Canine iPSC generation with non-integrative vectors

Abstract

s / Placenta 34 (2013) A1–A99 A61 P2.14. CANINE IPSC GENERATION WITH NON-INTEGRATIVE VECTORS Natalia J. Goncalves , Sehwon Koh, Fabiana F. Bressan , Daniele S. Martins , Flavio V. Meirelles , Jorge Piedrahyta , Carlos E. Ambrosio 1 Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, Pirassununga, SP, Brazil; Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA Episomal vector systems have the potential to avoid undesired side effects, since they behave as separate extrachromosomal elements in the nucleus of a target cell. The main goal of gene therapy is to treat loss-offunction genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels. Production of non-transgenic iPS cells is highly desirable especially in large animal models such as the canine model. Adult canine skin fibroblasts (cSFs) were seeded at 8E5 cells per 10-cm dish and maintained in DMEM/F12 and supplements. Episomal vectors containing the murine pluripotency transcription factors (Oct3/4, Sox2, Klf4 and c-Myc, OKSM) and episome plus each of the OKSM were used to generate independent ciPSC cell lines. Nucleofection of episomal plasmids was performed with Amaxa nucleofection protocol. Cells were transfected twice for eight hours, the vectors were removed and culture media was replaced. On day 6 post-infection, the transfected cells were replated into new MEFs, the next day, culture media was replaced by mTESR. Cell cultures were analysed after non-integrated vectors in each treatment and compared with cells transfected with episomal vector only. It was observed morphological changes in the c-myc transfected culture, resembling iPS colonies. These colonies presented alkaline phosphatase (AP) activity and we confirmed pluripotency marker expression using polymerase chain reaction (PCR). Our findings show the way for the generation of iPSC free of vector integration, excluding problems caused by viral integration, and increasing the therapeutic potential of these cells. The efficiency of canine iPSC in this study, however, was increased when c-myc alone was used in combination with OKSM vector. http://dx.doi.org/10.1016/j.placenta.2013.06.182 P2.15. PLACENTAL EVOLUTION IN THE ANTEATERS AND ARMADILLOS (EUTHERIA, XENARTHRA) Andrea Mess , Phelipe Favaron , Christiane Pfarrer , Maria Angelica Miglino 1 Department of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil; 2 Institute of Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria that was likely in a basal position and makes that their characteristics influence perceptions of evolution. Objectives: Since only 1% of eutherian species have been investigated in relations to the placentation the aim of this research was to investigate the fetomaternal interface in 2 species of anteaters (Myrmecophaga tridactyla and Tamandua tetradactyla) and 3 species of armadillos (Euphractus sexcinctus, Chaetophractus villosus, and Chaetophractus vellerosus). Methods: Placentae samples were investigated by histology, immunohistochemistry, and transmission and scanning electron microscopy. Results: Data indicated differences in gross anatomy: Euphractus and Chaetophractus villosus had extended, zonary placentas and Chaetophractus vellerosus had a disk. In anteaters the gross anatomy varied from an extended placenta from mid to a discoidal placenta in late stages. Data showed that in anteaters remnants of the maternal vessel endothelium inside the placental disc were completely absent, resulting in a fully haemochorial barrier throughout the placenta. Two structurally different parts, the villous and trabecular areas were complexly intermingled. The trabeculae were attached to the decidua and consisted of cellular, proliferative trophoblast and connective tissue. In contrast, all three armadillo taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that in armadillos the endothelium of the sinuses was largely intact. In both anteaters and armadillos, the villi contained fetal capillaries and hypertrophied mesenchymal cells that occurred near to the villi surface in advanced gestation. And flat, syncytial trophoblast, interspersed with proliferative trophoblast cells, formed the villi surface. Conclusion: Data indicated fundamental differences in the establishment of the fetomaternal area between anteaters and armadillos. Thus, placental evolution in xenarthrans was more complex than previously thought. http://dx.doi.org/10.1016/j.placenta.2013.06.183 P2.16. PLACENTAL DEVELOPMENT IN NECROMYS LASIURUS (RODENTIA, CRICETIDAE) FUNCTIONAL MORPHOLOGY USING STEREOLOGICAL APPROACH Phelipe Favaron , Andrea Mess , Moacir de Oliveira , Maria Angelica Miglino , Pascale Chavatte-Palmer , Anne Tarrade 3,4 Department of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil; Department of Animal Science, Universidade Federal Rural do Semi-Arido, Mossoro, Rio Grande do Norte, Brazil; 3 INRA, UMR 1198 Biologie du Developpement et Reproduction, Jouy-en-Josas, Paris, France; 4 ENVA, Maisons-Alfort F-94704, Maisons-Alfort, Paris, France The mature chorioallantoic placenta in rodents is organized in labyrinth zone (Lz), junctional zone (Jz), and decidua (Dd). Within rodents, the placental development of murids has been well described but that of cricetids remains less investigated. Objectives: We described the development of the main placental regions in a wild Brazilian rodent Necromys lasiurus. Methods: The volume fraction (VF) of different cell types which constitute the labyrinth was quantified by the one stop stereology using 31 placentae at different stages of gestation. In addition, we investigated the volume changes of the Lz, Jz, and Dd in samples of early gestation (10-11 days, n1⁄43), mid-gestation (15-16 days, n1⁄44), and near term (21 days, n1⁄43) using the Cavalieri principle. 5 mm sections were stained using H&E and fetal vessels were localized by immunohistochemistry using vimentin. Samples were analyzed using the Mercator software and statistical analysis (oneway ANOVA and Mann and Whitney test) was performed using Graphpad Prism . Results: There was a significant increase in absolute placental volume from early (4.427 0.282mm3) to mid-gestation (12.98 1.305mm3, p<0.01), followed by a reduction near term (7.52 0.155mm3, p<0.05). The absolute volume of the Lz, Jz, and Dd followed this same trend. The VF of the labyrinth components (fetal vessels, maternal compartment, giant cells and trophoblast) differed according to gestational age with a continuous increase in the proportion of fetal vessels from day 10 (7.38 0.49%) to 21 (34.05 0.876%, p<0.01) and in that of giant cells from mid(11.66 0.286%) to late gestation (19.26 1.344%, p<0.01). In contrast, the VF of trophoblast decreased from mid(37.29 1.013%) to late gestation (21.16 0.658%, p<0.01). There was no significant variation in the VF of maternal compartment throughout pregnancy. Conclusion: Although the placental volume in N. lasiurus is smaller than murine placenta, the dynamics of placental development appears similar between these rodents. http://dx.doi.org/10.1016/j.placenta.2013.06.184