Rock inhibitor

Primed-hiPSCs of both patients were maintained on irradiated mouse embryonic fibroblasts (MEFs) in DMEM/F12 GlutaMAX supplemented with 20% KSR (thermofisher), 1X non-essential amino acids, 50 µM β-mercaptoethanol (thermofisher), and 10 ng/ml of bFGF (thermofisher). Media were replaced every day. Cells were passaged as clumps every 4 to 6 days using trypsin and 10 µM of ROCK inhibitor (TEBU-Bio). Primed-like hiPSCs were converted into a naive state by cultivating them on MEFs feeders cells in knock-out DMEM medium (thermofisher) supplemented with 20% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0.1 mM MEM NEAA (thermofisher) and 50 µM β-mercaptoethanol (thermofisher) and containing basic fibroblast growth factor (b-FGF, thermofisher), human leukemia inhibitory factor (LIF, Peprotech), transforming growth factor-β1 (TGFβ, TEBU-Bio), and four small inhibitory molecules (4i), 3 μM CHIR99021 (R&D systems), 1 μM PD0325901 (R&D systems), 5 μM SB203580 (R&D systems), and 5 μM SP600125 (R&D systems)^{27 (link)}. Cells were maintained in 4i hiPSC medium for two weeks replacing daily with fresh medium. The growing naive-hiPSCs colonies were picked up and passaged every 2–3 days as single cells with accutase. For each passage, 10 μM of ROCK inhibitor (TEBU-Bio) were used.

Naive-hiPSCs colonies were separated as single cells with accutase and seeded in ultra-low attachment U-bottom 96 well plate (Corning, New York, USA) at a density of 8000–10,000 cells/well in PGC-LCs medium. Then naive-hiPSCs were centrifuged 120 g for 2 min to induce embryoid bodies (EBs) formation. Simultaneously of EBs formation, naive-hiPSCs differentiation into PGC-LCs was induced by culturing EBs in GMEM medium (thermofisher) supplemented with 15% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0,1 mM nonessential amino acid (thermofisher), 1 mM sodium pyruvate (Sigma), 50 µM mercaptoethanol (thermofisher), 100 U/ml Penicillin—100 U/ml Streptomycin (Sigma) and cytokines 500 ng/ml BMP4 (TEBU-Bio), 1 µg/ml LIF (Peprotech), 100 ng/ml SCF (TEBU-Bio), 50 ng/ml EGF (TEBU-Bio) and 10 µM ROCK inhibitor (TEBU-Bio). After 4 days, EBs were observed in the center of each well. Three to five EBs were formaldehyde-fixed and paraffin-embedded for each clone studied, then paraffin sections of EBs were used for immunohistochemistry experiments.