60 Chapter 3 Cardiac tissue generation. Engineered Heart Tissues were with small modifications generated as described previously (Hansen et al., 2010). Briefly, induced pluripotent stem cell-derived cardiomyocytes were detached and resuspended in culture medium containing bovine fibrinogen (5 mg/ml), aprotinin (2.5 μg/ml) and 10% Matrigel. EHTs of 1×106 cardiomyocytes each were casted by mixing the reconstitution mix (97 μl/EHT) with thrombin (3 μl/EHT, 100 U/ml) and pipetting it into the casting molds. Fibrin polymerization (37 °C, 7% CO2, 98% RH, 2 h) led to the formation of a muscle strip. The EHTs were transferred to culture medium (Dulbecco’s modified Eagle’s medium) containing 10% horse serum, 1% penicillin/ streptomycin, 10 μg/ml insulin and 33 μg/ml aprotinin and maintained at 21% oxygen, 7% CO2 and 37 °C in a humidified cell culture incubator for 25 days. Contractility measurements were performed in culture medium as described previously (Hansen et al., 2010). Small molecules / growth factors. PNU74654, MK2206, CHIR99021, C59 and Palbociclib were obtained commercially. CHIR99021 and C59 were used depending on the assay at 2.0 or 4.0 μM final concentration and PNU74654, MK2206 and Palbociclib were used at 32, 1.0 and 1.0 μM, respectively. For in vitro studies, recombinant scFv-DKK1c and RSPO were expressed and purified as previously described (Janda et al., 2017) and used in final concentrations of 200 and 25 nM, respectively. Recombinant Wnt3A protein was purchased commercially and added in 100 ng/mL final concentration. Protein expression analysis. Immunocytochemistry was performed with overnight incubation of primary antibodies (listed below) followed by 2-hour incubation with Alexaconjugated fluorescent secondary antibodies. Immunostaining images were captured using confocal (Zeiss LSM 710) or epifluorescence (Leica DM IL) microscopy. Kinase phosphorylation screening assays were performed using the Proteome Profiler Human Phospho-Kinase Array Kit containing 43 human kinases. Western blotting was performed to validate the kinase screen results. Total protein expression was measured with a gel imager and quantified based on pixel intensity (BioRad). Only lanes from the same gel were used for displaying and quantification. Luciferase assays Day 12 hiPSC-CMs were transfected for 48 hours with a TCF reporter plasmid or the mutant reporter plasmid using Lipofectamine. 72 h after transfection cells were treated with various small molecules for an additional 24 h and then lysed and mixed with luciferase substrate. The expression of firefly luciferase was finally measured using a 96-well micro plate reader. Real-time PCR expression analysis For quantitative analysis of gene expression, RNA was extracted with a lysis buffer from hiPSC-CMs and stored at −80 °C. Total RNA from each sample was purified from cell lysate using a RNA purification kit. cDNA was made using a cDNA synthesis kit. Quantitative PCR was performed using a 96-well thermocycler system (Biorad) with SYBR Green substrate for 40 cycles. All primers sequences were obtained from the PrimerBank (Massachusetts General Hospital / Harvard Medical School) online database. Oligos were synthesized at Stanford University.
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