The information of puchased reagents and cell line used in the study: LHpb-YaabC3 hiPSC (HNF-P30-P11, OSINGLAY BIO, China), BJ human foreskin fibroblast cell line (CRL2522, ATCC, USA), HN4 human embryonic stem cell (hESC) line (HES-P20-P9, OSINGLAY BIO, China), Cell culture media RPMI/1640 (11875093, Thermo Fisher Scientific, USA), and DMEM/F12 media (11320082, Thermo Fisher Scientific, USA), medium for hiPSC BioCISO (BC-PM0001, OSINGLAY BIO, China), GSK3 inhibitor CHIR99021 (S1263, Sigma, USA), FGF inhibitor PD (3044, Tocris Bioscience, UK), Wnt inhibitor IWP2 (3533, Tocris Bioscience, UK), ROCK inhibitor Y27632 (1254, Tocris Bioscience, UK), BMP activator BMP4 (120-05ET, PEPROTECH, USA), RA inhibitor BMS (SML1149, Sigma, USA), B-27 supplement with (17504-044, Thermo Fisher Scientific, USA) or without insulin, Matrigel (354277, Corning, UK), TRIzol Reagent (15596026, Thermo Fisher Scientific, USA), Real-time PCR reagents (208056, Qiagen, Germany). All primers/oligos were synthesized by Shenggong Biotech, China and listed in SUPPL-Table 1. All other reagents, unless specified otherwise, were products of Sigma.
Cardiomyocytes differentiation from hiPSC
Cardiomyocyte differentiation was performed in a growth factor and serum-free system by temporal modulation of the canonical Wnt signaling pathway with GSK3 inhibitor (Gi) and Wnt inhibitor (Wi), known as the GiWi protocol . Briefly, 80-90% confluent hiPSC was harvested using 0.5mM EDTA, and resuspended with hiPSC maintaining medium at 0.5 x 105 cells per ml. Two ml of the cell suspension was seeded per well in a 12-well matrigel-coated plate with 2 ml of hiPSC at minus day4. At day 0 the medium was refreshed with RPMI/B-27 containing CHIR99021 (10 μM, GSK3 inhibitor) without insulin and continued to incubate for 24 h. The medium was replaced with RPMI/B-27 without insulin for another 48 h. On day 3 of the differentiation (72 h after addition of CHIR99021), medium was refreshed with RPMI/B-27 containing IWP2 (5 μM, Wnt inhibitor) without insulin for 48 h, followed by RPMI/B-27 without insulin from day 5 to 7. From day 7, the medium was refreshed with RPMI/B-27 containing insulin every 3 days. The beating cardiomyocytes can be seen as early as on day 8. Enriched differentiation of SAN-like cardiomyocytes from hiPSC by BMP4 (B), PD (P) and BMS (M) treatment
Based on the GiWi protocol that it caused highly efficient pan-cardiomyocytes as shown above, different concentrations of BMP4 (0, 1.25, 2.5, 5 ng/ml), PD (0. 480, 720, 960 nM) and BMS (0, 1 μM) were added alone between day 5 and 7 after differentiation for 2 days. The qRT-PCR analysis was performed to evaluate the mRNA levels of SAN markers at day16 to determine the optimal concentration of each compound. To investigate the synergistical effect, the cells were treated with combinations of BMP4, PD and BMS with the optimal concentration from day 5 to 7 during the differentiation process. The markers of pacemaker cells were evaluated by analysis of qRT-PCR, immunofluorescence and flow cytometry at day 21. Electrophysiological characteristics were analyzed using Action potential (AP) recording at day 60. The schematical protocol was shown in Figure 4A.
RNA isolation and qPCR
Total RNA was isolated using Trizol method (15596026, Invitrogen, USA). One μg of total RNA was reversely transcribed in a total volume of 10μl with ReverTra Ace qPCR RT Master Mix kit (FSQ-201, TOYOBO, Japan) following manufacturer’s instructions. The cDNA was diluted 3 times, and 1 μl was used for real-time PCR in a 20 μl reaction using SYBR Green Real Time PCR Mix (204143, Qiagen, Germany). The PCR conditions were 95°C for 2min, followed by 40 cycles of 95°C for 20’’ and 60°C for 15’’. All primers were listed in SUPPL Table 1. The expression of target gene was normalized to that of GAPDH and calculated using 2-∆∆Ct method.
Single hiPSC cells and induced cardiomyocytes were seeded in a µ-Slide 8 well (80827, ibidi) coated with Matrigel at the density of 2 × 104 per well for 48 h. Cells were fixed with 4% (w/v) Paraformaldehyde (PFA) for 15 min at room temperature, permeabilized, and incubated with the following primary antibodies: anti-OCT4 antibody (#2750, Cell Signaling Technology, USA), anti-NANOG antibody (#3580, Cell Signaling Technology, USA), anti-SSEA4 antibody (#4755, Cell Signaling Technology, USA), anti-TRA-1-60 antibody (#4746, Cell Signaling Technology, USA), anti-Ki67 antibody (ab15580, abcam, USA), anti-NKX2.5 antibody (ab91196, abcam, USA), anti-cTNT antibody from mouse (MS-295-P1, Thermo Fisher Scientific, USA), anti-cTNT antibody from rabbit (15513-1-AP, Proteintech, China) anti-α-actinin antibody (A7811, Sigma, USA), anti-SHOX2 antibody (ab55740, abcam, USA), anti-TBX18 antibody (ab115262, abcam, USA), anti-COUPTFⅡ antibody (PP-H7147-00, R&D, USA), anti-MLC2V antibody (MABT180, Sigma, USA) and anti-TBX3 antibody (ab154828, abcam, USA), followed by the following species-specific fluoresce-conjugated secondary antibodies: alexa fluor 488 labeled goat anti-rabbit IgG (A-11008, Invitrogen, USA), alexa fluor 488 labeled goat anti-mouse IgG (A-11001, Invitrogen, USA), alexa fluor 594 labeled goat anti-rabbit IgG (R37177, Invitrogen, USA) and alexa fluor 594 labeled goat anti-mouse IgG (A-11005, Invitrogen, USA). The cells were then counterstained using 0.5 µg/ml of DAPI (4083, Cell Signaling Technology, USA) for 15 min at room temperature. After rinsing with PBS, the chambers were mounted and visualized under fluorescence microscopy (IX83, Olympus, Japan). Corresponding antibody isotype control, mouse IgG (ab205719, abcam, USA), rabbit IgG (ab205718, abcam, USA), mouse IgG (ab190369, abcam, USA), mouse IgG1 (#5415, Cell Signaling Technology, USA), mouse IgG3 (#37988, CST, USA) were used.
The induced cardiomyocytes in 12-well plate were digested with 0.25% trypsin with 0.5mM EDTA into single cell suspension and washed with PBS. Cells were fixed with 4% formaldehyde for 10 min at room temperature and chilled on ice for 1 min. Permeabilization was performed by adding one tenth of ice-cold 100% methanol slowly to the pre-chilled cells and continue to incubate on ice for 30 min. Cells were then blocked with blocking buffer (0.5% BSA in PBS) for 10 min, incubated with the following primary antibodies, anti-OCT4 antibody (#2750, Cell Signaling Technology, USA), anti-NANOG antibody (#3580, Cell Signaling Technology, USA), anti-SSEA4 antibody (#4755, Cell Signaling Technology, USA), anti-TRA-1-60 antibody (#4746, Cell Signaling Technology, USA), anti-Ki67 antibody (ab15580, abcam, USA), anti-cTNT antibody (MS-295-P1, Thermo Fisher Scientific, USA), anti-NKX2-5 antibody (ab91196, abcam, USA), and anti-SHOX2 antibody (ab55740, abcam, USA) for 1 h at room temperature, then washed with PBS, and followed by incubation with the corresponding species-specific fluorence-conjugated secondary antibodies, alexa fluor 488 labeled goat anti-mouse IgG (A-11029, Invitrogen, USA)，alexa fluor 488 labeled goat anti-rabbit IgG (A-11034, Invitrogen, USA), alexa fluor 647 labeled goat anti-mouse IgG (A-21235, Invitrogen, USA), and alexa fluor 647 labeled goat anti-rabbit IgG (A-32733, Invitrogen, USA) for 30 min at room temperature. Cells were analyzed using flow cytometry machine (651155, BD FACS Verse, BD Bioscience, USA) according to the manufacturer's protocol.
Action potential (AP) recording
AP recording was performed following El-Battrawy I’s protocol with some modifications . Briefly, on 60 days after cardiomyocytes differentiation, induced cardiomyocytes were dissociated into single cell suspension by 30 min’s typeⅠcollagenase (2 mg/ml) followed by 3 min’s Trypsin (0.25%) without EDTA. 1 × 104 cells were seeded into a 3.5 cm dish containing a lysine-treated glass coverslip and incubated for 3 days. AP was recorded using the whole cell patch clamp electrophysiology method. Briefly, the adherent cells on the coverslip were placed in the recording chamber and perfused with bath solution containing 140 mM NaCl, 1 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mM glucose, (The pH was adjusted to 7.40, and the osmolality to 301 ± 3 mOsm, respectively). The patch pipettes were pulled from borosilicate glass capillaries (7-000-0650-LHC, Drummond, USA) by a horizontal puller (PC100, NARISHIGE, Japan) and had resistances of 1.5–3 MΩ. Pipette solution consisted of 110 mM K-gluconate, 20 mM KCl, 1mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM ethylene glycol tetraacetic acidpotassium chloride (EGTA-KOH), 5 mM ATP‐Mg2+, 5 mM Na-phosphocreatine. The pH was adjusted to 7.2 by KOH and the osmolality to 290 ± 3 mOsm. A Multiclamp 700B amplifier was used to record APs, and data were analyzed using a custom software.
Experimental data are presented as ‘Mean ± SD’ with at least three repeats. Comparisons between multiple groups were performed using one-way analysis of variance (one-way ANOVA), with p < 0.05 was considered statistically significant.