Laminin

Samples were first sterilized by immersion in ethanol washed twice in sterile water, dried in a laminar flow hood and further sterilized by UV irradiation for 1 h. The day before dissection substrates were coated with 0.05–0.1% polyethyleneimine (PEI, MW 25.000, Sigma-Aldrich) solution for 45 min. Then, the PEI solution was removed and substrates were washed 4 times with sterile distilled water and stored at 4°C. To perform random neuronal growth on the samples, the day of dissection, substrates were coated with 100 μl laminin (Sigma-Aldrich) [20 μg/ml] and incubated at 37°C for 2 h. laminin was removed by aspiration prior to seeding dissociated neurons. All biosecurity and safety procedures were followed, as specifically required by the Health & Safety Office of the Istituto Italiano di Tecnologia (Genova, Italy).

hiPSC sensory co‐cultures were carried out as previously described,^[27
] with minor changes regarding cell types. 48‐well MEAs from Axion Biosystems (Atalanta, GA) were first pre‐treated overnight with 3 to 5 µl of 0.1% polyethyleneimine (PEI, Sigma‐Aldrich, St. Louis, MO), washed twice using DI water, allowed to dry, and then coated with 3 to 5 µl of 20 µg ml⁻¹ laminin (Sigma‐Aldrich, St. Louis, MO) for at least 1 h. laminin was aspirated immediately prior to seeding and substrates were not allowed to dry completely. hiPSC glia (BX‐0600, BrainXell, Madison, WI) and hiPSC SN (Anatomic, Minneapolis, MN) were thawed rapidly, separately diluted in 37 °C DMEM (ThermoFisher Scientific, Waltham, MA), centrifuged at 300× g for 5 min, and reconstituted in supplemented SN medium (Anatomic's Senso‐MM without Inhibitors plus 1:1000 BrainXell astrocyte supplement). Following a viable cell count, cell solutions were mixed and co‐cultures were seeded simultaneously at a 2:3 astrocyte:sensory neuron ratio in a 5 µl droplet placed at the center of each MEA well. After cells were visually confirmed to be attached, the wells were gently flooded with 200 µl of fresh, pre‐warmed supplemented SN medium (described above) and cultures were housed in a 37 °C, 10% CO₂ incubator at ≈90% humidity. 50% medium exchanges were performed every alternate day for the duration of the culture.

The NE cells from monkey ESCs were detached with TrypLE Select (Thermo Fisher Scientific) and suspended in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Thermo Fisher Scientific) supplemented with 20 ng/mL FGF2, 200 ng/mL SHH, 20 ng/mL PDGF-AA (R&D Systems), 1 × B-27 Supplement without vitamin A (Thermo Fisher Scientific), 1 × N-2 Supplement (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich), and were then allowed to grow in suspension as spheres for 44 days. The medium was changed twice weekly. The spheres were treated with 1 × Accutase (Thermo Fisher Scientific) for 5 min and centrifuged. The cell pellet was resuspended with the same medium and dissociated into single cells by pipetting. The cells were transferred to plates coated with 0.01% poly-L-ornithine (Sigma-Aldrich) and 10 μg/mL laminin (Sigma-Aldrich) (poly-L-ornithine/laminin) and cultured for 7 days. The medium was changed twice weekly. The cells were detached with Accutase and centrifuged. The cells were suspended in STEM-CELLBANKER (Takara Bio) and cryopreserved in -80°C refrigerator.

aNPCs were plated in default media (A-DMEM/F12, 1% P/S, 0.5% Glutamax, 0.5% N2, 0.2% B27, 2 µg/mL Heparin (Sigma)) on poly-D-lysine (Sigma), laminin (Sigma), fibronectin (Sigma) and matrigel coated coverslips for differentiation and fed twice a week. Default media was supplemented with 10 µM forskolin (Tocris) in weeks 2 and 3. From week 4 onwards forskolin was removed and default media was supplemented with 5 ng/mL BDNF and 5 ng/mL GDNF.
For motor neuron differentiation aNPCs were first caudalised in proliferation media with 0.3 µM RA and then plated down on poly-D-lysine (Sigma), laminin (Sigma) and fibronectin (Sigma) coated coverslips in Neurobasal, 0.1 µM RA, 2 µM Purmorphamine (Calbiochem), 1% N2, 1% P/S, 1% Glutamax, 5 ng/ml bFGF for 7–10 days to generate motor neuron presurcors. Motor neuron precursors were replated and media was gradually switched to Neurobasal, 0.5% N2, 0.2% B27, 1% P/S, 0.5% Glutamax, 10 ng/ml BDNF, 10 ng/ml GDNF.

Two lentiviral vectors, namely pLOX-Ttag-iresTK (addgene No. 12246) and Ef1a-Large T-Ires-Puro (addgene No. 18922), were purchased from addgene. Lentiviruses were produced by co-transfecting the either transfer vector and the 2^nd generation packaging vectors psPAX2 and pMD2.G into HEK293T cells, and were concentrated using ultracentrifugation. The porcine cells collected after differential plating were seeded to 12-well plates coated with laminin (20 μg/mL; Sigma-Aldrich), and transduced with the prepared lentiviruses by using “spinfection”. This method can be used to transduce SSCs with substantial efficiency [22 (link)]. Briefly, the cells exposed to 10 μg/mL polybrene (Sigma-Aldrich) and the concentrated virus supernatant at a multiplicity of infection (MOI) of 100 were centrifuged at 3,000×g for 1 h at 32 °C, followed by 16 h of incubation at 37 °C. One day after lentiviral transduction, cells were subjected to FACS employing an antibody against PLD6 (rabbit anti-PLD6; ab237612, Abcam). The sorted PLD6⁺ cells were seeded to 24-well plates coated with laminin (20 μg/mL; Sigma-Aldrich) for expansion.

Mice at the age of 12–16 weeks were sacrificed with CO₂, and their DRG neurons were collected and cultured in accordance with the procedures described in Cheng et al., (2010) [14 (link)] and Lin et al., (2016) [15 (link)]. The dissected DRGs were digested with 0.125% collagenase (type I, Sigma-Aldrich, St. Louis, MO, USA) and 2 units/mL dispase II (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. The segregated neurons were triturated using a flame-polished Pasteur pipette and seeded on laminin (Sigma-Aldrich, St. Louis, MO, USA)-coated polydimethylsiloxane (PDMS, UNI WARD, New Taipei City, Taiwan) substrate, which was prepared on a 12 mm coverslip with a base-to-curing-agent ratio of 35:1. Before the DRG neurons were seeded, the PDMS-covered coverslips were exposed to ultraviolet light for 15 min and coated with poly-L-lysine (0.01%, Sigma-Aldrich, St. Louis, MO, USA) for 10 min; then, they were coated with laminin (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The neurons were cultured in a 3.5 cm Petri dish with DMEM plus 10% fetal bovine serum and maintained in an incubator with 5% CO₂ at 37 °C for 2–3 days. The cultured DRG neurons were then subjected to electrophysiological recordings of acid-induced currents or mechanically activated currents.

For the assessment of cellular invasion, fluorescent MSCs and GBM cells and mixed spheroids were transferred into 24-well plates (Corning, Life Sciences, USA) and embedded in collagen type I (1 mg/mL; BD Biosciences) or Matrigel (6.35 mg/mL; BD Biosciences). To assess cellular invasion on laminin, the 96-well plates were coated with laminin (2 μg/cm²; Sigma-Aldrich). The spheroids were then placed in the middle of each well in the growth medium. Cell invasion was monitored using a fluorescence inverted microscope with the NIS-Elements software (Eclipse Ti-E; Nikon, Japan). Invasiveness was defined as the distance measured from the edge of the spheroids to the most distant cell population, divided by the spheroid diameter.