RNA Virus-Based Episomal Vector with a Fail-Safe Switch Facilitating Efficient Genetic Modification and Differentiation of iPSCs

Figures

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  Figure 1. Susceptibility of iPSCs to REVec and Its Effect on Cell Viability (A) Human iPSCs (110, 112, 116, BMI, Epi, 201B7, and 409B2) were transduced with replication-defective REVec at a MOI of 1.0. At 1 week post-transduction, expression of GFP and pluripotency marker SSEA4 was analyzed (top panels). The percentage of GFP-positive cells was determined and reported as compared to mock (bottom panels). Scale bars, 100 μm. (B) Cell viability was determined by WST-1 assay at 1 week post-transduction and presented as percentage compared to mock. Data are shown as averages of two independent experiments with error bars represending SEM.

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  Figure 2. Efficiency of iPSC Transduction by Various Viral Vectors (A) BMI and BM9 iPSCs were transduced with AAV2, Ad5, REVec, lentiviral, and SeV vectors encoding GFP. At 1 week post-transduction, the percentage of GFP-positive cells was assessed by FACS analysis. (B) Percentage of GFP-positive cell populations at 2 weeks post-transduction with REVec, lentiviral, and SeV vectors.

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  Figure 3. Differentiation Potentials of iPSCs Persistently Transduced with REVec and Lentiviral Vector (A) Embryoid bodies (EBs) were generated from BMI and Epi iPSCs transduced with REVec and lentiviral vector by culturing in suspension in DMEM containing 20% FCS. Scale bars, 100 μm. (B) EBs were dissociated into smaller clumps, plated on Matrigel-coated chamber slides, and analyzed by immunostaining with BRACHYURY, NESTIN, PAX6, and SOX17 antibodies. Cells were counterstained with DAPI. Scale bars, 100 μm. (C) Transcript levels of OCT4, BMP2, SOX1, and SOX17 in iPSCs and EBs were determined by real-time RT-PCR. The relative expression levels were normalized to β-actin and reported as compared to mock iPSCs. Data are shown as averages of three independent experiments with error bars representing SEM.

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  Figure 4. Myogenic Differentiation of iPSCs Using REVec (A) Genome organization of REVec encoding MyoD1 and GFP. (B) Expression of MyoD1 and GFP in vector-producing cells at 1 and 2 months post-transfection. Scale bars, 100 μm. (C) Schematic of myogenic differentiation of iPSCs using REVec. iPSCs were transduced with MyoD1 vector at a MOI of 1.0. Infected cells were cultured for 1 week in iPSC medium, followed by incubation in DMEM containing 2% FCS to further induce formation of myotubes. (D) 201B7 and 409B2 iPSCs were transduced with REVec-GFP or REVec-MyoD1 GFP. At 1 week post-transduction, GFP-positive cells were examined for differentiated morphology. Scale bars, 100 μm. (E) Differentiated cells after transfer to a plate without vitronectin coat. Scale bars, 100 μm. (F) Myogenic lineage of differentiated cells was assessed by immunostaining with MyoD1 and MHC antibodies. Scale bars, 50 μm. (G) Efficiency of myogenic differentiation by REVec. Ratio of MyoD1+DAPI+/DAPI+ cells and MHC+DAPI+/DAPI+ cells were determined and presented as percentage of MyoD1- and MHC-positive cells. Data are shown as averages of three independent experiments with error bars representing SEM. (H) One week…

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  Figure 5. T-705 Eliminates REVec from iPSC-Derived Myocytes (A) Time course of T-705 treatment for removal of REVec from myocytes. (B) Real-time RT-PCR analysis for REVec mRNA and gRNA levels in myocytes after treatment with indicated concentrations of T-705 for 14 days. (C) REVec mRNA and gRNA levels at 14 days post-removal of T-705 from the culture medium. Data are shown as averages of three independent expreiments with error bars representing SEM. (D) Expression of GFP and MHC was assessed by immunostaining after removal of T-705 from culture medium. Cells were counterstained with DAPI. Scale bars, 50 μm.