Date of Award:

5-2013

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Advisor/Chair:

S. Clay Isom

Abstract

In mammals, the trophoblast lineage of the embryo is specified before implantation. It is restricted to become the fetal portion of the placenta. We have isolated and cultured trophoblast-derived cells from day 10 and day 13 porcine embryos. These cells demonstrate morphological and biological characteristics that make them unique. We have demonstrated that these cells can grow in vitro in a defined, serum-replacement medium for over a year without showing any signs of senescence. Trophoblast-derived cells placed into serum-containing medium, however, rapidly senesce and fail to proliferate. Gene expression analysis by RT-PCR and Fluidigm analysis of cells in culture from 0-30 days confirmed expression of genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). These experiments revealed changes in gene expression over time and in response to serum-containing medium. We have demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. Also, immunofluorescence analysis results demonstrated that these cells do not only demonstrate epithelial characteristics by the expression of KRT18, but also they show expression of VIMENTIN which is a protein found in mesenchymal cells. These findings contradict studies done by Ramsoondar in 1993 and Flechon in 1995 which reported the negative expression of VIMENTIN in similar cells. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics which have taken us to the conclusion that they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

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