187 INCREASED EFFICIENCY OF DERIVING BOVINE STEM CELL-LIKE COLONIES USING VALPROIC ACID AND SMALL-MOLECULE COCKTAILS

Abstract

Nonviral reprogramming of bovine embryonic and adult fibroblasts was undertaken using electroporation of a polycistronic plasmid carrying human reprogramming factors Oct4, Sox2, Lin28, and Nanog (Addgene Plasmid #20922). Because of difficulties encountered in reliably and reproducibly reprogramming bovine cells to a pluripotent state, several small-molecule combinations have been tested to support reprogramming. Previously, a combination of three molecules, (sodium butyrate, PD0325901 and SB431542; NaB-PD-SB) in induced pluripotent stem (iPS) medium [Minimum Essential Medium Alpha, 20% FBS, 1x insulin-transferrin-selenium (ITS), 2 mM Glutamax (Gibco, Grand Island, NY, USA), 100 μM nonessential amino acids (NEAA), 50 U mol^–1 penicillin, 50 mg mL^–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL^–1 human leukaemia inhibitory factor, and 10 ng mL^–1 basic fibroblast growth factor] was found to accelerate the reprogramming process of bovine iPS cells, with colonies observed at 12 days post-electroporation, as opposed to 21 days without the addition of the small-molecule cocktail. The addition of 1 mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, in combination with the other small-molecule cocktail (NaB-PD-SB), was found to improve the reprogramming efficiency of bovine adult and embryonic fibroblasts, with colonies observed at Day 10 post-electroporation, and the average number of colonies present per 10-cm dish, rising from an average of 5 colonies observed in the NaB-PD-SB cocktail to 27 with the addition of VPA. Colonies grown in the presence of VPA had a consistent morphology, forming compact domed colonies consisting of small round cells with well-defined borders. It was observed that colonies growing in the presence of VPA tended to have a better defined border and grew larger in size than those grown in the presence of NaB-PD-SB alone. Colonies began to differentiate after 21 days and were no longer alkaline phosphatase positive after this time. Cells were either harvested for mRNA extraction or differentiated into embryoid bodies (EB). Expression of pluripotency genes, Oct4, Sox2, Klf4, Nanog, and Alkaline Phosphatase were significantly increased in the presence of VPA compared to nonreprogrammed somatic bovine fibroblasts, with expression profiles similar to those grown in the NaB-PD-SB cocktail. Embryoid bodies were analysed for gene expression of different germ layer markers, FOXA2 (endoderm), Nestin and TUBB3 (ectoderm), and Desmin (mesoderm) using quantitative RT–PCR. For both EBs derived from the NaB-PD-SB cocktail and those with VPA, at least one marker from each germ layer was present, demonstrating the potential potency of these cells. At least a 10-fold increase in expression of these was observed in comparison to somatic fibroblast cells. It is apparent that the addition of small molecules can assist in the reprogramming of bovine iPS cells, and addition of VPA to the cocktail results in more consistent putative bovine iPS colonies. Further work is needed to identify the causes for early differentiation of colonies in order to obtain fully reprogrammed pluripotent stem cells.