Ssea4

AP staining was performed according to the manufacturer's (Sidansai, China) instructions. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

For immunofluorescence staining, HF-MSCs or HF-iPSCs were fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 1% bovine serum albumin (Roche Diagnostics, France), and incubated with primary antibodies against CD90, CD105, CD31 (Bioscience, CA, USA), CD44 (R&D Systems, UK), CD73 (Life Technologies, USA), stage-specific embryonic antigen (SSEA) 3, SSEA4 (Developmental Studies Hybridoma Bank, USA), TRA-1-60, TRA-1-81 (Chemicon, USA), NANOG (R&D Systems), and OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The next day, Alexa Fluor 488-conjugated goat anti-mouse/rabbit antibodies were used to detect the primary antibodies (Cell Signaling Technology, Danvers, MA, USA). HF-MSCs were then counterstained with DAPI (Life Technologies, USA) and imaged using fluorescence microscopy (Olympus, Japan). For flow cytometry, HF-MSCs were collected by centrifugation, fixed with paraformaldehyde, blocked with bovine serum albumin, and incubated with primary and secondary antibodies as described above. HF-MSCs were then subjected to flow cytometry (FACS Calibur flow cytometer; BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software.

Single cardiomyocytes or hiPSCs were cultured on matrigel-coated tissue culture plates (Falcon), and subsequently fixed in 4% PFA for 15 minutes at room temperature. Cells were blocked and permeabilized in 2% BSA, 2% FBS, and 0.01% Triton for 1 h at room temperature. Primary antibodies anti-human cTNT (1∶100, ThermoScientific), MLC2v (1∶200, Synaptic Systems), OCT4 (1∶100, BioVision), and SSEA4 (1∶25, Developmental Studies Hybridoma Bank) were added to blocking/permeabilization buffer overnight at 4°C. Secondary antibodies were either goat-anti-mouse or anti-rabbit AlexaFluor 488 or 594 (1∶400, Invitrogen). For TRA1-81 live cell immunostaining, hiPSCs were incubated with anti-human TRA1-81-Biotin (1∶100, eBioscience) for 2 h at 37°C in hESC media. Primary antibody was detected by incubation with PE-conjugated streptavadin secondary antibody (1∶100, eBioscience) for 2 h at 37°C in hESC media. Fluorescence was detected on the EVOS FL digital inverted fluorescent microscope (Life Technologies).