Derivation and Characterization ofSleeping BeautyTransposon-Mediated Porcine Induced Pluripotent Stem Cells

Abstract

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over > 40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NA-NOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.

Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells

“…Passage 2 MEFs were either transduced or transfected using newly developed reprogramming lentiviral vectors (LV) [12,13] or Sleeping Beauty transposon (SB) [14,15] in conjunction with hyperactive transposase mutant SB100X [14] and then cultured for iPSC derivation. Further details can be found in Supporting Information Material and Methods.…”

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…The SB reprogramming transposon carried the murine cDNAs of Oct4, Sox2, Klf4, and cMyc, each separated by sequences coding for self-cleaving 2A peptide. The SB transposase (SB100X) helper plasmid has been described before Ivics et al, 2014;Kues et al, 2013;Talluri et al, 2014).…”

“…Total RNA was prepared from iPSC lines and tissues with TriReagent (Ambion), according to the manufacturer's instructions (Kues et al, 2013). Isolated total RNA was treated with RNase-free DNase (1 U/lg RNA) (Epicentre Biotechnologies) and 0.5 lg of RNA was used for cDNA synthesis.…”

“…A number of hyperactive transposase variants are available that approach the efficiency of retroviral or lentiviral gene transfer; the production costs are low and transposons are safe because they are noninfective and the removal of integrated transposons is possible. Transposons, specifically Sleeping Beauty (SB) and piggyBac (PB), have emerged as useful alternatives to virus-mediated reprogramming of somatic cells from different species, including human (Davis et al, 2013;Woltjen et al, 2009), murine Muenthaisong et al, 2012;Talluri et al, 2014;Tsukiyama et al, 2014), porcine (Kues et al, 2013), and equine somatic cells (Nagy et al, 2011). Until now, there are no reports on transposon-mediated reprogramming of bovine somatic cells to pluripotent iPSCs.…”

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…Gene targeting by homologous recombination is extremely inefficient in porcine somatic cells 3 , and porcine embryonic stem cells (ES) have not yet been established 15 . Recently, several groups described the derivation of porcine induced pluripotent cells (iPS) [16][17][18][19][20] , however, only low chimerism has been found after blastocyst complementation experiments 21,22 . The employment of porcine iPS cells in the SCNT method resulted in extremely low rates of born piglets 23 .…”