OBJECTIVE: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). MATERIALS AND METHODS: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. RESULTS: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.01, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. CONCLUSION: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.
Alkaline Phosphatase ; Cryopreservation ; Embryonic Stem Cells* ; Humans* ; Karyotype ; Octamer Transcription Factor-3 ; Seoul ; Vitrification*

OBJECTIVETo evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.

METHODSTwo 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.

RESULTSThe 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).

CONCLUSIONTwo 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.

Apoptosis ; Cell Proliferation ; Genetic Vectors ; Humans ; K562 Cells ; Lentivirus ; Octamer Transcription Factor-3 ; genetics ; Transfection

OBJECTIVETo study the expression of OCT4 protein in bladder cancer and its correlation to the clinicopathologic features and prognosis of bladder cancer.

METHODSOCT4 mRNA and protein expression was detected in 5 bladder cancer cell lines (RT-4, Tcc-Sup, KK47, T24, and 5637) and 1 normal bladder cell lines by real-time PCR and Western blotting, respectively. Immunohistochemical analysis was used to detect the expression of OCT4 protein in 46 bladder cancer samples.

RESULTSAll the 5 bladder cancer cell lines expressed detectable levels of OCT4 mRNA and proteins, whereas the normal bladder cell line SV-HUC-1 was negative for OCT4 expression. The clinical bladder cancer tissues showed a high positivity rate of OCT4 expression (76.1%), which was not detected in normal bladder tissues. Specific OCT-4 signals were localized mainly in the nuclei of the cancer cells. The expression rate of OCT4 protein was significantly higher in bladder cancer tissue than in normal bladder epithelium (P<0.05), and showed a positive correlation to the grade of tumor differentiation and metastasis (P<0.05) but not to the patients' age, gender or TNM stage.

CONCLUSIONOCT4 protein expression is associated with tumor differentiation and metastasis in bladder cancer and may play an important role in the early diagnosis and prognostic evaluation of bladder cancer.

Cell Line, Tumor ; Humans ; Neoplasm Metastasis ; Neoplasm Staging ; Octamer Transcription Factor-3 ; metabolism ; Prognosis ; Urinary Bladder Neoplasms ; diagnosis ; metabolism ; pathology

OBJECTIVE: To discuss the meanings of Oct-4's expression in thyroid adenoma, thyroid papillary carcinoma, thyroid follicular carcinoma, and medullary thyroid carcinoma. METHOD: We examined the expression of Oct-4 in 15 thyroid adenoma, 30 thyroid papillary carcinomas, 2 thyroid follicular carcinomas, and 3 medullary thyroid carcinomas using immunofluorescence. RESULT: Oct-4 expression was observed in all the thyroid-related diseases mentioned above. In thyroid papillary carcinomas, the expression of Oct-4 were higher than that in thyroid adenoma, and had no obvious relationship with the patients age, sex, the size and location of tumor and tumor metastasis. CONCLUSION The formation of the thyroid carcinomas may be concerned with the stem cells in thyroid. There are more stem cells in medullary thyroid carcinomas and follicular carcinomas.
Adenocarcinoma, Follicular ; metabolism ; pathology ; Carcinoma, Neuroendocrine ; Carcinoma, Papillary ; metabolism ; pathology ; Humans ; Octamer Transcription Factor-3 ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

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.
Adipose Tissue/cytology ; *Cell Differentiation ; *Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells/cytology/*metabolism/physiology ; Octamer Transcription Factor-3/genetics/*metabolism ; SOXB1 Transcription Factors/genetics/*metabolism

BACKGROUNDHuman embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study.

METHODSThree oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.

RESULTSFour ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.

CONCLUSIONSHES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

Blastocyst ; cytology ; Cell Differentiation ; Cell Line ; DNA-Binding Proteins ; analysis ; Female ; Fertilization in Vitro ; Humans ; Karyotyping ; Male ; Octamer Transcription Factor-3 ; Stem Cells ; cytology ; Tissue Donors ; Transcription Factors ; analysis

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.
Bone Marrow ; Humans ; Induced Pluripotent Stem Cells ; Mesenchymal Stem Cells ; Nanog Homeobox Protein ; genetics ; Octamer Transcription Factor-3 ; genetics ; Transcription Factors ; Up-Regulation

Animals ; Astrocytes ; cytology ; Cell Differentiation ; Cellular Reprogramming ; Induced Pluripotent Stem Cells ; cytology ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Mice ; Neurons ; cytology ; Octamer Transcription Factor-3 ; genetics ; metabolism

Animals ; Aquaporin 3 ; analysis ; Cell Separation ; Fluorouracil ; pharmacology ; Humans ; Lung ; cytology ; Membrane Proteins ; analysis ; Octamer Transcription Factor-3 ; genetics ; Stem Cells ; cytology ; Trachea ; cytology

OBJECTIVETo isolate, culture and identify of human endometrial stromal stem cells.

METHODSEndometrial tissue was obtained from 10 women undergoing hysterectomy. Purified stromal stem cell suspensions were then obtained by selecting cells with magnetic bead sorting and colony-forming. The surface markers of stromal cell were identified by flow cytometry. Proliferation of stromal stem cell was observed by MTT methods. The osteogenic potential was evaluated by alizarin red staining, the adipogenic potential by oil red O staining. Then the adipogenic and osteogenic specific markers of differentiated cells assayed by RT-PCR method. Expression of cell surface antigen OCT-4 was detected with immunocytochemical staining.

RESULTSEndometrial stem cells were successfully isolated from human endometrial tissue. They were stably proliferated and subcultured in vitro. Most of the passage cells expressed mesenchymal stem cell markers CD90, CD105 but not hemopoietic markers CD34, CD45. The analysis of cell cycle indicated that the percentage of G2-M phase and S phase cells increased. The growth curves of the third passage presented in "S" shape. After cultured in differentiation medium, the cells differentiated toward adipoblasts and osteoblasts as verified by positive staining with Oil Red O and alizarin red staining. Under induction,cells expressed osteogenic and adipogenic marker genes. The immunocytochemical staining of OCT-4 was positive.

CONCLUSIONHuman endometrium contains endometrial stromal stem cells, which present characteristics of mesenchymal stem cells and may be used as seed cells for tissue engineering.

Adult ; Cell Culture Techniques ; Cell Separation ; Cells, Cultured ; Endometrium ; cytology ; Female ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Octamer Transcription Factor-3 ; metabolism ; Stromal Cells ; cytology