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 mg/mL) or hygromycin B (300 mg/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.

Transcription Factors and Medium Suitable for Initiating the Differentiation of Human‐Induced Pluripotent Stem Cells to the Hepatocyte Lineage

“…Fewer cells survived in L15 than in the other three media, which was consistent with the findings in Figure . The fluorescence intensity of CherryPicker1 was stronger in 210B7 cells cultured in ReproFF or WE [Tomizawa et al, ]. These results suggested that 201B7 cells expressed the transfected gene in ReproFF or WE.…”

“…For fragments amplified by PCR, the sequences were confirmed by Bio Matrix Research, Inc. The fragments in pBluescriptII SK(−) were subcloned into pEBMulti‐Neo (Wako Pure Chemicals, Osaka, Japan), pEBMulti‐Hyg (Wako Pure Chemicals), pEBNK‐Neo [Tomizawa et al, ], or pEBNK‐Hyg [Tomizawa et al, ]. pEBMulti‐Neo, pEBMulti‐Hyg, pEBNK‐Neo, and pEBNK‐Hyg were episomal plasmids.…”

“…Plasmid construction is briefly presented in Table I [Tomizawa et al, 2014], or pEBNK-Hyg [Tomizawa et al, 2014]. pEBMulti-Neo, pEBMulti-Hyg, pEBNK-Neo, and pEBNK-Hyg were episomal plasmids.…”

“…Nicotinamide and proline were added because they are necessary for proliferation of hepatocytes [Nakamura et al, 1984;Mitaka et al, 1991]. For some experiments, the cells were also transfected with pEBNK-Hyg/Cherry [Tomizawa et al, 2014].…”

Differentiation of human induced pluripotent stem cells in William's E initiation medium supplemented with 3-bromopyruvate and 2-deoxy-d-glucose

“…Primer sequences. Inc.) according to the manufacturer's protocol(22). The reporter plasmids expressed Metridia luciferase, which is secreted into the medium; the pMetLuc2-control plasmid expresses Metridia luciferase driven by the cytomegalovirus (CMV) immediate early promoter.…”