111 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 Fisher Scientific, Waltham, MA, USA, 11020021), 1 × B27 and 1% KOSR (Thermo Fisher Scientific, Waltham, MA, USA, 10828028). The medium was refreshed every 3–4 days. Immunofluorescence. hiPSC-CMs after two days of expansion were seeded into a µ-Plate 96 well (Ibidi) with a density of ~5 × 104 cells/well for continuously culturing with RPMI/B27 and RPMI/B27 supplemented with CHIR99021 (3 µM) for six days. Media was refreshed every other day. At day 6, the cells were washed and fixed with 4% formaldehyde for 10 min at room temperature, permeabilized with 0.25% PBS-Triton for 5 min and then blocked with 10% donkey serum for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies for cTnT (TNNT2 [1C11], Ab8295, dilution 1:250, Abcam, Cambridge, UK) and Ki67 (D3B5, dilution 1:400, Cell Signaling Technology, Danvers, MA, USA). Next, the cells were incubated with the corresponding secondary Alexa fluor-555 and −647 antibodies (1:400 dilution, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Nuclear DNA was labeled with DAPI. Images were acquired by an Eclipse Ti2 inverted microscope system (Nikon, Tokio, Japan). Z-stacks function was performed with 0.2 um thickness containing 11 frames under the 40×/0.95 NA objective in wide-field mode. All the images were processed afterwards for 3D deconvolution by NIS elements analysis software. Analysis and quantifications were performed with a maximum intensity projection by Image J. Time-Lapse Imaging. Time-lapse sequence was implemented in order to keep track of hiPSCCMs single cell cycle phases. hiPSC-CMs were replated on a Matrigel-coated 8-well chamber (Ibidi, Grafelfing, Germany) with a density of 1 × 105 cells/well. The cells were treated with 3 µM CHIR99021 for 2 days, then subjected to time-lapse imaging on an Eclipse Ti2 inverted microscope system (Nikon, Tokio, Japan) for imaging under the objective of 10×/0.45 NA. Cells were kept at an Okolab enclosure with temperature 37 °C, 5% CO2 and 95% humidity. Each frame was acquired every 5 min until 12–24 h. Nuclear morphology varied in mitotic phases were captured from mononucleated or binucleated cells. To keep track of mCherry expression in hiPSC-CMs, The time-lapse sequence was developed 12 h post-transfection of hiPSC-CMs with the mCherry plasmid on an Eclipse Ti2 inverted microscope system (Nikon, Tokio, Japan) using bright-field mode/or confocal mode with a 10×/0.45 NA objective. Images were taken every 5 min for 12 h. RNA Sequencing for CM Proliferation Genes. hiPSC-CMs collected at passage 1–3 and during maturation (day 77, 84 and 97) were obtained from a healthy donor hiPSC-CMs. RNA was isolated using ISOLATE II RNA Mini Kit (Meridian Biosciences, Cincinnati, OH, USA) according to the manufacturers’ instructions with minor adjustments. After the isolation of mRNA, libraries were prepared using the NEXTflexTM Rapid RNA-seq Kit (Perkin Elmer, Waltham, MA, USA). Libraries were sequenced on the Nextseq500 platform (Illumina, San Diego, CA, USA), producing single-end reads of 75 bp. Reads were aligned to the human
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