Factors In Embryonic Stem Cells Research Articles

Nonsense-mediated mRNA decay: a 'nonsense' pathway makes sense in stem cell biology.

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional regulatory mechanism of gene expression in eukaryotes. Originally, NMD was identified as an RNA surveillance machinery in degrading ‘aberrant’ mRNA species with premature termination codons. Recent studies indicate that NMD regulates the stability of natural gene transcripts that play significant roles in cell functions. Although components and action modes of the NMD machinery in degrading its RNA targets have been extensively studied with biochemical and structural approaches, the biological roles of NMD remain to be defined. Stem cells are rare cell populations, which play essential roles in tissue homeostasis and hold great promises in regenerative medicine. Stem cells self-renew to maintain the cellular identity and differentiate into somatic lineages with specialized functions to sustain tissue integrity. Transcriptional regulations and epigenetic modulations have been extensively implicated in stem cell biology. However, post-transcriptional regulatory mechanisms, such as NMD, in stem cell regulation are largely unknown. In this paper, we summarize the recent findings on biological roles of NMD factors in embryonic and tissue-specific stem cells. Furthermore, we discuss the possible mechanisms of NMD in regulating stem cell fates.

Growth and Neurotrophic Factors in Embryonic Stem Cells.

In this chapter we illustrate protocols to investigate growth and neurotrophic factors in human and rodent (rat and mouse)-derived embryonic stem cells. The conventional two-dimensional cell monolayer system to grow embryonic stem cells is presented, focusing on the coating strategies also using extracellular matrix components. Then, different approaches for three-dimensional stem cell culture are presented, using hydrogels and scaffolds. Quantitative polymerase chain reaction, immunocytochemistry, immunoenzymatic ELISA assay, and multiparametric assays to quantify growth and neurotrophic factor production are presented.

Abstract DPOC-001: CANCER STEM CELLS: THE SEEDS FOR RECURRENT OVARIAN CANCER

Abstract PURPOSE: The treatment of ovarian cancer (OC) with chemotherapy leaves resistant cancer cells which in a short time re–grow as recurrent cancer. A diverse array of resistance mechanisms for chemotherapy in tumor cells has been described but none has proven to be a viable target in a clinical setting. Cancer stem cells (CSCs) are increasingly accepted as the putative mediators of chemoresistance and relapse of cancer. This study aimed to understand the molecular mechanisms involved with chemoresistance and recurrence by investigating the roles of CSCs and their associated pathways in OC cell lines and tumor cells isolated from the ascites of OC patients. METHODS: Tumor cells were collected from chemonaive (CN) and recurrent (CR) OC patients diagnosed with advanced–stage serous OC using a novel in vitro method to obtain a distinct population of epithelial tumor cells. Flow cytometry and immunofluorescence were used to characterize the tumor population. High–resolution label–free quantitative proteomic profiling was used to define significantly differentially expressed proteins between CN and CR tumor cells. KEGG and DAVID software's were used to determine pathways associated with CR cells. The mechanisms of survival of in vitro cisplatin or paclitaxel treated ascites–derived tumor cells as well as cultured OC cell lines were determined by in vitro assays and in mouse xenografts. In another approach, the expression of embryonic stem cell factor Oct4A was determined in primary ovarian tumors and its functional role was investigated using in vitro assays and in vivo mouse models with stable knockdown (shRNA) of Oct4A in OC cell lines. RESULTS: Proteomic profiling of CN and CR tumor cells showed significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell–cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism in CR tumor cells. The treatment of ascites–derived OC cells with chemotherapy in vitro resulted in a CSC–like residual population with increased activation of JAK2/STAT3 pathway. Both JAK2/STAT3 activation and CSC–like characteristics were suppressed by a low dose JAK2 specific inhibitor, Momelotinib, in vitro and in vivo. This also resulted in a significantly reduced tumor burden and increased disease–free survival periods in mice in vivo. In another approach, stable knockdown of Oct4A resulted in the decreased expression of CSCs in OC cells and was consistent with decreased cell proliferation, migration and chemoresistance in vitro. In vivo Oct4A knockdown cells produced a significantly reduced tumor burden in mice resulting in a significantly increased disease–free survival periods compared to vector control cells. CONCLUSION: The above studies suggest that targeting the CSCs may prove a therapeutic option for advanced–stage OC patients. Citation Format: Nuzhat Ahmed, Emily Chan, Chantel Samardzija, Khalid Abubaker, Ardian Latifi, George Kannourakis & Jock Findlay. CANCER STEM CELLS: THE SEEDS FOR RECURRENT OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-001.

Stem cell markers in glioma progression and recurrence.

Aggressive cancer cells show histological similarities to embryonic stem cells. As differentiated cells can re-acquire pluripotency and self-renewal by transfection with the transcription factors OCT4, SOX2, KLF4 and MYC, with Nanog as readout for success, we comprehensively investigated their occurrence and frequency in human astrocytomas of different malignancy grades, primary and matched recurrent glioblastomas, short- and long-term glioblastoma cultures and glioma cell lines. Among astrocytomas, mRNA expression of OCT4, MYC and (less robust) KLF4 increased with malignancy, while in recurrent glioblastomas MYC expression slightly decreased. Correlation analysis revealed distinct positive correlation between distinct stem cell markers, and this effect was most prominent in the recurrent glioblastoma cohort. Insitu, embryonic stem cell factors were found also in more differentiated tumor regions. Respective cells were rarely actively proliferating and showed single or combined expression signatures, which, at least in parts, corresponded to observed positive correlations of mRNA expression. However, a 'master-marker' defining the complete glioma stem cell subset could not be confirmed. In glioma cell lines, long- and short-term cultures, embryonic markers were detected at comparable levels. Upon exposure to temozolomide, increased expression of KLF4 (and lesser Nanog and OCT4) was observed. Experimental intrinsic overexpression of SOX2, KLF4 or OCT4 did not affect the other stem cell factors. The embryonic stem cell factors comprehensively investigated in this project can control self-renewal and pluripotency, and therefore tumorigenicity. They should be considered for the development of future diagnostic and therapeutic strategies.

Abstract 2487: Nicotine-mediated regulation of Sox2 and stemness in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer deaths in both men and women, and is highly correlated with cigarette smoking. Nicotine, the addictive component of tobacco smoke, cannot initiate tumors, but can promote proliferation, migration, and invasion of cells in vitro and promote growth and metastasis of tumors in vivo. More recently, nicotine has been implicated in the promoting self-renewal of stem-like side-population cells from lung cancers. This subpopulation of cancer stem-like cells has been implicated in tumor initiation, generation of entire heterogeneous tumor population, metastasis, dormancy, and drug resistance. We have shown that nicotine promotes the expression of genes that promote stemness, such as Stem Cell Factor (SCF/c-kit ligand), in a specific receptor dependent fashion. Here we demonstrate that nicotine can induce the expression of embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties in non-small cell lung adenocarcinoma cells. High level of Sox2 expression also correlates with poor patient survival outcome in lung cancer, and metastatic lung adenocarcinoma shows higher levels of Sox2 expression than primary tumor or normal tissue, implicating Sox2 in this disease. Nicotine was found to induce Sox2 in A549 cells after 18 and 24 hours of stimulation, an effect which was diminished by 48 hours. This was seen at the mRNA and protein level as assessed by transient transfection and luciferase assay, real-time PCR, western blot, and immunofluorescence respectively. Experiments were conducted to elucidate the mechanism by which nicotine induces Sox2; it was found that siRNA mediated depletion of α7 nicotinic acetylcholine receptor (nAChR), the Hippo pathway effector Yap1, or the transcriptional repressor ID1 could abrogate nicotine-mediated induction of Sox2. Further, treatment with inhibitors of Src kinase or PI3K additionally abrogated the effect, suggesting these proteins play a role in nicotine-mediate induction of Sox2. Interestingly, nicotine could induce Sox2 expression in human mesenchymal stem cells (hMSCs) after 24 hours of stimulation as seen by immunofluorescence and real-time PCR, and this effect was abrogated by treatment with inhibitors to Src kinase, PI3K, Yap1, or α7 nAChR. Additionally, studies are under way to elucidate whether e-cigarettes or flavored tobacco, newer and less studied alternatives to traditional cigarettes, have similar effects on stemness. These studies can be expected to have a direct impact on our understanding of the molecular mechanisms involved in the initiation, growth, and metastasis of non-small cell lung cancer, especially in smokers and tobacco users. Citation Format: Courtney Schaal, Srikumar Chellappan. Nicotine-mediated regulation of Sox2 and stemness in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2487.

Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells.

Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs.

Abstract A32: PBX1, a transcriptional regulator, promotes stemness and chemoresistance in ovarian cancer.

Abstract Background: Cancer recurrence and chemoresistance contribute greatly to the high mortality of ovarian cancers; however, molecular players involved in these processes remain under-determined. Several theories have been proposed to account for the development of resistance to chemotherapeutic agents. One of the emerging concepts is tumor heterogeneity, which argues for the presence of different cell subpopulations in bulk tumor with varying degrees of tumor initiating potential and drug sensitivity. The fractions of cells with enhanced tumor initiating potential are referred to as cancer stem-like cells (CSC). These cells have defining “stemness” phenotypes, including unlimited cell division, repopulation by a minimum cell number and resistance to cytotoxic agents and irradiation. New Findings: To identify embryonic stem cell factors that are potentially involved in tumor recurrence and chemoresistance, we have screened expression levels of four of these factors by immunohistochemistry in pairs of matched recurrent and primary tumor tissues from 41 patients. Our data demonstrated that PBX1 is significantly up-regulated in recurrent/chemoresistant ovarian tumors. Moreover, PBX1 overexpression in ascites tumors correlates with shorter overall survival in post-chemotherapy ovarian cancer patients. When PBX1 is ectopically expressed, it promotes cancer stem cell-like phenotypes, including increased side population and ALDH1 activity, enhanced tumorigenicity at low cell density, and increased resistance to platinum-based therapy. In platinum-resistant cell lines that overexpress PBX1, silencing PBX1 expression using RNA interference sensitizes cells to platinum treatment and reduces their stem cell-like phenotypes. Gene expression and chromatin immunoprecipitation analyses identified PBX1 direct target genes involved in multi-drug resistance, stem cell maintenance, immunomodulation, and DNA damage response. In the TCGA ovarian cancer recurrent series, expression of several PBX1 direct target genes, including ABCA1, Nanog, BMP3, and ATM, is tightly associated with PBX1 expression, further supporting the tissue-level transcriptional regulation of these genes by PBX1. Conclusion: The above findings establish PBX1 as an upstream regulator of key functional networks that mediate cancer stem-like and drug resistant phenotypes. Studies on the hematopoietic system have indicated that PBX1 maintains a viable pool of quiescent stem cells. Recently, in a renal cell carcinoma system, chemotherapy has been shown to induce damage signaling and to stimulate cell division and repopulation of quiescent CSCs. We speculate that chemotherapy in ovarian cancer may induce comparable injury responses and trigger PBX1 signaling, which in turn may activate downstream networks that support CSC survival and create a microenvironment niche that is essential for CSC repopulation at the tissue level. Collectively, the association between PBX1 and a cascade of stemness pathways points to a potential Achilles's heel critical to responding to chemotherapy and developing chemoresistance and argues for the development of antagonists of PBX1 signaling as anticancer agents. Citation Format: Jin-Gyoung Jung, Tae-Hoen Kim, Emily Gerry, Jen-Chun Kuan, Ayse Ayhan, Ben Davidson, Ie-Ming Shih, Tian-Li Wang. PBX1, a transcriptional regulator, promotes stemness and chemoresistance in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A32.

Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

Tgif1 Counterbalances the Activity of Core Pluripotency Factors in Mouse Embryonic Stem Cells

Core pluripotency factors, such as Oct4, Sox2, and Nanog, play important roles in maintaining embryonic stem cell (ESC) identity by autoregulatory feedforward loops. Nevertheless, the mechanism that provides precise control of the levels of the ESC core factors without indefinite amplification has remained elusive. Here, we report the direct repression of core pluripotency factors by Tgif1, a previously known terminal repressor of TGFβ/activin/nodal signaling. Overexpression of Tgif1 reduces the levels ofESC core factors, whereas its depletion leads to the induction of the pluripotency factors. We confirm the existence of physical associations between Tgif1 and Oct4, Nanog, and HDAC1/2 and further show the level of Tgif1 is not significantly altered by treatment with an activator/inhibitor of the TGFβ/activin/nodal signaling. Collectively, our findings establish Tgif1 as an integral member of the core regulatory circuitry of mouse ESCs that counterbalances the levels of the core pluripotency factors in a TGFβ/activin/nodal-independent manner.

Regulation of Nucleosome Architecture and Factor Binding Revealed by Nuclease Footprinting of the ESC Genome.

Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.

Network plasticity of pluripotency transcription factors in embryonic stem cells.

Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.

A critical role of Oct4A in mediating metastasis and disease-free survival in a mouse model of ovarian cancer.

BackgroundHigh grade epithelial ovarian cancer (EOC) is commonly characterised by widespread peritoneal dissemination and ascites. Metastatic EOC tumour cells can attach directly to neighbouring organs or alternatively, maintain long term tumourigenicity and chemoresistance by forming cellular aggregates (spheroids). Cancer stem-like cells are proposed to facilitate this mechanism. This study aimed to investigate the role of Oct4A, an embryonic stem cell factor and known master regulator of pluripotency in EOC progression, metastasis and chemoresistance.MethodsTo investigate the expression of Oct4A in primary EOC tumours, IHC and qRT-PCR analyses were used. The expression of Oct4A in chemonaive and recurrent EOC patient ascites-derived tumour cells samples was investigated by qRT-PCR. The functional role of Oct4A in EOC was evaluated by generating stable knockdown Oct4A clones in the established EOC cell line HEY using shRNA-mediated silencing technology. Cellular proliferation, spheroid forming ability, migration and chemosensitivty following loss of Oct4A in HEY cells was measured by in vitro functional assays. These observations were further validated in an in vivo mouse model using intraperitoneal (IP) injection of established Oct4A KD clones into Balb/c nu/nu mice.ResultsWe demonstrate that, compared to normal ovaries Oct4A expression significantly increases with tumour dedifferentiation. Oct4A expression was also significantly high in the ascites-derived tumour cells of recurrent EOC patients compared to chemonaive patients. Silencing of Oct4A in HEY cells resulted in decreased cellular proliferation, migration, spheroid formation and increased chemosensitivity to cisplatin in vitro. IP injection of Oct4A knockdown cells in vivo produced significantly reduced tumour burden, tumour size and invasiveness in mice, which overall resulted in significantly increased mouse survival rates compared to mice injected with control cells.ConclusionsThis data highlights a crucial role for Oct4A in the progression and metastasis of EOC. Targeting Oct4A may prove to be an effective strategy in the treatment and management of epithelial ovarian tumours.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0417-y) contains supplementary material, which is available to authorized users.

Abstract 2085: Nicotine-mediated regulation of Sox2 and its implications in the biology of non-small cell lung adenocarcinoma stem-like cells

Abstract Nicotine, the addictive component of tobacco smoke, is known to promote proliferation, migration, and invasion of multiple cancer cells, including those of the lung and the pancreas. In addition, nicotine can promote the growth and metastasis of such cancers in mouse xenograft models. More recently, nicotine has also been implicated in promotion of cancer stem cell properties such as self-renewal. Our lab has shown that stimulation of lung cancer cells with nicotine results in activation of Src kinase in a β-arrestin-1 dependent manner, resulting in inactivation of the Rb tumor suppressor protein and subsequently the activation of E2F-mediated transcription of proliferative and survival genes. In addition to proliferative and survival genes, the E2F family of transcription factors has been shown to activate genes like matrix metalloproteinases that are involved in invasion and migration, indicating a role for E2F in the growth and metastasis of cancers downstream of nicotine. Our recent studies have shown that E2F1 can also induce genes which promote stemness, such as Stem Cell Factor (SCF/c-kit ligand), promoting nicotine-mediated enhancement of self-renewal of stem-like cells. In addition to these findings, here we demonstrate the ability of nicotine to induce the embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties, as well as the ability of E2F1 transcription factor to regulate Sox2 gene expression in non-small cell lung cancer cells. Western blot analysis revealed an induction of Sox2 protein at 18 and 24 hours post nicotine stimulation, which diminished by 48 hours. Similarly, quantitative real time PCR analysis showed an increase in Sox2 mRNA levels at 18 and24, hours post nicotine stimulation. Analysis of a 1500bp region of the human Sox2 gene promoter revealed multiple predicted E2F binding sites. Chromatin immunoprecipitation assays demonstrated E2F transcription factors to associate with the Sox2 promoter at multiple predicted binding sites. Transient transfection experiments further demonstrated the ability of E2F1 to induce the expression of a Sox2-luciferase reporter. These results suggest that nicotine may enhance lung cancer stem cell properties in part through the induction of Sox2, and this could potentially be mediated via E2F transcriptional activation. Further studies are underway to elucidate the impact of nicotine mediated induction of Sox2 on stemness, the underlying mechanism of activation, and the implications this has for non-small cell lung cancer. These studies can be expected to have a direct impact on our understanding of the molecular mechanism involved in the initiation, growth and metastasis of non-small cell lung cancer, especially in smokers. Citation Format: Courtney Schaal, Namrata Bora Singhal, Smitha Pillai, Jonathan Nguyen, Srikumar Chellappan. Nicotine-mediated regulation of Sox2 and its implications in the biology of non-small cell lung adenocarcinoma stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2085. doi:10.1158/1538-7445.AM2015-2085

Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells

During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.

Partial inhibition of differentiation associated with elevated protein levels of pluripotency factors in mouse embryonic stem cells expressing exogenous EGAM1N homeoprotein

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

Retroviral transcriptional regulation and embryonic stem cells: war and peace.

Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.

Abstract SY35-03: Novel anti-tumor activity of targeted LSD1 inhibition

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in development. LSD1 can interact with pluripotency factors in human embryonic stem cells and is essential for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 dysregulation plays a key role in cancer and LSD1 is overexpressed in multiple tumor types, including acute myeloid leukemia (AML). Recent literature suggests inhibition of LSD1 reactivates the all-trans retinoic acid differentiation pathway in AML. Together, these observations suggest LSD1 is an important regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552). GSK2879552 is a potent, selective, mechanism-based irreversible inhibitor of LSD1 currently in clinical development. Screening of over 150 cancer cell lines revealed that while many tumor types appear resistant to LSD1 inhibition, specific tumor types were uniquely sensitive. For example, AML cells have a distinct requirement for LSD1, and LSD1 inhibition results in a prodifferentiation effect in this setting. Preclinical characterization of the anti-tumor activities of GSK2879552 both in vitro and in vivo in AML as well as the solid tumor setting will be discussed. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Ryan G. Kruger. Novel anti-tumor activity of targeted LSD1 inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY35-03. doi:10.1158/1538-7445.AM2014-SY35-03

Abstract 5067: An expressed retrogene of master embryonic stem cell factor OCT4 alters prostate cancer susceptibility

Abstract Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24, but a causal gene and a mechanism for cancer predisposition has remained elusive. The locus harbors the POU5F1B retrogene and several non-coding RNA genes, and is also proximal to the MYC oncogene. The open reading frame of the retrogene is preserved with 96% identity to that of the parent POU5F1 gene on chromosome 6, encoding the master embryonic stem cell factor Oct4. We observed that 8q24 risk alleles are significantly correlated with reduced expression of the POU5F1B retrogene in normal prostate tissue. The alleles were not correlated with the expression of MYC or other flanking genes in normal prostate tissue. Predicted deleterious POU5F1B missense germline variants were also associated with risk of prostate cancer. Thus, two independent lines of genetic evidence support a potential causal role for POU5F1B in the transformation of normal prostate tissue. A plausible hypothesis of POU5F1B function is dysregulation of Oct4; POU5F1 is transcriptionally regulated by the encoded Oct4 transcription factor. The POU5F1B retrogene on 8q24 and POU5F1 on 6p21 were significantly co-expressed. These results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer. Note: This abstract was not presented at the meeting. Citation Format: Joan Breyer, Daniel Dorset, Travis Clark, Kevin Bradley, Tiina Wahlfors, Kate McReynolds, William Maynard, Sam Chang, Michael Cookson, Joseph Smith, Johanna Schleutker, William Dupont, Jeffrey R. Smith. An expressed retrogene of master embryonic stem cell factor OCT4 alters prostate cancer susceptibility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2014-5067

Sox2 acts as a transcriptional repressor in neural stem cells.

BackgroundThe transcription factor, Sox2, is central to the behaviour of neural stem cells. It is also one of the key embryonic stem cell factors that, when overexpressed can convert somatic cells into induced pluripotent cells. Although generally studied as a transcriptional activator, recent evidence suggests that it might also repress gene expression.ResultsWe show that in neural stem cells Sox2 represses as many genes as it activates. We found that Sox2 interacts directly with members of the groucho family of corepressors and that repression of several target genes required this interaction. Strikingly, where many of the genes activated by Sox2 encode transcriptional regulators, no such genes were repressed. Finally, we found that a mutant form of Sox2 that was unable to bind groucho was no longer able to inhibit differentiation of neural stem cells to the same extent as the wild type protein.ConclusionsThese data reveal a major new mechanism of action for this key transcription factor. In the context of our understanding of endogenous stem cells, this highlights the need to determine how such a central regulator can distinguish which genes to activate and which to repress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2202-15-95) contains supplementary material, which is available to authorized users.

In vitro ciliary development in an epithelial stem equivalent cell line M3E3/C3 derived from the fetal Syrian hamster lung

Failure in airway regeneration after injury may result in profound consequences like a disease with bronchiolitis obliterans. Re-building of ciliated cells is a good marker for resumed airway activity. We decided to investigate whether M3E3C3,a fetal hamster stem equivalent cell line, is capable of in vitro ciliogenesis. Immunoreactivity and qRT-PCR have revealed that this cell line expresses a stem cell marker Sox2 and another marker, Clara cell secretory protein (CCSP) specific for the stem cells in the bronchiolar region. An embryonic stem cell factor Oct4 was negative.