Primed and Naive Human Stem Cell Culture

Conventional (primed) human iPSC line C1 (Whitehead Institute Center for Human Stem Cell Research, Cambridge, MA) (Hockemeyer et al., 2008 (link)) and human ESC lines WIBR2 and WIBR3 (Whitehead Institute Center for Human Stem Cell Research, Cambridge, MA) (Lengner et al., 2010 (link)) were maintained on mitomycin C inactivated MEF feeder layers and passaged mechanically using a drawn Pasteur pipette or enzymatically by treatment for 20 min with 1 mg/ml Collagenase type IV (GIBCO) followed by sequential sedimentation steps in human ESC medium (hESM) to remove single cells. Primed human ESCs and human iPSCs were cultured in hESM—DMEM/F12 (Invitrogen) supplemented with 15% FBS (Hyclone), 5% KSR (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), penicillin-streptomycin (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and 4 ng/ml FGF2 (R&D systems). Naive human ESCs/hiPSCs were cultured on mitomycin C-inactivated MEF feeder cells and were passaged every 5–7 days by a brief PBS wash followed by single-cell dissociation using 3–5 min treatment with Accutase (GIBCO) and centrifugation in fibroblast medium (DMEM [Invitrogen] supplemented with 10% FBS [Hyclone], 1 mM glutamine [Invitrogen], 1% nonessential amino acids [Invitrogen], penicillin-streptomycin [Invitrogen], and 0.1 mM β-mercaptoethanol). For conversion of preexisting primed human ESC lines, we seeded 2 × 10⁵ trypsinized single cells on an MEF feeder layer in hESM supplemented with ROCK inhibitor Y-27632 (Stemgent, 10 μM). One or two days later, medium was switched to 5i/L/A-containing naive hESM. Following an initial wave of widespread cell death, dome-shaped naive colonies appeared within 10 days and could be picked or expanded polyclonally using 3–5 min treatment with Accutase (GIBCO) on an MEF feeder layer. Naive human pluripotent cells were derived and maintained in serum-free N2B27-based media supplemented with 5i/L/A. Medium (500 ml) was generated by inclusion of the following: 240 ml DMEM/F12 (Invitrogen; 11320), 240 ml Neurobasal (Invitrogen; 21103), 5 ml N2 supplement (Invitrogen; 17502048), 10 ml B27 supplement (Invitrogen; 17504044), 10 μg recombinant human LIF (made in-house), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), penicillin-streptomycin (Invitrogen), 50 μg/ml BSA (Sigma), and the following small molecules and cytokines: PD0325901 (Stemgent, 1 μM), IM-12 (Enzo, 1 μM), SB590885 (R&D systems, 0.5 μM), WH-4-023 (A Chemtek, 1 μM), Y-27632 (Stemgent, 10 μM), and Activin A (Peprotech, 20 ng/ml). 0.5% KSR (GIBCO) can be included to enhance conversion efficiency. FGF2 (R&D systems, 8 ng/ml) enhanced the generation of OCT4-ΔPE-GFP+ cells from the primed state, but it was dispensable for maintenance of naive human ESCs. Additional chemicals described in this work include: CHIR99021 (Stemgent, 0.3–3 μM as indicated), SP600125 (R&D systems, 10 μM), PD173074 (Stemgent, 0.1 μM), SB431542 (Tocris, 5 μM), BIRB796 (Axon Medchem, 2 μM), and doxycycline (Sigma-Aldrich, 2 μg/ml). Tissue culture media were filtered using a low protein-binding binding 0.22 μM filter (Corning). Alternative formulations for naive human ESC culture were followed as described elsewhere (Chan et al., 2013; Gafni et al., 2013; Valamehr et al., 2014; Ware et al., 2014 ). All experiments in this paper were performed under physiological oxygen conditions (5% O₂, 3% CO₂) in the presence of a MEF feeder layer unless stated otherwise.