Au nano-cone array for SERS detection of associated miRNA in lymphoma patients.

Based on Au nano-cone array (Au-NCA) and a three-segment hybridization strategy, a novel SERS biosensor is proposed for the ultrasensitive detection of the microRNA miR-21. The uniform, stable, and reproducible Au-NCA was prepared by the single-layer colloidal ball template method. Subsequently, the target was hybridized with sequence 2. The resulting target-sequence 2 complex was then hybridized with sequence 1 anchored on Au-NCA. Thus, a three-segment sequence complex was formed. SERS measurements can be performed without the need for complex purification and amplification steps. Due to the ability of miR-21 to perform specific complementary hybridization with two sequences, SERS biosensors have superior specificity for miR-21 without interference from other miRNAs. Under the optimal conditions, the SERS biosensor was applied and the limit of detection (LOD) was as low as 3.02 aM. This method has been successfully used to the detection of miR-21 in the serum of lymphoma patients and healthy volunteers. The results are consistent with the traditional test methods. Therefore, this novel SERS biosensor shows excellent clinical translational potential in the detection of lymphoma.

Intergenic transcription in in vivo developed bovine oocytes and pre-implantation embryos.

Background: Intergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Transcription termination failure has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation. Results: Using Automatic Readthrough Transcription Detection (ARTDeco) and data of in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb up-stream of reference genes, extending up to 15 kb up-stream). Read-throughs (continued transcription from TES of expressed reference genes, 4-15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3,084 to 6,565 or 33.36-66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (P < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1,504 read-outs, 1,045 read-ins, and 1,021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log2 fold change ≥ 2, P < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and down-stream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing. Conclusion: In summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or down-stream.

Identification of New Compounds against PRRSV Infection by Directly Targeting CD163.

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 µM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos.

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

Transcriptomic Responses of Mycoplasma bovis Upon Treatments of trans-Cinnamaldehyde, Carvacrol, and Eugenol.

Mycoplasma bovis (M. bovis) is an insidious, wall-less primary bacterial pathogen that causes bovine pneumonia, mid-ear infection, mastitis, and arthritis. The economic losses caused by M. bovis due to culling, diminished milk production, and feed conversion are underestimated because of poor diagnosis/recognition. Treatment with common antibiotics targeting the cell wall is ineffective. Plant-derived antimicrobials (PDAs) such as food-grade trans-cinnamaldehyde (TC), eugenol (EU), and carvacrol (CAR) are inexpensive and generally regarded as safe for humans and animals yet possess strong anti-bacterial properties. In preliminary studies, we found that all three PDAs inhibited the growth of M. bovis in vitro. Through RNA sequencing, we report here that CAR affected the expression of 153 genes which included the downregulation of energy generation-related proteins, pentose phosphate pathway, and upregulation of ribosomes and translation-related proteins. Few differentially expressed genes were found when M. bovis was treated with TC, EU, or when the three PDAs were double or triple combined. Our results suggest that, as opposed to the effect of CAR, the growth-inhibitory effects of TC and EU at levels tested may be exerted through mechanisms other than gene expression regulations.

Effects of intramuscularly injected plant-derived antimicrobials in the mouse model.

With increasing antibiotic resistance, the use of plant derived antimicrobials (PDAs) has gained momentum. Here, we investigated the toxicity of trans-cinnamaldehyde, eugenol, and carvacrol after intramuscular injection in mice. Two doses of each PDA-300 and 500 mg/kg body weight-and vehicle controls were injected into the muscle of the right hind limb of CD-1 adult mice (n = 8/treatment). Ten physical/behavioral parameters were monitored hourly for 2 h and twice daily for 4 days post-injection together with postmortem examination of leg muscles and organs. Within the first 2 days of carvacrol treatment, one male died in each dose level and a third male receiving 500 mg/kg was removed from the study. No mortality was seen with any other treatment. Among all 81 parameters examined, significant higher relative liver weights (300 and 500 mg/kg eugenol groups; P < 0.05) and relative kidney weights (300 mg/kg carvacrol group; P < 0.001) were observed. Taken together, little to mild toxicity was seen for trans-cinnamaldehyde and eugenol, respectively, while carvacrol exerted more toxicity in males. This study lays the foundation for future extensive work with large sample size, varied treatment durations, and additional treatment levels.

Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium.

Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.

Establishment of Bovine-Induced Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

Cytotoxic effects on cancerous and non-cancerous cells of trans-cinnamaldehyde, carvacrol, and eugenol.

Essential oils and their active components, referred here as plant derived antimicrobials (PDAs), have been used for their antimicrobial, anti-inflammatory and antioxidant properties. Many reports also document PDAs' cytotoxic effects on cancerous cells, raising the hope that they could be used for cancer treatments. Due to the lack of specificity, we hypothesize that PDAs are cytotoxic to both cancerous and non-cancerous cells. Trans-cinnamaldehyde (TCA), carvacrol, and eugenol were assessed for their cytotoxicity on cancerous HeLa cells and normal skin fibroblasts (CCD-1123Sk, CCD) by MTT and LDH assays, flow cytometry, and reverse transcription quantitative PCR (RT-qPCR). After 24 h of treatment, carvacrol and TCA significantly decreased cell viability (by more than 50%) at 100 µg/ml, whereas eugenol was ineffective up to 400 µg/ml. Cell detachment and significantly increased apoptosis were observed with 100 µg/ml of TCA on both cell types. RT-qPCR for apoptotic genes (BCL2, CASP3 and CASP8) and necrosis genes (MLKL, RIPK1 and RIPK3) did not show significant differences between control and treated cells of both types, with the exception of eugenol-treated HeLa cells in which expression of BCL2, MLKL and RIPK1 was significantly higher than controls. Taken together, we conclude that the three PDAs studied here exhibited similar cytotoxic effects on both cancerous and non-cancerous cells.

Methylome Dynamics of Bovine Gametes and in vivo Early Embryos.

DNA methylation undergoes drastic fluctuation during early mammalian embryogenesis. The dynamics of global DNA methylation in bovine embryos, however, have mostly been studied by immunostaining. We adopted the whole genome bisulfite sequencing (WGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, immature oocytes, oocytes matured in vivo and in vitro, as well as in vivo developed single embryos at the 2-, 4-, 8-, and 16-cell stages. We found that the major wave of genome-wide DNA demethylation was complete by the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs), which were primarily intergenic, suggesting that these non-coding regions may play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes, suggesting environmental effects on epigenetic modifications. In addition, virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, by using RNA-seq data generated from embryos at the same developmental stages, we revealed a weak inverse correlation between gene expression and promoter methylation. This comprehensive analysis provides insight into the critical features of the bovine embryo methylome, and serves as an important reference for embryos produced in vitro, such as by in vitro fertilization and cloning. Lastly, these data can also provide a model for the epigenetic dynamics in human early embryos.

Dosage Compensation and Gene Expression of the X Chromosome in Sheep.

Ohno's hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1 In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno's hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and "dosage-sensitive".

Dosage Compensation of the X Chromosomes in Bovine Germline, Early Embryos, and Somatic Tissues.

Dosage compensation of the mammalian X chromosome (X) was proposed by Susumu Ohno as a mechanism wherein the inactivation of one X in females would lead to doubling the expression of the other. This would resolve the dosage imbalance between eutherian females (XX) versus male (XY) and between a single active X versus autosome pairs (A). Expression ratio of X- and A-linked genes has been relatively well studied in humans and mice, despite controversial results over the existence of upregulation of X-linked genes. Here we report the first comprehensive test of Ohno's hypothesis in bovine preattachment embryos, germline, and somatic tissues. Overall an incomplete dosage compensation (0.5 < X:A < 1) of expressed genes and an excess X dosage compensation (X:A > 1) of ubiquitously expressed "dosage-sensitive" genes were seen. No significant differences in X:A ratios were observed between bovine female and male somatic tissues, further supporting Ohno's hypothesis. Interestingly, preimplantation embryos manifested a unique pattern of X dosage compensation dynamics. Specifically, X dosage decreased after fertilization, indicating that the sperm brings in an inactive X to the matured oocyte. Subsequently, the activation of the bovine embryonic genome enhanced expression of X-linked genes and increased the X dosage. As a result, an excess compensation was exhibited from the 8-cell stage to the compact morula stage. The X dosage peaked at the 16-cell stage and stabilized after the blastocyst stage. Together, our findings confirm Ohno's hypothesis of X dosage compensation in the bovine and extend it by showing incomplete and over-compensation for expressed and "dosage-sensitive" genes, respectively.

Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters.

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells.

BACKGROUND: Male germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells. METHODS: Porcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis. RESULTS: We identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs). CONCLUSION: Collectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses.

Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.

DNA methylomes of bovine gametes and in vivo produced preimplantation embryos.

DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)-DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.

JAK/STAT3 regulated global gene expression dynamics during late-stage reprogramming process.

BACKGROUND: The generation of induced pluripotent stem cells (iPSCs) has underdefined mechanisms. In addition, leukemia inhibitory factor (LIF) activated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is the master regulator for naïve-state pluripotency achievement and maintenance. However, the regulatory process to attain naïve pluripotent iPSCs is not well understood. RESULTS: We performed transcriptome analysis to dissect the genomic expression during mouse iPSC induction, with or without blocking the JAK/STAT3 activity. We describe JAK/STAT3 signaling-specific biological events such as gametogenesis, meiotic/mitotic cell cycle, and DNA repair, and JAK/STAT3-dependent expression of key transcription factors such as the naïve pluripotency-specific genes, developmental pluripotency associated (Dppa) family, along with histone modifiers and non-coding RNAs in reprogramming. We discover that JAK/STAT3 activity does not affect early phase mesenchymal to epithelial transition (MET) but is necessary for proper imprinting of the Dlk1-Dio3 region, an essential event for pluripotency achievement at late-reprogramming stage. This correlates with the JAK/STAT3-dependent stimulation of Dppa3 and Polycomb repressive complex 2 (PRC2) genes. We further demonstrate that JAK/STAT3 activity is essential for DNA demethylation of pluripotent loci including Oct4, Nanog, and the Dlk1-Dio3 regions. These findings correlate well with the previously identified STAT3 direct targets. We further propose a model of pluripotency achievement regulated by JAK/STAT3 signaling during the reprogramming process. CONCLUSIONS: Our study illustrates novel insights for JAK/STAT3 promoted pluripotency establishment, which are valuable for further improving the naïve-pluripotent iPSC generation across different species including humans.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming.

The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

Akt3 is responsible for the survival and proliferation of embryonic stem cells.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.

WNT regulation of embryonic development likely involves pathways independent of nuclear CTNNB1.

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1 Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1.