IPSCs were maintained with Essential 8 Medium media (Life Technologies) on Geltrex (Life Technologies), and passaged using EDTA (Life Technologies, 0.5 mM). IPSC cultures were kept at 37°C and 5% carbon dioxide. IPSCs underwent differentiation into spinal cord MNs as described in Hall et al.12 (link) Full details can be found in the Supplementary Information .
Essential 8 medium media
Manufactured by Thermo Fisher Scientific
Essential 8 Medium is a cell culture media developed by Thermo Fisher Scientific for the maintenance and expansion of human pluripotent stem cells. It provides the necessary nutrients and growth factors to support the undifferentiated state of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Geltrex, by Thermo Fisher Scientific (6 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (5 mentions)
Dorsomorphin, by Merck Group (3 mentions)
Sb431542, by Bio-Techne (3 mentions)
Chir99021, by Miltenyi Biotec (3 mentions)
Compound e, by Enzo Life Sciences (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
6 protocols using essential 8 medium media
1
IPSC Differentiation into Spinal Cord MNs
2
Differentiation of Human iPSC-derived Astrocytes
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Human hiPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies) and passaged using EDTA (Life Technologies, 0.5 mM). For the in vitro assays and western blot three cultures of human AS were used for each group, which derived from three separate inductions of hiPSCs, using two healthy control lines and a patient carrying the SOD1D90A mutation (Sposito et al., 2015 (link)) (Table S2 ). For qPCR we used three independent AS cultures for each group deriving from hiPSCs of three healthy controls and two patients with the SOD1D90A mutation. These lines included an isogenic control line (SOD1D90D) and its mutant pair (Chen et al., 2014 (link)) (Table S2 ). Spinal AS differentiation was carried out as previously published (Hall et al., 2017 (link), Tyzack et al., 2017 (link)).
3
Generation of Astrocytes from hiPSCs
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hiPSCs were maintained using standard protocols and were differentiated into astrocytes as described previously, generating highly enriched (>90%) populations of astrocytes (Supplementary Figure S1A ) (8 (link),11 (link),48–51 (link)). hiPSCs were maintained on Geltrex (Life Technologies) with serum-free Essential 8 Medium media (Life Technologies), and passaged using EDTA. After neural conversion (7 days in a chemically defined medium containing 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience) and 3.3 μM CHIR99021 (Miltenyi Biotec), neural precursors were patterned for 7 days with 0.5 μM retinoic acid and 1 μM purmorphamine, followed by a 4-day treatment with 0.1 μM purmorphamine. After a propagation phase (60–120 days) with 10 ng/ml FGF-2 (Peprotech), astrocytes were terminally differentiated in presence of BMP4 (10 ng/ml, R&D) and LIF (10 ng/ml, Sigma-Aldrich) for 30 days. Informed consent was obtained from all patients and healthy controls in this study. Experimental protocols were all carried out according to approved regulations and guidelines by UCLH’s National Hospital for Neurology and Neurosurgery and UCL’s Institute of Neurology joint research ethics committee (09/0272).
4
Culturing Induced Pluripotent Stem Cells
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Induced PSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA (Life Technologies, 0.5 mM). All cell cultures were maintained at 37 C and 5% carbon dioxide.
5
Efficient MN Differentiation from iPSCs
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iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA (Life Technologies, 0.5mM). All cell cultures were maintained at 37°C and 5% carbon dioxide. MN differentiation was performed using an adapted version of a previously published protocol (Hall et al., 2017). Briefly, iPSCs were first differentiated from neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, L- Glutamine, N2 supplement, non-essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0 to day 7 was as follows: 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience), and 3 μM CHIR99021 (Miltenyi Biotec). On day 8, the neuroepithelial layer was enzymatically dissociated using dispase (GIBCO, 1 mg/mL), plated onto laminin coated plates and next patterned for 7 days with 0.5 μM retinoic acid and 1μM Purmorphamine. At day 14, MN precursors were treated with 0.1μM Purmorphamine for a further 4 days before being terminally differentiated in 0.1 μM Compound E (Enzo Life Sciences) to promote cell cycle exit.
6
Directed Differentiation of iPSCs to Motor Neurons
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Directed differentiation to human iPSC‐motor neurons was performed as previously reported (Hall et al, 2017 ). Briefly, iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium Media (Life Technologies) and passaged using EDTA (Life Technologies, 0.5 mM). All cell cultures were maintained at 37°C and 5% carbon dioxide. For motor neuron differentiation, iPSCs were differentiated to neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 GlutaMAX, Neurobasal, L‐Glutamine, N2 supplement, non‐essential amino acids, B27 supplement, β‐mercaptoethanol (Life Technologies) and insulin (Sigma). Treatment with the following small molecules from day 0–7: 1 µM dorsomorphin (Millipore), 2 µM SB431542 (Tocris Bioscience) and 3.3 µM CHIR99021 (Miltenyi Biotec). At day 8, cells patterned for 7 days with 0.5 µM retinoic acid and 1 µM purmorphamine. At day 14, spinal cord motor neuron precursors were treated with 0.1 µM purmorphamine for a further 4 days before being terminally differentiated for > 10 days in 0.1 µM Compound E (Enzo Life Sciences) to promote cell cycle exit. Throughout the neural conversion and patterning phase (D0‐18), the neuroepithelial layer was enzymatically dissociated twice (at D4‐5 and D10‐12) using dispase (GIBCO, 1 mg/ml).
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