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JSRM Code: 015020300004EPA170819    
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Increased mesodermal and mesendodermal populations by BMP4 treatment facilitates human iPSC line differentiation into a cardiac lineage
Kimura M1, Furukawa H1, Shoji M2, Shinozawa T1

Author Names in full: Maya Kimura1, Hatsue Furukawa1, Masanobu Shoji2, Tadahiro Shinozawa1

1Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, IPB University, Bogor, Indonesia; 2Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia; 3Center for Research and Development of Biomedical and Basic Health Technology, National Institute of Health Research and Development, Ministry of Health Republic of Indonesia

Supplementary Information

Fluorescent immunostaining for cardiac markers
Several beating EBs derived from hiPSCs on days 30–60 after the induction of differentiation were picked and dissociated using 10 mg/mL collagenase IV (MP Biomedicals, OH, USA) for 3−4 h at 37°C. The dissociated cells were seeded on collagen I-coated dishes. Two or three days after seeding, the cells were fixed in 4% formaldehyde for 30 min and subsequently permeabilized with 2.5% Tween 20 (WAKO, Kanagawa, Japan) at room temperature. The cells were incubated at 4°C overnight with the following primary antibodies diluted in Dulbecco’s PBS(-) buffer (PBS, Gibco): monoclonal anti-cardiac troponin T (cTnT, 1:100 dilution; Abcam, MA, USA, ab33589), polyclonal anti-GATA4 (1:100 dilution; Abcam, ab63398), and monoclonal anti-cardiac actin (1:50 dilution; Fitzgerald, MA, USA, 10R-C116a). Fluorescent dye-conjugated secondary antibodies, Texas red-conjugated goat anti-rabbit IgG (1:100; Vector, CA, A21202), or FITC-conjugated rabbit anti-mouse IgG (1:40; Dako, Denmark, A10042) were applied to the cells for 1 h at room temperature. After the reaction, the cells were washed three times with a washing buffer (PBS containing 0.5% Triton-X 100 [Sigma, MO, USA] and 0.5% bovine  serum  albumin  [Sigma]).  The nuclei  were  stained  with 5 µg/mL Hoechst 33342 (Sigma) for 10 min at room temperature, after which the cells were again washed three times with the washing buffer. The specimens were then observed under Biozero fluorescence microscope BZ-9000 (Keyence, Osaka, Japan).

Assessment of hiPS-CM electrophysiological function

Microelectrode array analysis was performed to investigate the electrophysiological characteristics of hiPS-CMs around day 30 using the MED 64 Quad system (Alpha MED Sciences, Osaka, Japan) as described previously [1]. Human iPS-CMs were plated on MED-probe dishes (Alpha MED Sciences) and cultured at 37°C for several days to allow attachment.



Supplementary Figure 1.:
Characterization of hiPSC-derived cardiomyocytes in 253G1. (A) Immunostaining of GATA4 (red) and cardiac troponin T (cTnT; green) and cardiac actin (green). The nuclei were counterstained with Hoechst 33342 (blue). Bars = 50 m. (B) Measurement of field potential of cardiomyocytes from 253G1 line.