Figure 2

Generation of induced pluripotent stem cell (iPSC)-like clones by MV(OCT4) in combination with three lentiviral vectors (LVs). (A) Level of BJ cells transduction with MV(OCT4). BJ cells were infected with MV(OCT4) and three LVs encoding SOX2, KLF4, and cMYC (left panel). Forty-eight hours after infection, pictures were taken under phase contrast (top panels) or fluorescence (middle panels). Cells were then collected, and the number of cells expressing GFP was quantified by flow cytometry (bottom panels). (B) Reprogramming of BJ cells. BJ cells were infected with MV(OCT4) and three LVs encoding SOX2, KLF4, and cMYC (left panels); four LVs encoding OCT4, SOX2, KLF4, and cMYC (middle panels); or three LVs encoding SOX2, KLF4, and cMYC (right panels). Cells were observed under light microscopy, and pictures of iPSC-like clones were taken at different time points, as indicated. Third row panels show iPSC clones in culture. (C) Loss of viral gene expression after passaging. Nucleoprotein (N) mRNA expression levels were analyzed in iPSC clones by reverse transcription-polymerase chain reaction at passages 6, 8, 10, 12, 15, and 18. Control (Cont): (+) BJ cells infected with MV(OCT4), (−) BJ mock infected, (w) water, (L) ladder. (D) Immunofluorescence analysis of N protein expression. iPSC clones were analyzed by immunostaining and confocal microscopy. The cells were fixed, permeabilized, and stained with antibody to N (fluorescein isothiocyanate, green). Nuclei were counterstained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars: 50 μm. MV, measles virus.