Expression Of Nanog Research Articles

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects

A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.

The Survey of the Fruit of Capparis spinosa Extracts Effects on the Expressional Profile of Essential Self-renewal Genes in MCF7 Cell Line

Background: Several drugs have been proposed for the treatment of breast cancer, but none has fully treated the disease so far. Methods: MCF7 cells were cultured in RPMI-1640 medium, consisting of different concentrations of aqueous-alcoholic extract of the fruit of Capparis spinosa (0.125, 0.25, 0.5, 1, 1.5, 2, 2.5, and 5 mg/mL) for 48 and 72 hours. MTT assay was used to determine the cell proliferation inhibition (IC50). The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin V/PI apoptosis detection kit. RT-PCR method was carried out to assess the fold changes of OCT4, NANOG, and SOX2 genes. Two-way ANOVA (tukeys) and t-test (repeated measure) were used for the statistical analysis of obtained data MTT assay and RT-PCR method, respectively. p < 0.05 was considered significant. Results: Our results have shown that cell death was induced by increasing fruit of Capparis spinosa extract concentration. IC50 was observed at 48 h culture period with 4.81 mg/mL, and 72 h with 2.72 mg/mL fruit extract, respectively. Flow cytometry results exhibited an obviously significant augmentation in apoptotic MF7 cells. According to RT-PCR findings on the fruit of Capparis spinosa extract-treated cells, the mean expression of OCT4, NANOG, and SOX2 genes decreased after 48 and 72 h of incubation with IC50 concentration compared to controls. Conclusion: The fruit of the Capparis spinosa species was able to decrease the expression of self-renewal genes in the MCF7 cell line. Therefore, the fruit of Capparis spinosa extract can be considered a promising candidate for the management of human breast cancer after clinical trials.

Mechanisms of Breast Cancer Stem Cell Specification and Self-Renewal Mediated by Hypoxia-Inducible Factor 1.

Many advanced human cancers contain regions of intratumoral hypoxia, with O2 gradients extending to anoxia. Hypoxia-inducible factors (HIFs) are activated in hypoxic cancer cells and drive metabolic reprogramming, vascularization, invasion, and metastasis. Hypoxia induces breast cancer stem cell (BCSC) specification by inducing the expression and/or activity of the pluripotency factors KLF4, NANOG, OCT4, and SOX2. Recent studies have identified HIF-1-dependent expression of PLXNB3, NARF, and TERT in hypoxic breast cancer cells. PLXNB3 binds to and activates the MET receptor tyrosine kinase, leading to activation of the SRC non-receptor tyrosine kinase and subsequently focal adhesion kinase, which promotes cancer cell migration and invasion. PLXNB3-MET-SRC signaling also activates STAT3, a transcription factor that mediates increased NANOG gene expression. Hypoxia-induced NARF binds to OCT4 and serves as a coactivator by stabilizing OCT4 binding to the KLF4, NANOG, and SOX2 genes and by stabilizing the interaction of OCT4 with KDM6A, a histone demethylase that erases repressive trimethylation of histone H3 at lysine 27, thereby increasing KLF4, NANOG, and SOX2 gene expression. In addition to increasing pluripotency factor expression by these mechanisms, HIF-1 directly activates expression of the TERT gene encoding telomerase, the enzyme required for maintenance of telomeres, which is required for the unlimited self-renewal of BCSCs. HIF-1 binds to the TERT gene and recruits NANOG, which serves as a coactivator by promoting the subsequent recruitment of USP9X, a deubiquitinase that inhibits HIF-1α degradation, and p300, a histone acetyltransferase that mediates acetylation of H3K27, which is required for transcriptional activation.

Pluripotency and embryonic lineage genes expression in the presence of small molecule inhibitors of FGF, TGFβ and GSK3 during pre-implantation development of goat embryos

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFβ inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2–7 post-insemination in goat.Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2.According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

β-CATENIN stabilizes HIF2 through lncRNA and inhibits intravenous immunoglobulin immunotherapy.

Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT). We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by β-CATENIN-downstream pathways. β-CATENIN activity protected TICs from IVIG effects. The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal. Inhibition of β-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both β-CATENIN and NANOG expression. The co-expression of constitutively active β-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through β-CATENIN-inducible lncRNAs EGLN3-AS1 and lnc-β-CatM. Co-expression of constitutively active β-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. β-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent β-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy. Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.

A functional crosstalk between the H3K9 methylation writers and their reader HP1 in safeguarding embryonic stem cell identity.

Histone H3 lysine 9 (H3K9) methylation, as a hallmark of heterochromatin, has a central role in cell lineage and fate determination. Although evidence of a cooperation between H3K9 methylation writers and their readers has started to emerge, their actual interplay remains elusive. Here, we show that loss of H3K9 methylation readers, the Hp1 family, causes reduced expression of H3K9 methyltransferases, and that this subsequently leads to the exit of embryonic stem cells (ESCs) from pluripotency and a reciprocal gain of lineage-specific characteristics. Importantly, the phenotypes of Hp1-null ESCs can be rescued by ectopic expression of Setdb1, Nanog, and Oct4. Furthermore, Setdb1 ablation results in loss of ESC identity, which is accompanied by a reduction in the expression of Hp1 genes. Together, our data support a model in which the safeguarding of ESC identity involves the cooperation between the H3K9 methylation writers and their readers.

The Stem Cell Expression Profile of Odontogenic Tumors and Cysts: A Systematic Review and Meta-Analysis.

Stem cells have been associated with self-renewing and plasticity and have been investigated in various odontogenic lesions in association with their pathogenesis and biological behavior. We aim to provide a systematic review of stem cell markers' expression in odontogenic tumors and cysts. The literature was searched through the MEDLINE/PubMed, EMBASE via OVID, Web of Science, and CINHAL via EBSCO databases for original studies evaluating stem cell markers' expression in different odontogenic tumors/cysts, or an odontogenic disease group and a control group. The studies' risk of bias (RoB) was assessed via a Joanna Briggs Institute Critical Appraisal Tool. Meta-analysis was conducted for markers evaluated in the same pair of odontogenic tumors/cysts in at least two studies. 29 studies reported the expression of stem cell markers, e.g., SOX2, OCT4, NANOG, CD44, ALDH1, BMI1, and CD105, in various odontogenic lesions, through immunohistochemistry/immunofluorescence, polymerase chain reaction, flow cytometry, microarrays, and RNA-sequencing. Low, moderate, and high RoBs were observed in seven, nine, and thirteen studies, respectively. Meta-analysis revealed a remarkable discriminative ability of SOX2 for ameloblastic carcinomas or odontogenic keratocysts over ameloblastomas. Stem cells might be linked to the pathogenesis and clinical behavior of odontogenic pathologies and represent a potential target for future individualized therapies.

NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency.

Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

Bazi Bushen alleviates skin senescence by orchestrating skin homeostasis in SAMP6 mice.

Bazi Bushen, a Chinese-patented drug with the function of relieving fatigue and delaying ageing, has been proven effective for extenuating skin senescence. To investigate the potential mechanism, senescence-accelerated mouse prone 6 (SAMP6) was intragastrically administered with Bazi Bushen for 9 weeks to induce skin homeostasis. Skin homeostasis is important in mitigating skin senescence, and it is related to many factors such as oxidative stress, SASP, apoptosis, autophagy and stem cell. In our study, skin damage in SAMP6 mice was observed using HE, Masson and SA-β-gal staining. The content of hydroxyproline and the activities of SOD, MDA, GSH-PX and T-AOC in the skin were measured using commercial assay kits. The level of SASP factors (IL-6, IL-1β, TNF-α, MMP2 and MMP9) in skin were measured using ELISA kits. The protein expressions of p16, p21, p53, Bax, Bcl-2, Cleaved caspase-3, LC3, p62, Beclin1, OCT4, SOX2 and NANOG were measured by western blotting. The expression of ITGA6 and COL17A1 was measured by immunofluorescence staining and western blotting. Our findings demonstrated that Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis, reducing the level of oxidative stress and the expression of SASP, regulating the balance of apoptosis and autophagy and enhancing the protein expressions of ITGA6 and COL17A1 to improve skin structure in SAMP6 mice. This study indicated that Bazi Bushen could serve as a potential therapy for alleviating skin senescence.

Nanog, Stat-3, and Sox-5 involvement in human fetal temporomandibular joint late development

Background and aimThe temporomandibular joint (TMJ) is a synovial joint that allows the complex movements essential for life. It connects the jawbone to the skull, working as a sliding hinge. Moreover, pluripotent stem cells are a source of precursors and tissue-specific cells in developing organisms, however, their biodistribution in developing fetal tissues is weakly studied. The aim of our study was analyse immunohistochemical expression of Nanog, Oct-4, Sox-2 and Stat-3 and Sox-5, in TMJ tissue samples from human fetuses aged between the 12th and 20th weeks of intrauterine life. Materials and methodsWe fixed and processed TMJ tissue samples from human fetuses, histological sections and immunohistochemical procedures were carried out. ResultsTMJ histological studies examination did not reveal any difference in the tissue organization between the samples in the studied periods. Immunohistochemical analysis demonstrated that Oct-4 and Sox-2 lack their expression in TMJ. In contrast, Nanog was expressed in nucleous of proliferative layer of mandibular condyle, Stat-3 was expressed in nuclear cells of articular disc, Stat-3 and Sox-5 showed positive nuclear and cytoplasmic immunostaining in codrocyte layers and in ossification areas. ConclusionsNanog acts in maintanence of pluripotency, Stat-3 in articular disc acts as a transcriptional factor. Stat-3 and Sox-2 act in chondrocyte and osteoblast diferentiation. Distribution of the cells, which express Nanog, Stat-3, and Sox-5 in TMJ tissue during fetal development, can help further understand its physiology, pathology, and repairing capacities.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

MPP8 Governs the Activity of the LIF/STAT3 Pathway and Plays a Crucial Role in the Differentiation of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) possess the remarkable characteristics of unlimited self-renewal and pluripotency, which render them highly valuable for both fundamental research and clinical applications. A comprehensive understanding of the molecular mechanisms underlying mESC function is of the utmost importance. The Human Silence Hub (HUSH) complex, comprising FAM208A, MPP8, and periphilin, constitutes an epigenetic silencing complex involved in suppressing retroviruses and transposons during early embryonic development. However, its precise role in regulating mESC pluripotency and differentiation remains elusive. In this study, we generated homogenous miniIAA7-tagged Mpp8 mouse ES cell lines. Upon induction of MPP8 protein degradation, we observed the impaired proliferation and reduced colony formation ability of mESCs. Furthermore, this study unveils the involvement of MPP8 in regulating the activity of the LIF/STAT3 signaling pathway and Nanog expression in mESCs. Finally, we provide compelling evidence that degradation of the MPP8 protein impairs the differentiation of mESC.

Caffeic Acid Inhibits Proliferation, Migration, and Stemness of DU-145 Prostate Cancer Cells

The issue of toxicity associated with existing cancer drugs necessitates the exploration of the anticancer potentials of natural products such as caffeic acid (CA). Here, we determined the effect of CA on the proliferation, migration, and stem cell-like properties of DU-145 prostate cancer cells. Tetrazolium-based colorimetric assay and flow cytometric analysis showed that CA decreased cell proliferation in a dose- and time-dependent manner without affecting cell cycle progression. CA also inhibited cell migration and repressed epithelial-to-mesenchymal transition by upregulating the expression of cadherin 1 (CDH1) and downregulating the expression of cadherin 2 (CDH2). Furthermore, CA reduced the cancer stem cell population from 95% to 63% and 47% at concentrations of 1.25 and 2.5 mg/mL, respectively; and inhibited stem cell-like properties by downregulating the expression of NANOG and octamer-binding transcription factor 4 (OCT4) genes. These findings suggest that CA could be considered in the development of improved chemotherapy against prostate cancer.

The Antiproliferative Activity of Adiantum pedatum Extract and/or Piceatannol in Phenylhydrazine-Induced Colon Cancer in Male Albino Rats: The miR-145 Expression of the PI-3K/Akt/p53 and Oct4/Sox2/Nanog Pathways.

Colon cancer is one of the most common types of cancer worldwide, and its incidence is increasing. Despite advances in medical science, the treatment of colon cancer still poses a significant challenge. This study aimed to investigate the potential protective effects of Adiantum pedatum (AP) extract and/or piceatannol on colon cancer induced via phenylhydrazine (PHZ) in terms of the antioxidant and apoptotic pathways and histopathologic changes in the colons of male albino rats. The rats were randomly divided into eight groups: control, AP extract, piceatannol (P), PHZ, PHZ and AP treatments, PHZ and P treatments, PHZ and both AP and P, and PHZ and prophylaxis with both AP and P. The results demonstrated that PHZ induced oxidative damage, apoptosis, and histopathological changes compared to the control group. However, the administration of AP or P or AP + P as therapy or prophylaxis significantly ameliorated these changes and upregulated the colonic mir-145 and mRNA expression of P53 and PDCD-4 while downregulating the colonic mRNA expression of PI3K, AKT, c-Myc, CK-20, SOX-2, OCT-4, and NanoG compared to the PHZ group. These findings suggest that the candidate drugs may exert their anti-cancer effects through multiple mechanisms, including antioxidant and apoptotic activities.

High NANOG expression correlates with worse patients’ survival in esophageal adenocarcinoma

BackgroundPatients diagnosed with esophageal cancer demonstrate a low overall survival even despite the established multimodal therapy as the current standard of care. Therefore, further biomarkers for patients with high-risk and additional therapy options are needed. NANOG is a transcription factor, which can be found in stem cells and is known to support tumorigenesis.MethodsSix hundred sixty patients with esophageal adenocarcinoma, who were operated at the University of Cologne with a curative intent, were included. Immunohistochemical stainings for NANOG were performed. The study population was divided into NANOG-positive and -negative subgroups.ResultsPositive NANOG expression correlates significantly with worse overall survival (p = 0.002) and could be confirmed as an independent risk factor for worse patient survival in multivariate analysis (HR = 1.40, 95%CI = 1.09–1.80, p = 0.006). This effect could be detected in the subgroup of primarily operated patients, but not in patients after neoadjuvant therapy.ConclusionsWe describe a NANOG-positive subgroup of patients with esophageal cancer, who exhibit worse overall survival in a large patient cohort. This discovery suggests the potential use of NANOG as a biomarker for both intensified therapy and stricter follow-up regimes. Additionally, NANOG-positive stem cell-like cancer cells could be used as a new antitumoral treatment target if validated in mechanistic and clinical studies.

The Combination of PD-L1 and CTLA-4 Suppression Significantly Decreased the Expression Levels of Cancer Stem Cell Factors in the Pancreatic Cancer Cell Line

Background: It is thought that a limited number of aberrant cancer stem cells (CSCs), which encourage carcinogenesis, tumor metastasis, and treatment resistance, are the cause of pancreatic cancer, a disease with a high mortality rate. Increasing evidence shows that CSCs use immunosuppressive properties to avoid immune system identification. In Pancreatic cancer, the therapeutic consequences of the relationship between the expression of immune checkpoints such as programmed death receptor ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the presence of CSCs are poorly known. As a result, in the present investigation, we examined how the expression levels of CSCs were affected by PD-L1 and CTLA-4 knockdown using specific siRNA. Methods: Independently and together, PD-L1 and CTLA-4 siRNA were transfected into MIA PaCa-2 cells. RNA extraction and cDNA synthesis were then performed. Finally, using qRT-PCR, the gene expression levels of CSCs markers, including Nanog, CD133, CD44, and Oct-4, in transfected groups were measured. Results: In the MIA PaCa-2 cell line, siRNA-mediated inhibition of PD-L1 and CTLA-4 decreased the expression of CSC factors. Moreover, combined suppression of these two stemness-related immune checkpoints significantly decreased Nanog, CD133, CD44, and Oct-4 expression compared to inhibition of PD-L1 and CTLA-4 separately. Conclusion: Considering that the combination of PD-L1 and CTLA-4 suppression using siRNA significantly decreased the expression levels of CSCs factors, this approach may be regarded as an effective therapy for this cancer.

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable

P-701 Human embryos diagnosed as mosaic and aneuploid after preimplantation genetic testing for aneuploidy are associated with increased levels of apoptosis in trophectoderm cells

Abstract Study question Are there cell lineage-related differences in the apoptotic rates of human blastocysts classified as euploid, mosaic and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer In human blastocysts, apoptosis is more frequent in trophectoderm (TE) cells and is associated with the presence of mosaic and uniform chromosomal alterations. What is known already Embryos diagnosed as mosaic after PGT-A can develop into healthy infants. However, the reason why these embryos achieve reproductive competence needs further research. One hypothesis is that aneuploid cells are negatively selected during embryo development through cell competition mechanisms, such as apoptosis or differential proliferation. While these mechanisms have been demonstrated in mouse embryos, in which apoptosis occurs more frequently in the inner cell mass (ICM), evidence in human embryos is scarce. In fact, only one previous study has shown an association between mosaicism and aneuploidy after PGT-A and increased levels of apoptosis, which was more frequent in TE cells. Study design, size, duration Prospective cohort study performing colocalization of cell-lineage and apoptotic expression markers by immunofluorescence (IF). A total of 53 vitrified human blastocysts with a previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 13 euploid embryos (n = 11 on D5; n = 2 on D6), n = 22 mosaic embryos (n = 16 on D5; n = 6 on D6), and n = 18 aneuploid embryos (n = 11 on D5; n = 7 on D6). All embryos were donated for research purposes. Participants/materials, setting, methods Following warming and re-expansion, blastocysts were fixed in 4% paraformaldehyde, processed for IF and imaged by confocal microscopy. Primary antibodies: 1:80 goat anti-human Nanog (AF1997, R&D-Systems), 1:100 mouse anti-human Gata3 (MAB6330, R&D-Systems) and 1:1000 rabbit anti-human Caspase-3 (NB10056113, Novus Biologicals). 165 μM DAPI was used for nuclei staining. Total cells: DAPI+ cells. TE: Gata3+ cells. ICM: Gata3-/Nanog+ cells. Incidence of apoptosis: % Caspase-3+ cells (per embryo). Data were analysed using Chi-square or ANOVA (p < 0.05 significant). Main results and the role of chance Total cell number was similar among euploid embryos (59.2±20.6 on D5; 78.5±0.7 on D6), mosaic embryos (49.6±15 on D5; 58.8±16.9 on D6) and aneuploid embryos (48.6±14.2 on D5; 59.6±23.5 on D6) (P > 0.05). Nanog and Gata3 expression evidenced the establishment of ICM and TE cell lineages during the blastocyst stage: while the proportion of Nanog+ cells decreased from 38.2% (755/1979) on D5 to 20.3% (188/927) on D6 (P < 0.0001), the proportion of Gata3+ cells increased from 77.8% (1540/1979) on D5 to 83.1% (770/927) on D6 (P < 0.01). The persistence of Nanog expression in TE cells was associated with the chromosomal status of the embryo, as evidenced by the significantly higher proportion of Gata3+/Nanog+ cells found in aneuploid embryos (27.9%=149/534 on D5; 4.6%=19/417 on D6) compared to mosaic embryos (13.1%=104/794 on D5; 3.4%=12/353 on D6) and euploid embryos (9.7%=63/651 on D5; 0%=0/157 on D6) (P < 0.05). The incidence of apoptosis in the TE was significantly higher in aneuploid embryos (45.9%±16.1 on D5; 49%±15.1 on D6) and mosaic embryos (44.1%±19.6 on D5; 43%±16.8 on D6), compared to euploid embryos (26.6%±16.6 on D5; 17.5%±14.8 on D6) (P < 0.05). Conversely, apoptosis was much less frequent in the ICM (2.2%±7.7), and no differences were found among the study groups (P > 0.05). Limitations, reasons for caution This is a descriptive, single-centre study with a limited sample size. The karyotype concordance between ICM and TE cells was not tested. Wider implications of the findings Our findings demonstrate that apoptosis is common in human blastocysts, particularly in TE cells. The increased apoptotic rates found in the TE of embryos with chromosomal abnormalities support its role as a mechanism to eliminate aneuploid cells. This mechanism may indeed be related to the reproductive potential of mosaic embryos. Trial registration number Not applicable

Suppression of NANOG Expression Reduces Drug Resistance of Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that harbors therapy-resistant cancer stem cells (CSCs). Due to the limited effectiveness of conventional chemotherapies and radiation treatments against CSCs, there is a critical need for the development of innovative therapeutic approaches. Our previous research revealed the significant expression of embryonic stemness genes, NANOG and OCT4, in CSCs, suggesting their role in enhancing cancer-specific stemness and drug resistance. In our current study, we employed RNA interference (RNAi) to suppress the expression of these genes and observed an increased susceptibility of CSCs to the anticancer drug, temozolomide (TMZ). Suppression of NANOG expression induced cell cycle arrest in CSCs, specifically in the G0 phase, and it concomitantly decreased the expression of PDK1. Since PDK1 activates the PI3K/AKT pathway to promote cell proliferation and survival, our findings suggest that NANOG contributes to chemotherapy resistance in CSCs through PI3K/AKT pathway activation. Therefore, the combination of TMZ treatment with RNAi targeting NANOG holds promise as a therapeutic strategy for GBM.