To generate dorsally patterned forebrain organoids, we modified the method previously described in Kadoshima et al.3 (link). We eliminated the need for growth under 40% O2, the need for cell aggregates to be periodically bisected, and the use of high O2 penetration dishes, by adapting the cultures to growth in spinner-flask bioreactors. Specifically, on day 0, feeder-free cultured human PSCs, 80–90% confluent, were dissociated to single cells with Accutase (Gibco), and 9,000 cells per well were reaggregated in ultra-low cell-adhesion 96-well plates with V-bottomed conical wells (sBio PrimeSurface plate; Sumitomo Bakelite) in Cortical Differentiation Medium (CDM) I, containing Glasgow-MEM (Gibco), 20% Knockout Serum Replacement (Gibco), 0.1 mM Minimum Essential Medium non-essential amino acids (MEM-NEAA) (Gibco), 1 mM pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Corning). From day 0 to day 6, ROCK inhibitor Y-27632 (Millipore) was added to the medium at a final concentration of 20 μM. From day 0 to day 18, Wnt inhibitor IWR1 (Calbiochem) and TGFβ inhibitor SB431542 (Stem Cell Technologies) were added at a concentration of 3 μM and 5 μM, respectively. From day 18, the floating aggregates were cultured in ultra-low attachment culture dishes (Corning) under orbital agitation (70 rpm) in CDM II, containing DMEM/F12 medium (Gibco), 2mM Glutamax (Gibco), 1% N2 (Gibco), 1% Chemically Defined Lipid Concentrate (Gibco), 0.25 μg/mL fungizone (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. On day 35, cell aggregates were transferred to spinner-flask bioreactors (Corning) and maintained at 56 rpm, in CDM III, consisting of CDM II supplemented with 10% fetal bovine serum (FBS) (GE-Healthcare), 5 μg/mL heparin (Sigma), and 1% Matrigel (Corning). From day 70, organoids were cultured in CDM IV, consisting of CDM III supplemented with B27 supplement (Gibco) and 2% Matrigel. A step-by-step protocol describing the long-term culture of dorsally patterned forebrain organoids is available at Protocol Exchange 33 . Over the time course of the differentiations (experimental batches) included in this paper, an estimated 13 batches of FBS, 4 batches of KSR, and 23 batches of Matrigel were used.
We observed that when starting with healthy viable hiPSCs (mycoplasma-free, karyotypically normal and below passage 50) with typical morphological features of undifferentiated cells (tightly packed colonies of round cells with large nuclei and nucleoli) at 80–90% confluency, the efficiency of forebrain cell type generation was ~90% (93/103 organoids, single-cell RNA-seq and IHC combined efficiency,Extended Data Table 1 ).
Self-patterned whole-brain organoids were generated as previously described3 (link),34 . Dorsal and ventral spheroids were generated using a modification of a previously described protocol15 (link). Specifically, spheres of pluripotent stem cells were grown for four additional days before neural induction; for ventral spheroids, specification of ventral telencephalic identity was obtained by treatment with the SHH agonist SAG (Selleckhem; 100nM) from days 7 to 20.
We observed that when starting with healthy viable hiPSCs (mycoplasma-free, karyotypically normal and below passage 50) with typical morphological features of undifferentiated cells (tightly packed colonies of round cells with large nuclei and nucleoli) at 80–90% confluency, the efficiency of forebrain cell type generation was ~90% (93/103 organoids, single-cell RNA-seq and IHC combined efficiency,
Self-patterned whole-brain organoids were generated as previously described3 (link),34 . Dorsal and ventral spheroids were generated using a modification of a previously described protocol15 (link). Specifically, spheres of pluripotent stem cells were grown for four additional days before neural induction; for ventral spheroids, specification of ventral telencephalic identity was obtained by treatment with the SHH agonist SAG (Selleckhem; 100nM) from days 7 to 20.
