Human bdnf

The use of all animals was approved by the Institutional Animal Care and Use Committee at the University of California, Irvine. Primary neuronal cultures were obtained from embryonic day 18 (E18) rat embryos. Dissociated cells were cultured on either pre-coated poly-L-Lysine 6-well plates (Biocoat plates, BD Bioscience) or poly-L-Lysine coated glass coverslips affixed to microfluidic chambers at 1 × 10⁶ cells/9.5 cm² and 5 × 10⁶ cells/ml (for microfluidic chamber), respectively. All cells were maintained in Neurobasal (Gibco) supplemented with B27, penicillin/streptomycin, and Glutamax at 37 ^oC, 5% CO₂, 95% humidity. Rat recombinant IL-1β (PeproTech) was used at a final concentration of 10 ng/ml. Human BDNF (PeproTech) was used at a concentration of 50 ng/ml and Ciliobrevin D (Sigma) was dissolved in sterile Me₂SO at 100 μM and used at 1 μM.

To confirm that both the BDNF antibodies bound specifically to BDNF we performed an absorption control test using recombinant BDNF protein (Human BDNF; PeproTech). First, BDNF protein was eluted in 2× Laemmli buffer [250 mm Tris, 10% (v/v) glycerol, 4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 0.005% (w/v) bromophenol blue (Sigma-Aldrich)] in double-deionized water (DDW; pH 6.8), separated by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Precast Gels; Bio-Rad) and transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer Pack; Bio-Rad). Recombinant proteins were detected by Western blot using 3% (w/v) skim milk powder in TBS-T [100 mm Tris, 154 mm NaCl, 0.1% (v/v) Tween 20, in DDW, pH 7.5] blocking buffer, antibodies to BDNF (rabbit; Biosensis R-172-20, 1:500 or Santa Cruz Biotechnology, SC-546; 1:500) and α-rabbit-HRP secondary antibody (1:10,000 dilution; Pierce Scientific). Proteins were visualized by chemiluminescence (Immun-Star; Bio-Rad) using the ChemiDoc system (Bio-Rad). In the absorption control, the anti-BDNF antibody (1 µg, rabbit; Biosensis R-172-20) was incubated with 10 μg of BDNF diluted in 100 µl blocking agent and incubated overnight at 4°C. The pre-absorbed primary antibody was then used to immunostain purified RGC cultures using the protocol described above.

TRE3G-NGN2 was integrated into the adeno-associated virus integration site (AAVS) of the hESCs and iPSCs as previously described^{50 (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).

Cells were differentiated to a striatal neuron fate using a published method [44 (link)]. Briefly, PSC monolayers were first directed to a forebrain neural progenitor fate by way of the presence of N2 and B27 supplements (Gibco, Thermo Fisher Scientific) and BMP and SMAD inhibitors (100 nM LDN193189 (Sigma-Aldrich, Merck); 200 nM dorsomorphin (Cambridge Biosciences); 10 μM SB431542 (Cambridge Biosciences)) over a 9-10 day period. Thereafter, patterning to a lateral ganglionic eminence/striatal fate was achieved by the addition of 25 ng/ml activin A (PeproTech), which was maintained throughout the remainder of the differentiation process. The addition of 10 ng/ml human BDNF (PeproTech), 10 ng/ml human GDNF (PeproTech) and vitamin A-containing B27 for 10 days from day 26 post-induction onwards aided maturation of these striatal precursors to a more mature striatal neuron identity, resulting in cultures expressing markers of striatal GABAergic neurons, including a proportion expressing a marker of full maturity, DARPP32.

iPSCs were differentiated into neurons following a previously validated and published protocol (Zhang et al., 2013 (link)). Briefly, iPSCs were cultured in mTesR1 medium added with Ngn2 and rtTA lentivirus on day -1, and then the next day (Day 0) the culture medium was replaced with DMEM/F12 medium containing N2 (Thermo Fisher), NEAA (Invitrogen), human BDNF (10 mg/l, PeproTech), and human NT-3 (10 mg/l, PeproTech). Doxycycline (2 g/l, Clontech) was added on day 0 to induce TetO gene expression. On day 2, a 24 hr puromycin (1 mg/l) selection period was started. On day 3, medium was changed to Neurobasal medium containing B27(Thermo Fisher), Glutamax (Invitrogen), BDNF and NT3; After day 7, 50% of the medium in each well was exchanged every 2 days. On day 14, iPSCs-derived neurons (iN) were collected and processed for RNA extraction and gene expression analysis.