235 EXPRESSION OF PLURIPOTENCY-ASSOCIATED GENES IN BOVINE FETAL FIBROBLAST CELLS AND ADIPOSE-DERIVED STEM CELLS

Abstract

The extraordinary nature of embryonic stem cells (ESC) lends them the remarkable ability to give rise to all the cell types of a mammalian organism, an attribute known as pluripotency. The pluripotent state of ESC is dependent on the expression of the genes Oct-4, Nanog, and Sox-2, which have also been identified as the key transcriptional regulators of pluripotency. Until recently, Oct-4, Nanog, and Sox-2 were believed to be expressed solely in ESC; however, studies have reported the expression of these genes in some sources of adult stem cells (ASC) of both the mouse and pig. In the current study, we examined cells derived from bovine adipose tissue and fetal fibroblast for the expression of Nanog and Sox-2. Cells were isolated from the adipose tissue of an adult cow and the skin of 2 male fetuses, approximately 70 and 80 d old, and cultured through 6 passages. Total RNA was isolated from each of the 3 cell lines at passages 2, 4, and 6 using TRI Reagent®. Using the Bio-Rad iScript™ cDNA Synthesis Kit, the resulting RNA products were transcribed into cDNA 3 times for separate RT-PCR reactions. RT-PCR was performed with primers for Nanog, Sox-2, and Poly A Polymerase (PAP), as a reference gene for normalization. Primer sets for Nanog and PAP have previously been verified to amplify their respective transcripts in bovine embryos. Reaction products of PCR were subjected to electrophoresis and analyzed by Quantity One software (Bio-Rad). The number of pixels in each electrophoresis band was used to determine the relative transcript levels and was expressed as a ratio of Nanog or Sox-2 to PAP (Table 1). Both Nanog and Sox-2 were present in all cell lines at all 3 passages. Because Oct-4 is known to function synergistically with Nanog and Sox-2 to confer pluripotency, we are currently examining these samples for the presence of Oct-4. The presence of transcripts for the pluripotency-associated genes Nanog and Sox-2 in cells that are not inherently pluripotent suggests that there might be another level of regulation at the translational level for these genes. Table 1.Transcript abundance of pluripotency-associated genes relative to PA P in bovine fetal fibroblast (BFF) and adipose-derived adult stem cells (ADAS)