroxylase (hTH) transcription in precursor cells, while it activates hTH expression in dopaminergic (DA) neuronal cells. Using proteomic analysis, we identified TFII-I as an interacting partner of Nurr1. Here we report that two alternative splicing forms of TFII-I acts in switching from repression to activation of hTH expression. TFII-I preferentially interacts with Nurr1 in Δneural stem cell F3, while TFII-I in SH-SY5Y cells. In addition, γeach TFII-I isoform majorly occupies hTH promoter in each cells. The transcriptional outcome of each isoform is totally different; repression for TFII-I and activation for TFII-I . Next, to investigate ∆γif SUMOylation of TFII-I represses Nurr1 transcriptional activity ∆via synergy control motif, we mutated SUMO sites of TFII-I isoforms as K221R and K240R. While de-SUMOylation of TFII-I ∆did not change nuclear localization, it loses transcriptional repression activities in F3 cells. Oligoprecipitation and ChIP experiments showed that deSUMOylation result in higher DNA-binding activity on hTH promoter. Last, TFII-I preferentially Δco-localizes with bivalent chromatin marks H3K4/K27me3 in F3 cells compared to SH-SY5Y cells. All our results demonstrated that two alternative splicing forms of TFII-I gene may play an important role in fine tuning of hTH gene expression during DA neurogenesis.Funding Source: This work was supported by the National Research Foundation of Korea, a grant funded by the Korean Government [2015M3A9C6028956].F-3008UNDERSTANDING THE DEVELOPMENT OF SPINAL SENSORY INTERNEURONS THROUGH IN VITRO DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLSGupta, Sandeep - Neurobiology, UCLA, University of California, Los Angeles (UCLA), Los Angeles, CA, USA
565POSTER ABSTRACTSSpinal cord injury (SCI) patients can lose somatosensation, the ability to sense the environment if sensory interneurons (dI1-dI6) are damaged. These six classes of interneurons arise in the developing dorsal spinal cord through the combined actions of the Bone Morphogenetic Protein (BMP) and retinoic acid (RA) signaling pathways. However, it remains unresolved how RA and BMP signaling direct a variety of dI fates in the spinal cord. Using developmental signals, we have identified the conditions to derive four classes of dI neurons (dI1-dI4) from mouse embryonic stem cells (mESCs). We have found that RA alone is sufficient to direct ESCs towards dI2 (unknown function) and dI4s (pain-sensing) neurons while RA with BMP4 suppresses dI2/dI4 fates and concomitantly induces dI1 (proprioception) and dI3 (mechanosensing) neurons. We hypothesize that RA and BMP4 activate different transcriptional networks to drive ESCs towards bipotential progenitors, which then differentiate into either dI2/dI4 or dI1/dI3 fates. We are evaluating this model using RNA-Seq analysis of differentiating dI neurons to identify the RA and BMP4 specific transcriptional networks. This information will enable us to design clinically relevant ESC protocols to generate specific dI neurons that can be used as cell replacement therapies to treat SCI.F-3010RECONSTRUCTION OF MOTOR FUNCTION IN PERIPHERAL NERVES BY TRANSPLANTATION OF HUMAN IPS CELLS-DERIVED MOTOR NEURON PROGENITORSNiwa, Satoshi - Neurology, Aichi Medical University, Nagakute, Japan Saeki, Masaomi - Neurology, Aichi Medical University, Nagakute, Japan Shinkai, Hiroki - Neurology, Aichi Medical University, Nagakute, Japan Kurimoto, Shigeru - Hand Surgery, Nagoya University, Nagoya, Japan Hirata, Hitoshi - Hand Surgery, Nagoya University, Nagoya, Japan Doyu, Manabu - Neurology, Aichi Medical University, Nagakute, Japan Okada, Yohei - Neurology, Aichi Medical University, Nagakute, JapanSkeletal muscles denervated by nerve injury or motor neuron disease often fall into irreversible motor dysfunction. To overcome this issue, regeneration of peripheral nerves has been attempted to reconstruct damaged motor functions. Previously, we reported that transplantation of motor neurons derived from spinal cord of rat embryos into peripheral nerves facilitated muscle contraction by electrical stimulation. In this study, human iPSC-derived motor neuron progenitors (hiPSC-MNPs) were transplanted into injured peripheral nerves and were evaluated for the effectiveness of functional neuromuscular reconstruction. Peripheral nerve injury model was generated by the transection of peroneal nerves of 8-week-old Nude rat (F344/NJcl-rnu/rnu). One week after the transection, one million hiPSC-MNPs stably expressing ffLuc (fusion protein of Venus and luciferase) as a fluorescent and luminescent marker (201B7-ffLuc), were transplanted into the distal portion of the transected nerves. Transplanted cells were evaluated over time by in vivo bioluminescent imaging (BLI), followed by the fixation at 12 weeks for histological evaluation. In BLI, luminescence intensity gradually increased until 12 weeks. Compared to the surgical control group, transplanted rats showed suppression of the atrophy of the tibialis anterior muscles, and functional contracture of tibialis anterior by electrical stimulation. Action potentials were also detected in electromyogram. Maximum muscle contraction force by tetanus stimulation was about 1/5 of the healthy side. Histological analysis has shown the cells stained for Venus (GFP) or human specific antigens in the grafted peroneal nerves, with axonal elongation to form a-BTX+ neuromuscular junctions (NMJs) in tibialis anterior muscle. In this study, we confirmed in vivo NMJ formation by hiPSC-derived motor neurons, as well as functional muscle contraction by electrical stimulation. This system could be applicable not only to the reconstruction therapy of motor function, but also to the in vivo analysis of neuromuscular pathology using disease specific iPSCs as a humanized model.F-3012EFFICIENT INDUCTION OF ASTROGLIOGENESIS ENABLES AUTOMATED, HIGH-TROUGHPUT GENERATION OF ASTROCYTES FROM HUMAN PLURIPOTENT STEM CELLSJovanovic, Vukasin M - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Malley, Claire - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Tristan, Carlos - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Ormanoglu, Pinar - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Austin, Cristopher - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Simeonov, Anton - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USA Singec, Ilyas - Stem Cell Translation Laboratory, NIH National Center for Advancing Translational Sciences (NCATS), Rockville, MD, USAAstrocytes play important roles in normal brain development, synaptic function, neurodegenerative diseases, and various pathological conditions (e.g. opioid addiction). Derivation of human astrocytes from a scalable source such as induced pluripotent stem cells (iPSCs) is an attractive approach for disease modeling and drug discovery; however, currently
566POSTER ABSTRACTSavailable protocols are variable, inefficient, and lengthy (lasting up to several months). Here, we developed a highly efficient and controlled astrocyte differentiation protocol that overcomes the limitations of previously published methods. By identifying and simultaneously manipulating several critical pathways, we directly induced astrogliogenesis from iPSCs with over 90% efficiency in less than 30 days. These cells displayed astrocyte morphologies and expressed typical markers such as GFAP, NF-IA and S100-B. Unlike previous protocols, our approach enabled the direct transition of pluripotent cells into PAX6+ neuroepithelia and then into BLBP+ radial glial cells in only 7 days. By day 14, radial glial cells differentiated into S100B+ astroglia, thereby largely bypassing neurogenesis, followed by NF-IA+ expression at day 21 as demonstrated by immunocytochemistry and time-course RNA-Seq experiments. Single-cell analysis and comparison of iPSC-derived neuroepithelia to astrocytes confirmed strong enrichment of astroglial genes and absence of genes indicative of other cell types (e.g. neurons, oligodendrocytes, microglia, endothelial cells, pluripotent cells). Importantly, iPSC-derived astrocytes were functional and capable of taking up the neurotransmitter glutamate, storing glycogen intracellularly, and promoting neuronal survival and synaptic activity when co-cultured with neurons. Finally, the differentiation protocol was automated using a robotic cell culture system, which now enables standardized production of large quantities of astrocytes for high-throughput screening and other translational applications.Funding Source: NIH Common FundF-3014CHARACTERIZATION OF FETAL BRAIN TISSUE AND VENTRAL MIDBRAIN-PATTERNED HUMAN EMBRYONIC STEM CELLS AT SINGLE CELL RESOLUTIONSharma, Yogita - Wallenberg Neuroscience Center, Lund University, Lund, Sweden Birtele, Marcella - Wallenberg Neuroscience Center, Lund University, Lund, Sweden Fiorenzano, Alessandro - Wallenberg Neuroscience Center, Lund University, Lund, Sweden Parmar, Malin - Wallenberg Neuroscience Center, Lund University, Lund, SwedenParkinson disease (PD) is the most common neurodegenerative disorder, characterized by progressive loss of dopamine neurons in midbrain. The relatively focal degeneration makes it a good candidate for cell-based therapies. Clinical trials using cells derived from human fetal brain have shown dopamine release restored to normal levels and in some PD patients produced substantial long-term clinical improvement. To move to large-scale clinical applications, the current challenge is to recreate authentic and functional dopamine neurons from human embryonic stem cells (hESCs) in vitro, thereby opening up unprecedented opportunities to gain access to a renewable source of cells potentially suitable for PD therapeutic applications. In this study we used unbiased single cell RNA sequencing to transcriptionally compare fetal VM tissue and VM-patterned hESCs after in vitro differentiation. Our analysis confirmed the diversity of cells types during midbrain development and revealed both developmental similarities and differences between the fetal VM cells and the ones differentiated from pluripotent stem cells.F-3016HUMAN STEM CELL-DERIVED NEURONS REPAIR CIRCUITS AND RESTORE NEURAL FUNCTIONChen, Yuejun - Institute of Neural Science, Chinese Academy of Science (CAS), Shanghai, China Xiong, Man - Institute of Pediatrics, Children’s Hospital, Fudan University, Shanghai, China Tao, Yezhen - Waisman Center, University of Wisconsin-Madison, Madison, WI, USA Feng, Ban - Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Gao, Qinqin - Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Haberman, Alexander - Waisman Center, University of Wisconsin-Madison, Madison, WI, USA Kotsonis, Thomas - Waisman Center, University of Wisconsin-Madison, Madison, WI, USA Xi, Jiajie - Waisman Center, University of Wisconsin-Madison, Madison, WI, USA Yan, Wei - Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China You, Zhiwen - Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Zhou, Wenhao - Institute of Pediatrics, Children’s Hospital, Fudan University, Shanghai, China Zhang, Su-Chun - Waisman Center, University of Wisconsin-Madison, Madison, ChinaThe adult mammalian brain has a limited capacity to regenerate after injury or diseases. Transplantation of neural cells to repair the damaged circuitry is a potential treatment, but whether and to what extent by which the function of the repaired circuit is restored is not known. By transplanting midbrain dopamine (mDA) neurons and forebrain glutamate neurons, derived from hESCs, into the substantia nigra (SN) of adult Parkinson’s disease model mice, we found that both neuronal types extended long axons but to different brain regions with mDA neurons predominantly projecting to the dorsal striatum via the nigra-striatal pathway. The grafted mDA neurons received area-specific synaptic inputs and these inputs became functional 3-6 months after transplantation. Transplantation with mDA neurons, but not forebrain glutamate neurons, resulted in motor functional recovery in the PD mice, which was abrogated or enhanced by regulating the activity of the grafted mDA neurons.
567POSTER ABSTRACTSThese results highlight the capacity of hESC-derived neurons for specific circuit integration both pre- and post-synaptically in the adult brain and the dependence on reconstruction of functional circuitry for therapeutic outcomes.Funding Source: the National Key Research and Development Program of China (2018YFA0108000), NIH-NINDS (NS045926, NS076352, NS086604), NIH-NIMH (MH099587, MH100031), NICHD (HD076892, U54 HD090256), the NSFC (81370030, 31771137, 31722024)NEURAL DISEASE AND DEGENERATIONF-3018DEVELOPMENT OF A NEUROLOGICAL DISEASE MODELING PLATFORM USING DIRECTLY INDUCED ASTROCYTIC CELLS FROM HUMAN NEURAL STEM CELLSTomooka, Ryo - Department of Physiology, Keio University School of Medicine, Shinjuku, Japan Zhou, Zhi - E-WAY Laboratory, Neurological business group, Eisai company, Tsukuba, Japan Sanosaka, Tsukasa - Department of physiology, Keio University School of Medicine, Tokyo, Japan Banno, Satoe - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Koya, Ikuko - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Chai, Muh Chyi - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Shimamura, Rieko - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Ando, Tomoko - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Okano, Hideyuki - Department of Physiology, Keio University School of Medicine, Tokyo, Japan Kohyama, Jun - Department of Physiology, Keio University School of Medicine, Tokyo, JapanIt has becoming apparent that glial cells play pivotal roles in the pathogenesis of neurodegenerative disease. To achieve pathologic elucidation and innovative drug development against the neurological disorders, it is important to establish disease-in-a-dish model. However, there is a limited robust methodology for the rapid and efficient preparation of disease-specific astrocytic cells so far. Here, we established a polycistronic episomal expression vector, which harbors astrocyte-inducing factors with the self-cleavage 2A peptide. This system enabled to generate astrocytic cells from human neural progenitors efficiently. Furthermore, as a proof-of-concept trial, we applied this system to generate in vitro model of Alzheimer’s disease. Then, we could generate astrocytic cells from Alzheimer’s disease patient-derived cells and found morphological changes and enhancement of disease-relevant cellular phenotype in Alzheimer’s disease patient-derived astrocytic cells. To further examine dysregulated astrocytic function of the cells, we performed functional analyses and transcriptome analysis. In the transcriptome analysis, we performed RNA-seq analysis and found that the pathways associated with inflammatory signaling were upregulated in Alzheimer’s disease-patient astrocytes. These results might add new layers of pathogenesis of Alzheimer’s disease, regarding neuroinflammatory response evoked by astrocytes. Taken together, our approach will pave the way to establish an in vitro system for disease-modeling of neurodegenerative disease.F-3020ESTABLISHING A CENTRALIZED REPOSITORY OF HUMAN PLURIPOTENT STEM CELLS FOR ALZHEIMER’S DISEASE AND RELATED DEMENTIASOhlemacher, Sarah K - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Gillespie, Kristin - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA White, Nicholas - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Kovarik, Madeline - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Sullen, Katharina - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Gregory, Domonique - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Nudelman, Kelly - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Schwantes-An, Tae-Hwi - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Marshall, Jeanine - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Faber, Kelley - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Mitchell, Colleen - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Edler, Michael - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Meyer, Jason - Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA Foroud, Tatiana - Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USAThe National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) biorepository was established in 1990 and currently houses more than 20,000 lymphoblastoid cell lines and peripheral blood mononuclear cells. Recently, NCRAD has been funded by the National Institute on Aging (NIA) to create a centralized repository of human pluripotent stem cells (hPSCs) and fibroblasts from patients with Alzheimer’s Disease and Related Dementias (ADRD), mild cognitive impairment as well as healthy controls. hPSCs provide a remarkable tool to study early human development, disease modeling, and drug development. However, much variability exists between hPSC lines, which is compounded by the diversity of culture conditions that exist among laboratories. Additionally, many labs
568POSTER ABSTRACTSdo not have the resources to perform thorough quality control measures on cell lines or to keep up with the high demand of requests. Due to these factors, NCRAD currently works with researchers to centralize hPSC and fibroblast cell lines in one location and perform rigorous quality control to ensure cell lines can be offered in a standardized manner to ADRD researchers. hPSCs are collected from researchers around the world and are screened for multiple species of mycoplasma and other sources of bacterial, fungal, and yeast contamination. A single nucleotide polymorphism (SNP) fingerprint is obtained using a panel of 94 unique sites to assign an identity to every cell line. G-band karyotype is performed to assess genetic stability. Pluripotency is confirmed by immunocytochemistry as well as qPCR analysis. hPSCs are directed to differentiate to each of the three germ layers. Differentiation efficiency is determined by qPCR analysis of more than 20 genes corresponding to each lineage. Reprogramming factors are measured to ensure residual elements are not present. Any pathogenic mutations are confirmed by PCR amplification followed by Sanger sequencing. Lines that pass all quality control measures are made available to any researcher interested in studying ADRD. Through these efforts, NCRAD has established a standardized facility to advance the study of ADRD through the distribution of hPSCs and fibroblasts.Funding Source: U24AG021886- NIAF-3022DEGENERATING NEURONS CONVERT ALS MICROGLIA FROM A NEURODEGENERATIVE TO A NEUROPROTECTIVE STATEHung, Shu-Ting - Department of Stem Cell Biology and Regenerative Medicine, University of Southern California (USC), Los Angeles, CA, USAAmyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder marked by severe loss of motor neurons in the central nervous system (CNS). The hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of ALS. Recent studies suggest that microglia, the resident immune cells in the CNS, modulate neurodegeneration. However, whether their behaviors are adaptive or maladaptive, and the mechanisms underlying these behaviors are not well understood due to the limited access of primary microglia from patients. Importantly, simultaneous gain- and loss-of-function mechanisms induced by C9ORF72 repeat expansion are hard to recapitulate in mice. To dissect the role of microglia in neurodegeneration, we established a human induced microglia (iMG) model using transcription factor-mediated reprogramming from patient iPSCs. iMG possess functional properties of microglia and have similar transcriptomic signatures to human primary microglia. iMG derived from C9ORF72 ALS patients display an abnormal gene expression profile that is similar to that found in microglia in multiple different models of neurodegenerative disease. In addition, C9ORF72 ALS iMG possess multiple vesicle trafficking abnormalities, a microglial phenotype that has been linked to several neurodegenerative diseases. Consistent with these findings, C9ORF72 ALS iMG induce the degeneration of control induced motor neurons (iMNs). In contrast, C9ORF72 ALS iMG surprisingly increase the survival of iMNs from C9ORF72 ALS patients, which normally degenerate more rapidly than control iMNs. Using cytokine profiling, we identified several cytokines as neuroprotective when C9ORF72 ALS iMG are co-cultured with C9ORF72 ALS instead of control iMNs. These results are consistent with a model in which the ALS causative C9ORF72 repeat expansion mutation induces microglia to adopt a neurotoxic state. However, the presence of degenerating C9ORF72 ALS motor neurons converts the innately neurotoxic C9ORF72 ALS microglia into a neuroprotective state that mediate its effects in part through cytokines release. These results suggest that in neurodegenerative diseases, microglia can initially act in a neuroprotective manner and maintaining this ability throughout the disease course could lead to new therapeutic approaches.F-3024ASTROCYTE CROSS-TALK WITH MICROGLIA STIMULATE PROLIFERATION AND M2-POLARIZATION OF RAT BRAIN MICROGLIAKim, Sumin - Tissue Engineering, Kyunghee University, Yongin, Korea Ahn, Woosung - Tissue Engineering, Kyunghee University, Yongin-si, Korea Zhang, Mingzi - Tissue Engineering, Kyunghee University, Yongin-si, Korea Son, Youngsook - Tissue Engineering, Kyunghee University, Yongin-si, KoreaAstrocytes are the most abundant cells in the brain that maintain homeostasis of CNS, provide structural support as well as regulate ion, nutrient, and gas concentration. Microglia are the resident immune cells of CNS. At the injury status, microglia become activated either classically activated or alternatively activated referred as pro-inflammatory M1 or anti-inflammatory M2 type. In this study, we aimed to establish whether astrocytes and microglia can cross-talk under the resting or activated status. For this approach, microglia only or mixed glial cells were prepared from brain of postnatal day (P) 1-3 inbred Lewis rat. After the 14 day culture, microglia were isolated from both cultures using Magnetic-activated cell sorting (MACS). Astrocytes co-culture promote the cell proliferation and phenotypic polarization of brain microglia; Cd163+Cd206+ microglia among total Cd11b + brain cells were increased from 56% at P0 to 98.6% at two weeks co-culture. This positive effect of astrocytes co-culture was confirmed by treating the conditioned medium to MACS-sorted Cd11b+ brain cells and recombination of purified GFAP+ astrocyte and Cd11b+ sorted P0 or 2 weeks microglia. In a various ratio of GFAP+ astrocytes and Cd11b+ microglia co-culture, approximately ten fold more iba1+ BrdU+ cells were obtained. In an attempt to identify soluble factors present in the GFAP+ astrocyte co-culture to promote M2 polarization and proliferation of microglia, cytokine arrays was performed. GFAP+ astrocytes are expressing high
569POSTER ABSTRACTSlevels of activin, agrin, CINC1,2,3, CNTF, TIMP1, MCP-1, INF- γand more cytokines. Further analysis of cross-talk between reactive astrocytes and microglia and classification of reactive astrocytes are under way. Taken together, GFAP+ astrocytes can stimulate the proliferation and M2-polarization of microglia, which suggests positive cross-talk of astrocyte under the brain injury for less tissue damage and better tissue repair.Funding Source: This study was supported by grants NRF-2016M3A9B4917320 and HI18C1492 given to Dr YSon.F-3026ELUCIDATING AXONAL PATHOPHYSIOLOGY OF IPSCS-DERIVED MOTOR NEURONS FROM FUSED IN SARCOMA (FUS)- ALS PATIENTAkiyama, Tetsuya - Neurology, Tohoku University, Sendai, Japan Suzuki, Naoki - Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan Ishikawa, Mitsuru - Physiology, Keio University School of Medicine, Shinjuku-ku, Japan Kawada, Jiro - Jiksak Bioengineering, Kawasaki, Japan Fujii, Teruo - Industrial Science, University of Tokyo, Meguro-ku, Japan Mitsuzawa, Shio - Neurology, Tohoku, Sendai, Japan Ikeda, Kensuke - Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan Funayama, Ryo - Cell Proliferation, Tohoku University Graduate School of Medicine, Sendai, Japan Nakayama, Keiko - Cell Proliferation, Tohoku University Graduate School of Medicine, Sendai, Japan Fujishima, Fumiyoshi - Anatomic Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan Mitsuhashi, Hiroaki - Applied Biochemistry, School of Engineering, Tokai University, Hiratsuka, Japan Warita, Hitoshi - Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan Okano, Hideyuki - Physiology, Keio University School of Medicine, Shinjuku-ku, Japan Aoki, Masashi - Neurology, Tohoku University Graduate School of Medicine, Sendai, JapanThe characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. Fused in sarcoma (FUS) is one of the most common ALS causative gene which have multifunction on neuronal axon via RNA metabolisms. We aim to elucidate the axon pathomechanism associated with FUS mutation. We constructed two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference in the FUS. We identified aberrant morphology in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. Moreover, we applied microfluidic devices that enable axon bundles to be produced for omics analysis and conducted RNA profiling of isolated axons. By culturing iPSCs derived MNs on microfluidic devices, the axon specific RNA could be extracted enough to RNA sequencing. Thus, we further revealed the entire in vitro RNA profile of the human MN axon and identified a causative factor of aberrant axon morphology in FUS-mutant MNs. Intervention in the novel factor revealed the reproducibility of the morphological change in axon even in vivo. Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed a key regulator of FUS-mutant MN axon morphology.F-3028HUMAN IPS CELL MODELS FOR THE VALIDATION OF SMALL MOLECULE INHIBITORS OF THE TRPM4 ION CHANNEL - A NEW POTENTIAL TARGET FOR THE TREATMENT OF MULTIPLE SCLEROSISHaferkamp, Undine - Fraunhofer Institut for Molecular Biology and Applied Ecology (IME), Fraunhofer Society, Hamburg, Germany Lam, Dennis - Fraunhofer Institut for Molecular Biology and Applied Ecology (IME), Fraunhofer Society, Hamburg, Germany Schaefer, Wiebe - Fraunhofer Institut for Molecular Biology and Applied Ecology (IME), Fraunhofer Society, Hamburg, Germany Binkle, Lars - Institute of Neuroimmunology and Multiple Sclerosis, Center for Molecular Neurobiology Hamburg, Hamburg, Germany Hornig, Soenke - Experimental Neuropediatrics, Center for Molecular Neurobiology Hamburg, Hamburg, Germany Neu, Axel - Experimental Neuropediatrics, Center for Molecular Neurobiology Hamburg, Hamburg, Germany Diecke, Sebastian - Pluripotent Stem Cell Core (PSCC), Max-Delbrueck-Center for Molecular Medicine (MDC), Berlin, Germany Gribbon, Philip - Fraunhofer Institut for Molecular Biology and Applied Ecology (IME), Fraunhofer Society, Hamburg, Germany Friese, Manuel - Institute of Neuroimmunology and Multiple Sclerosis, Center for Molecular Neurobiology Hamburg, Hamburg, Germany Pless, Ole - Fraunhofer Institut for Molecular Biology and Applied Ecology (IME), Fraunhofer Society, Hamburg, GermanyMultiple sclerosis (MS) is the most frequent chronic inflammatory disease of the central nervous system (CNS), leading to axonal demyelination and progressive neuronal degeneration. While progress has been made in treating the inflammatory processes of MS, neuronal loss in the CNS is not well understood and there is no curative treatment available yet. The ion channel transient receptor potential melastin 4 (TRPM4) has been shown to be involved in the process of inflammation-mediated neurodegeneration. Activation and misexpression of TRPM4 contributes to neuroaxonal damage in the CNS, without modulation of the immune response in the animal model of MS (experimental autoimmune encephalomyelitis;
570POSTER ABSTRACTSEAE). Furthermore, genetic knock out or unspecific blockage of TRPM4 led to an amelioration of the EAE disease course and an increased resistance of mouse neurons to glutamate-induced excitotoxicity. Previously, the suitability of TRPM4 as a therapeutic target was established and we have identified potent and selective small molecule inhibitors of the TRPM4 channel in a high-throughput screening approach. Confirmed hits have further been optimized in hit-to-lead programmes and are currently being extensively validated in various in vitro and in vivo assays using human cell lines and primary mouse neurons. We aim to demonstrate the neuroprotective effect of lead candidates targeting TRPM4 in human tissue specific cells generated from induced pluripotent stem (iPS) cells. For this purpose, we established efficient protocols for their differentiation into glutamatergic neurons. These neurons showed an upregulation of TRPM4 and NMDA receptors over time and exhibited the electrophysiological properties of functional neurons. However, 12 weeks of culturing was required to record spontaneous action potentials and synaptic activity via Patch-Clamp technique. In an alternative approach, we therefore used hiPSCs harbouring an inducible neurogenin 2 (NGN2) transgene that enables rapid conversion into defined and functional neurons. In combination with a TRPM4 knock out cell line generated using the CRISPR-Cas9 technology, we aim for validation of the potency and selectivity to prioritize compounds for consecutive preclinical development steps.Funding Source: German Federal Ministry of Education and Research (BMBF) - VIP+ grant 03VP01751F-3030DEVISING HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED EXCITATORY NEURAL NETWORK TISSUE FOR THE REPAIR OF DAMAGED NEURAL CIRCUITS AFTER SPINAL CORD INJURYZeng, Xiang - Department of Histology and Embryology, Sun Yat-sen University, Guangzhou, China Wei, Qing-Shuai - Department of Histology and Embryology, Sun Yat-sen University, Guangzhou, China Zeng, Yuan-Shan - Department of Histology and Embryology, Sun Yat-sen University, Guangzhou, ChinaWe have previously developed a tissue engineering methodology to construct adult stem cell-derived neural network scaffold in vitro and used it for treating the rats with completely transected spinal cord injury (SCI). However, the existing repair strategies can hardly deliver sufficient excitatory signals across the injured area of the spinal cord, leading to unsatisfactory recovery of neural function. We therefore hypothesized that the damaged neural circuits may function properly after replenishing excitatory neurons. To test this hypothesis, we adopted the established protocols to construct induced pluripotent stem cell (iPSC)-derived excitatory neural network tissue in vitro by tissue engineering approach. Two lines of human iPSCs were induced into high purity of neural progenitor cells before seeding to a three-dimensional (3D) gelatin sponge scaffold for 3D culture lasting from 7 days to 35 days. The immunocytochemistry results suggested that over 95% of the cell population was Map2 or NF200 positive neurons starting from 7 days of culture. Approximately 80% and 15% of the Map2 immunopositive neurons expressed glutamate and ChAT, respectively. The expression of synaptic marker, synapsin-1 and PSD95 became significant at 28 days of culture. The typical action potentials of the neurons were recorded at 28 days of culture. The synapses between neurons resembled excitatory synapse features as observed by transmission electron microscopy. Detection of robust excitatory postsynaptic currents at 28 days of culture may suggest formation of an excitatory neural network in the scaffold. After 14 days of culture, the neural network tissue was transplanted into the injured area of the spinal cord in a Sprague Dawley rat transected SCI model followed by daily administration of a triple-immunosuppressive therapy. The pilot data showed a significant earlier onset of motor function recovery starting from 4 weeks after the transplantation of iPSC-derived excitatory neural network tissue, when compared with the finding from our previous studies using mixed population of neurons. Pathological analysis showed that more than 65% of the donor cells survived up to 8 weeks after transplantation. This study suggests that iPSC-derived excitatory neural network tissue may hold promise for the functional repair of SCI.Funding Source: National Natural Science Foundation of China (31600780, 81891000), Guangdong Science and Technology Program (2017B020210012) and Guangzhou Health Care Cooperative Innovation Major Project (201704020221)F-3032MODELING ASPECTS OF THE CHROMOSOME 16P11.3 DUPLICATION IN NEURAL PROGENITOR CELL, NEURONS AND ASTROCYTES FROM PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLSJiang, Xueying - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USA Corona, Winston - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USA Detera-Wadleigh, Sevilla - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USA England, Bryce - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USA Johnson, Kory - Intramural IT and Bioinformatics Program, NINDS/NIH, Bethesda, MD, USA Kassem, Layla - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USA McMahon, Francis - Human Genetics Lab, NIMH/NIH, Bethesda, MD, USAA rare 650 kb duplication on chromosome 16p11.2 (dup16p11.2) is associated with neurodevelopmental disorders, schizophrenia, and bipolar disorder. This project aims to explore the use of induced pluripotent stem cell (iPSC) technology to study the biological impact of dup16p11.2 in neural cells and screen for therapeutic agents. Fibroblast samples were obtained from 3 carriers and 3 sex-matched non-carriers belonging to an
571POSTER ABSTRACTSextended family ascertained through a proband with bipolar disorder. We also obtained additional unrelated carriers from the Rutgers University Repository. All iPSCs were reprogrammed using lentiviral methods, then differentiated into neural progenitor cells, neurons, or astrocytes. Genome-wide gene expression was measured by microarray or RNA sequencing. Several genes within the duplicated region showed increased expression in carriers compared to non-carriers. In neurons, ALDOA, KCDT13, KIF22, PPP4C, QPRT, and TMEM219 showed the greatest increase (1.5- to 2-fold). Carriers showed more neurite formation during the first two weeks of neural progenitor cell differentiation, developed fewer MAP2-positive neurons after 4 weeks. MAP2-positive neuron counts remained lower in carriers even after more than 20 weeks of culture (ratio< 50%). Consistent with these observations, gene set enrichment analysis of the many genes differentially expressed in carriers revealed significant decreases in GO terms related to neuronal differentiation (P<2.29E-14, activation z-score, -2.2), brain development (P<2.69E-19, activation z-score -1.82), synaptic transmission (P<1.26E-08, activation z -score -2.0). VPA treatment for 5 wks during neuronal differentiation led to increased counts of mature neurons in carriers. These early data show that dup16p11.2 leads to increased expression of genes within the duplicated region and a marked reduction in the differentiation and survival of neurons that is partly rescued by VPA. The dup16p11.2 also perturbed expression of large sets of genes involved in important neurodevelopmental pathways. Patient-specific iPSC are a promising approach to the neurobiology of rare copy number variants associated with neuropsychiatric disorders and may provide an efficient platform for screening novel therapeutics.Funding Source: Funded by the NIMH Intramural Research Program, grant#1ZIAMH002843 and protocol 80-M-0082. Human materials collected by informed consent under protocol.F-3036EXPLORING MICROGLIA REPLACEMENT AS A TOOL FOR REGENERATIVE MEDICINEShibuya, Yohei - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA Wernig, Marius - Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USAThe brain is separated from the circulating blood by the blood-brain barrier (BBB), which represents one of the most significant limitations in the treatment of brain disorders. Microglia are a myeloid population in the central nervous system (CNS) that is developmentally and functionally distinct from circulating myeloid cells. However, upon depletion of endogenous microglia, circulating myeloid cells can cross the BBB and differentiate into long-lived microglia-like cells. Thus, replacement of endogenous microglia with exogenous myeloid cells expressing therapeutic genes is an attractive way to deliver therapeutic reagents to the entire CNS. Bone marrow transplantation (BMT) studies have revealed the potential of bone marrow-derived myeloid cells to migrate into the brain and become microglia-like cells. However, microglia reconstitution after BMT is a relatively slow and inefficient process compared with donor engraftment in the bone marrow. Therefore, it is clinically important to enhance microglia reconstitution with donor-derived myeloid cells after BMT to explore the potential of microglia replacement as a tool for regenerative medicine. In the current study, we have developed an optimized BMT protocol for rapid, consistent and robust engraftment of bone marrow-derived myeloid cells in the mouse brain. We demonstrate that BMT using the alkylating agent busulfan and the CSF1R inhibitor PLX5622 leads to a high (consistently ~90%) and stable (up to 9 months) brain chimerism in mice. We also show that donor-derived cells are distributed throughout the brain of recipient mice. Although engrafted bone marrow-derived cells using this method are morphologically distinct from endogenous microglia, they express several microglia-specific proteins including TMEM119 that has been reported to be a highly specific microglia marker that is not expressed in peripheral macrophages or other immune cells. The optimized BMT protocol we developed in the current study may provide a powerful approach to efficiently treat neurological disorders with global CNS pathology such as Alzheimer’s disease.F-3038HUMAN PLURIPOTENT STEM CELL-DERIVED PUTATIVE UPPER MOTOR NEURONS EXHIBIT PROGRESSIVE DEGENERATION ASSOCIATED WITH FAMILIAL ALSJordan, Zachary S - Biology, University of Texas at San Antonio, TX, USA Gomez, Jorge - Biology, University of Texas at San Antonio, TX, USA Hutchinson, Charles - Biology, University of Texas at San Antonio, TX, USA Thangamani, Kannan - Biology, University of Texas at San Antonio, TX, USA Johnson, Landry - Biology, University of Texas at San Antonio, TX, USA Maroof, Asif - Biology, University of Texas at San Antonio, TX, USAAmyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of both upper (cortical) motor neurons (MNs) and lower (spinal) MNs, leading to paralysis and eventual death. Using a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (SOD1G93A) progressive MN toxicity associated with ALS is accurately recapitulated. Many in vitro studies have also demonstrated that SOD1G93A astrocytes secrete neurotoxic factors which cause the death of co-cultured MNs while non-MN subgroups are spared. Therefore, we hypothesize that neurotoxicity will be observed in cortical MNs, analogous to spinal MNs. In addition, cortical inhibitory interneurons will be insensitive to the neurotoxic effects of SOD1G93A astrocytes. We used a novel differentiation
572POSTER ABSTRACTSprotocol to generate putative upper MNs. Using a FEZF2::GFP reporter in combination with antibodies to surface antigens specific to neuronal precursors, we isolated putative upper MNs via fluorescence activated cell sorting (FACS). We found that putative upper MNs exhibited survival deficits when co-cultured with SOD1G93A mouse astrocytes, analogous to survival deficits observed in lower MNs. Furthermore, we are testing whether various inflammatory cytokines produced by microglia are capable of inducing reactive astrogliosis, as defined by the expression of lipocalin 2 (LCN2), and assessing the resulting neurotoxicity associated with MN degeneration. Therefore, this co-culture system is a useful platform for studying the non cell-autonomous mechanisms of neurodegeneration caused by the interaction of reactive astrocytes with vulnerable neurons.Funding Source: This project was supported by a grant from the National Institute on Aging (R00AG047335). F-3040EFFECTS OF LIPOPOLYSACCHARIDE ON THE SELF-RENEWAL AND NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS AND NEURAL STEM CELLSHsu, Yi-chao - Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, TaiwanPrenatal infections in early brain development can engender adverse neurological outcomes in children. We hypothesized that prenatal infections may affect the neural differentiation of embryonic stem cells (ESCs) and further interfere with neural development and differentiation. To mimic the most common scenario of prenatal infection, mouse ESCs and ESC-derived neural stem cells (NSCs) were treated with lipopolysaccharide (LPS). In the ESC stage, 65% ± 1.3% of toll-like receptor 2 (TLR2)-positive cells and 1.6% ± 0.5% of TLR4-positive cells in ESCs were detected by flow cytometry. Notably, the percentage of TLR2(+) cells significantly decreased to 1.6% ± 0.4% and that of TLR4(+) cells significantly increased to 7.0% ± 0.9% after the ESC-to-NSC transition at days in vitro (DIV) 7. Furthermore, LPS did not affect the viability of mouse ESCs but significantly increased the number of ESC-derived neurospheres during the ESC-to-NSC transition. When LPS was administered during the ESC-to-NSC transition, we further observed that LPS significantly upregulated the mRNA expression levels of neuronal markers (Tuj1, Map2), astrocytic marker (Gfap), and oligodendrocyte marker (O4 and Oliog2) in the differentiated neural cells at DIV14, by using quantitative reverse transcription polymerase chain reaction analyses. Whole transcriptome analysis further revealed that significant upregulation of membrane dynamics of plasma, lysosomal and vacuole membranes and intracellular protein activities are involved. Notably, administration of LPS during the NSC-to-neural differentiation resulted in the downregulation of the above neural markers through significant upregulation of cell death pathways, such as p53 signalling pathway, and ferroptosis. In conclusion, this study revealed the dynamic expression profiles of TLRs during early neural development and that the timing of prenatal infection could be crucial for determining the types and severity of neural disorders developed in adulthood. Our findings may facilitate the development of preventative and therapeutic strategies for the adverse neurological side effects of prenatal infections.ORGANOIDSF-3042CAPTURING DIFFERENT DISEASE SERVERITIES OF LIS1-LISSENCEPHALY IN IPSC-DERIVED CEREBRAL ORGANOIDSKrefft, Olivia - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI) and German Cancer Research Center (DKFZ), Mannheim, Germany Maillard, Camille - Department of Pediatric Neurology, Université Paris Descartes, Imaging Institute, Paris, France Jabali, Ammar - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Marsoner, Fabio - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Bahi-Buisson, Nadja - Department of Pediatric Neurology, Universit é Paris Descartes, Imaging Institute, Paris, France Koch, Philipp - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Ladewig, Julia - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, GermanyThe development of the human cortex requires a precise choreography of progenitor proliferation, neurogenesis and neuronal migration, which can be disrupted in malformations of cortical development (MCD). In the past, most studies on MCD were performed in mouse models. Critical structural differences between human and mice might, however, necessitate the use of additional model systems. In this context, pluripotent stem cell (PSC)-derived three-dimensional (3D) cerebral organoids, which faithfully recapitulate certain aspect of human brain development in vitro, have emerged as an attractive alternative. Here we use forebrain specific cerebral organoids derived from human induced (i)PSCs to address the variable phenotypic severities of LIS1-lissencephaly, which is characterized by a smooth brain and a disorganized cortex. The LIS1-protein is one component of an intracellular multiprotein complex essential for the regulation of cytoplasmic dynein, centrosomal protein localization and microtubule dynamics. When applying our cortical organoid model to iPSCs derived from LIS1-patients exhibiting different severity grades within the LIS1-lissencephaly spectrum,wefounddisease-related,patient-specific
573POSTER ABSTRACTSphenotypes that capture the variable phenotypic severities. In particular, we observed that organoids from individuals with mild or severe disease show either mild or severe alterations in the organization of vRGCs’ microtubule networks, disruption of the architecture of the cortical niche and altered expression of cell adhesion molecules. These data indicate that iPSC based 3D cortical organoids represent a sensitive tool which allows to recapitulate variable and patient-specific disease severities, and can thus contribute to an advanced understanding of developmental mechanisms and disease-related changes caused by the dysfunction of single genes.Funding Source: The authors acknowledge the generous support of the Hector Stiftung II and STEM-MCD.F-3044SMALL INTESTINAL ENTEROIDS ON TRANSWELLS ARE IDEAL TO STUDY SWINE ENTERIC CORONA VIRUSESNelli, Rahul K - Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USA Todd, Atherly - Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA Allenspach, Karin - Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA Mochel, Jonathan - Biomedical Sciences, Iowa State University, Ames, IA, USA Jergens, Albert - Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA Gimenez-Lirola, Luis - Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USADespite several years of research efforts, the mechanisms defining enteric mucosal immunity against porcine epidemic diarrhea virus (PEDV) are poorly understood. In 2014, USDA dedicated US $26.2 million for domestic prevention of this very important swine enteric disease, which has caused devastating economic losses to the swine industry. PEDV, a member of family Coronaviridae, affects the epithelial lining of the intestine causing diarrhea and up to 100% mortality in neonatal pigs. Currently, there is a lack of well-established and reproducible intestinal infection models to evaluate the complex mechanisms underlying the mucosal immune response against PEDV and other swine enteric pathogens. Crypt stem cell–derived 3D enteroids cultures have recently emerged as a biologically and physiologically relevant ex vivo culture systems that resembles intestinal physiology in vivo. However, one of the limitations of 3D enteroids is accessing the apical surface of the enteroids during their growth in matrigel. Such methods would allow evaluation of mechanisms driving early innate immune responses towards PEDV and its receptor affinity. Thus, the objective of this study was to establish and subculture 3D porcine enteroids on transwell membranes exposing the apical surface of enteroids. We demonstrated that 3D porcine enteroids cultured in matrigel can be established on transwell culture system, replicate efficiently and can be sub-cultured and frozen for subsequent use. By day 5, the microscopic morphology of porcine enteroids on transwells (PETCs) resembled the cross-sectional epithelia of their ex vivo counterparts (duodenum, jejunum and ileum). Immuno-cytological characterization showed that PETCs expressed cell markers specific for different cell lineages of the intestinal mucosa, i.e., villin-1 (enterocyte), mucin-2 (goblet), lysozyme C (paneth), chromogranin A (enteroendocrine), Lgr5 , occludin, Ki67 and sialic acids receptors. Moreover, we demonstrated that PETCs are susceptible to PEDV infection and replication within enteroids grown on transwells. In conclusion, we have developed a novel PETCs culture system that closely resembles small intestinal epithelium of pigs and supports PEDV infection. The proposed PETC cell culture model can be extensible to study other swine enteric pathogens.F-3046A PERSONALIZED MODEL TO ASSESS INTESTINAL PERMEABILITY IN IBD AND VEOIBD PATIENTS USING HUMAN IPSC-DERIVED INTESTINAL AND COLONIC ORGANOIDSBarrett, Robert - Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Gleeson, John - Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Rabizadeh, Shervin - Division of Pediatric Gastroenterology, Cedars Sinai Medical Center, Los Angeles, CA, USA Estrada, Hannah - Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Li, Dalin - F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA McGovern, Dermot - F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Targan, Stephan - F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA Svendsen, Clive - Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, Los Angeles, CA, USAInflammatory bowel disease (IBD) refers to a spectrum of complex polygenic disorders that are thought to result from dysregulated immune responses to commensal microbes in genetically susceptible hosts. In IBD, the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of scientific approach and the profound genetic heterogeneity associated with this disease. Our aim is to develop a personalized in vitro intestinal permeability model using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) so that genetically defined subsets of IBD patients can be selected and ultimately allows for these genetic underpinnings to be defined. To confirm our approach was suitable for a wide range of individuals, iPSCs were generated from 2 healthy controls,3 IBD and 2 very early onset-IBD (VEOIBD) patients. HIOs and HCOs
574POSTER ABSTRACTSwere generated from each iPSC line by directing them to form definitive endoderm, hindgut tissue and ultimately HIOs or HCOs. There was a significant increase in the apparent permeability of FD4 across epithelial monolayers derived from HIOs generated from healthy controls and all IBD patients in response to TNF αand IFN (both 10ng/ml). HCO epithelium was generated from γa control individual and both VEO-IBD patients and there was significantly increased expression of colonic markers and significant increase in transepithelial resistance in all three lines in comparison their corresponding HIOs, confirming that colonic tissue was generated. There was also a significant increase in permeability in HCO tissue in response to inflammatory cytokines. We also demonstrate that E-cadherin expression was reduced and mislocalized in HIOs but not HCOs in response to these cyokines also. Overall we demonstrate that we can now measure intestinal permeability, and examine changes in tight/adherens junction proteins, in both the small and large bowel using iPSC-derived organoid technology. Subsets of genetically defined IBD patients can now be selected and an investigation into how single nucleotide polymorphisms associated with IBD influence intestinal permeability can now be carried out.Funding Source: NIH/NIDDK (R56DK106202-01) (S.R.T and C.N.S.), Board of Governors Regenerative Medicine Institute, the F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute and the Drown Foundation.F-3048ON-CHIP CONSTRUCTION OF A PERFUSABLE VASCULAR NETWORK WITH HUMAN IPS CELL-DERIVED KIDNEY ORGANOIDOkada, Ryu - Department of Micro Engineering, Kyoto University, Kyoto, Japan Kameda, Yoshikazu - Department of Micro Engineering, Kyoto University, Kyoto, Japan Araoka, Toshikazu - Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Takasato, Minoru - Center for Biosystems Dynamics Research, RIKEN, Kobe, Japan Yokokawa, Ryuji - Department of Micro Engineering, Kyoto University, Kyoto, JapanMicrofluidic devices can mimic the in vivo microenvironment three-dimensionally by controlling disposition of the cells, the extracellular matrix, and the flow of the medium. We have previously reported on the construction of a perfusable vascular network system using a spheroid as a tissue model. Here, we attempt to develop a microfluidic cell culture device to construct perfusable vascular networks using a human iPS cell-derived kidney organoid, and analyze the effects that the interaction of the organoid with endothelial cells and flow-induced shear stress in blood vessels give to kidney development. Co-culturing a kidney organoid with human umbilical vein endothelial cells (HUVECs) in the device revealed that the organoid at a mid-stage of kidney development can induce angiogenesis of HUVECs. The length of the angiogenic sprouts was the longest when the media for the organoid and HUVECs were mixed. However, the elongation of angiogenic sprouts was inhibited until after seven days of culture, and the vascular network did not have a lumen. To connect a perfusable vascular network into a kidney organoid, we developed a pre-constructed vascular bed in a new type of device by introducing HUVECs and human lung fibroblasts. Perfusion culture in the vascular bed showed connection with endogenous blood vessels in the organoid and the vascular bed. Now, we are aiming to analyze the developmental stage of kidney organoids using this culture system.Funding Source: This study was partially supported by AMED-MPS project; COI from MEXT and JST; Kyoto University Nano Technology Hub in “Nanotechnology Platform Project” sponsored by MEXT, Japan.F-3050IMPROVING CELL SURVIVAL TO CONTROL CEREBRAL ORGANOID FORMATION FROM HUMAN PLURIPOTENT STEM CELLSRyu, Seungmi - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Chen, Yu - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Chu, Pei-Hsuan - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Malley, Claire - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Simeonov, Anton - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA Singec, -Ilyas - National Center for Advancing Translational Sciences, NIH, Rockville, MD, USAGeneration of in vitro organoid models from pluripotent stem cell holds great promise for future advances in disease modeling, drug development, and tissue engineering. Cerebral organoids are complex three-dimensional (3D) structures that form by aggregation and self-organization of pluripotent cells, ultimately mimicking some aspects of the physiologically complex, multicellular, and layered architecture of the brain. However, significant variability and experimental challenges exist in organoid formation protocols. One such challenge is the large amount of cell death when pluripotent cells are dissociated and aggregated into 3D structures. Currently, the ROCK inhibitor Y-27632 is the most widely used reagent to improve cell survival. Nevertheless, poor cell survival and emergence of debris are evident even after treatment with Y-27632 suggesting that uncontrolled cell stress introduces an inherent systematic shortcoming in currently used protocols. Here, we used a novel small molecule cocktail, recently developed at NCATS, that greatly enhances cell survival during cerebral organoid formation. Improved cell survival at the onset of sphere formation generated larger, healthier and better controlled organoids. Ongoing molecular and functional experiments are aimed at demonstrating that optimal cell survival enhances morphogenesis, differentiation, and reproducibility of cerebral organoids.
575POSTER ABSTRACTSF-3052GENERATION OF OLFACTORY PLACODAL/EPITHELIAL ORGANOIDS FROM HUMAN PLURIPOTENT STEM CELLSNakano, Tokushige - Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan Takahashi, Yasuhiko - Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan Kitamoto, Sachiko - Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan Tomigahara, Yoshitaka - Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd, Osaka, JapanThe olfactory epithelium lies the roof of nasal cavity and is involved in smell. The organogenesis of olfactory systems were mainly investigated with in vivo animal models, and the olfactory epithelial differentiation methods from pluripotent stem cells have not been established. Here we challenged to recapitulate the organogenesis of olfactory systems in a dish. In this study, we present our Serum-free Floating culture of Embryoid Bodies-like aggregates (SFEBq)-based human olfactory epithelial organoids (hOlfOs) differentiation method and discuss the features of generated organoids. As well as lens, pituitary, ear and trigeminal ganglia, the olfactory epithelium is derived from specific region of non-neural ectoderm called placode. Previously generation of pituitary and otic organoids from pluripotent stem cells were reported, and we modified these protocols for olfactory placode differentiation. In the process of differentiation, single cell layer of non-neural ectoderm appeared at the surface of cell aggregates and gradually got thicker. The thickened epithelium took on distinctive phase-bright appearance, and the surface of epithelium was smooth as well as retinal organoids. The thickened epithelium were pseudostratified, and expressed non-neural ectoderm/placodal (Six1/Dlx5/Sox2/E-Cadherin/EpCAM/Cytokeratins) and olfactory region (Otx2/Sp8) markers. In addition, Ebf2 (Olf2)/NeuroD1/Lhx2/Tuj1/NCAM/Calretinin+ immature olfactory sensory neurons (iOSNs) exists in these epithelium. Our preliminary data indicates that modified SFEBq method could recapitulate the self-formation of olfactory epithelium from pluripotent cells in vitro, and will provide valuable insights to investigation of olfactory system development, behavior of olfactory stem cells and genetic diseases such as Kallmann syndrome.F-3054MODELING HSV-1 INFECTIONS IN HUMAN CNS NEURONAL CELLS USING TWO-DIMENSIONAL AND THREE-DIMENSIONAL CULTURES DERIVED FROM INDUCED PLURIPOTENT STEM CELLSZheng, Wenxiao - Western Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Bloom, David - Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA Naciri, Jennifer - Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA Smith, Adam - Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA Edwards, Terri - Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA McClain, Lora - Research, Magee-Women’s Research Institute, PA, USA Callio, Jason - Department of Pathology, University of Pittsburgh, PA, USA Jessup, Morgan - Cell Biology, University of Pittsburgh, PA, USA Wood, Joel - Psychiatry, University of Pittsburgh, PA, USA Chowdari, Kodavali - Psychiatry, University of Pittsburgh, PA, USA Demers, Matthew - Psychiatry, University of Pittsburgh, PA, USA Abrahamson, Eric - Neurology, University of Pittsburgh, PA, USA Ikonomovic, Milos - Neurology, University of Pittsburgh, PA, USA Viggiano, Luigi - Biology, University of Bari, Aldo Moro, Bari, Italy DeZio, Roberta - Biology, University of Bari, Aldo Moro, Bari, Italy Watkins, Simon - Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Kinchington, Paul - Ophthalmology, University of Pittsburgh, PA, USA Nimgaonkar, Vishwajit - Psychiatry, University of Pittsburgh, PA, USA D’Aiuto, Leonardo - Psychiatry, University of Pittsburgh, PA, USAHSV-1 establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcome of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced human pluripotent stem cells (hiPSCs) and neurons derived from them as tools to study aspects of neuropathogenesis are documented but few have focused on modeling infections of the (CNS). Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that: i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; iv) the organoids support reactivation of HSV, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.
576POSTER ABSTRACTSFunding Source: Leonardo D’Aiuto NS096405 David C. Bloom T32AI007110 Vishwajit L. Nimgaonkar MH063480 Vishwajit L. Nimgaonkar 07R-1712 Simon C. Watkins P30CA047904 Paul R. Kinchington AI122640 Paul R. Kinchington NS064022F-3056DEVELOPING A HUMAN EMBRYONIC STEM CELLS BASED HIGH-THROUGHPUT PLATFORM TO SCREEN FOR DEVELOPMENTAL TOXICANTSChen, Yichang - National Toxicology Program Laboratory, National Institute of Environmental Health Sciences/National Toxicology Program, Durham, NC, USA Luz, Anthony - National Toxicology Program laboratory, National Institute of Environmental Health Sciences/National Toxicology Program, Durham, NC, USA Tokar, Erik - National Toxicology Program Laboratory, National Institute of Environmental Health Sciences/National Toxicology Program, Durham, NC, USAEvery year, millions of infants worldwide are born with a serious birth defect, which not only raises the risk for lifelong disabilities to those who survive but also increases the economic burden to their families and society. Besides genetic or hereditary factors, many of these defects can be caused by environmental chemical exposure, such as alcohol, smoking, and drugs. While there are over 80,000 chemicals registered for use in the United States, many of them have undergone little safety testing. Therefore, a rapid and accurate method for predicting developmental toxicants in the environment to humans and understanding their toxic mechanisms is strongly desired. Pluripotent human embryonic stem cells (hESCs) possess the capacity to differentiate into any cell type which makes them an ideal in vitro model to investigate developmental toxicity. In this study, we aim to develop a cost-effective transcriptomic-based high-throughput platform using hESCs to screen for environmental chemicals and pharmaceutical compounds with embryotoxic potential. In general, three-dimensional embryoid bodies (EBs), which recapitulate many developmental processes of early embryogenesis, were formed from hESCs. 30 chemicals with known or suspected teratogenicity (i.e. thalidomide, sodium arsenate, and tretinoin) were administered to EBs for seven days at concentrations causing a minimal cell viability loss (i.e. LC10). Pluripotency and embryonic differentiation of EBs were assessed by measuring the expression of 16 hallmark genes of these processes. The impacts of tested chemicals on key signaling pathways (Wnt, Notch, Sonic hedgehog, and TGF- ) βrequired for the early embryogenesis were investigated as well. Hierarchical clustering analysis of our preliminary data allowed us to separate embryonic toxicants apart from the negative controls. Furthermore, consistent with previous reports, our results also indicate that tretinoin, benomyl, and perfluorooctanoic acid possess neural developmental toxicity since they drastically decrease the expression of genes associated with ectoderm formation ( 2-fold). Together, these results indicate that our ≥screening platform could be successfully applied for identifying developmental toxicants and understanding their etiology.F-3058A PROTEIN ASSAY TO MEASURE R-SPONDIN LEVELS IN CONDITIONED MEDIUM FOR ORGANOID CULTURESWang, Hsu-Kun - Research and Development, MilliporeSigma, Temecula, CA, USA Abai, Anna - Research and Development, MilliporeSigma, Temecula, CA, USA Chu, Vi - Research and Development, MilliporeSigma, Temecula, CA, USA3D organoid culture systems are increasingly employed as powerful tools for the study of human diseases. R-spondin-1 (RSPO1) is one of the most extensively used niche factors for culturing 3D organoids and has been used to establish organoid cultures from the stomach, small intestine, colon, pancreas and liver from both mouse and human sources along with intestinal organoids from various large & small animals. R-spondin-1 conditioned medium (RSPO1 CM) is widely used as a potent and inexpensive alternative to purified recombinant RSPO1 protein. However, most protocols for the generation of RSPO1 CM do not monitor RSPO1 protein levels and thus the timing for conditioned media collection may be suboptimal. Batch-to-batch variations may also exist which may affect the quality and subsequent analyses of the different organoid culture systems. We describe a semi-quantitative assay to measure the levels of RSPO1 protein in medium conditioned with a 293T cell line stably transfected with a modified mouse R-spondin-1 gene. Results from the protein assay directly correlated with the ability of different batches of RSPO1 CM to culture and expand mouse intestinal organoids over multiple passages. With the aid of the assay, we were able to scale-up the manufacture of RSPO1 CM, ascertain the optimal time to harvest the CM, ensure batch-to-batch consistency and determine the stability of the RSPO1 CM in long-term storage conditions (>1 year). RSPO1 CM was subsequently used to develop an optimized supplement that could be used to isolate and maintain mouse intestinal organoids. We show that in the optimized RSPO1 CM supplement, relatively primary mouse intestinal organoids (<p5) initiated budding within 3 days after passage versus 10 days with an alternative commercial source of RSPO1-supplemented medium. Moreover, neglected and overgrown preparations of mouse intestinal organoids could be rescued by culture in the optimized RSPO1 CM supplement. We anticipate that these findings may be extended to other organoid systems that require R-Spondin-1.
577POSTER ABSTRACTSTISSUE ENGINEERINGF-3060EFFECT OF INCUBATION TIME ON THE VIABILITY AND FUNCTION OF POST TOOTH EXTRACTED HUMAN DENTAL PULP STEM CELLSAryal A C, Smriti - Wound Healing and Oral Diagnosis/Sharjah Institute of Medical Research, University of Sharjah, University City Road, United Arab Emirates Khan, Amir Ali - Applied Biology/Biotechnology, University of Sharjah, University City Road, United Arab Emirates Samsudin, Ab Rani - Wound Healing and Oral Diagnosis, University of Sharjah, University City Road, United Arab EmiratesHuman teeth contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Human dental pulp stem cells (HDPSCs) are stem cells present in the dental pulp, the soft living tissue within the teeth. They are multipotent, so they have the potential to differentiate into a variety of cell types and possess the potentiality or infinite applications in reconstruction, regenerative therapies and tissue repair. HDPSCs are easily accessible and histocompatible. Daily, many live teeth are extracted for many reasons, such as avulsion, exfoliation or during orthodontic treatments. These teeth are usually discarded rather than being put to use. The infinite potential of HDPSCs, their accessibility, and their multipotency to differentiate into different cell lineage makes them a promising source for tissue regeneration and stem cell banks. However, there haven’t been studies about the effect of the post tooth extraction incubation time on the viability and function of HDPSCs. It is very important to establish how long pulp cells are viable and functional in order to construct a pulp cell preservation protocol for stem cell banking. Considering the above problems the aim of this study was to investigate the viability and function of HDPSCs isolated from the extracted teeth immediately and after 6 and 24 hours (hrs) postextraction. Cell proliferation was analyzed using MTT and XTT assay both of which showed slight reduction in the proliferation of the HDPCS collected after 24 hrs post extraction compared to immediate and 6 hrs group. There was no change in cell morphology and cell viability between groups as evidenced by light microscopy, crystal violet staining and trypan blue hemocytometer cell counting. The HDPSCs isolated immediately,6 and 24 hrs post extraction were characterized by flow cytometry using mesenchymal stem cells (MSC) specific and unspecific markers, the result showed that all the HDPSCs groups were more than 80% positive for MSC positive markers such as CD44,CD106, CD90 and more than 80% negative for MSC negative markers such as CD45 and CD11b. Taken together the data suggests that a delay of up to 24 hrs for tooth processing and HDPSCs collection does not inhibit the establishment of dental pulp cell cultures or affect the cell morphology and the viability of the HDPSCs.Funding Source: University of SharjahF-3062LOW OXYGEN GRAPHENE AS A PLATFORM FOR OSTEOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLSNewby, Steven D - Large Animal Clinical Sciences / University of Tennessee, Knoxville, College of Veterinary Medicine, Knoxville, TN, USA Masi, Tom - Surgery, University of Tennessee Graduate School of Medicine, Knoxville, TN, USA Griffin, Chris - Center of Integrative Nanotechnology Sciences, University of Arkansas, at Little Rock, AR, USA King, William - Center of Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, , AR, USA Stephenson, Stacy - Surgery, University of Tennessee Graduate School of Medicine, Knoxville, TN, USA Biris, Alexandru - Center of Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, , AR, USA Anderson, David - Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA Bourdo, Shawn - Center of Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, AR, USA Dhar, Madhu - Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USAThe effects of low oxygen graphene can be utilized as both a cell-adhesion substrate and a delivery platform for the osteogenic differentiation of human mesenchymal stem cells (MSCs). Graphene, a 2D high surface area lattice monolayer is composed of hexagonally arranged carbon atoms, and can be functionalized by chemical modifications to generate nanocomposites with specific physicochemical properties. In this study, we first identified the mass concentration of graphene that showed a significant increase in osteogenesis, by Alizarin red staining and quantitation, and subsequently, the expression and cellular localization of extracellular matrix (ECM) glycoproteins was assessed in vitro by immunofluorescence. The expression of ECM proteins laid down by human MSCs was evaluated in undifferentiated and osteogenic cells. ECM is a network structure composed of various biomolecules, controls the cellular behavior and provides a favorable microenvironment for the growth and differentiation of cells. The results show that graphene is cytocompatible towards MSCs and the amount of graphene coated on a surface modulates cell adhesion, spreading, growth, and osteogenic differentiation. We also observed that human MSCs express various integrin subunits and lay down the matrix i.e. rich in fibronectin, suggesting the interaction between the two, to play a major role in adhesion and osteogenic differentiation of human MSCs on graphene surfaces. In this study, we will discuss (1) bone ECM composition and key integrin proteins implicated in osteogenic differentiation, (2) the use of graphene alone and graphene modified with ECM–mimetic peptides/protein fragments that will promote the
578POSTER ABSTRACTSmechanical fixation of implants to bone and to enhance bone healing within large defects. This study suggests that a layer of graphene as a potential bone scaffold will be beneficial for osteoblast attachment, proliferation, and differentiation.Funding Source: Supported by NIH R15 National Institute of Arthritis and Musculoskeletal and Skin DiseasesF-3064DERMAL PAPILLA CELLS-ENCAPSULATED HAIR BEADS FOR HAIR REGENERATIVE MEDICINEKageyama, Tatsuto - Faculty of Engineering, Kanagawa Institute of Industrial Science and Technology and.Yokohama National University, Yokohama, Japan Fukuda, Junji - Faculty of Engineering, Yokohama National University and Kanagawa Institute of Industrial Science and Technology, Yokohama, JapanHair loss generally occurs due to various causes such as genetics, aging, hormonal imbalances, autoimmune reactions, and anti-cancer drug medications, and this is linked to the loss of stem cells responsible for normal hair formation and hair cycling. Recently, hair regeneration by co-transplantation of epithelial-derived follicular stem cells and dermal papilla cells (DPCs) have emerged as a promising approach for treating hair loss. DPCs provide signals to hair follicle stem cells, specifying the size, shape, and pigmentation of hair shafts, and epithelial-derived follicular stem cells differentiate and eventually form a hair shaft. However, the hair induction ability of both cell types is gradually lost after isolation from in vivo tissues and during expansion culture. Latest studies have shown that three-dimensional culture of DPCs could recover expression of hair induction markers and efficiently induce hair follicles when transplanted with hair follicle stem cells. In this study, we propose an effective approach to fabricate collagen-enriched cell aggregates, named hair beads (HBs), through the spontaneous constriction of cell-encapsulated collagen drops. DPCs were encapsulated in 2-μl collagen microgels, which were spontaneously constricted and concentrated >10-fold in volume during 3 days of culture. Interestingly, HB constriction was attributed to attraction forces driven by myosin II and involved the upregulation of follicular genes. Gene expression of hair induction markers in DPCs cultured in HBs was greater than that in typical spheroid culture at 7 days of culture. The DPCs cultured in HBs were mixed with epithelial-derived follicular stem cells, and subcutaneously transplanted into a pocket surgically created on the back skin of nude mice and shown to be capable of efficient hair follicle and shaft generation at 3 weeks after transplantation. Compared to spheroid culture, hair numbers generated were more than double in HB culture. These results suggested that the HB culture provides a more suitable approach for recapitulating the in vivo dermal papilla niche.F-3066METABOLICALLY-DRIVEN MATURATION OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED HEART-ON-A-CHIPHuebsch, Nathaniel - Bioengineering, University of California, Berkeley, Saint Louis, MO, USA Charrez, Berenice - Bioengineering, University of California, Berkeley, CA, USA Siemons, Brian - Bioengineering, University of California, Berkeley, CA, USA Boggess, Steven - Chemistry, University of California, Berkeley, CA, USA Wall, Sam - Simula Research Laboratory, Oslo, Norway Charwat, Verena - Bioengineering, University of California, Berkeley, CA, USA Jaeger, Karoline - Simula Research Laboratory, Oslo, Norway Lee Montiel, Felipe - Bioengineering, University of California, Berkeley, CA, USA Jeffreys, Nicholas - Bioengineering, University of California, Berkeley, CA, USA Deveshwar, Nikhil - Bioengineering, University of California, Berkeley, CA, USA Edwards, Andrew - Simula Research Laboratory, Oslo, Norway Serrano, Jonathan - Pathology, New York University, NY, USA Snuderl, Matija - Pathology, New York University, NY, USA Stahl, Andreas - Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA Tveito, Aslak - Simula Research Laboratory, Oslo, Norway Miller, Evan - Chemistry, University of California, Berkeley, CA, USA Healy, Kevin - Bioengineering, University of California, Berkeley, CA, USAHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are a promising in vitro testbed for drug development. However, their immature electrophysiology limits their utility. For in vitro diagnostics, approaches involving aligned 3D culture enhance hiPSC-CM structural, but not electrophysiological maturation. We hypothesized that recapitulating the heart’s ability to use fatty acids as an energy source could enhance electrophysiological maturation of hiPSC-CM cultured in 3D Microphysiological Systems (MPS). We formed cardiac MPS by adding a defined cocktail of 80% hiPSC-CM and 20% isogenic stromal into microfabricated tissue chips. We assessed micro-tissue function by tracking contractile motion and calcium flu , and voltage. Cardiomyocytes in MPS aligned rapidly and exhibited robust sarcomere assembly. However, the Action Potential Duration (APD) of hiPSC-CMs within the MPS was prolonged when the system was cultured in standard cardiac media. To identify medias that could enhance electrophysiologic maturation, we used a Design of Experiments approach to study combinatorial effects of albumin, glucose, galactose, oleate and palmitate. In MPS, optimal fatty-acid based media yielded APDs similar to that of adult human left ventricle. However, the same media did not alter the APD of hiPSC-CM monolayers. Matured cardiac MPS was superior to both hiPSC-CM monolayers and
579POSTER ABSTRACTSstandard MPS in terms of its ability to predict safety margins for drugs that exhibit false positive toxicity (Verapamil), or false negative toxicity (Alfusozin). In silico modeling of specific ion channel contributions to action potential and calcium flux, together with gene expression analysis, suggested enhanced Ca2+ handling as the mechanism underlying action potential shortening.by fatty acid enriched media.Funding Source: This work was supported by NIH-NCATS UH2TR000487 and UH3TR000487.F-3068SILK-BASED SCAFFOLDS FOR SKIN WOUND HEALINGMohanty, Sujata - Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India Jain, Krishan Gopal - All India Institute of Medical Sciences Delhi (AIIMS) Dutta, Mayuri - Stem Cell Facility CoE SCR, All India Institute of Medical Sciences, New Delhi, India Kaur, Amtoj - Stem Cell Facility CoE SCR, All India Institute of Medical Sciences, New Delhi, India Midha, Swati - Stem Cell Facility CoE SCR, All India Institute of Medical Sciences, New Delhi, India Rajput, Bitesh - Stem Cell Facility CoE SCR, All India Institute of Medical Sciences, New Delhi, IndiaSkin wounds have a large socio-economic effect on the society. The global wound care market is expected to increase from 18.35 million USD in 2017 to 22.01 million USD by 2022. Full-thickness wounds, especially caused by 3rd degree burns, > 10 mm in diameter require skin grafting to fully heal and prevent related complications. In spite of a range of products available, complete skin regeneration is still considered as an unmet clinical need. Till date, there is no such skin substitute which can recapitulate the anatomy and functionality of native human skin. Thus, this proposal intends to fabricate functional skin substitute that could; (i) target efficient wound healing, (ii) possess optimal mechanical properties, (iii) validation of protocols as per GMP compliance for translational purpose. Different ratios of silk are being combined with Polycaprolactone (PCL)/Pluronic F-127/gelatin to optimize inks in terms of rheology, printability, stiffness and stability for bioprinting. In vitro optimization and characterization of various bioinks and commercial bioinks (as positive control) was conducted. Confocal micrographs demonstrated that majority of encapsulated mesenchymal stem cells were live (Live/Dead staining) in the 3D stack and possessed characteristic morphology as observed using phalloidin/DAPI. Nanofibrous electrospun mats of silk-based biocomposites (in formic acid) were fabricated having nanofibers in the range of 200-600 nm. Lyophilization was also performed to obtain different scaffold geometries resulting in robust structures with pore sizes in the range of 50-500 um allowing infiltration of MSCs and subsequent ECM synthesis. Biocompatibility assessment is currently underway using human mesenchymal stem cells for cell viability assays (MTS/Live/Dead staining), proliferation and differentiation to compare the most optimal scaffold surface for regenerating the different layers of the skin. The in vivo studies conducted on excision wound rat model showed enhanced healing and regeneration in groups transplanted with the 3D printed scaffolds. This research aims to develop transplantable customized scaffolds for faster and efficient wound healing restoring the native functionality of the skin.Funding Source: We would like to acknowledge Department of Biotechnology (DBT, Government of India) for their financial support (Grant No BT/01/COE/07/03).F-3070GENERATION OF HIGHLY VASCULARIZED INDUCED HEPATIC-LIKE TISSUES VIA DIRECT REPROGRAMMINGJin, Yoonhee - Department of Biotechnology, Yonsei University, Seoul, Korea Cho, Seung-Woo - Department of Biotechnology, Yonsei University, Seoul, Korea Min, Sungjin - Department of Biotechnology, Yonsei University, Seoul, KoreaInduced hepatic (iHep) cells generated by direct cellular reprogramming have been proposed to replace primary hepatocytes for drug screening industry and regenerative medicine. However, the practical use of a three-dimensional (3D) hepatic tissue culture comprised of iHep cells for drug screening and toxicology testing has not been explored in detail yet. In this study, we demonstrate a 3D vascularized liver organoid composed of non-virally generated iHep cells and a liver extracellular matrix (LEM) cultured in a microfluidic system. iHep cells were generated by transfection with polymer nanoparticles and plasmids expressing transcription factors important in a hepatic lineage. Here, the iHep cells were co-cultured with endothelial cells in the 3D LEM hydrogel in a microfluidic-based cell culture device with a continuous dynamic flow of media that mimicked the blood circulation. The resultant 3D vascularized liver organoids maintained under this physiologically relevant culturemicroenvironmentexhibitedimprovedhepaticfunctionalities, metabolic activity, and drug responses. Finally, we confirmed the feasibility of using the iHep-based 3D liver organoid as a high-throughput drug testing platform, as well as its use in an integrated drug testing platform comprised of multiple internal organoids. Our study suggests that a combined strategy of direct reprogramming, matrix engineering, and microfluidics could be used to develop a highly functional, drug screening and toxicological analysis platform.Funding Source: This study was supported by the National Research Foundation (NRF) of Korea grants funded by the Korea government (MSIT) (2018R1D1A1B07042768 and 2018M3A9H1021382).
580POSTER ABSTRACTSF-3072TUNABLE PARATHYROID HORMONE-RELATED PROTEIN RELEASE FOR MODULATION OF HUMAN MESENCHYMAL STEM CELL HYPERTROPHY TOWARD STABILIZED CHONDROGENESISYang, Yueh-Hsun Kevin - School of Engineering, City University of New York - The City College, New York, NY, USA Chang, Tsui-Yun - School of Engineering, City University of New York - The City College, New York, NY, USA Barabino, Gilda - Biomedical Engineering, City University of New York - The City College, New York, NY, USAStable chondrogenesis of mesenchymal stem cells (MSCs) has not been achieved since their implantation leads to rapid formation of endochondral bone, which is attributed to strong hypertrophic chondrocyte phenotype expressed by the implanted cells. This study aimed to suppress cell hypertrophy via tunable delivery of parathyroid hormone-related protein (PTHrP) to human MSCs undergoing TGF- 3 induced chondrogenic βdifferentiation. To this end, soluble PTHrP was encapsulated within poly lactic-co-glycolic acid (PLGA; PLA-to-PGA, 75:25) microspheres using a water-in-oil-in-water double emulsion method. PLGA with an ester-terminated (capped) or carboxylic acid-terminated (uncapped) end group was used to generate different PTHrP release profiles. While no significant difference in physical properties was identified between PTHrP-loaded capped and uncapped PLGA microspheres, the initial burst release of PTHrP decreased from 30% in the ester-terminated group to 10% in the acid-terminated group. Moreover, most of encapsulated PTHrP molecules were retained within acid-terminated microspheres for more than 6 days, indicating that PLGA with an uncapped end group tends to interact firmly with encapsulated biomolecules. Each of cell pellets was then prepared by mixing 106 MSCs with 1 mg of either type of PTHrP-loaded microspheres, followed by centrifugation. PTHrP-free uncapped microparticles were used as the control. MSC pellets were cultured with TGF- 3-encriched chondrogenic medium βfor 4 weeks. We found that PTHrP-loaded ester-microspheres promoted gene expression of type II collagen (a chondrogenic marker) whose level remained similar in the other two groups. With the delayed PTHrP release, however, acid-terminated microspheres significantly reduced synthesis of hypertrophic markers (type X collagen and MMP-13) by chondrogenically differentiated MSCs at both gene and protein levels. In summary, our findings suggest that delayed PTHrP treatment during MSC chondrogenesis can effectively suppress hypertrophy of the cells without compromising their chondrogenic potential. This work not only substantiates the importance of PTHrP release profile in modulating chondrocyte hypertrophy, but represents significant advancement toward our ultimate goal of achieving stable MSC chondrogenesis.Funding Source: PSC-CUNY Award #61584-00 49ETHICAL, LEGAL AND SOCIAL ISSUES; EDUCATION AND OUTREACHF-3074BONE MARROW STROMAL CELLS AND THEIR STEMNESS PARADIGMGutierrez, Maria L - Department of Morphology, Pontificia Universidad Javeriana, Bogota, Colombia Calixto, Camilo - Morphology, Pontificia Universidad Javeriana, Bogota, Colombia Gomez, Ana - Morphology, Pontificia Universidad Javeriana, Bogota, ColombiaThe prevailing definition of a stem cell is a cell with unlimited self-renewal and potential to produce highly differentiated cells. However, after decades of debate stem cell definition remains argumentative. Even though today claimed stem cells are widely used in the clinic, most of the public is unaware of their true nature. Some clinicians promote their indiscriminate use for multiple conditions, without any scientific support. We performed a literature review about adult stem cells, how they became famous for assumed properties and their possible role in therapies. The field of adult stem cells emerged in the 1950s’ in an effort to develop strategies for radiation protection with bone marrow transplantation in humans. In 1988, Friedenstein and Owen described non-hematopoietic bone marrow-derived stromal cells. In 1998, isolation of human blastocysts cell lineages served as a catalyst in this field. To avoid the controversies established by embryonic cell line use, Arnold Caplan et al. isolated human bone marrow stromal cells and named them mesenchymal stem cells (MSCs), suggesting properties not yet proven. Soon the literature regarding MSCs grew strikingly. Since then many claim MSCs have regenerative properties. However, most studies lack cell-tracing demonstrating persistence in tissue after implantation. It is vexing that a supposed stem cell isolated from a particular tissue can regenerate tissue from a different lineage. Understanding of embryonic lineages is fundamental to unravel their possible differentiation potential. Despite increasing numbers in publications and therapies, there is no standardized protocol for their manufacturing, with important implications on quality and function. Studies have shown ex vivo serial replications lead MSCs into replicative senescence, affecting their quality. At present, only therapies for bone marrow transplantation and grafting of bone for treating injuries, which rely on adult stem cells have proven to be successful. In conclusion, although MSCs have regenerative properties in certain scenarios, it is unclear whether they are true stem cells. There is still much to be learned about bone marrow stromal cells for bank purposes and their expansion for therapies before they are used in patients.
581POSTER ABSTRACTSF-3076GLOBAL TRENDS IN STEM CELL RESEARCH SINCE 1998Negoro, Takaharu - Department of Regenerative Medicine Support Promotion Facility, Center for Research Promotion and Support, Fujita Health University, Osaka, Japan Okura, Hanayuki - Department of Regenerative Medicine Support Promotion Facility, Center for Research Promotion and Support, Fujita Health University, Toyoake, Japan Maehata, Midori - Department of Regenerative Medicine, School of Medicine, Fujita Health University, Osaka, Japan Matsuyama, Akifumi - Department of Regenerative Medicine, School of Medicine, Fujita Health University, Toyoake, JapanPreviously, we comprehensively analyzed the trends of research using induced pluripotent stem cells (iPSCs) and reported how it had made a phase transition since 2006 over a 10-year span. Including iPSCs, various stem cells that are ethically acceptable such as embryonic stem cells (ESCs) and somatic stem cells, have been used for the research. It is important for the study design to keep track of trends for whole stem cell research. Here, we broaden the range of analysis of the stem cell study as a whole, including targeting ESC and somatic stem cell studies which were carried out from 1998 through present day to determine which cells have been used for a specific kind of study. To investigate stem cell research trends, we used MeSH term “Stem cells” and “Humans” to search for publications published in the PubMed database from 1998 to 2018. From the reports obtained, secondary information, articles without abstract, or articles not written in English were excluded. Over 50,000 original articles were obtained and tagged for 7 stem cell types used and 19 research targeted disease areas, using MeSH term. The articles were also classified by country based on the first authors’ affiliations. When classified data were categorized by country, the United States was top-ranked by the total number of articels, followed by China, Japan, Germany and United Kingdom. In regard to stem cell-type analysis, the number of articles for ESC has increased from 2006. Comparatively, iPSC research has increased from 2010 and has continued to grow, surpassing the number of studies for ESC in 2016. Although the number of articles for multipotent stem cells began to increase around 2005, it has gradually declined after reaching a maximum of 300 studies in 2012. Regarding the number of articles using each stem cell-type, the United States was always ranked in first place, but the second place varied by country-specific characteristics. In addition, by using the accumulated data, we analyzed the focused disease areas of each country. This analysis revealed that ESC (since 2006) and iPSC (since 2010) have been the leading engine in facilitating and promoting various research for diseases.Funding Source: This study was supported by the Highway Program for Realization of Regenerative Medicine of The Japan Agency for Medical Research and Development (AMED) under Grant Number JP18bm0504009.F-3078PUBLIC ATTITUDES IN THE UNITED STATES TOWARDS HUMAN-ANIMAL CHIMERIC EMBRYO RESEARCH USING HUMAN INDUCED PLURIPOTENT STEM CELLS TO GENERATE HUMAN ORGANS FOR TRANSPLANTATIONLow, Walter C - Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA Shen, Francis - Law School and Graduate Program on Neuroscience, University of Minnesota, Minneapolis, MN, USA Brown, Jennifer - Graduate Program in Neuroscience, Law School, University of Minnesota, Minneapolis, MN, USA Ruiz-Estevez, Mercedes - Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA Voth, Joseph - Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA Sawai, Tsutomu - 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, Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto, Japan Crane, Andrew - Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USAPublic attitudes are a contributing factor in determining how stem cell research is funded and governed. Yet research remains very limited on whether the American public supports the creation of human–animal chimeric embryos (HACEs) for basic research, drug discovery, and organ transplantation. To fill this gap and robustly assess American public attitudes on HACE research, we conducted a national survey of Americans, replicating an approach pioneered in a 2017 study of Japanese public attitudes on HACE research using iPS cells. Our new data suggest that the American public strongly supports HACE research. But we also find that this support is mediated by political ideology, religious affiliation, and the organ to which the human cells contribute. The study was conducted from April to August 2018. 328 survey participants, from 40 different states, were recruited via Amazon’s Mechanical Turk service to take a survey hosted on the Qualtrics online platform. The survey asked a series of questions to elicit participant attitudes related to HACE research. Notably, participants were asked a multi-part question to assess their support for each of several steps required for the creation of HACEs. Overall, nearly 75% of the participants reported a willingness to provide their own cells for blastocyst complementation, and over 80% of the participants felt it socially acceptable to allow for iPS cells to be injected into swine embryos. Further analysis revealed additional important trends. First, while almost 75% of the respondents accepted growing livers in swine, less than 50% of participants supported growing brain cells and less than 40% supported growing
582POSTER ABSTRACTShuman cells in sperm/ovum. Second, the majority of both non-religious and religious participants support human-animal chimera research. Over 90% of non-religious participants, and about 70% of religious participants, support at least some steps toward creating HACEs. These findings are timely because the NIH is currently reviewing its funding moratorium on HACE research. Given the important role of public attitudes on science policy, the results of our study will contribute to on-going debate in this area.Funding Source: University of Minnesota Office of the Provost Internal FundingCLINICAL TRIALS AND REGENERATIVE MEDICINE INTERVENTIONSF-3080EFFECTS OF TYPE I COLLAGEN ON THE GROWTH OF HUMAN DENTAL PULP STEM CELLS IN A XENOGENEIC SERUM-FREE MEDIUM FOR THE ESTABLISHMENT OF A PRACTICAL CULTURE METHODMochizuki, Mai - Life Science Dentistry, The Nippon Dental University School of Life Dentistry, Tokyo, Japan Nakahara, Taka - Developmental and Regenerative Dentistry, The Nippon Dental University School of Life Dentistry, Tokyo, JapanHuman dental pulp stem cells (DPSCs) are useful tools in regenerative medicine. We recently reported that xenogeneic serum-free culture (XFC) is preferable for the clinical applications of DPSCs because XFC prevents contamination with pathogens and can be used to obtain a large number of DPSCs in a short period of time. However, it was also found that the XFC cells develop a multilayered structure upon reaching over-confluence, and this abnormal change induces apoptosis/cell death resulting in the reduction of the number of cells. The aim of this study was to establish an appropriate XFC condition in order to suppress the unfavorable death of DPSCs. We hypothesized that over-production of type I collagen (COL1), a major part of the extracellular matrix, contributes to the formation of an abnormal multilayered structure by DPSCs. We first confirmed that the XFC cells abundantly produced COL1 protein upon reaching over-confluence. To investigate the effects of COL1 on XFC, the culture dishes were pre-coated with COL1 protein. Upon seeding the cells, the COL1-XFC cells adhered to the culture dishes significantly earlier than those cultured in fibronectin- or non-coated dishes. On the other hand, the DPSCs cultured in a serum-containing medium showed no significant difference with the different types of culture dishes. Moreover, the COL1-XFC cells showed higher growth than those cultured in fibronectin- or non-coated dishes. Interestingly, although the COL1-XFC cells formed a proper stratified structure upon over-confluence, the cells did not show apoptosis and reduction in their number. Collectively, these findings suggest that COL1 secreted by the XFC cells is involved in the stratified/multilayered formation, and that pre-coating of culture dishes with COL1 could prevent apoptosis/cell death within the stratified XFC. Therefore, the use of COL1-precoated dishes facilitates an effective large-scale expansion of DPSCs under XFC conditions. These findings might be useful in the establishment of a safe and highly predictive xenogeneic serum-free culture method for handling DPSCs.F-3082ALLOGENEIC CORD BLOOD CELL THERAPY COMBINED WITH ERYTHROPOIETIN IN CHILDREN WITH CEREBRAL PALSY: A RANDOMIZED PLACEBO CONTROLLED TRIALKim, MinYoung - Rehabilitation Medicine/CHA Bundang Medical Center, CHA University College of Medicine, Seongnam, Korea Cho, Kye Hee - Rehabilitation Medicine, CHA University College of Medicine, Seongnam, Korea Min, Kyunghoon - Rehabilitation Medicine, CHA University College of Medicine, Seongnam, Korea Suh, Mi Ri - Rehabilitation Medicine, CHA University College of Medicine, Seongnam, KoreaRecently, stem cell therapy has been highlighted as a new treatment option for cerebral palsy (CP). In our previous study, children with CP treated by allogeneic UCB with erythropoietin (EPO) showed higher improvements in motor and cognitive aspects, yet it lacked to identify individual and synergistic efficacies of UCB and EPO. This factorial designed study aimed to identify individual and synergistic efficacies of UCB and EPO in children with CP. Children with 1) a diagnosis of CP, 2) age between 10 months and 6 years, 3) appropriate UCB units, 4) written informed consents from parents were included as study candidates. Participants were randomly assigned into four groups: A) UCB + EPO, B) UCB + placebo EPO (pEPO), C) placebo UCB (pUCB) + EPO), and D) pUCB + pEPO groups. Allogeneic UCB units were selected including at least 3 ´ 107/kg total nucleated cells, matched for at least 4 of 6 human leukocyte antigen (HLA) types A, B, and DRB1. A single infusion of UCB or pUCB was delivered intravenously and 500 IU/kg of EPO or pEPO were injected subcutaneously for 6 times every 3 days. Group A and B received oral cyclosporine. Primary outcomes such as gross motor function measure (GMFM), gross motor performance measure (GMPM), Bayley scales of infant development II (BSID-II) were assessed at baseline, 1, 3, 6 and 12 months post intervention. Baseline and post-intervention MRI and PET/CT were also acquired. All adverse events were monitored during 12 months. Eighty-eight children with CP were included as final subjects in this study (n=22, 24, 20, and 20 for Group A, B, C, and D, respectively). There were no significant differences of baseline characteristics among four groups. Group A showed meaningful improvement in the ratio of GMPM change at 12 months post-therapy compared to group D (p=0.021). Most of the parameters in four groups showed improvements in primary outcomes, although the changes were not significant. More HLA-matched UCB presented better
583POSTER ABSTRACTSenhancement in change of GMFM at 1 month (p=0.036) and 3 months (p=0.050) post-intervention. Ten serious adverse events were reported, although these cases were all resolved and the distribution of events did not differ among four groups. These results suggest that UCB therapy is safe and effective treatment for children with CP, and its combination with EPO can bring more synergistic effects.Funding Source: This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI13C1204).F-3084STRATEGIES TO IDENTIFY MESENCHYMAL STEM CELLS IN FRESH HUMAN BONE MARROW ASPIRATE CONCENTRATE LACK CONSENSUS AT THE SINGLE CELL TRANSCRIPTOME LEVELRuoss, Severin - Department of Orthopaedic Surgery, University of California, San Diego (UCSD), La Jolla, CA, USA Walker, James - Department of Orthopaedic Surgery, University of California San Diego (UCSD), La Jolla, CA, USA Paez, Conner - Department of Orthopaedic Surgery, University of California San Diego (UCSD), La Jolla, CA, USA Nasamran, Chanond - Department of Medicine, University of California San Diego (UCSD), La Jolla, CA, USA Fisch, Kathleen - Department of Medicine, University of California San Diego (UCSD), La Jolla, CA, USA Ahmed, Sonya - Department of Orthopaedic Surgery, University of California San Diego (UCSD), La Jolla, CA, USA Ward, Samuel - Department of Orthopaedic Surgery, University of California San Diego (UCSD), La Jolla, CA, USAGiven the current regulatory landscape in the United States, the most convenient and applicable use of mesenchymal stem cells (MSCs) as a therapeutic agent to treat orthopedic conditions involves aspiration and re-injection of autologous bone marrow aspirate concentrate (BMAC) into the target site within the same surgical procedure. As MSCs are suggested to be the primary active compound of BMAC, identification and quantification of MSCs are prerequisites to define necessary BMAC doses and dose-dependent outcomes. Current strategies to identify MSCs in human BMAC have been established in cultured cell populations. However, as both surface marker and gene expression change in these cells upon culture, this study investigated whether the current strategies are well-suited to identify non-cultured MSCs in a source that is of direct clinical relevance, i.e. fresh BMAC. Running single cell RNA sequencing (scRNA-seq) on nine orthopedic patients’ BMAC, we hypothesized overlap of “MSCs” identified by different strategies is relatively low. The proposed gold standard (according to the International Society for Cellular Therapy, ISCT) identified zero cells since fresh BMAC-MSCs lack expression of CD90. A few CD271+ cells were detected, but these cells clustered with B cells and monocytes. Only 116 of 386 genes that were previously reported to be expressed by both cultured and non-cultured MSCs were detected in our patient BMAC samples. 35 of 12850 BMAC cells expressed at least 9 of these 116 genes and 66% of these cells were not identified as MSCs by any of the other strategies. As a control, we pooled cultured MSCs with the patient BMAC samples and all strategies identified the cultured MSC cluster, with the ISCT guidelines being the best (1354 of 2978 cultured MSCs detected, no detections of other cells), and CD271+ being the worst strategy (14 of 2978 cultured MSCs detected, 9 of 23 CD271+ cells clustered with patient B cells and monocytes). In conclusion, novel strategies to identify MSCs in fresh BMAC are urgently needed so that orthopedic researchers can design meaningful clinical trials using autologous, minimally manipulated BMAC injections, and investigate dose-response relationships.F-3086INTRATHECAL INJECTIONS OF AUTOLOGOUS MESENCHYMAL STEM CELL-DERIVED NEURAL PROGENITORS IN PATIENTS WITH PROGRESSIVE MULTIPLE SCLEROSIS: RESULTS FROM PHASE I/II TRIALSHarris, Violaine K - Regenerative Neurology, Tisch MS Research Center of New York, NY, USA Stark, James - Clinical Research, Tisch MS Research Center of New York, NY, USA Yang, Sophia - Clinical Research, Tisch MS Research Center of New York, NY, USA Clague, Madison - Clinical Research, Tisch MS Research Center of New York, NY, USA Sadiq, Saud - Regenerative Neurology, Tisch MS Research Center of New York, NY, USAMSC-NPs are an autologous bone marrow-derived population of cells currently under investigation as a novel MS treatment targeting CNS repair. Preclinical evidence suggests that MSC-NPs function through immunoregulatory/trophic mechanisms. The open-label phase I clinical trial in 20 patients with progressive MS demonstrated safety and tolerability of the treatment, and was associated with functional neurological improvement, particularly in ambulatory patients. Whether or not these improvements are sustained and quantifiable against a placebo control remains unknown. The objective of this study is to evaluate therapeutic efficacy of repeated dosing of intrathecal mesenchymal stem cell-derived neural progenitor (IT-MSC-NP) treatment in patients with progressive MS. Bone marrow MSCs were isolated, expanded, and cultured with neural progenitor media to generate MSC-NPs. In the phase I trial, IT-MSC-NPs were injected with 3 doses of up to 10 million cells every 3 months with follow-up EDSS assessments performed 12 and 30 months following the start of the treatment. The phase II trial is a double-blind, placebo-controlled, randomized crossover study in 50 patients with progressive MS, consisting of 6 doses of IT-MSC-NPs every 2 months. Eight of the 20 subjects in the phase I trial showed EDSS improvements (range 0.5 to 3.5 point improvement) after 12 months. The 30 month follow-up in 18 out of 20 subjects demonstrated that 7 subjects (39%) showed
584POSTER ABSTRACTScontinued sustained improvement in EDSS. One subject who had previously demonstrated 1.0 improvement, demonstrated slight worsening in year 2. In the remaining subjects, 33% had continued stable EDSS, and 22% showed continued disease progression. These results suggest that additional treatments may be required to sustain the full effect of IT-MSC-NP treatment. A phase II trial is underway to investigate the safety and efficacy of 6 separate IT-MSC-NP treatments compared to a placebo IT-saline control. IT-MSC-NP therapy is a promising regenerative therapy for progressive MS.GERMLINE, EARLY EMBRYO AND TOTIPOTENCYF-3088CULTURE AND ANALYSIS OF HUMAN SPERMATOGONIAL STEM CELLSThompson, Merlin - Department of Biology, San Diego State University, La Mesa, CA, USA Song, Hye-Wong - Department of Reproductive Sciences, University of California: San Diego, La Jolla, CA, USA Tan, Kun - Department of Reproductive Sciences, University of California: San Diego, La Jolla, CA, USA Wilkinson, Miles - Department of Reproductive Sciences, University of California: San Diego, La Jolla, CA, USASpermatogonial Stem Cells (SSCs) are essential for the generation of sperm and have therapeutic potential. In mice, it has been shown that SSC transplants can rescue fertility in infertile males, providing a possible future therapeutic option in humans. “SSC therapy” has potential therapeutic value for various forms of male infertility (which afflicts >100 million men world-wide), including as a means to restore fertility to pre-pubescent males that have been rendered infertile by chemotherapy. A major bottleneck limiting SSC therapy is the lack of reproducible methods to culture and enrich for human SSCs. Here, we report on our progress on this topic using human testes biopsies obtained from fertile donors. In organ culture, we observed a proliferative expansion of spermatogonia, including cells expressing SSC markers, and dramatic loss of more differentiated germ cells. Cultured dissociated germ cells exhibited a similar pattern of marker expression; clusters of proliferative cells were also consistently observed. FACS analysis with the human SSC marker, SSEA4, revealed that both culture conditions increased the number of SSEA4+ cells, confirming SSC expansion. Lentivirus infection with a shRNA against the germ cell gene, RHOXF2, successfully depleted RHOXF2 expression, demonstrating our ability to manipulate gene expression in these SSC cultures. To define the transcriptome of enriched SSCs cultured under different conditions, we have employed RNAseq analysis. By comparison with the transcriptomes of freshly isolated primitive spermatogonia (highly enriched for SSCs) and differentiating spermatogonia, each purified with specific antibodies, we are comparing how different culture conditions enrich for human SSCs. Our focus is on newly described stage-specific spermatogonial markers defined by our recent single-cell RNA sequencing analysis of neonatal and adult testes. Our study reveals conditions that allow for the survival and expansion of human SSCs and it provides information on growth factors and cell signaling pathways that drive the survival and expansion of SSCs for future clinical applications.F-3090MESENCHYMAL STEM CELLS PROTECT TESTICULAR SPERMATOGONIAL STEM CELL NICHE IN VITROOnen, Selin - Hacettepe University Institute of Health Sciences Department of Stem Cell Sciences and Atilim University Faculty of Medicine Department of Medical Biology, Hacettepe University Institute of Health Sciences Department of Stem Cell Sciences and Atilim University, Ankara, Turkey Korkusuz, Petek - Department of Histology and Embryology, Hacettepe University Faculty of Medicine, Ankara, Turkey Kose, Sevil - Atilim University Faculty of Medicine Department of Medical Biology, Atilim University Faculty of Medicine Department of Medical Biology, Ankara, TurkeyIn childhood cancers, 46% infertility rate has seen in male patients because of the damage in germ cells during chemo or radiotherapy. In males, because the spermatogenesis starts with puberty, it is not possible to collect mature sperms from children prior to cancer therapy. To be able to provide sustainability of the fertility, viability of spermatogonial stem cells (SSC) should be provided in in vitro conditions. Normally, SSCs locate in a very complex, physical and chemical 3D niche. Therefore, it is not easy to provide the longterm viability of the SSCs by conventional cell culture methods. Multi-potent, adult and somatic mesenchymal stem cells (MSC) support several niches due to their immune regulator, proliferative and anti-apoptotic characteristic. Moreover, MSCs coming from the same origin with the Sertoli Cells which are the cells giving chemical and physical support tod the SSC homeostasis in testis. Therefore, it is hypothesized that MSC secretions ensure the viability of SSCs by supporting the Sertoli Cells. With this hypothesis, MSCs isolated from bone marrow (BM) of the mice and characterised. Then, the cells were co-cultured with the testes isolated from newborn mice for 1, 2, 4 and 6 week time periods. As control group, the testes which were cultured alone are used. After end of each period, the testes were taken out, then quantitative histomorphometric analysis was performed on HE, MT and PAS stained tissue sections under light microscope according to tubule diameter , number of living/death cells and SSCs. The SSCs have been quantitatively evaluated by flow cytometric and immune fluorescent (IF) labelling. The integrity of the seminiferous tubules in co-cultured testis group exhibited a better sustainability in comparison to the control group (p<0.05). A decrease was noted in the apoptotic index in MSC co-cultured testes compared to the control group according to TUNEL assay (p<0.05). The number of SSCs in co-culture group increased according to IF labelling. Thus, it is concluded that the co-culture with BM-MSCs contribute the protection of the
585POSTER ABSTRACTSmicroenvironment of SSCs during the ex vivo culture period of the mouse testis. The outputs gathered from this research are important for elucidating the relation between MSCs and the continuity of SSCs in in vitro conditions.Funding Source: The Scientific and Technological Research Council of Turkey -TUB TAK (grant number 218S421) and İHacettepe University Research Fund (grant number TYL-2018-17531) funded the study.F-3092MAINTENANCE OF GENOMIC IMPRINTING IN PLURIPOTENT STEM CELLSLi, Xiajun - School of Life Science and Technology, ShanghaiTech University, Shanghai, China Jiang, Weijun - School of Life Science and Technology, ShanghaiTech University, Shanghai, China Liu, Yuhan - School of Life Science and Technology, ShanghaiTech University, Shanghai, China Chen, Fenghua - School of Life Science and Technology, ShanghaiTech University, Shanghai, ChinaGenomic imprinting is essential in mammals. Dysregulation of genomic imprinting causes many major human diseases including cancer, diabetes, cardiovascular diseases and neurological disorders. Previously, we and others have shown that genomic imprinting is not stably maintained in pluripotent stem cells, in particular the nuclear transfer-derived embryonic stem (ntES) cells and induced pluripotent stem (iPS) cells. Since loss of genomic imprinting can cause cancer and other human diseases, it is important to find out how genomic imprinting is maintained in the pluripotent stem cells before they can be used for cell-based therapies. We identified a maternal-zygotic effect gene Zfp57 as a key regulator in genomic imprinting. To elucidate the molecular mechanisms for the maintenance of genomic imprinting, we have generated Zfp57 mutant embryonic stem (ES) cells by gene editing and analyzed its functions in ES cells. We found that ZFP57 maintains genomic imprinting at most imprinted regions examined in ES cells, similar to its roles in mouse embryos. We have also applied gene editing to examine the functions of DNA methyltransferases, PGC7, UHRF1 and other related factors that may be involved in maintaining genomic imprinting in ES cells. To test if the maintenance of DNA methylation imprint is a dynamic process in ES cells, we eliminated TET proteins that are known to play important roles in active DNA demethylation. Indeed, DNA methyltransferases, PGC7 and UHRF1 are important for maintaining genomic imprinting in ES cells. Our preliminary results also suggest that genomic imprinting may be dynamically maintained in ES cells that requires ZFP57 and other multiple factors involved in DNA methylation. Dissecting the functions of these proteins in genomic imprinting will shed light on the molecular mechanisms underlying the maintenance of DNA methylation imprint in ES cells. This will help to obtain therapeutically suitable pluripotent stem cells with relatively stable genomic imprinting in the future.Funding Source: This research is partly funded by ShanghaiTech University, Department of Science and Technology of Shanghai Municipal Government (Grant# 18PJ1407700) and Ministry of Science and Technology of China.F-3094TRIM28 HAPLOINSUFFICIENCY IN MOUSE GERMLINE CAUSES INFERTILITY IN ADULT MALETao, Yu - MCDB, University of California, Los Angeles, CA, USA Clark, Amander - MCDB, University of California, Los Angeles, CA, USA Tan, Yao Chang - MCDB, University of California, Los Angeles, CA, USATrim28 is a chromatin regulator required to repress retrotransposons in somatic cells and the germline. A null mutation in Trim28 causes embryonic lethality, with haploinsufficiency leading to complex metastable epigenetic phenotypes in somatic cells of mice and in men. In order to evaluate Trim28 haploinsufficiency in the germline, we created a null mutation at the Trim28 locus in mouse PGCs using Cre-LoxP recombination with Blimp1-Cre. We discovered that Trim28 haploinsufficiency beginning in the PGC stage leads to age-related changes in spermatogonial stem cell self renewal and differentiation. Specifically, we discovered a progressive loss of germ cells in seminiferous tubes of adult male mice starting from 2 months of age. By 4-6 months, the majority of seminiferous tubules were composed of sertoli cells, and devoid of germline cells. A clinical phenotype in men referred to as sertoli cell only syndrome. Fertility testing from 8 weeks of life confirmed that the Trim28 PGC haploinsufficient mice were indeed infertile. Using immunofluorescence we discovered that a Trim28 haploinsufficiency phenotype starting in the PGC stage lead to a loss of SALL4 positive spermatogonial stem cells by 4 months, and this was accompanied by exhaustion of the differentiated cells. Taken together, our data suggests that the acute mutation of TRIM28 in one allele during early PGC development results in problems with balancing spermatogonial stem cell self renewal and differentiation which eventually leads to infertility.CHROMATIN AND EPIGENETICSF-3096RONIN SAFEGUARDS SISTER CHROMATID COHESION IN PLURIPOTENT STEM CELLSRamamoorthy, Mahesh - Huffington Center for Cell-based Research in Parkinson’s Disease, Black Family Stem Cell Institute, Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Brosh, Ran - Huffington Center for Cell-based Research in Parkinson’s Disease, Black Family Stem Cell Institute, Department of Cell, Developmental and Regenerative Biology,
586POSTER ABSTRACTSIcahn School of Medicine at Mount Sinai, New York, NY, USA Dejosez, Marion - Huffington Center for Cell-based Research in Parkinson’s Disease, Black Family Stem Cell Institute, Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Zwaka, Thomas - Huffington Center for Cell-based Research in Parkinson’s Disease, Black Family Stem Cell Institute, Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine, New York, NY, USARecent advances in understanding the genomic architecture and its spatial organization in stem cells have been restricted mostly to coding regions. Despite the abundance of repetitive sequences, such as those found in the centromeric and pericentromeric domains, we do not yet understand the detailed principles governing their structural arrangement. Pluripotent stem cells are characterized by the presence of relatively few voluminous chromocenters that upon differentiation become smaller, condensed and more numerous. Exactly how and why these regions are formed, maintained and change with differentiation is an open question. Here, we report that the stem cell factor, Ronin (Thap11), is essential for the integrity of these nascent chromocenters in pluripotent stem cells and, as a result, for proper cohesion between sister chromatids. Ronin directly binds to both major and minor satellite repeats and is associated with chromocenters. Upon conditional Ronin knockout, chromocenters lose their constitutive heterochromatinsignatures.Theresultingphenotypesrange from an increased number of chromocenters and/or chromocenter shattering to an eventual loss of chromocenters. Thereafter, sister chromatid cohesion deteriorates specifically at centromeric and pericentromeric regions, delaying mitotic progression and promoting segregation errors. Mechanistically, Ronin requires its cofactor, HCF-1, to enable its translocation to the pericentromeric heterochromatin. This allows for the binding of HDAC3, which was previously identified as a regulator of sister chromatid cohesion. Together, these data shed light on the importance of Ronin in early development and prompt us to propose that Ronin configures the unique chromocenters and other heterochromatin structures seen in pluripotency.F-3098UTX/KDM6A MAINTAINS IDENTITY AND FUNCTION OF HUMAN PLURIPOTENT AND NEURAL STEM CELLSSalie, Muneeb - Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Xu, Beisi - Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA Mulvey, Brett - Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Yang, Xiaoyang - Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Matsui, Yurika - Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Fan, Yiping - Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA Peng, Jamy - Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USAEpigenetic mechanisms execute gene expression programs for cell fate decisions and organismal development. UTX/KDM6A is a demethylase of methylated lysine 27 in histone H3 (H3K27me). In humans, mutations in UTX have been linked to the Kabuki Syndrome and myriad cancer types. To fully understand the role of UTX in human development and disease, we aim to elucidate the mechanism by which UTX executes developmental gene regulation during the differentiation of human embryonic stem cells (hESCs) along the neural lineage. We used CRISPR-Cas9 to generate UTX-knockout (KO) hESCs, which lost pluripotency marker expression and had a morphology similar to human neural stem cells (hNSCs). Comparison of marker expression and transcriptomes (by RNA-seq) of UTX-KO cells, wild-type hESCs, hNSCs, and differentiated neurons revealed that UTX-KO cells most resembled hNSCs. Combined chromatin immunoprecipitation-deep sequencing (ChIP-seq) with RNA-seq showed that UTX targets and promotes the expression of pluripotency factors OCT4 and HMGA1 as well as suppressors of neurogenesis NOTCH1, WDR62, KDM2B and DNMT3B. Other UTX target genes modulate the self-renewal property of hNSCs. During directed differentiation toward neurons, UTX-KO hNSCs had markedly reduced neuronal differentiation but instead preferentially differentiated towards the glial lineage. These data suggest that UTX is required for neuronal differentiation and suppressing the glial lineage. We conclude that UTX binds and promotes activators and suppressors of cell lineages and is therefore able to directly regulate self-renewal as well as concomitant specification of cell lineages. Comprehensive chromatin profiling of control and UTX-KO hNSCs discovered an unexpected influence of UTX in chromatin dynamics and gene regulation. We will also report our current effort in using UTX-KO stem cells in a pharmacological screen to suppress stem cell defects.F-3100GLOBAL CHARACTERIZATION OF X CHROMOSOME INACTIVATION IN HUMAN PLURIPOTENT STEM CELLSBar, Shiran - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Hebrew University of Jerusalem, Jerusalem, Israel Seaton, Lev - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Hebrew University, Jerusalem, Israel Weissbein, Uri - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Hebrew University, Jerusalem, Israel Eldar-Geva, Talia - IVF Unit, Division of Obstetrics and Gynecology, Shaare Zedek Medical Center, Jerusalem, Israel Benvenisty, Nissim - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Hebrew University, Jerusalem, Israel
587POSTER ABSTRACTSX chromosome inactivation (XCI) is an essential epigenetic silencing process established during early embryonic development of female mammals, guaranteeing a proper dosage compensation of X-linked genes between males and females. Activation of the lncRNA XIST, which triggers XCI, occurs early during differentiation, therefore human pluripotent stem cells (hPSCs) are potentially a major model for studying this process. While XCI was extensively studied in the mouse, there are significant differences when compared to humans, both in vivo and in vitro. Previous studies demonstrated variable states of XCI in hPSCs, many of them exhibit aberrant XCI patterns also after differentiation, thus impeding the use of these cells in therapeutic applications. These studies however focused on only a limited number of cell lines grown in the same lab. Here, we performed a large-scale characterization of XCI in 785 hPSCs from various sources, using RNA sequencing (RNA Seq) data. The analysis combined inputs on XIST expression, allelic polymorphism quantification and assessing the global expression levels of X-linked genes. These genome-wide analyses provide significantly higher coverage compared to other methods which interrogate individual genes, thereby enabling a sensitive and accurate classification of XCI state. We identify a striking difference between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in their XCI landscape, in which most iPSCs maintain the inactive X of their parental somatic cells, whereas ESCs silence the expression of XIST and upregulate distal regions of the X-chromosome. We suggest this to be the most significant epigenetic difference between ESCs and iPSCs, globally dividing them to distinct groups based on their XCI status. We further show that this erosion is slightly reduced upon differentiation, in a XIST-independent manner. Altogether, we propose a model in which XCI variations in hPSC lines are affected by epigenetic memory of their different parental sources, as well as by dynamic changes occurring during derivation and culture of hPSCs. Collectively, our research may serve as a platform to analyze the status of XCI for the study of human development and disease modeling and to characterize the cells prior to their transplantation. *Under-revision in Cell ReportsF-3102CANCER MUTATIONS TO THE COHESIN COMPLEX IMPAIR TRANSCRIPTIONAL INSULATION IN A MURINE EMBRYONIC STEM CELL MODEL SYSTEMCarico, Zachary M - Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Yimit, Askar - Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Dowen, Jill - Integrative Program in Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAThe Cohesin complex has been implicated in transcriptional regulation through the formation and maintenance of two classes of chromatin loops. Enhancer-promoter loops drive gene expression by bringing active enhancers directly into contact with target promoters, while insulated loops restrict enhancer activity to discrete units of the genome flanked by loop boundary sequences that bind the architectural protein CTCF. In stem cells, insulated loops are essential regulators of genes that encode master transcription factor (TFs), supporting robust expression of pluripotency master TFs and maintaining suppression of developmentally-silenced lineage-specific master TFs. In cancer settings, mutational inactivation of insulated loop boundary elements has been implicated in activation or re-expression of silenced oncogenes. Notably, the genes that encode the subunits of the Cohesin complex are also recurrently mutated across many cancers, but the mechanisms through which this may drive malignancy are not understood. Here, we examine whether these mutations might cause altered cell identity through global interference with insulated loop formation or function. We used CRISPR/Cas9 genome editing to generate murine embryonic stem (ES) cell lines with cancer mutations in endogenous SMC1A and STAG2 Cohesin subunits, and assayed those cells for transcriptional changes using RT-qPCR and RNA-seq. We identified missense mutations in SMC1A and inactivating mutations in STAG2 that reduced transcriptional insulation across numerous insulated loop boundaries. Strikingly, the ES cell transcriptional program was broadly dysregulated, reducing expression of pluripotency-controlling TFs and increasing expression of differentiation TFs. Surprisingly, the SMC1A and STAG2 mutations act through distinct mechanisms to impair insulation: preliminary data suggest that SMC1A mutation reduces binding of Cohesin to chromatin, while STAG2 loss allows for normal binding and localization of Cohesin on chromatin despite reduced capacity for insulation. Together, these results indicate that proper insulated loop function is indispensable for maintenance of stem cell identity, and that cancer-linked mutations to the Cohesin complex impair the ability of cells to properly maintain transcriptional insulation.PLURIPOTENCYF-3104PROTECTING EARLY EMBRYOGENESIS AND PLURIPOTENT STEM CELLS AGAINST GENETIC PARASITES THROUGH A PRIMITIVE IMMUNE SYSTEMSkowronska, Marta - Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Wagner, Ryan - Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Jin, Xiao - Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Dejosez, Marion - Cell, Developmental and Regenerative
588POSTER ABSTRACTSBiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Zwaka, Thomas - Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USARetrotransposons are expressed in early ontogeny and may induce structural rearrangements and mutations that seriously damage the genome of the nascent embryo, resulting in diseases and infertility. At present, cellular protection against retrotransposons is believed to rely solely on a cell autonomous system involving epigenetic silencing. Here, we report that pluripotent cells can sense the retrotransposable load of their neighboring cells and, if it is higher than their own, act to kill and remove them. To determine how cells detect neighbors with more retrotransposon activity, we performed transcriptome analysis; this revealed that the sensing mechanism is based on a dsRNA response that triggers a negative feedback loop. We then used super-resolution microscopy paired with lysosomal activity assays, and found that cells were employing non-professional phagocytosis, most likely induced by NF-kB signaling, to remove their unhealthy, retrotransposon-infested neighbors. Finally, experiments performed in stressed cells revealed that this defense system is conditional, as its activating threshold was subject to change. Our study shifts attention away from solely cell-intrinsic defense strategies towards considering a much more nuanced system in which cells constantly monitor one another for endogenous retrotransposon-related health. Our line of investigation will provide new targets for genetic diagnosis and interventions targeting pregnancy loss and infertility.F-3106GENE EXPRESSION ANALYSIS OF PAPIO ANUBIS STEM CELLS REVEALS UNIQUE METABOLIC STATE THAT APPROXIMATES HUMAN STEM CELL METABOLISMMahlke, Megan - Department of Biology, University of Texas at San Antonio, TX, USA Chaudhari, Shital - Biology, University of Texas at San Antonio, TX, USA Cheng, Keren - Biology, University of Texas at San Antonio, TX, USA McCarrey, John - Biology, University of Texas at San Antonio, TX, USA Navara, Christopher - Biology, University of Texas at San Antonio, TX, USAA major hurdle impeding clinical implementation of stem cell-based therapies is the failure to translate therapeutic approaches from typical model organisms to human patients. Validating non-human primate (NHP) models for use in stem cell-based therapies has the potential to increase successful translation of therapies to the clinic by offering a model that better represents humans. To that end, we performed RNA-Seq analysis on embryonic and induced pluripotent stem cells derived from Papio anubis (Olive baboon) and compared the data to human and mouse embryonic and induced pluripotent RNA-Seq datasets. Overall, P. anubis pluripotent cells were a closer representation of human pluripotent cells than were mouse pluripotent cells. Gene ontology analysis revealed that the most highly dysregulated genes between baboons and humans and between mice and humans were related to metabolism. We found that the oxidative phosphorylation pathway was relatively downregulated in baboon stem cells when compared to human stem cells, but significantly upregulated in mouse stem cells when compared to human stem cells. Notably, in mouse pluripotent stem cells, mitochondrial genes for subunits of the NADH+ dehydrogenase complex are actively transcribed while being suppressed in human and baboon pluripotent cells. Because a unique metabolic state is critical to maintaining pluripotency, distinct metabolic states between pluripotent cells of different species may diminish translatability of stem cell therapies from typical model organisms to human patients.F-3108THE BCL-2 FAMILY MODULATES STEM CELL IDENTITY BY REGULATING MITOCHONDRIAL DYNAMICSJoshi, Piyush - Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA Rasmussen, Megan - Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA Bodnya, Caroline - Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA Gama, Vivian - Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USAOver the past decades, the function of the mitochondria has evolved from being a mere powerhouse to a complex signaling hub. Mitochondrial dynamics, which includes fusion and fission, have been implicated in many diseases including aging, neurodegeneration, and tumorigenesis. Recent studies from our laboratory suggest that proteins of the BCL-2 (B-cell lymphoma 2) family, known as regulators of cell death, can also modulate mitochondrial dynamics in human pluripotent stem cells (hPSCs). The myeloid cell leukemia 1 (MCL-1) protein, a known anti-apoptotic protein of the BCL-2 family, localizes not only to the outer mitochondrial membrane, where it exerts its anti-apoptotic function, but also to the mitochondrial matrix, where its function is not clear. Inhibition of MCL-1 in hPSCs via a small molecule also leads to loss of pluripotency markers (NANOG and OCT4), independently from its anti-apoptotic function. In addition, MCL-1 inhibition results in a fused and elongated mitochondrial network, which suggests that MCL-1 is crucial in maintaining fission in hPSCs. We will also present our preliminary studies describing how other proteins of the BCL-2 family, known to interact with MCL-1, are also capable of modulating mitochondrial dynamics in homeostatic and stress conditions. Overall, our data suggest that BCL-2 family plays a major role in stem cell identity and this is accomplished by the regulation of mitochondrial dynamics.
589POSTER ABSTRACTSFunding Source: 5 T32 ES 7028-44 (PJ), 1 R35 GM128915-01 NIGMS (VG), NIH-R00 (NCI) grant (4R00CA178190 to V.G). The Vanderbilt Cell Imaging Shared Resource is supported by NIH grants 1S10OD012324-01 and 1S10OD021630-01.F-3110PODOCALYXIN-LIKE PROTEIN 1 REGULATES HUMAN PLURIPOTENT STEM CELL SELF-RENEWAL THROUGH CHOLESTEROL BIOSYNTHESIS PATHWAYChen, Wei-Ju - Genomics Research Center, Academia Sinica, Taipei, Taiwan Lin, Hsuan - Department of Pediatrics, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan Lu, Jean - Genomics Research Center, Academia Sinica, Taipei, TaiwanThe transmembrane signals maintain the pluripotency of human pluripotent stem cells (hPSCs) are rarely investigated. A transmembrane glycoprotein, podocalyxin-like protein 1 (PODXL, also called PCLP1, Gp200/GCTM-2, MEP21, and Thrombomucin), aside from its well-known roles in tumor malignancy, very little is known about its functions in hPSCs. Here, we report that the downregulation of PODXL in undifferentiated hPSCs significantly altered the selfrenewal and survival abilities and lead to a decrease in c-MYC and telomerase proteins. Of note, the formation of induced pluripotent stem cells (iPSCs) colonies was hampered upon the knockdown of PODXL. Consistently, overexpression of PODXL promoted hPSC selfrenewal, the expressions of c-MYC and telomerase, and iPSC formation. In a transcriptomic analysis, forcing PODXL expression revealed that PODXL can activate HMGCR expression and thus control the cholesterol biosynthesis. Meanwhile, we found that PODXL also regulates master regulators of cholesterol synthesis genes, SREBP1/SREBP2, in accordance with alteration of c-MYC expression. Importantly, compared to fibroblasts, hPSCs are more sensitive to cholesterol synthesis dysregulation blocked by chemical inhibitors statin and AY9944, and lipid raft disruption by M CD treatment which lead to impairments of βself-renewal and survival abilities. Of note, cholesterol can fully restore PODXL knockdown-mediated pluripotency loss and apoptosis phenotype in a dose-dependent manner. Cholesterol also notably restores TERT, c-MYC, and HMGCR protein expression. Furthermore, we found PODXL regulated the sizes of lipid raft and may transmit signals through controlling CD49B (integrin 2) mobilization into lipid rafts. The knockdown of αCD49B blocks ESC renewal. Our data highlight the important roles of PODXL in controlling cholesterol metabolism to achieve hPSC selfrenewal.F-3112HUMAN NAÏVE PLURIPOTENT STEM CELLS ACQUIRE CANCER-RELATED MUTATIONS DURING THEIR ESTABLISHMENT FROM PRIMED CELLSAvior, Yishai - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel Eggan, Kevin - Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Benvenisty, Nissim - The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, IsraelHuman pluripotent stem cells (hPSCs) have been previously shown to harbor several types of genetic aberrations that accumulate during culturing. These aberrations include chromosomal abnormalities and copy number variations, which can affect the tumorigenic potential of the cells. More recently, point-mutations in the gene coding for the p53 tumor suppressor (TP53) have been found in early passaged hPSCs using whole exome sequencing (WES) And late passaged cells using RNA sequencing. These mutations were shown to gradually take over the culture, suggesting they provide a growth advantage in vitro. However, it remained unclear if in other cancer-related mutations appear in hPSC cultures. We therefore established a new strategy to compare early and late passaged hPSCs to identify mutations listed in the census Catalogue of Somatic Mutations in Cancer (COSMIC Census). We found that alongside mutations in TP53, recurrent mutations appear in at least 22 other cancer-related verified genes and that these mutations appear in both embryonic and induced-pluripotent stem cells. We then looked for mutations in these genes in both primed hPSCs and their naïve derivates from eight independent studies. Naïve cells were found to harbor 4-times more cancer-related mutations on average, significantly more than their primed counterparts. We also found indications that these mutations accumulate throughout the naïve-state induction process, suggesting this procedure imposes a substantial selective pressure. Together, our results suggest that hPSCs gain validated cancer-related mutations and that selective pressures could enhance this accumulation. These mutations should be taken into consideration in future applications, specifically in clinical ones.F-3114METABOLIC NETWORK-CENTRIC ANALYSIS OF SINGLE CELL RNA-SEQ DATA REVEALS NOVEL DYNAMIC CHANGES IN ENZYME EXPRESSION AND NUTRIENT UTILIZATION IN IMPLANTATION DEVELOPMENTWang, Yuliang - Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA
590POSTER ABSTRACTSRecent studies have revealed that changes in metabolism regulate cell fate decisions in a wide range of developmental processes. However, these metabolic changes are often measured in heterogeneous cell populations, potentially masking cell type-specific and rich dynamic changes. Single cell RNA-seq have been extensively used to study cell fate decisions, but few have been analyzed from a metabolic perspective. The massive amount of single cell RNA-seq data of developmental processes is both a significantly untapped resource and unprecedented opportunity to study metabolism with high cellular and temporal resolution. Using monkey pre- and post-implantation epiblast development as a model system, we combined single cell RNA-seq data with genome-scale metabolic network models to systematically reveal dramatic metabolic dynamics in this early developmental stage. Our predictions recapitulated many known metabolic shifts, such as changes in glycolysis, fatty acid oxidation, fatty acid and lipid synthesis, OXPHOS, vitamin C/L-proline ratio and the effects of aKG in naïve and primed pluripotency (corresponding to pre- and post-implantation, respectively). Furthermore, we further revealed many novel metabolic shifts, such as anti-oxidant response, amino acid utilization, co-factor and mineral uptake. We also identified novel transient dynamics of metabolic enzymes that would be missed by bulk gene expression assays, and discovered differential temporal trajectories of isozymes localized in the mitochondrial vs. cytoplasmic compartments. By analyzing trophoblast development in parallel, we also identified unique enzyme dynamics and nutrient requirements in each lineage. These computational predictions can potentially lead to culture medium metabolically optimized to maintain naïve pluripotent stem cells or trophoblast stem cells. The computational framework is generally applicable to many other developmental processes where single cell RNA-seq data is available, such as hematopoiesis, neural stem cell development etc.Funding Source: Institute for Stem Cells and Regenerative Medicine, University of WashingtonPLURIPOTENT STEM CELL DIFFERENTIATIONF-3122DEVELOPMENT OF AN EVALUATION SYSTEM THAT CAN PREDICT THE OSTEOGENIC POTENTIAL OF HUMAN MESENCHYMAL STEM CELLS EASILY AND PROMPTLYSawada, Rumi - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Kono, Ken - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Tanaka, Kazusa - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Sato, Yoji - Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Kawasaki, Japan Kidoaki, Satoru - Institute for Materials Chemistry and Engineering, Kushu University, Fukuoka, JapanHuman mesenchymal stem cells (hMSC) are promising candidate cells as raw materials for regenerative medical products. When hMSC are differentiated to produce target products, selection of hMSC strain with high differentiation potential is important, and development of an efficient screening method is required. In this study, focusing on the osteogenic potential of hMSC, we aimed to develop a system capable of evaluating bone differentiation ability at an early stage in bone differentiation induction which takes about 3 weeks. We investigated the relationship between the mRNA expression levels of 5 genes widely used as markers of bone differentiation and the actual bone differentiation potential of each hMSC strain.10 lots of hMSC derived from bone marrow of different donors were induced to osteogenic differentiate and the each degree of bone differentiation of those hMSCs was determined by measurement of calcium deposits by Alizarin Red staining. These 10 lots of hMSC contained 5 lots each of a strain with high bone differentiation ability and a strain with low. In order to investigate whether each osteogenic potential can be predicted according to the gene expression level, mRNA expression levels of five genes (Runx2, Alkaline phosphatase (ALP), Type I Collagen (COL1), Osteopontin (OPN), Osteocalcin (OCN)) which are often used as markers of osteogenic differentiation were measured before and one week after induction of bone differentiation. We found that mRNA expression level of ALP was significantly higher in hMSCs with higher bone differentiation potential than in hMSCs with lower, both before induction of bone differentiation and one week after induction. On the other hand, the mRNA expression levels of the other four genes (Runx2, COL1, OPN, OCN) were not related to the bone differentiation potential of hMSC. ALP mRNA expression level is related to the osteogenic potential of hMSC. Its potential can be predicted by the expression level of ALP even before induction of bone differentiation. By measuring mRNA expression of ALP, it was found that osteogenic differentiation ability of hMSC can be predicted at the latest at one week in bone differentiation induction which takes about 3 weeks.F-3124MATURATION AND ACTIVITY-DEPENDENT RESISTANCE OF HUMAN IPSC-DERIVED NEURONS TO PRO-APOPTOTIC STIMULIWilkens, Ruven - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI) and German Cancer Research Center (DKFZ), Mannheim, Germany Kleinsimlinghaus, Karolina - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Bohl, Bettina - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Ladewig, Julia - Hector Institute for Translational Brain
591POSTER ABSTRACTSResearch (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, Germany Koch, Philipp - Hector Institute for Translational Brain Research (HITBR), Central Institute of Mental Health (ZI), University of Heidelberg/ Medical Faculty Mannheim and German Cancer Research Center (DKFZ), Mannheim, GermanyNeurons represent a highly specialized cell population of the central and peripheral nervous system responsible for communicating information throughout the body. In humans, most neurons are born during embryonic development and persist throughout the entire life span of an individual. Presumably, they rely on preemptive strategies to protect against stress and accidental cell death. During early development neurons are produced in excess numbers which are later reduced during the establishment of mature neuronal circuitries. Thus, cell death pathways should be essential at earlier stages of neuronal network formation to enable trimming of surplus connections while they might become dispensable or even detrimental once specialized mature neuronal networks have been established. We set out to decipher the pathways associated with increased resistance of mature human neurons to stress and apoptosis using human induced pluripotent stem cell (iPSC)-derived neuronal cultures. We show that during the time course of maturation, iPSC-derived neurons become increasingly resistant to several types of cellular stressors. This is accompanied by a global downregulation of initiator and effector caspases and a shift in the balance of pro- and anti-apoptotic proteins. Contemporaneously, the neurons possess an increased activation of the AKT pro-survival pathway acting upstream of the identified changes. This activation of AKT can be modified by interfering with neuronal activity indicating that recurring synaptic stimulation of neurons within a complex network reinforces their survival and boosts network resilience.Funding Source: The authors acknowledge the generous support of the Hector Stiftung II.F-3126DYNAMICS OF CELL FATE PATTERNING IN LIVE HUMAN EMBRYONIC STEM CELLSWolff, Samuel C - Genetics, University of North Carolina at Chapel Hill, NC, USA Beltran, Adriana - Human Pluripotent Stem Cell Core, UNC-Chapel Hill, NC, USA Smiddy, Nicole - Chemistry, UNC-Chapel Hill, NC, USA Kedziora, Katarzyna - Genetics, UNC-Chapel Hill, NC, USA Redick, Margaret - Genetics, UNC-Chapel Hill, NC, USA Daugird, Timothy - Biological and Biomedical Sciences Program, UNC-Chapel Hill, NC, USA Allbritton, Nancy - Biomedical Engineering, UNC-Chapel Hill, NC, USA Purvis, Jeremy - Genetics, UNC-Chapel Hill, NC, USALive-cell reporters of pluripotency transcription factors in human embryonic stem cells (hESCs) allows the study of protein dynamics and the critical role they play in maintaining pluripotency and differentiation. However, generating clonal cell lines expressing fluorescently tagged genes is difficult not only due to the relative inefficiency of homologous recombination, but also to the high sensitivity of hESCs to perturbations, their spontaneous differentiation, and recalcitrance to being cultured as single cells. Historically, most approaches to selecting modified hESCs clones have relied on an iterative process of enriching a population for positively expressing cells (“cell pruning”). This process is tedious, time-consuming, and limits the timely development of new reporter cell lines. Here, we describe the use of microraft array cell sorting technology and high-content imaging to identify and isolate undifferentiated reporter hESC lines. The microraft arrays consist of 12,000 microwells each containing a magnetic releasable cell culture element (“microraft”). The arrays were populated with hESCs targeted through CRISPR-mediated homologous recombination to introduce fluorescent reporters into the endogenous loci of genes of interest. Each microraft cell carrier was rapidly screened for successfully gene-edited cells via fluorescence imaging. Microrafts identified as containing potential clones were gently released from the array and collected with a magnet for expansion of their adhered cells in order to screen them for the correct insertion. This approach was applied to develop endogenous reporters for three core human pluripotency factors: OCT4, SOX2 and NANOG. Using time-lapse imaging to track the real-time expression of these factors in single hESCs, we describe the dynamics of transcription factor patterning in stem cell colonies as a function of cell growth and differentiation.Funding Source: This work was supported by NIH grant DP2-HD091800-01, the W. M. Keck Foundation, and the Loken Stem Cell Fund.F-3128NOVEL CROSSTALK BETWEEN VPS26A AND NOX4 SIGNALING DURING NEUROGENESISChoi, Seon-A - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Korea An, ju-Hyun - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Kyung-Min - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Mun-Hyeong - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Yang, Hae-Jun - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Jeong, Pil-Soo - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea
592POSTER ABSTRACTSCha, Jae-Jin - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Yoon, Seung-Bin - Primate Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup-si, Korea Lee, Seung Hwan - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lim, Kyung-Seob - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Jong-Hee - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Park, Young-Ho - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Song, Bong-Seok - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Sim, Bo-Woong - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Huh, Jae-Won - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Young-Hyun - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Sang-Rae - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Ji-Su - Primate Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup-si, Korea Jin, Yeung Bae - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Sun-Uk - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, KoreaDespite numerous studies on the molecular switches governing the conversion of stemness to differentiation in embryonic stem cells (ESCs), little is known about the involvement of the retromer complex. Under neural differentiation conditions, Vps26a deficiency (Vps26a-/-) or knockdown suppressed the loss of stemness and subsequent neurogenesis from ESCs or embryonic carcinoma cells, respectively, as evidenced by the long-lasting expression of stemness markers and the slow appearance of neuronal differentiation markers. Interestingly, relatively low reactive oxygen species (ROS) levels were generated during differentiation of Vps26a-/- ESCs, and treatment with an antioxidant or inhibitor of NADPH oxidase (Nox), a family of ROS-generating enzymes, led to restoration of stemness in wild-type cells to the level of Vps26a-/- cells during neurogenesis. Importantly, a novel interaction between Vps26a and Nox4 linked to the activation of ERK1/2 depended highly on ROS levels during neurogenesis, which were strongly suppressed in differentiating Vps26a-/- ESCs. Moreover, inhibition of phosphorylated ERK1/2 (pERK1/2) resulted in decreased ROS and Nox4 levels, indicating the mutual dependency between pERK1/2 and Nox4-derived ROS during neurogenesis. These results suggest that Vps26a regulates stemness by actively cooperating with the Nox4/ROS/ERK1/2 cascade during neurogenesis. Our findings have important implications for understanding the regulation of stemness via crosstalk between the retromer molecule and redox signaling, and may contribute to the development of ESC-based therapeutic strategies for the mass production of target cells.Funding Source: KRIBB Research Initiative Program (KGM4251824) and the Bio and Medical Technology Development Program through the NRF funded by the Ministry of Education, Science and Technology (MEST) (No. 2018M3A9H1023142), Republic of Korea.F-3130A CHEMICALLY-DEFINED AND ANIMAL-ORIGIN FREE CULTURE MEDIUM STEMFIT AS400 IS USEFUL FOR TRILINEAGE DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS FOR CLINICAL APPLICATIONSOgawa, Shimpei - Institute for Innovation, Ajinomoto, Inc.,., Kawasaki, Japan Ito, Kenichiro - Institute for Innovation, Ajinomoto, Inc., Kawasaki, Japan Jessica, Chang - Institute for Innovation, Ajinomoto, Inc., Kawasaki, Japan Kobayashi, Tsuyoshi - Institute for Innovation, Ajinomoto, Inc., Kawasaki, Japan Konishi, Atsushi - Institute for Innovation, Ajinomoto, Inc., Kawasaki, Japan Wagatsuma, Hirotaka - Institute for Innovation, Ajinomoto, Inc., Kawasaki, JapanHuman pluripotent stem cells (hPSCs) such as iPS cells and ES cells are expected to be a valuable source for regenerative medicine. Although it is possible to expand hPSCs under chemically-defined and animal origin-free (AOF) medium, most of differentiation medium contains animal-derived or undefined components such as FBS and B27. Undefined and animal component-containing medium hampers clinical applications of hPSC-derived cells/tissues because of higher risks of viral infection and larger lot-to-lot variation. Therefore, it is necessary to develop a differentiation medium for clinical application without affecting differentiation efficiency. We have developed a novel differentiation nutrient supplement “StemFit AS400” (AS400), which is utilized by addition to basal medium, differentiation factors, and other reagents. All ingredients of AS400 are chemically-defined, and none of the ingredients originate from animal sources. In this study, hPSCs were spontaneously differentiated via embryonic body (EB) formation using our medium containing AS400 for 2 weeks and evaluated trilineage differentiation potential with TaqMan® hPSC ScorecardTM. We found that differentiation medium
593POSTER ABSTRACTScontaining AS400 supported sufficient trilineage differentiation. Furthermore, we succeeded in making neural stem cells (ectoderm), cardiomyocytes (mesoderm), and hepatocyte (endoderm) from hPSCs by using AS400 on defined and xeno-free extracellular matrix-coated dishes with comparable efficiency to conventional medium. These results suggest that AS400 is useful for trilineage differentiation of hPSCs in various cases. This nutrient supplement is expected to promote clinical applications of hPSC-derived target cells/tissues by establishing the chemically-defined and AOF culture systems of both hPSC expansion and differentiation.F-3132HUMAN IPSC-DERIVED M1/M2 MACROPHAGES: NOVEL OFF-THE-SHELF CELL THERAPEUTICS FOR CANCER AND REGENERATIVE MEDICINEPouyanfard, Somayeh - Regenerative Medicine, University of California , San Diego, CA, USA Cruz, Luis - Regenerative Medicine, University of California, San Diego, CA, USA Kaufman, Dan - Regenerative Medicine, University of California, San Diego, CA, USADistinct populations of macrophages show significant contribution to human disease pathogenesis, as well as repair of damaged tissues. Our studies provide a method to routinely generate a large number of macrophages from human induced pluripotent stem cells (hiPSCs). Moreover, these iPSC-derived macrophages can be polarized to specific M1 and M2 phenotypes. Briefly, hematopoietic progenitor cells (HPCs) are derived from undifferentiated hiPSCs using a well-defined serum-free and stromal-free protocol. These HPCs then cultured in stage II conditions in serum-free media supplemented with M-CSF and IL-3. Using these conditions, macrophage progenitor cells (CD14, CD11b, CD36, SIRP- and CD68) are continuously αproduced from a starting cell population of approximately 105 undifferentiated hiPSCs for more than 8 weeks. During this time, we collect 0.5 × 106 – 106 macrophage progenitor cells per well per week. These cells can then be further differentiated in stage III conditions to mature macrophages (hiPSCs-M ) in presence φof M-CSF for 7 days. HiPSCs-M exhibited morphology, φsurface marker expression (CD14, CD11b, CD36, SIRP- αand CD68) and latex bead phagocytic activity that are similar to peripheral blood-derived macrophages (PB-M ). We can φfurther polarize these hiPSCs-M toward M1 pro-inflammatory φmacrophages by culture with LPS and IFN- . M1 macrophages γare characterized by expression of CD80, TNF- , and IL-6. αWe can also produce M2 anti-inflammatory macrophages by culture with IL-4 and IL-13. M2 macrophages are characterized by expression of CD206, CCL17, and CCL22. Both hiPSCs-M φand PB-M expressed high level of CD80 upon stimulation in φpro-inflammatory conditions although the M1 differentiation was more efficient in hiPSCs-M . Conversely, the M2 polarization φof PB-M resulted in more pronounced expression of CD206 φcompared to hiPSCs-M . hiPSCs-M1 expressed high level φof TNF- and IL-6 while hiPSCs-M2 showed elevated level αof CCL17 and CCL22 in culture supernatant. Collectively, these studies provide an efficient system for rapid generation of distinct human macrophage populations, helping to better understand their biology and facilitating their use for cell-based therapy of human diseases.F-3134KUF11 TREATMENT LED TO ENHANCED HEMATOPOIETIC STEM CELL DIFFEREENTIATION FROM URINE CELL- OR BLOOD CELL-DERIVED PLURIPOTENT STEM CELLSCho, Ssang-Goo - Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Korea Kim, Kyeongseok - Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Korea Abdal Dayem, Ahmed - Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Korea Yang, Gwang-Mo - Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, KoreaAutologous urine-derived stem cells (USCs), which can be noninvasively obtained from urines, are thought to be ideal for applications in regenerative medicine. Here, we developed a method for efficient isolation and culture of USCs, which showed enhanced migration and colony forming capacities. We also efficiently produced human USC-derived induced pluripotent stem cells (hU-iPSCs). Efficient maintenance of the undifferentiated status of pluripotent stem cells (PSCs) may be crucial for producing a high-quality cell source that could be successfully applied in stem cell research. Here we tried to screen various natural compounds to find the materials enhancing of the quality of human pluripotent stem cells (hPSCs). Among the tested compounds, treatment of KUF11 showed increases in cell proliferation, cell cycle (S phase), and phosphorylation of extracellular signal–regulated kinases (ERKs) signaling pathway, and survival rate. Of note, KUF-exposed hPSCs showed upregulation in the level of glutathione (GSH) and generation of high population of GSH-high cells when measured by FreSHtracer system. hPSCs treated with KUF11 showed enhanced mesodermal differentiation efficiency, in particular, into hematopoietic stem cells (HSC) differentiation. Taken together, our data afford a novel natural compound possessing high efficiency in enhancing hPSC proliferation, pluripotency, and HSC differentiation that can be applied for production of high-quality hPSC for the clinical applications in therapy and hematology in the future.F-3136EFFICIENT, LARGE-SCALE PRODUCTION AND FUNCTIONAL CHARACTERIZATION OF NOCICEPTORS DERIVED FROM HUMAN PLURIPOTENT STEM CELLSDeng, Tao - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USA
594POSTER ABSTRACTSOrmanoglu, Pinar - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USA Chu, Pei-Hsuan - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USA Tristan, Carlos - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USA Malley, Claire - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USA Austin, Christopher - NCATS, NIH, Rockville, MD, USA Simeonov, Anton - NCATS, NIH, Rockville, MD, USA Singec, Ilyas - Stem Cell Translation Laboratory, NIH/NCATS, Rockville, MD, USADevelopment of new non-addictive analgesics would greatly benefit from directed differentiation of hPSCs into relevant cell types. Here, we devised a highly efficient step-wise protocol that differentiates human pluripotent stem cells (hPSCs) exclusively into nociceptors under fully defined conditions. By manipulating critical cell signaling pathways using small molecule inhibitors, hPSCs were first converted into SOX10+ neural crest stem cells followed by differentiation into bona fide nociceptors. Time-course RNA-Seq analysis (Day 0-28) and immunocytochemistry experiments confirmed that nociceptors expressed typical neuronal markers, transcription factors, neuropeptides, and ion channels. Focusing on pain-relevant receptors expressed by hPSC-derived nociceptors (e.g. P2RX3, opioid receptors), we could demonstrate robust functional activities in multi-electrode array experiments and differential responses to nociceptive stimuli and specific drugs including natural and synthetic opioids. The nociceptor differentiation protocol was then automated by using a robotic cell culture system (CompacT SelecT™) enabling multiple high-throughput projects that require large numbers of cells. In summary, the scalable human nociceptor platform developed here will aid in the discovery of new pain medications as part of the effort to tackle the opioid crisis.Funding Source: NIH Common Fund, NIH HEAL Initiative, NCATS Intramural ResearchF-3138HETEROGENEITY IN THE SURFACE MARKER EXPRESSION AND THE DIFFERENTIATION POTENTIAL OF HAEMATOPOIETIC STEM CELL PROGENITORS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLSThamodaran, Vasanth - Centre for Stem Cell Research, Christian Medical College, Vellore, India Nandy, Krittika - Centre for Stem Cell Research, Christian Medical College, Vellore, India Meharwade, Thulaj - Center for Stem cell Research, Christian Medical College, Vellore, India Palani, Dhavapriya - Centre for Stem Cell Research, Christian Medical College, Vellore, India Srivastava, Alok - Centre for Stem Cell Research and Department of Haematology, Christian Medical College, Vellore, India Velayudhan, Shaji - Department of Haematology and Centre for Stem Cell Research, Christian Medical College, Vellore, IndiaAs human induced pluripotent stem cells (hiPSCs) can be differentiated to haematopoietic stem and progenitor cells (HSPCs) and further to erythroid cells, hiPSCs generated from patients with red cell diseases are valuable tools for studying the pathogenesis of these diseases. Two different types of iPSC derived HSPCs (iPSC-HSCs) with different expression of surface makers, CD34+CD235a+CD41+ and CD34+CD45+, have been reported in the literature, and both have been found to have the potential to differentiate to erythroid cells. We carried out a study to know whether these two types of iPSC-HSCs are generated on different stages of differentiation of iPSCs and whether there is a difference in their differentiation potential to erythroid lineage. We differentiated three iPSC lines to HSPCs using a previously reported protocol, and the suspension cells containing HSPCs were analyzed for the expression of CD34, CD235a, CD41 and CD45 on days 11, 15 and 17 of differentiation. We found that CD34+CD235a+CD41+ HSPCs were generated from the early stage (day 11) and CD34+CD235- CD45+ HSPCs from the late stage (days 15 and day 17) of differentiation. Colony forming unit assay showed that early and late HSPCs predominantly formed colonies with granulocyte, macrophage and megakaryocyte lineage, but the late HSPCs formed 3 times more erythroid colonies compared to the early HSPCs (26% Vs 7%). Erythroid differentiation of iPSC-HSPCs in liquid culture also showed 10-fold increase in the number of erythroid cells generated from the late HSPCs, compared to the early HSPCs. However, expression kinetics of the erythroid specific markers, CD36, CD71 and CD235a, showed that the early HSPCs underwent rapid differentiation than the late HSPCs. RNA sequencing analysis of the early and the late HSPCs showed significant difference in the expression of haematopoietic genes. Altogether, our results showed that two types of HSPCs are generated from iPSCs at different time points of differentiation, and they have significant difference in their erythroid differentiation potential. For studying the disease mechanisms of red cell diseases, the HSPCs that express high levels of CD45 are more suitable due to their higher erythroid differentiation potential.Funding Source: We acknowledge research funding from Department of Biotechnology, Government of India.F-3140A PLURIPOTENT STEM CELL DERIVED, 3-DIMENSIONAL MODEL OF THE HUMAN ENDOMETRIUMChu, Virginia - Neurology, Northwestern University, Chicago, IL, USA Marinic, Mirna - Human Genetics, University of Chicago, Chicago, IL, USA Peng, Chian-yu - Neurology, Northwestern University, Chicago, IL, USA Lynch, Vincent - Human Genetics, University of Chicago, Chicago, IL, USA
595POSTER ABSTRACTSKessler, John - Neurology, Northwestern University, Chicago, IL, USAThe human endometrium is a highly dynamic organ – cyclically proliferating, differentiating, and shedding in response to ovarian hormones. Precise endometrial responses are necessary to enable implantation, pregnancy, and birth. While mouse models of pregnancies exist, there are fundamental differences that make it a poor model for the human endometrium. Cells from the human endometrium can be obtained by biopsy but have limited proliferative capacities and involve an invasive procedure. Consequently, little is known about how the human endometrium differs in healthy versus diseased pregnancies and births. To address this, we developed a model of the human endometrium that combines human pluripotent stem cells (PSCs)-derived endometrial mesenchyme (EM) and endometrial epithelial cells (EEC) in a 3-dimensional organoid system. We developed a protocol for differentiation of PSCs into EM, precursors of endometrial stromal fibroblasts (ESF). The PSC progress through developmental stages: first differentiating to cells of the late primitive streak, intermediate mesoderm, Müllerian mesenchyme, and finally EM. The progression of differentiation through these stages was characterized by gene expression and immunocytochemistry. Notably, these cells expressed anti-mullerian hormone receptor 2 (AMHR2), which is specific to reproductive tract mesenchyme, and the PSC-EM demonstrated regional specificity characterized by expression of HOXA10 and HOXA11, but not HOXA9 or HOXA13. The EMs then were co-cultured with EEC organoids derived from the adult uterus. The PSC-EM progenitors interacted with the EEC organoids to form a structure in which the PSC derived cells organized in a layer surrounding the EECs. This model of the endometrium will enable the study of how these two human cell types interact and will enable the study of defects in pregnancy as well as endometrial diseases such as endometriosis and endometrial cancer.Funding Source: March of DimesF-3142THE ROLE OF ACIDIFICATION IN HUMAN PLURIPOTENT STEM CELL DIFFERENTIATIONLu, Vivian - Pathology and Laboratory Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA, USA Roy, Irena - Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA Dahan, Perrine - Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA Teitell, Michael - Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USAHuman pluripotent stem cells (hPSCs) hold profound medical promise because they can differentiate into any cell type, yet, for clinical applications, improved differentiation protocols are required. The embryogenesis process is initiated by formation of the three embryonic germ layers— mesoderm, ectoderm, and endoderm—which ultimately constitute all cell types in the body. Current differentiation strategies using chemical agents, cytokines, and growth factors produce suboptimal terminal cells of interest. Recently, the facilitating role of metabolic flux and metabolite levels in reconfiguring epigenetic regulation programs to improve hPSC differentiation has been established. Our findings show that induction of distinct metabolic programs is necessary during embryonic germ lineage differentiation; these metabolic activities result in differential extracellular acidification levels during early cell type specification. Yet, acidification has not been examined as a microenvironment stimulus controlling hPSC differentiation despite its known roles in driving differentiation during pathogenic and physiological development. Our findings suggest that low pH skews embryonic tri-lineage progenitor cell fate, possibly revealing another aspect of developmental regulation thus far unexplored. This confirms a direct connection between metabolism and acidification, and further suggests a role for pH in early cell fate determination. Success in these studies will open new pathways through pH manipulations for generating superior in vitro models of early human development for numerous promising applications in health and disease.Funding Source: This study was supported by Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, CA90571, CA156674, GM073981, CA185189, GM114188, and the Air Force Office of Scientific Research FA9550-15-1-0406F-3144SINGLE-CELL TRANSCRIPTOME APPROACH TO INVESTIGATE THE MECHANISM OF SPECIFYING MESODERM LINEAGES USING HUMAN INDUCIBLE PLURIPOTENT STEM CELLSZhao, Wei - Laboratory for Human Organogenesis, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Takasato, Minoru - Laboratory for Human Organogenesis, RIKEN Center for Biosystems Dynamics Research, Kobe, JapanHuman inducible pluripotent stem cells (iPSCs) is a useful tool for studying the development of human embryo. Mesoderm population including kidney, which organoid we have successfully generated, are all derived from primitive streak (PS) during gastrulation. Interestingly, the 2D cultured iPSCs did not uniformly differentiate into one type of mesoderm cell, instead, they consist of heterogeneous progenitor cells including ureteric bud (UB) and metanephric mesenchyme (MM) precursors expressing either anterior or posterior makers of intermediate mesoderm (IM). In the present study, we aimed to investigate the mechanism of heterogeneity in mesoderm development using directed differentiation of iPSCs. Recently, single-cell RNA sequence (scRNA-seq) already became a powerful method to describe heterogenous cell populations expressing variability of genes. We made use of this technique to trace the dynamics in genes expression. To this end, we used the culture condition that could give rise to the heterogeneous mesoderm cells containing anterior, posterior, paraxial, and lateral plate mesoderm. iPSCs undergoing differentiation were harvested every day from day 0 to 7 and performed scRNA-seq. The result showed highly
596POSTER ABSTRACTSexpressed CDX2, a marker for undifferentiated tail bud, indicating cells were not fully differentiated into posterior and lateral mesoderm cells by day 7. In order to obtain better mediolateral and anteroposterior balance at more differentiated stage, here, we optimized the culture condition and confirmed the induction of each mesoderm region by qPCR and immunofluorescent.F-3146STANDARDIZED QC WORKFLOW FOR ANALYZING THE QUALITY AND DIFFERENTIATION POTENTIAL OF HUMAN IPSCSChen, Xiuqing - Department of Neurology and Neurosurge, Montreal Neurology Institute, Montreal, QC, Canada Abdian, Narges - Department of Neurology and Neurosurgery, Montreal Neurology Institute, Montreal, QE, Canada Maussion, Gilles - Department of Neurology and Neurosurgery, Montreal Neurology Institute, Montreal, QE, Canada Fon, Edward - Department of Neurology and Neurosurgery, Montreal Neurology Institute, Montreal, QE, Canada Durcan, Thomas - Department of Neurology and Neurosurgery, Montreal Neurology Institute, Montreal, QE, CanadaHuman induced pluripotent stem cells (hiPSCs) derived from human somatic have given scientists new opportunities to model and investigate human diseases, as well as to develop new therapeutics. The routine application of hiPSCs and functional cells derived from hiPSCs in high throughput applications for drug discovery will require a constant supply of pluripotent, well characterized and quality controlled cell stocks. However, the absence of standardized quality control of IPSC in many groups and execution challenges laboratory efficiency and experimental reproducibility. Here, we established a workflow to monitor the hiPSCs morphology and proliferation using longitudinal time-lapse imaging, coupled with an assessment of pluripotency through immunofluorescence staining and q-PCR analyse of pluripotency markers, assessment EB formation and differentiation to 3 germ layers by q-PCR and IF staining, assessment of karyotypic abnormalities by qPCR. Following this workflow, 10 healthy hiPSCs lines cultured in different medium (mTeSR1 and Essential 8) were monitored using this process. Cell cycle time, cell attachment, spontaneous differentiation and cell viability in passage 1 and 7 post thaw were evaluated. No significant differences were observed between two media but slightly different exist from line to line, at both early and late passage. All hiPSC lines showed a homogenous morphology with high expression of pluripotency markers SSEA-4, Nanog, Oct3-4 and also retained genomic stability. EB formation and q-PCR analysis provided further validation that these cell-lines possessed an intrinsic ability to differentiate into any of the three germ layers. Together, the workflow outlined here provides a simple, standardized characterization for routine quality control of hiPSC to ensure researchers can adapt for their own labs, to ensure high quality hiPSCs for their research and translational needs.F-3148FUNCTIONAL SCREEN REVEALS ESSENTIAL ROLES OF RNA BINDING PROTEINS IN THE EXIT FROM PLURIPOTENCYWang, Xue - Genetics, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Huang, Yue - Genetics, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaRNA-binding proteins (RBPs) play crucial roles in various cellular processes mainly through post-transcriptional control of RNAs, including polyadenylation, splicing, stabilization, localization and mRNA translation. However, the functions of RBPs in the onset of differentiation of mammalian embryonic stem (ES) cells remain largely unknown. Here, by functional screening dozens of pre-selected RBPs in ES cells, we identified a few crucial RBPs required for exit from pluripotency and further explored the underlying molecular mechanisms. Firstly, a number of candidate RBPs were picked by bioinformatics analysis based on the RBPs database of mouse ES cells and published literatures. Then, we disrupted these candidates by using CRISPR/Cas9 system in mouse ES cells respectively, and evaluated the onset of differentiation in these mutant cell lines thoroughly. A few candidate RBP-null cell clones, including Rbm34, Hnrnpll and Rbpms, showed significantly differentiation defect compared with wide-type ES cells. Further mechanism research showed that the RBPs regulate mRNA splicing and stability. The RNA-seq analysis revealed substantial changes in pre-mRNA alternative splicing (AS) at each examined time point during ES cell differentiation, revealing distinct temporal patterns of AS underlying cell-fate changes. Combined with RIP-seq data, we found the long transcript of two important transcription factors cannot be decreased accordingly without the RBP. These results indicated that RBPs played an important role in the exit from pluripotency.F-3150TRISOMY 12 COMPROMISES THE MESENDODERMAL DIFFERENTIATION PROPERTY OF HUMAN PLURIPOTENT STEM CELLSHayashi, Yohei - iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, Tsukuba, Japan Yanagihara, Kana - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Liu, Yujung - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Yamaguchi, Tomoko - Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Kokunugi, MInako - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan
597POSTER ABSTRACTSWakabayashi, Mari - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Uchio-Yamada, Kozue - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Fukumoto, Ken - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Suga, MIka - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Terada, Satoshi - Department of applied biochemistry, University of Fukui, Fukui, Japan Nikawa, Hiroki - Department of Oral Biology and Engineering, Hiroshima University, Hiroshima, Japan Kawabata, Kenji - Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan Furue, Miho - Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, JapanTrisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. hPSC sublines carrying trisomy 12 showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in serum-free medium. BMP4- or Activin A-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, hematopoietic and hepatic differentiation were also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.F-3152COMPARATIVE EVALUATION OF HORMONES AND HORMONE-LIKE MOLECULES IN LINEAGE SPECIFICATION OF HUMAN INDUCED PLURIPOTENT STEM CELLSAn, Ju-Hyun - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Korea Choi, Seon-A - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Kyung-Min - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Cha, Jae-Jin - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Jeong, Pil-Soo - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Yang, Hae-Jun - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Sanghoon - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Seung Hwan - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Park, Young-Ho - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Song, Bong-Seok - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Sim, Bo-Woong - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Kim, Sun-Uk - Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, Korea Lee, Jong-Hee - National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, KoreaProficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidence has implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17- (E2), progesterone β(P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34+CD45+ cells with progenitor functions, even in the CD43– population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision. Our findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.Funding Source: This study was supported by grant from the KRIBB Research Initiative Program (KGM4251824), Republic of Korea.
598POSTER ABSTRACTSF-3154PROTEIN DISCOVERY PLATFORM USING DEER ANTLER AS A MODEL OF MAMMALIAN ORGAN REGENERATIONDong, Zhen - Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand Haines, Stephen - Proteins and Biomaterials, AgResearch Lincoln Research Centre, Christchurch, New Zealand Coates, Dawn - Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New ZealandAnimal models can provide important insights into how to activate and control stem cells during tissue repair and regeneration. The robust regenerative capacity of some lower-order animals, such as planarian and zebrafish, has been well documented. Stem cell niches in mammals that drive complete organ regeneration are rare. Deer antler, however, is the only known mammalian model of complete organ regeneration based on stem cells. Antler neural crest-derived stem cells can be used as a model system to study the maintenance of a stem cell niche and activation and differentiation of stem cells. This model allows the discovery of proteins and mechanisms underlying the activation of stem cell niches involved in regeneration of a complex organ. In the present study, label-free quantification was used to investigate the protein profiles of antler stem cells under different stages of activation and included: dormant pedicle periosteum (N=3), growth centre (N=3), middle beam periosteum (N=3), and, as a control tissue, deer facial periosteum (N=3). PEAKS and Ingenuity Pathway Analysis software were utilized to interpret the proteomics data. Pleiotrophin (PTN), one of the key regulators during antler regeneration obtained by in-situ hybridization, with its main receptors were studied using immunohistochemistry (IHC). PTN’s expression levels in antler stem cells with/without osteogenic media were measured by ELISA. Moreover, the location of the three commonly-accepted markers of mesenchymal stem cells (MSCs) - CD73, CD90 and CD105 - was also examined by IHC. Our research firstly confirmed the central role of stem cell activation in development of this mammalian organ by localizing the MSCs’ markers within the antler growth centre. The functions played by PTN during rapid antler regeneration on both tissue and cell levels were preliminarily investigated. In addition, this protein discovery platform revealed that multiple biological processes and signalling pathways involving functional molecules participate in antler regeneration. This work sheds new light on the underlying mechanisms in mammalian stem cell-driven organ regeneration and provides further directions for mammal organ regeneration research.Funding Source: This work was supported by the “Healing mechanisms in stem-cell driven regeneration of deer antler” project funded by Velvet Antler Research New Zealand.PLURIPOTENT STEM CELL: DISEASE MODELINGF-3158THE EFFICIENT PRODUCTION OF FUNCTIONAL CHOLANGIOCYTES FROM HUMAN PLURIPOTENT STEM CELLSOgawa, Mina - McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada Yang, Donghe - McEwen Stem Cell Institute, Toronto, ON, Canada Hernandez, Marcela - McEwen Stem Cell Institute, Toronto, ON, Canada Cui, Changyi - McEwen Stem Cell Institute, Toronto, ON, Canada Higuchi, Yuichiro - Laboratory Animal Research Department, Central Insitute for Experimantal Animals, Kawasaki, Japan Suemizu, Hiroshi - Laboratory Animal Research Department, Central Institute for Experimental Animals, Kawasaki, Japan Dorrell, Craig - Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR, USA Grompe, Markus - Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR, USA Bear, Christine - Programme in Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada Keller, Gordon - McEwen Stem Cell Institute, Toronto, ON, Canada Ogawa, Shinichiro - McEwen Stem Cell Institute, Toronto, ON, CanadaCholangiocytes, the epithelial cells that form the bile ducts of the liver express the cystic fibrosis transmembrane conductance regulator (CFTR), the chloride channel mutated in cystic fibrosis (CF). Dysfunction of CFTR in in the cholangiocytes of CF patients leads to impaired secretion and deposition of bile resulting in cholestasis. The ability to generate functional cholangiocytes from human pluripotent stem cells (hPSCs) offers the opportunity to model CF disease and to test the function of new CF drugs. We have previously described a protocol for the generation of hPSC-derived cholangiocytes in 3D organoids (Ogawa et al. Nat. Biotechnol. 2015) that yielded mature cholangiocytes, but was not amenable to cell expansion or high throughput drug screening. To improve the differentiation efficiency in monolayer cultures, we screened cytokines and small molecules based on flow cytometric analysis with the DHC5-4D9 antibody that stains mature cholangiocytes in the adult liver. We identified a combination of factors that promoted the development of hPSCs-derived DHC5-4C9+ cholangiocytes that express high levels of CFTR and contain primary cilia. Cholangiocytes generated from three different CF patient iPSC lines generated with this approach showed differential rescue of CFTR function in response to CFTR modulators using a high throughput FLIPR assay indicating that cells produced under these conditions can be used for drug efficacy studies. 3D cysts/ organoids were efficiently generated from monolayer cultures to apply the CFTR mediated swelling assay. As the secretory activity of mature
599POSTER ABSTRACTScholangiocytes is regulated by Ca2+ signaling, we next analyzed intracellular Ca2+ flux in the hPSC-derived cells to determine if they display this capacity. hPSC-derived cholangiocytes showed Ca2+ Influx in response to ATP or flow stimuli. Finally, to evaluate their potential to generate biliary structures in recipient livers, iPSCs-derived cholangiocytes were transplanted into TK-NOG mice. Six weeks following transplantation, multiple ductal structures comprised of human cholangiocytes were detected throughout the liver, indicating that these cells have some capacity to regenerate the biliary tree. Future studies will focus on evaluating the function of the engrafted ductal structures in these animals.F-3160MODELING ISCHEMIC HEART CONDITION WITH HUMAN EMBRYONIC STEM CELLSKomarovsky Gulman, Nelly - The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel Shainberg, Asher - The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel Urbach, Achia - The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, IsraelIschemic heart disease is characterized by reduced of blood and oxygen supply to the heart muscle and it is the most common cause of death in most western countries. In 1986, Charles Murry discovered a phenomenon called pre-conditioning that causes adaptive changes in heart cells and protects them from extended ischemia damages. Here we aimed to use human pluripotent stem cells (hPSCs) in order to generate a human cell based model for ischemic heart disease and pre-conditioning. The first stage in our study was to differentiate hPSCs into beating cardiomyocytes. Next, we tested the effect of hypoxia on the differentiated cardiomyocytes cells. For this purpose, we grew the cells under hypoxic conditions for 4 hours (0% oxygen) or for 24 hours (1% oxygen). We found that the hypoxic conditions lead to the around seven fold increase in the secretion of the enzyme creatine kinase (Which is one of the markers to detect muscle damage and Myocardial infarction) compared to control cells (under normal oxygen conditions). Consequently, we showed that our system cells behave in the same way as cardiomyocytes from animal models and, in general, mimic the ischemic heart diseases. We will use this model to examine how to induce cardiomyocyte pre-conditioning by pharmacological materials in order to “rescue” the cells from an extended hypoxia. We hope that this system will serve in the future as a tool for screening various materials for pre-conditioning induction in people at high risk for ischemic heart diseases.F-3162KCNQ2-ENCEPHALOPATHY IN A DISH: MECHANISTIC INSIGHT THROUGH 2D AND 3D NEURONAL MODELSDirkx, Nina - CMN-Laboratory of Neurogenetics, University of Antwerp, Berchem, Belgium Asselbergh, Bob - VIB, University of Antwerp, Wilrijk, Belgium Verstraelen, Peter - Laboratory of Cell Biology and Histology, University of Antwerp, Wilrijk, Belgium Deconinck, Tine - CMN-laboratory of Neurogenetics, University of Antwerp, Wilrijk, Belgium Heeman, Bavo - CMN-VIB, University of Antwerp, Wilrijk, Belgium De Vos, Winnok - Laboratory of Cell Biology and Histology, University of Antwerp, Wilrijk, Belgium De Jonghe, Peter - Neurology, University Hospital Antwerp, Edegem, Belgium Guigliano, Michele - Theoretical Neurobiology and Neuroengineering lab, University of Antwerp, Wilrijk, Belgium Weckhuysen, Sarah - CMN-laboratory of Neurogenetics, University of Antwerp, Wilrijk, BelgiumMutations in the gene KCNQ2 are associated with both severe KCNQ2-encephalopathy (KCNQ2-E), characterised by neonatal seizures and developmental delay, and a self-limiting epilepsy syndrome called Benign Familial Neonatal Epilepsy (KCNQ2-BFNE), where development is completely normal. Interestingly, dominant negative (DN) or gain-of-function (GOF) variants are the underlying cause of KCNQ2-E, whereas haploinsufficiency results in KCNQ2-BFNE. KCNQ2 encodes for a subunit of the M-channel which is responsible for the resting membrane potential and controlling neuronal excitability. While the role of KCNQ2 in epilepsy is evident, as dysfunction of the M-channel affects neuronal excitability, its role in neurodevelopment is less well understood. To understand how KCNQ2 affects neuronal development, we generated 2D and 3D neuronal cultures (brain organoids) derived from human induced pluripotent stem cells (hiPSC). We compare 1 known GOF (R201H) and 3 known DN KCNQ2-E variants (D290G, A294V and R560W), as well as 2 KNCQ2-BFNE variants (K327G, R547W). By studying 2D neuronal co-cultures of excitatory and inhibitory neurons, generated via a fast overexpression protocol we aim to (1) discover a read-out system for both the epileptic and the neurodevelopmental features of KCNQ2-E, and to (2) develop a platform that can be used for future drug screening. The brain organoids are used to (3) study the effect of the mutations on the more heterogeneous and complex neuronal network, to discover the affected cell types and to find possible new drug targets. Currently used therapy for KCNQ2-E consists of symptomatic treatment, mainly directed at the (often difficult to treat) seizure activity. So far, no therapies influencing the developmental outcome of KCNQ2-E exist. To develop such a therapy, it is of utmost importance to shed light on the mechanism underlying the neurodevelopmental delay observed in these patients.Funding Source: Nina Dirkx is holder of a PhD grant strategic basic research of the Research Foundation Flanders (60745)
Search
Read the Text Version
- 1
- 2
- 3
- 4
- 5
- 6
- 7
- 8
- 9
- 10
- 11
- 12
- 13
- 14
- 15
- 16
- 17
- 18
- 19
- 20
- 21
- 22
- 23
- 24
- 25
- 26
- 27
- 28
- 29
- 30
- 31
- 32
- 33
- 34
- 35
- 36
- 37
- 38
- 39
- 40
- 41
- 42
- 43
- 44
- 45
- 46
- 47
- 48
- 49
- 50
- 51
- 52
- 53
- 54
- 55
- 56
- 57
- 58
- 59
- 60
- 61
- 62
- 63
- 64
- 65
- 66
- 67
- 68
- 69
- 70
- 71
- 72
- 73
- 74
- 75
- 76
- 77
- 78
- 79
- 80
- 81
- 82
- 83
- 84
- 85
- 86
- 87
- 88
- 89
- 90
- 91
- 92
- 93
- 94
- 95
- 96
- 97
- 98
- 99
- 100
- 101
- 102
- 103
- 104
- 105
- 106
- 107
- 108
- 109
- 110
- 111
- 112
- 113
- 114
- 115
- 116
- 117
- 118
- 119
- 120
- 121
- 122
- 123
- 124
- 125
- 126
- 127
- 128
- 129
- 130
- 131
- 132
- 133
- 134
- 135
- 136
- 137
- 138
- 139
- 140
- 141
- 142
- 143
- 144
- 145
- 146
- 147
- 148
- 149
- 150
- 151
- 152
- 153
- 154
- 155
- 156
- 157
- 158
- 159
- 160
- 161
- 162
- 163
- 164
- 165
- 166
- 167
- 168
- 169
- 170
- 171
- 172
- 173
- 174
- 175
- 176
- 177
- 178
- 179
- 180
- 181
- 182
- 183
- 184
- 185
- 186
- 187
- 188
- 189
- 190
- 191
- 192
- 193
- 194
- 195
- 196
- 197
- 198
- 199
- 200
- 201
- 202
- 203
- 204
- 205
- 206
- 207
- 208
- 209
- 210
- 211
- 212
- 213
- 214
- 215
- 216
- 217
- 218
- 219
- 220
- 221
- 222
- 223
- 224
- 225
- 226
- 227
- 228
- 229
- 230
- 231
- 232
- 233
- 234
- 235
- 236
- 237
- 238
- 239
- 240
- 241
- 242
- 243
- 244
- 245
- 246
- 247
- 248
- 249
- 250
- 251
- 252
- 253
- 254
- 255
- 256
- 257
- 258
- 259
- 260
- 261
- 262
- 263
- 264
- 265
- 266
- 267
- 268
- 269
- 270
- 271
- 272
- 273
- 274
- 275
- 276
- 277
- 278
- 279
- 280
- 281
- 282
- 283
- 284
- 285
- 286
- 287
- 288
- 289
- 290
- 291
- 292
- 293
- 294
- 295
- 296
- 297
- 298
- 299
- 300
- 301
- 302
- 303
- 304
- 305
- 306
- 307
- 308
- 309
- 310
- 311
- 312
- 313
- 314
- 315
- 316
- 317
- 318
- 319
- 320
- 321
- 322
- 323
- 324
- 325
- 326
- 327
- 328
- 329
- 330
- 331
- 332
- 333
- 334
- 335
- 336
- 337
- 338
- 339
- 340
- 341
- 342
- 343
- 344
- 345
- 346
- 347
- 348
- 349
- 350
- 351
- 352
- 353
- 354
- 355
- 356
- 357
- 358
- 359
- 360
- 361
- 362
- 363
- 364
- 365
- 366
- 367
- 368
- 369
- 370
- 371
- 372
- 373
- 374
- 375
- 376
- 377
- 378
- 379
- 380
- 381
- 382
- 383
- 384
- 385
- 386
- 387
- 388
- 389
- 390
- 391
- 392
- 393
- 394
- 395
- 396
- 397
- 398
- 399
- 400
- 401
- 402
- 403
- 404
- 405
- 406
- 407
- 408
- 409
- 410
- 411
- 412
- 413
- 414
- 415
- 416
- 417
- 418
- 419
- 420
- 421
- 422
- 423
- 424
- 425
- 426
- 427
- 428
- 429
- 430
- 431
- 432
- 433
- 434
- 435
- 436
- 437
- 438
- 439
- 440
- 441
- 442
- 443
- 444
- 445
- 446
- 447
- 448
- 449
- 450
- 451
- 452
- 453
- 454
- 455
- 456
- 457
- 458
- 459
- 460
- 461
- 462
- 463
- 464
- 465
- 466
- 467
- 468
- 469
- 470
- 471
- 472
- 473
- 474
- 475
- 476
- 477
- 478
- 479
- 480
- 481
- 482
- 483
- 484
- 485
- 486
- 487
- 488
- 489
- 490
- 491
- 492
- 493
- 494
- 495
- 496
- 497
- 498
- 499
- 500
- 501
- 502
- 503
- 504
- 505
- 506
- 507
- 508
- 509
- 510
- 511
- 512
- 513
- 514
- 515
- 516
- 517
- 518
- 519
- 520
- 521
- 522
- 523
- 524
- 525
- 526
- 527
- 528
- 529
- 530
- 531
- 532
- 533
- 534
- 535
- 536
- 537
- 538
- 539
- 540
- 541
- 542
- 543
- 544
- 545
- 546
- 547
- 548
- 549
- 550
- 551
- 552
- 553
- 554
- 555
- 556
- 557
- 558
- 559
- 560
- 561
- 562
- 563
- 564
- 565
- 566
- 567
- 568
- 569
- 570
- 571
- 572
- 573
- 574
- 575
- 576
- 577
- 578
- 579
- 580
- 581
- 582
- 583
- 584
- 585
- 586
- 587
- 588
- 589
- 590
- 591
- 592
- 593
- 594
- 595
- 596
- 597
- 598
- 599
- 600
- 601
- 602
- 603
- 604
- 605
- 606
- 607
- 608
- 609
- 610
- 611
- 612
- 613
- 614
- 615
- 616
- 617
- 618
- 619
- 620
- 621
- 622
- 623
- 624
- 625
- 626
- 627
- 628
- 629
- 630
- 631
- 632
- 633
- 634
- 635
- 636
- 637
- 638
- 639
- 640
- 641
- 642
- 643
- 644
- 645
- 646
- 647
- 648
- 649
- 650
- 651
- 652
- 653
- 654
- 1 - 50
- 51 - 100
- 101 - 150
- 151 - 200
- 201 - 250
- 251 - 300
- 301 - 350
- 351 - 400
- 401 - 450
- 451 - 500
- 501 - 550
- 551 - 600
- 601 - 650
- 651 - 654
Pages:
