Iwp 2

hiPSCs (253G1; Riken) were used in this study (Nakagawa et al., 2008 (link)). Cardiomyogenic differentiation was induced as previously described with some modifications (Matsuura et al., 2012 (link)). Briefly, the cells were cultured in StemPro 34 medium (Thermo Fisher Scientific) containing 2 mM l‐glutamine, 50 μg/ml ascorbic acid, and 400 μM 1‐thioglycerol. hiPSCs were dissociated using Accumax (Nacalai Tesque), transferred to a bioreactor, and supplemented with several human recombinant proteins, including BMP4, activin A, bFGF, and VEGF, and small molecules such as IWR‐1 and IWP‐2 (Sigma‐Aldrich/Merck) on Days: 0–1, BMP4; 1–4, activin A, BMP4, and bFGF; 4–6, IWR‐1 and IWP‐2; and after Day 6, VEGF and bFGF. hiPSC‐CMs were cultured in DMEM with 10% FBS and then in serum‐free medium for 24 h before use.

On day 0, passage 2 hAMSCs were seeded at a density of 3 × 10⁴ cells/m² in 6-well plates. The culture medium was then changed to EFD medium supplemented with 10 μM 5-AZA (Sigma, A2385), 7.5 μM CHIR99021 (Sigma, SML1046), 10 ng/mL Activin A, 5 ng/mL BMP4, and 5 μM IWP2 (Sigma, 5.06072) on day 0 to Day 5, and with only 5 μM IWP2 on days 6 to Day 9 (Fig. 1). After day 9, the culture medium was changed to EFD medium without any supplements twice a week, and cells were cultured until day 17. To investigate the effects of each supplement, the experiments were divided into 16 groups. BMP4 and Activin A were combined as one condition to reduce the number of groups. The corresponding groups and the added chemicals are listed in Table 2. All supplements were stored at −20 °C, and 5-AZA (100 mM), CHIR99021 (12 mM), and IWP2 (10 mM) were dissolved in DMSO. BMP4 and Activin A were dissolved in 1x DPBS and diluted with EFD medium before use.Fig. 1

The schedule of cardiac differentiation in hAMSCs.

Fig. 1Table 2

The groups correspond to the adding of chemicals.

Table 2

Group No.	BMP4&ActivinA	5-AZAcytidine	CHIR99021	IWP2
1	–	–	–	–
2	–	+	–	–
3	–	–	+	–
4	–	–	–	+
5	–	+	+	–
6	–	+	–	+
7	–	–	+	+
8	–	+	+	+
9	+	–	–	–
10	+	+	–	–
11	+	–	+	–
12	+	–	–	+
13	+	+	+	–
14	+	+	–	+
15	+	–	+	+
16	+	+	+	+

Human-induced pluripotent stem cells (hiPSCs) (253G1; Riken, Tsukuba, Japan) were cultured in a primate embryonic stem cell medium (ReproCELL, Kanagawa, Japan) supplemented with the basic fibroblast growth factor (bFGF; ReproCELL, Kanagawa, Japan) at 37 °C. Mitomycin Ctreated mouse embryonic fibroblasts (ReproCELL, Kanagawa, Japan) were used as feeder cells. Cardiomyogenic induction was performed in the StemPro 34 medium (Thermo Fisher Scientific, Waltham, MA) containing 2 mM l-glutamine (Thermo Fisher Scientific, Waltham, MA), 50 mg/mL ascorbic acid (Wako, Pure Chemical Industries, Tokyo, Japan), and 400 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO), as previously described [18 (link)].
To obtain hiPSC-CMs, hiPSCs were dissociated using Accmax (Nacalai Tesque, Kyoto, Japan), and induced in a bioreactor (ABLE Corporation & Biott Co., Tokyo, Japan). Human recombinant bone morphogenetic protein 4 (BMP4), activin A, bFGF, and vascular endothelial growth factor (VEGF) (R&D Systems, Minneapolis, MN), along with the small-molecule compounds IWR-1 and IWP-2 (Sigma-Aldrich, St. Louis, MO) were used for induction as follows: BMP4 from day 0–1; activin A, BMP4, and bFGF from days 1–4; IWR-1 and IWP-2 from days 4–6; and VEGF and bFGF after day 6. Flow cytometry analysis was performed on day 14 using a FACS Canto II (BD, Franklin Lakes, NJ) as previously reported [18 (link)].