hiPSCs were treated with 0.5 mM EDTA and plated as dissociated cells in Essential 8 medium containing 5 μM Y-27632 (MedChemExpress, USA) at a density of 105 cells/mL onto vitronectin-coated dishes on day −1. On day 0, the culture medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM)/F12 containing N-2 supplement, non-essential amino acids (NEAA), human brain-derived neurotrophic factor (BDNF) (10 μg/L, PeproTech, USA), human neurotrophin-3 (NT-3) (10 μg/L, PeproTech), mouse laminin (0.2 mg/L, Invitrogen) and doxycycline (2 mg/L). On day 1, a 24 hr puromycin selection (1 mg/L) period was started. On day 2, the transfected cells were replated in Neurobasal medium supplemented with B27/Glutamax (Invitrogen) containing BDNF and NT-3. On day 5, Ara-C (2 μM, Sigma-Aldrich, USA) was added to the medium for 24 hr to inhibit the proliferation of undifferentiated cells. After this, half of the medium in each dish was replaced every 3–4 days. The iN cells were assayed on day 28 during most experiments.
Human neurotrophin 3 nt3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human Neurotrophin-3 (NT3) is a recombinant protein that plays a role in the survival, growth, and differentiation of specific neuronal populations. It is a member of the neurotrophin family of growth factors.
Automatically generated - may contain errors
Lab products found in correlation
Mouse laminin, by Thermo Fisher Scientific (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
Human brain derived neurotrophic factor bdnf, by Thermo Fisher Scientific (1 mentions)
Ara c, by Merck Group (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Y 27632, by MedChemExpress (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Human bdnf, by Thermo Fisher Scientific (1 mentions)
4 protocols using human neurotrophin 3 nt3
1
Generation of Induced Neuronal Cells
2
Induced Neuronal Differentiation of Human ES Cells
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A detailed version of this protocol is available at dx.doi.org/10.17504/protocols.io.br9em93e . Briefly, human ES cells (H9, WiCell Institute) with TRE3G-NGN2 integrated into the AAVS site have been previously described (Ordureau et al., 2020 (link)) and were cultured in E8 medium on Matrigel coated plates. To generate induced neurons (i3-neurons) from ES cells, cells were plated at 2 × 105 cells/ml on Day 0 on plates coated with Matrigel in ND1 medium (DMEM/F12, 1× N2 (thermo), human brain-derived neurotrophic factor (BDNF) (10 ng/ml, PeproTech)), human Neurotrophin-3 NT3 (10 ng/ml, PeproTech), 1× nonessential amino acids, Human Laminin (0.2 μg/ml) ,and doxycycline (2 μg/ml). The media was replaced with ND1 the next day. On the next day, the medium was replaced with ND2 neurobasal medium, 1× B27, 1× Glutamax, BDNF (10 ng/ml), NT3 (10 ng/ml), and doxycycline at 2 μg/ml. On Days 4 and 6, 50% of the media was changed with fresh ND2. On Day 7, cells were replated at 4 × 105 cells/well in ND2 medium supplemented with Y27632 (rock inhibitor – 10 μM). The media was replaced the next day with fresh ND2 and on Day 10 onwards 50% media change was performed until the experimental day (Day 14 of differentiation unless otherwise noted).
3
Differentiation of Stem Cells to Induced Neurons
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TRE3G-NGN2 was integrated into the adeno-associated virus integration site (AAVS) of the hESCs and iPSCs as previously described50 (link). To start differentiation to iNeurons from stem cells (day 0), cells were plated at 2 × 105 cells ml−1 onto Matrigel-coated plates into ND1 medium (DMEM/F12, 1X N2 (Thermo Fisher Scientific), human BDNF (10 ng ml−1; PeproTech), human neurotrophin-3 (NT3, 10 ng ml−1; PeproTech), 1X NEAA, human laminin (0.2 μg ml−1) and doxycycline (2 mg ml−1) also containing Y27632 (ROCK inhibitor, 10 mM). The medium was replaced with ND1 without Y27632 the next day. The following day, the medium was replaced with ND2 (neurobasal medium, 1X B27, 1X GlutaMAX, BDNF (10 ng ml−1), NT3 (10 ng ml−1) and doxycycline at 2 mg ml−1. On days 4 and 6, 50% of the medium was changed with fresh ND2. On any day in the day 4–7 range, cells were replated at 4 × 105 cells well−1 in ND2 medium with Y27632. The medium was replaced the next day with fresh ND2 (without Y27632). Every other day, 50% of the medium was changed with ND2. At day 9 and onwards, doxycycline was removed from the ND2 mixture. iNeurons were fed every other day with 50% medium change until the experimental day (day 12 of differentiation, unless otherwise noted).
4
Differentiation of hESCs into iNeurons
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TRE3G-NGN2 was integrated into the AAVS site of the hESCs as previously described (Ordureau et al., 2020). To start differentiation to induced neurons (i-Neurons) from ES cells (day 0), cells were plated at 2x105 cells/ml onto Matrigel coated plates into ND1 medium (DMEM/F12, 1X N2 (thermo), human Brain-derived neurotrophic factor BDNF (Brain derived neurotrophic factor) (10 ng/ml, PeproTech), human Neurotrophin-3 NT3 (10 ng/ml, PeproTech), 1X NEAA (Non-essential amino acids), Human Laminin(0.2 ug/ml) and Doxycycline (2 mg/ml)) also containing Y27632 (rock inhibitor-10 mM)). The media was replaced with ND1 without Y27632 the next day. The following day media was replaced with ND2 (Neurobasal medium, 1X B27, 1X Glutamax, BDNF (10 ng/ml), NT3 (10 ng/ml) and doxycycline at 2 mg/ml. On Days 4 and 6, 50% of the media was changed with fresh ND2. On any day in the Day 4–7 range, cells were replated at 4x105 cells/well in ND2 medium with Y27632. The media was replaced the next day with fresh ND2 (without Y27632) Every other day 50% of the media was changed with ND2. At Day9 and onwards doxycycline was removed from the ND2 mixture. iNeurons were fed every other day with 50% media change until the experimental day (Day12 of differentiation unless otherwise noted).
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