H9 wa09

Human iPS cell line, HPS0077, was received from Riken Cell Bank (Tsukuba, Japan). Human ES cell line WA09 (H9) was purchased from the WiCell Research Institute (Madison, WI). Human iPS and ES cells were cultivated on Matrigel-coated dishes in the chemically defined medium, Essential 8 (A1517001, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) at 37.0 °C and 5.0% CO₂ with daily medium exchange using the conventional culture method for human ES and iPS cells38 (link).

Human embryonic stem cell (WA09: H9, WiCell Research Institute, Madison, WI, USA) and human induced pluripotent stem cell lines [21 (link),22 (link)] were maintained in iPSC-brew (Miltenyi biotechnology, #130-104-368) with 0.1% gentamycin (Gibco, Waltham, MA, USA, #15750-060) on a matrigel (Corning, Corning, NY, USA, #354277)-coated cell culture dish at 37 °C and humidified to 5% in a CO₂ incubator. Quercetin (#Q4951), luteolin (#L9283), kaempferol (#11852), chrysin (#C80105), apigenin (#10798), and the glycoside analogues were purchased from Sigma–Aldrich and kindly provided by Prof. Sang–Hyun Sung (College of Pharmacy, Seoul National University). The human perivascular progenitor cells (PVPCs) derived from hESCs [23 (link)] (kindly gifted from Dr. Moon Sung–Hwan, T&R Biofab Co., Siheung, Korea) and human smooth muscle cell derived from human iPSCs [21 (link)] (kindly gifted from Dr. Tae–Hee Lee, Sejeong University) were maintained in EBM2 medium (Lonza, #CC-3156) with 0.1% gentamycin at 37 °C humidified to 5% in a CO₂ incubator. Concerning cardiomyocyte differentiation and culture, RPMI-1640 (#R8758) and advanced-MEM (Gibco, #12492013) were used.

hESCs were used according to the guidelines provided by the ethical committee of Kyoto University (Approved# ES3-9). WA09 (H9) (RRID:CVCL_9773, hPSCreg Name WAe009-A) hESCs used in this study were purchased from WiCell Research Institute (Madison, WI, USA). Before culturing, hESC-certified Matrigel (Corning, Corning, NY, USA) was diluted with Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Sigma-Aldrich, St. Louis, MO, USA) at a 1:75 (v/v) ratio and coated onto a culture dish. The Matrigel was incubated in the culture dish for 24 h at 4 °C. Then, excess Matrigel was removed, and the coated dish was washed with fresh DMEM/F12 medium. We used mTeSR-1-defined medium (Stem Cell Technologies, Vancouver, Canada) for daily culturing of hPSCs. For passaging, cells were dissociated with TryPLE Express (Thermo Fisher Scientific, Tokyo, Japan) for 3 min at 37 °C and then harvested. A cell strainer was used to remove undesired cell aggregates from the cell suspension, and cells were then centrifuged at 200×g for 3 min and resuspended in mTeSR-1 medium. Live/dead cells were counted using a NucleoCounter NC-200 (Chemetec, Baton Rouge, LA, USA). We used mTeSR-1 medium containing 10 µM of the ROCK inhibitor Y-27632 (Wako, Osaka, Japan) to prevent apoptosis of dissociated hPSCs on day 1. On subsequent days, we used mTeSR-1 medium without the ROCK inhibitor with daily medium changes.