208 REPROGRAMMING OF BOVINE SOMATIC CELLS WITH CELLULAR EXTRACTS FROM ADIPOSE-DERIVED ADULT STEM CELLS AND HUMAN EMBRYONIC STEM CELLS

Abstract

The differentiation potential of adult stem cells (ASC) has long been considered limited to the cell types present in the organ or tissue from which they are derived. Several studies have challenged this notion by demonstrating that ASC exhibit a high degree of plasticity; therefore, it is likely that these cells possess an epigenomic regulatory pattern more like pluripotent cells than somatic cells. In the present study, we used quantitative RT-PCR (qRT-PCR) to analyse bovine adipose-derived adult stem cells (ADAS) and bovine fetal fibroblasts (BFF) for the presence of Oct-4, Nanog and Sox-2. The ADAS cells were isolated from the stromal fraction of bovine adipose tissue from adult cows and BFF were isolated from 50-d-old fetuses. Both cell types were expanded through 3 passages in DMEM supplemented with 10% fetal bovine serum. Transcripts for these pluripotency-associated genes were detected in all samples, supporting the hypothesis that ADAS and BFF cells are less differentiated than other somatic cell types. To induce reprogramming we exposed BFF cells to ADAS cell extracts and human ESC (hESC) extracts. Cellular components of ADAS cells and hESC were extracted using cell lysis buffer and Dounce homogenization. Individual quanta of BFF cells were permeabilized with streptolysin O and allowed to incubate with 100 μL of ADAS cell extracts or hESC extracts containing an ATP regenerating system. BFF cells were then resealed with complete culture medium containing 2 mM CaCl₂ and cultured for 72 h before mRNA isolation. Quantitative RT-PCR was performed to determine transcript levels for Oct4, Sox2, Nanog and poly adenylate polymerase (PAP) as a reference gene in untreated BFF cells, ADAS cells and treated BFF cells. Transcript levels were quantified by relative quantification using the ΔΔCT method and expressed as ratios of the target genes (Oct4, Sox2 and Nanog) to the reference gene (PAP) and normalized against a calibrator consisting of untreated BFF cells. Differences between normalized ratios were analysed by Student's t-test. We did not detect a difference in Oct-4 and Nanog transcript levels between untreated BFF cells and BFF cells exposed to ADAS cell extracts. Comparison between untreated BFF cells and BFF cells treated with ADAS cell extracts revealed a higher (P ≤ 0.05) Sox-2 expression in the extract-treated cells. The BFF cells were then exposed to extracts derived from hESC. Unlike other studies that have reported the up-regulation of Oct-4, Nanog and Sox-2 in cells treated with hESC extracts, we did not detect a difference in Oct-4, Nanog and Sox-2 expression levels between untreated BFF cells and BFF cells treated with hESC extracts. This may be due to expression differences between species. Currently, we are assessing the ability of mouse ESC extracts to reprogram bovine ADAS cells.

This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.