Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 104 cells per cm2 in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml−1 activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml−1 ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml−1 ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml−1 BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml−1 ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml−1 FGF10 (Stemcell Technologies), 10 ng ml−1 FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.
Sb431542
Manufactured by Miltenyi Biotec
Sourced in United States
SB431542 is a small molecule that selectively inhibits the transforming growth factor-beta (TGF-β) type I receptor ALK5. It is a useful tool for studying the TGF-β signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Dorsomorphin, by Abcam (3 mentions)
Bdnf and nt3, by Fujifilm (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Y 27632, by Selleck Chemicals (3 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (3 mentions)
Su5402, by Merck Group (3 mentions)
Ldn 193189, by Miltenyi Biotec (3 mentions)
Facsaria 3, by BD (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Monothioglycerol, by Merck Group (2 mentions)
Dorsomorphin, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Thermo Fisher Scientific (2 mentions)
Monothioglycerol, by Thermo Fisher Scientific (2 mentions)
Monothioglycerol, by STEMCELL (2 mentions)
Activin a, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Merck Group (2 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions)
Fgf10, by STEMCELL (2 mentions)
Chir99021, by Merck Group (2 mentions)
12 protocols using sb431542
1
Directed Differentiation of iPSCs into Endoderm
2
Cortical Spheroids Generation from hiPSCs
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Cortical spheroids were produced following Pasca et al. [52 (link)]. Briefly, enzymatically lifted hiPSCs were transferred to ultra-low attachment plates and cultured in DMEM supplemented with Knockout Serum Replacement (Gibco) supplemented with 5 μM Dorsomorphin (BioVision, Milpitas, CA, USA), 10 μM SB431542 (Miltenyi Biotec) for 6 days. Then, 10 μM Y27632 (Selleck Chemicals) was added during the first 48 h. The resulting spheroids were then maintained in neurobasal media until day 90: Neurobasal A medium, 2% B-27 supplement without vitamin A, GlutaMAX (Gibco) and penicillin/ streptomycin (Gibco). Spheroids were supplemented with 20 ng/mL of FGF and EGF (PeproTech) from day 6 to 25, and 20 ng/mL of BDNF and NT3 (Shenandoah Biotechnology) from day 26 to 42. Spheroids were harvested beginning at day 90 for analysis. Once a month, the cultures were evaluated to ensure they were free of mycoplasma.
3
Derivation and Differentiation of FUS-Mutant iPSCs
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Derivation and maintenance conditions of human iPSCs carrying the P525L mutation in both FUS alleles (FUSP525L, male) and their isogenic FUS wild-type control (FUSWT, male) used in this study are described in Lenzi et al. (2015) (link). The MN differentiation protocol is detailed in De Santis et al. (2017) (link) and depicted in Figure S1 A. In brief, cells were differentiated in N2B27 medium supplemented with 1 mM all-trans retinoic acid (Sigma-Aldrich) and 1 mM SAG (Merck Millipore) for 12 days in the presence of 10 mM SB431542">SB431542 and 100 nM LDN-193189 (both from Miltenyi Biotec) from day 0 to 6, and 5 mM DAPT and 4 mM SU-5402 (both from Sigma-Aldrich) from day 6 to 12. Cells were sorted at day 12-13 using a FACSAria III (BD Biosciences) and re-plated on poly-L-ornithine- and laminin-coated dishes (both from Sigma-Aldrich) in Neural Medium as described in De Santis et al. (2017) (link).
4
Directed Differentiation of iPSCs into Endoderm
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Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 104 cells per cm2 in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml−1 activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml−1 ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml−1 ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml−1 BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml−1 ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml−1 FGF10 (Stemcell Technologies), 10 ng ml−1 FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.
5
Mesenchymal Stem Cell Differentiation
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MSC differentiation was induced by exchanging StemMACS iPS-Brew XF medium with differentiation medium containing 20% KSR, 1% GlutaMAX, 1% NEAA, and 0.1 mM beta-mercaptoethanol supplemented with 10 μM SB431542 (Miltenyi Biotec). Fresh medium was applied every other day and after 7 days, cells were transferred in a 1:3 ratio to a non-coated tissue culture-treated plate with MSC expansion medium (Miltenyi Biotec). Fresh medium was applied daily before splitting the cells at differentiation day 14. Process control of MSC differentiation was performed by flow cytometry and RT-qPCR at day 21. At day 21, MSCs were differentiated to adipocytes or osteocytes using StemMACS AdipoDiff Media or StemMACS OsteoDiff Media (both Miltenyi Biotec), respectively. Fresh medium was applied every 3 days for 20 days before process control by OilRed O or Alizarin Red staining, respectively.
6
Generation of Cortical Spheroids from hiPSCs
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Cortical spheroids were produced following an adaption to Pasca methods, Nature Methods 201541 (link). Briefly, enzymatically lifted hIPSCs were transferred to ultra-low attachment plates and cultured in DMEM supplemented with Knockout Serum Replacement (Gibco) supplemented with 5 μM Dorsomorphin (BioVision), 10 μM SB431542 (Miltenyi Biotec), 10 μM Y27632 (Selleck Chemicals) for 6 days. The resulting spheroids were then maintained in neurobasal media until day 90: Neurobasal A medium, 2% B-27 supplement without vitamin A, GlutaMAX L-glutamine supplement (Gibco) penicillin/ streptomycin (Gibco). Spheroids were next supplemented with 20 ng/mL of bFGF and EGF (PeproTech) from day 6 to 25, and 20 ng/mL of BDNF and NT3 (Shenandoah Biotechnology) from day 26 to 42. Spheroids were harvested beginning at day 90 for analysis (refer to Fig. 3 for experimental details).
7
Efficient Generation and Maintenance of iPSC-Derived MNs
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Generation and maintenance of iPSC lines is described in Lenzi et al. (2015) (link). The MN differentiation protocol has been modified from Hill et al. (2016) (link). In brief, cells were differentiated in N2B27 medium supplemented with 1 μM all-trans retinoic acid (Sigma-Aldrich) and 1 μM SAG (Merck Millipore) for 12 days in the presence of 10 μM SB431542 and 100 nM LDN-193189 (both from Miltenyi Biotec) from day 0 to 6, and 5 μM DAPT and 4 μM SU-5402 (both from Sigma-Aldrich) from day 6 to 12. Cells were sorted at day 12 using a FACSAria III (BD Biosciences) and re-plated on poly-L-ornithine-coated dishes and laminin- coated dishes (both from Sigma-Aldrich) in Neural Medium. The Hb9:GFP reporter was stably integrated in the AAVS1 locus, as described previously (Wainger et al., 2014 (link)).
8
Endothelial Cell Differentiation from iPSCs
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Endothelial differentiation was performed as described previously with some modifications.[12 (link)] The iPSCs at subconfluency were detached using CTK solution consisting of 0.1 mg/ml collagenase IV (Invitrogen), 0.25% trypsin (Invitrogen), 0.1 mM CaCl2 (Nacalai tesque) and 20% KSR and seeded onto Matrigel-coated dishes at a ratio of 1:5 to 1:10. We treated iPSCs with 50 ng/mL bone morphogenetic protein 4 (BMP4; R & D Systems, Minneapolis, MN) and 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan) for the first 24 h; 40 ng/mL vascular endothelial growth factor (VEGF; Invitrogen, Waltham, MA) and 50 ng/mL bFGF for 2 days; and 40 ng/mL VEGF, 50 ng/mL bFGF, and 20 μmol/L SB431542 (Miltenyi Biotec, Teterow, Germany) for 3–4 days. On days 6–7, ECs were purified using FITC-conjugated anti-CD31 antibody (1 μg/1 × 106 cells, WM59; BioLegend, San Diego, CA) and APC-conjugated anti-CD144 antibody (0.5 μg/1.0 × 106cells; 16B1, eBioscience) with a FACSAria III (BD Biosciences, San Jose, CA).
9
Cortical Spheroid Production Protocol
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Cortical spheroids were produced following the methods described by Pasca et al. [38 (link)]. Briefly, enzymatically lifted hIPSCs were transferred to ultra-low attachment plates and cultured in DMEM supplemented with Knockout Serum Replacement (Gibco) supplemented with 5 μM Dorsomorphin (BioVision, Milpitas, CA, USA), 10 μM SB431542 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 μM Y27632 (Selleck Chemicals) for 6 days. The resulting spheroids were then maintained in neurobasal media until day 90: Neurobasal A medium, 2% B-27 supplement without vitamin A, GlutaMAX L-glutamine supplement (Gibco) penicillin/streptomycin (Gibco). Spheroids were next supplemented with 20 ng/mL of bFGF and EGF (PeproTech, Cranbury, NJ, USA) from day 6 to 25, and 20 ng/mL of BDNF and NT3 (Shenandoah Biotechnology, Warminster, PA, USA) from day 26 to 42. Spheroids were harvested beginning at day 90 for analysis.
10
Differentiation of iPSCs into ECs
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