281 USE OF SMALL MOLECULES ENHANCES REPROGRAMMING SUCCESS IN BOVINE DERMAL FIBROBLASTS

Abstract

Reprogramming of differentiated cells to induced pluripotency holds tremendous potential for livestock reproduction. Here we report use of small molecules to support the generation of stem cell-like colonies from bovine dermal fibroblasts that show characteristics of pluripotent cells. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc through a lentiviral drug-inducible polycistronic vector. Several small molecules were tested to support reprogramming. A combination of 3 small molecules (sodium butyrate, PD0325901, and SB431542; NaB-PD-SB) in iPS medium [Minimum Essential Medium Alpha, 20% fetal bovine serum, 1× insulin-transferrin-selenium (ITS), 2 mM Glutamax, 100 µM NEAA, 50 U mol^–1 penicillin, 50 mg mL^–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL^–1 human leukemia inhibitory factor, and 10 ng mL^–1 basic fibroblast growth factor] was found to accelerate the kinetics of the reprogramming process. Colonies appeared at 12 days of culture compared with 21 days in iPS medium alone (P < 0.01, n = 5). Colonies in NaB-PD-SB iPS medium had consistent morphology, with small cells in compact dome formations with well-defined colony borders. Cells were not passaged but were either harvested for mRNA extraction or differentiated into embryoid bodies. Gene expression of the reprogramming factors in stem cell-like colonies was confirmed by qRT-PCR, and further gene expression analysis revealed activation of Nanog and ALPL mRNA expression (P < 0.01, n = 2). Embryoid bodies were analysed for gene expression by quantitative RT-PCR. Silencing of the transgene was not observed in embryoid bodies despite withdrawal of the inducer doxycycline. Expression of the endoderm marker FOXA2, ectoderm markers NES and TUBB3, and mesoderm marker DES was strongly increased in embryoid bodies compared with non-reprogrammed bovine fibroblasts (P < 0.01, n = 2), confirming expression of marker genes for the 3 germ layers.