307 REPROGRAMMING OF PIG SOMATIC CELLS TO PLURIPOTENCY WITH SLEEPING BEAUTY TRANSPOSON VECTORS CONTAINING THE PORCINE TRANSCRIPTION FACTOR SEQUENCES

Abstract

Induced pluripotent stem cells (iPSC), developed by Yamanaka and co-workers (Takahashi et al., 2006), hold significant potential for the development of regenerative therapies due to the possibilities of deriving patient-specific pluripotent cells. In this aspect, the pig is an important animal model for testing iPSC-based applications for the human medicine. However, even though significant progress has been made, the derivation of porcine iPSC lines fully equivalent to those from mouse and human has been elusive. To date, most of the reported putative pig iPSC lines have been derived with the use of lentiviral or retroviral vectors harboring the mouse or human transcription factor sequences. Here, we report the construction of Sleeping Beauty (SB) transposon vectors with porcine cDNA sequences coding for OCT4, SOX2, NANOG, C-MYC, and KLF4, in addition to the human LIN28. By using standard cloning techniques, we produced 2 polycistronic SB-CAG-pOSMK-ires-Tomato and SB-Ef1a-pNANOG-ires-hLIN28 transposon vectors and we transfected them together with the SB100X transposase into pig fetal fibroblasts (pFF) harboring a mouse OCT4-GFP reporter construct (Nowak-Imialek et al., 2010). Both the basic transposon and transposase vectors were generously provided by Dr. Zoltan Ivics from Paul Ehrlich Institute, Langen, Germany. In each experiment, 2 × 10⁶ pFF were electroporated with 3 µg of each transposon together with 0.5 µg of SB100X. Two days after transfection, the cells were transferred to mouse embryonic fibroblast (MEF) feeders and cultured with iPSC medium [DMEM with antibiotics, nonessential amino acids, 20% Knockout serum replacement, 5 ng mL^–1 human recombinant basic fibroblast growth factor (bFGF), and 1000 U mL^–1 ESGRO]. Two weeks post-transfection, multiple compact colonies were apparent (mean = 2195; SEM = 166; n = 3), which were 95% alkaline phosphatase-positive and ~80% expressed the OCT4-GFP reporter. Reverse transcription-PCR showed that these colonies expressed high levels of endogenous OCT4, SOX2, NANOG, REX1, UTF1, CDH1, and TDH. The cultures were passaged by trypsin disaggregation, followed by seeding on fresh feeders at density 10 × 10³ cells cm^–2. The established cell lines proliferated as compact, mouse iPSC-like colonies that retained their OCT4 reporter expression as well as the expression of the endogenous pluripotency genes for at least 30 passages. The expression of the transgenes was persistent and showed that no silencing had occurred, even in long-term culture. When subjected to in vitro differentiation protocols, the putative iPSC formed mainly large trophectodermal (TE) vesicles (positive for TE markers CDX2, PAG, and HAND1), fibroblast-like, and neuronal-like cells. These cells still expressed the transgenes as well as most endogenous pluripotency markers, demonstrating limited differentiation capacity. Because the stable transgene expression and the suboptimal culture conditions are the most likely causes of this limited differentiation potential, we are currently working on generating transgene-free iPSC lines under improved cell culture conditions.