Endogenous Nanog Expression Research Articles

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods.

331 ABNORMAL DNA METHYLATION PATTERNS AND ALLELE-SPECIFIC EXPRESSION OF IMPRINTED GENES IN BOVINE-INDUCED PLURIPOTENT STEM CELLS

Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (NT) to oocytes and, more recently, through induction with pluripotency-related factors (iPS cells). However, the epigenetic reprogramming process that enables the derivation of both NT-derived cloned animals and iPS cells is usually incomplete, leading to unhealthy offspring and poorly reprogrammed iPS cell lines. These unfavourable outcomes result in part from abnormal genome DNA methylation that leads to aberrant gene expression patterns. For instance, differentially methylated regions (DMR) and monoalleleic expression of imprinted genes, essential for normal cellular commitment and early development, are thought to be severely disturbed by reprogramming techniques. Indeed, H19 and SNRPN, imprinted genes, were disturbed in bovine NT-derived embryos and fetuses. Herein we investigated whether the DMR and parent-of-origin expression of the imprinted genes H19 and SNRPN are also perturbed in iPS lines. To analyse the DMR methylation patterns and allelic expression of H19 and SNRPN using parental-specific polymorphisms, we derived multiple clones of bovine iPS (biPS) cells from an interspecies (Bos indicus × Bos taurus) fetal fibroblast (bFF) using transduction with a policystronic lentivirus containing mouse Oct4, Sox2 c-Myc, and Klf-4 transcription factors. The DNA methylation patterns were evaluated by bisulfite sequencing and allelic expression by designing allele-specific PCR probes. We also quantified transcript expression by RT-PCR of H19, IGF2, SNRPN, OCT4, and NANOG by normalization with 3 housekeeping genes (GAPDH, NAT1, and ACTB). The biPS lines were characterised by a high nuclear : cytoplasmic ratio, dome-shaped colonies, positive AP activity, embryoid body formation, in vitro and in vivo (teratoma) formation, and expression of pluripotency-related genes. Compared to the bFF cells, methylation analyses of H19 showed partial hypomethylation of the paternal DMR on 1 iPS cell line and partial demethylation of the CTCF-binding region in the DMR of 2 other biPS lines, indicating abnormal demethylation of 3 out of the 4 biPS lines analysed. Methylation analyses of SNRPN revealed a partial hypomethylation in the maternal DMR and partial hypermethylation of the paternal DMR in 2 iPS lines. Gene expression analyses revealed the biallelic expression of H19 and decreased global expression of both H19 and IGF2, as well as the exclusively monoallelic paternal expression and significant increase in global expression of SNRPN. Interestingly, although OCT4 was substantially overexpressed in biPS lines, we identified a hypermethylation of the CG-rich region of the OCT4 exon 1. Endogenous NANOG expression was observed in 2 biPS clones. We conclude that imprinting errors are observed in biPS clones, suggesting that these epigenetic anomalies are related to the reprogramming process and could be directly responsible for the variable phenotypes and low success rates of both cloning and iPS derivation procedures.Financial support was from NSERC, FAPESP (13/13686-8, 11/08376-4, 57877-3/2008, 08.135-2/2013), CNPq (573754/2008-0, 482163/2013-5).

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal.

Single-Cell Analysis Reveals that Expression of Nanog Is Biallelic and Equally Variable as that of Other Pluripotency Factors in Mouse ESCs

The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in embryonic stem cell (ESC) self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Gata4 Blocks Somatic Cell Reprogramming By Directly Repressing Nanog

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of the four factors Oct4, Klf4, Sox2, and Myc. Here, we investigated the role of Gata4 in the reprogramming process and present evidence for a negative role of this family of transcription factors in the induction of pluripotency. Coexpression of Gata4 with Oct4, Klf4, and Sox2 with or without Myc in mouse embryonic fibroblasts greatly impaired reprogramming and endogenous Nanog expression. The lack of Nanog upregulation was associated with a blockade in the transition from the initiation phase of reprogramming to the full pluripotent state characteristic of iPS cells. Addition of Nanog to the reprogramming cocktail blocked the deleterious effects observed with Gata4 expression. Downregulation of endogenous Gata4 by short hairpin RNAs during reprogramming both accelerated and increased the efficiency of the process and augmented the mRNA levels of endogenous Nanog. Using comparative genomics, we identified a consensus binding site for Gata factors in an evolutionary conserved region located 9 kb upstream of the Nanog gene. Using chromatin immunoprecipitation, gel retardation, and luciferase assays, we found that Gata4 bound to this region and inhibited Nanog transcription in mouse embryonic stem cells. Overall, our results describe for first time the negative effect of Gata4 in the reprogramming of somatic cells and highlight the role of Gata factors in the transcriptional networks that control cell lineage choices in the early embryo.

Abstract LB-292: NANOG promotes chemoresistance in prostate carcinoma cells

Abstract NANOG promotes chemoresistance in prostate carcinoma cells Hongmei Jiang, Man-Tzu Wang, Daotai Nie The emergence of drug resistant prostate cancer cells is a leading cause of chemotherapy failure in patients with prostate cancer. The mechanisms behind the acquisition of chemoresistance in prostate cancers are, however, poorly understood. NANOG is a transcription factor associated with the self-renewal of embryonic stem cells. Expression of NANOG has been detected in a number of cancer cells, and its expression linked with those positive for stem/progenitor markers. In the present studies, several lines of evidence are presented to suggest that NANOG regulates tumor responses to chemotherapy. First, in the surviving fractions of tumor cells after chemotherapy, there was an elevated level of NANOG expression and activities. Second, prospective enrichment of tumor cells with endogenous NANOG expression led to enrichment of tumor cells resistant to taxol, vinblastine, and doxorubicin. Knockdown of NANOG expression via small hairpin RNAs (shRNAs) sensitized tumor cells toward chemotherapeutics. Real-time PCR array identified an enhanced expression of the ATP-binding cassette efflux transporter B1 (ABCB1) and G2 (ABCG2) in tumor cells enriched with endogenous NANOG expression. Inhibition of ABCB1 activities or knockdown of ABCB1 expression attenuated, but not abolished, the increased resistance in NANOG expressing cells toward taxol and vinblastine. Knockdown of ABCG2 attenuated the resistance of NANOG-expressing tumor cells toward doxorubicin. Together, our results suggest that NANOG confers tumor cells with increased resistance to chemotherapy partially through regulating the expression of ABC transporters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-292. doi:1538-7445.AM2012-LB-292

Reprogramming human endometrial fibroblast into induced pluripotent stem cells

ObjectiveA recent breakthrough demonstrated that ectopic expression of four genes is sufficient to reprogram human fibroblasts into inducible pluripotent stem cells (iPSCs). However, it remains unknown whether human endometrial fibroblasts (EMFs) are capable of being reprogrammed into EMF-derived iPSCs (EMF-iPSCs). MethodsEMFs were obtained from donors in their third and fourth decade of life and were reprogrammed into iPSCs using retroviral transduction with Oct-4, Sox2, Klf4, and c-Myc. ResultsThe EMF-iPSCs displayed the accelerated expression of endogenous Nanog and OCT-4 during reprogramming compared with EMFs. As a result, EMF-iPSC colonies that could be subcultured and propagated were established as early as 12 days after transduction. After 2 weeks of reprogramming, the human endometrial cells yielded significantly higher numbers of iPSC colonies and formed more 3D spheroid bodies than the EMFs. We have shown that human EMF-iPSCs are able to differentiate into neuronal-like cells, adipocytes, and osteocyte-like cells that express specific osteogenic genes. ConclusionHuman EMFs can undergo reprogramming to establish pluripotent stem cell lines in female donors by the retroviral transduction of Oct-4, Sox2, Klf4, and c-Myc.

RNA interference-mediated silencing of NANOG reduces cell proliferation and induces G0/G1 cell cycle arrest in breast cancer cells

Since the processes of normal embryogenesis and neoplasia share many of similar pathways, tumor development has been interpreted as an abnormal form of organogenesis. NANOG is a homeodomain-containing transcription factor that functions to maintain self-renewal and proliferation of embryonic stem cells (ESCs). Aberrant expression of NANOG has been observed in many types of human malignancies. However, its potential implication in tumorigenesis has not been fully clarified. In this study, we have employed small interference RNA (RNAi) technology to silence endogenous NANOG expression in breast cancer cells and successfully selected three independent clones with stably inhibited NANOG expression of MCF-7 cells. Functional analysis revealed that down-regulation of NANOG reduced cell proliferation, colony formation and migration ability of MCF-7 cells. Consistently, proliferation of breast cancer MDA-MB-231 cells was also significantly inhibited after the knockdown of NANOG expression. Interestingly, we found that the expression levels of cyclinD1 and c-myc were markedly down-regulated and the cell cycle were blocked at the G0/G1 phases after the knockdown of NANOG, while the expression of cyclinE and signal transducers and activators of transcription3 (STAT3) remained unaffected. In addition, the expression of NANOG and cyclinD1 can be rescued after the transfection of pcDNA3.1 (-)-NANOG expression vector into the three clones. Finally, our chromatin immunoprecipitation (ChIP) experiment showed that NANOG protein can bind to the promoter region of cyclinD1 and regulate cells cycle. Taken together, our findings may not only establish a molecular basis for the role of NANOG in modulating cell cycle progression of breast cancer cells but also suggest a potential target for the treatment of at least some subtypes of breast cancer.

Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos

Nanog as an important transcription factor plays a pivotal role in maintaining pluripotency and in reprogramming the epigenome of somatic cells. Its ability to function on committed somatic cells and embryos has been well defined in mouse and human, but rarely in pig. To better understand Nanog's function on reprogramming in porcine fetal fibroblast (PFF) and nuclear transfer (NT) embryo, we cloned porcine Nanog CDS and constructed pcDNA3.1 (+)/Nanog and pEGFP-C1/Nanog overexpression vectors and transfected them into PFFs. We studied the cell biological changes and the expression of Nanog, Oct4, Sox2, Klf4, C-myc, and Sall4 in transfected PFFs. We also detected the development potential of the cloned embryos harboring Nanog stably overexpressed fibroblasts and the expression of Oct4, Sox2, and both endogenous and exogenous Nanog in these embryos. The results showed that transient overexpression Nanog in PFF could activate the expression of Oct4 (5-fold), C-myc (2-fold), and Sall4 (5-fold) in somatic cells, but they could not be maintained during G418 selection. In NT embryos, although Nanog overexpression did not have a significant effect on blastocyst development rate and blastocyst cell number, it could significantly activate the expression of endogenous Nanog, Oct4, Sox2 to 160-fold, 93-fold, and 182-fold, respectively (P < 0.05). Our results demonstrate that Nanog could interact with and activate other pluripotent genes both in PFFs and embryos.

Abstract 4379: Role of Nanog in resistance of prostate cancer cells toward chemotherapy

Abstract Drug resistance is a major barrier to efficacious chemotherapy. Cancer stem cells (CSCs) may pose resistance to chemo- or radio-therapy, but the mechanism remains unknown. Nanog is a transcription factor essential for self-renewal of embryonic stem cells. The expression of Nanog has been reported in some cancer cells but its role in resistance to chemotherapy has not been convincingly demonstrated. Herein, we report that Nanog is expressed in tumorigenic prostate cancer cells and further it contributes to chemoresistance. Nanog expression was detected in prostate cancer cells by Western blot and RT-PCR and confirmed by sequencing. In the surviving fractions of prostate cancer cells after chemo-treatments, we found increased levels of Nanog expression and activities when compared to vehicle control-treated group. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells had enhanced endogenous Nanog expression, increased positivity for stem/progenitor markers (CD44 and CD133), and presented increased resistance to Taxol, vinblastine, and doxorubicin, but not camptothecin (CPT). In addition, Nanog-enriched DU145 cells showed reduced apoptosis in the response to Taxol treatment, whereas control cells had increased activation of effector caspases after treatment with Taxol. Colony formation assay revealed that prostate tumor cells with enriched Nanog expression displayed increased recurrent ability after chemotherapy. Knockdown of Nanog by tet-inducible shRNA sensitized prostate tumor cells to multiple drug treatments, suggesting its essential role in prostate cancer chemo-resistance. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA levels of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities or knockdown of ABCB1 expression sensitized Nanog expressing cells toward Taxol and vinblastine, but not doxorubicin. Knockdown of ABCG2 sensitized Nanog expressing cells toward doxorubicin, and to lesser extent, vinblastine, but not Taxol. Knockdown of Nanog reduced the expression of ABCB1 and ABCG2 and reduced the resistance of tumor cells toward chemotherapy. In summary, Nanog expression is associated with tumor cells positive for stem/progenitor markers and resistant to chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to resist chemotherapy via regulating the expression of ABC transporters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4379. doi:10.1158/1538-7445.AM2011-4379

Human Endometrial Cells Express Elevated Levels of Pluripotent Factors and Are More Amenable to Reprogramming into Induced Pluripotent Stem Cells

The human endometrium is a tissue with remarkable plasticity and regenerative capacity. Additionally, endometrial cells can be retrieved using minimally invasive procedures, which makes them an ideal source for reprogramming into a pluripotent state. Endometrial cells were obtained from donors in their fifth decade and reprogrammed into induced pluripotent stem (iPS) cells using retroviral transduction with SOX2, OCT4, KLF4, and MYC. The human endometrial cells displayed accelerated expression of endogenous NANOG and OCT4 during reprogramming compared with neonatal skin fibroblasts. As a result, iPS cell colonies that could be subcultured and propagated were established as early as 12 d after transduction rather than the usually reported 3-4 wk for other cell types. After 3 wk of reprogramming, the human endometrial cells also yielded significantly higher numbers of iPS colonies in comparison with the neonatal skin fibroblasts. Although the efficiency of iPS colony formation varied depending on the donor, the basal level of endogenous expression of the defined factors was positively correlated with reprogramming efficiency. The reprogramming resulted in an average colony-forming efficiency of 0.49 ± 0.10%, with a range from 0.31-0.66%, compared with the neonatal skin fibroblasts, resulting in an average efficiency of 0.03 ± 0.00% per transduction, with a range from 0.02-0.03%. Our studies show that the human endometrium expresses elevated levels of pluripotent factors, which with additional defined factors, results in significantly more efficient and accelerated generation of induced pluripotent stem cells compared with conventional somatic cells.

Abstract 3330: Nanog, cancer stem cells, and resistance to chemotherapy

Abstract It has been increasingly appreciated that cancer stem cells are responsible for initiating tumor formation as well as resistance to chemoradiotherapy, but the mechanisms involved remain unknown. Nanog is a transcription factor essential for self-renewal of embryonic stem cells. The expression of Nanog has been reported in some cancer cells but its role in tumor initiation and resistance to chemotherapy has not been convincingly demonstrated. Herein, we report that Nanog is expressed in tumorigenic prostate cancer cells and further it plays an essential role of Nanog in tumor formation and resistance to chemotherapy. Enrichment for Nanog-expressing tumor cells led to an increase in the number of cells positive for stem/progenitor markers, increased clonogenic growth, and increased ability to form prostaspheres. When injected into mice, enriched Nanog-expressing cells formed tumors earlier and formed larger tumors than the vector control cells did. Knockdown of Nanog in tumorigenic cancer cells, via small hairpin RNAs, reduced clonogenic growth, stimulated epithelial differentiation, and significantly compromised their tumor initiating ability. Prospective enrichment of tumor cells with endogenous Nanog expression also led to enrichment of tumor cells resistant to Taxol, vinblastine, and doxorubicin, but not camptothecin (CPT). The increased resistance in Nanog expressing cells was attenuated, but not abolished, by inhibitors of p-glycoprotein, the ATP-bind cassette efflux transporter B1 (ABCB1), or by knockdown of ABCB1 via small hairpin RNAs (shRNAs). Expression of ABCB1 was increased in tumor cells enriched with endogenous Nanog expression while knockdown of Nanog reduced the expression of ABCB1 and sensitized tumor cells toward chemotherapeutics. Taken together, the data from gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to self-renew or propagate, to initiate tumor formation, and to resist chemotherapy via regulating the expression of ABC transporters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3330.

Abstract A160: Regulation of resistance of prostate cancer cells to chemotherapy by nanog, a transcription factor for self-renewal of stem cells

Abstract Nanog is a transcription factor usually expressed in embryonic stem cells that plays essential role in the stem cell self-renewal. Expression of Nanog has been detected in a number of cancer cells, especially in those positive for stem/progenitor markers. In the present studies, it was found that there was elevated expression of Nanog in the surviving fractions of tumor cells after chemotherapy. Prospective enrichment of tumor cells with endogenous Nanog expression led to enrichment of tumor cells resistant to Taxol, vinblastine, and doxorubicin, but not camptothecin (CPT). The increased resistance in Nanog expressing cells was attenuated, but not abolished, by inhibitors of p-glycoprotein, the ATP-bind cassette efflux transporter B1 (ABCB1), or by knockdown of ABCB1 via small hairpin RNAs (shRNAs). Expression of ABCB1 was increased in tumor cells enriched with endogenous Nanog expression while knockdown of Nanog reduced the expression of ABCB1 and sensitized tumor cells toward chemotherapeutics. Together, our results suggest that Nanog confers tumor cells with increased resistance to chemotherapy via regulating the expression of ABC transporters. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A160.

Regulation of the Pluripotency Marker Rex-1 by Nanog and Sox2

Rex-1 (Zfp-42) is a known marker for undifferentiated embryonic stem cells and teratocarcinoma cells. However, the mechanism by which Rex-1 is regulated in pluripotent cells remains unresolved. Here we report that Nanog, an Nk-2 homeodomain protein known for its role in maintaining stem cell pluripotency, is a transcription activator for the Rex-1 promoter. Knockdown of Nanog in embryonic stem cells resulted in a reduction of Rex-1 expression, whereas forced expression of Nanog in P19 stimulated Rex-1 expression. Employing a Rex-1 reporter, we demonstrate that Nanog transactivates Rex-1 directly. Serial deletion studies mapped the Nanog-responsive element between -187 and -286 of the Rex-1 promoter. Although Oct-3/4 and Sox2 can both transactivate Rex-1 promoter, only Sox2 cooperates with Nanog in up-regulating Rex-1. Furthermore, we demonstrate that the C terminus of Nanog is responsible for transactivating the Rex-1 promoter, a function that can be substituted for by a viral transactivator Vp16 efficiently in NIH3T3 cells but less so in P19 cells. Taking these findings together, we conclude that Rex-1 is a direct target of Nanog, which is augmented by Sox2 and Oct-3/4.