Human neural stem cells isolation and culture
To produce the hNSCs lines starting from the foetal brain specimen, two main steps have been performed: i) Primary culture, ii) Intermediate Product preparation and cryopreservation. (i) Primary culture. Human brain tissue was immediately transferred under strict sterile conditions to the GMP facility in a controlled environment. Then brain specimen was washed in a PBS solution (Dulbecco’s PBS 1X, Carlo Erba Reagent) supplemented with 50 µg/ml of gentamicin and dissociated to reach a monocellular suspension mechanically. Cells were seeded at a density of 104 cells/cm2 in a chemically defined culture medium with EGF 20 ng/ml and bFGF 10 ng/ml as previously described(74). Cultures were maintained in a humidified incubator at 37°C, 5% O2 and 5% CO2 and cells were allowed to proliferate as free floating clusters named neurospheres according to ‘neurosphere assay technique’. This technique allows to obtain a hNSCs culture with the very same characteristic independently from donor gestation age. Approximately 7–10 days after the primary cell seeding, neurospheres were collected in 15 mL tube, centrifuged, the supernatant discharged, and the cell pellet mechanically dissociated using p200 micropipette. The obtained single-cell suspension was stained with 0.4% Trypan Blue stain solution (Invitrogen, cat #T10282) and counted using a Burker chamber. A cell viability near 95–98% was measured in each neurospheres passage. Throughout these passages aliquots of cells were frozen as neurospheres and cryopreserved in culture medium with 10% dimethyl sulfoxide(50). When adhesion culture was necessary glass supports were coated with Cultrex (BME, Pathclear cat #3432-005-01) 1:50 in hNSC-medium for 1 hour at 37°C in according to manual instruction. In the monoculture experiments, a seeding density of 1x104 cells/cm2 was used. In both homotype and heterotype co-culture experiments, a ratio of 1:1 was respected, seeding a total of 2.5x104 cells/cm2.
SH-SY5Y cells culture and differentiation
Human SH-SY5Y neuroblastoma cells (ATCC, CRL-2266™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM F-12, Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin (Invitrogen) and 0.1 mg/ml streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% CO2.
For the experiments, the cover glasses were pretreated with Cultrex™ and cells were seeded at a density of 2,5 × 104 cells per 12 mm (Ø) cover glass in 24-well plates or at the density of 5 x 104 cells per 15 mm (Ø) cover glass in 12-well plates. Immediately after the adhesion of the cells the culture media was substituted with the neuron differentiation medium which consisted of Dulbecco’s Modified Eagle’s Medium (DMEM F-12, Invitrogen) supplemented with 1% fetal bovine serum, 100 U/ml penicillin (Invitrogen), 0.1 mg/ml streptomycin and 1µM retinoic acid (RA, S-R2625-, Sigma)(75) (76). The cells were differentiated for 7 days before their use, with the differentiating medium being replaced daily. The RA was freshly added to the medium immediately before the use. The correct differentiation of human SH-SY5Y was evaluated by whole-cell patch clamp experiments as the ability to generate evoked action potentials (75, 76). Correctly differentiated human SH-SY5Y were indicated in the main text as human neurons.
Immunofluorescence in neurospheres and 2D-culture
Neurospheres at 7–10 days were collected, resuspended in PBS and fixed in 4% paraformaldehyde for 30 minutes. After fixation, neurospheres were washed in PBS and cryoprotected in 30% sucrose in PBS overnight at 4°C. The day after, neurosphere were gently collected by centrifugation and the sucrose/PBS solution was removed. Fixed and cryoprotected neurospheres were resuspended in OCT-medium, frozen in liquid nitrogen and cryosection in 50µm depth sections were prepared. Sectioned neurospheres were permeabilized by 0.3% Triton X-100 in PBS for 30min, blocked in 3% BSA, 0.3% Triton X-100 in PBS for 30 minutes, and incubated with primary antibody diluted in 0.3% Triton X-100, 3% BSA in PBS for 24 hours at 4°C. The day after, were extensively washed with 1.5% BSA, 0.15% Triton X-100 in PBS and incubated in the same solution with secondary antibodies for 24h. The day after sections were washed many times in PBS and mounted in Vectashield antifade mounting medium (Vector Laboratories cat. #H-1000-10) for confocal microscopy.
In 2D-culture, to minimize breakage of the TNTs, fixation with 4% paraformaldehyde was performed for 10 min at room temperature, gently replacing the culture medium with it. Afterward, samples were carefully washed with PBS, permeabilized by 0.3% Triton X-100 in PBS for 15min, blocked with 3% BSA in PBS for 30 minutes, and incubated with primary antibody in 3% BSA in PBS for 24 hours at 4°C. After the incubation the samples were gently washed with 3% BSA in PBS multiple times and then incubated with secondary antibodies for one hours at room temperature in 3% BSA in PBS, finally washed many times and mounted in Vectashield antifade mounting medium (Vector Laboratories cat. #H-1000-10).
Antibodies and working dilution
CD15/SSEA1 (MC480) Mouse mAb (Cell Signaling, cat #4744) 1:500. SOX2 (L1D6A2) Mouse mAb (Cell Signaling, cat #4900) 1:400. Nestin (10C2) Mouse mAb #33475 (Cell Signaling, cat #33475) 1:1000. Musashi-1 Antibody (R&D Systems, cat #AF2628) 1:100.
Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (ThermoFisher, cat. #A-21202) 1:1000. Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 546 (ThermoFisher, cat. #A-11056) 1:1000.
Electron Mircoscopy
For Trasmission Electron Microscopy (TEM), after 12 days in culture hNSCs were collected, washed in PBS 1X (w/o Ca/Mg) and fixed with 2.5% glutaraldehyde in 0.1M PBS, pH 7.4, at 4°C for 2 h, washed, post-fixed with 1% OsO4 at 4°C for 1 h. After rinsing in 0.1M PBS, the samples were dehydrated in graded concentrations of acetone and embedded in a mixture of Epon and Araldite (Electron Microscopic Sciences, Fort Washington, PA, USA). Ultrathin sections were cut at 70 nm thickness on an Ultracut E ultramicrotome (Reichert-Jung, Heidelberg, Germany), contrasted with lead citrate and observed on a Philips Morgagni 268 D electron microscope (Fei Company, Eindhoven, The Netherlands), equipped with a Megaview III camera for acquisition of digital images.
For Scanning Electron Microscopy (SEM) analysis, after 12 days in culture hNSCs were collected, washed in PBS 1X (w/o Ca/Mg) and fixed in 2,5% glutaraldehyde in 0.1 M PB for 2h at 4°C, washed in the same buffer, post-fixed with 1% OsO4 at 4°C for 1 h, and dehydrated with graded concentrations of ethanol (Fluka). The samples were then treated using a drying agent hexamethyldisilazane (HMDS), mounted on metallic specimen stubs and sputter-coated with gold (MED 010 Balzers). SEM imaging was performed by an XL30 ESEM (FEI-Philips).
Staining of mitochondria and F-actin for live-cell fluorescence microscopy
For live-imaging, neurospheres were incubated in suspension with 1uM CellMask™ Green Actin Tracking Stain (Invitrogen, cat. #A57243) diluted in hNSCs medium, for 30 min at 37°C. After 30min of incubation, cells were centrifugated and resuspended in fresh prewarmed hNSC-medium at least two times. The day after were seeded on confocal dish (Greiner Bio-One, cat. #627870) previously coated with cultrex as described above.
For live imaging of mitochondria in the neurosphere, spheres were incubated with 100 nM Mito-Tracker Green FM (MT; Invitrogen, cat. #M7514) or 100 nM MitoTracker™ Red CMXRos (Invitrogen, cat. #M7512) diluted in hNSC-medium. After 30min of incubation, cells were centrifugated and resuspended in fresh prewarmed hNSC-medium at least two times, left overnight in culture and centrifugated. The cell pellet was washed once to clear the excess dye completely. The day after were seeded on confocal dish previously coated with cultrex as described above.
For live-imaging of mitochondria, hNSCs single-cell suspension was incubated with 100 nM Mito-Tracker Green FM (MT; Invitrogen, cat. #M7514) or with 100 nM MitoTracker™ Red CMXRos (Invitrogen, cat. #M7512) diluted in hNSC-medium. After 30min of incubation, cells were centrifugated and resuspended in fresh prewarmed hNSC-medium at least two times, left overnight in culture and centrifugated. The cell pellet was washed once to clear the excess dye completely and seeded on cultrex-caoted glass.
Staining of mitochondria and F-actin for fixed-cell based microscopy analysis
For fixed-cell based analysis, single-cell suspension of hNSCs was incubated with 100 nM Mito-Tracker Deep-Red FM (MT; Invitrogen, cat. #M22426) diluted in hNSC-medium. After 30min of incubation, cells were centrifuged e resuspended in fresh prewarmed hNSC-medium at least two times, left overnight in the hNSC-medium and centrifuged. The cell pellet was washed once to clear the excess dye completely and seeded on cultrex-coated glass.
To reduce the breakdown of TNT and maintain the fluorescence of MitoTracker Deep-Red, cells were fixed directly in the medium by adding formaldehyde to a final concentration of 3.7% v/v for 15min at 37°C. Afterward, samples were carefully washed with PBS, permeabilized by 0.3% Triton X-100 in PBS for 15min, blocked with 3% BSA in PBS, and incubated with AlexaFluor488- Phalloidin (1:500; Thermo Fisher, cat. #A12379) for 60min at room temperature in order to stain F-actin. After careful washing, the samples were mounted in Vectashield antifade mounting medium (Vector Laboratories cat. #H-1000-10).
Live-cells time lapse fluorescence microscopy
For live-cell imaging, single cells or neurospheres were seeded on confocal dishes (Greiner Bio-One, cat. #627870), and images were acquired by Biostation IM-Q at 37°C, humidity level of 95% and 5% CO2. To correctly visualize the mitochondria’ dynamics, images were acquired every 5–7 seconds with a low-intensity light source to reduce phototoxicity and photobleaching of the dye. Timelapse images were analyzed using NIS ELEMENTS AR software and FiJi software.
Confocal microscopy and Tau-STED microscopy of hNSCs in 2D-culture and sectioned neurospheres
Routine confocal microscopy was performed using a Leica TCS SP5 microscope. All acquisitions were performed by serial acquisition mode between frames. XYZ-series were acquired with a raster size of 1024x1024 pixels in the X–Y planes and a Z-step of 0.2 µm between optical slices. For super-resolution Tau-STED microscopy acquisitions Leica Stellaris 8 STED microscope was used.
Three dimensional (3D) images and projections from z-stack were constructed and processed using Leica Application Suite X software (LASX). Images were analyzed using LASX and FIJI software. The identification of TNTs between human neural stem cells and between hNSCs and differentiated SH-SY5Y was performed as already reported(25).
Measure of Nestin Cluster area and TNT-diameter
Super-resolved confocal X-Y images obtained using Leica Stellaris 8 STED microscope were analyzed for nestin cluster areas using Fiji software. TNTs-diameter were measured using confocal X-Y images obtained by Leica TCS SP5 microscope through the analysis of F-actin and Nestin fluorescence intensity profile (PSF) followed by the measure of the full width at half-maximum (FWHM).
Oxygen glucose deprivation/reoxygenation of differentiated SH-SY5Y and coculture with hNSCs
The day before the treatment, glucose- and serum-free DMEM (DMEM no glucose, Gibco cat. #11966025) was conditioned in a hypoxic gas mixture (see below) for 24 h (OGD medium), and differentiated SH-SY5Y were seeded on glass, coated with cultrex 1:50 for 1 hour at 37°C (BME, Pathclear cat #3432-005-01) with a seeding density of 2,5x10^4 cells/cm2. The day after seeding, differentiated SH-SY5Y were incubated with 1:500 Vybrant DiI Cell-Labelling Solution (Thermofisher, cat. #V22885) in complete growth media for 45 minutes. Then, cells were washed with glucose- and serum-free DMEM (DMEM no glucose, Gibco cat. #11966025) and left for one hour in incubator at 37°C. After this treatment cells were gently washed with OGD medium, placed in a hypoxic chamber conditioned with 0.2% O2, 5% CO2, 95% N2 (20 L/min for 4 min) in OGD medium, and incubated at 37°C for 24 h (OGD). Then, the cells were washed and reoxygenated with or without healthy hNSCs previously stained with the ∆Ψ-dependent MitoTracker Deep Red (hNSC-Mito) cocultured in direct contact or non-contact co-culture system. For the non-contact co-culture Corning Transwell® insets with 0.4 µm pore size membrane were used. Cocultures were placed in normoxic incubator at 37°C, 5%CO2 in hNSCs-medium for 24h (OGD/R).
Analysis of mitochondria activity
Normoxic and OGD/R differentiated SH-SY5Y were stained with AM 100nM Mito-Tracker Green (Invitrogen, cat. #M7514) and 100 nM ΔΨ-dependent MitoTracker Red CMXRos (Invitrogen, cat. #M7512) diluted in hNSC-medium for 30 minutes, carefully washed and images were acquired using Biostation IM-Q. The Mitotracker Red/Green fluorescent ratio was measured using Fiji software as semi-quantitative analysis of mitochondria activity (52, 53).
Caspase-3/7 activity assay
Cells were incubated with 1 µL of the CellEvent™Caspase-3/7Green detection reagent (Thermofisher, cat. #C10423) added directly in the 24-well in hNSC-medium for 30 min in incubator at 37°C, 5%CO2. After 30 min cells were fixed adding 37% formaldehyde to a final concentration of 3.7% at 37°C for 15 minutes. After the fixation cells were gently washed with PBS and immediately analysed. The quantification of apoptotic differentiated SH-SY5Y was performed by confocal microscopy using Leica TCS SP5 microscope as double positive DiI/Caspase 3–7 positive cells. Random areas were acquired per biological replicate, and apoptotic differentiated SH-SY5Y were counted as percentage of total differentiated SH-SY5Y.
Analysis of reactive oxygen species (ROS)
Cells were incubated with 5 µM CellROX Green Reagent (C10444, www.thermofisher.com) in hNSC-medium for 30 min at incubator at 37°C, 5%CO2. After 30 min cells were fixed adding 37% formaldehyde to a final concentration of 3.7% at 37°C for 15 minutes. After the fixation cells were gently washed with PBS and immediately analysed by confocal microscopy using Leica TCS SP5 microscope. ROS production was measured in DiI positive differentiated SH-SY5Y using FiJi software as mean fluorescence intensity per cell.
Whole-cell patch clamp analysis of differentiated SH-SY5Y
Whole-cell patch clamp experiments were conducted under the optical guidance of an Olympus B51WI microscope using the Multiclamp 700B amplifier (Axon CNS-Molecular Devices, Sunnyvale, CA, USA) interfaced with the Axon Digidata 1500 (Axon Instrument-Molecular Devices, Sunnyvale, CA, USA). Currents were sampled at 10 kHz and low-pass filtered at 5 kHz. Borosilicate patch pipettes were pulled to achieve tip resistances of 5–7 MΩ with a P-1000 Pipette puller (SUTTER INSTRUMENT, Novato, CA 94949, USA) and filled with a pipette solution containing (in mM): 130 K-gluconate, 10 NaCl, 1 CaCl2, 1 EGTA, 10 Hepes, 2 ATP-Na2, 2 MgCl2, with a pH of 7.2 adjusted with KOH and an osmolarity of 280 mmol/kg. The recordings were conducted at room temperature on SHSY5Y cells plated on Cultrex®-coated coverslips (15 mm Ø) using a bath solution containing (in mM): 140 NaCl, 2.8 KCl, 1 CaCl2, 0.01 EDTA, 10 Hepes, with a pH of 7.2 adjusted with NaOH and an osmolarity of 283–284 mmol/kg. The biophysical profiles of four different experimental conditions were investigated: i) Normoxic differentiated SHSY5Y cells; II) OGD/R differentiated SHSY5Y cells;
III) OGD/R SHSY5Y differentiated cells cocultured with hNSCs in direct contact; IV) OGD/R SHSY5Y differentiated cells cocultured with hNSCs in non-contact coculture.
The identification of differentiated SH-SY5Y in the direct contact co-culture was achieved through both a morphological evaluation of the two cell types and DiI fluorescence labelling of the differentiated SH-SY5Y before co-culturing. The co-colture experiments were performed with hNSCs obtained by three different spontaneously aborted fetuses.
Current-clamp experiments, in Gap -free mode without current injection, were performed to monitor the resting membrane potential. Additionally, depolarizing steps of current (of 500 ms in duration, with increments of 5 pA) were injected to investigate the cells' ability to generate action potentials. The criterion used to distinguish an evoked action potential was the presence of the overshoot in the evoked event. A voltage-clamp protocol composed of depolarizing steps of current of 10mv from − 100 mV to 70 mV, with a holding potential of -85 mV, was applied to investigate the evoked current. The recorded currents were normalized for the cell capacitance.
Experimental design and statistical analysis
All data represent at least three replicates from three independently hNSCs donors. Statistical analyses were conducted using GraphPad Prism 6 software (GraphPad Prism). All data are reported as the mean ± SEM. Statistically significant differences were computed using the Student’s t test for unpaired data and two-way Anova with Tukey’s multiple comparisons test for multiple statistical comparisons between groups. The significance level was set at p < 0.05.
Supplementary information is available at Cell Death and Disease’s website