Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

Stem Cells Dev. 2013 Jan 1;22(1):124-35. doi: 10.1089/scd.2012.0382. Epub 2012 Nov 2.

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, NANOG, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Differentiation / metabolism
  • Cell Transformation, Neoplastic
  • Cells, Cultured
  • Coculture Techniques
  • DNA Transposable Elements / genetics*
  • Fibroblasts / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Induced Pluripotent Stem Cells / physiology*
  • Induced Pluripotent Stem Cells / transplantation
  • Induced Pluripotent Stem Cells / ultrastructure
  • Kruppel-Like Factor 4
  • Mice
  • Mice, Nude
  • Microscopy, Fluorescence
  • Neurogenesis
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism
  • Promoter Regions, Genetic
  • SOXB1 Transcription Factors / metabolism
  • Sus scrofa
  • Teratoma / pathology
  • Transcriptome
  • Transgenes

Substances

  • Antigens, Differentiation
  • DNA Transposable Elements
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Octamer Transcription Factor-3
  • SOXB1 Transcription Factors
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins