Towards a 3D spheroid system for modelling leukodystrophies 135 4 to an adapted version of the Marton protocol (Marton et al., 2019). Prior to differentiation, EB formation was started from single cells suspension generated by accutase dissociation (Merck Millipore, A6964) for 5-7 minutes. Cells were collected by centrifugation (5 min, 400 rcf) in E8 medium (Thermo Fisher, A1517001). Pellets were resuspended in E8 medium supplemented with ROCK inhibitor Y-27632 (10 µM, SIGMA, Y0503). Uniform spheroids were obtained by dropwise plating of 3 x 106 cells in each well of an AggreWell™800 (STEMCELL Technologies, 34815) which was pretreated with Anti-Adherence Rinsing Solution (STEMCELL Technologies 07010). After plating, cells were captured in the bottom by centrifugation at 100 rcf for 3 minutes and placed in an incubator (37°C, 5% CO2). After 24 hours, spheroids of approximately 300 µm had formed in each of the 300 microwells. Spheroids were collected by pipetting up and down the medium with sterile wide-bore tips (Thermo Fisher, 11374245) and transferred to low attachment 10 cm petridishes. To initiate the differentiation, at day 1 of the protocol, spheroids were plated in E6 medium (Thermo Fisher, A1516401) supplemented with SMAD pathway inhibitors dorsomorphin (2.5 µM, Sigma-Aldrich, P5499) and SB-431542 (10 µM, Sigma-Aldrich, S4317). The medium was refreshed daily on the next two days. On day 4 and 5 also Wnt pathway inhibitor IWP-2 (5 µM, Selleckchem, S7085) was used in the medium. The following six days medium was still refreshed daily but with Differentiation and Maintenance Medium (DMM), which consisted of DMEM/F12 1:1 (Gibco) (Thermo Fisher Scientific, 11-330-057) with 1x B-27 supplement without vitamin A (Thermo Fisher Scientific, 12587010), 1x N2 supplement (Invitrogen, 17502048), 1x MEM NEAA (Invitrogen, 11140035), 1x Glutamax (Invitrogen, 35050038), 25 µg/ml Human Insulin (Sigma-Aldrich, I9278-5ML), 0.1 mM β-mercaptoethanol (Invitrogen, 31350010), 100 U/ml P/S and supplemented with 20 ng/ml EGF (Peprotech, AF10015B) 20 ng/ml FGF-2 (Peprotech, 167100-18B) and 5 µM IWP-2 (Selleckchem, S7085). Between day 12 and 17, also 1 µM of Smoothened agonist (Cayman, 11914) was supplemented to the daily refreshed medium. From day 18 onward, frequency of medium changes was reduced to every other day. On day 25, DMM was supplemented with 60 ng/ml T3 (Sigma-Aldrich, T2877), 100 ng/ml Biotin (Sigma-Aldrich, B4639), 20 ng/ml NT-3 (Peprotech, 450-03), 20 ng/ml BDNF (Peprotech, 450-02), 1 μM cAMP (Sigma-Aldrich, D0627), 5 ng/ml HGF (Peprotech, 315-23), 10 ng/ml IGF-1 (VWR, 100-11), 10 ng/ml PDGF-AA (R&D Systems, 221AA). On day 37, DMM was supplemented with 60 ng/ml T3, 100 ng/ml Biotin, 1 μM cAMP and 20 μg/ml Ascorbic Acid (SIGMA, A4403). Spheroids were maintained in this medium until the end of the experiment, from day 44 onwards, the medium changes were reduced to twice a week.
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