In order to determine the actual fate commitment of the generated NPCs, we performed further differentiation in mature neurons. Since the two NPC generation protocols showed no statistically significant difference, we selected the 1323–2 line generated using the double BMP/SMAD inhibition protocol and the WT3 mouse NPCs to further differentiate into mature neurons following the procedure described by Gunhanlar et al. [29 ]. Briefly, NPCs at passage 7 were dissociated using TrypLE and plated on poly-ornithine/laminin-coated plates and cultured using neural differentiation medium (neurobasal medium, 1% N2 supplement, 2% B27-RA supplement, 1% NEAA, 20 ng/ml BDNF, 20 ng/ml GDNF, 1 μM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich), 200 μM ascorbic acid (Sigma-Aldrich), 2 μg/ml laminin, and 1% penicillin/streptomycin) for 35 days before immunostaining.
Dibutyryl cyclic adenosine monophosphate
Manufactured by Merck Group
Sourced in United States
Dibutyryl cyclic adenosine monophosphate is a chemical compound used in research and laboratory settings. It is a structural analog of the secondary messenger molecule cyclic AMP, which plays a crucial role in various cellular signaling pathways. This product is primarily utilized as a research tool to study the effects of increased cyclic AMP levels in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Ascorbic acid, by Merck Group (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 13 dibutyrate, by Merck Group (1 mentions)
Isobutylmethylxanthine, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
α mem, by Welgene (1 mentions)
Insulin, by Merck Group (1 mentions)
Insulin transferrin selenium supplement, by Thermo Fisher Scientific (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Transforming growth factor β3, by R&D Systems (1 mentions)
L proline, by Merck Group (1 mentions)
Ascorbic acid, by R&D Systems (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Dexamethasone dexa, by Merck Group (1 mentions)
5 protocols using dibutyryl cyclic adenosine monophosphate
1
Neuronal Differentiation of NPCs
2
Differentiation of HT22 cells into neurons
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HT22 cells were purchased from the National Laboratory of Molecular Oncology, Cancer Institute and Hospital (Chinese Academy of Medical Sciences and the Peking Union Medical College, Beijing, China) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco), in a humidified atmosphere of 5% CO2/95% air at 37°C.
A 24-hour incubation with modified DMEM containing 100 mM dibutyryl cyclic adenosine monophosphate (Sigma, St. Louis, MO, USA), 100 mM phorbol 12,13-dibutyrate (Sigma), 50 ng/mL nerve growth factor-beta (Sigma), and 1× N2 supplement (Gibco) was used to induce the differentiation of HT22 cells into cells with neuronal characteristics and functions (Zhao et al., 2016).
A 24-hour incubation with modified DMEM containing 100 mM dibutyryl cyclic adenosine monophosphate (Sigma, St. Louis, MO, USA), 100 mM phorbol 12,13-dibutyrate (Sigma), 50 ng/mL nerve growth factor-beta (Sigma), and 1× N2 supplement (Gibco) was used to induce the differentiation of HT22 cells into cells with neuronal characteristics and functions (Zhao et al., 2016).
3
Multilineage Differentiation of ADMSCs
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For adipogenic differentiation, ADMSCs were differentiated using growth medium containing 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, St. Louis, MO), 10 μg/mL insulin (Sigma-Aldrich), 100 nM dexamethasone (DEXA; Sigma-Aldrich), and 100 μM indomethacin (Sigma-Aldrich) for 2 weeks. Adipogenic differentiation was confirmed by staining cells with Oil Red O (Sigma-Aldrich). For osteogenic differentiation, ADMSCs were incubated in α-minimum essential medium (α-MEM; Welgene, Daegu, Korea) containing 10% fetal bovine serum, 1% amino acids, 10 mM β-glycerophosphate (Sigma-Aldrich), 50 μg/mL ascorbic acid (Sigma-Aldrich), 10 nM DEXA, and 1 mM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich) for 2 weeks. Fixed cells were stained with Alizarin Red S (Sigma-Aldrich). For chondrogenic differentiation, cells were pellet-cultured in chondrogenic differentiation medium, which was α-MEM containing 1% amino acids, 50 ng/mL ascorbic acid, 100 nM DEXA, 10 ng/mL transforming growth factor-β3 (R&D Systems, Minneapolis, MN), 1× insulin-Transferrin-Selenium Supplement (Gibco), and 40 μg/mL L-proline (Sigma-Aldrich) for 3 weeks. Fixed pellets were embedded in optimal cutting temperature compound medium (Sakura Finetek, Torrance, CA). Cryostat sections (8-μm thick) were stained with Alcian Blue (Sigma-Aldrich).
4
Directed Differentiation of Neural Cells
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For the generation of neurons, astrocytes and oligodendrocytes, reprogrammed DPSC were seeded at 1.5 × 104 cells/cm2 onto 0.1 mg/mL poly-D-lysine (Sigma-Aldrich) (overnight at room temperature) and 2 µg/mL laminin (Gibco) (overnight at 37 °C) (neurons and oligodendrocytes) or gelatin (astrocytes) coated glass coverslips or tissue-culture treated 6-well plates, and differentiation media were added. For neurons, DMEM (5 mM glucose) (Gibco) supplemented with 2 mM L-glutamine, 2% (v/v) B-27™ supplement, 1% (v/v) N-2 supplement, 1 mM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich), and 30 ng/mL neurotrophin-3 (Prospec), was added for 4 weeks [25 (link), 54 (link), 55 (link)]. For astrocytes, DMEM (Gibco) supplemented with 1% (v/v) FBS, 2 mM L-glutamine, and 1% (v/v) N-2 supplement, was added for 2 weeks (ThermoFisher Scientific). For oligodendrocytes, DMEM/F12 supplemented with 2 mM L-glutamine and 2% (v/v) B-27™ supplement was used, with 10 ng/mL basic fibroblast growth factor, 10 ng/mL platelet-derived growth factor (ImmunoTools) and 10 nM forskolin (Sigma-Aldrich) added for the first 5 days, then 200 nM L-ascorbate 2-phosphate and 30 ng/mL triiodothyronine (Sigma-Aldrich) added for the next 5 days [56 (link)]. The media were refreshed every 2–3 days.
5
Differentiation of HDNPCs to Neurons
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HDNPCs were purchased from ABMGood (T4034, Richmond, BC, Canada). As per vendor, cells are human-derived precursor cells [P2 fetal tissue (14–16weeks gestation)] obtained from the brain, and the isolation method used included a tyrosine free medium supplemented with a mixture of growth factors. These cells were cultured in plates coated with poly-L -lysine (PLL) at a density of 1 × 104 cells/cm2 using proprietary PriGrow IV culture medium (TM004) supplemented with 5% fetal bovine serum (TM999). At confluence, 10 ng/mL basic fibroblast growth factor (R&D Systems 4114TC), 10 ng/mL epidermal growth factor (R&D Systems 236-EG-01 M) and 100 µM dibutyryl-cyclic adenosine monophosphate (Sigma D0627) were added to PriGrow IV. Cells were maintained in this media for 7 days to achieve full differentiation to neurons prior to assay performance.
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