title: Dual gene expression in embryoid bodies derived from human induced pluripotent stem cells using episomal vectors.
abstract: Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 μg/mL) or hygromycin B (300 μg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6×10(3),×10(4), and×10(5) cells/mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6×10(5) cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.
authors: 1 Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital , Yotsukaido City, Japan .
authors: Minoru
authors: M
authors: Tomizawa
authors: Fuminobu
authors: F
authors: Shinozaki
authors: Yasufumi
authors: Y
authors: Motoyoshi
authors: Takao
authors: T
authors: Sugiyama
authors: Shigenori
authors: S
authors: Yamamoto
authors: Makoto
authors: M
authors: Sueishi
mesh_terms: D002460:Cell Line; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D010957:Plasmids / Q000235:genetics*