117 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 and non-proliferative hiPSC-CMs in the presence or absence of CHIR99021. We found that in the presence of CHIR99021, non-proliferative CMs expressed mCherry in 12 ± 0.6%, while in the absence of CHIR99021, matured CMs were mCherry positive in 8.3 ± 0.8% (p = 0.89) (Figure 4B). Additionally, we compared ViaFect with Lipofectamine 3000 transfection reagent and found that CHIR99021 treatment increased the transfection efficiency in both reagents by more than 50% (Figure S3D). To confirm general transfection efficiency and potential non-cell cycle-dependent effects of CHIR99021, we utilized a common source of indefinitely proliferating HEK293 cells and transfected these with and without GSK3β inhibition. In HEK293 cells we observed no significant increase in transfection efficiency when CHIR99021 was added to the media versus the DMSO carrier control (33.8% vs. 35.8%, p = 0.93) (Figure 4C). Next, we confirmed the expression of mCherry in the fraction of CMs, which were treated with CHIR99021 via immunohistochemistry for α-actinin and endogenous mCherry fluorescence (Figure 4D). These data illustrate that CHIR99021 promotes non-viral vector uptake in hiPSCCMs predominantly via a cell cycle-dependent way. Figure 4. Transfection efficiency in immature hiPSC-CMs vs. mature hiPSC-CMs and HEK293 cells. (A) Quantitative flow-cytometry graph indicating high transfection efficiency of up to 34% in immature and up to 20% in mature hiPSC-CM treated with 2 µM of CHIR99021, 100 ng/mL mCherry plasmid and with ViaFect transfection reagent. (B) Bar graph displaying mean mCherry transfection efficiency in immature (CM) and mature (MM) hiPSC-CMs and HEK293 cells (HEK) treated with CHIR99021 or DMSO carrier control in the presence of ViaFect or Lipofectamin transfection reagent. Mean flow-cytometry values of 3 biological replicates are represented and error bars indicate standard deviation. (C) Representative immunofluorescence for Hoechst (blue), mCherry (red) and α-actinin (green) after 72 h of transfection treatment (ViaFect) in hiPSC-CMs and HEK293 cells treated with CHIR99021. (D) Quantitative bar graph representing mean transfection efficiency in a fraction of hiPSC-CMs or HEK293 cells by mCherry expression of high transfection efficiency. Experiments performed 3 biological replicates. Error bars indicated standard deviation. * indicates p < 0.05, ** indicates p < 0.01).
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