Ssea4

Cells were fixed with 4% PFA (Solarbio, China) for 10–15 min at room temperature, permeabilized with 0.3% Triton X-100 (Sigma) for 10–15 min at room temperature, blocked with 3% bovine serum albumin (Solarbio) for 45–60 min at room temperature, and then incubated with primary antibodies against OCT4 (1:100; Santa Cruz Biotechnology), SSEA4 (1:100; Santa Cruz Biotechnology), SOX2 (1:100; Santa Cruz Biotechnology), NESTIN (1:100; Sigma), β3-tubulin (1:100; Abcam), and GFAP (1:100; Abcam) overnight at 4 °C. And then cells were incubated with secondary antibodies: Goat anti-Rabbit IgG Alexa Fluor 488 (1:200; Invitrogen) and Goat anti-Mouse IgG Alexa Fluor 594 (1:200; Invitrogen) for 1 h at 37 °C. Wash with PBS three times before each step. Nuclei were stained with DAPI (300 nM, Invitrogen) for 15 min at room temperature. Fluorescence images were captured by Leica DMI 4000B fluorescence microscope and Leica TCS SP5 MP confocal laser scanning microscope (Leica, Germany).

To confirm pluripotency, the newly generated colonies were immunostained with OCT3/4 (1:50), NANOG (1:50), SSEA4 (1:50), and TRA-1-81 (1:200) (Santa Cruz Biotechnologies, Dallas, Texas, USA) at the given concentrations. The colonies were fixed in 3% paraformaldehyde for 30 minutes, washed with phosphate-buffered saline (PBS) and permeabilized with PBS and 0.5% Triton. After blocking with 5% bovine serum albumin, the colonies were incubated with primary antibodies at appropriate concentrations overnight, followed by appropriate secondary antibody treatment. For multilineage cell staining, the cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton. After blocking with bovine serum albumin, the cells were incubated overnight with β-tubulin III (ectoderm marker, 1:50), nestin (endoderm marker, 1:50) and alpha smooth muscle actin (αSMA; mesoderm marker, 1:50) (Santa Cruz Biotechnologies), followed by appropriate secondary antibody treatment. The results were evaluated using a Leica Fluroscence DMI 4000-B (Leica Microsystems Heerbrugg, St Gallen, Switzerland).

hiPSCs or kidney organoids were fixed with 4% paraformaldehyde. The following antibodies were used: NANOG (1E6C4, 1:100; Santa Cruz Biotechnology), SSEA-4 (813-70, 1:100; Santa Cruz Biotechnology), TRA-1-81 (TRA-1-80, 1:100; Santa Cruz Biotechnology), biotinylated Lotus Lectin (LTL, B-1323,1:100; Vector Laboratories), E-Cadherin (ab11512, 1:50; Abcam), CD77 (551352, 1:100; BD Biosciences), and Podocalyxin (BAF1658, 1:100; R&D Systems). Antibody staining was visualized using the following secondary antibodies: Alexa Fluor 488-donkey anti-rat IgG (1:250; Invitrogen), Alexa Fluor 488-donkey anti-mouse IgG (1:250; Invitrogen), Alexa Fluor 647-donkey anti-goat IgG (1:250; Invitrogen), or Cy3-streptavidin (1:1000; Jackson ImmunoResearch). Cell nuclei were then counterstained with 4,6-diamidino-2-phenylindole (DAPI; Roche, Mannheim, Germany). Stained sections were observed using a confocal microscope (LSM700; Carl Zeiss Co. Ltd., Oberkochen, Germany). Images were converted to TIFF format and contrast levels were adjusted using Adobe Photoshop v. 13 (Adobe System, San Jose, CA, USA).

For the immunofluorescence staining, hiPSCs were fixed in PBS containing 4% paraformaldehyde for 20 min at RT and then washed with PBS 3 times. The cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT and then subjected to blocking with 1% bovine serum albumin (BSA) (MP Biomedicals, Santa Ana, CA, USA) for 1 h at RT (25 °C). Afterwards, the cells were incubated with the primary Abs including stage-specific embryonic antigen (SSEA)-4 (1:100, Santa Cruz Biotechnology), tumor-related antigen (TRA)-1-60 (1:100, Santa Cruz Biotechnology), OCT4 (1:100, Santa Cruz Biotechnology), NANOG (1:100, Santa Cruz Biotechnology), or SOX2 (1:100, Santa Cruz Biotechnology) overnight at 4 °C and then washed 3 times with PBS. Following this process, the cells were incubated with the corresponding secondary Abs for 1 h at RT. Finally, hiPSCs were washed 3 times and stained with TOPRO3 (Invitrogen) for 10 min in order to visualize the nuclei. Fluorescent signals were examined using confocal microscope equipment (FV-1000 spectral).

Immunophenotypic analysis was performed using flow cytometry. For this, 1 × 10⁵ cells/ml were trypsinized, centrifuged and fixed in 10% paraformaldehyde. The cells were then incubated with primary antibodies (dilution 1:100) for 30 minutes at 4 °C. After this period, the cells were incubated with the secondary antibody (dilution 1:500) for 30 minutes at 4 °C. Finally, the cells were washed and analysed by a BD FACSariaIIu flow cytometer (Becton Dickinson, San Jose, CA, USA). Controls were performed using unmarked cells exposed only to the non-specific secondary antibody. For each sample, 10,000 events were counted. The primary antibodies used were as follows: Nanog (n-17, sc30331), Sox-2 (sc-17320), vimentin (sc-73259), cytokeratin 18 (RGE53, sc-32329), Stro-1 (sc-47733), PCNA-3 (sc-46), β-tubulin (sc-47751), SSEA-4 (sc-59368), TRA-1-60 (sc-21705) and CD73 (sc-14684) (all purchased from Santa Cruz Biotechnology, Inc., Europe); in addition to CD105 (ab53321; Abcam, Cambridge, UK), CD117 (A4502; Dako Cytomation, Carpinteria, CA, USA) and CD45 (MHCD4501; Invitrogen, Carlsbad, CA, USA). For nuclear antibodies such as PCNA-3, Nanog and Sox-2 we added 0.1% of triton (catalogue number 13-1315-05; LGC Biotecnologia) in order to permeabilize the cell membranes.