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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #299429

Research Project: DEVELOPING GENETIC BIOTECHNOLOGIES FOR INCREASED FOOD ANIMAL PRODUCTION, INCLUDING NOVEL ANTIMICROBIALS FOR IMPROVED HEALTH & PRODUCT SAFETY

Location: Animal Biosciences & Biotechnology Laboratory

Title: Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors

Author
item Donovan, David
item Ealy, Alan
item Powell, Anne
item Caperna, Thomas
item Blomberg, Le Ann
item Garrett, Wesley
item Sparks, Wendy
item Talbot, Neil

Submitted to: Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2017
Publication Date: 3/23/2017
Citation: Donovan, D.M., Ealy, A.D., Powell, A.M., Caperna, T.J., Blomberg, L., Garrett, W.M., Sparks, W.O., Talbot, N.C. 2017. Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors. Reproduction. 84(6):468-485.

Interpretive Summary: Stem cell technology could greatly influence our animal improvement goals by helping to enable the fine genetic engineering of farm animals. It would also be useful in the genetic analysis of livestock and would constitute another means by which to preserve breeds and species. The study describes the results of attempts to create embryonic stem cells of cattle by an alternative means, i.e., inducing the stem cell state by over expressing master control genes in the ordinary connective tissue cells of cattle. While this approach has worked in mouse and human cells, its successful application to farm animals is still debatable. In our hands, this technique of inducing cells into a stem-cell-like state produced a few specific cell types, and the study describes the identification of one of those cell types. The cell type, called trophectoderm, was characterized by analysis of its specific cell structure and its expression of trophectoderm-specific proteins and genes. The trophectoderm tissue is a very important tissue in that it is a key component of the placenta. The work will enable comparative studies to be made between these “induced-trophectoderm cells” and natural trophectoderm cells, and this may produce information useful to the stem cell inducing technique and to understanding better the reproductive biology of cattle and related ungulates, such as goats and sheep.

Technical Abstract: Bovine trophectoderm (TE) cells were induced [induced bovine trophectoderm-like (iBT)] from bovine fetal liver-derived fibroblasts, and other bovine fetal fibroblasts, after viral-vector transduction with either four or six reprogramming factors (RF), including POU5F1, KLF4, SOX2, C-MYC, SV40 large T antigen, or hTert. Light and electron microscopic analysis showed the induced epithelial cells had morphology typical of bovine TE cells. The cells formed epithelial monolayers with interspersed domes, often had lipid droplets, and displayed cell polarity with apical microvilli and lateral cell unions of desmosomes and tight junction-like elements. Semi-quantitative RT-PCR and antiviral activity assay showed that all of the independently colony-cloned cell lines expressed interferon-tau (IFN-t) at passage1 or 2. Subsequent measurement at later passage levels (= passage 8) of IFN-t expression, including by immunoblot assay, showed that more than half of the iBT cells lines had stopped expressing IFN-t. Messenger RNAs specific to TE differentiation and function were found in the iBT cell lines and 2D-gel analysis of cellular proteins showed an expression pattern similar to that of TE cell lines derived from bovine blastocysts. Integration of the virally transduced human RFs, POU5F1, KLF4, SOX2, and NANOG were detected by PCR in the iBT cell lines and their expression was variable, except human MYC was expressed in all the cell lines. Assay of endogenous bovine RF expression showed POU5F1 and MYC were expressed in all iBT cell lines, SOX2 in none, and KLF4 and NANOG in half of the cell lines. The results demonstrate that bovine TE can be induced via RF expression from bovine liver-derived fibroblasts, although other fibroblast populations, e.g., derived from fetal muscle, could also result in TE, but at lower frequencies.