A GBA1–/–hESC line was previously generated and characterized [8 (link)]. Neural precursor cells (NPCs) were generated from the hESCs using a modified dual SMAD inhibition protocol [9 (link)], as previously described [10, 11 (link)]. For neuronal differentiation, NPC were dissociated with 0.05% trypsin-EDTA (ThermoFisher, Cat. 25300054) and plated on poly-L-ornithine/laminin (PLO)-coated dishes at a density of 10,000–15,000 cells/cm2 in N2B27 medium supplemented with 100 ng/ml FGF-8 (PeproTech, Cat.100-25), 200 ng/ml sonic hedgehog (PeproTech, Cat. 100-45), and 100μM ascorbic acid 2-phosphate (Sigma, Cat. A8960-5G) for seven days. Finally, the NPCs were plated on PLO-coated plates at a density of 50,000 cells/cm2 in BGAA medium, which was composed of N2B27 medium supplemented with 20 ng/ml BDNF (R&D System, Cat. 248-BDB-01M/CF), 10 ng/ml GDNF (PeproTech, Cat. 450-10), 500μM Dibutyryl-cAMP (STEMCELL Technologies, Cat. 100-0244), and 100μM ascorbic acid 2-phosphate (Sigma, Cat. A8960-5G). Neuronal cultures were maintained in BGAA medium for 6 weeks, and the cell culture medium was changed every 4 days.
Sonic hedgehog
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sonic hedgehog is a recombinant protein that functions as a signaling molecule in cellular development. It is involved in the regulation of various processes, including embryonic patterning and tissue homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Ascorbic acid 2 phosphate, by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Brain derived neurotrophic factor (bdnf), by R&D Systems (2 mentions)
Dibutyryl camp, by STEMCELL (2 mentions)
Dibutyryl cyclic amp, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
N2b27 medium, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Glial cell derived neurotrophic factor gdnf, by Thermo Fisher Scientific (1 mentions)
9 protocols using sonic hedgehog
1
Differentiation of GBA1-/- hESCs into Neurons
2
Neural Stem Cell Differentiation Protocol
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Neural stem cells (NSCs) were generated and differentiated into neural cultures as described previously72 (link). For patterning, NSCs were plated on polyornithin-/laminin-coated culture flasks at 1E4 cells/cm² in DMEM/F-12 with GlutaMax (#31331093) and neurobasal medium (#21103049) supplemented with 1X B27 (ThermoFisher #12587010), 1X N2 (ThermoFisher #17502048), 50 µM 2-mercaptoethanol (ThermoFisher #31350010) and 100 ng/ml FGF-8 (Peprotech), 200 ng/ml sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma) and cultured for one week. For differentiation, the resultant progenitors were plated at 5E4 cells/cm² in basal medium supplemented with 20 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF; Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), and 100 μM ascorbic acid 2-phosphate.
3
Neural Differentiation of Stem Cells
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Neural precursors were seeded at a concentration of 5 × 104 cells/cm2 and the inducing medium composed of a neurobasal medium (Gibco® BRL, Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10 ng/mL of bFGF (Peprotech®, East Windsor, NJ, USA) and 10 ng/mL EGF (Peprotech®, East Windsor, NJ, USA) was added. Every 3 days of culture, 50% of the medium was replaced with the neurobasal medium supplemented with 10 ng/mL of platelet-derived growth factor—PDGF-AA (Peprotech®, East Windsor, NJ, USA), 10 ng/mL of bFGF (Peprotech ®, East Windsor, NJ, USA) and 10 ng/mL of Sonic Hedgehog (Peprotech®, East Windsor, NJ, USA). After six days of cultivation, the inducing medium was entirely replaced by a fresh inducing medium. Finally, the final differentiation medium was composed of DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA) supplemented with 3% FBS (Sigma-Aldrich®, St. Louis, MO, USA) and 15 nM of F3 (R&D System®, Minneapolis, MN, USA) [20 (link)].
4
Differentiation of LiPS Cells into Diverse Lineages
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Embryoid body (EB) formation was induced by culturing human LiPS in clumps in the absence of FGF in bacterial plates to avoid attachment to the bottom of the plates. To allow spontaneous endoderm formation, after 3–4 days, embryoid bodies were transferred onto 0.2% gelatin-coated glass six-well plates and cultured in differentiation medium (DMEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 0.1 mM 2-mercaptoethanol, nonessential amino acids, and penicillin-streptomycin, all from Invitrogen) for 2 to 3 weeks. The medium was changed every other day. For mesoderm/cardiomyocyte differentiation, LiPS cells were maintained on gelatin-coated plates in differentiated medium supplemented with 100 µM ascorbic acid (Sigma).
For ectoderm differentiation, EBs were cultured on laminin (Stemgent) coated six-well plates. Briefly, after 4 days in EB medium as a floating culture, the cells were plated on laminin (Stemgent) coated plates in N2B27 medium (Invitrogen), Sonic Hedgehog (0.1 µg/µl), and FGF8 (100 ng/ml) (both from Peprotech), and maintained for 3 to 5 weeks in the absence of FGF2. The medium was changed every other day.
For ectoderm differentiation, EBs were cultured on laminin (Stemgent) coated six-well plates. Briefly, after 4 days in EB medium as a floating culture, the cells were plated on laminin (Stemgent) coated plates in N2B27 medium (Invitrogen), Sonic Hedgehog (0.1 µg/µl), and FGF8 (100 ng/ml) (both from Peprotech), and maintained for 3 to 5 weeks in the absence of FGF2. The medium was changed every other day.
5
Neuronal Differentiation Media Formulations
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N2B27 medium is composed of DMEM/F12 supplemented with modified N2 supplement and Neurobasal medium supplemented with B27 supplement minus vitamin A, in a 1:1 ratio and 50 μM β-mercaptoethanol (Thermo Scientific). FEB medium is N2B27 medium supplemented with 10 ng/mL FGF-2 (Peprotech), 10 ng/mL EGF (R&D Technologies), and 20 ng/mL BDNF (Peprotech). SFA medium is N2B27 medium supplemented with 100 ng/mL FGF-8 (Peprotech), 200 ng/mL sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma). BGAA medium is N2B27 medium supplemented with 20 ng/mL BDNF, 10 ng/mL GDNF (Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin (Pen-Strep, Thermo Scientific), and 2 μg/mL Laminin (Roche).
Cyno NPC differentiation medium is composed of Neurobasal medium supplemented with B27 supplement (Thermo Scientific) and GlutaMAX supplement (Thermo Scientific), 20 ng/mL BDNF, 10 ng/mL GDNF, 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 μg/mL Laminin.
Cyno NPC differentiation medium is composed of Neurobasal medium supplemented with B27 supplement (Thermo Scientific) and GlutaMAX supplement (Thermo Scientific), 20 ng/mL BDNF, 10 ng/mL GDNF, 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 μg/mL Laminin.
6
Differentiation of LiPS Cells into Diverse Lineages
7
Neuronal Differentiation Assay Protocol
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The neuronal differentiation assay was performed following our previous work (4 (link)). Briefly, cells were seeded into 24- or 6-well plates at a density of 1×104 or 2×105 cells/well with neurogenic medium consisting of 10% FBS (GE Healthcare Life Sciences), 2% B27 (Gibco; Thermo Fisher Scientific, Inc.), 1 µg/ml ATRA (Merck KGaA), 50 ng/ml sonic hedgehog (PeproTech, Inc.) and 50 ng/ml NT-3 (PeproTech, Inc.) and placed at 37°C in a humidified incubator containing 5% CO2 for two weeks; the medium was replaced every three days. These differentiated cells were fixed with 4% PFA for immunofluorescence staining at 4°C for 12 h, digested using a 0.25% trypsin solution, and washed several times with PBS according to the aforementioned protocol. The primary antibodies were purchased from Chemicon International; Thermo Fisher Scientific, Inc. and comprised of mouse anti-β III Tubulin (Tuj-1; rabbit polyclonal; cat. no. ab1827; Abcam; 1:300), mouse anti-growth associated protein-43 (GAP-43; rabbit polyclonal; cat. no. ab16053; Abcam; 1:300), mouse anti-microtubule-associated protein 2 (MAP2; mouse monoclonal; cat. no. ab11267; Abcam; 1:300) and mouse anti-actin (mouse monoclonal; cat. no. BM5422; Wuhan Boster Biological Technology, Ltd.; 1:400).
8
Differentiation of GBA1-/- hESCs into Neurons
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A GBA1−/− human embryonic stem cell (hESCs) line was previously generated and characterized [9 (link)]. Neural precursor cells (NPCs) were generated from the hESCs using a modified dual SMAD inhibition protocol [28 (link)], as previously described [29 (link),30 ]. For neuronal differentiation, NPC were dissociated with 0.05% trypsin-EDTA (ThermoFisher, Cat. 25300054) and plated on poly-L-ornithine/laminin (PLO)-coated dishes at a density of 10,000–15,000 cells/cm2 in N2B27 medium supplemented with 100 ng/ml FGF-8 (PeproTech, Cat.100–25), 200 ng/ml sonic hedgehog (PeproTech, Cat. 100–45), and 100 μM ascorbic acid 2-phosphate (Sigma, Cat. A8960–5G) for seven days. Finally, the NPCs were plated on PLO-coated plates at a density of 50,000 cells/cm2 in BGAA medium, which was composed of N2B27 medium supplemented with 20 ng/ml BDNF (R&D System, Cat. 248-BDB-01M/CF), 10 ng/ml GDNF (PeproTech, Cat. 450–10), 500 μM Dibutyryl-cAMP (STEMCELL Technologies, Cat. 100–0244), and 100 μM ascorbic acid 2-phosphate (Sigma, Cat. A8960–5G). Neuronal cultures were maintained in BGAA medium for 6weeks, and the cell culture medium was changed every 4 days.
9
Dopaminergic Neuron Differentiation Protocol
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NSCs were grown at high confluence (70%) for 7 days on Matrigel-coated dishes in N2B27 medium supplemented with 200 ng/ml Sonic Hedgehog and 100 ng/ml FGF8 (100-25; PreproTech). This first culture step was required to pattern NPCs as DAn progenitors. For terminal differentiation, DAn progenitors were plated on polyornitine/laminin-coated dishes in N2B27 medium supplemented with 20 ng/ml BDNF (450-02, PeproTech), 20 ng/ml GDNF (450-10, PeproTech), 0.5 mM db-cAMP (A6885-25MG, Sigma-Aldrich) and 5 µM DAPT (565770; Calbiochem, San Diego, CA) for the indicated time points.
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