Lentiviral transduction of epigenetically modified bovine adult stem cells

Bovine adipose-derived stem cells (ADS), a form of adult stem cells, are somatic cells that have similar characteristics of embryonic stem (ES) cells. Bovine ADS cells possess multipotent capabilities and have been found to express pluripotency genes associated with ES cells. The unique properties of ADS cells make them a desirable source for reprogramming experiments. The goal of reprogramming experiments is to transform somatic cells from a differentiated state to a pluripotent state. When somatic cells reprogram, there are certain epigenetic changes or modifications that must occur in order to successfully reprogram the nucleus. Epigenetic modifications will change the chromatin configuration without changing the DNA sequence. Somatic cells can be exposed to small molecules that may be able to reduce the chances of having incomplete chromatin modification. Two epigenetic modifying factors are a DNA methyltranferase inhibitor, zebularine (Zeb), and a histone deacetylase inhibitor, valproic acid (VPA). By inducing gene expression with the epigenetic modifiers, the cells may be stimulated to reprogram more efficiently than cells with lower gene expression. In the first experiment, three bovine ADS cell lines were treated with VPA or Zeb to observe the changes in expression levels of Oct4, Sox2, and Nanog (pluripotency-associated genes). The cells were treated for a period of 5, 7,10, or 14 days. VPA led to the highest increase of the pluripotency genes; however, both treatments may have produced a partial reprogramming. This partial reprogramming may result in the bovine ADS cells reaching complete pluripotency when combined with a reprogramming technique. In the second experiment, three bovine ADS cell lines were treated with VPA or Zeb for five days then followed with transduction using lentivirus. Oct4, Sox2, and Nanog were increased the highest when using epigenetic modifiers. Statistical differences for expression of the pluripotency-associated genes were found for cells treated with zebularine. While it was thought that viral transduction in combination with epigenetic modifiers would produce higher expression levels of the pluripotency-associated genes, this was not found to be true in this experiment