Factors Oct4 Research Articles

A Comparison of m-Carboxycinnamic Acid Bishydroxamide with Trichostatin A as Histone Deacetylase Inhibitor on the Developmental Competence of Somatic Cell Nuclear Transfer Porcine Embryos

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

343 DIRECT EXPRESSION OF PLURIPOTENCY MARKERS IN CULTURED SOMATIC CELLS BY SMALL REPROGRAMMING MOLECULES

The differentiate state of the cell may be reversed by a process called reprogramming. To date, a totipotent status is conferred to a somatic cell by nuclear transfer (SCNT) and a condition of pluripotency is conferred by induced expression of defined factors (iPSC). While the restoration of full totipotency by SCNT is rarely achieved, pluripotency by Yamanaka's factors (Oct4, c-myc, Sox-2, Klf4) is inducible, although with low efficiency, in a large set of cell in different animal models. However, the isolation of iPSC requires complex technical skills and time-consuming protocols. In our laboratory we have observed that the simple expansion of fibroblasts in culture switches on pluripotency markers such as Oct4 and Nanog (Anzalone et al. 2015 Reprod. Fertil. Dev. IETS Abstract 344). CHIR99021 is a small molecule, targeting the Wnt/β-catenin signalling pathway, which is used for stem cell culture (Li et al. 2009). CHIR99021 acts as selective inhibitor of both isoform of GSK3 α/β regulating cellular proliferation and differentiation. In this work we tested the hypothesis that the exposure to a small reprogramming molecule (CHIR99021) induces pluripotency marker expression in primary cultures of somatic cells. Sheep and mouse primary fibroblasts cultured in low oxygen and induced to enter GO (low serum, 0.5% FBS for 5 days, &lt;3% cell proliferation in our conditions) were treated with different CHIR concentrations (from 2.5 to 5 µM) for different time periods (from 1 to 5 days) in order to test the proper concentration and to exclude any cytotoxic effects. Nuclear reprogramming was assessed in treated and control cells by analysing β-catenin and oct4, nanog, sox2, klf4, and c-myc expression by immuno-detection and PCR. We found that CHIR interferes with β-catenin pathway in both sheep and mouse fibroblast in a time- and dose-dependent manner; the best results were obtained using 3 µM of CHIR for 3 days. Western blot analysis confirmed that CHIR treatment leads to an increased cellular level of β-catenin; furthermore, pluripotency marker expression (protein and mRNA) was increased (P = 0.023 nonparametric Mann-Whitney test) in CHIR-treated cells compared to controls. These observations, confirmed in both the experimental models, indicate that treatment with a small molecule inhibitor interfering with glucose metabolism induces the expression of pluripotency marker in somatic cells.

The immunoglobulin heavy chain VH6-1 promoter regulates Ig transcription in non-B cells.

BackgroundNon-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.MethodsThe promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.ResultsOur data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.ConclusionThe regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

Abstract 4123: Exploiting sonic hedgehog pathway in pancreatic carcinogenesis as a potential target for chemoprevention

Abstract Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. The lethal nature of pancreatic cancer stems from its propensity to rapidly disseminate accompanied by resistance to current therapies contributes to the high mortality in patients with pancreatic cancer. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. We also show here for the first time, that sulforaphane treatment resulted in a significant reduction in the tumor growth, of orthotopically implanted primary pancreatic CSCs isolated from human pancreatic tumors into the pancreas of NOD/SCID/IL2Rgamma mice, which is mediated through the modulation of Sonic hedgehog-GLI signaling. Hedgehog pathway blockade by SFN at a dose of 20 mg/kg resulted in a 45% reduction in growth of pancreatic cancer tumors. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN treatment resulted in a significant reduction in EMT markers Zeb-1, which correlated with increase in E-Cadherin expression suggesting the blockade of signaling involved in early metastasis. Interestingly, SFN downregulated the expression of Bcl-2 and XIAP to induce apoptosis. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. Such information will not only allow rational design of SFN-based strategies for prevention and/ or treatment of pancreatic cancer but could also facilitate development of mechanism-driven protocols for optimal clinical effects. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer. Citation Format: Rakesh Srivastava, Sharmila Shankar. Exploiting sonic hedgehog pathway in pancreatic carcinogenesis as a potential target for chemoprevention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2014-4123

Synergistic transcriptional and post-transcriptional regulation of ESC characteristics by core pluripotency transcription factors in protein-protein interaction networks.

The molecular mechanism that maintains the pluripotency of embryonic stem cells (ESCs) is not well understood but may be reflected in complex biological networks. However, there have been few studies on the effects of transcriptional and post-transcriptional regulation during the development of ESCs from the perspective of computational systems biology. In this study, we analyzed the topological properties of the “core” pluripotency transcription factors (TFs) OCT4, SOX2 and NANOG in protein-protein interaction networks (PPINs). Further, we identified synergistic interactions between these TFs and microRNAs (miRNAs) in PPINs during ESC development. Results show that there were significant differences in centrality characters between TF-targets and non-TF-targets in PPINs. We also found that there was consistent regulation of multiple “core” pluripotency TFs. Based on the analysis of shortest path length, we found that the module properties were not only within the targets regulated by common or multiple “core” pluripotency TFs but also between the groups of targets regulated by different TFs. Finally, we identified synergistic regulation of these TFs and miRNAs. In summary, the synergistic effects of “core” pluripotency TFs and miRNAs were analyzed using computational methods in both human and mouse PPINs.

Generation of rat-induced pluripotent stem cells from a new model of metabolic syndrome.

We recently characterized DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive rats and Zucker rats, as a new animal model of metabolic syndrome (MetS). Although the phenotype of DS/obese rats is similar to that of humans with MetS, the pathophysiological and metabolic characteristics in each cell type remain to be clarified. Hence, the establishment of induced pluripotent stem cells (iPSCs) derived from MetS rats is essential for investigations of MetS in vitro. Reports of rat iPSCs (riPSCs), however, are few because of the difficulty of comparing to other rodents such as mouse. Recently, the advantage of using mesenchymal stromal cells (MSCs) as a cell source for generating iPSCs was described. We aimed to establish riPSCs from MSCs in adipose tissues of both DS/obese rats and their lean littermates, DahlS.Z-Lepr+/Lepr+ (DS/lean) rats using lentivirus vectors with only three factors Oct4, Klf4, and Sox2 without c-Myc. The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas). Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone. Real-time PCR analysis also revealed that both riPSCs and the adipose tissue from DS/obese and DS/lean rats possess similar expression patterns of adipocyte differentiation-related genes. We succeeded in generating riPSCs effectively from MSCs of both DS/obese and DS/lean rats. These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics.

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.

Abstract 77: Endothelial LOX-1 Protects Against Arterial Thrombosis via Activation of the Oct-1/SIRT1 Pathway

Background: The lectin-like oxLDL receptor-1 (LOX-1) promotes the endothelial uptake of oxidized low-density lipoprotein (oxLDL). However, LOX-1 is involved in several other biological processes and its role in arterial thrombus formation remains unknown. The present study was designed to investigate whether LOX-1 activation plays a role in thrombus formation in vivo. Methods and Results: Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). While plasma levels of oxLDL were comparable, carotid tissue oxLDL content was markedly increased in LOX-1TG as compared to wild type (WT). Arterial thrombus formation was assessed using an in vivo photochemical injury model. Time to arterial occlusion was prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity were reduced by 50% in the carotid arteries of LOX-1TG mice. This effect was mediated by the activation of the transcription factor Oct-1 leading to upregulation of mammalian deacetylase SIRT1 via binding to its promoter and subsequent inhibition of NF-κB signaling as demonstrated by siRNA experiments. This was further confirmed in LOX-1TG endothelial cells (EC) where expression of Oct-1 and SIRT1 was increased upon exposure to oxLDL. Increased expression of SIRT1 was further associated with decreased DNA-binding of RelA/p65 subunit of NF-κB. Conclusions: LOX-1 activates a novel compensatory pathway which protects against arterial thrombus formation in vivo. These unexpected findings suggest that Oct-1/SIRT1 signaling may represent a novel target for the prevention of arterial thrombus formation in the setting of hyperlipidemia and atherosclerosis.

Recent Advances in Understanding the Etiology and Pathogenesis of Pediatric Germ Cell Tumors

Pediatric germ cell tumors (GCTs) are rare neoplasms arising predominantly in the gonads and sacrococcygeal, mediastinal, and intracranial localizations. In this article, we review current knowledge of pathogenesis of pediatric GCTs, which differs from adult/adolescent GCTs. One distinctive feature is the absence of a progenitor stage, such as carcinoma in situ or gonadoblastoma, which are seen in adult/adolescent GCTs, except spermatocytic seminoma. The primordial germ cell (PGC) is the suggested origin of all GCTs, with variations in histology reflecting differentiation stage. Expression of pluripotency transcription factors OCT-3/4, NANOG, and AP-2γ in germinomas/seminomas/dysgerminomas is consistent with retaining a germ cell phenotype. Teratomas, in contrast, develop through a pathway of aberrant somatic differentiation of immature germ cells, and the yolk sac tumors and choriocarcinomas result from abnormal extraembryonic differentiation. In pediatric GCTs, origin is suggested at an earlier developmental stage because of predisposing genetic factors, although responsible genes remain largely unknown. Some extragonadal GCTs have been linked to overexpression of the KIT/KITLG system, allowing for survival of aberrantly migrated ectopic PGCs. Infant gonadal/sacrococcygeal GCTs may be caused by apoptosis-related pathways, consistent with an association with polymorphisms in BAK1. Although recent advances have identified candidate pathways, further effort is needed to answer central questions of pathogenesis of these fascinating tumors.

CHRNB3 c.-57A>G functional promoter change affects Parkinson's disease and smoking

Cigarette smoking is protective in Parkinson's disease (PD), possibly because of nicotine action on brain nicotinic-acetylcholine receptors. The β3 nicotinic-acetylcholine receptor subunit (encoded by CHRNB3) is depleted in the striatum of PD patients and associated with nicotine dependence. Herein, the CHRNB3 gene was sequenced, and the c.-57G allele frequency was 0.31 and 0.26 among patients (n = 596) and controls (n = 369), respectively (p = 0.02, odds ratio = 1.33, 95% confidence interval = 1.03–1.73). The c.-57G allele was strongly associated with smoking in patients, as 48.4% of c.-57G carriers compared with 32.6% of noncarriers reported smoking history (p < 0.0001). The transcription factor Oct-1 binding was almost eliminated in lymphoblasts with the c.-57G/G genotype, to only 6.5% percent, and the CHRNB3 promoter activity was reduced in cells with the c.-57G/G genotype by 96%–70%. These findings suggest that the CHRNB3 c.-57A>G alteration affects the promoter activity and is associated with PD and smoking in PD patients. It is therefore possible that nicotine may be valuable for patients who carry this alteration and beneficial in PD only for patients with specific genotypes.

Establishment and Characterization of Novel Porcine Induced Pluripotent Stem Cells Expressing hrGFP

Induced pluripotent stem (iPS) cells have been established in various animal species since 2006. The pig is a potentially useful model in human regenerative medicine, and the characters of porcine embryonic stem (pES) cells were much similar with human embryonic stem (hES) cells. In present study, the traceable humanized recombinant green fluorescent protein expressing porcine induced pluripotent stem (piPS/hrGFP+) cells were generated from porcine ear fibroblasts (pEF) by introducing four human transcription factors (Sox2, Oct4, Klf4, and c-Myc) constructed in lentivirus vectors. The piPS/hrGFP+ cells continuously and steadily expressed hrGFP signal continuously and steadily for more than 90 passages over 20 months despite repeated subcultures. They also retained the typical defined characteristics including continuous proliferation with undifferentiated status, expression of ES pluripotent pluripotency markers (Oct4, AP, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and maintenance of a normal karyotype (36+XY). Three embryonic germ layers were also successfully revealed from in vitro differentiation by EB formation. Various histological analysis and immunohistochemical staining of the teratomas revealed various tissues derived from three embryonic germ layers, including neural tissues, keratin-containing epidermal tissues, skeletal muscle, smooth muscle, cartilage, adipose tissues, and glandular structures. These results support that piPS/hrGFP+ cells can be generated from pEF by direct reprogramming, and these traceable piPS/hrGFP+ cells would be beneficial for future application on cell transplantation and tissue regeneration.

TATκ Fusion Protein of OCT-3/4 and KLF-4: Stable Mixed Population Cell Lines Capable of Delivering Fusion Proteins to Target Cells

A rapidly growing body of evidence has shown that enforced expression of a small panel of genes, including Oct-3/4, KLF4, Sox2 and c-Myc, can induce the reprogramming of previously differentiated cells, to generate induced Pluripotent Stem Cells (iPSCs). However, the generation of iPSCs by genetic modifications raises the risk of malignant transformation. Therefore, efficient in vitro reprogramming of differentiated cells, without irreversible genetic modification is highly desirable. The improved trans-activator of transcription kappa (TATκ), a synthetic TAT-HIV, confers the ability to deliver several proteins into target cells, thus, making it a potential alternative delivery mechanism for transcription factors or therapeutic agents. Using Green Fluorescence Protein (GFP) and Apoptin as model proteins, we have recently described a strategy to generate cell lines that secrete proteins carrying a modified HIV-TAT Protein Transduction Domain (PTD) for subsequent protein transduction mediated uptake by target cells. Using this strategy we have generated 293T cells secreting the pluripotent factors Oct-3/4 or KLF4 in fusion with TATκ. Oct-3/4 and KLF4 were detected in the culture medium of the transduced 293T cell and uptake of Oct-3/4 by haematopoietic cell lines JURKAT and FDCP-1 was confirmed by Western blot analysis. KLF4 was present in the culture medium of the producer 293T cell but we are still unable to demonstrate uptake by target cells. Based on results obtained, we hope that these stable mix population cell lines can play an important role in generation of iPSCs for therapeutic purposes.

Aberrant Neural Stem Cell Proliferation and Increased Adult Neurogenesis in Mice Lacking Chromatin Protein HMGB2

Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs, suggesting that it may regulate NSC maintenance. We report now that Hmgb2−/− mice exhibit SVZ hyperproliferation, increased numbers of SVZ NSCs, and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB) granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM), along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2−/− SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation, and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration.

Reprogramming to Pluripotency through a Somatic Stem Cell Intermediate

Transcription factor-based reprogramming can lead to the successful switching of cell fates. We have recently reported that mouse embryonic fibroblasts (MEFs) can be directly reprogrammed into induced neural stem cells (iNSCs) after the forced expression of Brn4, Sox2, Klf4, and Myc. Here, we tested whether iNSCs could be further reprogrammed into induced pluripotent stem cells (iPSCs). The two factors Oct4 and Klf4 were sufficient to induce pluripotency in iNSCs. Immunocytochemistry and gene expression analysis showed that iNSC-derived iPSCs (iNdiPSCs) are similar to embryonic stem cells at the molecular level. In addition, iNdiPSCs could differentiate into cells of all three germ layers, both in vitro and in vivo, proving that iNdiPSCs are bona fide pluripotent cells. Furthermore, analysis of the global gene expression profile showed that iNdiPSCs, in contrast to iNSCs, do not retain any MEF transcriptional memory even at early passages after reprogramming. Overall, our results demonstrate that iNSCs can be reprogrammed to pluripotency and suggest that cell fate can be redirected numerous times. Importantly, our findings indicate that the induced pluripotent cell state may erase the donor-cell type epigenetic memory more efficiently than other induced somatic cell fates.

Transcriptional regulation of the HMGA1 gene by octamer-binding proteins Oct-1 and Oct-2.

The High-Mobility Group AT-Hook 1 (HMGA1) protein is an architectural transcription factor that binds to AT-rich sequences in the promoter region of DNA and functions as a specific cofactor for gene activation. Previously, we demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR) gene and an important downstream target of the INSR signaling cascade. Moreover, from a pathogenic point of view, overexpression of HMGA1 has been associated with human cancer, whereas functional variants of the HMGA1 gene have been recently linked to type 2 diabetes mellitus and metabolic syndrome. However, despite of this biological and pathological relevance, the mechanisms that control HMGA1 gene expression remain unknown. In this study, to define the molecular mechanism(s) that regulate HMGA1 gene expression, the HMGA1 gene promoter was investigated by transient transfection of different cell lines, either before or after DNA and siRNA cotransfections. An octamer motif was identified as an important element of transcriptional regulation of this gene, the interaction of which with the octamer transcription factors Oct-1 and Oct-2 is crucial in modulating HMGA1 gene and protein expression. Additionally, we demonstrate that HMGA1 binds its own promoter and contributes to its transactivation by Oct-2 (but not Oct-1), supporting its role in an auto-regulatory circuit. Overall, our results provide insight into the transcriptional regulation of the HMGA1 gene, revealing a differential control exerted by both Oct-1 and Oct-2. Furthermore, they consistently support the hypothesis that a putative defect in Oct-1 and/or Oct-2, by affecting HMGA1 expression, may cause INSR dysfunction, leading to defects of the INSR signaling pathway.

32 PREGNANCY OF EQUINE CLONED EMBRYOS MICROINJECTED WITH PLURIPOTENCY INDUCING GENES (Oct4, Sox2, c-Myc, K1f4)

Animal cloning is a high impact tool for scientific and economical production, but still with inefficient results. The efficiency of the cloning process depends on the state of differentiation of the donor cell. An adult equine somatic cell can be differentiated to a pluripotent stem cell (iPSC) inducing the expression of certain transcription factors (Oct4, Sox2, c-Myc, and K1f4; Breton et al. 2013). The objective of this work was to assess the effect of the intracytoplasmic injection of pluripotency inducing genes on embryo development and pregnancy rates of equine cloned embryos. Cumulus–oocyte complexes (COC) were obtained from slaughterhouse ovaries. Oocyte collection and maturation procedure were performed as described by Lagutina et al. (2007). After the removal of cumulus cells, oocytes showing first polar body were microinjected with a mixture 1/3 of plasmids/liposomes (Mi group). The plasmid used was the pEP4-E02s-EM2k, which encodes the human genes Oct4, Sox2, Myc, and K1f4. The DNA concentration was adjusted to 0.5 μg mL–1. Microinjected oocytes were enucleated using the zona free method. Adult male skin fibroblasts from the same animal were used as donor nucleus cells. These fibroblasts were attached to the ooplasts with phytohemagglutinin and then fused with an electric pulse. Activation was performed using 8.7 mM ionomycin for 4 min, followed by culture for 4 h in a combination of 1 mM 6-DMAP and 5 mg mL–1 cycloheximide. Zona free reconstructed embryos (ZFRE) were cultured for 7 to 8 days in DMEM-F12 in the well of the well (WOW) system, aggregating 3 embryos per well. A control group (CC group) of not microinjected embryos was included. Cleavage and blastocyst development was assessed at Days 2 and 7, respectively. Transcervical transfer of 49 Day 7 to 8 blastocysts was performed 6 days after ovulation. The mares received 2 blastocysts per transfer. Pregnancy was diagnosed by transrectal ultrasonography 15 days after ovulation. Cleavage and blastocyst rates were analysed by Chi-squared test and pregnancy rate by Fisher test (P &lt; 0.05). Cleavage was 92.1% (n = 58/63) for the Mi group and 90.4% (n = 868/960) for the CC group. Blastocyst rate was statistically higher per well, 28.6% (n = 6/21) v. 13.4% (n = 43/320) but not per oocyte, 9.5% (n = 6/63) v. 4.5% (n = 43/960), for the Mi and CC groups, respectively. Pregnancy rate was 17% (n = 1/6) for the Mi group and 7% (n = 3/43) for the CC group. No twin pregnancies were found and all the pregnancies are still ongoing. The higher blastocyst rates obtained with the embryos microinjected with pluripotency inducing genes compared with the control group showed an improvement in embryo quality. In conclusion, the data presented indicate that the intracytoplasmic microinjection of pluripotency inducing genes in equine zona free cloned embryos improved blastocyst rates on a per well basis and showed a tendency to improve the pregnancy rates. The expression of the Oct4, Sox2, c-Myc, and K1f4 genes could be probably generating better reprogrammed donor nucleus compared with adult differentiated cells used in conventional cloning.

197 INDUCED PLURIPOTENT STEM CELLS (iPS) DERIVED FROM EQUINE UMBILICAL CORD CELLS USING LENTIVIRUS VECTOR Stemcca

Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5 mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 105 cells, these cells were transduced with 50 μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8 ng mL–1 polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12 h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n = 10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.

190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P &lt; 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.

Abstract 19248: Endothelial Overexpression of LOX-1 Protects From in vivo Arterial Thrombosis and Modulates Oct-1 and SIRT1 Expression

Background: The hallmark of the initiation of atherosclerotic lesion is foam cell formation, and oxidized LDL (oxLDL) is believed to play a key role in the initiation of the atherosclerotic process. OxLDL is internalized by several receptors, such as SR-AI/II, SR-BI, CD36, and CD68. OxLDL is also internalized by EC, but this uptake depends on receptors other than the classic scavenger receptors. In 1997, a lectin-like oxidized LDL receptor-1 (LOX-1, OLR1) was identified in bovine aortic endothelial cells. LOX-1 is a type II membrane glycoprotein with an apparent molecular weight of 50 kDa. It has a C-terminal extracellular C-type lectin-like domain. This lectin-like domain is essential for binding to oxLDL. Binding of oxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO, which may trigger the onset of cardiovascular events. Methods and Results: We generated endothelial-specific LOX-1 transgenic mice using the Tie2 promoter ( LOX-1TG ). 12-wk-old male LOX-1TG and wild-type (WT) mice were applied for carotid artery thrombosis model. LOX-1TG mice developed carotid artery thrombosis within a mean occlusion time of 36.96±4.83 min, while WT mice occluded within a mean time period of 22.75±3.87 min (n=10, P &lt; 0.05). Initial blood flow in carotid artery did not differ between both groups. Decreased occlusion time in LOX-1TG mice was further associated with decreased tissue factor expression and surface activity as shown by qPCR and ELISA. Furthermore, LOX-1TG mice showed increased mRNA and protein expression of transcription factor Oct-1 and histone deacetylase SIRT1 in carotid artery, pointing out that Oct-1 and SIRT1 both may be involved in the observed downregulation of tissue factor through its known target NF-kB. Indeed, in vivo knockdown of either Oct-1 or SIRT1 by siRNA further confirmed their role in arterial thrombosis. Conclusions: Thus, our data suggest that LOX-1 plays a protective role in the arterial thrombosis and that both Oct-1 and SIRT1 are involved representing new LOX-1-mediating signaling pathway. Hence, modulation of LOX-1 may represent novel therapeutic options for targeting arterial thrombosis.

The Protein Kinase A Regulatory Subunit R1A (Prkar1a) Plays Critical Roles in Peripheral Nerve Development

Signaling through cAMP has been implicated in Schwann cell (SC) proliferation and myelination, but the signaling pathway components downstream of cAMP required for SC function remain unknown. Protein kinase A (PKA) is a potential downstream effector of cAMP. Here, we induced loss of Prkar1a, the gene encoding the type 1A regulatory subunit of PKA, in SC to study its role in nerve development; loss of Prkar1a is predicted to elevate PKA activity. Conditional Prkar1a knock-out in mouse SC (Prkar1a-SCKO) resulted in a dramatic and persistent axonal sorting defect, and unexpectedly decreased SC proliferation in Prkar1a-SCKO nerves in vivo. Effects were cell autonomous as they were recapitulated in vitro in Prkar1a-SCKO SC, which showed elevated PKA activity. In the few SCs sorted into 1:1 relationships with axons in vivo, SC myelination was premature in Prkar1a-SCKO nerves, correlating with global increase in the cAMP-regulated transcription factor Oct-6 and expression of myelin basic protein. These data reveal a previously unknown role of PKA in axon sorting, an unexpected inhibitory role of PKA on SC cell proliferation in vivo and define the importance of Prkar1a in peripheral nerve development.