Ciliary neurotrophic factor

RGCs from retinas of rat pups post natal 4–7 days were purified by a Thy-1.1 antibody-panning method [35 (link)] as described by Barres et al. [36 (link)]. Timed-pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA, USA). All procedures were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee (IACUC) at University of North Texas Health Science Center at Fort Worth, TX, USA. Briefly, retinas were separated from the enucleated eyeballs of postnatal day 4 to 7 rat pups and dissociated using papain treatment. The cell suspension was panned with a rabbit anti-macrophage antibody (Cedarlane, Burlington, Ontario, Canada) to exclude macrophages followed by a panning with an anti-Thy1.1 antibody to selectively bind RGCs. The collected RGCs were seeded on glass coverslips coated with mouse-laminin and poly-D-lysine in serum-free Dulbecco’s modified Eagle’s medium containing brain-derived neurotrophic factor (50 ng/mL; Peprotech, Rocky Hill, NJ, USA), ciliary neurotrophic factor (10 ng/mL; Peprotech), and forskolin (5 ng/mL; Sigma-Aldrich Corp.). Cells were incubated at 37°C in a humidified atmosphere of 10% CO₂ and 90% air. One-half volume of the culture medium was changed every two days.

Mixed ventral horn cultures were prepared as previously described [6 (link)–8 (link)]. Briefly, ventral horns from E11.5–13.5 WT and SOD1^G93A mice were dissociated, centrifuged at 380 × g for 5 min, seeded into two-chambered microfluidic devices (Fig. 3A) [7 (link)], and maintained in motor neuron media (Neurobasal (Gibco) with 2% B27 (Gibco), 2% heat-inactivated horse serum, 1% Glutamax (Invitrogen), 24.8 µM β-mercaptoethanol, 10 ng/ml ciliary neurotrophic factor (Peprotech, 450–13), 0.1 ng/ml GDNF (Peprotech, 450–10), 1 ng/ml BDNF (Peprotech, 450–02) and 1 × penicillin streptomycin (Thermo Fisher; 15140122)) at 37 °C and 5% CO₂. After 6 days in vitro (DIV6), 30 nM H_CT-555 and ± 50 ng/ml of BDNF was added to existing media for 45 min, then all media was replaced with fresh MN media containing 20 mM HEPES–NaOH (pH 7.4) ± 50 ng/ml of BDNF for time-lapse microscopy. Live imaging was performed on an inverted LSM780 confocal microscope at 37 °C using a 40x, 1.3 NA DIC Plan-Apochromat oil- immersion objective (Zeiss). Videos were taken at 2 frames/s for > 2.5 min. Videos were manually tracked using TrackMate [54 (link)] to determine endosome track dynamics (Fig. 3). The breakdown of each experimental group can be found in Additional file 2: Table S2.

Mouse OPCs were isolated from the cortices of pups at postnatal days 3–8 as described previously.^[30] Briefly, cortical tissues were dispersed into single cells and the cell suspension was then subjected to immunopanning with antibodies against GalC and O4 sequentially. The enriched Galc‐negative O4‐positive OPCs were plated into poly‐D‐lysine coated dishes and cultured with Mouse OPC growth medium (DMEM/F‐12 (GIBCO, Cat# 11330‐032) supplemented with 1% N2 supplement (GIBCO, Cat# A1370701), 2% B27 supplement (GIBCO Cat# A3582801), penicillin–streptomycin solution (MP Biomedicals, Cat# 0916700), 1% sodium pyruvate (GIBCO, Cat# 11360070), 1% L‐glutamine (Hyclone, Cat# SH30034), 10 ng mL⁻¹ platelet‐derived growth factor‐aa (Peprotech, Cat#100‐13A), 10 ng mL⁻¹ ciliary neurotrophic factor (Peprotech, Cat# 450‐13), 20 ng mL⁻¹ human basic fibroblast growth factor (Sino Biological, Cat# 10014HNAE), 0.5 mg mL⁻¹ insulin (Sigma, Cat# 91077), 5 mg mL⁻¹N‐acetyl cysteine (Sigma, Cat# A8199), 10 ng mL⁻¹ D‐biotin (Sigma, Cat# B4639), 5 mm forskolin (Sigma, Cat# F3917), and 0.1% Trace Elements B (Corning, Cat# 25‐022‐CI)). OPC differentiation medium contained the same components as growth medium except human basic fibroblast growth factor and platelet‐derived growth factor‐aa, but supplemented with 40 ng mL⁻¹ triiodo‐thyronine (Sigma, Cat# T2877).

HBG3 Hb9::EGFP mouse ESCs (a gift from H. Wichterle, Columbia University) and newly generated Hb9::GFP and Hb9::Foxp1; Hb9::GFP mouse ESCs were maintained and differentiated into MNs as previously described16 52 (link). Briefly, mouse ESCs were first plated on gelatin to remove MEFs prior to differentiation, and then plated in 60 mm bacterial Petri dishes in core MN medium to induce EB formation. Core MN medium consisted of a 1:1 mixture of DMEM/F12 and Neurobasal Medium supplemented with 10% knockout serum replacement, 1% Glutamax, 2-mercaptoethanol (560 nM), 1% penicillin/streptomycin and Primocin. Except as noted, media components were obtained from Invitrogen. About 2 days later, N2 supplement (1 × , Invitrogen), RA (1 μM; Sigma), and SAG (1 μM; Calbiochem) were added to the EBs. After 5–6 days, the EBs were either dissociated and plated on matrigel-coated coverslips for immunostaining or used for transplantation and muscle assays. For culturing the MNs past day 6, RA and SAG were removed from the medium and replaced with GDNF (10 ng ml⁻¹; Peprotech), Brain-Derived Neurotrophic Factor (10 ng ml⁻¹; Peprotech), and Ciliary Neurotrophic Factor (10 ng ml⁻¹; Peprotech).

Spinal cord motoneurons were prepared from E12.5 OF1 mice embryos as described by Henderson et al. [14 ] with minor modifications. Briefly, anterior horn of the embryo were dissected in HBSS supplemented with 4.5 g/l glucose and 7 mM HEPES (invitrogen). Motoneurons were purified by using a 6% OptiPrep density gradient medium (D1556, Sigma). Then, motoneurons were resuspended in supplemented Neurobasal medium (Invitrogen) containing 1 ng/ml brain-derived neurotrophic factor (Peprotech), 1 ng/ml glial cell line–derived neurotrophic factor (Peprotech), and 10 ng/ml ciliary neurotrophic factor (Peprotech) and were seeded on polyornithin/laminin-coated glass coverslips (P8638 and L2020, Sigma). After two days in vitro, plasmid transfections were done by Magnetofection following the manufacturer recommendations (OZBiosciences). Two or four days later, motoneurons were fixed using 4% paraformaldehyde for 20 min, and washed with PBS.