Human embryonic stem cell culture
The SNUhES31 hESC line was obtained from the Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University Hospital, South Korea. hESCs were cultured on mouse embryonic fibroblasts treated with 10 μg/mL mitomycin-C (Roche, Mannheim, Germany) and were maintained in hESC medium comprising 20% knockout serum replacement (Life Technologies, Carlsbad, CA, USA), 1% Minimum Essential Medium-Nonessential Amino Acids (MEM-NEAA; Life Technologies), 1% Glutamax (Life Technologies), and 7 μL/L β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) in -12 (Life Technologies) containing 20 ng/mL of bFGF (R&D Systems, Minneapolis, MN, USA). For feeder-free hESC culture, cells were detached from the feeder cells using 1 mg/mL dispase (Life Technologies) and cultured in Essential-8 Medium (Life Technologies) on Geltrex (Life Technologies)-coated culture plates. Cells were subcultured as small clusters every 4 days using 0.5 mM EDTA solution.
Genetic regulation of YAP expression by lentiviral vectors
YAP shRNA expression vectors were used to specifically downregulate YAP. YAP target sequences (5¢–TGACTCAGGATGGAGAAATTT–3¢ for shYAP and 5¢–GACTCAGGATGGAGAAATTTTA–3¢ for shYAP #2) were acquired from the GPP web portal (http://portals.broadinstitute.org/gpp/public/) and cloned into the pLKO.1-TRC cloning vector. The pLKO.1-TRC shRNA vector was used as a control. Both vectors were gifts from Dr. David Root, University of Coloado in Boulder (Addgene plasmids #10878 and #10879) . For overexpression of YAP, we used YAP5SA that was mutated five serine amino acid to alanine to prevent protein degradation by phosphorylation. To construct a lentiviral vector overexpressing YAP5SA, we subcloned the YAP5SA gene into the lentiviral vector with regulation under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter. The YAP5SA vector was a gift from Dr. Kunliang Guan, University of California, San Diego (Addgene plasmid #33093) . The lentiviral vectors were kindly provided by Dr. Yibing Qyang (Yale Cardiovascular Research Center, Yale School of Medicine). For the production of lentiviral particles, subcloned lentiviral plasmids were co-transfected with the lentivirus-packaging plasmids (VSV-G-expressing envelope plasmid and another plasmid containing the gag, pol, and rev genes (kindly provided Dr. Yibing Qyang) into HEK293T cells using the X-tremeGene HP DNA transfection reagent (Roche Applied Science, Penzburg, Germany) at 37°C in 5% CO2 for 24 h. The virus-containing medium was collected daily for 3 days after transfection and concentrated by ultracentrifugation at 55,200 × g at 4°C for 2 h (Hitachi, Ltd., Tokyo, Japan). To transfect hESCs with lentiviral particles, 2.5 × 105 hESCs dissociated into single cells were plated in a 24-well plate with concentrated virus-containing medium at a low titer (4 × 106 IU/mL) or a high titer (2 × 107 IU/mL) for 24 h at 37°C, followed by 2 days of culture. Cells were selected by treatment with 2 μg/mL of puromycin (Life Technologies).
Western blot analysis
Cells were lysed in lysis buffer (iNtRON Biotechnology, Seongnam, Korea) by sonication (Vibra-Cell, Sonics, Newtown, CT, USA) on ice. Cell lysates were separated by 10%–12% SDS-PAGE and transferred PVDF membranes (Millipore, Burlington, MA, USA). Blots were washed with TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20; Affymetrix, Santa Clara, CA, USA), blocked with 5% skim milk (Millipore) for 1 h, and incubated with the primary antibodies. The following primary antibodies were used: rabbit anti-YAP (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-p-YAP (cell signaling technology, Danvers, MA, USA), rabbit anti-Oct4 (Santa Cruz Biotechnology), rabbit anti-Sox2 (cell signaling technology), rabbit anti-Nanog (cell signaling technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Primary antibodies were detected using goat anti-rabbit (Santa Cruz Biotechnology) or goat anti-mouse (Santa Cruz Biotechnology) IgG conjugated with horseradish peroxidase (HRP). The bands were visualized using an enhanced chemiluminescence solution (Thermo Scientific, Waltham, MA, USA). Images were acquired using an ImageQuant LAS 4000 Mini system (GE Healthcare, Chicago, IL, USA).
Cell counting kit-8 (CCK-8) assay
hESCs were plated at a density of 1 × 104 cells/well in 96-well plates (BD Biosciences, Franklin Lakes, NJ, USA) and cultured under each condition. CCK-8 solution was added to each well at a 1:10 dilution, followed by further incubation at 37°C for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
Real‐time quantitative polymerase chain reaction (RT‐qPCR)
Total RNA was extracted using the RNeasy Plus RNA Extraction Kit (Qiagen, Germantown, MD, USA). Reverse transcription was performed using the iScript™ cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA). Reverse transcription products (2.5 ng cDNA) were amplified using the FastStart Essential DNA Green Master PCR Kit (Roche Applied Science, Penzburg, Germany) and primers. The primers used here were as follows: 5¢–GTTGGAGAAGGTGGAACCAA–3¢ (forward) and 5¢–CTCCTTCTGCAGGGCTTTC–3¢ (reverse) for Oct‐4; 5¢–CAGAAGGCCTCAGCACCTAC–3¢ (forward) and 5¢–ATTGTTCCAGGTCTGGTTGC–3¢ (reverse) for Nanog; 5¢–CCTAAGGAACCACCGGTCA–3¢ (forward) and 5¢–AGCATGGACAGACAAGCAGA–3¢ (reverse) for Brachyury T; 5¢–GATGGAGCCAAGCCCAC–3¢ (forward) and 5¢–CACAGAGACGGCGTCAGT–3¢ (reverse) for MESP1; 5¢–GTTGTCCGCCTCTGTCTTCT–3¢ (forward) and 5¢–TCTATCCACGTGCCTACAGC–3¢ (reverse) for Nkx2.5; and 5¢–TTCACCAAAGATCTGCTCCTCGCT–3¢ (forward) and 5¢–TTATTACTGGTGTGGAGTGGGTGT–3¢ (reverse) for TNNT2. Samples were cycled 45 times using a LightCycler® 96 Real‐Time System (Roche Applied Science). RT‐qPCR was performed using the following parameters: 5 min at 95°C, 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. All RT‐qPCR experiments were performed in triplicates. The cycle threshold was calculated using default settings with real‐time sequence‐detection software (Roche Applied Science).
Neural differentiation was induced by transferring approximately 9 × 103 cells in 20 μL of hESC medium without bFGF and supplemented with 50 μM Y-27632, 5 μM dorsomorphin (DM) (Sigma-Aldrich), and 5 μM SB431542 (Sigma-Aldrich) onto the lid of a 100 mm petri dish followed by culture for 2 days as a hanging drop, to form embryoid bodies (EBs). The day of hanging-drop preparation was defined as EB day 0. On EB day 2, the EBs were transferred to a 100-mm petri dish and cultured for an additional 4 days in suspension in medium with same composition. On EB day 6, the EBs were attached to a 60-mm tissue-culture dish coated with Matrigel (BD Biosciencesplate, San Jose, CA, USA) and cultured in neural induction medium [1× N2 supplement (Life Technologies) and 1× nonessential amino acids (Life Technologies) in DMEM/F12 medium] for 4 days. The cells were analyzed on EB day 10.
Cardiac differentiation was induced as previously described . Feeder-free hESCs were dissociated into single cells by incubation with Accutase® for 5–8 min, then plated onto Matrigel-coated plates at a density of 1.5 × 105 cells/cm2 in mTeSR™1 supplemented with 5 μM Y27632, which is a Rho-associated protein kinase inhibitor. We set this day as day −4. The following day, we changed the medium to mTeSR™1 without Y27632, and refreshed the medium every day for an additional 2 days. At day −1, we changed the medium to mTeSR™1 containing Matrigel (1:60 dilution). To induce cardiac differentiation, we replaced the mTeSR™1 medium with RPMI/B-27 minus insulin medium (RPMI1640 and B-27 minus insulin supplement) supplemented with 10 μM CHIR99021, which is a GSK-3 inhibitor (Day 0), followed by culture for 24 h. The culture medium was then replaced with RPMI/B-27 minus insulin medium without supplement (day 1) and cultured for 2 days. At day 3, we replaced the medium with RPMI/B-27 minus insulin medium supplemented with 7 μM XAV939, which is a tankyrase inhibitor, and 5 μM IWP2, which is a porcupine inhibitor, followed by culture for 48 h. The culture medium was replaced with RPMI/B-27 minus insulin medium and cultured starting at day 5. At day 7, the medium was replaced with RPMI/ B-27 (RPMI1640 and 50X B-27 supplement), and was changed every other day thereafter. Analyses were performed at day 10.
Cells were dissociated with Accutase® for 10 min and fixed with 3.2% paraformaldehyde in PBS. Subsequently, cells were blocked and permeabilized with 10% normal goat serum in PBST for 1 h at room temperature. Cells were then incubated with the primary antibody overnight at 4°C, followed by the Alexa 488-conjugated secondary antibody for 3 h at room temperature. Samples were analyzed using a FACS Aria III Flow cytometer (BD Biosciences, NJ, USA).
The results were reported as means ± S.E.M. Differences between mean values were analyzed using Student's t-test. Significance was set at P < 0.05.