Oryza sativa derived recombinant human albumin

Single cell culture was achieved by subjecting iPSC (>p20) to versene treatment. The cells were plated again and allowed to grow to >75% confluency. The medium was changed to cardiac differentiation medium—RPMI 1640 (Thermo Fisher Scientific), 500 μg × mL⁻¹ Oryza sativa–derived recombinant human albumin (Sigma-Aldrich, Oakville, ON, Canada), and 213 μg × mL⁻¹ L-ascorbic acid 2-phosphate (Sigma-Aldrich), the medium was replenished at 48 h. The small molecules were added to initiate cardiac differentiation in the following sequence—days 0–2 (CHIR99021, 6 μM) and days 2–4 (Wnt-C59, 2 μM). On day 4, cardiac differentiation medium without any small molecules was used to maintain the differentiation. The day 7 onwards, the cultures were observed for contracting cells and the batch with cardiac differentiation efficiency over 90% was used for further experiments. Immunostaining was carried out to ensure cardiac lineage using cardiac troponin (CTNT) (Cat#AB8295, Abcam), Homeobox protein NKX-2.5 (Cat#8792S, Cell signaling technologies).

When PSCs were grown for 2 days, at which time they reached 70~80% confluence, namely at day 0 of induction of differentiation, adherent cells were washed with PBS (Hyclone, South Logan, UT, USA), and then enzymatically dissociated using 0.5 mM EDTA. Cells were seeded at a density of 8000–13,000 cells/cm² in 1:200 growth factor-reduced matrigel (9 μg/cm²)-coated 12-well plates. Medium was changed to CDM3-UA, consisting of RPMI 1640 (Life Technologies Corporation, Gaithersburg, MD, USA), 500 μg/mL Oryza sativa-derived recombinant human albumin (Sigma-Aldrich, St. Louis, MO, US), and 7.5 mg/dl UA (Sigma-Aldrich). Medium was changed after every 48 h. For d0–d2, medium was supplemented with 6 μM CHIR99021 (Selleck Chemicals, Houston, TX, USA). On d2, medium was changed to CDM3-UA supplemented with 2 μM Wnt-C59 (Selleck Chemicals). Medium was changed on d4 and every day for CDM3-UA. Cardiomyocyte was derived from NKX2–5-GFP hESCs using the cardiac differentiation process above. When we further identified the effect of AA in cardiac differentiation, UA was replaced with 213 μg/mL L-ascorbic acid-2-phosphate magnesium (Selleck Chemicals) in the medium CDM3-AA during experimental procedures, and other differentiation procedure was retained.