Anti tra 1 60

HEK293T cells were cultured in DMEM with 15% FBS and 1% Penicillin-Streptomycin and dissociated with 1X TrypLE. H1 human ESCs (WA01, WiCell Research Institute) were maintained in a feeder-free mTesR1 medium (StemCell Technologies, Inc.). HEK293T and H1 cells were cultured at 37°C and with 5% CO2. hESCs (passage 26) were dispersed with 1U/mL Dispase and collected for single-cell sorting or nuclei isolation. For the sorting of single H1 and HEK293T cells, equal amounts of H1 and HEK293T cells were mixed and stained with anti-TRA-1-60 (Biolegend, Cat#330610) antibody.

hiPSCs were dissociated into single cells and washed three times with D-PBS. A total of ~ 2 × 10⁵ cells were stained with the following PE-conjugated antibodies diluted in 500 µL 3% BSA/PBS blocking buffer: anti-SSEA-4 (mouse monoclonal IgG3, 1:250; 330406), anti-SSEA-1 (mouse monoclonal IgG1, 1:250; 323006), anti-TRA-1-60 (mouse monoclonal IgM, 1:500; 330610) or the corresponding isotype controls, all purchased from BioLegend (San Diego, CA, USA). After incubation in the dark at room temperature for 30 min, hiPSCs were washed three times with PBS and assessed by immune-positive cell surface markers using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A minimum of 10,000 cell events were acquired during each assay, and the data obtained were analyzed using FlowJo (v10, Tree Star).

Cells were analyzed by flow cytometry for identity and purity markers using the following antibodies: anti-A2B5 (1:20; Miltenibiotec), anti-GLAST (1:20; Miltenibiotec), anti-CD44 (1:20; BD Pharmingen), anti-CXCR4 (1:20; Biolegend), anti-TRA-1-60 (1:50; Biolegend), anti-EPCAM (1:50; Biolegend), anti-SSEA4 (1:50; Biolegend), anti-GFAP (1:2000; Sigma), Nestin (1:500; BD Pharmingen) and anti-AQP-4 (1:2000; Abcam). The Flow Cytometer FACS Canto II (BD) was operated with FACSDIVA software (BD). At least 10,000 events were collected per sample.