Overexpression Of Oct4 Research Articles

Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation

Although Oct4 is known as a critical transcription factor involved in maintaining “stemness”, its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression.

Wnt/β-catenin signalling maintains self-renewal and tumourigenicity of head and neck squamous cell carcinoma stem-like cells by activating Oct4.

Accumulating evidence suggests that a distinct subpopulation of cancer stem cells (CSCs) is responsible for tumour initiation and progression in head and neck squamous cell carcinoma (HNSCC). Wnt/β-catenin signalling is essential for stem cell regulation and tumourigenesis, but its molecular mechanism in HNSCC CSCs remains unknown. We investigated whether Wnt/β-catenin signalling regulates self-renewal and tumourigenicity of HNSCC stem-like cells in vitro and in vivo. Cytoplasmic/nuclear β-catenin, a major effector of Wnt/β-catenin signalling, was expressed in a subpopulation of tumour cells in primary HNSCC tissue but in none of normal head and neck tissues. Overexpression of β-catenin increased proliferation of HNSCC cells and induced dedifferentiation of these cells to cells with stem-like features. Knockdown of β-catenin in HNSCC stem-like cells blocked their self-renewal capacity, stemness-associated gene expression, chemoresistance, and in vivo tumourigenicity. Furthermore, β-catenin directly regulates Oct4 transcription in HNSCC stem-like cells. In addition, the effect of shRNA-mediated repression of β-catenin on CSC traits in HNSCC stem-like cells was reversed by overexpression of Oct4. In patients with HNSCC, higher levels of both cytoplasmic/nuclear β-catenin and Oct4 correlated with the worst prognosis. These results suggest inhibition of Wnt/β-catenin signalling as a novel therapeutic strategy for targeting HNSCC stem-like cells.

High Expression of SOX2 and OCT4 Indicates Radiation Resistance and an Independent Negative Prognosis in Cervical Squamous Cell Carcinoma

Radiotherapy (RT) as a preoperative or postoperative adjuvant or primary treatment is the most common management modality for locally advanced cervical cancer. Radioresistance of tumor cells remains a major therapeutic problem. Consequently, we aimed to explore if the stem cell biomarkers SOX2 and OCT4 protein could be used to predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). These 132 patients were divided into two groups (radiation-resistant and radiation-sensitive groups) according to progress-free survival (PFS). Using pretreatment paraffin-embedded tissues, we evaluated SOX2 and OCT4 expression using immunohistochemical staining. The percentage of overexpression of SOX2 and OCT4 in the radiation-resistant group was much higher than that in the radiation-sensitive group (p<0.001 and p <0.001, respectively). The patients with high expression of SOX2 and OCT4 showed a shorter PFS than those with low expression. Our study suggests that the expression of SOX2 and OCT4 in tumor cells indicates resistance to radiotherapy and that these two factors were important predictors of poor survival in patients with LACSCC (hazard ratio [95% CI], 2.294 [1.013, 5.195] and 2.300 [1.050, 5.037], respectively; p=0.046 and p=0.037, respectively).

MP34-12 TARGETING IGF-BP2 AND 5 WITH THE BISPECIFIC ANTISENSE OLIGODEOXYNUCLEOTIDE OGX-225 IN CHEMORESISTANT BLADDER CANCER

observed in chemo-resistant patients (**p< 0.01), which also showed correlation to prognosis. Similar results obtain by qPCR array. Furthermore, Oct4 and Nanog expression were also upregulated in multidrug resistant (253J-ADM) cell compared with parental cell, the higher tumorigenesis ability were also observed by in vitro and in vivo tumorigenesis assay(*p<0.05). Knocking down of Oct4 or Nanog in multidrug resistant BCa cell line dramatically suppressed cell renew ability (*p<0.05) and enhanced chemosensitivity to chemo-drugs (mitomycin/ pirarubicin) (*p<0.05). CONCLUSIONS: These data indicate that cancer stem-like properties were expanded during the acquisition of multidrug resistance in BCa. Clinically, BCa stemness markers (Oct4 and nanog) overexpression may promote the BCa recurrence to resist multidrugs, which could be a potential therapeutic target for chemoresistant BCa treatment.

MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2014MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA Yi Sun, Liang Liang, Yule Chen, Yongyi Cheng, Yonggang Xu, and Dalin He Yi SunYi Sun More articles by this author , Liang LiangLiang Liang More articles by this author , Yule ChenYule Chen More articles by this author , Yongyi ChengYongyi Cheng More articles by this author , Yonggang XuYonggang Xu More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1024AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer (BCa) is the most prevalent and easy recurrent cancer worldwide, high propensity for recurrence and progression are still serious problems leading to high costs and life-treatment. Multidrug resistant always induced metastasis and treatment failure. Cancer stem cells (CSC) model theoretically contribute to BCa metastasis and mulitidrug resistantce. In this study, we screened BCa stemness specific marker and further detected the potential roles during multidrug resistant process. METHODS 65 pairs of primary and relapsed chemo-resistant tumors were collected during past 10 years from shaanxi provincial people¡¯s hospital, differential expression profile of stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) were screened by IHC staining and qPCR assay. The specific stemness markers were further confirmed with newly constructed multidrug resistant (253J-ADM) and their parental cell by WB, qPCR ,in vitro and in vivo tumorigenesis assay. We finally evaluated the self-renewal ability and chemosensitivity change after knocking down specific markers by siRNA in multidrug resistant BCa cell line. RESULTS Highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) was detected in relapsed chemo-resistant tumors vs primary tumor (*p<0.05 for Bmi1, CD117, CD133, and ABCG2). Oct4 and Nanog expression was significantly observed in chemo-resistant patients (**p< 0.01), which also showed correlation to prognosis. Similar results obtain by qPCR array. Furthermore, Oct4 and Nanog expression were also upregulated in multidrug resistant (253J-ADM) cell compared with parental cell, the higher tumorigenesis ability were also observed by in vitro and in vivo tumorigenesis assay(*p<0.05). Knocking down of Oct4 or Nanog in multidrug resistant BCa cell line dramatically suppressed cell renew ability (*p<0.05) and enhanced chemosensitivity to chemo-drugs (mitomycin/ pirarubicin) (*p<0.05). CONCLUSIONS These data indicate that cancer stem-like properties were expanded during the acquisition of multidrug resistance in BCa. Clinically, BCa stemness markers (Oct4 and nanog) overexpression may promote the BCa recurrence to resist multidrugs, which could be a potential therapeutic target for chemoresistant BCa treatment. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e365-e366 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yi Sun More articles by this author Liang Liang More articles by this author Yule Chen More articles by this author Yongyi Cheng More articles by this author Yonggang Xu More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Counteracting Activities of OCT4 and KLF4 during Reprogramming to Pluripotency

SummaryDifferentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.

EMT and Acquisition of Stem Cell-Like Properties Are Involved in Spontaneous Formation of Tumorigenic Hybrids between Lung Cancer and Bone Marrow-Derived Mesenchymal Stem Cells

The most deadly phase in cancer progression is metastatic conversion. Epithelial-to-mesenchymal transition (EMT) is a key process by which cancer cells acquire invasive and metastatic phenotypes. In order to spawn macroscopic metastases, disseminated cancer cells would seem to require self-renewal capability. However, the underlying mechanism defining these processes is poorly understood. One possible mechanism underlying metastasis is fusion between myeloid cells and cancer cells. In this study, we found that spontaneously-formed tumorigenic hybrids between bone marrow-derived mesenchymal stem cells (MSCs) and three different non-small cell lung cancer (NSCLC) cell lines contributed to highly malignant subpopulations with both EMT and stem cell-like properties. Hybrids lost their epithelial morphology and assumed a fibroblast-like appearance. Up-regulation of vimentin, α-smooth muscle actin (α-SMA), and fibronectin, and down-regulation of E-cadherin and pancytokeratin were observed in tumorigenic hybrids. These cells also exhibited increased expression of the stem cell marker prominin-1 (CD133) and over-expression of transcription factors OCT4, Nanog, BMI1, Notch1, ALDH1 as well as Sox2, all genes responsible for regulating and maintaining the stem cell phenotype. In addition, in spontaneously-formed tumorigenic hybrids, increased pneumosphere-forming capacity and tumor-forming ability in NOD/SCID mice were detectable. Thus, cell fusion between lung cancer cells and MSCs provides a nonmutational mechanism that could contribute to aberrant gene expression patterns and give rise to highly malignant subpopulations both capable of EMT and with properties of cancer stem cells (CSCs).

Oct4 Mediates Tumor Initiating Properties in Oral Squamous Cell Carcinomas through the Regulation of Epithelial-Mesenchymal Transition

BackgroundOverexpression of Oct4, an important transcription factor of embryonic stem cells (ESC), has been reported in several cancers. The aim of this study was to determine the emerging role of Oct4 in oral squamous cell carcinoma (OSCC) both in vitro and in vivo.Methodology/Principal FindingTumourigenic activity and molecular mechanisms of Oct4 overexpression or knockdown by lentiviral infection in OSCC was investigated in vitro and in vivo. Initially, we demonstrated that Oct4 expression was increased in OSCC cell lines as compared to a normal oral epithelial cell line SG. Overexpression of Oct4 was demonstrated to enhance cell proliferation, invasiveness, anchorage-independent growth and xenotransplantation tumourigenicity. These findings were coupled with epithelial-mesenchymal transition (EMT) transformation in OSCCs. In contrast, the silence of Oct4 significantly blocked the xenograft tumorigenesis of OSCC-derived cancer stem cells (OSCC-CSCs) and significantly improved the recipient survival. Clinically, the level of Oct4 expression was higher in recurrent and metastatic OSCC specimens but lower in primary OSCC specimens.Conclusion/SignificanceOur results suggest that Oct4-mediated tumorigenecity is associated with the regulation of EMT. Oct4 might be a therapeutic target for OSCC.

MiRNAs promote generation of porcine-induced pluripotent stem cells

The pigs have similarities of organ size, immunology and physiology with humans. Porcine-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. Here, we established piPSCs induced from porcine fetal fibroblasts by the retroviral overexpression of Oct4, Sox2, Klf4, and c-Myc. The piPSCs not only express pluripotent markers but also have the capacity for differentiation in vivo and in vitro, including EB and teratoma formation. We supplemented microRNAs during the induction process because miR-302a, miR-302b, and miR-200c have been reported to be highly expressed in human and mouse embryonic stem cells and in iPSCs. In this study, we found that the overexpression of miR-302a, miR-302b, and miR-200c effectively improved the reprogramming efficiency and reduced the induction time for piPSCs in the OSKM and OSK induction systems. Due to the similar induction efficiency of 4F-induced piPSCs or of three factors combined with miR-302a, miR-302b, and miR-200c (3F-miRNA-induced piPSCs), we recommend the addition of miRNAs instead of c-Myc to reduce the tumorigenicity of piPSCs.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells

CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications.

Effect of Exogenous Oct4 Overexpression on Cardiomyocyte Differentiation of Human Amniotic Mesenchymal Cells

Regenerative therapy is a new strategy for the end-stage heart failure; however, the ideal cell source has not yet been established for this therapy. We expected that the amnion might be an ideal cell source for cardiac regenerative therapy and that the differentiation potency of the human amnion mesenchymal cells (hAMCs) could be improved by overexpression of Oct4, a key factor that maintains the undifferentiated state. A plasmid vector was made by insertion of the Oct4 open reading frame (ORF) under control of a cytomegalovirus (CMV) promoter (pCMV-hOct4) and transfected into hAMCs by electroporation. The optimum induction time was investigated by comparing the quantity of stem cell-specific mRNAs, cardiac-specific mRNAs, and cardiac-specific proteins with time. hAMCs already expressed cardiac-specific proteins such as Nkx2.5 and Connexin43. After pCMV-hOct4 transfection, endogenous Oct4 mRNA and other stem cell markers showed a transient increase. With 5-azacytidine treatment, quantities of the cardiac-specific mRNAs, such as GATA4 and myosin light-chain-2v (Mlc-2v), were increased significantly. After Oct4 overexpression, the highest expression of cardiac-specific mRNAs and stem cell makers was seen at almost the same time. Furthermore, more mature myocardial contraction proteins were observed when hAMCs were induced at specific optimal times after gene transfection. In conclusion, hAMCs were activated to an undifferentiated state by overexpression of Oct4, and their cardiac differentiation potency was improved. Thus, the single-time transfection of the Oct4 expression vector may be a useful strategy for effective cell therapy. The use of cryopreserved hAMCs in cell therapy still requires more investigation.

Follicle-stimulating hormone inhibits apoptosis in ovarian cancer cells by regulating the OCT4 stem cell signaling pathway

OCT4, a stem cell marker, is overexpressed in several types of human cancer and can induce resistance to chemotherapy and inhibition of apoptosis. We previously demonstrated that human follicle stimulating hormone (FSH) can inhibit ovarian cancer cell apoptosis. However, the role of OCT4 in FSH-induced inhibition of apoptosis has not been reported in detail. Here, we profiled OCT4 protein expression in ovarian epithelial cancer (OEC) with benign cystadenoma, borderline tumor and carcinoma tissues as well as different ovarian cancer cell lines and normal ovarian epithelial cells. Furthermore, the effects of FSH on OCT4 expression and related signaling pathways were evaluated. The overexpression of OCT4 in ovarian carcinoma and OEC cell lines suggest that OCT4 plays a critical role in OEC carcinogenesis. Moreover, FSH-induced apoptosis inhibition was confirmed and FSH stimulation induced the expansion of CD44+CD117+ cells with a stem cell-like phenotype. Re-expression of OCT4 enhanced the expression of Notch, Sox2 and Nanog molecules that play critical roles in cancer stem cell proliferation and differentiation. FSH upregulated the expression of Notch, Sox2 and Nanog and these effects were abolished by knocking down OCT4, suggesting that several cancer stem cell pathways are involved in FSH regulation. We also examined OCT4 expression in surgical specimens of ovarian cancer. Immunohistostaining revealed that OCT4 expression was increased in ovarian carcinoma compared with benign cystadenomas and borderline tumors, and OCT4 expression was significantly correlated with histological grade. Staining for OCT4 was increased in serous cystadenocarcinoma, when compared with clear cell carcinoma. In summary, the OCT4 cancer stem cell signaling pathway may mediate FSH-induced inhibition of apoptosis and could provide a target for treatment of ovarian cancer.

Reprogramming of Human Fibroblasts to Pluripotency with Lineage Specifiers

Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.

Oct-4 is required for an antiapoptotic behavior of chemoresistant colorectal cancer cells enriched for cancer stem cells: Effects associated with STAT3/Survivin

Cancer stem cells (CSCs) have been implicated in multidrug resistance, a phenomenon responsible for the failure of cancer chemotherapy. However, the underlying mechanisms are still unclear. In our study, we established two oxaliplatin-resistant colorectal cancer cell lines displaying some CSCs characteristics. Oct4 overexpression was observed in these two lines. We performed Oct4 knock down by lentiviral vector-mediated specific shRNA. Knockdown increased apoptosis, decreased CSCs marker expression and weakened tumorigenicity in drug-resistant cell lines. In conclusion, we show that these events can be at least in part attributed to the STAT3/Survivin pathway.

PSCA and Oct-4 Expression in the Benign and Malignant Lesions of Gallbladder: Implication for Carcinogenesis, Progression, and Prognosis of Gallbladder Adenocarcinoma

PSCA and Oct-4 have been thought as markers of cancer stem cells. Although overexpression of PSCA and Oct-4 in cancer has been reported, little is known about the clinical and pathological significance with PSCA and Oct-4 expression in gallbladder adenocarcinoma. In this study, overexpression of PSCA and Oct-4 was detected in gallbladder adenocarcinoma (54.6% and 55.6%). Less expression of PSCA and Oct-4 was detected in the pericancerous tissues (19.6% and 21.7%), gallbladder polyps (13.3% and 13.3%), and gallbladder epithelium with chronic cholecystitis (14.3% and 14.3%). The overexpression of PSCA and Oct-4 was significantly associated with differentiation, tumor mass, lymph node metastasis, invasion of gallbladder adenocarcinoma, and decreased overall survival. Our study suggested that overexpression of PSCA and Oct-4 might be closely related to the carcinogenesis, progression, metastasis, or invasive potential and prognosis of gallbladder carcinoma.

Oct4 and Sox2 overexpression improves the proliferation and differentiation of bone mesenchymal stem cells in Xiaomeishan porcine

Mesenchymal stem cells derived from bone marrow (BMSCs) are a population of self-renewing multipotent cells that are capable of differentiating into various cellular lineages, and are widely employed in tissue engineering and cell therapy. Recently, clinical research involving BMSCs has become increasingly popular. In order to conduct appropriate research, it is first necessary to amplify large amounts of functional BMSCs in vitro. However, after several passages of expanding in vitro, the proliferation and differentiation potential of BMSCs gradually decline. To determine whether overexpression of Oct4 or Sox2 might prevent this decline, we transfected Oct4 or Sox2, which are essential for the pluripotency and self-renewal of embryonic stem cells, into BMSCs of Xiaomeishan porcine by a lentivirus. The results showed that overexpression of Sox2 or Oct4 BMSCs in culture media containing a basic fibroblast growth factor resulted in higher proliferation and differentiation compared to controls, suggesting that genetic modification of stemness-related genes is an efficient way to maintain the proliferation and differentiation potential of BMSCs.

The Regulation of Downstream Target Gene AP-2γ by Transcription Factor OCT-4

In recent years,OCT-4 and AP-2γ have been widely used as clinical markers of testicular germ cell tumors.Transcription factor OCT-4,which plays an important role in embryonic development,gives full play to a variety of biological functions in different developmental periods and differentiations.The effect of OCT-4 is realized through the regulation of the target genes.In this study,we found OCT-4 binding sites within the sequence of AP-2γ promoter region by a variety of bionemerics.AP-2γ,a novel target gene of OCT-4,was identified by chromatin immunoprecipitation(ChIP)-PCR.A combination of sequence analysis,reporter gene assays,Western blot,immunofluorescence assay and mouse cryptorchidism model experiment further confirmed that AP-2γ was the target gene of OCT-4.OCT-4 inhibited the transcriptive activity of AP-2γ.The expression of AP-2γ gene was confirmed to be significantly altered by silencing or overexpression of OCT-4.This new discovery is conductive to study the malignant process of germ cell tumors at the molecular level.

Exploring the Neural Reprogrammome using Bioinformatics Approaches

Recent studies demonstrate that mammalian cells can be artificially reprogrammed by ectopic expression of transcription factors in an unforeseen straightforward manner. Patient-derived reprogrammed cells hold great potential for biomedical applications such as cell replacement therapy, drug toxicity studies and disease modeling. Somatic cells such as fibroblasts can be dedifferentiated into so-called induced pluripotent stem cells (iPSCs) that are similar to embryonic stem cells (ESCs) by overexpression of Oct4, Sox2, Klf4 and cMyc. However, clinical applications using iPSCs carry the risk of tumor formation due to incomplete differentiation. More recently, it has been demonstrated that transcription factor-driven reprogramming enables direct conversion of fibroblasts into neurons, cardiomyocytes, hepatocytes as well as neural progenitors. Various groups elaborated protocols for the direct conversion of fibroblasts into multipotent induced neural stem cells (iNSCs) using different combinations of transcription factors and media conditions. These studies have shown that iNSCs exhibit morphology, gene expression and self-renewing capacity similar to NSCs derived from primary tissue. Moreover, these iNSCs differentiated into neurons, astrocytes and oligodendrocytes indicating multipotency of these cells. Here, we compare the gene expression profile of reprogrammed cells reported in these studies to determine the similarity in expression profile between the generated iNSCs using bioinformatics approaches. We provide a general workflow that can be applied to evaluate the status of reprogrammed cell populations. Using hierarchical clustering analysis and principal component analysis (PCA), we show that iNSCs reported by Thier et al. resemble more closely to those of Han et al. On the other hand, iNSCs generated by Ring et al. are relatively similar to 4F iNSC (late) of the study by Han et al. as judged by hierarchical clustering analysis. Our study demonstrates that bioinformatics approaches are particularly valuable to robustly assess the transcriptional status of reprogrammed cells and to anticipate their cellular functionality.

Conversion of genomic imprinting by reprogramming and redifferentiation

Induced pluripotent stem cells (iPSCs), generated from somatic cells by overexpression of transcription factors Oct4, Sox2, Klf4 and c-Myc have the same characteristics as pluripotent embryonic stem cells (ESCs). iPSCs reprogrammed from differentiated cells undergo epigenetic modification during reprogramming, and ultimately acquire a similar epigenetic state to that of ESCs. In this study, these epigenetic changes were observed in reprogramming of uniparental parthenogenetic somatic cells. The parthenogenetic pattern of imprinted genes changes during the generation of parthenogenetic maternal iPSCs (miPSCs), a process referred to as pluripotent reprogramming. We determined whether altered imprinted genes are maintained or revert to the parthenogenetic state when the reprogrammed cells are redifferentiated into specialized cell types. To address this question, we redifferentiated miPSCs into neural stem cells (miPS-NSCs) and compared them with biparental female NSCs (fNSCs) and parthenogenetic NSCs (pNSCs). We found that pluripotent reprogramming of parthenogenetic somatic cells could reset parthenogenetic DNA methylation patterns in imprinted genes, and that alterations in DNA methylation were maintained even after miPSCs were redifferentiated into miPS-NSCs. Notably, maternally methylated imprinted genes (Peg1, Peg3, Igf2r, Snrpn and Ndn), whose differentially methylated regions were fully methylated in pNSCs, were demethylated and their expression levels were found to be close to the levels in normal biparental fNSCs after reprogramming and redifferentiation. Our findings suggest that pluripotent reprogramming of parthenogenetic somatic cells followed by redifferentiation leads to changes in DNA methylation of imprinted genes and the reestablishment of gene expression levels to those of normal biparental cells.