Pluripotency Factors Research Articles

Serial Xenotransplantation in NSG Mice Promotes a Hybrid Epithelial/Mesenchymal Gene Expression Signature and Stemness in Rhabdomyosarcoma Cells.

Serial xenotransplantation of sorted cancer cells in immunodeficient mice remains the most complex test of cancer stem cell (CSC) phenotype. However, we have demonstrated in various sarcomas that putative CSC surface markers fail to identify CSCs, thereby impeding the isolation of CSCs for subsequent analyses. Here, we utilized serial xenotransplantation of unsorted rhabdomyosarcoma cells in NOD/SCID gamma (NSG) mice as a proof-of-principle platform to investigate the molecular signature of CSCs. Indeed, serial xenotransplantation steadily enriched for rhabdomyosarcoma stem-like cells characterized by enhanced aldehyde dehydrogenase activity and increased colony and sphere formation capacity in vitro. Although the expression of core pluripotency factors (SOX2, OCT4, NANOG) and common CSC markers (CD133, ABCG2, nestin) was maintained over the passages in mice, gene expression profiling revealed gradual changes in several stemness regulators and genes linked with undifferentiated myogenic precursors, e.g., SOX4, PAX3, MIR145, and CDH15. Moreover, we identified the induction of a hybrid epithelial/mesenchymal gene expression signature that was associated with the increase in CSC number. In total, 60 genes related to epithelial or mesenchymal traits were significantly altered upon serial xenotransplantation. In silico survival analysis based on the identified potential stemness-associated genes demonstrated that serial xenotransplantation of unsorted rhabdomyosarcoma cells in NSG mice might be a useful tool for the unbiased enrichment of CSCs and the identification of novel CSC-specific targets. Using this approach, we provide evidence for a recently proposed link between the hybrid epithelial/mesenchymal phenotype and cancer stemness.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells from the Tasmanian Devil (Sarcophilus harrisii) Express Immunomodulatory Factors and a Tropism Toward Devil Facial Tumor Cells.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.

SOX2 Expression and Transcriptional Activity Identifies a Subpopulation of Cancer Stem Cells in Sarcoma with Prognostic Implications

Background: Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cell (CSC) subpopulations. The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Methods: shRNA and cDNA lentiviral constructions were used to deplete and overexpress SOX2 in a model of undifferentiated pleomorphic sarcoma (UPS). In addition, we introduced a reporter system (SORE6) which allows to detect and isolate viable cells expressing SOX2 and/or OCT4 in the UPS model and patient-derived chondrosarcoma lines. Functional CSC characterization included tumorsphere formation, in vivo xenograft growth assays, and pharmacologic testing of the effects of doxorubicin, placlitaxel, trabectedin or the mithramycin analogue EC8042. Immunohistochemical analysis of a cohort of 88 formalin-fixed and paraffin-embedded sarcoma samples was performed to correlate SOX2 expression with clinical parameters. Findings: SOX2 depletion dramatically reduced the ability of sarcoma cells to form tumorspheres and to initiate tumor growth in vivo. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. In sarcoma patients, SOX2 expression was associated to advanced disease stages, aggressive phenotypes and lower survival. Finally, SORE6+ cells were significantly more tumorigenic than the SORE6- population and EC-8042 effectively targeted SORE6+ cells in vitro and in vivo. Interpretation: SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations. Funding Statement: This work was supported by the Agencia Estatal de Investigacion (AEI) [MINECO/Fondo Europeo de Desarrollo Regional (FEDER) (SAF-2016-75286-R to R.R.), ISC III/FEDER (Miguel Servet Program CPII16/00049 to R.R., Sara Borrell Program CD16/00103 to S.T.M. and PI16/00280 and PI19/00560 to J.M.G-P) and Consorcio CIBERONC CB16/12/00390)] and the Plan de Ciencia Tecnologia e Innovacion del Principado de Asturias/FEDER (IDI/2018/155) to J.P.R. PharmaMar and EntreChem also provided support. Finally, the authors acknowledge the Principado de Asturias BioBank (PT17/0015/0023), financed jointly by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundacion Bancaria Cajastur and integrated in the Spanish National Biobanks Network, for its collaboration. Declaration of Interests: The authors declare they have no competing interests. Ethics Approval Statement: All experimental protocols have been performed in accordance with institutional review board guidelines and were approved by the Institutional Ethics Committee of the Principado de Asturias. All samples from human origin were obtained upon signed informed consent. All experimental protocols were carried out in accordance with the institutional guidelines of the University of Oviedo and were approved by the Animal Research Ethical Committee of the University of Oviedo prior to the study.

SOX2 upregulates side population cells and enhances their chemoresistant ability by transactivating ABCC1 expression contributing to intrinsic resistance to paclitaxel in melanoma.

Paclitaxel is the last choice for the treatment of advanced melanoma as a second-line chemotherapeutic agent, but there are still many cases of intrinsic resistance to paclitaxel in melanoma and the reasons that cause paclitaxel resistance remain unclear. Here, we identified that high expression of SRY-box transcription factor 2 (SOX2) and high ratio of side population (SP) cells reduced the sensitivity to paclitaxel in melanoma cells. The knockout and the induction of SOX2 completely depleted and significantly upregulated the ratios of melanoma SP cells, respectively. These data suggest that SOX2, a pluripotent transcription factor for inducing cancer stem cells in melanoma, is also sufficient and necessary for the induction of melanoma SP cells. ATP-binding cassette (ABC) subfamily C member 1 (ABCC1) is one of ABC transporters which causes SP cells to be resistance to chemotherapeutic agents by efficiently pumping drugs out of cells. The knockout and the induction of ABCC1 significantly increased and decreased the sensitivity of melanoma cells to paclitaxel. High expression of ABCC1 was identified in melanoma cell lines with high expression of SOX2 and in their SP cells. SOX2 was identified to induce ABCC1 transcription. Taken together, SOX2 upregulates SP cells and enhances their chemoresistant ability by increasing ABCC1 expression, which contributes to intrinsic resistance to paclitaxel in melanoma. Our findings will lead to new insights into melanoma biology and therapy resistance, and eventually to new therapeutic targets.

Pro- and Antioxidant Effects of Vitamin C in Cancer in correspondence to Its Dietary and Pharmacological Concentrations.

Vitamin C is an antioxidant that may scavenge reactive oxygen species preventing DNA damage and other effects important in cancer transformation. Dietary vitamin C from natural sources is taken with other compounds affecting its bioavailability and biological effects. High pharmacological doses of vitamin C may induce prooxidant effects, detrimental for cancer cells. An oxidized form of vitamin C, dehydroascorbate, is transported through glucose transporters, and cancer cells switch from oxidative phosphorylation to glycolysis in energy production so an excess of vitamin C may limit glucose transport and ATP production resulting in energetic crisis and cell death. Vitamin C may change the metabolomic and epigenetic profiles of cancer cells, and activation of ten-eleven translocation (TET) proteins and downregulation of pluripotency factors by the vitamin may eradicate cancer stem cells. Metastasis, the main reason of cancer-related deaths, requires breakage of anatomical barriers containing collagen, whose synthesis is promoted by vitamin C. Vitamin C induces degradation of hypoxia-inducible factor, HIF-1, essential for the survival of tumor cells in hypoxic conditions. Dietary vitamin C may stimulate the immune system through activation of NK and T cells and monocytes. Pharmacological doses of vitamin C may inhibit cancer transformation in several pathways, but further studies are needed to address both mechanistic and clinical aspects of this effect.

CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma.

Background: Oral squamous cell carcinoma (OSCC), with high mortality rates, is one of the most diagnosed head and neck cancers. Epithelial-to-mesenchymal transition (EMT) and the generation of cancer stem cells (CSCs) are two keys for therapy-resistance, relapse, and distant metastasis. Accumulating evidence indicates that aberrantly expressed cluster of differentiation (CD)47 is associated with cell-death evasion and metastasis; however, the role of CD47 in the generation of CSCs in OSCC is not clear. Methods: We investigated the functional roles of CD47 in OSCC cell lines SAS, TW2.6, HSC-3, and FaDu using the bioinformatics approach, immunoblotting, immunofluorescence staining, and assays for cellular migration, invasion, colony, and orosphere formation, as well as radiosensitivity. Results: We demonstrated increased expression of CD47 in OSCC patients was associated with an estimated poorly survival disadvantage (p = 0.0391) and positively correlated with the expression of pluripotency factors. Silencing CD47 significantly suppressed cell viability and orosphere formation, accompanied by a downregulated expression of CD133, SRY-Box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and c-Myc. In addition, CD47-silenced OSCC cells showed reduced EMT, migration, and clonogenicity reflected by increased E-cadherin and decreased vimentin, Slug, Snail, and N-cadherin expression. Conclusion: Of therapeutic relevance, CD47 knockdown enhanced the anti-OSCC effect of radiotherapy. Collectively, we showed an increased CD47 expression promoted the generation of CSCs and malignant OSCC phenotypes. Silencing CD47, in combination with radiation, could provide an alternative and improved therapeutic efficacy for OSCC patients.

Multiple novel hepatocellular carcinoma signature genes are commonly controlled by the master pluripotency factor OCT4

BackgroundWorldwide, hepatocellular carcinoma (HCC) is a common solid tumor with a poor prognosis. HCC is often due to hepatitis B virus (HBV) infection. As yet, efficacious HCC treatment regimens for late-stage HCC patients are lacking. Therefore, the identification of more specific and sensitive biomarkers for its early diagnosis and treatment remains an urgent need.MethodsTotal RNAs from paired HBV-derived HCC tumors and adjacent peritumor tissues (APTs) were subjected to RNA sequencing (RNA-seq), and differentially expressed genes (DEGs) between HCC tumors and APTs were selected and verified.ResultsWe identified 166 DEGs and found that eight top-ranked and verified DEGs (TK1, CTTN, CEP72, TRIP13, FTH1, FLAD1, CHRM2, AMBP) all contained putative OCT4 binding motifs in their promoter regions. TK1, TRIP13 and OCT4 were found to exhibit concurrent higher expression levels in HCC tumors than in APTs. The mRNA levels of TK1, TRIP13 and OCT4 in a cohort of 384 HCC samples from the TCGA database were all found to be negatively correlated with patient overall survival, relapse-free survival and progression-free survival, underscoring the HCC biomarker status of TK1 and TRIP13 on one hand, and implicating their association with OCT4 on the other hand. Furthermore, OCT4 proteins were found to bind to the promoters of both genes in vitro and in vivo. Knocking out OCT4 in HCC-derived cell lines reduced the expression of TK1 and TRIP13 and significantly decreased their tumorigenicity.ConclusionsUsing RNA-seq, we identified several novel HCC signature genes that may serve as biomarkers for its diagnosis and prognosis. Their common transcriptional regulation by OCT4 suggests key roles in the development of HCC, and indicates that OCT4 may serve as a potential therapeutic target.

Abstract A50: IMPACT inhibits metastatic outgrowth in pancreatic cancer by restraining GCN1-ATF4 signaling

Abstract Introduction: Although liver metastasis is the major cause of pancreatic cancer mortality, the mechanisms by which KRAS-driven pancreatic tumor cells initiate metastatic outgrowth in the liver are unknown. High-throughput next-generation sequencing studies have revealed that driver mutations and copy number alterations are highly concordant between matched primary tumors and metastases in pancreatic cancer. This suggests that the mechanisms leading to the metastatic outgrowth and colonization involve the cooperation of many other cellular factors in addition to driver mutations. We hypothesize that in the absence of metastasis-specific driver mutations, the initial adaptation and outgrowth steps of metastasis are driven by metabolic reprogramming, which is induced or activated in vivo under physiologic conditions of the target organ like nutrient deprivation. Method: To identify the metabolic drivers, a retroviral cDNA library was generated from a highly metastatic pancreatic cell line (M-4964Liv) derived from a KPC mouse that forms macroscopic liver lesions upon splenic injection in mice. The cDNA library was then transduced into dormant pancreatic KPC cells (D-4964Liv) cells that do not give liver lesions upon splenic injection in mice. This methodology will allow all the cDNAs from the metastatic cells to express in the dormant cells, but only those cDNAs that can trigger the dormant cells to outgrow will give rise to a metastatic liver lesion. Results: The screening strategy led to the identification of the translational regulator general control of amino-acid synthesis 1-like 1 (GCN1). We show that activation of GCN1 at physiologic glutamine activates the master regulator of metabolic homeostasis ATF4, oncogenic mTORC1 signaling, and pluripotency factor SOX2. Consistent with this, knockdown of GCN1 not only inhibits ATF4, mTORC1, and expression of Sox2 but dramatically abrogates liver metastasis in immunocompetent mice upon intrasplenic injection, suggesting that GCN1-ATF4 signaling is indispensable for metastatic outgrowth. Mechanistic insights reveal that the GCN1-ATF4 signaling pathway is governed by a competitive inhibitor, IMPACT, which is induced at physiologic glutamine and inhibits GCN1 function. Surprisingly, the expression of IMPACT is lost in highly metastatic pancreatic cell lines, leading to activation of the GCN1-ATF4 pathway and mTORC1 at physiologic glutamine, while overexpression of IMPACT effectively inhibits the pathway and reduces metastatic outgrowth of pancreatic cancer cells in the liver in immunocompetent mice. Conclusions: The loss of expression of IMPACT leading to GCN1-dependent activation of ATF4 and mTORC1 at physiologic levels of glutamine provides metabolic plasticity during the initial steps of metastatic outgrowth. Thus, inhibitors targeting the GCN1-ATF4 pathway or mTORC1 will greatly benefit pancreatic adenocarcinoma patients by inhibiting the outgrowth of their metastatic disease. Citation Format: Surajit Sinha, Reed I Ayabe, Alok Ranjan, Michael Wach, Samantha Ruff, Kirsten Remmert, Elke Van Beek, Imani Alexander, James D. McDonald, Shreya Gupta, Eileen Li, Jonathan Hernandez. IMPACT inhibits metastatic outgrowth in pancreatic cancer by restraining GCN1-ATF4 signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A50.

A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional(3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

218 Urine samples as a noninvasive source of induced pluripotent cells in the swine model

Noninvasive collection of cells used for generation of induced pluripotent stem cells (iPSC) generation would simplify its use for regenerative and reproductive purposes in veterinary medicine, although it is still unpublished in species other than humans. This study aimed to derive urine progenitor cells (UPCs) invitro from urine samples collected from swine, and then to reprogram them into iPSCs. For that, urine samples were collected from three females, and cells were isolated and cultured from each following the human UPCs protocol (Steichen et al. 2017 Curr. Protoc. Hum. Genet. 21, 7.1-21.7.22; https://doi.org/10.1002/cphg.26). Approximately 200mL of urine samples were collected in sterile flasks and centrifuged at 300×g; the pellet was washed in Dulbecco's PBS, resuspended, and cultured in 45% Dulbecco's modified Eagle's medium (DMEM)-high glucose, 5% fetal bovine serum (FBS), 50% Renal Epithelial Cell Growth basal medium (REBM) supplemented with 1% glutamine, 1% nonessential amino acids (NEAA), penicillin/streptomycin (P/S), and REGM supplements hEGF, insulin, hydrocortisone, GA-1000, FBS, transferrin, triiodothyronine, epinephrine (Lonza), and 10ngmL−1 basic fibroblast growth factor (bFGF). The UPCs first colonies were observed approximately 1 week after and resembled epithelial-like cells. At passage 2, one cell line was transduced with murine OCT4, SOX2, KLF4, and C-MYC cDNAs (OSKM) using a lentiviral vector. After 5 days, cells were plated onto mouse embryonic fibroblasts and cultured in knockout DMEM/F12, 20% knockout serum replacement, NEAA, L-glutamine, 2-mercaptoethanol, and P/S supplemented with 10ngmL−1 bFGF. Efficiency of reprogramming was 8.45%, measured by analysing the number of typical iPSC colonies relative to the transduced cells plated, after ~12 days. Three clonal lines (C1, C4, and C6) were maintained invitro and characterised regarding pluripotency markers for at least 30 passages. All three lines were positive for alkaline phosphatase activity in passages 15 and also 22. Immunocytochemistry analysis revealed that C6 (passage 22) was positive for the pluripotency genes OCT4 (1:100, SC), SOX2 (1:500, AB), SSEA1 (1:50, SC), TRA1- 81 (1:50, Millipore), and NANOG (1:100, AB), whereas C1 and C4 (passages 23 and 22, respectively) were positive only for OCT4, SOX2, and SSEA1. The expression of exogenous and endogenous pluripotency factors (OCT4, SOX2, and NANOG) was evaluated by qRT-PCR, comparing the three clonal lines at passages 16/17 and 21/22 and comparing different passages (10, 11, 14, 17, and 22) on C1 line. No statistical difference was observed between cell lines when compared in different passages, perhaps because of the great variation between lines. However, analysis of C1 line over time showed that pluripotency genes increased and exogenous vector expression decreased during early passages (±10 passages); however, after passage 17, OCT4 and NANOG decreased whereas SOX2 and exogenous vector expression increased. In conclusion, it was possible to reprogram UPCs into iPSCs and maintain them in culture for at least 30 passages; however, iPSCS were dependent on exogenous factors. These results represent the partial reprogramming of UPCs to iPSCs in animals for the first time, enabling the generation of invitro disease models using a noninvasive method. Financial support for this study was received from FAPESP (2019/02811-2, 2015/26818-5), CNPq (433133/2018-0), and CAPES.

Nerve growth factor inhibits TLR3-induced inflammatory cascades in human corneal epithelial cells

BackgroundIn herpes simplex epithelial keratitis, excessive TLR3-induced cellular responses after virus infection evoke inflammatory cascades that might be destructive to the host cornea. Nerve growth factor (NGF), a pluripotent neurotrophic factor with immune regulatory effect, was proved to be effective in Herpes simplex keratitis (HSK) treatment, although the detailed mechanisms remain unclear. This study aims to investigate the effects of NGF on modulating inflammatory responses triggered by TLR3 activation in human corneal epithelial cells (HCECs) in vitro.MethodsHCECs were stimulated with TLR3 agonist, poly(I:C), in the absence or presence of NGF. Cell viability and cytotoxicity were measured by a CCK-8 assay and LDH release assay, respectively. The activation of NF-κB signaling pathway was examined using immunofluorescence staining and western blotting. Levels of proinflammatory cytokines were determined by ELISA or RT-qPCR. ROS generation and 8-OHdG positive cells were examined by a fluorometric analysis.ResultsIt was shown that NGF significantly inhibited the generation of proinflammatory cytokines in HCECs triggered by TLR3 activation (P < 0.05), probably via suppressing NF-κB activation. NGF also impeded the upstream signal to initiate NF-κB activation by scavenging ROS by approximately 50% (P < 0.05). In addition, 8-OHdG positive cells were substantially attenuated by NGF treatment (P < 0.01).ConclusionsTaken together, this study indicates that NGF could inhibit TLR3-induced inflammatory cascades in HCECs, suggesting NGF as a potential therapeutic agent for HSK.

Mammalian germ cells are determined after PGC colonization of the nascent gonad

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

Genetic Deletion of Hesx1 Promotes Exit from the Pluripotent State and Impairs Developmental Diapause.

SummaryThe role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos.

PDTM-22. A C19MC-LIN28A-MYCN ONCOGENIC CIRCUIT DRIVEN BY HIJACKED SUPER-ENHANCERS IS A DISTINCT THERAPEUTIC VULNERABILITY IN ETMRS – A LETHAL BRAIN TUMOR

Abstract Embryonal tumors with multi-layered rosettes (ETMR) are aggressive brain cancers with characteristic C19MC oncomiR amplification and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a potent C19MC-LIN28A-MYCN circuitry driven by multiple regulatory loops and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. C19MC and LIN28A targets respectively converge on critical cell cycle tumor suppressors and neo-embryonic DNMT3A/B isoforms. We identify a MYCN driven, core transcriptional network, conserved in early neural stem cells, that is potently abrogated by treatment with bromodomain inhibitor JQ1, leading to ETMR cell death. Our collective data suggest C19MC oncomiRs drive a malignant primitive cell state in ETMRs via entrapment of an early neural lineage network, which represents a critical therapeutic vulnerability for this recalcitrant disease.

Generation of an induced pluripotent stem cell line (UBCi001-A) from a presymptomatic individual carrying the R418X progranulin gene mutation.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood-derived erythroid progenitor cells obtained from a presymptomatic female carrying the heterozygous R418X progranulin (GRN) nonsense mutation, known to cause autosomal dominant frontotemporal lobar degeneration. Erythroid progenitor cells were reprogrammed into iPSCs using integration free episomal plasmids which enables exogenous expression of the Yamanaka factors. The pluripotent potential of the iPSCs was validated through expression of pluripotency factors and their capacity to differentiate into the three primary germ layers. The cells were confirmed to carry the described mutation and shown to have a normal karyotype.

Chlorogenic acid inhibits esophageal squamous cell carcinoma growth in vitro and in vivo by downregulating the expression of BMI1 and SOX2

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China, accompanied by an extremely high mortality rate. Chlorogenic acid (CGA) is a small-molecule compound, that has been shown to have a wide range of biological activities, including antitumor. However, the efficacy and molecular mechanism of CGA on ESCC remains unknown. In this study, we confirmed the inhibition of proliferation by CGA in ESCC cells, as well as the reduction of ESCC xenograft volume by CGA in vivo. In addition, CGA also suppressed both the migration and invasion of ESCC cells in vitro. In a carcinogen-induced murine model of ESCC, hyperplasia of the esophagus was slowed by CGA, while mice suffering from ESCC that were treated with CGA had longer survival times than mice in the control group. The measurement of pluripotency factors (BMI1, SOX2, OCT4 and Nanog) that are related to poor prognosis revealed reduced expression of both BMI1 and SOX2, but not of OCT4 or Nanog, in ESCC cells, in both a dose- and time-dependent manner. Together, our initial findings demonstrate that CGA suppresses ESCC progression, downregulates the expression of BMI1 and SOX2, and provide an anti-tumor candidate for ESCC therapy.

Dynamic lineage priming is driven via direct enhancer regulation by ERK.

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.

Tex10 promotes stemness and EMT phenotypes in esophageal squamous cell carcinoma via the Wnt/β‑catenin pathway.

A previous study by our group suggested that testis expressed 10 (Tex10) contributes to tumor progression by promoting stem cell-like features in hepatocellular carcinoma. However, the relevance of pluripotency factor Tex10 in esophageal squamous cell carcinoma (ESCC) has remained elusive. The objective of the present study was to investigate the role of Tex10 in ESCC. For this purpose, the mRNA and protein expression of Tex10 was detected by reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry. In a loss-of-function experiment, EC109 cells were transfected with lentiviral vectors containing Tex10 short hairpin RNA or negative control. Cell proliferation was assessed using a Cell Counting kit-8, and flow cytometry was used to analyze apoptosis and the cell cycle. Transwell assays were employed to examine the migratory and invasive capacity, and a sphere formation assay was performed to assess the clonogenicity of the EC109 cells. The results revealed that the elevated expression of Tex10 was positively associated with malignancy and with epithelial-mesenchymal transition (EMT)-associated mesenchymal markers in human ESCC specimens. The knockdown of Tex10 led to the inhibition of cell proliferation, the induction of apoptosis and cell cycle arrest, and decreased the stemness, migratory and invasive capacity of the EC109 cells. Furthermore, the silencing of Tex10 enhanced the sensitivity of the ESCC cells to 5-fluorouracil. In addition, the present study revealed that Tex10 plays an essential role in regulating EMT via the activation of Wnt/β-catenin signaling. On the whole, the findings of the present study suggest that the downregulation of Tex10 in ESCC specimens is significantly associated with tumor malignancy, and that Tex10 promotes stem cell-like features and induces the EMT of ESCC cells through the enhancement of Wnt/β-catenin signaling.

TNF-α suppresses sweat gland differentiation of MSCs by reducing FTO-mediated m6A-demethylation of Nanog mRNA.

An effect of inhibition of tumor necrosis factor-α (TNF-α) on differentiation of mesenchymal stromal cells (MSCs) has been demonstrated, but the exact mechanisms that govern MSCs differentiation remain to be further elucidated. Here, we show that TNF-α inhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment, accompanied with reduced expression of Nanog, a core pluripotency factor. We elucidated that fat mass and obesity-associated protein (FTO)-mediated m6A demethylation is involved in the regulation of MSCs differentiation potential. Exposure of MSCs to TNF-α reduced expression of FTO, which demethylated Nanog mRNA. Reduced expression of FTO increased Nanog mRNA methylation, decreased Nanog mRNA and protein expression, and significantly inhibited MSCs capacity for differentiation to sweat gland cells. Our finding is the first to elucidate the functional importance of m6A modification in MSCs, providing new insights that the microenvironment can regulate the multipotency of MSCs at the post-transcriptional level. Moreover, to maintain differentiation capacity of MSCs by regulating m6A modification suggested a novel potential therapeutic target for stem cell-mediated regenerative medicine.

Cellular reprogramming by single-cell fusion with mouse embryonic stem cells in pig.

Fusion of differentiated somatic cells with pluripotent stem cells can be used for cellular reprogramming, but the efficiency to obtain hybrid cells is extremely low. Here, we explored a novel cell fusion system, termed single-cell fusion, the efficiency was significantly improved verified by fusion of mouse embryonic stem cells (mESCs), comparing to traditional polyethylene glycol fusion. Then, we employed the optimized system to perform cell fusion of porcine embryonic fibroblasts (PEFs) and porcine pluripotent stem cells (pPSCs) with mESCs. The hybrid cells showed both red and green fluorescence and expressed species-specific genes of mouse and pig to evidence that the fusion was successful. The hybrid cells displayed characteristics similar with mESCs, including colony morphology, alkaline phosphatase positive and formation of embryoid body, and the expressions of core pluripotent factors OCT4, NANOG, and SOX2 of the pig were induced in the mESC/PEF hybrid cells. The results indicate PEFs and pPSCs could be reprogrammed by mESCs via the single-cell fusion. Taking advantage of the hybrid cells to investigate the signaling pathways depended on the pluripotency of pig, we suggest the transforming growth factor-β signaling pathways may play important roles. In summary, the single-cell fusion is highly efficient, and we believe in the future it will be widely used in the application and fundamental research.