Human embryonic stem cells (line H7, WiCell) were grown in mTesR1 media and oligocortical spheroids were generated with minor modifications to the protocol previously described (Madhavan et al., 2018 (link)). Briefly, in the first step of generating oligocortical spheroids, CloneR (Stem Cell Technologies, 5889) was used instead of Y-27632 and Dorsomorphin was replaced with 150nM LDN193189 (Sigma, SML0559). Spheroids were treated with 150nM LDN193189 and 10μM SB-43152 (Sigma, S4317) for the first 6 days followed 20ng/ml FGF-2 (R&D Systems, 233-FB-25/CF) and 20ng/ml EGF (R&D Systems, 236-EG-200) from day 7 to 25.
This was followed by 10ng/ml BDNF (R&D Systems, 248-BD) and 20ng/ml NT-3 (R&D Systems, 267-N3) treatment every other day between days 27 and 40. For OPC development and oligodendrocyte differentiation cultures 10ng/ml PDGF-AA (R&D Systems, 221-AA) and 10ng/ml IGF (R&D Systems, 291-GF-200) were added to cultures every other day between days 51 and 60 an 40n/ml T3 (Sigma, T6397) every other day between days 61 and 70. Spheroids were treated every other day with vehicle DMSO or 300nM MEKi between days 70 and 74 and harvested on day 90. Spheroids were treated with 200μM Hypoxyprobe-1 two hours prior to harvesting for IHC (pimonidazole, Hypoxyprobe Inc, Burlington MA, HP1–100Kit).
This was followed by 10ng/ml BDNF (R&D Systems, 248-BD) and 20ng/ml NT-3 (R&D Systems, 267-N3) treatment every other day between days 27 and 40. For OPC development and oligodendrocyte differentiation cultures 10ng/ml PDGF-AA (R&D Systems, 221-AA) and 10ng/ml IGF (R&D Systems, 291-GF-200) were added to cultures every other day between days 51 and 60 an 40n/ml T3 (Sigma, T6397) every other day between days 61 and 70. Spheroids were treated every other day with vehicle DMSO or 300nM MEKi between days 70 and 74 and harvested on day 90. Spheroids were treated with 200μM Hypoxyprobe-1 two hours prior to harvesting for IHC (pimonidazole, Hypoxyprobe Inc, Burlington MA, HP1–100Kit).