B27 insulin

Prior to differentiation, cells were dissociated with Accutase™ (StemCell Technologies, Vancouver, BC, Canada) and passaged to six-well plates. Until 100% confluence of iPSCs, NutriStem™ (Biological Industries) was refreshed. On day 0, the medium was changed to 3 mL of RPMI (Biological Industries), supplemented with 0.5% L-glutamine (Biological Industries), B27-Insulin (Invitrogen, Carlsbad, CA, USA) and 4.5 µM CHIR-99021 (Tocris, Bristol, UK). On day 2, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine, B27-Insulin, and 5 µM IWP-2 (Tocris). On day 4, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27-Insulin. This medium was refreshed on day 6. On day 8, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27, and this medium was refreshed on day 10. From day 12, the medium was changed to M-199 (Biological Industries), supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological Industries), 0.6 mM CuSO₄·5 H₂O, 0.5 mM ZnSO₄·7 H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich, Darmstadt, Germany). This medium was refreshed every other day.

Prior to differentiation,
cells were passed to 6-well plates. NutriStem was refreshed daily
until iPSCs reached 100% confluence. At that point (Day 0), the medium
was changed to 3 mL of RPMI (Biological Industries), supplemented
with 0.5% l-glutamine (Biological Industries), B27-Insulin
(Invitrogen, Carlsbad, California), and 4.5 μM CHIR-99021 (Tocris,
Bristol, UK). On Day 2, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine, B27-Insulin, and 5 μM IWP-2
(Tocris). On Day 4, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine and B27-Insulin, and this medium was
refreshed on Day 6. On Days 8 and 10, the medium was changed to 3
mL of RPMI supplemented with 0.5% l-glutamine and B27. From
Day 12, the medium was changed to M-199 (Biological Industries), supplemented
with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological
Industries), 0.6 mM CuSO₄·5H₂O, 0.5 mM
ZnSO₄·7H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich).
This medium was refreshed every other day.^{74 (link)}