209 EPIGENETIC MODIFICATION WITH ZEBULARINE AND VALPROIC ACID AND EXPRESSION OF PLURIPOTENCY GENES IN BOVINE ADIPOSE STEM CELLS

Abstract

Bovine adipose-derived stem cells (ASC), a form of adult stem cells, are somatic cells that have similar characteristics of embryonic stem (ES) cells. ASC are multipotent and have been found to express genes associated with pluripotency, Oct4, Sox2 and Nanog. The unique properties of ASC make them a desirable source for reprogramming experiments. When somatic cells are reprogrammed, there are certain epigenetic changes or modifications that must occur. Epigenetic modifications will change the chromatin configuration without changing the DNA sequence. Somatic cells can be exposed to small molecules that induce some of these changes. Two epigenetic modifying factors are a DNA methyltranferase inhibitor, zebularine (Zeb) and a histone deacetylase inhibitor, valproic acid (VPA). By altering gene expression with these epigenetic modifiers, the cells may be stimulated to reprogram more efficiently than untreated cells. Three lines of bovine ASC were isolated from the stromal fraction of adipose tissue and expanded through 3 passages in DMEM (high glucose) supplemented with 10% fetal bovine serum. Cells were cultured by addition of 5 mM VPA or 100 μM Zeb to the culture medium and cultured for 5, 7, 10 or 14 days before mRNA was isolated and converted to cDNA. Control groups, consisting of untreated bovine ASC from the same cell lines, were cultured for the same time periods as the treatment groups. Quantitative RT-PCR was performed to determine transcript levels for Oct4, Sox2, Nanog and poly adenylate polymerase (PAP) as a reference gene. 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 bovine ADS cells. Normalized ratios were log-transformed before analysis by ANOVA to correct for lack of normality. A one-way ANOVA was completed to test the statistical significance among the control and treatment groups. Pairwise comparison was conducted to test expression levels between individual treatments. When cells were treated for 5 days, Oct4 and Nanog expression levels were increased (P ≤ 0.05) between the control and both treatment groups. Expression of Sox2 was not different (P = 0.06) across treatment groups. Based on pair wise comparisons gene expression in VPA and Zeb treated groups were not different. Treatment for 7, 10, or 14 days did not result in any difference in transcript levels between the treatment groups and control for any of the genes analysed. VPA and Zeb treatment for 5 days may have produced a partial reprogramming. This partial reprogramming could aid in the bovine ASC reaching complete pluripotency when combined with other reprogramming techniques.

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