Fig 1.
Scheme of optimized CM differentiation protocol.
Fig 2.
Induction and maintenance of hiPSC-derived CMs with high-purity.
(A) Representative flow cytometry analysis of cTnT (and side scatter plot (SSC)) on d21 induced by combinations of Wnt signal inhibitors (XAV939 and IWP4). The percentages of cTnT+ and cTnT- cells are indicated. (B) Quantitative analysis of cTnT+ CMs on d21. Data are means ± SD. ***p < 0.001 by one-way ANOVA followed by Tukey-Kramer post hoc test (n = 4). (C) CM yield on d21. Data are means ± SD. ***p < 0.001 by one-way ANOVA followed by Tukey-Kramer post hoc test (n = 4). (D) Scheme of CM long-term maintenance protocol. CMs were re-cultured under αMEM medium with 10% FBS and Y27632, for the initial 2 days, followed by a serum-free condition (RPMI1640+B27) for 18 days. CMs underwent passage every 20 days. (E) Representative flow cytometry analysis of cTnT in 836 B3 cell line on d61, and d91. Percentages of cTnT+ cells are indicated. (F) Quantitative analysis of cTnT+ CMs in 836 B3 cell line on d61, and d91. (n = 4) (G) Representative flow cytometry analysis of cTnT in 836 B3 cell line on d201. Percentages of cTnT+ cells are indicated. (H) Representative flow cytometry analysis of cTnT in 253G1 and 1201C1 cell lines on d21, d61, and d91. Percentages of cTnT+ cells are indicated. (I) Quantitative analysis of cTnT+ CMs in 253G1 and 1201C1 cell lines on d21, d61, and d91. (253G1; n = 4, 1201C1; n = 5).
Fig 3.
Proliferation capacity of hiPSC-derived CMs.
(A) Growth profile of CMs from d21 to d101. CMs on d21 were seeded at 2.0 × 105 cells/cm2 (1.9 × 106 cells per well of a 6-well plate). Cells were passaged every 20 days and plated at the same cell density. The accumulated cell number was calculated from cell counts during subsequent passages. Data are means ± SD. (n = 4). (B) Representative flow cytometry analysis of cTnT and Ki67 on d21 and d101. Numbers in each quadrant represent the respective percentages of cells. (C) Quantitative analysis of Ki67+/cTnT+ CMs. Data are means ± SD. *p < 0.05 by Mann-Whitney test (n = 4). (D) Representative immunostaining of d21 and d100 CMs for cTnT (Green), Ki67 (Red), and DAPI (Blue). Scale bars, 50 μm.
Fig 4.
Structural maturation and chamber specification of hiPSC-derived CMs after long term culture.
(A, B) Immunostaining of MLC-2a and MLC-2v. (A) Immunofluorescent staining of MLC-2a (Green), MLC-2v (Red), and DAPI (Blue) in d21 and d91 CMs. Scale bar, 50 μm. (B) Quantification of MLC2a and MLC2v immunostaining of d21 and d91 CMs. Data are means ± SD. (n = 4). A+V+: MLC-2a-positive MLC-2v-positive, ventricular CMs; Ahigh+ Vlow+: MLC-2a-high positive MLC-2v-low positive, immature ventricular CMs; Alow+ Vhigh +: MLC-2a-low positive MLC-2v-high positive, relatively mature ventricular CMs; A+ V-: MLC-2a-positive MLC-2v-negative, atrial CMs; ND: not detected. (C) qPCR of relative mRNA expressions of Mlc2a and Mlc2v in CMs on d21 and d91. The mean of d21 was set as 1. **p<0.01, *p<0.05, n = 6 with technical triplicates for each. (D) Double immunofluorescent staining of cTnT (green) and α-actinin (red) on d39 and d155. Right panels: higher magnification views of boxed areas in the left panels. Scale bars in the left panels: 10 μm. Scale bars in the right panels: 5 μm. (E-J) Transmission electron microscopy images of CMs. (E) CMs on d24. CMs contained myofibrils (MF) that lacked clear alignment or an organized sarcomeric pattern. Z-bodies (Zb), CMs connected by desmosomes (Ds) and mitochondria (Mt) can be observed. (F) CMs on d91. Sarcomeres show clearly aligned Z-disks (Z) and organized A- and I-bands with a clear H-zone (H). Mitochondria (Mt) are located adjacent to sarcomere structures. (G) CMs on d140. Faint M-lines starting to form. (H) CMs on d231. Clear muscle-like alignment of myofibers and mitochondria can be observed. (I) CMs at d231. Clear sarcomeric structures with Z-, H-, I-, A-, and M-bands and well established mitochondria can be observed. T?: T-tubule-like structure. (J) CMs on d24. T-tubule-like structures along Z-bands (T?). Scale bars: 1μm (E, F, H), 400 nm (G, I), 600 nm (J). (K) Sarcomere length in CMs on d24, d91, and d231. ***p<0.001, n = 24 (d24), 21 (d91), 23 (d231), respectively.
Fig 5.
Electrophysiological characterization and maturation of CMs after long-term culture.
(A) Criteria for CM subtype classification. (B) Relationship between APD30-40/APD70-80 ratio and dV/dt max on d21 and d91 CMs. (C) Pie charts illustrating the distribution of early ventricular-, late ventricular-, atrial- and nodal-like APs on d21 (n = 31 from three independent experiment), and d91 (n = 23 from three independent experiment). (D) Representative action potentials of early and late ventricular-like and nodal-like CMs. (E) Comparison of action potential parameters between early ventricular-like and late ventricular-like CMs on d21(n = 28 from three independent experiments) and d91 (n = 22 from three independent experiments). (n = 3). *p < 0.05, ***p < 0.001 by one-way ANOVA followed by Tukey-Kramer post hoc test. (F) Administration of E4031 (Ikr blocker) prolonged FPD in a dose-dependent manner at 0–30 nM in CMs. (G) Administration of Chromanol (Iks blocker) prolonged FPD in a dose-dependent manner at 0–30 μM in CMs. (H) Homogeneous response to Chromanol in a time-dependent manner. d31 (n = 16), over d91 (n = 8).