RNAi mediated silencing of Nanog expression suppresses the growth of human colorectal cancer stem cells.

Colorectal cancer (CRC) is the third most common cancer in the world known for its poor recurrence-free prognosis. Previous studies have shown that it is closely linked with cancer stem cells (CSCs), which have self-renewal potential and the capacity to differentiate into diverse populations. Nanog is an important transcription factor that functions to maintain the self-renewal and proliferation of embryonic stem cells; however, many recent studies have shown that Nanog is also highly expressed in many cancer stem cells. To investigate whether Nanog plays a crucial role in maintaining the stemness of colorectal CSCs, RNA interference was used to downregulate Nanog expression in the CRC stem cell line, EpCAM+CD44+HCT-116 cells (CCSCs). We examined the anti-tumor function of Nanog in vitro and in vivo, using small interfering RNA. Our results revealed that the Nanog mRNA expression level in CCSCs was higher than that in HCT-116 cells. We found that the depletion of Nanog inhibited proliferation and promoted apoptosis in CCSCs. In addition, the invasive ability of CCSCs was markedly restricted when Nanog was silenced by small interfering RNA. Furthermore, we found that the silencing of Nanog decreased tumor size and weight and improved the survival rate of tumor-bearing mice. In conclusion, these findings collectively demonstrate that Nanog, which is highly expressed in CRC stem cells, is a key factor in the development of tumor growth, and it may serve as a potential marker of prognosis and a novel and effective therapeutic target for the treatment of CRC.

Nanog promotes stem-like traits of glioblastoma cells.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor progrnosis and a high recurrence rate after surgery. To this end, we examined the role of Nanog that is highly expressed in this tumor. NANOG is a transcription factor involved in the pluripotency of embryonic stem cells (ESCs) and the induction of malignancy in cancer stem-like cells (CSCs). Bioinformatic analysis revealed that NANOG may be associated with the development of stem-like traits in GBM. Forced expression of NANOG markedly increased the expression of cancer stem cell markers and promoted the sphere formation and migration of GBM cells. Nanog enhanced the expression of SHH which is required for the maintenance of the positive feedback loop of Hedgehog signaling pathway. Treatment of GBM cells with SANT-1 and GANT61 significantly reduced the tumor progression. These data support a view that reduction of Nanog might have therapeutic benefits in GBM.

MicroRNAs organize intrinsic variation into stem cell states.

Pluripotent embryonic stem cells (ESCs) contain the potential to form a diverse array of cells with distinct gene expression states, namely the cells of the adult vertebrate. Classically, diversity has been attributed to cells sensing their position with respect to external morphogen gradients. However, an alternative is that diversity arises in part from cooption of fluctuations in the gene regulatory network. Here we find ESCs exhibit intrinsic heterogeneity in the absence of external gradients by forming interconverting cell states. States vary in developmental gene expression programs and display distinct activity of microRNAs (miRNAs). Notably, miRNAs act on neighborhoods of pluripotency genes to increase variation of target genes and cell states. Loss of miRNAs that vary across states reduces target variation and delays state transitions, suggesting variable miRNAs organize and propagate variation to promote state transitions. Together these findings provide insight into how a gene regulatory network can coopt variation intrinsic to cell systems to form robust gene expression states. Interactions between intrinsic heterogeneity and environmental signals may help achieve developmental outcomes.

Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells.

Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs.

The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1.

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.

Rabbit induced pluripotent stem cells retain capability of in vitro cardiac differentiation.

Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.

Functional characterization of NANOG in goat pre-implantation embryonic development.

Nanog as a novel pluripotent cell-specific gene plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in inner cell mass (ICM) and embryonic stem cells (ESC) in mouse. The molecular features and transcription regulation of NANOG gene in domestic animals are not well defined. In this study, we performed knockdown of NANOG mRNA in goat embryos and examined its effect on early embryonic development. Presumptive zygotes were injected with a volume of 8-10 pl NANOG or scrambled (SCR) siRNA, and subsequently cleavage and blastocyst formation rate were assessed. Furthermore, gene expression analysis was carried out in 6-8 cell and blastocyst derived embryos from non-injected controls, SCR - and siRNA-injected presumptive zygotes. Cleavage and blastocyst rates in siRNA groups were insignificantly lower than the control and SCR groups. Embryos with reduced expression of NANOG showed decrease in number of trophectoderm (TE) and total cells in blastocysts. Analysis of expression of developmentally important genes (SOX2, OCT4 and NANOG), which work as a network, showed that NANOG knockdown results in significant increase in expression of SOX2 and OCT4 and among the possible target genes (CDX2, REX1 and GATA4) of this network, only GATA4 showed increased expression. Our results suggest that NANOG is likely to be required for proliferation of trophoblastic cells.

Stem Cell Therapies for Neurodegenerative Diseases.

Stem cell therapies have been proposed as a treatment option for neurodegenerative diseases, but the best stem cell source and therapeutic efficacy for neuroregeneration remain uncertain. Embryonic stem cells (ESCs) and neural stem cells (NSCs), which can efficiently generate neural cells, could be good candidates but they pose ethical and practical issues. Not only difficult to find the good source of those cells but also they alway pose immunorejection problem since they may not be an autologous cells. Even if we overcome the immunorejection problem, it has also been reported that transplantation of ESCs develop teratoma. Although adult stem cells are more accessible, they have a limited developmental potential. We developed technologies to increase potency of mesenchymal stem cells, which allow them to develop into neural cells, by over expression of the ESC gene, nanog. We also developed a small molecule compound, which significantly increases endogenous NSCs by peripheral administration, eliminating even the necessity of stem cell injection to the brain. These novel technologies may offer neuroregenerative therapies for Alzheimers disease (AD). However, we found that AD pathological condition prevent neurogenesis from NSCs. This chapter discusses how to overcome the problem associated stem cell therapy under AD pathology and introduces exosome as a tool to improve the modification of adult stem cells. These new technologies may open a door for the new era for AD therapy.

Nanog induces suppression of senescence through downregulation of p27KIP1 expression.

A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated ß-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27(KIP1) (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27(KIP1) gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene.

The significance of the pluripotency and cancer stem cell-related marker NANOG in diagnosis and treatment of ovarian carcinoma.

Ovarian cancer is among the most common gynecologic cancers and unfortunately the most common cause of death from gynecologic malignancies. Due to few early symptoms and insufficient screening programs, an early diagnosis of ovarian cancer is very difficult and new biomarkers related to early ovarian carcinogenesis are needed. In the last years a growing scientific knowledge about cancer stem cells and their markers opened a new perspective on screening and early diagnosis of ovarian cancer. The transcription factor NANOG is not only a pluripotency and cancer stem cell-related marker, but also promotes cancer stem cell-like characteristics of tumor, tumor growth, dissemination, immune evasion, and resistance to conventional therapy. The recent data showed that small stem cells resembling very small embryonic-like stem cells are present in the ovarian surface epithelium of adult human ovaries. These cells expressed several genes related to primordial germ cells, germinal lineage, and pluripotency, including NANOG, therefore their involvement in the manifestation of ovarian cancer are not excluded. As majority of cancer cells within a tumor are non tumorigenic, the therapies targeting these cells cause tumor regression, but the survived cancer stem cells regenerate the tumor, so tumor relapse or reoccur. The eradication of cancer actually requires the elimination of cancer stem cells, therefore new strategies in treatment that specifically target cancer stem cells are urgently needed. Although the therapeutic efficacy of targeting NANOG as a cancer treatment method is still in experimental phase, the gene therapy with small interfering RNA or short hairpin RNA have already shown some promising therapeutic potential. The authors can conclude that NANOG represents a promising diagnostic marker and agent for target therapy of ovarian cancer.