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Background: Cancer stem cell biomarkers SRY (sex-determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (Oct4) account for radioresistance in cervical squamous cell cancers (CSCCs). Their clinical implications are limited and contradictory., Methods: In this prospective cohort study, we recruited patients with FIGO IB2-IVA CSCC treated with primary chemoradiotherapy on regular follow-up. Tissue biopsy specimens were evaluated for SOX2 and Oct4 expression by immunohistochemistry, quantified by a product of proportion and intensity scores., Results: A total of 59 patients were included. Most had a moderately differentiated (81%), keratinizing (59%) CSCC, and ≥FIGO stage IIB disease (95%). SOX2 expression (high:low 21:38 patients) and Oct4 expression (high:low 4:55 patients) had a significant interrelation (p = 0.005, odds ratio (95% CI) - 1.23 (1.004-1.520)). At a median follow-up of 36 months, the 3-year overall survival (OS) was 60% and 53% for low and high SOX2 expression (p = 0.856), and 54% and 100% for low and high Oct4 expression (p = 0.114). The 3-year disease-frese survival (DFS) was 65% and 50% in the low and high SOX2 expression (p = 0.259), and 59% and 75% for low and high Oct4 expression (p = 0.598). SOX2 expression was the only variable significantly associated with a lower OS and DFS on regression analysis., Conclusion: Our study demonstrated a trend toward improved OS and DFS with low SOX2 and high Oct4 expression in CSCC patients undergoing chemoradiotherapy., (© 2024 John Wiley & Sons Australia, Ltd.)

We studied the expression of pluripotency factor Oct-4 and the intensity of apoptosis in the uterus during spontaneous and immune abortions in mice. Increased expression of factor Bax and reduced protein Bcl-2 synthesis in cells of the decidual membrane and decreased Oct-4 expression in the myometrium and perimetrium were detected. Thus, both spontaneous and immune-dependent abortions impair the apoptosis processes in the decidua and the formation of a pool of Oct-4 + cells in the uterus. In immune-dependent abortions, the intensity of apoptosis of decidual cells was lower than in spontaneous abortion. Low expression of the transcription factor Oct-4 in the myometrium and perimetrium characterizes pregnancy failure irrespective of its mechanisms., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Cancer stem cell is considered as an important cause to exacerbate the prognosis. NANOG and POU5F1 are markers for cancer stem cells. The associations between NANOG and POU5F1 expressions with the sorafenib anticancer effects in primary cultured hepatocellular carcinoma (HCC) cells were investigated. Eight primary cultured HCC parent cell lines and 13 subgroups established by flow cytometric sorting using NANOG and POU5F1 as targets were investigated with clinically achievable sorafenib plasma concentrations (5 and 10 μg/mL). Sorafenib showed obvious downregulation of RAF/MEK/ERK signaling pathways and dose-dependent anti-proliferative effects only on s003 parent cell line, which showed the lowest expression of NANOG among all tested cell lines except one downregulated NANOG with upregulated POU5F1 s020 subgroup. Sorafenib also inhibited proliferation in this s020 subgroup but promoted proliferation in its parent cell line. For the only one downregulated NANOG alone s015 subgroup, sorafenib which had no influence on its parent cell line inhibited proliferation in this subgroup. Only the above three cell lines could demonstrate sorafenib antiproliferative effects. On the contrary, sorafenib promoted proliferation in three (s003, s015, s071) out of four upregulated NANOG alone subgroups. On the other hand, Sorafenib showed diverse influence on proliferation among four upregulated POU5F1 alone subgroups. In conclusion, NANOG rather than POU5F1 expression is a critical marker for the anticancer effects of sorafenib on HCC. The sorafenib anticancer effects on HCC cells with high NANOG expression were limited. Sorafenib should be combined with other drug able to target cancer cells with high NANOG expression., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Environmental changes can stress and alter biology at the molecular and cellular level. For example, metal-protein interaction is a classic physic and biological property of nature, which is fundamentally influenced by acidity. Here, we report a unique cellular reprogramming phenomenon in that a brief strong acid treatment induced the expression of pluripotent stem cell (PSC) markers. We used strong acid to briefly challenge mix-cultured gastric cells, and then subcultured survived cells in a normal cell culture medium. We found that survival acid-treated cells expressed PSC markers detected by commonly used pluripotent antibodies such as SSEA-4 and Oct4. In addition, we observed that the survived cells from the acid challenge grew faster during the second and third weeks of subculture and had a relative short doubling time (DT) than the controls. PSC marker-labeled 'older' cells also presented immature cell-like morphology with some having marker Oct4 in the nucleus. Finally, the expression of the markers appeared to be sensitive to metal ion chelation. Removal of the metals during a brief acid treatment reduced pluripotent marker-positive cells, suggesting the dissociation of metals from metal-binding proteins may be a factor involved in the induction of stem cell markers. Our findings reveal that somatic cells appear to possess a plasticity feature to express pluripotent marker proteins or to select cell subpopulations that express pluripotent marker proteins when cells are transiently exposed to strong acid. It opens new directions for understanding conserved regulatory mechanisms involved in cellular survival under stressful stimulation.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT + /MYHC-B + /ISL1 + could mark immature cardiomyocytes and CTNT + /ISL1 - mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.

Background: Endometriosis is an estrogen-dependent inflammatory disease that often causes infertility and chronic pelvic pain. Although endometriosis is known as a benign disease, it has demonstrated characteristics of malignant neoplasms, including neoangiogenesis, tissue invasion, and cell implantation to distant organs. Octamer-binding protein 4 (Oct-4) is a molecular marker for stem cells that plays an essential role in maintaining pluripotency and self-renewal processes in various types of benign and malignant tissues. CD44 is a multifunctional cell surface adhesion molecule that acts as an integral cell membrane protein and plays a role in cell-cell and cell-matrix interactions. E-cadherin is an epithelial cell-cell adhesion molecule that plays important role in the modulation of cell polarization, cell migration, and cancer metastasis. The aim of this study was to investigate the expression patterns of Oct-4, CD44, and E-cadherin in eutopic and ectopic endometrial tissues from women with endometrioma compared to control endometrial tissues from women without endometrioma., Methods: In the present study, Oct-4, CD44, and E-cadherin expressions were evaluated in eutopic and ectopic endometrial tissue samples from women with endometrioma (n = 32) and compared with those of control endometrial tissue samples from women without endometrioma (n = 30)., Results: Immunohistochemical expression of Oct-4 was significantly higher in the ectopic endometrial tissue samples of women with endometrioma than in the control endometrial tissue samples (p = 0.0002). Conversely, CD44 and E-cadherin expressions were significantly lower in the ectopic endometrial tissue samples of women with endometrioma than in the control endometrial tissue samples (p = 0.0137 and p = 0.0060, respectively). Correlation analysis demonstrated significant correlations between Oct-4 expression and endometrioma cyst diameter (p = 0.0162), rASRM stage (p = 0.0343), and total rASRM score (p = 0.0223). Moreover, CD44 expression was negatively correlated with the presence of peritoneal endometriotic lesions (p = 0.0304) while E-cadherin expression was negatively correlated with the presence of deep infiltrating endometriosis (p = 0.0445)., Conclusions: Increased expression of Oct-4 and decreased expression of adhesion molecules in endometriotic tissues may contribute to the development and progression of endometriosis.

Introduction: Astrogliosis and glial scar formation following spinal cord injury (SCI) are viewed as major obstacles that hinder axonal regeneration and functional recovery. Regulating the glial scar and axonal regeneration in the lesion site is important for treating SCI., Aims: Considering the important role of astrocyte in glial scar formation and subsequent axonal regeneration, we intended to investigate the effect of the transcription factors OCT4 and KLF4 on astrocyte and the underlying mechanism after spinal cord contusion injury in transgenic mice., Results: Western blotting, q-PCR, immunofluorescence, and functional evaluation suggested that glial fibrillary acidic protein (GFAP) expression decreased in the lesion area, the porosity of the scar increased, and remyelination enhanced. Mice overexpressing the transcription factors OCT4 and KLF4 had higher Basso Mouse Scale scores than did the control mice. Moreover, using immunofluorescence and Western blotting, we discovered that some astrocytes expressed nestin and sox2 protein, suggesting that these astrocytes were reprogrammed into neural stem cell-like cells. Furthermore, a cell scratch assay showed that the migration ability of the astrocytes was significantly inhibited in the presence of the transcription factors OCT4 and KLF4. In addition, we demonstrated that the Hippo/Yap pathway was activated after these two transcription factors overexpressed in astrocytes., Conclusions: In summary, these results suggest that overexpression of the transcription factors OCT4 and KLF4 could induce astrocyte reprogramming, which subsequently improves remyelination and functional recovery after SCI., (© 2020 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The cancer stem cells deliver uncontrolled proliferative capacity within the tumor imparting to increasing size while epithelial mesenchymal transition adds to the invasive potential. Studies using specific markers elucidating the role of these phenomena may bring advancement in the targeted therapy of tumor. SOX2 and OCT4 are two among few stem cell markers indicative of proliferative potential and WNT5A is an epithelial mesenchymal transition marker indicative of invasive potential. We aimed to determine the association between expression of SOX2, OCT4 and WNT5A in oral epithelial dysplasia, oral squamous cell carcinoma and normal oral mucosa. 20 cases of oral squamous cell carcinoma, 20 cases of oral epithelial dysplasia (leukoplakia with dysplasia) and 25 normal oral mucosa tissues specimens were immunohistochemically stained to assess SOX2, OCT4 and WNT5A expression. SOX2 expression was higher in oral squamous cell carcinoma than in oral epithelial dysplasia and very low in normal oral mucosa. OCT4 was very low in oral squamous cell carcinoma and oral epithelial dysplasia when compared to SOX2, while negative in normal tissues. Co-expression of SOX2 and OCT4 showed statistically non-significant difference for tumor proliferation. WNT5A expression was found to be increasing from normal oral mucosa to oral epithelial dysplasia and oral squamous cell carcinoma. In conformity with present study, SOX2 itself can act as a potential marker for proliferation in tumor cells while OCT4 has non-significant role in regulation of tumor behavior in oral squamous cell carcinoma as well as in oral epithelial dysplasia. WNT5A can be a putative marker in studying invasive potential of oral squamous cell carcinoma.

Aim: Cancer stem-like cell (CSC) markers and the role of CSCs derived from papillary thyroid carcinoma (PTC) in pathogenesis are unclear. This study aimed to investigate CSC properties using tumor spheres from passaged PTC cells but without sorting CSCs., Materials and Methods: To identify the properties of CSCs derived from PTC, the expression of SRY-box transcription factor 2(SOX2), octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), thyroglobulin (TG), thyroid-stimulating hormone receptor (TSHR), E-cadherin, YES-associated protein 1 (YAP1), and signal transducer and activator of transcription 3 (STAT3) was investigated in tumor spheres serially passaged without sorting CSCs., Results: The cultured tumor spheres had cancer stemness; high expression of OCT4, SOX2, NANOG, and YAP1; low expression of E-cadherin; and varied expression of TG, TSHR, and STAT3. PTC tumor spheres transfected with small interfering RNA targeting YAP1 had fewer CSC properties than the non-transfected tumor spheres did., Conclusion: Tumor spheres derived from PTC cells by passaging without sorting CSCs have more stem-like cell properties, and less differentiation potential. Thus, this simple and cost-effective method can be used for the enrichment of PTC stemness for employment in cell-based models, reducing the need for use of animal models., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Tamoxifen is the most important treatment component in estrogen receptor positive (ER+) breast carcinoma patients. Tamoxifen resistance incidence presents an important obstacle in clinical treatment. Mechanisms underlying tamoxifen refractory are not completely understood. Although elevated expression of Gankyrin (P28GANK) and stem cell markers Nanog, Oct-4 and Sox-2 have been reported in breast carcinoma, their role in tamoxifen resistance progression has not been explored. In the present study, P28GANK and stem cell markers Nanog, Oct-4 and Sox-2 expression were evaluated using quantitative RT-PCR and immunohistochemical technology in 72 breast carcinoma patients who received tamoxifen as adjuvant anti-hormone treatment. Expression data were correlated with the clinical outcome and survival of patients. Data analysis showed that P28GANK, Oct-4 and Sox-2 transcripts were significantly overexpressed in tamoxifen resistance patients. Immunohistochemical staining indicated that protein expression of P28GANK and Oct-4 were also significantly higher in tamoxifen resistance patients. We have shown a positive correlation between mRNA and protein expression of P28GANK, Oct-4 and Sox-2. Multivariate logistic regression analysis indicated that P28GANK (P = 0.002) and Oct-4 (P = 0.013) overexpression could be negative independent factors of disease outcome. Additionally, in the whole study group, multivariate Cox regression analysis revealed that high expression of P28GANK and Oct-4 remained significant and unfavorable predictive factors for patients' survival. These findings suggest that Gankyrin and Oct-4 overexpression could promote tamoxifen refractory in breast cancer patients. More studies are warranted to clarify the predictive role of these potential biomarkers for patients who don't benefit from tamoxifen treatment and their possible application as prognostic markers in ER + tamoxifen-treated breast carcinoma patients.

Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell. Methods: We have prepared (3-hydroxybutyrate- co -3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR. Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.

Octamer binding transcription factor-4 (Oct4), is highly expressed in stem cells and has indispensable roles in pluripotency and cellular reprogramming. In contrast to other factors used for cellular reprogramming, a role for Oct4 outside embryonic stem cells has been elusive and highly controversial. Emerging evidence implicates Oct4 in the carcinogenic process, but the mechanism through which Oct4 may be functioning in cancers is not fully appreciated. Here, we provide evidence that Oct4 is expressed in human cervical cancer and this expression correlates with the presence of the human papillomavirus (HPV) oncogenes E6 and E7. Surprisingly, the viral oncogenes can complement exogenously provided Oct4 in reprogramming assays, providing functional validation for their ability to activate Oct4 transcription in Mouse Embryonic Fibroblasts (MEFs). To interrogate potential roles of Oct4 in cervical cancers we knocked-down Oct4 in HPV(+) (HeLa & CaSki) and HPV(-) (C33A) cervical cancer cell lines and found that Oct4 knockdown attenuated clonogenesis, only in the HPV(+) cells. More unexpectedly, cell proliferation and migration, were differentially affected in HPV(+) and HPV(-) cell lines. We provide evidence that Oct4 interacts with HPV E7 specifically at the CR3 region of the E7 protein and that introduction of the HPV oncogenes in C33A cells and human immortalised keratinocytes generates Oct4-associated transcriptional and phenotypic patterns, which mimic those seen in HPV(+) cells. We propose that a physical interaction of Oct4 with E7 regulates its activity in HPV(+) cervical cancers in a manner not seen in other cancer types., Competing Interests: The authors have declared that no competing interests exist.

Cancer stem-like cells (CSCs) contribute to the tumorigenicity, progression, and chemoresistance of cancers. It is not known whether CSCs arise from normal stem cells or if they arise from differentiated cancer cells by acquiring self-renewal features. These CSCs share stem cell markers that normal stem cells express. There is a rising interest in octamer-binding transcription factor 4 (OCT4), one of the stem cell factors that are essential in embryogenesis and pluripotency. OCT4 is also overexpressed in CSCs of various cancers. Although the majority of the studies in CSCs reported a positive association between the expression of OCT4 and chemoresistance and an inverse correlation between OCT4 and clinical prognosis, there are studies rebuking these findings, possibly due to the sparsity of stem cells within tumors and the heterogeneity of tumors. In addition, post-translational modification of OCT4 affects its activity and warrants further investigation for its association with chemoresistance and prognosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background/aims: Acute kidney injury (AKI) is a common clinical condition that can lead to chronic kidney failure. Although mesenchymal stem cell-derived extracellular vesicles (MSC EVs) are regarded as a potent AKI treatment, the mechanisms underlying their beneficial effects remain unclear. Oct-4 may play an important role in tissue injury repair. We thus hypothesized that oct-4 overexpression might enhance the therapeutic effects of MSC EVs in AKI treatment., Methods: Renal tubular epithelial cells were cultured in a low oxygen environment, then cocultured with MSC EVs or control medium for 48 h. BrdU and transferase-mediated dUTP nick-end labeling (TUNEL) staining were used to assess cell proliferation and apoptosis. Mice subjected to ischemia reperfusion were randomly divided into 4 groups, then injected with either phosphate-buffered saline (vehicle), EVs, EVs overexpressing oct-4 (EVs+Oct-4), and EVs not expressing Oct-4 (EVs-Oct-4). Blood creatinine (CREA) and urine nitrone levels were assessed 48 h and 2 weeks after injection. After ischemia reperfusion, renal tissues from each group were stained with TUNEL and proliferating cell nuclear antigen (PCNA) to determine the degree of apoptosis and proliferation. Masson trichrome staining was used to evaluate renal fibrosis progression. Snail gene expression was assessed using polymerase chain reaction (PCR)., Results: At 48 h after hypoxic treatment, TUNEL and BrdU staining indicated that the EVs+Oct-4 group had the least apoptosis and the most proliferation, respectively. Treatment with EVs overexpressing Oct-4 significantly decreased serum Crea and blood urea nitrogen levels and rescued kidney fibrosis, as indicated by the low proportion of Masson staining, high number of PCNA-positive cells, and low number of TUNEL-positive cells. PCR analysis indicated that Snail was most upregulated in the vehicle group and least upregulated in the EVs+Oct-4 group., Conclusions: MSC EVs had a pronounced therapeutic effect on ischemic reperfusion injury-related AKI, and Oct-4 overexpression enhanced these therapeutic effects. Our results may inspire a new direction for AKI treatment with MSC EVs., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Background: Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit., Methods: The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics-to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique-to generate induced pluripotent stem cells (iPSCs)., Results: In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system-the FlpO recombinase, knocked into 3'UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4 + cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs., Conclusions: The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

The paper presents an evaluation of the POU5F1 gene expression in mesenchymal stem cells derived from Wharton's jelly within the umbilical cord, collected from 36 patients during labor. The study is the first one to show that the expression of POU5F1 in mesenchymal stem cells has been dependent on maternal age, birth order, route of delivery, and use of oxytocin. Our research proves that the POU5F1 gene expression in mesenchymal stem cells decreases with each subsequent pregnancy and delivery. Wharton's jelly stem cells obtained from younger women and during their first delivery, as well as patients treated with oxytocin, show higher POU5F1 gene expression when compared with the subsequent deliveries. This leads to a conclusion that they are characterized by a lower level of differentiation, which in turn results in their greater plasticity and greater proliferative potential. Probably, they are also clinically more useful., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Paulina Gil-Kulik et al.)

Background: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear., Methods: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo., Results: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells., Conclusions: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b-mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically "open" state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.

OCT4 is a key regulator in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Human OCT4 gene has three mRNA isoforms, termed OCT4A, OCT4B and OCT4B1. The 190-amino-acid protein isoform (OCT4B-190) is one of the major products of OCT4B mRNA, the biological function of which is still not well defined. Recent evidence suggests that OCT4B-190 may function in the cellular stress response. The glycogen synthase kinase-3β (GSK-3β) and histone deacetylase 6 (HDAC6) are also key stress modulators that play critical roles in the ischemic cascades of stroke. Hence, we here further investigated the effects of OCT4B-190 in the experimental stroke, and explored the underlying roles of GSK-3β and HDAC6. We found that OCT4B-190 overexpression enhanced neuronal viability at 24 h after oxygen-glucose deprivation (OGD) treatment. Moreover, in male C57BL/6 mice subjected to transit middle cerebral artery occlusion (MCAO), OCT4B-190 overexpression reduced infarct volume and improved neurological function after stroke. Notably, we found spatio-temporal alterations of GSK-3β and HDAC6 in the ischemic cortex and striatum, which were affected by adenovirus-mediated OCT4B-190 overexpression. OCT4B-190 demonstrated similar impacts on neuronal cultures in vitro, downregulating OGD-induced GSK-3β activity and HDAC6 expression. In addition, we found that GSK-3β and HDAC6 were co-expressed in the cytoplasm of neurons, and OCT4B-190 had an effect on interactions between GSK-3β and HDAC6 in neuronal cultures subjected to OGD treatment. These findings suggest that OCT4B-190 exerts neuroprotection in the experimental stroke potentially by regulating actions of GSK-3β and HDAC6 simultaneously, which may be an attractive therapeutic strategy for ischemic stroke., (Copyright © 2019. Published by Elsevier Inc.)

BACKGROUND The POU domain class 5 transcription factor 1B (POU5F1B), is a pseudogene that is homologous to octamer-binding transcription factor 4 (OCT4), and is located adjacent to the MYC gene on human chromosome 8q24. POU5F1B has been reported to be transcribed in several types of cancer, but its role in cervical cancer remains unclear. This study aimed to investigate the expression and function of POU5F1B in tissue samples of human cervical cancer and in cervical cancer cell lines in vitro. MATERIAL AND METHODS Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify POU5F1B expression in cervical cancer tissues and in SiHa, HeLa, CaSki, and C33A human cervical cancer cell lines. Functional in vitro studies included analysis of the effects of POU5F1B expression on cervical cancer cell proliferation, migration, and apoptosis using a Cell Counting Kit-8 (CCK-8) assay, cell migration assays, and flow cytometry. Luciferase activity assays, qRT-PCR, and Western blot were performed to confirm the expression of POU5F1B. RESULTS POU5F1B was significantly upregulated in cervical cancer tissues and cell lines. Interference with the expression of POU5F1B significantly inhibited cell proliferation, apoptosis, migration and invasion, and induced apoptosis in vitro. Western blot demonstrated that POU5F1B could modulate the expression of the OCT4 protein. CONCLUSIONS POU5F1B was upregulated in cervical cancer and down-regulation inhibited cell proliferation and migration and induced apoptosis in cervical cancer cell lines by modulating OCT4. Further studies are required to determine whether POU5F1B might be a diagnostic or prognostic biomarker or therapeutic target in cervical cancer.

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16-22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs.

Beiging of white adipose tissue (WAT) is a particularly appealing target for therapeutics in the treatment of metabolic diseases through norepinephrine (NE)-mediated signaling pathways. Although previous studies report NE clearance mechanisms via SLC6A2 on sympathetic neurons or proinflammatory macrophages in adipose tissues (ATs), the low catecholamine clearance capacity of SLC6A2 may limit the cleaning efficiency. Here, we report that mouse organic cation transporter 3 (Oct3; Slc22a3) is highly expressed in WAT and displays the greatest uptake rate of NE as a selective non-neural route of NE clearance in white adipocytes, which differs from other known routes such as adjacent neurons or macrophages. We further show that adipocytes express high levels of NE degradation enzymes Maoa, Maob, and Comt, providing the molecular basis on NE clearance by adipocytes together with its reuptake transporter Oct3. Under NE administration, ablation of Oct3 induces higher body temperature, thermogenesis, and lipolysis compared with littermate controls. After prolonged cold challenge, inguinal WAT (ingWAT) in adipose-specific Oct3-deficient mice shows much stronger browning characteristics and significantly elevated expression of thermogenic and mitochondrial biogenesis genes than in littermate controls, and this response involves enhanced β-adrenergic receptor (β-AR)/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (Creb) pathway activation. Glycolytic genes are reprogrammed to significantly higher levels to compensate for the loss of ATP production in adipose-specific Oct3 knockout (KO) mice, indicating the fundamental role of glucose metabolism during beiging. Inhibition of β-AR largely abolishes the higher lipolytic and thermogenic activities in Oct3-deficient ingWAT, indicating the NE overload in the vicinity of adipocytes in Oct3 KO adipocytes. Of note, reduced functional alleles in human OCT3 are also identified to be associated with increased basal metabolic rate (BMR). Collectively, our results demonstrate that Oct3 governs β-AR activity as a NE recycling transporter in white adipocytes, offering potential therapeutic applications for metabolic disorders., Competing Interests: The authors have declared that no competing interests exist.

Background: Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved., Methods: A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCs OCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected., Results: The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCs OCT4 and hHFMSCs EGFP , including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCs OCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCs OCT4 , and triggered cell senescence., Conclusions: OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Objectives: In vitro differentiation of oocytes from female germline stem cells (FGSCs) has exciting potential applications for reproductive medicine. Some researchers have attempted to reveal the in vitro differentiation capacity of FGSCs. However, no systematic comparative study of in vitro differentiation conditions has been performed for murine FGSCs (mFGSCs)., Materials and Methods: mFGSCs line was cultured under five different conditions for in vitro differentiation. RT-PCR was performed to detect the expression of Oct4, Fragilis, Blimp1, Mvh, Scp3 and Zp3. Immunofluorescence was carried out to test the expression of Mvh, Fragilis and Zp3. Two-photon laser-scanning microscope was used to analyze nucleus-plasma ratio, and the proportion of chromatin of GV oocytes differentiated from mFGSCs in vitro (IVD-GVO), GV oocytes from in vivo (GVO) and mFGSCs., Results: RT-PCR and immunofluorescence showed that mFGSC line expressed germ cell-specific markers, but not a meiosis-specific marker. By evaluating five different in vitro differentiation conditions, condition 5, which included a hanging drop procedure and co-culture of mFGSCs with granulosa cells, was shown to be optimal. mFGSCs could be successfully differentiated into germinal vesicle (GV) -stage oocytes under this condition. 3D observation revealed that both the nucleus-plasma ratio and proportion of chromatin were not significantly different between IVD-GVO and GVO., Conclusion: We evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV-stage oocytes using a three-step differentiation process., (© 2018 The Authors Cell Proliferation Published by John Wiley & Sons Ltd.)

Objectives: Soluble IL-33 (interleukin (IL)-1-like cytokine) acts as endogenous alarm signal (alarmin). Since alarmins, besides activating immune system, act to restore tissue homeostasis, we investigated whether IL-33 exerts beneficial effects on oral stem cell pull., Materials and Methods: Clonogenicity, proliferation, differentiation and senescence of stem cells derived from human periodontal ligament (PDLSCs) and dental pulp (DPSCs) were determined after in vitro exposure to IL-33. Cellular changes were detected by flow cytometry, Western blot, immunocytochemistry and semiquantitative RT-PCR., Results: IL-33 stimulated proliferation, clonogenicity and expression of pluripotency markers, OCT-4, SOX-2 and NANOG, but it inhibited ALP activity and mineralization in both PDLSCs and DPSCs. Higher Ki67 expression and reduced β-galactosidase activity in IL-33-treated cells were demonstrated, whereas these trends were more conspicuous in osteogenic medium. However, after 7-day IL-33 pretreatment, differentiation capacity of IL-33-pretreated cells was retained, and increased ALP activity was observed in both cell types. Results showed that IL-33 regulates NF-κB and β-catenin signalling, indicating the association of these molecules with changes observed in IL-33-treated PDLSCs and DPSCs, particularly their proliferation, pluripotency-associated marker expression and osteogenesis., Conclusions: IL-33 treatment impairs osteogenesis of PDLSCs and DPSCs, while increases their clonogenicity, proliferation and pluripotency marker expression. After exposure to IL-33, osteogenic capacity of cells stayed intact. NF-κB and β-catenin are implicated in the effects achieved by IL-33 in PDLSCs and DPSCs., (© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Objective To investigate the correlation of octamer-binding transcription factor 4 (OCT4) expression with clinicopathological features and its predictive value for treatment response as well as survival profiles in de novo acute myeloid leukaemia (AML) patients. Method One hundred fifty-two de novo AML patients and 52 non-hematologic malignancy patients were recruited in this prospective cohort study. OCT4 expression was determined in bone marrow sample collected before treatment. Complete response (CR), event free survival (EFS) and overall survival (OS) were evaluated. Results Compared with the controls, OCT4 mRNA expression was higher in AML patients (P < .001), and higher OCT4 expression was correlated with presence of complex karyotype (CK) (P = .037), FLT3-ITD mutation (P = .012) and poorer risk stratification (P < .001) in AML patients. As to predictive value, OCT4 mRNA expression was decreased in patients achieved CR compared to non-CR patients (P = .022). Kaplan-Meier (K-M) curves showed that shorter EFS (9.0 (95% CI (7.7-10.3)) months vs. 25.0 (95% CI (17.5-32.5)) months, P < .001) and shorter OS (20.0 (95% CI (17.8-22.2) months vs. 33.0 months, P < .001) were observed in OCT4 mRNA high expression patients compared to OCT4 mRNA low expression patients. Multivariate Cox's proportional hazards regression analyses revealed that OCT4 mRNA high expression was an independent predictive factor for shorter EFS and OS in AML patients. Conclusion OCT4 correlates with presence of CK, FLT3-ITD mutation and poorer risk stratification, and it could be served as a convincing biomarker for predicting unfavourable prognosis in AML patients.

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine's actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFβ, and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4, and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage.

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs before differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG, and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG, and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. Stem Cells 2018;36:1514-1524., (© AlphaMed Press 2018.)

With the enhancement of commercial manifestation of silver nanoparticles, concerned has risen on their accumulation in aquatic system and consequent effects on fish development and metabolism. In this study, experiments were conducted to assess the impacts of silver nanoparticles on early life cycles of fish considering Zebrafish (Danio rerio) as experimental model. Silver nanoparticles were synthesized through chemical reduction method and characterized through UV-visible spectroscopy, dynamic light scattering (DLS), and HR-TEM. Different sub lethal doses of nanosilver were applied (13.6, 21.6, 42.4, 64, and 128 μgL -1 ) to post-fertilization phases of Zebrafish embryos and their interaction effects were monitored up to six days period. No significant morphological variations were observed at 13.6, 21.6, 42.4 μgL -1 dose of silver nanoparticles, whereas 64 and 128 μgL -1 exposure dose exhibited bending in myotome, deformity in tail region, somites, notochord and swelling in anterior and posterior region of embryos and larva. Hatching performances analysis elicited highest hatching success in 13.6 and 21.6 μgL -1 doses of silver nanoparticles followed by positive and negative control, whereas exposure dose of 64 and 128 μgL -1 exhibited comparatively lower success. Western blot analysis were conducted on developing hatchlings with Oct4 antibody and at 13.6 and 21.6 μgL -1 dose,it showed over expression elucidating stimulatory role of nanosilver in these mentioned doses. In silico analysis depicted a firm interaction of nanosilver with Oct4 revealing their key role in growth stimulation of developing embryos. The study demonstrates the function of nanosilver as a growth promoter rather only as a toxicant in fish metabolic system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most common tumors, so far, there still aren't good therapeutic methods. Looking for new targets makes a top priority. In this study, we found a OCT4 pseudogene, POU5F1B was significantly upregulated in HCC cells and tissues, patients with high POU5F1B had shorter survival time compared to patients with low POU5F1B expression. POU5F1B overexpression promoted HCC proliferation, while its knockdown inhibited HCC proliferation determined by MTT assay, soft agar growth assay, BrdU incorporation assay, and cell cycle assay. Mechanism analysis suggested POU5F1B could activate AKT, AKT inhibition in HCC cells with POU5F1B overexpression significantly inhibited cell proliferation compared to cells only with POU5F1B overexpression, suggesting POU5F1B promoted HCC proliferation by activating AKT. Finally, we analyzed the relationship between POU5F1B expression and AKT activity in HCC tissues, and found POU5F1B expression was positive correlated with activated AKT. Taken together, our findings suggested POU5F1B promoted HCC proliferation by activating AKT, and provided a new target for HCC therapy., (Copyright © 2018. Published by Elsevier Masson SAS.)

Background: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development., Methods: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer., Results: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin., Conclusions: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells., General Significance: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We demonstrate that OCT4 expression is regulated by germ cell nuclear factor (GCNF) via its interactions with three nuclear receptor (NR) binding sites within OCT4 promoter conserved regions (CRs) in human embryonic carcinoma (EC) NCCIT cells. OCT4 expression is gradually reduced during the retinoic acid-induced differentiation, while GCNF temporarily increased after 2 days and then significantly decreased. In addition, OCT4 expression is significantly reduced by overexpression of exogenous GCNF, but increased by GCNF shRNA-mediated knockdown. The transcriptional activity of OCT4 is significantly inhibited by dose-dependent overexpression of GCNF. While mutants at each of the NR binding sites retain the repressive effects of GCNF on OCT4 promoter activity, the repressive effect was completely eliminated in the reporter construct with all binding sites mutated even in the presence of GCNF. Furthermore, the transcriptional activity of native minimal promoter (CR1-Luc) containing the first NR binding site was significantly reduced by GCNF overexpression, while the mutant retained basal activity to some extent. Next, an exogenous minimal ti promoter-inserted CR2 reporter construct containing the second and third NR binding sites (CR2-ti-Luc) was co-transfected with GCNF expression vector. The transcriptional activity of CR2-ti-Luc was significantly decreased by GCNF overexpression, while mutation of both binding sites retained the transcriptional activity of the reporter construct. Binding assays confirmed the direct interaction of GCNF with all three NR binding sites cooperatively. Taken together, GCNF acts as a transcriptional repressor in the regulation of OCT4 gene expression through cooperative interaction with three NR binding elements in pluripotent NCCIT cells., (© 2017 Wiley Periodicals, Inc.)

OCT4 is known as the gatekeeper of pluripotent embryonic state as it is responsible for maintenance of pluripotency via self-renewal of embryonic stem cells and acquisition of induced pluripotency via somatic cell reprogramming. OCT4 is responsible for oncogenic transformation by disrupting pre-scheduled differentiation programs and in general, favoring evolution of cancer cells into a more aggressive cancer stem cell phenotype. In this study, we have investigated in details, the epigenetic regulatory mechanisms responsible for over-expression and subsequent aberrant function of OCT4 in breast cancer. Expression of OCT4 in breast cancer tissues and cell lines was determined by qRT-PCR, Western blot, immunohistochemistry and immunocytochemistry followed by investigation of pro-tumorigenic properties such as cell proliferation, migration and apoptosis by gene knockdown and treatment with epigenetic modulators. Ectopic treatment of epigenetic modulators was done followed by MS-PCR and chromatin immunoprecipitation. OCT4 is over-expressed in both of the breast cancer cell lines; MCF7 and MDA-MB-231 and its inhibition resulted in drastic decrease in rate of cell proliferation, metastatic ability and induced apoptosis. After treatment with epigenetic modulators, general increase in expression of OCT4 was observed at both gene and protein levels; however, changes of promoter DNA methylation was not found to be significant during OCT4 over-expression. Active histone marks especially H3K4me3 and H3K9acS10p were enriched in the promoter region with very low levels of repressive marks H3K9me3 and H3K27me3 indicating that active histone modifications are the deciding factor in inducing over-expression of OCT4 during breast carcinogenesis. These findings could provide the basis on which epigenetic therapy, targeting reversible epigenetic modifications and their perpetuating enzymes, can be designed for effective treatment of aggressive breast cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Pluripotent stem cells hold great promise for regenerative medicine. However, before clinical application, reproducible protocols for pluripotent stem cell differentiation should be established. Extracellular signal-regulated protein kinase (ERK) signaling plays a central role for the self-renewal of epiblast stem cells (EpiSCs), but its role for subsequent germ layer differentiation is still ambiguous. We proposed that ERK could modulate differentiation of the epiblast., Methods: PD0325901 was used to inhibit ERK activation during the differentiation of embryonic stem cells and EpiSCs. Immunofluorescence, western blot analysis, real-time PCR and flow cytometry were used to detect germ layer markers and pathway activation., Results: We demonstrate that the ERK phosphorylation level is lower in neuroectoderm of mouse E7.5 embryos than that in the primitive streak. ERK inhibition results in neural lineage commitment of epiblast. Mechanistically, PD0325901 abrogates the expression of primitive streak markers by β-catenin retention in the cytoplasm, and inhibits the expression of OCT4 and NANOG during EpiSC differentiation. Thus, EpiSCs differentiate into neuroectodermal lineage efficiently under PD0325901 treatment. These results suggest that neuroectoderm differentiation does not require extrinsic signals, supporting the default differentiation of neural lineage., Conclusions: We report that a single ERK inhibitor, PD0325901, can specify epiblasts and EpiSCs into neural-like cells, providing an efficient strategy for neural differentiation.

Aim: The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells., Methods: We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells., Results: Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5., Conclusion: The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors., (© 2016 John Wiley & Sons Australia, Ltd.)

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.

OCT4 is a crucial transcription factor that maintains self-renewal and pluripotency of embryonic stem and embryonic carcinoma cells. The human OCT4 gene can generate at least three variants (OCT4A, OCT4B, and OCTB1) via alternative splicing and alternative promoters. It has been previously reported that OCT4A is the main isoform, retaining stemness state in embryonic stem and embryonic carcinoma cells. There are several reports on the expression of OCT4A, OCT4B, and OCT4B1 in some cancers and tumor cells. The expression of OCT4 in cancer tissues and cell lines appeared to be highly controversial since it was believed that OCT4 is exclusively expressed in embryonic stem/embryonic carcinoma cells. Here, we are reporting the detection of a novel alternatively spliced variant of OCT4, OCT4B2, in several pluripotent and tumor cell lines. Moreover, the expression pattern of OCT4B2 in the course of neural differentiation of NT2 and NCCIT, embryonic carcinoma cells, was similar to that of OCT4A. OCT4B2 was highly expressed in undifferentiated cells; however, its expression was sharply downregulated upon induction of differentiation. Overexpression of OCT4B2 did not affect the distribution of cells in different cell-cycle phases of transfected cells, compared to the mock transfected cells. Interestingly, the expression of OCT4B2 transcript was elevated under the heat-shock induction. In conclusion, we are reporting a new variant of OCT4, which is expressed under different physiological conditions. The finding shed more light on complexity of OCT4 expression and functions.

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing., (© 2017 Japanese Society of Developmental Biologists.)

Objective: In this study, we aimed to investigate the downstream effector of GLI2 in gastric cancer (GC) and their regulative effect on cancer stem cell (CSC) properties of GC., Materials and Methods: Bioinformatic data mining was performed in TCGA-Stomach Adenocarcinoma (STAD), as well as in Kaplan-Meier plotter. Moderate-differentiated GC cell line SGC-7901 and poor-differentiated GC cell line MKN-45 were used as in-vitro model to investigate the regulative effect of GLI2 on PDGFRB expression. MKN-45 cells were further used to explore the effect of GLI2 shRNA or PDGFRB shRNA on CSC properties of the cells., Results: Bioinformatic results showed that GLI2 is usually upregulated in GC tissues than in normal tissues, and high GLI2 expression is associated with unfavorable first progression free survival (PFS) and also worse overall survival (OS) in patients with GC. PDGFRB is co-upregulated with GLI2 in GC and its promoter region contains a putative GLI2 binding site. The results of dual luciferase assay confirmed this binding site. Enforced GLI2 expression elevated PDGFRB expression at both mRNA and protein level. GLI2 or PDGFRB knockdown showed similar effect on reducing spheroid colony formation and on reducing the expression of CSC related genes, including CD44, Nanog, and Oct4 in MKN-45 cells., Conclusions: High GLI2 or PDGFRB expression is associated with unfavorable survival in GC patients. GLI2 can induce PDGFRB expression in GC cells via directly binding to its promoter. In addition, the GLI2-PDGFRB axis might be an important signaling pathway modulating CSC properties of GC cells.

Purpose: Circulating tumor cells (CTCs) with epithelial-to-mesenchymal transition (EMT) phenotypes might be related to tumor progression while OCT4 expression is involved in tumor metastasis and poor prognosis. But the possible clinical significance of EMT phenotypes of CTCs from non-small-cell lung cancer (NSCLC) patients has still to be demonstrated. Furthermore, none has been investigated the expression of OCT4 in CTCs. We therefore identified the EMT phenotype-based subsets of CTCs and determined the OCT4 expression status of CTCs in NSCLC patients, to explore their possible clinical relevance., Methods: 37 NSCLC patients and ten healthy volunteers were enrolled, respectively. The Canpatrol™ CTC enrichment technique was used to isolate and identify the EMT phenotype-based subsets of CTCs. OCT4 expression in each CTC was also determined. Results were correlated with patients' clinico-pathological features., Results: CTCs were detected in 33 of 37 (89.2%) NSCLC patients, and no CTCs were identified in ten healthy volunteers. Three CTCs phenotypes, including epithelial, biophenotypic, and mesenchymal CTCs were identified based on the expression of EMT markers. Mesenchymal CTCs were more commonly found in patients with distant metastasis. Patients with distant metastasis tended to have a higher median CTCs number. OCT4-positive was observed in 21 of 28 (75.0%) patients. High expression of OCT4 tended to occur in advanced patients as well as in distant metastatic patients., Conclusions: The findings suggest that identification of CTCs by EMT markers as well as evaluation of OCT4 expression status by assessment of OCT4 expression in CTCs could serve as potential adjuncts for evaluating metastasis and prognosis in NSCLC patients.

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34 + CD38 - and CD34 + CD38 + ) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34 + CD38 - than in CD34 + CD38 + subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. In particular, the individual definitive endoderm (DE) cells have not been characterized in vivo Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (FGF) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the premesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6 Analysis of single-cell RNA-sequence data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

Epstein-Barr virus (EBV) infection is closely related to carcinogenesis of various cancers, and is also associated with the development of drug resistance in cancer stem cells. However, in EBV-positive cancer cells, the mechanistic details of the downstream signaling and the connection of PI3K with the NF-κB pathway for development of drug resistance remain controversial. Diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM) cells infected by EBV display drug resistance-related proteins (MDR1, MRP1 and MRP2) and stem cell markers (OCT4 and SOX2). EBV-infected HT (HT/EBV) and H929 (H929/EBV) cells activated p110δ expression, but downregulated the expression of p110α and p110β. A combination of CAL-101, a p110δ-specific inhibitor, with bortezomib treatment of HT/EBV cells synergistically suppressed proliferation, reduced levels of drug resistance-related proteins, activated caspase cleavage and recovered expression of p110α/p110β. Additionally, co-treatment with CAL-101 and bortezomib attenuated the expression of OCT4 and SOX2 via inhibition of activated NF-κB. Co-treatment with CAL-101 and bortezomib also attenuated drug resistance and NF-κB activity of EBV-infected H929 cells. Our results provide supportive evidence for the clinical application of CAL-101 and bortezomib to treat EBV-infected hematologic cancer.

Purpose: Cisplatin is commonly used in non-small-cell lung cancer (NSCLC) chemotherapy; however, chemoresistance to cisplatin remains a great clinical challenge. Octamer-binding protein 4 (OCT4) has been reported to be overexpressed in NSCLC. In this study, we aimed to investigate the potential role of OCT4 in NSCLC with chemoresistance to cisplatin., Methods: Expressions of OCT4 was detected in NSCLC tissues and cell lines. We utilized siRNA to knock down OCT4 expression in human NSCLC cells and analyzed their phenotypic changes., Results: We found that the difference of OCT4 expression between NSCLC and the adjacent non-tumourous tissues was statistically significant. Knockdown of OCT4 in NSCLC cells could decrease cell proliferation, and potentiate apoptosis induced by cisplatin, suggesting OCT4 may contribute to cisplatin resistance in NSCLC., Conclusion: Our findings indicate that targeting OCT4 could improve cisplatin effect in NSCLC, confirming their role in modulating cisplatin sensitivity.

In pancreatic ductal adenocarcinoma (PDAC), mutant KRAS stimulates the translation initiation factor eIF5A and upregulates the focal adhesion kinase PEAK1, which transmits integrin and growth factor signals mediated by the tumor microenvironment. Although eIF5A-PEAK1 signaling contributes to multiple aggressive cancer cell phenotypes, the downstream signaling processes that mediate these responses are uncharacterized. Through proteomics and informatic analyses of PEAK1-depleted PDAC cells, we defined protein translation, cytoskeleton organization, and cell-cycle regulatory pathways as major pathways controlled by PEAK1. Biochemical and functional studies revealed that the transcription factors YAP1 and TAZ are key targets of eIF5A-PEAK1 signaling. YAP1/TAZ coimmunoprecipitated with PEAK1. Interfering with eIF5A-PEAK1 signaling in PDAC cells inhibited YAP/TAZ protein expression, decreasing expression of stem cell-associated transcription factors (STF) including Oct4, Nanog, c-Myc, and TEAD, thereby decreasing three-dimensional (3D) tumor sphere growth. Conversely, amplified eIF5A-PEAK1 signaling increased YAP1/TAZ expression, increasing expression of STF and enhancing 3D tumor sphere growth. Informatic interrogation of mRNA sequence databases revealed upregulation of the eIF5A-PEAK1-YAP1-TEAD signaling module in PDAC patients. Taken together, our findings indicate that eIF5A-PEAK1-YAP signaling contributes to PDAC development by regulating an STF program associated with increased tumorigenicity. Cancer Res; 77(8); 1997-2007. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Poor cell survival and limited functional benefits have restricted mesenchymal stem cell (MSC) efficacy for treating myocardial infarction (MI), suggesting that a better understanding of stem cell biology is needed. The transcription factor HIF-2α is an essential regulator of the transcriptional response to hypoxia, which can interact with embryonic stem cells (ESCs) transcription factor Oct4 and modulate its signaling. Here, we obtained very small embryonic-like mesenchymal stem cells (vselMSCs) from MI patients, which possessed the very small embryonic-like stem cells' (VSELs) morphology as well as ESCs' pluripotency. Using microarray analysis, we compared HIF-2α-regulated gene profiles in vselMSCs with ESC profiles and determined that HIF-2α coexpressed Oct4 in vselMSCs similarly to ESCs. However, this coexpression was absent in unpurified MSCs (uMSCs). Under hypoxic condition, vselMSCs exhibited stronger survival, proliferation and differentiation than uMSCs. Transplantation of vselMSCs caused greater improvement in cardiac function and heart remodeling in the infarcted rats. We further demonstrated that HIF-2α and Oct4 jointly regulate their relative downstream gene expressions, including Bcl2 and Survivin; the important pluripotent markers Nanog, Klf4, and Sox2; and Ang-1, bFGF, and VEGF, promoting angiogenesis and engraftment. Importantly, these effects were generally magnified by upregulation of HIF-2α and Oct4 induced by HIF-2α or Oct4 overexpression, and the greatest improvements were elicited after co-overexpressing HIF-2α and Oct4; overexpressing one transcription factor while silencing the other canceled this increase, and HIF-2α or Oct4 silencing abolished these effects. Together, these findings demonstrated that HIF-2α in vselMSCs cooperated with Oct4 in survival and function. The identification of the cooperation between HIF-2α and Oct4 will lead to deeper characterization of the downstream targets of this interaction in vselMSCs and will have novel pathophysiological implications for the repair of infarcted myocardium.