Hypoxic Chondrocyte Differentiation of hESCs

HUES7 hESCs (Howard Hughes Medical Institute/Harvard University, USA) were cultured at 5% O₂ (hypoxia) on Matrigel coated plates as described previously^{40 (link)}. Cells were passaged at ~ 80% confluency using Collagenase IV. hESCs were differentiated into chondrocytes at either 20% O₂, or 5% O₂ in medium (DMEM: F12 containing 1 × non-essential amino acids, 1 × B27 supplement, 90 μM β-mercaptoethanol and 1X ITS supplement: 10 μg/ml insulin, 5.5 μg/ml transferrin and 5 ng/ml selenite premix) supplemented with growth factors over a 14-day period. Medium was replenished daily. Growth factors were added as follows: Day 1, 25 ng/ml WNT3A (R&D) and 50 ng/ml Activin-A (Peprotech); Day 2, 25 ng/ml WNT3A, 25 ng/ml Activin-A and 20 ng/ml FGF2 (Invitrogen); Day 3, 25 ng/ml WNT3A, 10 ng/ml Activin-A, 20 ng/ml FGF2 and 40 ng/ml BMP4 (Peprotech); Days 4–7, 20 ng/ml FGF2, 40 ng/ml BMP4, 100 ng/ml Follistatin (Sigma) and 2 ng/ml NT4 (Peprotech); Day 8, 20 ng/ml FGF2, 40 ng/ml BMP4 and 2 ng/ml NT4; Day 9 and 10, 20 ng/ml FGF2, 20 ng/ml BMP4, 20 ng/ml GDF5 (Peprotech), 2 ng/ml NT4 and 10 ng/ml TGF-β3 (Peprotech); Days 11–14, 20 ng/ml FGF2, 40 ng/ml GDF5, 2 ng/ml NT4 and 10 ng/ml TGF-β3. Cells were dissociated using collagenase IV and passaged at a ratio of 1:2 or 1:3 on days 4 and 9 of the protocol.