NANOG Protein Expression Research Articles

Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3–5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.

KLF4 and PBX1 Directly Regulate NANOG Expression in Human Embryonic Stem Cells

Insight into the regulation of core transcription factors is important for a better understanding of the molecular mechanisms that control self-renewal and pluripotency of human ESCs (hESCs). However, the transcriptional regulation of NANOG itself in hESCs has largely been elusive. We established a NANOG promoter luciferase reporter assay as a fast read-out for indicating the pluripotent status of hESCs. From the functional cDNA screens and NANOG promoter characterization, we successfully identified a zinc finger transcription factor KLF4 and a homeodomain transcription factor PBX1 as two novel transcriptional regulators that maintain the pluripotent and undifferentiated state of hESCs. We showed that both KLF4 and PBX1 mRNA and protein expression were downregulated during hESC differentiation. In addition, overexpression of KLF4 and PBX1 upregulated NANOG promoter activity and also the endogenous NANOG protein expression in hESCs. Direct binding of KLF4 on NANOG proximal promoter and PBX1 on a new upstream enhancer and proximal promoter were confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assay. Knockdown of KLF4/PBX1 or mutation of KLF4/PBX1 binding motifs significantly downregulated NANOG promoter activity. We also showed that specific members of the SP/KLF and PBX family are functionally redundant at the NANOG promoter and that KLF4 and PBX1 cooperated with OCT4 and SOX2, and transactivated synergistically the NANOG promoter activity. Our results show two novel upstream transcription activators of NANOG that are functionally important for the self-renewal of hESC and provide new insights into the expanded regulatory circuitry that maintains hESC pluripotency.

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro. AF was harvested during routine pre-natal amniocentesis at 14-16 weeks of pregnancy. AF sample pellets were plated in alpha-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction. Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na(+) channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na(+) channels. These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.

Overexpression of NANOG in Gestational Trophoblastic Diseases: Effect on Apoptosis, Cell Invasion, and Clinical Outcome

Gestational trophoblastic disease includes choriocarcinoma, a frankly malignant tumor, and hydatidiform mole (HM), which often leads to the development of persistent gestational trophoblastic neoplasia and requires chemotherapy. NANOG is an important transcription factor that is crucial for maintaining embryonic stem cell self-renewal and pluripotency. We postulated that NANOG is involved in the pathogenesis of gestational trophoblastic disease. In this study, significantly higher NANOG mRNA and protein expression levels, by quantitative PCR and immunoblotting, respectively, were demonstrated in HMs, particularly those that developed persistent disease, when compared with normal placentas. In addition, significantly increased nuclear NANOG immunoreactivity was found by immunohistochemistry in HMs (P < 0.001) and choriocarcinoma (P = 0.002). Higher NANOG expression levels were demonstrated in HMs that developed persistent disease, as compared with those that regressed (P = 0.025). Nuclear localization of NANOG was confirmed by confocal microscopy and immunoblotting in choriocarcinoma cell lines. There was a significant inverse correlation between NANOG immunoreactivity and apoptotic index assessed by M30 CytoDeath antibody (P = 0.012). After stable knockdown of NANOG in the choriocarcinoma cell line JEG-3 by an shRNA approach, increased apoptosis was observed in relation to with enhanced caspases and poly(ADP-ribose) polymerase activities. NANOG knockdown was also associated with decreased mobility and invasion of JEG-3 and down-regulation of matrix metalloproteases 2 and 9. These findings suggest that NANOG is involved in the pathogenesis and clinical progress of gestational trophoblastic disease, likely through its effect on apoptosis, cell migration, and invasion.

The prognostic role of the human embryonic stem cell protein, NANOG, in breast cancer

Breast cancer is a heterogenous disease. Recent cDNA microarray studies classified breast cancer into three main groups, which have been shown to be of prognostic importance: luminal (usually estrogen receptor (ER) and progesterone receptor (PR) positive), HER2 and basal-like subtypes. Basal-like tumors have the worst prognosis and are usually triple negative (ER\PR\HER2 negative). NANOG is a transcription factor that plays an important role in human embryonic stem cell pluripotency and self-renewal (Ezeh, 2005). It is located at chromosome 12p13, a region frequently gained in basal-like breast tumors (Bergmaschi, 2006). Given that NANOG is upregulated by SOX2, which is preferentially expressed in basal-like breast carcinomas (Rodriguez-Pinilla, 2007), the objective of our study was to assess whether NANOG protein expression was related to basal-like breast cancers, tumor recurrence and patient survival. Cohort study. Sporadic node-negative invasive breast carcinomas were analyzed by immunohistology and tissue microarrays for ER, PR, HER2 and NANOG protein expression (n = 117). Tumors were considered to have basal-like phenotype if they were triple negative (ER\PR\HER2-). We correlated NANOG tissue expression with breast cancer outcome. Kaplan-Meier survival curves (NANOG+ vs. NANOG-) for disease free and overall survivals were compared using Log-rank test. Preliminary analysis of 22 (13 NANOG+, 9 NANOG-) triple negative samples revealed longer mean disease-free (7.0 vs. 4.9 years) and overall (12.1 vs. 9.0 years) survival in the NANOG + group. The difference did not reach statistical significance (Figure 1, Figure 2). Preliminary data indicate that NANOG protein expression may be of prognostic importance in basal-like (ER, PR, and HER2 -) breast cancer. We intend to confirm the significance of this study in a larger cohort.

Developmental expression of pluripotency determining factors in caprine embryos: Novel pattern of NANOG protein localization in the nucleolus

Transcription factors, POU5F1/OCT4 and NANOG, whose expression is restricted to the inner cell mass (ICM) in mouse and human blastocysts, are used to characterize undifferentiated embryonic stem cells (ESC) in vitro. However, POU5F1 may not be a useful marker in domestic animals due to its expression in both ICM and trophectoderm (TE), while NANOG mRNA and protein expression have only been described fully in mice. In an effort to identify ESC markers for domestic animals, expression patterns of NANOG, POU5F1, and the cell surface markers (SSEA1, SSEA4, TRA-1-60, TRA-1-81) were examined in preimplantation goat embryos, a species that has proven to be a superior choice for the production of transgenic proteins in milk (biopharming). Our results indicate that while goat embryos express POU5F1, SSEA1, and SSEA4 proteins, their expression is not strictly restricted to the ICM. In a unique staining pattern, NANOG protein was localized to the nucleoplasm and nucleoli in ICM cells, but was localized strictly to nucleoli in TE. This pattern may reflect down-regulation of protein by sequestration/degradation utilizing a nucleolar mechanism known to operate in stem cells. Furthermore, NANOG mRNA in TE was also significantly down-regulated as compared with that in ICM. Taken together, this novel expression pattern of NANOG in goat preimplantation embryos suggests that NANOG could serve as marker of pluripotency in goats and may be useful in derivation and characterization of caprine ESC. This study is the first to characterize both NANOG mRNA and protein expression in any species other than the mouse.

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours

NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens. We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis. NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.