Dorsomorphin

As a control for our AMOs, we generated cortical hiPSC organoids from the same hiPSC line used to derive smNPCs for AMO line 2 (Reinhardt et al., 2013b (link)). After manual 2D culture of hiPCs, all steps were fully automated using our liquid handling system (Beckman Coulter) with attached incubator (Thermo Fisher). Generally, we followed the protocol previously published by Paşca et al., 2015 (link). (and also described in more detail by Sloan et al., 2018 (link)), with adaptations for our automation pipeline (see Figure 7—figure supplement 1). Starting with 90–100% confluent cultures, we detached hiPSCs with accutase and seeded 10,000 cells per well in ultra-low attachment U-bottom plates (Corning). Cortical organoid medium consisted of DMEM F-12, 20% Knock-out Serum replacement (GIBCO), 1% penicillin/streptomycin/glutamine, 1% Non-essential amino acids (Sigma-Aldrich), and 0.2% 2-Mercaptoethanol (Thermo Fisher). For the first 6 days, we supplemented the cortical organoid medium with 5 μM dorsomorphin (Enzo Life Sciences) and 10 μM SB-431542 (Biomol). During seeding only, we also added 10 μM ROCK inhibitor Y-27632. Aggregates were fed every 3 days using an automated liquid handling system. From day 6 to 24, culture medium supplements were exchanged to EGF and FGF2 (both 20 ng/ml, PeproTech) and afterwards BDNF and NT3 (metabion) (both 20 ng/ml).

Transfection, plating and starving of cells were done as described in the supplementary information. Cells were treated for 48 hrs with either vehicle (0.1% BSA in ddH₂O), 6 μg/mL recombinant murine Shh-N (Pepro Tech) reconstituted in vehicle solution, 1 μM Dorsomorphin (Enzo Life Sciences), or 400 nM Smoothened Agonist (Alfa Aesar). Fixed and immunostained samples were imaged with an upright Zeiss LSM 780 confocal and an EC Plan-Neofluar 40x/1.3 oil objective. Only cells with comparable RFP/mCherry fluorescence intensity were used for measurements of EGFP fluorescence intensity within the soma. The mean intensity of EGFP was quantified by drawing a free-hand region of interest (ROI) around the cell soma using ZEN 2012 SP1 software. All samples were blinded before imaging and quantification.

MPCs and ACs were cultured as 3D pellets (5 × 10⁵ cells/pellet) in chondrogenic medium (DMEM high-glucose; Gibco, Life Technologies, Germany), 0.1 μM dexamethasone, 0.17 mM ascorbic acid-2 phosphate, 5 μg/mL transferrin, 5 ng/mL selenous acid, 2 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/mL bovine serum albumin (all from Sigma Aldrich, Germany), 5 μg/mL insulin (Lantus, Sanofi-Aventis, Germany), or ITS + premix (Corning, Germany), 1% penicillin/streptomycin (Biochrom, Germany), 10 ng/mL recombinant human TGF-β1 (PeproTech or Miltenyi, Germany) for up to 6 weeks at 37°C, 6% CO₂ with medium changes three times a week. For indicated time points, chondrogenic medium was supplemented with the BMP-inhibitor dorsomorphin (10 μM in 0.1% DMSO, day 14–42; ENZO Life Science, United States), the WNT-inhibitor IWP-2 (2 μM in 0.04% DMSO, day 14–35; Tocris Bioscience, United Kingdom), the FGF-inhibitor PD173074 (250 nM in 0.02% DMSO, day 7–42; Sigma Aldrich, Germany), or the respective solvent. Where indicated, TGF-β was withdrawn from day 21 onward. One group received daily PTHrP(1-34) treatment for 6 h (2.5 nM in water; Bachem, Germany) as described before (Fischer et al., 2016 (link)).