61 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 Single cell gene expression analysis. For single cell gene expression analysis, day 12 hiPSCCMs were treated with DMSO, CHIR99021 or C59 for 24 hours. At the end of treatment hiPSCCM aggregates were dispersed with TrypLE Select Enzyme as described above and dissociated with gentle trituration. Single cells were washed and re-suspended in ice cold RPMI 1640 with B27 supplement and then captured using the 10x Genomics platform. RNA amplification and generation of cDNA expression libraries were performed prior to sequencing using HiSeq4000 (Illumina). The three data files (CTR, CHIR and C59) (GEO accession number: GSE148586) were merged and subsequent analysis was performed using Seurat software. Cells with poor quality and doublets/multiplets were filtered out based on aberrant gene numbers, total transcript counts, percentage of reads to mitochondrial genes, and housekeeping gene counts (GAPDH, SOD1, BLVRB, PSMB3). To remove batch effect, Seurat integration tool was employed (Stuart et al., 2019). Flow cytometry. Freshly isolated hiPSC-CMs were fixed in PFA 4% for 5 min and incubated with primary antibody against TnT for 1 hr. After multiple washings, an Alexa488-conjugated mouse secondary antibody was added for 30 min of incubation followed by washings to remove unbound antibodies. Samples were then analyzed using the FACSCalibur® flow cytometer (BD Biosciences) and data processed using the FlowJo® software (TreeStar). Electrophysiological studies. hiPSC-CM treated with DMSO, CHIR, or CHIR>C59 were passaged 3 times in RPMI 1640 + B27 Supplement for an equivalent number of days and seeded sparsely on Matrigel© coated 8 mm coverslips. Cells on coverslips were immersed in extracellular solution containing 140 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM glucose, at pH 7.4. Patch electrodes were filled with an intracellular solution containing 140 mM potassium gluconate, 10 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na-GTP, at pH 7.3, hence, giving resistances of ~2–5 M’Ω. Spontaneous CM action potentials were recorded at room temperature using a sharp current clamp mode. For calcium transient recording, iPSC-CMs were loaded with 5 μM Fluo-4 AM for 5–10 mins at 37 °C and were washed 3 times afterward. Spontaneous calcium transients were observed at 37 °C with a Carl Zeiss LSM 510 confocal microscopy (Göttingen, Germany) and a 63× objective (Plan-Apochromat 63×/1.40 Oil DIC M27). Calcium signalings were recorded using line-scanning mode, and the Imaging data were analyzed with a custom-made Mat lab algorithm. Transient amplitude was expressed as ΔF/F0. Single cell patterning and contractility. For single cell contractile assays, hiPSC-CMs were plated on polyacrylamide hydrogels of 10 kPa stiffness with micro-patterned Matrigel© to achieve a final hiPSC-CM aspect ratio of 7:1 (Lee et al., 2020). After 3–6 days of culture in the presence of DMSO, CHIR, or CHIR>C59, contractility of hiPSC-CMs were measured by recording contractile motion of at least 10 individual cells per group using high speed microscope
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