1a.Objectives (from AD-416)
1. Improve the efficiency of somatic cell nuclear transfer (NT). Protein expression of the tissues of pre-implantation NT, IVF (in vitro fertilized), and parthenogenic (P) 8-day and 16-day embryos (blastocysts) will be compared to identify techniques or conditions that can be altered to create NT embryos that will develop to term at higher frequency. 2. Establishment and characterization of ungulate embryonic stem cell (ESC) lines. Porcine or bovine epiblast tissue from the preimplantation embryo will be assayed for stem cell pathway gene expression and will be cultured under novel experimental conditions. 3. Establishment and characterization of ungulate somatic stem cell (SSC)lines. Porcine or bovine bone marrow or umbilical cord tissue will be cultured to establish cell lines of cells expressing SSC markers.

1b.Approach (from AD-416)
Cellular protein expression IVF-, NT-, and P-derived embryos will be compared by 2-D gel electrophoretic analysis of bovine trophectoderm and endoderm cell lines derived from blastocyst stage embryos. A composite 2-D gel and protein data base defining both overall protein expression and differential protein expression (potential reprogramming “markers”) from bovine NT and IVF preimplantation embryo-derived trophectoderm cell lines will be produced. These results will be compared to the protein profiles of in vivo-derived 16-day pre-implantation bovine embryos acquired from superovulated and artificially inseminated (AI) heifers. Tests for real-time assessment of the proper reprogramming of NT embryos will be devised based on detection of reprogramming “markers” by immunocytochemical assay, anti-viral assay, and ELISA. The undefined problems inherent to the establishment of ungulate ESC lines will be investigated by candidate-gene-directed RT-PCR and immunocytochemical analysis of the tissues of early preimplantation porcine embryos, particularly the cells constituting the epiblast (in vivo ESC equivalent). Novel culture methods, condition, and constituents will be tested for the continuous culture of ungulate ESC. The establishment of ungulate SSC lines will be attempted from the culture of bovine or porcine bone marrow or umbilical cord cells. Specific markers for the demonstration of pluripotency of putative SSC populations, i.e., differentiation into multiple cell types of one germ layer or more, will be assayed by immunocytochemistry.

3.Progress Report
During the 2011 fiscal year, two types of “feeder-cells”, used for the creation and maintenance of embryonic stem (ES) cell lines or induced pluripotent stem cell (iPSC) lines, were analyzed for the growth factors that they produce by several protein detection methods. The results showed that the feeder-cells produced numerous growth factors important for the maintenance of ES cells as ES cells, but also produced factors important in inducing the differentiation of the ES cell into specific cells of the body.

Cell culture analysis of putative pig induced pluripotent stem cell lines (piPSCs) was undertaken to verify their potential usefulness for the genetic engineering of pigs. So far the analysis indicates that two types of induced stem cell lines may have been created, neural stem cells and extra-embryonic stem cells. Also, the characterization of bovine fetal liver cell lines was begun to see if they are immortal cell lines, and, therefore, could be useful for cattle genetic engineering via nuclear cloning, and also to see if they are a good in vitro model of cow liver function. An in vitro model of cow liver does not presently exist and could be very useful for making predictions of how a cow’s liver would respond to changes in its food, drug regimens, or even changes in the genes expressed its own liver. This research coincided with target areas through the development of genome-enabling tools and reagents, specifically, ES cell lines and induced pluripotent stem cell lines of ungulates.

4.Accomplishments
1. Analysis of feeder-cell secreted proteins. Feeder-cells are fibroblastic cells that are used to grow and maintain embryonic stem (ES) cells in culture. Proteins produced by two frequently employed feeder-cell types need to be identified so that a better understanding of how they support the growth and maintenance of ES cells can be gained. ARS researchers at Beltsville, MD used proteomic methods to show that the two feeder-cells were producing several extracellular matrix proteins, collagen being the most abundant. Semi-quantitative protein array analysis of 105 specific growth factor proteins and inflammatory-response proteins clearly demonstrated differences in kind and quantity of the factors expressed between the two feeder-cell lines analyzed. Enzyme-linked immunosorbent assays (ELISA) showed statistically significant differences in the expression of 17 growth factor proteins between feeder-cell types. The results directly impact research to create ES cell lines of ungulates so that the fine genetic engineering of cattle and pigs can be routinely performed to improve production efficiency, product quality, animal welfare, and national food security.

Review Publications
Caperna, T.J., Blomberg, L., Garrett, W.M., Talbot, N.C. 2011. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media. In Vitro Cellular and Developmental Biology - Animals. 47(3):218-233.