Chemically defined lipid mixture

Cells were cultured for 3-5 days in hESC media until 80% confluent prior to initiating differentiation. Once at desired confluency, differentiation media was added (Easley et al. 2012 (link); Easley et al. 2015 (link); Greeson et al. 2020 (link)). Differentiation media consisted of MEM-Alpha + L-glutamine (Gibco, Montgomery County, MD), 0.2% bovine serum albumin (Millipore Bio, Burlington, MA), 0.2 mg/ml ascorbic acid (Sigma, St. Louis, MO), 0.2% chemically defined lipid mixture (Sigma, St. Louis, MO), 10μg/ml transferrin in water (Sigma, St. Louis, MO), 5μg/ml insulin in water (Sigma, St. Louis, MO), 20ng/ml recombinant hGDNF (Peprotech, Rocky Hill, NJ), 1ng/ml human recombinant bFGF in 5mM tris, 150mM NaCl buffer at pH 7.5 (Peprotech, Rocky Hill, NJ), 30nM sodium selenite (Sigma, St. Louis, MO), 10 mM HEPES (Gibco, Montgomery County, MD) and 0.5X Penicillin/Streptomycin (Gibco, Montgomery County, MD). The differentiation media was gassed with a blood gas mixture (Airgas, Durham, NC) of 5% CO₂, 5% O₂, balanced with N₂ for 30 seconds, and inverted several times to mix. Media was changed every other day and was gassed for 30 seconds each time prior to use. Differentiation occurred over a ten-day period, where day one was the first day that differentiation media was introduced (Easley et al. 2012 (link); Easley et al. 2015 (link); Greeson et al. 2020 (link)).