194 EFFECT OF SMALL MOLECULE INHIBITORS ON Oct-4, Nanog, AND Sox-2 EXPRESSION IN BOVINE FETAL FIBROBLAST CELLS

Abstract

Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. However, the majority of cells that undergo reprogramming become trapped in a partially reprogrammed state that is characterised by the down-regulation of somatic cell marker genes, incomplete reactivation of pluripotency genes, maintenance of viral expression, and the inability to form chimeras. Several small molecules, which act on specific signaling pathways or chromatin modifications, have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the roadblocks encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and reactivate the embryonic stem cell genome. Chemical treatment of cells before reprogramming can remodel the epigenetic landscape to be more like that of embryonic stem cell by removing the repressive epigenetic marks and relaxing chromatin structure to allow the reprogramming factors easier access to target genes. In the present study, we assessed the effect of pretreatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen-activated protein kinase inhibitor; CHIR99021, a glycogen synthase kinase-3 inhibitor; and NuP0178 (NuPotential, Baton Rouge, LA, USA), a G9a histone methyltransferase inhibitor. Cells were seeded at a density of 0.7 × 10⁶ and expanded before being divided evenly into 2 groups. Control cells were cultured in complete culture medium (DMEM with 10% fetal bovine serum), whereas treatment cells were cultured in complete culture medium containing 0.5 mM PD0325901, 3 mM CHIR99021, and 1.8 mM NuP0178. Messenger RNA was isolated from cell cultures using Dynabeads® mRNA DIRECT™ Kit (Dynal Inc., Lake Success, NY, USA) on Day 7 and 14, and the resulting RNA products were transcribed into cDNA using Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative RT–PCR was performed to measure transcript levels of Oct-4, Nanog, Sox-2, and poly adenylate polymerase in treated and untreated cells at Days 7 and 14. Transcript levels were quantified by relative quantification using the ΔΔCt method and expressed as ratios of target genes (Oct-4, Nanog, Sox-2) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine fetal fibroblast cells. No difference in expression levels between untreated and treated cells was detected at either Day 7 or 14. Currently, we are utilising chromatin immunoprecipitation to examine chromatin and DNA methylation patterns around the promoters of Oct-4, Nanog, and Sox-2 to further elucidate the effects of treatment with this combination of small molecule inhibitors.