Human recombinant basic fgf

Mouse embryonic fibroblasts and Venus porcine neonatal fibroblasts were cultured in complete DMEM AQ (high‐glucose Dulbecco's modified Eagle's medium [D0819; Sigma–Aldrich, St. Louis, MO] supplemented with 1% penicillin/streptomycin [Pen/Strep] [Sigma–Aldrich] and 10% fetal bovine serum [FBS] [Hyclone]). HEK‐293T cells were cultured in DMEM with high glucose (1965; Invitrogen, Waltham, MA), supplemented with 1× Glutamax (Invitrogen), 1× Pen/Strep, and 10% FBS (In vitro A/S, Denmark). Derived Venus piPSCs were cultured in piPSC medium (DMEM/F12 medium [Sigma–Aldrich] supplemented with 20% Knockout Serum Replacement [Invitrogen]), 1× Pen/Strep [Sigma–Aldrich], 1× non‐essential amino acids [Sigma–Aldrich], and 100 μM β‐mercaptoethanol [Life technologies, Waltham, MA]) supplemented with 10 ng/mL LIF (Millipore, Billerica, MA) or 20 ng/mL human recombinant basic FGF (Prospec, East Brunswick, NJ), 2i (1 μM PD0325901 [Sigma–Aldrich], 3 μM CHIR99021 [Sigma–Aldrich]), and 2 μg/mL doxycycline (Sigma–Aldrich).

Venus neonatal fibroblasts were harvested using 0.25% Trypsin‐EDTA solution, and 2 × 10⁴ cells were plated per well of 0.1% gelatin‐coated 6‐well plates. Cells were transduced 24 hr after plating using a total MOI of 20 for both viruses (pOSMK and rTA) and 8 μg/mL polybren (Sigma–Aldrich). Forty‐eight hours after addition of lentivirus, the medium was replaced with complete DMEM containing 2 μg/mL doxycycline (Sigma–Aldrich) to induce transgene transcription. Fours days later, the transduced Venus neonatal fibrobalsts were transferred onto mitomycin C–treated (Sigma–Aldrich) mouse embryonic fibroblasts (Millipore). On Day 5, the medium was replaced with piPSC medium containing 10 ng/mL LIF (Millipore) or 20 ng/mL human recombinant basic FGF (Prospec, East Brunswick, NJ), 2i (1 μM PD0325901 [Sigma–Aldrich], 3 μM CHIR99021 [Sigma–Aldrich]), and 2 μg/mL doxycycline (Sigma–Aldrich). On Day 17, colonies were visible and were manually picked to establish clonal lines. The Venus piPSCs were maintained in reduced oxygen (5% O₂, 5% CO₂ in N₂) in a humidified chamber at 38.5°C. Cells were dissociated with 1× TrypLE (Gibco Thermo Fisher Scientific, Waltham, MA), and passaged 1:6 every 7 days.