Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.

Uterine nourishment of embryos by the placenta is a key feature of mammals. Although a variety of placenta types exist, they are all derived from the trophectoderm (TE) cell layer of the developing embryo. Egg-laying mammals (platypus and echidnas) are distinguished by a very short intrauterine embryo development, in which a simple placenta forms from TE-like cells. The Pou5f1 gene encodes a class V POU family transcription factor Oct3/4. In mice, Oct3/4 together with the highly conserved caudal-related homeobox transcription factor Cdx2, determines TE fate in pre-implantation development. In contrast to Cdx2, Pou5f1 has only been identified in eutherian mammals and marsupials, whereas, in other vertebrates, pou2 is considered to be the Pou5f1 ortholog. Here, we show that platypus and opossum genomes contain a Pou5f1 and pou2 homolog, pou2-related, indicating that these two genes are paralogues and arose by gene duplication in early mammalian evolution. In a complementation assay, we found that platypus or human Pou5f1, but not opossum or zebrafish pou2, restores self-renewal in Pou5f1-null mouse ES cells, showing that platypus possess a fully functional Pou5f1 gene. Interestingly, we discovered that parts of one of the conserved regions (CR4) is missing from the platypus Pou5f1 promoter, suggesting that the autoregulation and reciprocal inhibition between Pou5f1 and Cdx2 evolved after the divergence of monotremes and may be linked to the development of more elaborate placental types in marsupial and eutherian mammals.

Enzyme-catalyzed, post-translational modifications of core histones have been implicated in the complex changes in gene expression that drive early mammalian development. However, until recently the small number of cells available from the preimplantation embryo itself has prevented quantitative analysis of histone modifications at key regulator genes. The possible involvement of histone modifications in the embryo's response to extracellular signals, or as determinants of cell fate or lineage progression, remains unclear. Here we describe the use of a recently-developed chromatin immunoprecipitation technique (CChIP) to assay histone modification levels at key regulator genes (Pou5f1, Nanog, Cdx2, Hoxb1, Hoxb9) as mouse embryos progress from 8-cell to blastocyst in culture. Only by the blastocyst stage, when the embryonic (Inner Cell Mass) and extra-embryonic (Trophoblast) lineages are compared, do we see the expected association between histone modifications previously linked to active and silent chromatin, and transcriptional state. To explore responses to an environmental signal, we exposed embryos to the histone deacetylase inhibitor, anti-epileptic and known teratogen valproic acid (VPA), during progression from 8-cell to morula stage. Such treatment increased H4 acetylation and H3 lysine 4 methylation at the promoters of Hoxb1 and Hoxb9, but not the promoters of Pou5f1, Nanog,Cdx2 or the housekeeping gene Gapdh. Despite the absence of detectable Hoxb transcription, these VPA-induced changes were heritable, following removal of the inhibitor, at least until the blastocyst stage. The selective hyperacetylation of Hoxb promoters in response to a histone deacetylase inhibitor, suggests that Hox genes have a higher turnover of histone acetates than other genes in the preimplantation embryo. To explain the heritability, through mitosis, of VPA-induced changes in histone modification at Hoxb promoters, we describe how an epigenetic feed-forward loop, based on cross-talk between H3 acetylation and H3K4 methylation, might generate a persistently increased steady-state level of histone acetylation in response to a transient signal.

We analyzed the transcriptional profiles of differentiating mouse embryonic stem cells (mESCs) and show that embryoid bodies (EBs) sequentially expressed genes associated with the epiblast, primitive streak, mesoderm and endoderm of the developing embryo, validating ESCs as a model system for identifying cohorts of genes marking specific stages of embryogenesis. By comparing the transcriptional profiles of undifferentiated ESCs to those of their differentiated progeny, we identified 503 mESC and 983 hESC genes selectively expressed in undifferentiated ES cells. Over 75% of the mESC genes were expressed in hESC and vice versa, attesting to the underlying similarity of mESCs and hESCs. The expression of a cohort of 68 genes decreased greater than 2-fold during differentiation in both mESCs and hESCs. As well as containing many validated ESC genes such as Oct4 [Pou5f1], Nanog and Nodal, this cohort included an uncharacterised gene (FLJ30046), which we designated SLAIN1/Slain1. Slain1 was expressed at the stem cell and epiblast stages of ESC differentiation and in the epiblast, nervous system, tailbud and somites of the developing mouse embryo. SLAIN1 and its more widely expressed homologue SLAIN2 comprise a new family of structurally unique genes conserved throughout vertebrate evolution.

Dax1 (Nr0b1) is an atypical orphan nuclear receptor that has recently been shown to play a role in mouse embryonic stem (mES) cell pluripotency. Here we describe a mechanism by which Dax1 maintains pluripotency. In steroidogenic cells, Dax1 protein interacts with the NR5A nuclear receptor steroidogenic factor 1 (Nr5a1) to inhibit transcription of target genes. In mES cells, liver receptor homolog 1 (LRH-1, Nr5a2), the other NR5A family member, is expressed, and LRH-1 has been shown to interact with Dax1. We demonstrate by coimmunoprecipitation that Dax1 is, indeed, able to form a complex with LRH-1 in mES cells. Because Dax1 was historically characterized as an inhibitor of steroidogenic factor 1-mediated transcriptional activation, we hypothesized that Dax1 would inhibit LRH-1 action in mES cells. Therefore, we examined the effect of Dax1 on the LRH-1-mediated activation of the critical ES cell factor Oct4 (Pou5f1). Chromatin immunoprecipitation localized Dax1 to the Oct4 promoter at the LRH-1 binding site, and luciferase assays together with Dax1 overexpression and knockdown experiments revealed that, rather than repress, Dax1 accentuated LRH-1-mediated activation of the Oct4 gene. Similar to our previously published studies that defined the RNA coactivator steroid receptor RNA activator as the critical mediator of Dax1 coactivation function, Dax1 augmentation of LRH-1-mediated Oct4 activation is dependent upon steroid receptor RNA activator. Finally, utilizing published chromatin immunoprecipitation data of whole-genome binding sites of LRH-1 and Dax1, we show that LRH-1 and Dax1 commonly colocalize at 288 genes (43% of LRH-1 target genes), many of which are involved in mES cell pluripotency. Thus, our results indicate that Dax1 plays an important role in the maintenance of pluripotency in mES cells through interaction with LRH-1 and transcriptional activation of Oct4 and other genes.

BACKGROUND

OCT3/4 (POU5F1) is an established diagnostic immunohistochemical marker for specific histological variants of human malignant germ cell tumours (GCTs), including the seminomatous types and the stem cell component of non-seminomas, known as embryonal carcinoma. OCT3/4 is crucial for the regulation of pluripotency and the self-renewal of normal embryonic stem- and germ cells. Detection of expression of this transcription factor is complicated by the existence of multiple pseudogenes and isoforms. Various claims have been made about OCT3/4 expression in non-GCTs, possibly related to using nonspecific detection methods. False-positive findings undermine the applicability of OCT3/4 as a specific diagnostic tool in a clinical setting. In addition, false-positive findings could result in misinterpretation of pluripotency regulation in solid somatic cancers and their stem cells. Of the three identified isoforms--OCT4A, OCT4B and OCT4B1--only OCT4A proved to regulate pluripotency. Up until now, no convincing nuclear OCT4A protein expression has been shown in somatic cancers or tissues.

METHODS

This study investigates expression of the various OCT3/4 isoforms in GCTs (both differentiated and undifferentiated) and somatic (non-germ cell) cancers, including representative cell lines and xenografts.

RESULTS

Using specific methods, OCT4A and OCT4B1 are shown to be preferentially expressed in undifferentiated GCTs. The OCT4B variant shows no difference in expression between GCTs (either differentiated or undifferentiated) and somatic cancers. In spite of the presence of OCT4A mRNA in somatic cancer-derived cell lines, no OCT3/4 protein is detected. Significant positive correlations between all isoforms of OCT3/4 were identified in both tumours with and without a known stem cell component, possibly indicating synergistic roles of these isoforms.

CONCLUSIONS

This study confirms that OCT4A protein only appears in seminomatous GCTs, embryonal carcinoma and representative cell lines. Furthermore, it emphasises that in order to correctly assess the presence of functional OCT3/4, both isoform specific mRNA and protein detection are required.

Primary intracranial germ cell tumours are rare neoplasms that occur in children and adolescents. This study examined both the biology and the origin of these tumours, as it has been hypothesized that they originate from a totipotent primordial germ cell. We applied recent knowledge from gonadal germ cell tumours and analysed expression of a wide panel of stem cell-related proteins (C-KIT, OCT-3/4 (POU5F1), AP-2gamma (TFAP2C), and NANOG) and developmentally regulated germ cell-specific proteins (including MAGE-A4, NY-ESO-1, and TSPY). Expression at the protein level was analysed in 21 children and young adults with intracranial germinomas and non-germinomas, contributing to a careful description of these unusual tumours and adding to the understanding of pathogenesis. Stem cell related proteins were highly expressed in intracranial germ cell tumours, and many similarities were detected with their gonadal equivalents, including a close similarity with primordial germ cells. A notable difference was the sex-specific expression of TSPY, a gene previously implicated in the origin of gonadoblastoma. TSPY was only detected in germ cell tumours in the central nervous system (CNS) from males, suggesting that it is not required for the initiation of malignant germ cell transformation. The expression of genes associated with embryonic stem cell pluripotency in CNS germ cell tumours strongly suggests that these tumours are derived from cells that retain, at least partially, an embryonic stem cell-like phenotype, which is a hallmark of primordial germ cells.

Testicular germ cell tumors (TGCT) are the most common malignancy in young men. While most TGCT are potentially curable, approximately 5% of patients with TGCT may develop chemoresistance and die from the disease. This review article summarizes current knowledge in genetics underlying the development, progression and chemoresistance of TGCT. Most post-pubertal TGCT originate from intratubular germ cell neoplasia unclassified (IGCNU), which are transformed fetal gonocytes. Development of IGCNU may involve aberrantly activated KITLG/KIT pathway and overexpression of embryonic transcription factors such as NANOG and POU5F1, which leads to suppression of apoptosis, increased proliferation, and accumulation of mutations in gonocytes. Invasive TGCT consistently show gain of chromosome 12p, typically isochromosome 12p. Single gene mutations are uncommon in TGCT. KIT, TP53, KRAS/NRAS, and BRAF are genes most commonly mutated in TGCT and implicated in their pathogenesis. Different histologic subtypes of TGCT possess different gene expression profiles that reflect different directions of differentiation. Their distinct gene expression profiles are likely caused by epigenetic regulation, in particular DNA methylation, but not by gene copy number alterations. Resistance of TGCT to chemotherapy has been linked to karyotypic aberrations, single-gene mutations, and epigenetic regulation of gene expression in small-scale studies. The study of TGCT genetics could ultimately translate into development of new molecular diagnostic and therapeutic modalities for these tumors and improve the care of patients with these malignancies.

A 30-node signed and directed network responsible for self-renewal and pluripotency of mouse embryonic stem cells (mESCs) was extracted from several ChIP-Seq and knockdown followed by expression prior studies. The underlying regulatory logic among network components was then learned using the initial network topology and single cell gene expression measurements from mESCs cultured in serum/LIF or serum-free 2i/LIF conditions. Comparing the learned network regulatory logic derived from cells cultured in serum/LIF vs. 2i/LIF revealed differential roles for Nanog, Oct4/Pou5f1, Sox2, Esrrb and Tcf3. Overall, gene expression in the serum/LIF condition was more variable than in the 2i/LIF but mostly consistent across the two conditions. Expression levels for most genes in single cells were bimodal across the entire population and this motivated a Boolean modeling approach. In silico predictions derived from removal of nodes from the Boolean dynamical model were validated with experimental single and combinatorial RNA interference (RNAi) knockdowns of selected network components. Quantitative post-RNAi expression level measurements of remaining network components showed good agreement with the in silico predictions. Computational removal of nodes from the Boolean network model was also used to predict lineage specification outcomes. In summary, data integration, modeling, and targeted experiments were used to improve our understanding of the regulatory topology that controls mESC fate decisions as well as to develop robust directed lineage specification protocols.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

The pig represents an ideal large-animal model, intermediate between rodents and humans, for the preclinical assessment of emerging cell therapies. As no validated pig embryonic stem (pES) cell lines have been derived so far, pig induced pluripotent stem cells (piPSCs) should offer an alternative source of undifferentiated cells to advance regenerative medicine research from bench to clinical trial. We report here for the first time the derivation of piPSCs from adult fibroblast with only three transcription factors: Sox2 (sex determining region Y-box 2), Klf4 (Krüppel-like factor 4), and c-Myc (avian myelocytomatosis viral oncogene homolog). We have been able to demonstrate that exogenous Pou5f1 (POU domain class 5 transcription factor 1; abbreviated as Octamer-4: Oct4) is dispensable to achieve and maintain pluripotency in the generation of piPSCs. To the best of our knowledge, this is also the first report of somatic reprogramming in any species without the overexpression, either directly or indirectly, of Oct4. Moreover, we were able to generate piPSCs without the use of feeder cells, approaching thus xeno-free conditions. Our work paves the way for the derivation of clinical grade piPSCs for regenerative medicine.

This article serves three purposes. We summarize current knowledge of the origin and characteristics of EPI-NCSC, review their application in a mouse model of spinal cord injury, and we present new data that highlight aspects of pluripotency of EPI-NCSC. EPI-NCSC are multipotent stem cells, which are derived from the embryonic neural crest and are located in the bulge of hair follicles. EPI-NCSC can undergo self-renewal and they are able to generate all major neural crest derivatives, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. Despite their ectodermal origin, neural crest cells can also generate cell types that typically are derived from mesoderm. We were therefore interested in exploring aspects of EPI-NCSC pluripotency. We here show that EPI-NCSC can fuse with adult skeletal muscle fibers and that incorporated EPI-NCSC nuclei are functional. Furthermore, we show that adult skeletal muscle represents an environment conducive to long-term survival of neurogenic EPI-NCSC. Genes used to create induced pluripotent stem (iPS) cells are present in our EPI-NCSC longSAGE gene expression library. Here we have corroborated this notion by real-time PCR. Our results show similarities in the expression of Myc, Klf4, Sox2 and Lin28 genes between EPI-NCSC and embryonic stem cells (ESC). In contrast there were major differences in Nanog and Pou5f1 (Oct-4) expression levels between EPI-NCSC and ESC, possibly explaining why EPI-NCSC are not tumorigenic. Overall, as embryonic remnants in an adult location EPI-NCSC show several attractive characteristics for future cell replacement therapy and/or biomedical engineering: Due to their ability to migrate, EPI-NCSC can be isolated as a highly pure population of multipotent stem cells by minimally-invasive procedures. The cells can be expanded in vitro into millions of stem cells/progenitors and they share some characteristics with pluripotent stem cells without being tumorigenic. Since the patients' own EPI-NCSC could be used for autologous transplantation, this would avoid graft rejection.

The transcription factor POU5F1 is a key regulator of embryonic stem (ES) cell pluripotency and a known oncoprotein. We have developed a novel high-throughput binding assay called MEGAshift (microarray evaluation of genomic aptamers by shift) that we use to pinpoint the exact location, affinity, and stoichiometry of the DNA-protein complexes identified by chromatin immunoprecipitation studies. We consider all genomic regions identified as POU5F1-ChIP-enriched in both human and mouse. Compared with regions that are ChIP-enriched in a single species, we find these regions more likely to be near actively transcribed genes in ES cells. We resynthesize these genomic regions as a pool of tiled 35-mers. This oligonucleotide pool is then assayed for binding to recombinant POU5F1 by gel shift. The degree of binding for each oligonucleotide is accurately measured on a custom oligonucleotide microarray. We explore the relationship between experimentally determined and computationally predicted binding strengths, find many novel functional combinations of POU5F1 half sites, and demonstrate efficient motif discovery by incorporating binding information into a motif finding algorithm. In addition to further refining location studies for transcription factors, this method holds promise for the high-throughput screening of promoters, SNP regions, and epigenetic modifications for factor binding.

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.

BACKGROUND

Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development.

METHODS

KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers.

RESULTS

The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression.

CONCLUSIONS

From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

POU5F1(OCT3/4) is a sequence-specific transcription factor that is essential for keeping germ cells and embryonic stem cells in an immature and pluripotent status. In this article, we report that POU5F1 was fused to EWSR1 in a case of undifferentiated sarcoma derived from pelvic bone with chromosomal translocation t(6;22)(p21;q12). The EWSR1-POU5F1 chimera consists of exons 1-6 of EWSR1 and exons 2-5 and a part of exon 1 of POU5F1. The predicted amino acid sequence indicates that the chimera is composed of the N-terminal QSY domain of EWS that functions as a transcriptional activation domain and the C-terminal POU DNA-binding domains derived from POU5F1. The t(6;22) tumor does not belong to any known categories of bone and soft-tissue tumors (BSTs). It is suggested that EWS-POU5F1 may act as an oncogenic transcription factor and that its expression may contribute to undifferentiated and immature phenotypes of BST.

Immunohistochemistry has become an important tool in the diagnosis of ovarian tumors. This article reviews the role of immunohistochemistry in the differential diagnosis of the three main categories of ovarian tumors, with emphasis on recently developed antibodies. In the surface epithelial stromal category the most common problem is its discernment from metastasis. The use of differential cytokeratins, primarily CK7 and CK20, as well as Cdx-2, beta-catenin, and P504S in differentiating between metastatic adenocarcinoma, particularly of colorectal origin, and primary ovarian carcinoma is discussed. Dpc4 may be useful in distinguishing pancreatic from ovarian mucinous carcinomas, because up to 55% of pancreatic carcinomas lack Dpc4 expression, whereas the differential expression of mucin genes may be helpful in distinguishing between primary ovarian mucinous and metastatic tumors. Urothelial markers (thrombomodulin and uroplakin III) and renal cell carcinoma markers (CD10 and renal cell carcinoma marker) can be helpful in the diagnosis of metastatic urothelial and renal cell tumors to the ovary. The roles of inhibin, calretinin, CD99, and other recently described markers in the diagnosis of sex cord-stromal tumors are reviewed. The uses of OCT-4 (POU5F1) (a new highly sensitive and specific marker of dysgerminoma and embryonal carcinoma), CD30, and c-kit are also discussed.

Germ cell tumors (GCTs) of the testis are the predominant cancer among young men. We analyzed gene expression profiles of 50 GCTs of various subtypes, and we compared them with 443 other common malignant tumors of epithelial, mesenchymal, and lymphoid origins. Significant differences in gene expression were found among major histological subtypes of GCTs, and between them and other malignancies. We identified 511 genes, belonging to several critical functional groups such as cell cycle progression, cell proliferation, and apoptosis, to be significantly differentially expressed in GCTs compared with other tumor types. Sixty-five genes were sufficient for the construction of a GCT class predictor of high predictive accuracy (100% training set, 96% test set), which might be useful in the diagnosis of tumors of unknown primary origin. Previously described diagnostic and prognostic markers were found to be expressed by the appropriate GCT subtype (AFP, POU5F1, POV1, CCND2, and KIT). Several additional differentially expressed genes were identified in teratomas (EGR1 and MMP7), yolk sac tumors (PTPN13 and FN1), and seminomas (NR6A1, DPPA4, and IRX1). Dynamic computation of interaction networks and mapping to existing pathways knowledge databases revealed a potential role of EGR1 in p21-induced cell cycle arrest and intrinsic chemotherapy resistance of mature teratomas.

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

BACKGROUND

Intracranial pediatric germ cell tumors (GCTs) are rare and heterogeneous neoplasms and vary in histological differentiation, prognosis and clinical behavior. Germinoma and mature teratoma are GCTs that have a good prognosis, while other types of GCTs, termed nongerminomatous malignant germ cell tumors (NGMGCTs), are tumors with an intermediate or poor prognosis. The second group of tumors requires more extensive drug and irradiation treatment regimens. The mechanisms underlying the differences in incidence and prognosis of the various GCT subgroups are unclear.

RESULTS

We identified a distinct mRNA profile correlating with GCT histological differentiation and prognosis, and also present in this study the first miRNA profile of pediatric primary intracranial GCTs. Most of the differentially expressed miRNAs were downregulated in germinomas, but miR-142-5p and miR-146a were upregulated. Genes responsible for self-renewal (such as POU5F1 (OCT4), NANOG and KLF4) and the immune response were abundant in germinomas, while genes associated with neuron differentiation, Wnt/beta-catenin pathway, invasiveness and epithelial-mesenchymal transition (including SNAI2 (SLUG) and TWIST2) were abundant in NGMGCTs. Clear transcriptome segregation based on patient survival was observed, with malignant NGMGCTs being closest to embryonic stem cells. Chromosome copy number variations (CNVs) at cytobands 4q13.3-4q28.3 and 9p11.2-9q13 correlated with GCT malignancy and clinical risk. Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5) within 4q13.3-4q28.3 were more abundant in germinomas.

CONCLUSIONS

Our results integrate molecular profiles with clinical observations and provide insights into the underlying mechanisms causing GCT malignancy. The genes, pathways and microRNAs identified have the potential to be novel therapeutic targets.

The transcription factor Yin Yang 1 (YY1) is frequently overexpressed in cancerous tissues compared to normal tissues and has regulatory roles in cell proliferation, cell viability, epithelial-mesenchymal transition, metastasis and drug/immune resistance. YY1 shares many properties with cancer stem cells (CSCs) that drive tumorigenesis, metastasis and drug resistance and are regulated by overexpression of certain transcription factors, including SOX2, OCT4 (POU5F1), BMI1 and NANOG. Based on these similarities, it was expected that YY1 expression would be associated with SOX2, OCT4, BMI1, and NANOG's expressions and activities. Data mining from the proteomic tissue-based datasets from the Human Protein Atlas were used for protein expression patterns of YY1 and the four CSC markers in 17 types of cancer, including both solid and hematological malignancies. A close association was revealed between the frequency of expressions of YY1 and SOX2 as well as SOX2 and OCT4 in all cancers analyzed. Two types of dynamics were identified based on the nature of their association, namely, inverse or direct, between YY1 and SOX2. These two dynamics define distinctive patterns of BMI1 and OCT4 expressions. The relationship between YY1 and SOX2 expressions as well as the expressions of BMI1 and OCT4 resulted in the classification of four groups of cancers with distinct molecular signatures: (1) Prostate, lung, cervical, endometrial, ovarian and glioma cancers (YY1(lo)SOX2(hi)BMI1(hi)OCT4(hi)) (2) Skin, testis and breast cancers (YY1(hi)SOX2(lo)BMI1(hi)OCT4(hi)) (3) Liver, stomach, renal, pancreatic and urothelial cancers (YY1(lo)SOX2(lo)BMI1(hi)OCT4(hi)) and (4) Colorectal cancer, lymphoma and melanoma (YY1(hi)SOX2(hi)BMI1(lo)OCT4(hi)). A regulatory loop is proposed consisting of the cross-talk between the NF-kB/PI3K/AKT pathways and the downstream inter-regulation of target gene products YY1, OCT4, SOX2 and BMI1.

In mammalian embryos, PGCs (primordial germ cells) are specified from a pluripotent epiblast cell population after implantation. In this study, we demonstrated an essential role for the germline-specific transcription factor Oct3/4 in PGC specification. We generated chimeric embryos with ZHBTc4 ES cells lacking both alleles of the Oct3/4 gene (pou5f1). Pluripotency was maintained by an Oct3/4 transgene, and its expression was suppressed by doxycycline (Dox). Transcription of the Oct3/4 transgene in the ES-derived cells unexpectedly suffered constitutive suppression in chimeric embryos without Dox, and the ES-derived cells contributed to PGC precursor-like cells, but failed to form PGCs. We then attempted to rescue Oct3/4 expression in the ES-derived cells in the chimeric embryos by introducing an additional Oct3/4 transgene. The ES cell-derived cells indeed recovered Oct3/4 transcription in these chimeric embryos, and were successfully specified to PGCs. We further confirmed the requirement of Oct3/4 by using another derivative of ZHBTc4 ES cells in which a Dex (dexamethasone)-dependent Oct3/4 transgene was introduced. In the presence of Dox, Oct3/4 protein was absent in the nuclei of the ES-derived cells, which failed to form PGCs. In contrast, the ES-derived cells could be specified to PGCs after activation of Oct3/4 function in the presence of Dex.

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS-ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS-ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T-E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.

OBJECTIVE

To elucidate the pathogenesis of HAART-associated lipodystrophy, by investigating the effects of antiretroviral drugs on adipocyte differentiation and gene expression profile.

METHODS

Analysis of gene expression profile by DNA microarrays and quantitative RT-PCR of 3T3-L1 preadipocytes treated with the nucleoside reverse transcriptase inhibitors (NRTI) lamivudine, zidovudine, stavudine, and zalcitabine, and with the protease inhibitors (PI) indinavir, saquinavir, and lopinavir during maturation into adipocytes.

RESULTS

Under standard adipogenic differentiation protocols, PI significantly inhibited adipocyte differentiation, as demonstrated by cell viability assay and Oil Red O staining and quantification, whereas NRTI had mild effects on adipogenesis. Gene expression profile analysis showed that treatment with NRTI modulated the expression of transcription factors, such as Aebp1, Pou5f1 and Phf6, which could play a key role in the determination of the adipocyte phenotype. PI also modulated gene expression toward inhibition of adipocyte differentiation, with up-regulation of the Wnt signaling gene Wnt10a and down-regulation of the expression of genes encoding master adipogenic transcription factors (e.g., C/EBPalpha and PPARgamma), oestrogen receptor beta, and adipocyte-specific markers (e.g., Adiponectin, Leptin, Mrap, Cd36, S100A8).

CONCLUSIONS

This study identifies new genes modulated by PI and NRTI in differentiating adipocytes. Abnormal expression of these genes, which include master adipogenic transcription factors and genes involved in lipid metabolism and cell cycle control, could contribute to the understanding of the pathogenesis of HAART-associated lipodystrophy.