288 INDUCING PLURIPOTENCY IN BOVINE SOMATIC STEM CELLS

Abstract

Embryonic stem cells are pluripotent cells that can self-replicate indefinitely. There is an interest in producing pluripotent cells by reprogramming somatic cells. Porcine, murine, and human somatic cells are able to reprogram after exposure to viral vectors expressing Sox2, Oct-4, Klf4, and c-Myc. Exposure of somatic cells to these epigenetic modifying factors has been shown to increase expression of endogenous pluripotency factors, especially Oct-4 and Nanog. There have been limited attempts to induce pluripotency in bovine somatic cells. Bovine adipose-derived stem (ADS) cells were infected with a lentiviral vector expressing murine Sox2, Oct-4, Klf4, and c-Myc. In addition, bovine ADS cells were exposed to valproic acid (VPA), a histone deacetylase inhibitor, and zebularine (Zeb), a DNA methyltransferase 1 inhibitor. When two cell lines (‘A’ and ‘B’) of bovine ADS cells (between passages 2–6) were infected with the lentiviral vector, cells were able to form embryonic-stem-cell-like colonies on BD Matrigel (BD, Franklin Lakes, NJ, USA). Bovine ADS cells were exposed to lentiviral particles for 24 h and then changed to a Matrigel-coated culture dish after 3 days. These infected cells were stained on days 10 and 15 with an alkaline phosphatase stain, which resulted in positive staining cell colonies. The RNA was isolated from these cells, and expression of endogenous Oct-4 and Nanog was analysed by semiquantitative RT-PCR. The intensity of electrophoresis bands was compared between treatments as ratios of the gene of interest to an internal control gene, Poly A. The infected cells from each cell line had higher Oct-4 and Nanog expression than did noninfected cells from the same lines. For cell lines A and B, the ratios of Oct-4 increased from 0.87 to 1.27 and 2.38 to 8.77, respectively, for each line. The ratios for Nanog were also increased for A and B from 0.78 to 1.17 and 1.71 to 14.26, respectively. Furthermore, bovine ADS cells exposed to VPA and Zeb were also analysed for expression of endogenous Oct-4 and Nanog by semiquantitative RT-PCR. Bovine ADS cells from lines A and B described above were plated and allowed to culture overnight. After 24 h, the treatment group was changed to a culture medium containing 100 μM Zeb. Valproic acid (5 mM) was added to the treatment group 24 h later. The medium was changed for treatment and controls every 3 days (with the treatment group receiving Zeb and VPA). On day 14, RNA was isolated from cells of the control and treatment groups, and expression of endogenous Oct-4 and Nanog was analysed as described above. In cell line B, the expression of Oct-4 and Nanog was four times that of the control (with ratios of Oct-4 increasing from 0.20 to 0.82). However, in cell line A, the levels were slightly lower than those of the control (Oct-4 ratios decreased from 0.80 to 0.56). We hypothesise that increasing levels of Oct-4 and Nanog with epigenetic modifiers such as VPA or Zeb in conjunction with lentiviral expression vectors will result in higher induced pluripotency rates. This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.