Esg1106

OSCs were isolated from 6-week-old mice using the methods described previously [9 (link), 10 (link), 16 (link), 17 (link)]. Briefly, the ovaries from female mice were dissected and minced into slurry in the collagenase/Dnase I solution (Worthington, USA) and then incubated at 37°C for 20 minutes which was repeated once or followed by trypsin treatment for 5–10 min and finally the trypsin was neutralized by 10% fetal bovine serum (FBS). After centrifugation of suspension and removal of supernatant, the pellet was placed onto 6-well plate without STO feeder layer. Two or three days later, the cells were trypsinized and purified by MACS using Fragilis antibody and goat anti-rabbit IgG microbeads [18 (link)]. The sorted cells were cultured onto feeder cells with the medium which consisted of minimum essential medium α medium (MEM-α) (32561-102, Invitrogen), 10% FBS (06902, Stemcell), 1 mM sodium pyruvate (P2256-25, Sigma), 1 mM nonessential amino acids (11140-050, Gibco), 0.1 mM β-mercaptoethanol (ES-007-E, Millipore), 1000 units/mL LIF (ESG1106, Millipore), 1 ng/mL bFGF (13256-029, Gibco), 10 ng/mL EGF (PHG0311L, Gibco), 20 ng/mL human GDNF (212-GD-010, R&D), 1×-concentrated N2-supplement (AR009, R&D), and 1×-concentrated penicillin-streptomycin. Subculture of oogonial stem cells (OSCs) was performed according to reports published previously.

All blastocysts were generated by natural mating of Tubgcp4 heterozygous mice. The morning of the day on which a vaginal plug was detected was designated day 0.5. Blastocysts were collected on E3.5 by flushing the uterus with M2 medium (Sigma-Aldrich) and cultured in ES medium (DMEM, 15% FBS (SH30070.02E, HyClone), 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 55 µM 2-mercaptoethanol (Invitrogen), and 10³ units/ml leukemia inhibitory factor (ESG1106, Millipore, Darmstadt, Germany)). Outgrowths were photographed under an inverted microscope (DM IRB, Leica) each day and harvested for genotyping.

All cell culture reagents were supplied from Invitrogen unless otherwise specified. Mouse E14 (ATCC, CRL-1821) ES cells were cultured in 5% CO₂/95% O₂ in DMEM supplemented with 10% ES cell qualified FBS, 2 mM l-glutamax, 1 mM sodium pyruvate, 100 μM non-essential amino acid, 0.1 mM β-mercaptoethanol, 1000 U mL⁻¹ mouse leukemia inhibitory factor (LIF, Millipore, ESG1106), and 100 U mL⁻¹ penicillin/100 μg mL⁻¹ streptomycin. For differentiation, 35 mm Petri dishes (Nunclon) were coated with fibronectin solution, where undifferentiated mouse ES cells were seeded at a moderate density (10³ cells cm⁻²) in culture medium without LIF.

R1 embryonic stem cells, obtained from the Nagy Lab at the ES cell core facility at the Samuel Lunenfeld Research Institute Mount Sinai Hospital, were cultured as previously described [44 (link)]. Cells were cultured on 0.1% gelatin treated plates in feeder-free conditions with embryonic stem cell media containing LIF (DMEM (Invitrogen 11965–118), 15% FBS (Millipore ES-009-B) 1x Non-Essential Amino Acids (Invitrogen 11140–050), 1x Sodium Pyruvate (Invitrogen 11360–070), 1x L-Glutamine (Invitrogen 25030–081), 100μM Beta mercaptoethanol (Sigma M7522-100ML), 1x Penicillin/Streptomycin (Invitrogen 15140–122), 10³ U/mL LIF (Millipore ESG1106)).

V6.5 mouse ES cells were cultured on 0.2% gelatin-coated plates at 37 °C with mES media: 500 ml Knockout DMEM (Gibco), 90 ml FBS, 6 ml non-essential amino acid (NEAA, 100×, Gibco), 6 ml glutamine or glutamax (200 mM stock solution), 6 ml Pen/Strep, 1 ml BME, and 60 μlLIF (Millipore, ESG1106). Mouse embryonic fibroblast (MEF) cells were cultured at 37 °C and 5% CO2 in 450 ml DMEM, 50 ml FBS, 5 ml Pen/Strep, 5 ml NEAA, 5 ml pyruvate, and 4 μl beta-mercaptoethanol. Mouse Neural Precursor cells (NPCs) were cultured in N2B27 medium (DMEM/F12 (Invitrogen, 11320-033), Neurobasal (Gibco, 21103-049), NDiff Neuro-2 Medium Supplement (Millipore, SCM012), B27 Supplement (Gibco, 17504-044)) supplemented with EGF and FGF (10 ng/ml, each) (315-09 and 100-18B, Peprotech). Cells were passaged using Accutase (SCR005, Millipore) and cultured on 0.2% gelatin-coated plates. H9 human embryonic stem cells were seeded in a feeder-free system using Matrigel hESC-Qualified Matrix (354277, Corning) and were maintained in Essential 8 media (A1517001, Thermo Fisher Scientific) as described previously [69 (link)]. Cells were passaged every 3 days as clumps with 0.5 mM EDTA. Human Female Fibroblasts (HFF) were cultured at 37 °C and 5% CO2 in DMEM supplemented with 1% pen/strep and 10% FBS.

Mouse ESC were grown on 0.1% gelatine-coated plates at 37 °C and 5% CO₂, in a serum-free medium made of KnockOut Dulbecco’s modified Eagle medium (KO-DMEM, Gibco 10829018), supplemented with 15% KnockOut Serum Replacement (KSR, Gibco 10828028), 2 mM L-Glutamine (Gibco 25030081), 1 mM non-essential amino acids (NEM NEAA, Gibco 11140076), 100 µM β-mercaptoethanol (Gibco 21985023) and 1x Antibiotic-Antimycotic (Gibco 15240062). They were maintained in a pluripotent state by using ESGRO Leukemia Inhibitory Factor (LIF 1 µM, Millipore ESG1106) and two inhibitors (called 2i), PD0325901 (MEK inhibitor 1 µM, Selleckchem S1036) and CHIR99021 (GSK-3 inhibitor 3 µM, Selleckchem S2924) to block the MAPK/ERK and glycogen synthase kinase 3β (GSK-3β) pathways.