Cell culture, differentiation, and MN spheroid formation of ALS-iPSC– and hESC-derived cells

Motor neurons. hESC (H9, WA09)–derived NSCs (Gibco) and ALS-iPSC–derived NSCs (from the peripheral blood of a 55-year-old Caucasian woman with sporadic ALS, iXCells Biotechnologies) were maintained on Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific) in StemPro NSC SFM medium supplemented with 2 mM GlutaMAX-I supplement (Gibco), human recombinant bFGF (20 ng/ml), epidermal growth factor (20 ng/ml), and 2% StemPro neural supplement. Neuro spheroids were formed using a spindle-shaped bottom 96-well plate (PrimeSurface 96M) for 24 hours by seeding the hESC-derived NSCs and ALS-iPSC–derived NSCs at a density of 1.0 × 104 cells per well. The differentiation protocol was previously described in the literature (56). Briefly, after 24 hours of seeding, the culture medium was replaced with 100 μl per well of StemPro hESC medium supplemented with RA (50 μM), sonic hedgehog (200 ng/ml), bFGF (8 ng/ml), and activin A (10 ng/ml) for cell fate determination via caudalization and ventralization (MN differentiation medium). Then, culture medium was changed to 100 μl per well of MN differentiation medium excluding activin A. After culturing for 20 days, the culture medium was replaced again using 100 μl per well of StemPro hESC medium supplemented with RA (50 μM), BDNF (10 ng/ml), and GDNF (10 ng/ml) for maturation of MNs and cultured for 7 to 8 more days (MN medium). To remove the neural progenitor cells and NSCs from the spheroids, CultureOne Supplement (Thermo Fisher Scientific) was treated before 24 hours of injection into the devices. The culture medium was then switched to MN medium without RA for an additional 14 days in the left medium reservoir of the microfluidic devices. Karyotype, donor, and reprogramming information of iPSCs used in this study is available at the following links: parental iPSCs of iCell skeletal myoblast and iCell ECs (https://hpscreg.eu/cell-line/CDIi001-A), parental ES cells of NSC (https://hpscreg.eu/cell-line/WAe009-A), and parental iPSCs of ALS-NSCs [www.ixcellsbiotech.com/image/data/Human%20iPSC%20(ALS)/Human%20iPS%20Cell%20Line%20(ALS).pdf]. G-band karyotyping analysis of parental ALS-iPSCs (performed by Cell Line Genetics) is shown in fig. S6D.

iPSC-derived skeletal muscle cells. Human iPSC-derived skeletal myoblasts (iCell Skeletal Myoblasts, Cellular Dynamics Technology) were maintained on fibronectin coating (10 μg/ml) in minimum essential medium–α (MEM-α) supplemented with 8-bromo-cyclic AMP (1 mM), CHIR99021 (2 μM), dorsomorphin (1 μM), and 5% knockout serum replacement (SkMM). For differentiation into mature myocytes in the microfluidic devices, 24 hours after cell seeding with SkMM, 200 μl of culture medium was injected into the right medium reservoir with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% horse serum, human recombinant IGF-1 (50 ng/ml), and 1% penicillin/streptomycin (SkDM). After several days, the injected culture medium invaded the left medium reservoir owing to the formation of muscle fiber bundle and capillary action. Then, SkDM was also injected to both the right and left medium reservoir (200 μl of each) up to 14 days of culture.

iPSC-derived ECs. Human iECs (iCell ECs, Cellular Dynamics Technology) were maintained on fibronectin coating (50 μg/ml) with a VascuLife VEGF Endothelial Cells Medium Complete Kit (Lifeline Cell Technology) and iCell growth supplement.

Mouse myoblasts. Mouse C2C12 myoblasts (American Type Culture Collection) were cultured to <70% confluency in growth medium consisting of DMEM with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin. None of the cells were used beyond a passage number of 20. All cells were kept in incubators at 37°C and 5% CO2.

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