BACKGROUND

Bone morphogenetic protein 4 (BMP4) has a significant role in primordial germ cells (PGCs) differentiation from mouse embryonic stem cell (mESC). The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC.

METHODS

To differentiate PGCs, embryoid bodies (EBs) from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 (Pou5f1), Stella (Dppa3) and Mvh (Ddx4) were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTS

Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups.

CONCLUSIONS

Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC.

OBJECTIVE

The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.

METHODS

Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.

RESULTS

Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival.

CONCLUSIONS

The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

POU5F1 is essential for maintaining pluripotency in embryonic stem cells (ESCs). It has been reported that the constitutive activation of POU5F1 is sustained by the core transcriptional regulatory circuitry in ESCs; however, the means by which POU5F1 is epigenetically regulated remains enigmatic. In this study a fluorescence-based reporter system was used to monitor the interplay of 5 reprogramming-associated TFs and 17 chromatin regulators in the transcription of POU5F1. We show the existence of a stoichiometric effect for SOX2, POU5F1, NANOG, MYC and KLF4, in regulating POU5F1 transcription. Chromatin regulators EP300, KDM5A, KDM6A and KDM6B cooperate with KLF4 in promoting the transcription of POU5F1. Moreover, inhibiting HDAC activities induced the expression of Pou5f1 in mouse neural stem cells (NSCs) in a spatial- and temporal- dependent manner. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) shows that treatment with valproic acid (VPA) increases the recruitment of Kdm5a and Kdm6a to proximal promoter (PP) and proximal enhancer (PE) of Pou5f1 whereas enrichment of Ep300 and Kdm6b was seen in PP but not PE of Pou5f1 promoter. These findings reveal the interplay between the chromatin regulators and histone modifications in the expression of POU5F1.

The objective of this study was to examine the effect of treating pig oocytes during in vitro maturation (IVM) with a proteasome inhibitor, MG132, on oocyte maturation and embryonic development. In one series of experiments, oocytes from medium-sized follicles (3-8 mm in diameter) were untreated (MCO) or treated with MG132 during 0-22 hr (M0-22) or 30-42 hr (M30-42) of IVM. There was no significant effect of MG132 on nuclear maturation or cytoplasmic maturation (as assessed by intracellular amounts of glutathione and p34cdc2 kinase activity). Blastocyst formation after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), however, was increased for M30-42 (65.2% and 27.7% for PA and SCNT, respectively) compared to MCO (42.6% and 13.6%, respectively) and M0-22 (45.3% and 19.5%, respectively; P<0.05). Expression of PCNA and ERK2 was increased in M30-42 for IVM oocytes while transcript abundance for POUF51, DNMT1, FGFR2, and PCNA was increased in M30-42 for 4-cell SCNT embryos. When oocytes derived from small follicles (<3 mm in diameter) were untreated (SCO) or treated with MG132 during 0-22 hr (S0-22), 30-42 hr (S30-42) of IVM, or 0-22 and 30-42 hr of IVM (S0-22/30-42), expression of POU5F1, DNMT1, FGFR2, and PCNA and blastocyst formation were increased for SCNT embryos derived from S30 to 42 (16.5%) and S0-22/30-42 oocytes (20.8%) as compared to embryos from SCO (8.7%) or S0-22 oocytes (8.8%; P<0.05). Results demonstrate that treatment of oocytes with MG132 during the later stage of IVM improves embryonic development and alters gene expression in pigs.

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.

Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

BACKGROUND

The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.

RESULTS

Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.

CONCLUSIONS

Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.

Lung adenocarcinomas are a heterogeneous set of diseases with distinct genetic and histologic characteristics. Besides the discovery of oncogenic mutations and introduction of the histologic classifications (2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society and 2015 WHO), increasing evidence has linked this intertumor heterogeneity to the lung lineage-specific pathways and lineage genes. Therefore, in this study, we assessed the gene expression of identified lung lineage genes to study their role in distinguishing lung adenocarcinoma diversities.

A total of 278 surgically resected lung adenocarcinomas were included. Each case was evaluated for genetic mutations and histologic classification. Lineage genes associated with respiratory tract differentiation (NK2 homeobox 1 gene [NKX2-1], GATA protein binding 6 gene [GATA6], foxhead box J1 gene [FOXJ1], and SAM pointed domain containing ETS transcription factor gene [SPDEF]) and stem/basal-like status (inhibitor of DNA binding 2, HLH protein gene [ID2], POU class 5 homeobox 1 gene [POU5F1], SRY-box 2 gene [SOX2], and v-myc avian myelocytomatosis viral oncogene homolog gene [MYC]) were selected. mRNA expression of these genes in each tumor sample was assessed by quantitative real-time polymerase chain reaction and normalized to paired normal lung tissue.

Distinct lineage gene expressions were found on the basis of genetic and histologic diversities. Expression of NKX2-1, GATA6, FOXJ1, and POU5F1 exhibited a significant linear relationship across histologic subgroups that was independent of genetic mutation status. Expression levels of NKX2-1 and POU5F1 were also associated with EGFR mutation status, independent of histologic subtypes. Further analysis revealed that the overexpression of SPDEF defined longer relapse-free survivals, especially in stage I disease.

For the first time, we showed the unique lineage backgrounds of different histologic subtypes and oncogenic mutations. Assessing this added parameter might be beneficial in discriminating intertumor heterogeneity, advancing target exploration, developing theranostic/prognostic biomarkers, and designing clinical trials.

Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17β-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 µM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 µM of α-ZEA groups. Only the 100 µM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 µM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding.

Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

The epiblast represents the final embryonic founder cell population with the potential for giving rise to all cell types of the adult body. The pluripotency of the epiblast is lost during the process of gastrulation. Large animal species have a lack of specific markers for pluripotency. The aim of the present study was to characterize the bovine epiblast cell population and to provide such markers. Bovine Day 12 and Day 14 embryos were processed for transmission-electron microscopy or immunohistochemistry. In Day 12 embryos, two cell populations of the epiblast were identified: one constituting a distinctive basal layer apposing the hypoblast, and one arranged inside or above the former layer, including cells apposing the Rauber layer. Immunohistochemically, staining for the octamer-binding transcription factor 4 (OCT4, also known as POU5F1), revealed a specific and exclusive staining of nuclei of the complete epiblast. Colocalization of vimentin and OCT4 was demonstrated. Only trophectodermal cells stained for alkaline phosphatase. Staining for the proliferation marker Ki-67 was localized to most nuclei throughout the epiblast. A continuous staining for zonula occludens-1 protein was found between cells of the trophectoderm and hypoblast but was not evident in the epiblast. A basement membrane, detected by staining for laminin, formed a "cup-like" structure in which the epiblast was located. The ventrolateral sides of the cup appeared to be incomplete. In conclusion, the bovine epiblast includes at least two cell subpopulations, and OCT4 was shown, to our knowledge for the first time, to be localized exclusively to epiblast cells in this species.

Nuclear receptor subfamily 6, group A, member 1 (NR6A1), also known as germ cell nuclear factor/retinoid receptor-related testis-associated receptor and neuronal cell nuclear factor, is a member of the nuclear orphan receptor superfamily. NR6A1 has been cloned in various species including humans and mice, but it has been scarcely investigated in avian species. In the present study, we cloned the chicken NR6A1 (cNR6A1) from a testis cDNA library. The cloned cNR6A1 sequence was mapped to chromosome 17 and contained an open reading frame of 1.4 kb encoding 445 amino acids. Multiple alignment analysis of the cNR6A1 protein-coding sequence with NR6A1s from humans, mice, boars, rats, zebrafish, and Xenopus showed high degrees of homology, 89%, 90%, 89%, 88%, 83%, and 87%, respectively. Using RNA interference, changes in the expression of pluripotency-, germ cell-, and differentiation-related key genes by silencing of cNR6A1 were validated in chicken blastoderm-derived embryonic stem cells. Among those genes, the relative expression levels of POU5F1, CRIPTO, DAZL, DDX4, BMP15, GSC, and SOX7 changed significantly compared to the control group. We also confirmed that the activity of alkaline phosphatase, known as a pluripotency marker, was maintained by cNR6A1 gene silencing in chicken blastodermal cells. Collectively, our data suggest that cNR6A1 may play an important role during chicken embryonic development and differentiation.

Immunoperoxidase and molecular genetic analysis showed that retinal pigment epithelial cells from adult human eye undergo morphogenetic changes in vitro. They lose expression of tissue-specific protein RPE65 and start to express stem cell markers: Oct4 (POU5F1), Nanog, Prox1, Musashi 1, and Pax6, which indicates their differentiation. Expression of Musashi 1 and Pax6 attest to neural differentiation, which is also confirmed by the expression of βIII-tubulin, a neuroblast marker, and markers of differentiated neuronal cells, tyrosine hydroxylase and neurofilament proteins. These findings attest to the capacity of retinal pigment epithelium from adult human eye to transdifferentiation into neural lineage cells, which makes them an interesting object for cell therapy in neurodegeneration.

Pericardial fibrocalcification (PF) is a prominent feature of human pericardial pathology, including constrictive pericarditis and, to a lesser extent, degenerated autologous pericardial substitutes. However, the role of pericardial interstitial cells (PICs) in the pathogenesis of PF has yet to be established. Using a combination of histology and immunohistochemistry, we showed that the critical cellular event in PF in situ was the transdifferentiation of PICs into myofibroblasts/osteoblasts and that the percentage of myofibroblasts/osteoblasts correlated positively with the severity of PF. In vitro studies demonstrated that PICs, similar to mesenchymal stem cells, had the potential to differentiate along adipogenic, osteogenic, chondrogenic or myogenic lineages. However, PICs exhibited a more limited self-renewal capacity and a lower expression of Oct4 (POU5F1) and Kruppel-like transcription factor Klf4, underwent earlier senescence and spontaneously transdifferentiated into myofibroblasts/osteoblasts. Quantitative-real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed that the mRNA levels of α-smooth muscle actin (α-SMA), alkaline phosphatase (ALP), core-binding factor α1/runt-related transcription factor2 (Cbfa1/Runx2), transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 were upregulated as the passage number increased. The mRNA level of platelet-derived growth factor (PDGF)-AA was also significantly upregulated with higher levels at passage 3. Ectopic expression of Oct4 and Klf4 enhanced the colony formation of PICs and selectively impaired induction of genes involved in transdifferentiation into myofibroblasts/osteoblasts (α-SMA, ALP, Cbfa1/Runx2, PDGF-AA and BMP-2). These data, while offering new insights into the biology of PICs, reinforce the central role of these cells in cell-mediated PF and may assist in future strategies to treat fibrocalcific pericardial diseases.

The POU-domain transcription factor Pou5f1 (Oct-4) is involved in transcriptional regulation during early embryonic development and cell differentiation. Despite highly conserved genomic organization of Oct-4 gene in mammals, expression pattern of Oct-4 is highly variable in different species. In the present study, expression pattern of Oct-4 in buffalo blastocyst, trophoectoderm (TE), and embryonic stem cells (ESCs) was investigated. For the derivation and characterization of buffalo ESCs, inner cell masses (ICMs) were isolated from 18 hatched and 21 expanded in vitro produced buffalo blastocyst and cultured over mitomycin-C-treated buffalo fetal fibroblast feeder layer. Alkaline phosphatase (AP) activity, SSEA-1 and 4, TRA 1-60 and 1-81, and Oct-4 proteins were localized in ICM, TE, and ESCs. Quantification of Oct-4 was done by amplifying a transcript of 125 base pairs by real-time polymerase chain reaction. Primary cell colony formation was higher (P < 0.05) in hatched blastocyst (83.33%, 15/18) compared to mechanically isolated ICMs from expanded blastocyst (52.38%, 11/21). Undifferentiated buffalo ESCs were positive for AP and expressed Oct-4, SSEA-1 and 4, TRA-1-60, and TRA-1-81 proteins. Oct-4 transcripts and proteins were detected in the ICM, TE cells and were invariably present in ESCs; however, expression level of Oct-4 transcript were significantly higher in ICM and ESCs as compared to TE cells. In conclusion, expression of Oct-4 is not only restricted to the ICM and ESCs but its expression was also detected in TE cells suggesting that instead of using Oct-4 as a single marker, it is better to have other flanking molecular markers for the identification of buffalo pluripotent embryonic stem cells.

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

POU5F1 and NANOG play important roles in the maintenance of embryonic stem cell pluripotency. Recently, we isolated cat embryonic stem (ES)-like cells from cat blastocysts generated in vivo. In an effort to identify genetic markers for the characterization of cat ES-like cells, we have determined the coding sequences (CDSs) of cat POU5F1 (cPOU5F1) and NANOG (cNANOG). The sequence identities of cPOU5F1 with orthologous genes of the human and mouse were 92 and 82%, respectively, at the nucleotide level and 94 and 83%, respectively, at the amino acid level. We identified POU-specific and POU homeodomain sequences in the CDS of cPOU5F1. The sequence identities of cNANOG with its human and mouse orthologs were 69 and 68%, respectively, at the nucleotide level and 69 and 58%, respectively, at the amino acid level. We identified a homeodomain, SMAD4 domain and tryptophan repeat domain (W/QXXXX) in the CDS of cNANOG. We examined the expression of cPOU5F1 and cNANOG mRNA in ES-like cells and fibroblast feeder cells by RT-PCR. Transcripts of cPOU5F1 and cNANOG were detected at a high level in ES-like cells. However, these two genes were undetectable in cat fibroblast feeder cells and 6 adult tissues. We also examined ES-like cells by immunocytochemistry and demonstrated that cPOU5F1 and cNANOG are present at high levels in cat ES-like cells and are undetectable in cat fibroblast feeder cells. These results confirmed that cat ES-like cells can be successfully isolated from in vivo-produced blastocysts and that the expression of cPOU5F1 and cNANOG can be used as a biomarker for characterization of cat ES-like cells.

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 μg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation (IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1 (BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01) expression (P<0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.

Incomplete transgene-silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids-a critical family in biomedical and biodiversity conservation science. In this study doxycycline-inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline-phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription-quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells.