Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer.

RESUMO: A geleia de Wharton é uma fonte de células tronco mesenquimais (CTMs) que ainda não havia sido testada para a produção de embriões bovinos por transferência nuclear (TN). O objetivo deste estudo foi isolar, caracterizar e testar as CTMs derivadas da geleia de Wharton para produção de embriões e gestações por transferência nuclear em bovinos. O cordão umbilical foi coletado durante o nascimento e as células derivadas da geleia de Wharton (CGWs) foram isoladas por explante e cultivadas em Dulbecco?s Modified Eagle Medium. Fibroblastos (FB) da pele foram isolados após 6 meses de vida. As análises morfológicas foram realizadas pelas microscopias de campo claro e eletrônica de varredura durante o cultivo celular. Caracterização fenotípica e genotípica por citometria de fluxo, imunocitoquímica, RT-PCR e indução da diferenciação em linhagens celulares foi realizada com as CGWs. No procedimento de TN, ovócitos no estágio de metáfase II foram enucleados usando micromanipuladores, fusionados com CGWs ou FB e então ativados artificialmente. Micrografias de microscopia de varredura revelaram que CGWs tiveram forma variada sob cultivo. Os marcadores mesenquimais de CTMs (CD29+, CD73+, CD90+ and CD105+) foram expressos em cultura de CGWs bovina, como evidenciado por citometria de fluxo, imunocitoquímica e RT-PCR. Quando induzidas, estas células diferenciaram-se em osteócitos, condrócitos e adipócitos. Após classificação, as CGWs foram utilizadas na TN. A taxa de formação de blastocistos por TN com CGWs no sétimo dia de cultivo foi de 25,80±0,03%, similar a produção de blastócitos por TN com fibroblastos de pele (19,00±0,07). Gestações foram obtidas e mostraram que CGWs constituem um novo tipo celular para ser usado na clonagem animal. ABSTRACT: Wharton?s jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton?s jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton?s jelly (WJCs) were isolated by explant and cultured in Dulbecco?s Modified Eagle Medium. Skin Fibroblasts (FB) were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM) during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+) were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%). Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

Numeric model to estimate pesticide deposition and losses from aerial spraying.

Pesticides applications have been described by many researches as a very inefficient process. In some cases, there are reports that only 0.02% of the applied products are used for the effective control of the problem. The main factor that influences pesticides applications is the droplet size formed on spraying nozzles. Many parameters affects the dynamic of the droplets, like wind, temperature, relative humidity, and others. Small droplets are biologically more active, but they are affected by evaporation and drift. On the other hand, the great droplets do not promote a good distribution of the product on the target. In this sense, associated with the risk of non target areas contamination and with the high costs involved in applications, the knowledge of the droplet size is of fundamental importance in the application technology. When sophisticated technology for droplets analysis is unavailable, is common the use of artificial targets like water-sensitive paper to sample droplets. On field sampling, water-sensitive papers are placed on the trials where product will be applied. When droplets impinging on it, the yellow surface of this paper will be stained dark blue, making easy their recognition. Collected droplets on this papers have different kinds of sizes. In this sense, the determination of the droplet size distribution gives a mass distribution of the material and so, the efficience of the application of the product. The stains produced by droplets shows a spread factor proportional to their respectives initial sizes. One of methodologies to analyse the droplets is a counting and measure of the droplets made in microscope. The Porton N-G12 graticule, that shows equaly spaces class intervals on geometric progression of square 2, are coulpled to the lens of the microscope. The droplet size parameters frequently used are the Volumetric Median Diameter (VMD) and the Numeric Median Diameter. On VMD value, a representative droplets sample is divided in two equal parts of volume, in such away one part contains droplets of sizes smaller than VMD and the other part contains droplets of sizes greater that VMD. The same process is done to obtaining the NMD, which divide the sample in two equal parts in relation to the droplets size. The ratio between VMD and NMD allows the droplets uniformity evaluation. After that, the graphics of accumulated probability of the volume and size droplets are plotted on log scale paper (accumulated probability versus median diameter of each size class). The graphics provides the NMD on the x-axes point corresponding to the value of 50% founded on the y-axes. All this process is very slow and subjected to operator error. So, in order to decrease the difficulty envolved with droplets measuring it was developed a numeric model, implemented on easy and accessfull computational language, which allows approximate VMD and NMD values, with good precision. The inputs to this model are the frequences of the droplets sizes colected on the water-sensitive paper, observed on the Porton N-G12 graticule fitted on microscope. With these data, the accumulated distribution of the droplet medium volumes and sizes are evaluated. The graphics obtained by plotting this distributions allow to obtain the VMD and NMD using linear interpolation, seen that on the middle of the distributions the shape of the curves are linear. These values are essential to evaluate the uniformity of droplets and to estimate the volume deposited on the observed paper by the density (droplets/cm2). This methodology to estimate the droplets volume was developed by 11.0.94.224 Project of the CNPMA/EMBRAPA. Observed data of herbicides aerial spraying samples, realized by Project on Pelotas/RS county, were used to compare values obtained manual graphic method and with those obtained by model has shown, with great precision, the values of VMD and NMD on each sampled collector, allowing to estimate a quantities of deposited product and, by consequence, the quantities losses by drifty. The graphics of variability of VMD and NMD showed that the quantity of droplets that reachs the collectors had a short dispersion, while the deposited volume shows a great interval of variation, probably because the strong action of air turbulence on the droplets distribution, enfasizing the necessity of a deeper study to verify this influences on drift.