Submitted to: Annual Biomedical Research Conference for Minority Students
Publication Type: Abstract Only
Publication Acceptance Date: 5/16/2014
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: The value of stem cells has become increasingly evident in recent years with the advent of genetic engineering tools that allow site-specific modifications to the genome. The use of stem cells to induce modifications has several potential benefits for the livestock industry including improving animal health, enhancing production traits, animal pharming, improving food safety, and generating research models for biomedical applications. Among the livestock species the domestic pig is a preferred model. The full potential of the pig as a research model has not been met however due to the lack of authentic embryonic stem cells (ESC) of inner cell mass (ICM) origin. Murine ESCs have elevated the mouse into the role as the foremost biomedical model species. In this study, as an alternative to ESC, we sought to generate porcine induced pluripotent stem cells (iPSC) an immortal cell type amenable to in vitro genetic modifications. Candidate pig iPSC lines were generated from fetal fibroblasts using cleavable (floxed) lentiviral vectors coding for tri-cistronic expression of OCT4, SOX2, KLF4, MYC, LIN28, and NANOG as well as a MIR302/367 cluster. Following lentiviral transduction, the fetal fibroblasts were cultured in a serum-free environment with FGF and LIF, or LIF with two small molecule inhibitors (2i), and produced lines with two distinct morphologies: those resembling mouse (naïve) ESC and those resembling human (primed) ESC. Both of these established lines exhibited a phenotype consistent with ESCs, including morphology, alkaline phosphatase activity, a high nuclear to cytoplasmic ratio, a short cell-cycle interval, and expression of endogenous pluripotency markers OCT4, SOX2, and NANOG. The derivation of these iPSC-like cells represents a critical first step toward generating foreign DNA-free, robust stem cells in the pig – an agriculturally and economically important species. Engineered pig iPSC may prove appropriate for regenerative medicine applications.