388 OPTIMIZATION OF INDUCED PLURIPOTENT STEM CELL GENERATION FROM BOVINE FIBROBLASTS

Abstract

Difficulties associated with isolation and culturing bovine embryonic stem (ES) cells has led to the exploration of alternative methods for generating pluripotent stem cells. The viral delivery of reprogramming factors Oct4, Sox2, cMyc, and Klf4 has resulted in generation of induced pluripotent stem cells (iPS) in rodent, human, rhesus monkey, and pig somatic cells. In the current study, we improved the efficiency of retroviral transduction of bovine adult fibroblasts (BAF) for the generation of bovine iPS cells. Bovine adult fibroblasts were transduced with 4 human factors: Oct 4, Sox 2, cMyc, and Klf4. To determine transfection efficiency, pMXs-GFP was used as a reporter. The effect of change in cell density of the Platinum A retroviral packaging cell line (Plat A), cell densities of the target BAF, and infection regimes on the transfection rates was examined. A reduction in Plat A cell density from 8 × 10⁶ to 2 × 10⁶ did not alter transfection rates. Reduced target cell density from 4 × 10⁵ to 4 × 10⁴ (10-fold) improved the transfection rates from 0.31 to 7.06%, P < 0.001 (n = 3). Subjecting the BAF to 2 sequential rounds of viral transduction further improved the transfection rates to 13.88%, P < 0.001 (n = 3). These preliminary results suggest that optimizing the density of target cells can greatly improve transduction outcomes. Following viral induction with the 4 reprogramming factors, putative bovine iPS colonies were observed when the transfection rate was >1%. The putative bovine iPS cells were cultured in alpha-minimal essential medium supplemented with 20% FCS and 10 ng mL^-1 human leukemia inhibitory factor. These putative bovine iPS colonies had mouse ES-like morphology, were multilayered, and had high nucleus-to-cytoplasmic ratio. They stained positive for alkaline phosphatase activity. The colonies were manually passaged onto mitomycin C-inactivated mouse embryonic fibroblasts every 5 to 7 days but could only be expanded for a limited number of passages. Other strategies are currently being explored to improve stable reprogramming of BAF such as epigenetic modification of cells, lentivirus-mediated transduction, and investigation of media suitable to maintain putative bovine iPS colonies for further characterization including RT-PCR or immunohistochemical detection for pluripotent markers and in vivo differentiation ability.

Acknowledgments are given to Dairy Australia.