Ssea4

Cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature and permeabilized with 1% Triton X-100/PBS (Sigma) for 10 min. After blocking with 10% donkey serum in PBS (Jackson ImmunoResearch) for 1 h at room temperature, cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with appropriate fluorescent-conjugated secondary antibodies for 1 h at room temperature the next day. Primary antibodies were as follows: OCT4 (Abcam), SOX2 (Abcam), SSEA4 (Abcam), TFAP2C (Santa Cruz), PRDM14 (Abcam), and NANOS3 (Abcam). Secondary antibodies were as follows: AlexaFluor 488 conjugated donkey anti-rabbit IgG and AlexaFluor 594 conjugated donkey anti-mouse IgG (all Life Technologies). The nuclei were counterstained with DAPI (Thermo Fisher Scientific). The cells were observed with a Zeiss inverted confocal microscope.

The cells in the culture dish were fixed in 4% paraformaldehyde for 15 min, then permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 10% bovine serum albumin (BSA). Next, the cells were incubated with specific primary antibodies overnight at 4 °C and then incubated with secondary antibodies in 10% BSA. Nuclei were counterstained with DAPI for 5 min. Images were acquired with a fluorescence microscope (Olympus, IX73, Japan). The primary anti-stem cell markers were Map2 (Millipore), NANOG (Cell Signaling Technology), Nestin, Pax6, SSEA4 (Abcam, Massachusetts, USA), and PSD95 (Invitrogen, Massachusetts, USA).

The main reagents include DMEM/F12 (1:1) medium, knockout serum replacement (KSR), valproic acid, fetal bovine serum, Essential 8™ Flex Medium, Geltrex™, KSFM medium, BMP‐4, bovine pituitary extract (BPE), (Gibco), NANOG, OCT4, SOX2, SSEA‐4, TRA‐1‐81, TRA‐1‐60, Krt19, Integrinβ1, CD200, CD31 and VEGF‐A antibodies (Abcam), PDGF‐B and Ang2 antibodies (Santa Cruz), recombinant human EGF (R&D), RA, valproic acid, sodium alginate (Sigma), Matrigel^® Matrix (Corning), mTeSRTM1 medium (Stem Cell), Astragalus polysaccharide (Solarbio), silk fibroin and collagen (Hefei Bomei Bio), and Dextran Texas red™ (Invitrogen). Primers were designed using Primier 6.0 software, and all primers were synthesized as shown in Table 1.

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton-X100 followed by goat serum blocking. H7 and hiPSC colonies were stained with pluripotency marker antibodies OCT3/4 (Santa Cruz), SOX2 (Abcam), Nanog (Santa Cruz), and SSEA-4 (Abcam), whereas hPSC-derived cardiomyocytes were stained with antibodies for cTNT (Abcam), Sarcomeric α-actinin (Abcam), MYL2 (Proteintech), MYL7 (Synaptic system), or EGFP (Proteintech) for 24 h at 4 °C respectively. Cells were then incubated with Alexa Fluor 594 or 488 at 37 °C for 1 h and subsequently counterstained with DAPI. For rat hearts, heart tissues were paraffin-embedded and sectioned, followed by H&E staining. The remaining tissues were embedded with optimal cutting temperature compound (OCT; Sakura Finetek, Japan) and sectioned into sections 8 mm thick. The slides were then labeled with antibodies for Laminin (Thermo Fisher Scientific), fibronectin (Abcam), and collagen III (Abcam). Images were captured with a fluorescence microscope Leica DMi8 (Leica).

The cultured cells were placed on 12 mm cover slips, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min at room temperature, washed three times with phosphate-buffered saline (PBS, Geno, China), and treated with a permeabilizing and blocking buffer (10% donkey serum, 0.225% Triton X-100) for 1 hour at room temperature. Then, the cells were incubated with the following primary antibodies: OCT4 (1 : 200, Abcam), Nanog (1 : 250, Abcam), SSEA4 (1 : 250, Abcam), TRA-1-60 (1 : 300, Abcam), SOX1 (1 : 300, Boster), SOX2 (1 : 300, Boster), Nestin (1 : 300, Abcam), Olig2 (1 : 500, Millipore), Pax6 (1 : 100, DSHB), HB9 (1 : 50, DSHB), Islet1 (1 : 250, Abcam), ChAT (1 : 100, Millipore), and TuJ1 (1 : 250, Abcam). All antibodies were diluted in antibody dilution buffer (2% donkey serum, 0.05%Triton X-100), and the cells were incubated with the antibodies overnight at 4°C. After three washing steps, the cells were incubated for 1 hour at room temperature with secondary antibodies: Alexa Fluor (488 or 555) donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat. For the detection of AChR, the cocultured cells were incubated with Alexa Fluor 555-conjugated α-BTX (1 μg/ml, Invitrogen) for 1 hour at 37°C before fixation. The cells were then washed 2 times in PBS and fixed with 4% PFA. All cell samples were observed using an Olympus fluorescence microscope.