Ascorbic acid

Human-induced pluripotent stem cells (hiPSCs; 253G1; Riken, Ibaraki, Japan) were used in this study. Undifferentiated hiPSCs were cultured and maintained in primate embryonic stem cell medium (ReproCELL, Kanagawa, Japan) with 5 ng/mL basic fibroblast growth factor (bFGF; ReproCELL) on mitomycin C-treated mouse embryonic fibroblast cells (ReproCELL). Cardiomyogenic differentiation was induced as previously described with specific modifications (Matsuura et al., 2012 (link)). Briefly, cardiac differentiation was induced in StemPro 34 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mM L-glutamine (Thermo Fisher Scientific), 50 μg/mL ascorbic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 400 μM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO, USA). The medium was also supplemented with several human recombinant proteins, including bone morphologic protein 4, activin A, bFGF (R&D Systems, Minneapolis, MN, USA), and VEGF (FUJIFILM Wako Pure Chemical Corporation), and small molecules, including IWR-1 and IWP-2 (Sigma-Aldrich). hiPSCs were dissociated using Accumax (Nacalai Tesque, Kyoto, Japan) and transferred to a bioreactor (ABLE Corporation & Biott, Tokyo, Japan). hiPSC-CMs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).

Parasites at 2% parasitemia were prepared in 24-well culture plates containing either 0, 250, or 500 μM of ascorbic acid (Wako, Osaka, Japan) and treated with the active compounds at its IC₅₀ concentration. The culture plates were incubated at 37°C for 24 h before the thin-smeared Giemsa slides were made for each well. The number of pRBCs in 3000 RBCs was determined under a light microscope at 1000 times of magnification. Each treatment group was made in triplicate, and the experiment was performed three times.

The capacities of passage 3 ASCs to differentiate into adipogenic lineages and osteogenic lineages were evaluated using a previously reported method [23] (link). For adipogenesis, the medium was switched to an adipogenic medium consisting of a complete medium supplemented with 0.5 μmol/L isobutyl-1-methyl xanthine (Sigma–Aldrich, St. Louis, USA), 0.5 μmol/L dexamethasone (Fuji Pharma, Tokyo, Japan), and 50 μmol/L indomethacin (Wako Pure Chemical Industries, Osaka, Japan). After 14 days, the cells were fixed with 4% PFA and stained with fresh Oil Red O solution (Wako Pure Chemical Industries). For osteogenesis, the medium was switched to a calcification medium consisting of a complete medium supplemented with 50 μmol/L ascorbic acid (Wako Pure Chemical Industries), 10 mmol/L β-glycerophosphate (Sigma–Aldrich), and 100 nmol/L dexamethasone. The cells were incubated for 21 days, and then stained with 1% alizarin red S solution. The proliferation capacities of passage 3 ASCs were evaluated according to the previously reported colony-forming unit assay method [23] (link). Briefly, 100 cells were cultured in 60-cm² dishes for 9 days and stained with crystal violet. Then, proliferation capacity was measured.

MSCs isolated from each tissue at passage five were seeded onto temperature-responsive culture dishes (35-mm diameter, UpCell, Cell Seed, Tokyo, Japan) at a density of 2 × 10⁵ cells/dish. The MSCs were cultured in α-MEM GlutaMAX (Invitrogen) with 10, 20, or 30% FBS (Moregate Biotech) and 1% penicillin/streptomycin (Sigma–Aldrich) with 82 μg/mL ascorbic acid (Wako) for 5–9 days. The temperature of the culture dishes was reduced to room temperature, and subsequently the medium was removed to produce MSC sheets (Fig. 1).