Immunohistochemical expression of OCT4 and CD44 in major and minor salivary gland neoplasms

Abstract The aim of this study was to identify tumor parenchyma cells exhibiting immunohistochemical profile of stem cells by evaluating the immunoreactivity of OCT4 and CD44 in a number of cases of salivary gland neoplasms. The sample consisted of 20 pleomorphic adenomas, 20 mucoepidermoid carcinomas, and 20 adenoid cystic carcinomas located in major and minor salivary glands. The expression of OCT4 and CD44 was evaluated by the percentage of positive cells and the intensity of expression. All studied cases showed positive expression of OCT4 and CD44 and higher values than the control groups. For OCT4, luminal and non-luminal cells were immunostained in the case of pleomorphic adenomas and adenoid cystic carcinomas. Moreover, the immunoreactivity of CD44 was particularly evident in the non-luminal cells of these lesions. In mucoepidermoid carcinomas, there was immunoreactivity for both markers in squamous and intermediate cells and absence of staining in mucous cells. For both markers, a significantly higher immunostaining was verified in neoplasms located in the major salivary glands compared with lesions in minor salivary glands (p<0.001). In the total sample and in minor salivary glands, malignant neoplasms exhibited higher immunoreactivity for OCT4 than pleomorphic adenoma. A significant moderate positive correlation (r = 0.444 and p ≤ 0.001) was found between OCT4 and CD44 immunoexpression in the total sample. The high expression of OCT4 and CD44 may indicate that these proteins play an important role in identifying tumor stem cells.

Effect of OCT4 Gene Expression Upregulation on the Expression of iPSC-related Transcription Factors in Human Bone Marrow Derived Mesenchymal Stem Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC，KLF4，LIN28，NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future.@*METHODS@#Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4（hBMMSCs-OCT4）were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs.@*RESULTS@#Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426).@*CONCLUSION@#Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2.

Expression relationship of Hippo signaling molecules and ovarian germline stem cell markers in the ovarian aging process of women and mice / 生理学报

The present study was aimed to investigate the expression relationship of Hippo signaling molecules and ovarian germline stem cell (OGSC) markers in the development schedule of OGSCs during ovarian aging in women and mice. The ovaries of 2-month-old mature (normal control) and 12-month-old (physiological ovarian aging) KM mice were sampled, and the ovarian cortex samples of young (postpuberty to 35 years old), middle age (36-50 years old) and menopausal period (51-60 years old) women were obtained with consent. The mice model of pathological ovarian aging was established by intraperitoneal injection of cyclophosphamide/busulfan (CY/BUS). HE staining was used to detect the changes of follicles at different stages, and the localization and expression changes of Hippo signaling molecules and OGSCs related factors (MVH/OCT4) were detected by immunohistochemistry and immunofluorescence staining. Western blot was used to detect the protein expression levels of the major molecules in the Hippo signaling pathway and OGSCs related factors. The results showed that there were not any normal follicles, but a few atresia follicles in the ovaries from physiological and pathological ovarian aging mice. Compared with the normal control mice, both the physiological and pathological ovarian aging mice showed decreased protein expression levels of the main Hippo signaling molecules (pYAP1) and MVH/OCT4; Whereas only the pathological ovarian aging mice showed increased ratio of pYAP1/YAP1. In comparison with the young women, the middle age and menopausal women showed looser structure of ovarian surface epithelium (OSE) and less ovarian cortical cells. The protein expression level of LATS2 in the OSE was the highest in young women, MST1 expression was the lowest in the menopausal period women, and the expression levels of YAP1 and pYAP1 were the highest in middle age women. Compared with the young women, the middle age and menopausal period women exhibited significantly decreased ratio of OSE pYAP1/YAP1, whereas there was no significant difference between them. The expression level of MVH protein in OSE from the young women was significantly higher than those of the middle age and menopausal period women. These results indicate that there is an expression relationship between the main molecules of Hippo signaling pathway and OGSCs related factors, which suggests that Hippo signaling pathway may regulate the expression levels of OGSCs related factors, thus participating in the process of physiological and pathological degeneration of ovarian.

Correlation of Cancer Stem-Cell Markers OCT4, SOX2, and NANOG with Clinicopathological Features and Prognosis in Operative Patients with Rectal Cancer

PURPOSE: To investigate the association of cancer stem-cell markers [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homebox (NANOG)] expression with clinicopathological properties and overall survival (OS) in operative rectal cancer (RC) patients receiving adjuvant therapy. MATERIALS AND METHODS: 153 patients with primary RC receiving surgery were enrolled. Tumor tissue and paired adjacent normal tissue sample were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunofluorescent staining. The median follow-up duration was 5.2 years, and the last follow-up date was August 2016. RESULTS: Tumor tissue OCT4 (p < 0.001), SOX2 (p=0.003), and NANOG (p < 0.001) expressions were higher than those in adjacent tissue. OCT4 expression was positively correlated with pathological grade (R=0.185, p=0.022), tumor size (R=0.224, p=0.005), and N stage (R=0.170, p=0.036). NANOG expression was positively associated with tumor size (R=0.169, p=0.036). Kaplan-Meier suggested that OCT4+ was associated with worse OS compared with OCT4− (p < 0.001), while no association of SOX2 (p=0.121) and NANOG expressions (p=0.195) with OS was uncovered. Compared with one or no positive marker, at least two positive markers were associated with shorter OS (p < 0.001), while all three positive markers were correlated with worse OS compared with two or less positive markers (p < 0.001). Multivariate Cox's analysis revealed that OCT4+ (p < 0.001) and N stage (p=0.046) were independent factors for shorter OS. CONCLUSION: Tumor tissue OCT4 expression was correlated with poor differentiation, tumor size, and N stage, and it can serve as an independent prognostic biomarker in operative patients with RC receiving adjuvant therapy.

Effect of OCT4A Gene on the Biological Characteristics of K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.</p><p><b>METHODS</b>Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.</p><p><b>RESULTS</b>The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).</p><p><b>CONCLUSION</b>Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.</p>

Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides

Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.

Isolation of breast cancer stem cell from MDA-MB231 cell line using vincristine / Aislamiento de células madre de cáncer de mama de la línea celular MDA-MB231 utilizando vincristina

Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs' viability (P> 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P>0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.

El cáncer ha sido considerado como una enfermedad de células madre. Recientemente se ha propuesto cultivo en suspensión en combinación con medicamentos contra el cáncer para aislamiento de las células madre del cáncer (CMC). En este estudio se utilizó la vincristina como fármaco anticanceroso combinado con cultivo en suspensión para el aislamiento y purificación de las células madre cancerosas, de la línea celular de cáncer de mama humano (MDA-MB231). Las células se trataron con diferentes concentraciones de vincristina (0, 2, 4, 6 y 8 ng/ml). Las células madre se identificaron mediante la expresión de los genes OCT4, Nanog, SOX2 y nucleostemin por RT-PCR. La unidad de formación mammosphere se midió a través de cultivo en suspensión que contenía EGF, bFGF, LIF, B27, insulina y BSA. Los mammospheres aislados fueron estudiados para la expresión de CD44. Los resultados mostraron que 4 ng/ml de vincristina durante 72 horas podrían ser utilizados como la mejor y más fiable dosis que permite eliminar alrededor del 80 % de las células madre no cancerosas, sin causar un efecto destructivo sobre la viabilidad de las CMC (P> 0,05). La RT-PCR mostró que en las células tratadas con él fármaco hubo expresión de los genes OCT4, Nanog, SOX2 y nucleostemin. La unidad de formación de las células pretratadas con vincristina fue significativamente más alta que las unidades sin tratamiento previo (P>0,05). La inmunofluorescencia para CD44 muestró una alta expresión del marcador de CMC entre mammospheres aisladas. La vincristina en combinación con el cultivo en suspensión puede ser considerado como un método apropiado para aislar CMC.

Expressions of OCT4, Notch1 and DLL4 and their clinical implications in epithelial ovarian cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the correlations among OCT4, Notch1 and DLL4 and their association with the clinicopathological features of patients with epithelial ovarian cancer (EOC).</p><p><b>METHODS</b>A total of 207 specimens of EOC and 65 specimens of benign ovarian epithelial tumor tissues were examined for expressions of OCT4, Notch1 and DLL4 proteins using immunohistochemistry.</p><p><b>RESULTS</b>The positivity rates of OCT4, Notch1 and DLL4 in EOC tissues were 60.0%, 61.8% and 60.9%, respectively, significantly higher than the rates in benign epithelial tumor tissues (9.2%, 6.2%, and 0, respectively; P<0.05). The expressions of OCT4, Notch1 and DLL4 in EOC were significantly correlated with tumor differentiation, FIGO stage, and lymph node metastasis (P<0.05). DLL4 was positively correlated with OCT4 and Notch1 expressions (r=0.758 and 0.704, respectively, P<0.001), and the latter two were also positively correlated (r=0.645, P<0.001). Overexpressions of OCT4, Notch1 and DLL4 were associated with a poor prognosis, and the survival rate was significantly lower in patients positive for OCT4, Notch1, and DLL4 than in the negative patients (P<0.05). FIGO stage and expressions of OCT4 and DLL4 were independent prognostic factors of EOC (P<0.05).</p><p><b>CONCLUSION</b>The expressions of OCT4, Notch1 and DLL4 are correlated with the differentiation, lymph node metastasis, clinical stage and prognosis of EOC. Combined detection of the 3 proteins has an important value in predicting the progression and prognosis of EOC.</p>

Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation / 华中科技大学学报（医学）（英德文版）

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.

Effects of Leukemia Inhibitory Factor Combined with Basic Fibroblast Growth Factor on Self-maintenance and Self-renewal of Human Umbilical Cord Mesenchymal Stem Cells In Vitro / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.</p><p><b>METHODS</b>Experiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.</p><p><b>RESULTS</b>The cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.</p><p><b>CONCLUSION</b>LIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.</p>

Effect of Guishen Pill on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Guishen Pill (GSP) on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve (DOR).</p><p><b>METHODS</b>Totally 40 female C57BL/6J mice were randomly divided into 4 groups, the normal control group, the model group, the GSP group, and the dehydroepiandrosterone (DHEA) group, 10 in each group. Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG), and prostaglandin F2α (PGF2α) were sequentially administrated to produce superovulation. The DOR model was established by exposing to ozone inhalation. Mice in the GSP group were intragastrically administered with GSP at 0.3 mL. Those in the DHEA group were intragastrically administered with DHEA at 0.3 mL. Equal volume of normal saline was intragastrically administered to mice in the normal control group and the model group. All mice wer treated for 21 days. Serum levels of estrogen (E2), progestogen (P), and anti-Müllerian hormone (AMH) were measured by ELISA. Changes of Oct-4, anti-AMH, and early growth response gene-1 (Egr-1) mRNA in ovaries were dtected by Real-time PCR.</p><p><b>RESULTS</b>Compared with the model group, serum levels of E2, P, and AMH, as well as contents of estrogen receptor (ER), progestogen receptor (PR), MVH, and Oct-4 mRNA significantly increased in the GSP group and the DHEA group (P < 0.05).</p><p><b>CONCLUSION</b>GSP could improve expression levels of Oct-4, MVH, and Egr-1 mRNA in DOR mice and their ovarian function.</p>

The potential role of COX-2 in cancer stem cell-mediated canine mammary tumor initiation: an immunohistochemical study

Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.

Comparing the effects of small molecules BIX-01294, Bay K8644, RG-108 and valproic acid, and their different combinations on induction of pluripotency marker-genes by Oct4 in the mouse brain

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid [VPA] are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction [PCR] was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell [NSC] markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog [p<0.05]. Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc [p<0.001]. VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc [p<0.01], Pax6 and Sox1 [p<0.001]. These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

Transient folate deprivation in combination with small-molecule compounds facilitates the generation of somatic cell-derived pluripotent stem cells in mice / 华中科技大学学报（医学）（英德文版）

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.

Sphere-forming-like cells (squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma / 国际口腔科学杂志·英文版

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell (CSC) traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas (SCCs) and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation (CD) 44, CD133, acetaldehyde dehydrogenase 1 (ALDH1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), Nestin, octamer-binding transcription factor 4 (Oct4) and reduced expression protein-1 (Rex-1) expression with reverse transcription-polymerase chain reaction (RT-PCR); chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers (CD44, Bmi-1, Nestin, Oct4 and Rex-1), capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts (with histological features resembling those of the original VX2 rabbit buccal SCC) from the transplantation of 10(3) undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.

Expression level of pluripotent genes in incomplete reprogramming

OBJECTIVE@#To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.@*METHODS@#Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR.@*RESULTS@#In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level.@*CONCLUSIONS@#It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.

Epigenetic reprogramming, gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor--m-carboxycinnamic acid bishydroxamide (CBHA)

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.

Construction of lentiviral vector carrying human OCT4A gene and establishment of leukemic cell line K562 stably expressing OCT4A / 中国实验血液学杂志

This study was purposed to construct a lentiviral vector carrying human OCT4A gene and green fluorescent protein (GFP) , and infect the leukemic cell line K562, observe the expression of OCT4A in K562 cells. According to the sequence of OCT4A mRNA which was found in GenBank, the special primer sequences were synthesized. The OCT4A gene was amplified by RT-PCR, and then cloned into the pCR-Blunt vector. The OCT4A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4A-ZsGreen1. The sequence of the recombinant plasmid was identified by DNA sequencing. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000 and transfected into K562 cells. Real-time PCR and Western blot were applied to detect the expression of OCT4A mRNA and protein, CCK-8 and colony formation assay were performed to evaluate the effects of OCT4A on proliferation of K562 cells. The results showed that the recombinant lentiviral vector pLVX-OCT4A-ZsGreen1 was successfully constructed. The virus titers were (1.43 ± 0.25) × 10(8) U/ml. After infection of K562 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of OCT4A gene and protein according the real-time PCR and Western blot detection results. CCK-8 and colony formation assay showed that exogenous OCT4A gene could significantly promote cell growth and the colony formation of K562 cells. It is concluded that the recombinant lentiviral vector pLVX-OCT4A- ZsGreen1 carrying human OCT4A gene is successfully constructed; K562 cells which stably up-regulates the expression of OCT4A mRNA are obtained, the results of this study provide fundamental basis for further study on mechanism of OCT4A in human leukemia development.

[ long non-coding RNA, PSORS1C3, located upstream of the human Oct4 gene is expressed in pluripotent and tumor cell lines]

Long non-coding RNAs [lncRNAs], a vast class of recently discovered noncoding genes in the human genome, have been implicated in the regulation of several biological processes, including the maintenance of stem cell pluripotency and neurogenesis. New evidences have emerged that some long IncRNAs act as enhancers for their neighboring genes. Oct4, also known as POU5F1 and Oct3/4, functions as a master regulator in maintaining the properties of pluripotency and self-renewal of embryonic stem [ES] cells and embryonal carcinoma [EC] cells. Oct-4 expression must be tightly regulated; too much or too little expression can lead to cell differentiation. PSORS1C3, an IncRNA, is located upstream of the Oct4 gene. This IncRNA could potentially impact the level of Oct4 expression. Here, we have investigated potential expression of PSORS1C3 on 23 different human pluripotent and cancer cell lines by means of RT-PCR. Our results revealed a noticeable expression of PSORS1C3 both in a well-known pluripotent cell line [NTera2/NT2] and five different cancer cell lines [AGS, 5637, Ht-29, HepG2 and PC3]. We detected the expression of PSORS1C3 for the first time in both cancer cell lines and stem cells